RU2798990C2 - Agents for the treatment of claudine-expressing cancer diseases - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions including a bispecific agent that binds claudin 6 (CLDN6) and CD3, a recombinant nucleic acid, a host cell for the expression of a bispecific agent, the use of a bispecific agent, a recombinant nucleic acid or a host cell for the manufacture of a pharmaceutical composition for the treatment or prevention of cancer, characterized by cancer cells expressing CLDN6, a pharmaceutical composition for treating or preventing cancer characterized by cancer cells expressing CLDN6 and a method for treating or preventing a cancer characterized by cancer cells expressing CLDN6 are described.

EFFECT: invention expands the arsenal of agents binding to claudin 6 (CLDN6) and CD3.

21 cl, 54 dwg, 12 tbl, 34 ex

Description

Claudins are integral membrane proteins localized in tight junctions between epithelium and endothelium. It is assumed that claudins contain four transmembrane segments with two extracellular loops, and N- and C-termini located in the cytoplasm. Claudin (CLDN), a family of transmembrane proteins, plays an important role in maintaining epithelial and endothelial tight junctions and may also be involved in cytoskeletal maintenance and cell signaling.

The claudin 18 (CLDN18) molecule is an integral transmembrane protein (tetraspanin) with four transmembrane hydrophobic regions and two extracellular loops (loop 1 located between hydrophobic region 1 and hydrophobic region 2; loop 2 located between hydrophobic regions 3 and 4). CLDN18 exists in two different spliced variants that have been described in mice and humans (Niimi, Mol. Cell. Biol. 21:7380-90, 2001). Spliced variants (Genbank accession number: spliced version 1 (CLDN18.1): NP_057453, NM_016369, and spliced version 2 (CLDN18.2): NM_001002026, NP_001002026) have a molecular weight of approximately 27.9/27.72 kDa. Spliced variants of CLDN18.1 and CLDN18.2 differ in the N-terminal portion, which contains the first transmembrane (TM) region and loop 1, while the primary protein sequence at the C-terminus is identical.

There is no detectable expression of CLDN18.2 in normal tissues, with the exception of the stomach, where CLDN18.2 is expressed exclusively on short-lived differentiated gastric epithelial cells. Expression of CLDN18.2 is conserved during malignant transformation and thus is often expressed on the surface of human gastric cancer cells. Moreover, this pan-tumor antigen is largely ectopically activated in esophageal, pancreatic, and lung adenocarcinama. The CLDN18.2 protein is also localized in lymph node metastases of gastric adenocarcinoma and distant metastases, especially in the ovaries (so-called Krukenberg tumors).

CLDN6 is expressed in a number of different human cancer cells, with expression in normal tissues being restricted to the placenta.

The differential expression of claudins such as CLDN18.2 and CLDN6 in cancer and normal cells, their membrane localization, and the absence of toxicity to relevant normal tissues in the vast majority of them make these molecules attractive targets for cancer immunotherapy, and the use of drugs based on antibodies to target claudins in cancer therapy promises a high level of therapeutic specificity.

Approaches that exploit the potential of T cells for cancer treatment include vaccination with tumor-derived proteins; RNA or peptide antigens; infusion of tumor-derived, ex-vivo enriched T cells (referred to as adoptive transfer); transfer of the T-cell receptor gene or direct capture of T-cells by bi- or trispecific antibodies. Similarly, many T-cell response stimulants have been clinically tested in combination or as monotherapies, such as ligands for Toll-like receptors; antibodies that block CTLA-4 on T cells; cytokines that stimulate the immune response; or antibodies that neutralize molecules involved in cancer cells escaping the immune response, such as TGF-beta or B7-H1. The intensive development of T-cell based therapies is driven by the observation that patients seem to live significantly longer if their tumors are infiltrated with T cells. Moreover, numerous mouse models have shown that trapping T cells in various ways can eliminate even large tumors, and a number of T cell therapies have recently made significant progress in treating a variety of cancer symptoms.

The aim of the invention is to provide new agents and methods for cancer therapy.

The solution to the problem underlying the invention is based on the concept of creating a binding agent containing a binding domain that is specific for a tumor-associated claudin molecule, ie cancer cells. The other binding domain is specific for CD3, allowing binding to T cells and entry into the complex, thus allowing the cytotoxic effect of T cells to target cancer cells. The formation of this complex can induce signal transduction in cytotoxic T cells, either alone or in combination with A cells, resulting in the release of cytotoxic mediators.

The authors report for the first time that binding agents targeting claudin and CD3 can induce potent T cell-mediated lysis and are effective in the treatment of neoplastic diseases.

Brief description of the invention

In one aspect, the invention provides a binding agent comprising at least two binding domains, wherein the first binding domain binds to claudin and the second binding domain binds to CD3. The binding agent of the invention can bind to a cytotoxic cell (involving the CD3 receptor) and a cancer cell expressing CLDN, which is the target to be destroyed.

In one embodiment, the binding agent is a bispecific molecule, such as a bispecific antibody, in particular a bispecific single chain antibody. In one embodiment, said claudin is expressed in a cancer cell. In one embodiment, said claudin is expressed on the surface of a cancer cell. In one embodiment, said claudin is selected from the group consisting of claudin 18.2 and claudin 6. In one embodiment, said first binding domain binds to the extracellular domain of said claudin. In one embodiment, said first binding agent binds to native CLDN epitopes present on the surface of living cells. In one embodiment, said first binding domain binds to the first extracellular loop of the CLDN. In one embodiment, said second binding domain binds to the CD3 epsilon chain. In one embodiment, said CD3 is expressed on the surface of a T cell. In one embodiment, binding of said binding agent to CD3 on T cells results in proliferation and/or activation of said T cells, wherein said activated T cells preferentially release cytotoxic factors, e.g., perforins and granzymes, and initiate cytolysis and apoptosis of cancer cells. cells. In one embodiment, said claudin binding and/or said CD3 binding is a specific binding.

In one embodiment, the binding agent is in the form of a full length antibody or antibody fragment. In one embodiment, the binding agent comprises four antibody variable domains with at least two binding domains, wherein at least one binding domain binds to claudin and at least one binding domain binds to CD3. In one embodiment, the binding agent comprises an immunoglobulin heavy chain (VH) variable domain with specificity for claudin antigen (VH(CLDN)), an immunoglobulin light chain variable domain (VL) with specificity for claudin antigen (VL(CLDN)), variable an immunoglobulin heavy chain (VH) domain with specificity for CD3 (VH(CD3)); and an immunoglobulin light chain (VL) variable domain with specificity for CD3 (VL(CD3)).

In one embodiment, the binding agent is in the format of a diabody that contains a heavy chain variable domain coupled to a light chain variable domain on the same polypeptide chain such that the two domains do not pair. In one embodiment, the diabody contains two polypeptide chains, with one polypeptide containing VH(CLDN) and VL(CD3) and the other polypeptide chain containing VH(CD3) and VL(CLDN).

In one embodiment, the binding agent is in the format of a bispecific single chain antibody that consists of two scFv molecules connected via a linker peptide, with the heavy chain variable regions (VH) and the corresponding light chain variable regions (VL) preferably located in the direction from N- end-to-C-terminus in the order VH(CLDN)-VL(CLDN)-VH(CD3)-VL(CD3), VH(CD3)-VL(CD3)-VH(CLDN)-VL(CLDN) or VH(CD3 )-VL(CD3)-VL(CLDN)-VH(CLDN). In one embodiment, said heavy chain variable regions (VH) and corresponding light chain variable regions (VL) are connected via a long peptide linker, preferably a peptide linker containing the amino acid sequences (GGGGS)3 or VE(GGGGS)2GGVD. In one embodiment, said two VH-VL or VL-VH scFv units are connected via a short peptide linker, preferably a peptide linker containing the amino acid sequence SGGGGS or GGGGS.

In one embodiment, said CLDN is CLDN18.2 and said VH(CLDN) contains the amino acid sequence represented by SEQ ID NO: 8, or a fragment thereof, or a variant of said amino acid sequence or fragment, and the VL(CLDN) contains an amino acid sequence, represented by SEQ ID NO: 15, or a fragment thereof, or a variant of the specified amino acid sequence or fragment.

In one embodiment, said CLDN is CLDN18.2 and said VH(CLDN) contains the amino acid sequence represented by SEQ ID NO: 6, or a fragment thereof, or a variant of said amino acid sequence or fragment, and the VL(CLDN) contains an amino acid sequence, represented by SEQ ID NO: 11, or a fragment thereof, or a variant of the specified amino acid sequence or fragment.

In one embodiment, said CLDN is CLDN6 and said VH(CLDN) contains the amino acid sequence represented by SEQ ID NO: 22, or a variant thereof, or a variant of said amino acid sequence or fragment, and VL(CLDN) contains the amino acid sequence represented by SEQ ID NO: 23, or a fragment thereof, or a variant of the specified amino acid sequence or fragment.

In one embodiment, said CLDN is CLDN6 and said VH(CLDN) contains the amino acid sequence represented by SEQ ID NO: 22, or a fragment thereof, or a variant of said amino acid sequence or fragment, and VL(CLDN) contains the amino acid sequence represented by SEQ ID NO: 97, 98, 99 or 100, or a fragment thereof, or a variant of the specified amino acid sequence or fragment.

In one embodiment, the specified VH(CD3) contains the amino acid sequence represented by SEQ ID NO: 36, 94 or 95, or a fragment thereof, or a variant of the specified amino acid sequence or fragment, and VL(CD3) contains the amino acid sequence represented by SEQ ID NO: 37 or 96, or a fragment thereof, or a variant of the specified amino acid sequence or fragment.

In one embodiment, the specified VH(CD3) contains the amino acid sequence represented by SEQ ID NO: 36, or a fragment thereof, or a variant of the specified amino acid sequence or fragment, and VL(CD3) contains the amino acid sequence represented by SEQ ID NO: 37, or its fragment, or variant of the specified amino acid sequence or fragment.

In one embodiment, said VH(CD3) contains the amino acid sequence represented by SEQ ID NO: 95, or a fragment thereof, or a variant of said amino acid sequence or fragment, and VL(CD3) contains the amino acid sequence represented by SEQ ID NO: 96, or a fragment thereof , or a variant of the specified amino acid sequence or fragment.

In one aspect, the binding agent of the invention is presented in the format of a bispecific single chain antibody that contains two scFv molecules connected via a linker peptide, with the heavy chain variable regions (VH) and the corresponding light chain variable regions (VL) located in the direction from the N-terminus to the C-terminus in the order VH(CLDN)-VL(CLDN)-VH(CD3)-VL(CD3). In one embodiment, said VH(CD3) and VL(CD3) are linked via a peptide linker consisting of 15-20, preferably 15 or 20 amino acids, preferably glycine and/or serine, and preferably linked via a peptide linker containing the amino acid sequence (GGGGS )4. In one embodiment, said VH(CLDN) and VL(CLDN) are connected via a peptide linker consisting of 15-20, preferably 15 or 20 amino acids, preferably glycine and/or serine, and preferably connected via a peptide linker containing the amino acid sequence (GGGGS )4. In one embodiment, the two VH-VL scFv units are connected via a linker peptide containing the amino acid sequence SGGGGS. One or both of these VH-VL scFv units may contain one or more linking disulfide bridges.

In one aspect, the binding agent of the invention is in the format of a bispecific single chain antibody that contains two scFv molecules connected via a linker peptide, with the heavy chain variable regions (VH) and the corresponding light chain variable regions (VL) located in the direction from the N-terminus to the C-terminus in the order VL(CLDN)-VH(CLDN)-VH(CD3)-VL(CD3). In one embodiment, said VH(CD3) and VL(CD3) are linked via a peptide linker consisting of 15-20, preferably 15 or 20 amino acids, preferably glycine and/or serine, and preferably linked via a peptide linker containing the amino acid sequence (GGGGS )4. In one embodiment, said VL(CLDN) and VH(CLDN) are linked via a peptide linker consisting of 15-20, preferably 15 or 20 amino acids, preferably glycine and/or serine, and preferably linked via a peptide linker containing the amino acid sequence (GGGGS )5. In one embodiment, said VL-VH and VH-VL scFv units are connected via a linker peptide containing the amino acid sequence SGGGGS. One or both of these two units VL-VH or VH-VL scFv may contain one or more linking disulfide bridges.

In one aspect, the binding agent of the invention is in the format of a bispecific single chain antibody that contains two scFv molecules connected via a linker peptide, with the heavy chain variable regions (VH) and the corresponding light chain variable regions (VL) located in the direction from the N-terminus to the C-terminus in the order VH(CLDN)-VL(CLDN)-VL(CD3)-VH(CD3). Preferably, said VL(CD3)-VH(CD3) scFv unit contains one or more linking disulfide bridges. In one embodiment, said VL(CD3) and VH(CD3) are linked via a peptide linker consisting of 20-25, preferably 20 or 25 amino acids, preferably glycine and/or serine, and preferably linked via a peptide linker containing the amino acid sequence (GGGGS )5. In one embodiment, said VH(CLDN) and VL(CLDN) are linked via a peptide linker consisting of 15-20, preferably 15 or 20 amino acids, preferably glycine and/or serine, and preferably linked via a peptide linker containing the amino acid sequence (GGGGS )4. In one embodiment, said VH-VL and VL-VH scFv units are connected via a linker peptide containing the amino acid sequence SGGGGS. Said VH(CLDN)-VL(CLDN) scFv unit may contain one or more linking disulfide bridges.

In one aspect, the binding agent of the invention is in the format of a bispecific single chain antibody that contains two scFv molecules connected via a linker peptide, with the heavy chain variable regions (VH) and the corresponding light chain variable regions (VL) located in the direction from the N-terminus to the C-terminus in the order VL(CLDN)-VH(CLDN)-VL(CD3)-VH(CD3). Preferably, said VL(CD3)-VH(CD3) scFv unit contains one or more linking disulfide bridges. In one embodiment, said VL(CD3) and VH(CD3) are linked via a peptide linker consisting of 20-25, preferably 20 or 25 amino acids, preferably glycine and/or serine, and preferably linked via a peptide linker containing the amino acid sequence (GGGGS )5. In one embodiment, said VL(CLDN) and VH(CLDN) are connected via a peptide linker consisting of 20-25, preferably 20 or 25 amino acids, preferably glycine and/or serine, and preferably connected via a peptide linker containing the amino acid sequence (GGGGS )5. In one embodiment, the two VL-VH scFv units are connected via a linker peptide containing the amino acid sequence SGGGGS. Said link VL(CLDN)-VH(CLDN) scFv may contain one or more linking disulfide bridges.

In one embodiment of any of the above aspects, said CLDN is CLDN18.2. Preferably, said VH(CLDN) contains the amino acid sequence represented by SEQ ID NO: 8, or a fragment thereof, or a variant of said amino acid sequence or fragment, and VL(CLDN) contains the amino acid sequence represented by SEQ ID NO: 15, or a fragment thereof, or a variant of said amino acid sequence or fragment. Alternatively, the specified VH(CLDN) contains the amino acid sequence represented by SEQ ID NO: 6, or a fragment thereof, or a variant of the specified amino acid sequence or fragment, and VL(CLDN) contains the amino acid sequence represented by SEQ ID NO: 11, or a fragment thereof, or a variant of said amino acid sequence or fragment. In one embodiment, the specified VH(CD3) contains the amino acid sequence represented by SEQ ID NO: 95, or a fragment thereof, or a variant of the specified amino acid sequence or fragment, and VL(CD3) contains the amino acid sequence represented by SEQ ID NO: 96, or its fragment, or variant of the specified amino acid sequence or fragment.

In one aspect, the binding agent of the invention is in the format of a bispecific single chain antibody that contains two scFv molecules connected via a linker peptide, with the heavy chain variable regions (VH) and the corresponding light chain variable regions (VL) located in the direction from the N-terminus to the C-terminus in the order VH(CLDN)-VL(CLDN)-VH(CD3)-VL(CD3); or in the order VH(CD3)-VL(CD3)-VH(CLDN)-VL(CLDN). In one embodiment, said VH(CLDN) and VL(CLDN) are connected via a peptide linker consisting of 15-20, preferably 15 or 20 amino acids, preferably glycine and/or serine, and preferably connected via a peptide linker containing the amino acid sequence (GGGGS )3. In one embodiment, said VH(CD3) and VL(CD3) are linked via a peptide linker consisting of 15-20, preferably 15 or 20 amino acids, preferably glycine and/or serine, and preferably linked via a peptide linker containing the amino acid sequence GGGGS( GGS-3GGGS. In one embodiment, the two VH-VL scFv units are connected via a linker peptide containing the amino acid sequence SGGGGS.

In one embodiment of the above aspect, said CLDN is CLDN6. Preferably, the specified VH(CLDN) contains the amino acid sequence represented by SEQ ID NO: 22, or a fragment, or a variant of the specified amino acid sequence or fragment. Preferably, the specified VL(CLDN) contains the amino acid sequence represented by SEQ ID NO: 98, 99 or 100, or a fragment, or a variant of the specified amino acid sequence or fragment. Most preferably, the specified VL(CLDN) contains the amino acid sequence represented by SEQ ID NO: 99, or a fragment, or a variant of the specified amino acid sequence or fragment. In one embodiment, the specified VH(CD3) contains the amino acid sequence represented by SEQ ID NO: 95, or a fragment thereof, or a variant of the specified amino acid sequence or fragment, and VL(CD3) contains the amino acid sequence represented by SEQ ID NO: 96, or its fragment, or variant of the specified amino acid sequence or fragment.

In one embodiment, said CLDN is CLDN18.2 and said binding agent of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40, and 41, or fragments or variants thereof.

In one embodiment, said CLDN is CLDN18.2 and said binding agent of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 103, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, and 93, or a fragment or variant thereof. In one embodiment, said CLDN is CLDN18.2 and said binding agent comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 103, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 and 93, or a fragment or variant thereof, wherein the specified amino acid sequence lacks secretion signals, such as N-terminal secretion signals, especially the sequence according to SEQ ID NO: 51, and/or lacking His tags, such as C-terminal His tags, especially the sequence Gly-Gly-Ser-( His) ₆ or (His) ₆ if present.

In one embodiment, said CLDN is CLDN6 and said binding agent of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44 and 45, or a fragment or variant thereof.

In one embodiment, said CLDN is CLDN6 and said binding agent of the invention comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 101, 102, 60, 61, 62, 63, 64, and 65, or a fragment thereof, or option. In one embodiment, said CLDN is CLDN6 and said binding agent comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 101, 102, 60, 61, 62, 63, 64, and 65, or a fragment or variant thereof, wherein said amino acid sequence is devoid of secretion signals, such as N-terminal secretion signals, in particular the sequence according to SEQ ID NO: 51, and/or is devoid of His tags, such as C-terminal His tags, in particular the sequence Gly -Gly-Ser-(His) ₆ or (His) ₆ if present.

In one embodiment, said cancer cells expressing CLDN18.2 are cancer cells selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), breast cancer , ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer, and their metastases, Krukenberg tumor, peritoneal metastases and / or metastases to the lymph nodes.

In one embodiment, said cancer cells expressing CLDN6 are cancer cells of a cancer selected from the group consisting of bladder cancer; ovarian cancer, in particular ovarian adenocarcinoma and ovarian teracarcinoma; lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), in particular squamous cell lung cancer and adenocarcinoma; stomach cancer; breast cancer; liver cancer; pancreatic cancer; skin cancer, in particular basal cell carcinoma and squamous cell carcinoma; malignant melanoma; head and neck cancer, in particular malignant pleomorphic adenoma; sarcomas, in particular synovial sarcomas and carcinosarcomas; bile duct cancer; bladder cancer, in particular transitional cell carcinoma and papillary cancer; kidney cancer, in particular squamous cell cancer, including clear cell kidney cancer and papillary carcinoma of the kidney; colon cancer; cancer of the small intestine, including cancer of the ileum, in particular adenocarcinoma of the small intestine and adenocarcinoma of the ileum; embryonic testicular carcinoma; placental choriocarcinoma; cervical cancer; testicular cancer, in particular testicular seminoma, testicular teratoma and fetal testicular cancer; uterine cancer; germinomas such as teratocarcinoma or embryonic carcinoma, in particular testicular germ cell tumors and their metastatic forms.

In one embodiment, the binding agent comprises an N-terminal secretion signal and/or a C-terminal his-tag epitope tag, preferably a six-histidine epitope tag.

In one aspect, the invention provides a recombinant nucleic acid that encodes a binding agent of the invention. In one embodiment, the recombinant nucleic acid is in the form of a vector. In one embodiment, the recombinant nucleic acid is RNA.

In one aspect, the invention relates to a host cell containing a recombinant nucleic acid according to the invention.

In one aspect, the invention provides a binding agent of the invention, a recombinant nucleic acid of the invention, or a host cell of the invention for use in therapy, in particular for use in the treatment and prevention of cancer.

In one aspect, the invention relates to a pharmaceutical composition comprising a binding agent of the invention, a recombinant nucleic acid of the invention, or a host cell of the invention.

In one aspect, the invention relates to a method for treating or preventing a cancer comprising administering to a patient a pharmaceutical composition of the invention.

In one embodiment, said cancer cells express claudin to which said binding agent is able to bind.

In one embodiment, said claudin is CLDN18.2 and said cancer is selected from the group consisting of gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), breast cancer, ovarian cancer, colon cancer, liver cancer, head and neck cancer, gallbladder cancer and their metastases, Krukenberg tumor, peritoneal metastases and / or metastases to the lymph nodes.

In one embodiment, said claudin is CLDN6 and said cancer is selected from the group consisting of bladder cancer; ovarian cancer, in particular ovarian adenocarcinoma and ovarian teracarcinoma; lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), in particular squamous cell lung cancer and adenocarcinoma; stomach cancer; breast cancer; liver cancer; pancreatic cancer; skin cancer, in particular basal cell carcinoma and squamous cell carcinoma; malignant melanoma; head and neck cancer, in particular malignant pleomorphic adenoma; sarcomas, in particular synovial sarcomas and carcinosarcomas; bile duct cancer; bladder cancer, in particular transitional cell carcinoma and papillary cancer; kidney cancer, in particular squamous cell carcinoma, including clear cell carcinoma of the kidney and papillary carcinoma of the kidney; colon cancer; cancer of the small intestine, including cancer of the ileum, in particular adenocarcinoma of the small intestine and adenocarcinoma of the ileum; embryonic testicular carcinoma; placental choriocarcinoma; cervical cancer; testicular cancer, in particular testicular seminoma, testicular teratoma and fetal testicular cancer; uterine cancer; germinomas such as teratocarcinoma or embryonic carcinoma, in particular testicular germ cell tumors and their metastatic forms.

In one aspect, the invention provides a binding agent or nucleic acid encoding a binding agent or host cell as described herein for use in the methods of treatment described herein. In one embodiment, the invention provides a pharmaceutical composition described herein for use in the methods of treatment described herein.

According to the invention, CLDN18.2 preferably contains the amino acid sequence according to SEQ ID NO: 1, and CLDN6 preferably contains the amino acid sequence according to SEQ ID NO: 2 or 3.

Other features and advantages of the present invention will be apparent from the following detailed description and claims.

Brief description of the drawings

FIGURE 1. Block diagram illustrating the design of recombinant bi-scFv proteins targeting CLDN18.2 TAA.

Design of bi-scFvs at (A) N-terminal and (B) C-terminal positions in relation to anti-TAA variable regions. The V _H and V _L regions of anti-CLDN18.2 are based on the sequence of the monoclonal antibody CLDN18.2 (mCLDN18.2ab). Anti-CD3 is shared by the V _H and V _L regions generated from the following anti-CD3 monoclonal antibody sequences: UCHT1-HU (humanized mAB), UCHT1, CLB-T3, TR66, 145-2C11. Bi-scFv is a bispecific single chain variable fragment; His, a histidine label containing 6 histidine residues; HU, humanized; LL, long linker (15-18 amino acids); Sec, secretion signal; SL, short linker (5-6 amino acids); TAA, tumor-associated antigen; V, heavy (H) and light (L) chain variable region of an antibody.

FIGURE 2. Effect of domain orientation and anti-CD3-scFv selection on specific target cell lysis: 5'-mCLDN18.2ab ^V _H ^-V _{L_} TR66 ^V _H ^-V _L -3' 1BiMAB bi-scFvs and no.15 are the most strong options.

Several bi-scFv variants directed against CLDN18.2 and CD3 were transiently expressed in HEK293T cells and purified on a small scale on Ni-NTA columns to compare their potency in a cytotoxicity assay. NugC4 cells endogenously expressing CLDN18.2 and stably expressing luciferase were used as target cells. Human T cells and target cells were incubated at a 5:1 E:T ratio with 5 ng/ml of each bi-scFv protein in a 96-well format. No.35, which targets unexpressed TAA, no.l1 and no.16, both targeting murine but not human T cells, were used as negative controls. Each test sample was sown in six replications, the control sample for L _min was sown in nine replications. The co-incubation time before the start of the analysis was 8 h, 16 h, and 24 h. After the addition of the luciferin solution at the given time points, the luminescence was measured on an Infinite M200 TECAN reader. Specific lysis of target cells was calculated by normalization to samples with control no.35 (L _min ) bi-scFv. The strongest bi-scFv proteins, 1BiMAB and no.l5, shared the same domain orientation and origin of anti-CD3 mAB TR66, but differed in their codon optimization (HS and CHO, respectively) and long linker sequences. CHO stands for Chinese Hamster Ovary Cells; mAB, monoclonal antibody; HU, humanized; TAA, tumor-associated antigen.

FIGURE 3. Coomassie staining and Western blot analysis of the 1BiMAB bi-scFv protein.

The FCS-free supernatant of HEK293 monoclonal cells stably expressing 1BiMAB was purified by affinity chromatography (IMAC) on a Ni-NTA column. Aliquots of the various purification steps were loaded onto 4-12% Bis-Tris gels. (A) Coomassie staining of cell supernatant, passage and eight eluate fractions. Fractions of the first elution peak were removed, fractions of the second elution peak were pooled for subsequent studies, dialyzed against PBS and then against 200 mm arginine buffer. (Lane 1: ΗΕΚ293/1ΒiΜΑΒ SN; lane 2: IMAC flow-through fraction; lanes 3-4: Elution peak fractions 1 (removed); lanes 5-10: Elution peak fractions 2 (combined)); (B) Western blot analysis of 0.5 g 1BiMAB from three independent purifications (lane 1, 2, 3). Detection was performed with a primary anti-His monoclonal and a peroxidase-conjugated secondary anti-mouse antibody. IMAC stands for immobilized metal affinity chromatography; PBS, phosphate buffered saline; SN, supernatant; WB, Western blotting.

FIGURE 4. Bi-scFv 1BiMAB protein binds efficiently and specifically to CLDN18.2-expressing target cells and human T cells.

(A) 2.5×10 ⁵ NugC4 cells endogenously expressing CLDN18.2 were incubated with 50 μg/ml 1BiMAB or 10 μg/ml mCLDN18.2ab as a positive control and the corresponding APC-conjugated secondary antibodies. Control stains included only secondary APC-conjugated antibodies (gah, gam), anti-His and gam APC, or 1BiMAB and gam APC. Analysis was performed by flow cytometry. The MFI of the APC signal was calculated using the FlowJo software. (B) l×l0 ⁵ NugC4 cells endogenously expressing CLDN18.2 were stained with increasing concentrations of 1BiMAB (20 pg/ml - 20 μg/ml), anti-His and gam APC. As a negative control, cells were incubated with anti-His and gam APC. mCLDN18.2ab and gah APC were used as positive controls. The MFI of the APC signal was calculated using the FlowJo software. (C) l×l0 ⁶ human T cells were incubated with increasing concentrations of 1BiMAB (2 ng/ml - 2 μg/ml), anti-His and gam APC. As a negative control, cells were incubated with anti-His and gam APC or gam APC alone. The MFI of the APC signal was calculated using the FlowJo software. (D) l×l0 ⁵ CLDN18.2-negative PA-1 cells were incubated with increasing concentrations of 1BiMAB (10 ng/ml - 10 μg/ml), anti-His and gam APC. As a negative control, cells were stained with anti-His and gam APC or gah APC alone. 10 μg/ml mCLDN18.2ab and gah APC were used to confirm cells were negative for CLDN18.2. Gah means goat-anti-human; gam, goat-anti-mouse; MFI, mean fluorescence intensity; TL, T-lymphocyte.

FIGURE 5. Bi-scFv 1BiMAB protein induces T cell clustering on CLDN18.2 positive target cells.

NugC4 cells endogenously expressing CLDN18.2 were incubated for 24 h with 1 ng/mL and 1 μg/mL 1BiMAB and human T cells at a 5:1 effector to target ratio in 6-well plates. Only T cells (TL), only target cells (NugC4) and human T cells with target cells (-Control) were selected as controls. After 24 hours, the samples were photographed using a Nikon Eclipse Ti microscope at a magnification of 200x. White arrows indicate clusters of T cells on target cells. TL stands for T lymphocyte.

FIGURE 6. 1BiMAB mediates T-cell activation in a dose-dependent manner.

NugC4 cells endogenously expressing CLDN18.2 were incubated for 24 h and 48 h with increasing concentrations of 1BiMAB bi-scFv protein (0.001 - 1000 ng/mL) and human T cells at a 5:1 effector to target ratio in duplicate in 24-well format. As a control, human T cells were incubated with 1-1000 ng/ml 1BiMAB without NugC4 target cells to confirm target dependent T cell activation mediated by 1BiMAB. After 24 hours (A) and 48 hours (B), T cells were harvested and labeled with anti-CD3-FITC, anti-CD25-PE and anti-CD69-APC and analyzed by flow cytometry. TL stands for T-lymphocyte.

FIGURE 7. 1BiMAB mediates highly target-dependent T cell activation, even after prolonged incubation with high, low, and non-CLDN18.2-expressing cell lines.

(A) Shown are RT-PCR data obtained from total RNA from six tumor cell lines. Ct values of CLDN18.2 expression normalized to the housekeeping gene HPRT were calculated from two independent experiments. The breast cancer cell line MCF7 (gray bar) not expressing CLDN18.2 was chosen as a control. (B) Cancer cell lines (A) were incubated for 144 h with 5 ng/ml 1BiMAB bi-scFv protein with or without human T cells in a 5:1 effector to target ratio in duplicate in a 6-well format. T cells were labeled with anti-CD3-FITC, anti-CD25-PE and anti-CD69-APC to analyze the total population of T cells (CD3), early activation (CD69) and late activation (CD25) T cells by flow cytometry. TL stands for T-lymphocyte.

FIGURE 8. 1BiMAB induces T cell proliferation and upregulation of granzyme B only in the presence of CLDN18.2 positive target cells.

(A) Human T cells were stained with CFSE and grown alone (TL) or in the presence of 1 ng/ml 1BiMAB (TL + 1 ng/ml 1BiMAB), NugC4 cells (TL + NugC4), or NugC4 cells and 1 ng/ml 1BiMAB (TL + 1 ng/ml 1BiMAB + NugC4) for 120 h. An effector to target ratio of 5:1 was chosen. Decreased CFSE signal indicative of T cell proliferation was analyzed by flow cytometry. (B) Human T cells were incubated with or without NugC4 target cells and with or without 5 ng/ml bi-scFv 1BiMAB protein. The effector to target ratio was 5:1 in a 6-well format. After 96 hours of co-incubation, T cells were harvested and intracellularly stained with anti-GrB-PE and analyzed by flow cytometry. MFI signal of anti-GrB-PE was calculated using FlowJo software. The signal of the unstained sample TL + NugC4 + 5 ng/ml 1BiMAB was subtracted from all samples. CFSE is an ester of succinimidyl and carboxyfluorescein; GrB, granzyme B; MFI, mean fluorescence intensity; PE, phycoerythrin; TL, T-lymphocytes.

FIGURE 9. The value of EC ₅₀ 1BiMAB for specific lysis of target cells after 48 h was approximately 10 pg/ml.

NugC4 cells endogenously expressing CLDN18.2, which stably express luciferase, were incubated for 24 h and 48 h with 1BiMAB bi-scFv protein at increasing concentrations (0.001-1000 ng/mL) with human T cells at an effector to target ratio of 5 :1 in triplicate in 96-well format. As minimal lysis (L _min ), control effector cells and target cells were seeded without 1BiMAB bi-scFv. Maximum lysis (L _max ) to normalize to spontaneous number of luminescence pulses was achieved by adding Triton X-100 to control wells containing effector and target cells in the absence of bi-scFv just prior to the addition of luciferin. After adding _the luciferin solution, luminescence was measured on an Infinite M200 Tecan microplate _reader after 24 _h and 48 _h . )] × 100. Values were plotted as a function of log 10 1BiMAB concentration. The value of EC ₅₀ means half of the maximum effective concentration; L, lysis.

FIGURE 10. 1BiMAB shows in vivo therapeutic efficacy in an advanced SC tumor model.

NOD.Cg-Prkdscid IL2rgtmlWjl/Sz.T (NSG) mice were injected subcutaneously (SC) with l×l0 ⁷ HEK293 cells stably expressing CLDN18.2. Five days later, human PBMCs as effector cells were injected intraperitoneally (IP) at 2×l0 ⁷ to groups G3 and G4, control groups (Gl and G2) received only PBS. The next day, daily IP administration of 5 μg bi-scFv 1BiMAB protein was started in each animal or vehicle as a control. Therapy was carried out for 22 days, tumor volume was measured using a caliper and calculated by the formula mm ³ = length mm × width mm × (width mm/2). (A) Single mouse tumor volume and mean per group are shown for treatment days 0 and 15 (top row), and days 3 and 13 post-treatment (bottom row). (B) Average tumor volume of two treatment groups inoculated with human effector cells is shown. A dash indicates dead animals. (C) Kaplan-Meier survival curve showing all groups from day of tumor inoculation to day 41. Animals were sacrificed after reaching a tumor volume of 500 mm ³ . After day 41, the remaining animals were sacrificed for engraftment assay of human effector cells in mouse spleens. (D) Splenocytes from all mice were isolated and stained with anti-CD45-APC and anti-CD3-FITC to detect human T cells by flow cytometry. The median engraftment is displayed on a boxplot. G means group; IP, intraperitoneally; PBMC, peripheral blood mononuclear cells; PBS, phosphate buffered saline; SC, subcutaneously.

FIGURE 11. Block diagram illustrating the design of recombinant bi-scFv proteins targeting CLDN6 TAA.

Design of bi-scFvs at (A) N-terminal (B) C-terminal position in relation to anti-TAA variable regions. The V _H and V _L regions of anti-CLDN6 are based on the sequence of the CLDN6 monoclonal antibody (mCLDN6ab). The V _H and V _L regions of anti-CD3 are based on the sequence of the monoclonal antibody CD3 TR66. Bi-scFv is a bispecific single chain variable fragment; His, a histidine tag containing six histidine residues; LL, long linker (15-18 amino acids); Sec, secretion signal; SL, short linker (5 amino acids); TAA, tumor-associated antigen; V, heavy (H) and light (L) chain variable region of an antibody.

FIGURE 12. Proteins 6PHU5 and 6PHU3 bi-scFv cause the formation of clusters of T cells on CLDN6-positive target cells.

PA-1 cells endogenously expressing CLDN6 were incubated for 24 h with 50 ng/ml 6PHU5 or 6PHU3 and human T cells at a 5:1 effector to target ratio in 6-well plates. Only T cells (TL), only target cells (PA-1) and human T cells with target cells (-contr) were selected as controls. After 24 hours, images of the samples were taken on a Nikon Eclipse Tj microscope at a magnification of 200×. White arrows indicate T-cell clusters on target cells. TL stands for T-lymphocytes.

FIGURE 13. Effect of domain targeting on efficiency: bi-scFv protein 6PHU3 is slightly more efficient in inducing T cell activation compared to 6PHU5.

PA-1 cells endogenously expressing CLDN6 were incubated for 44 h with increasing concentrations (5-200 ng/mL) of 6PHU5 or 6PHU3 and human T cells in a 5:1 effector to target ratio in duplicate in a 6-well format. As controls, human human T cells were incubated with 100 and 200 ng/ml 6PHU5 or 6PHU3 without target cells. After 44 hours, T cells were harvested and labeled with anti-CD3-FITC, anti-CD25-PE and anti-CD69-APC. Dose-dependent T-cell activation was analyzed by flow cytometry. Hu means human; TL, T-lymphocytes.

FIGURE 14. Coomassie staining and Western blot analysis of the 6PHU3 protein.

The FCS-free supernatant of HEK293 polyclonal cells stably expressing 6PHU3 was purified by Ni-NTA affinity chromatography (IMAC). Aliquots of the various purification steps were applied to 4-12% Bis-Tris gels. (A) Coomassie staining of cell supernatant, passage and nine eluate fractions. Fractions of the first elution peak were removed, fractions of the second and third elution peaks were combined for further studies, dialyzed against PBS and then against 200 mm arginine buffer. (Lane 1: HEK293/6PHU3 SN; lane 2: IMAC flow-through fractions; lanes 3-5: Elution peak fractions 1 (removed); lanes 6-11: Elution peak fractions 2 and 3 (combined)) (B) Western blot analysis of 0.5 μg 6PHU3 from two independent purifications. Detection was performed with a primary anti-His monoclonal and a peroxidase-conjugated secondary anti-mouse antibody. IMAC stands for immobilized metal affinity chromatography; PBS; phosphate buffered saline; SN, supernatant; WB, Western blotting.

FIGURE 15. The 6PHU3 bi-scFv protein binds efficiently and specifically to CLDN6-expressing target cells and human T cells.

(A) 1xl0 5 PA- ¹ cells endogenously expressing CLDN6 and OV-90 cells were incubated with increasing concentrations of 6PHU3 or control 1BiMAB bi-scFv (10 ng/mL - 10 μg/mL) and 10 μg/mL mCLDN6ab or control mAB mCLDN18.2ab with the appropriate APC-conjugated secondary antibodies. Control stains were only secondary APC-conjugated antibodies (gah, gam). The analysis was performed using flow cytometry. The MFI of the APC signal was calculated using the FlowJo software. (B) 5×l0 ⁵ human T cells were incubated with increasing concentrations of 6PHU3 (100 ng/ml - 10 μg/ml), anti-His and gam PE. As a negative control, cells were incubated with anti-His and gam PE or gam PE alone. The MFI of the PE signal was calculated using the FlowJo software. (C) l×l0 ⁵ CLDN6-negative NugC4 cells were incubated with increasing concentrations of 6PHU3 and 1BiMAB (10 ng/ml - 10 μg/ml), anti-His and gam APC. As a negative control, cells were incubated with gam APC alone. 10 μg/ml mCLDN6ab and gah APC were used to confirm the negativity of CLDN6 cells. mCLDN18.2ab and gah APC were used as positive controls. The MFI of the APC signal was calculated using the FlowJo software. APC means allophycocyanin; gah, goat-anti-human; gam, goat-anti-mouse; mAB, monoclonal antibody; MFI, mean fluorescence intensity; PE, phycoerythrin; TL, T-lymphocyte.

FIGURE 16. 6PHU3 mediates T cell activation in a dose-dependent manner

PA-1 cells endogenously expressing CLDN6 were incubated for 24 h and 48 h with increasing concentrations of 6PHU3 bi-scFv protein (0.001 - 1000 ng/mL) and human T cells at a 5:1 effector-to-target ratio in duplicate. 24-well format. As a control, human T cells were incubated with 1-1000 ng/ml 6PHU3 without PA-1 target cells to confirm target dependent T cell activation mediated by 6PHU3. After 24 hours (A) and 48 hours (B), T cells were harvested and labeled with anti-CD3-FITC, anti-CD25-PE and anti-CD69-APC and analyzed by flow cytometry. TL stands for T-lymphocytes.

FIGURE 17. The EC50 value of 6PHU3 for specific target cell lysis after 48 hours was approximately 10 pg/ml.

PA-1 cells endogenously expressing CLDN6, which stably express luciferase, were incubated for 24 h and 48 h with 6PHU3 protein at increasing concentrations (0.001–1000 pg/mL) with human T cells in an effector to target ratio of 5:1 in three replicates in 96-well format. As a minimum lysis control (L _min ), effector and target cells were seeded without 6PHU3 bi-scFv. Maximum lysis (L _max ) to normalize in terms of the number of spontaneous luminescence pulses was achieved by adding Triton X-100 to control wells containing effector and target cells in the absence of bi-scFv just prior to the addition of lyciferin. After addition of _the luciferin solution, luminescence was measured on an Infinite M200 Tecan _microplate reader after 24 _h and 48 _h . )] × 100. Values were plotted as a function of log10 6PHU3 concentration. The EC50 value means half of the maximum effective concentration; L, lysis.

FIGURE 18. 6PHU3 shows in vivo therapeutic efficacy in an advanced stage SC tumor model.

Mice NOD.Cg-Prkd ^scid IL2rg ^tm1Wj1 /SzJ (NSG) were injected subcutaneously (SC) with l×l0 ⁷ PA-1 cells endogenously expressing CLDN6. After 15 days, groups G3 and G4 were intraperitoneally (IP) injected with human PBMC cells in an amount of 2×l0 ⁷ , control groups (Gl and G2) received only PBS. Daily IP administration of 5 μg of 6PHU3 to each animal, either 1BiMAB bi-scFv control or vehicle control alone, was started five days after PBMC injection. The therapy was carried out for 25 days, the tumor volume was measured using a caliper and calculated by the formula mm ³ = length, mm × width, mm × (width, mm/2). (A) Single mouse tumor volume and mean per group are shown for days 0 and 14 of therapy (top row), and 21 and 25 (bottom row). (B) Average tumor volume of all treatment groups is shown. Dotted lines represent slaughtered animals. (C) The Kaplan-Meier survival curve is shown for all groups from day of tumor inoculation to day 45. Animals were sacrificed at tumor volume >1500 mm ³ . After 45 days, all remaining animals were sacrificed for engraftment assay of human effector cells in mouse spleens. (D) Splenocytes from all mice were isolated and stained with anti-CD45-APC and anti-CD3-FITC to detect human T cells by flow cytometry. The median engraftment is shown in the box plot. IP means intraperitoneally; PBMC, peripheral blood mononuclear cells; PBS, phosphate buffered saline; SC, subcutaneously.

FIGURE 19. Increased T cell infiltration in SC PA-1 tumors in response to 6PHU3 therapy.

NSG mice were injected subcutaneously (SC) with 1×10 ⁷ PA-1 cells endogenously expressing CLDN6. After 15 days, groups G3 and G4 were intraperitoneally (IP) injected with 2×10 ⁷ human PBMC cells, control groups (Gl and G2) received only PBS. Daily IP administration of 5 μg of 6PHU3 to each animal, either 1BiMAB bi-scFv control or vehicle control alone, was started five days after PBMC injection. Tumors were excised at 1500 mm ³ or at the end of the experiment, and preserved in 4% formaldehyde buffer for paraffin embedding.

Paraffin-embedded tumor tissues of SC PA-1 tumors were subjected to immunohistochemical stainings. Serial sections were stained with anti-Claudin 6 polyclonal primary antibody or anti-human CD3. Primary antibodies were detected using secondary anti-rabbit antibodies conjugated with HRP peroxidase. Top rows A-E show CLDN6 staining, bottom rows CD3 staining. Images were obtained using a Mirax scanner. (A) and (B) show Gl and G2 PBS control groups that received no human effector cells and received vehicle or 6PHU3 bi-scFv, respectively, (C) shows G3 control group that received human effector cells and vehicle as therapy, (D) shows the G4 group that received human effector cells and 6PHU3 bi-scFv as therapy, and (E) shows the G5 control group that received human effector cells and control 1BiMAB bi-scFv. Positive signals appear as red coloration. Black arrows indicate examples of CD3 signals. IP means intraperitoneally; PBMC, peripheral blood mononuclear cells; PBS, phosphate buffered saline; SC, subcutaneously.

FIGURE 20. Schematic representation of IVT-RNA molecules encoding bi-scFv antibodies that target CLDN18.2 TAA.

In vitro scheme of transcribed RNA sequences encoding bi-scFv anti-CLDN18.2 antibodies. (A) IVT mRNA at the 5' and 3' positions relative to the anti-TAA variable regions. (B) IVT alphavirus replicon at the 5' position relative to the anti-TAA variable regions. The V _H and V _L regions of anti-CLDN18.2 were generated based on the sequence of the monoclonal antibody CLDN18.2 (mCLDN18.2ab). "Cap" is equally used for ARCA, beta-S-ARCA (Dl), or beta-S-ARCA (D2). In (A), "anti-CD3" means the V _H and VL regions generated from the sequences of the following monoclonal CD3 antibodies: UCHT1-HU (humanized mAB), UCHT1, CLB-T3, TR66, 145-2C11, in (B)"anti-CD3" describes only V _H and V _L from TR66. A means adenine; bi-scFv, bispecific single chain variable fragment; hAg, human alpha globin 5'-UTR; hBg, human beta globin 3'-UTR; His, a histidine tag containing six histidine residues; IVT transcribed in vitro; LL, long linker (15-18 amino acids); nsPl-4, non-structural proteins 1-4; Sec, secretion signal; sgP, subgenomic promoter; SL, short linker (5-6 amino acids); TAA, tumor-associated antigen; UTR, untranslated region; V, heavy (H) chain and light (L) chain variable region of an antibody.

FIGURE 21. Effect of domain targeting and anti-CD3-scFv selection on target-dependent T cell activation and specific target cell lysis.

NugC4 cells endogenously expressing CLDN18.2 were transiently transfected with several bi-scFv variants directed against CLDN18.2 and CD3 to compare their potency in a cytotoxicity assay. 5×10 ⁶ NugC4 cells per variant were electroporated with 20 μg/ml IVT-mRNA. Transfected target cells were counted, 1×10 ⁵ cells were seeded in each 6-well plate and incubated with human cytotoxic T cells (selected CD8 ⁺ T cells) in an E:T ratio of 5:1. Bi-scFv IVT mRNA targeting unexpressed TAA (counter) and parental IgG mAB chCLDN18.2ab (counter IgG) targeting CLDN18.2 but not T cells were used as negative controls. Protein 1BiMAB at a concentration of 5 ng/ml served as a positive control. As reference background dead cells, electroporated cells were seeded without T cells and, as reference background activation, T cells were seeded without target cells. Each sample was seeded in duplicate. After 48 hours, T cells and target cells were harvested and labeled with anti-CD3-FITC, anti-CD25-PE, anti-CD69-APC and 7-AAD to stain live and dead cells and analyzed by flow cytometry. (A) TAA-dependent, bi-scFv-mediated T cell activation was observed for all anti-CLDN18.2 bi-scFv variants. (B) Specific target cell lysis was determined by subtracting the 7-AAD reference population from the target cell population of the 7-AAD samples. The bi-scFv antibodies causing slightly higher target cell lysis - 1BiMAB and no.5 - share the same domain orientation and anti-CD3 origin of mAB TR66, but differ in their codon optimization (HS and CHO, respectively) and long linker sequences. Bi-scFv is a bispecific single chain variable fragment; counter, control; IgG, immunoglobulin G; IVT transcribed in vitro; mRNA, messenger RNA; TL, T-lymphocyte.

FIGURE 22. Co-incubation of 1BiMAB IVT-mRNA-transfected target cells and human T cells results in the formation of T cell clusters.

NugC4 cells endogenously expressing CLDN18.2 were transiently transfected by electroporation with 80 μg/ml 1BiMAB IVT-mRNA and co-incubated with human cytotoxic T cells (CD8 ⁺ T cells selected) at a 5:1 effector to target ratio in 96-well tablets. As a negative control, NugC4 target cells transfected with bi-scFv IVT mRNA targeting unexpressed TAA (-contra) co-incubated with human cytotoxic T cells (upper row, left) were used. Bottom row shows NugC4 cells transfected with control bi-scFv (left) or IVT-mRNA 1BiMAB (right) without human T cells. After 24 hours of co-incubation, images were taken of the samples using a Nikon Eclipse Ti microscope at 200x magnification. White arrows indicate T-cell clusters on target cells. CTL means cytotoxic T-lymphocytes; counter, control; hu, human.

FIGURE 23. 1BiMAB secreted from target cells after transfection with IVT-mRNA mediates T cell activation in a concentration dependent manner.

NugC4 cells endogenously expressing CLDN18.2 were transiently transfected by electroporation with a total of 40 µg/mL IVT mRNA containing 0.4-40 µg/mL 1BiMAB IVT mRNA plus appropriate amounts of luciferase IVT mRNA. Transfected target cells were co-incubated with human cytotoxic T cells (CD8 ⁺ T cells selected) at a 5:1 effector to target ratio in 6-well plates in duplicate. As T cell activation, reference human T cells were co-incubated with NugC4 target cells transfected with 40 μg/ml luciferase IVT-mRNA (0.0 μg/ml 1BiMAB IVT-mRNA). After 24 hours (A) and 48 hours (B), T cells were harvested and labeled with anti-CD3-FITC, anti-CD25-PE and anti-CD69-APC and analyzed by flow cytometry. The charts show the percentage of positive stained human cytotoxic T cells determined using the FlowJo software. IVT means in vitro transcribed; mRNA, messenger RNA; TL, T-lymphocyte.

FIGURE 24. 1BiMAB secreted by target cells after transfection with IVT-mRNA leads to concentration-dependent lysis of target cells.

NugC4 cells endogenously expressing CLDN18.2 were transiently transfected by electroporation with IVT mRNA at a total of 40 µg/mL containing 0.4-40 1BiMAB IVT mRNA plus appropriate amounts of lyciferase IVT mRNA or 40 µg/mL IVT mRNA alone luciferase as a reference sample. Transfected target cells were seeded with human cytotoxic T cells (CD8 ⁺ T cells selected) at a 5:1 effector to target ratio or without effector cells to determine the percentage of background dead target cells by individual electroporation. All samples were grown in 6-well plates in duplicate. After 24 hours (A) and 48 hours (B), T cells were harvested, labeled with propidium iodide (PI) for live/dead cell staining, and analyzed by flow cytometry. The percentage of dead (PI ⁺ ) target cells was determined using FlowJo software. The values were then normalized against each individual background sample and reference sample. IVT means in vitro transcribed; mRNA, messenger RNA; TL, T-lymphocyte.

FIGURE 25. T cell proliferation specifically induced in response to 1BiMAB secretion by target cells in the presence of CLDN18.2

Human T cells were stained with CFSE for analysis. T cells were grown without target cells (T cells) in combination with 5 µg/ml OKT3 and 2 µg/ml αCD28 as a positive activation control (+cont), with 5 ng/ml non-targeted control bi-scFv (protein - contr) or with 5 ng/ml of 1BiMAB protein (1BiMAB protein). T cells and NugC4 target cells overexpressing CLDN18.2 were incubated together (T cells + CLDN18.2 positive target cells) without anything (mock control) or with 5 ng/ml 1BiMAB protein (1BiMAB protein) . For IVT mRNA testing, NugC4 cells were transfected with 20 μg/ml of 1BiMAB IVT mRNA (1BiMAB mRNA) or bi-scFv IVT mRNA targeting unexpressed TAA (mRNA-counter) and incubated with T cells. In addition, NugC4 cells transfected with bi-scFv IVT mRNA targeting unexpressed TAA were combined with 5 ng/ml 1BiMAB protein (mRNA-contr + 1BiMAB protein). As an additional specificity control, samples containing the CLDN18.2 non-expressing target cell line MDA-MB-231 together with T cells (T cells + CLDN18.2 negative target cells) were included. MDA-MB-231 was used untreated and incubated without anything (mock control), with 5 ng/ml bi-scFv protein control (protein -contr) or 5 ng/ml 1BiMAB protein (1BiMAB protein), or MDA-MB- 231 were transfected with 20 g/ml 1BiMAB IVT-mRNA (1BiMAB mRNA) or bi-scFv IVT-mRNA targeting unexpressed TAA (mRNA-counter). The assay was performed at a 5:1 effector to target ratio in a 96-well plate, each sample was prepared in triplicate, and the incubation time was 72 h. FlowJo software and plotted as % proliferating T cells. CFSE is an ester of succinimidyl and carboxyfluorescein; IVT transcribed in vitro; mRNA, messenger RNA.

FIGURE 26. T cell activation and T cell mediated target cell lysis in response to 1BiMAB secretion begins at an effector to target ratio of 0.3:1.

NugC4 cells endogenously expressing CLDN18.2 were transiently transfected by electroporation with 40 μg/ml 1BiMAB IVT-mRNA. Transfected target cells were co-incubated with human cytotoxic T cells (CD8 ⁺ T cells selected) at the indicated effector to target ratios ranging from 0.3:1 to 10:1 in 6-well plates in duplicate. As reference samples, human T cells were grown in the absence of target cells ((A) 1:0) and target cells transfected with control IVT mRNA were grown in the absence of effector cells ((B) 0:1). As a negative control, human T cells were co-incubated with NugC4 target cells transfected with 40 μg/ml luciferase IVT-mRNA (counter-IVT-mRNA) at an E:T ratio of 10:1, ((A) and (B) counter IVT-mRNA 10:1). After 48 hours, cells were harvested and labeled with anti-CD3-FITC, anti-CD25-PE, anti-CD69-APC, and propidium iodide (PI) for live/dead cell staining and analyzed by flow cytometry. (A) shows the percentage of positively stained cytotoxic human T cells. (B) shows the percentage of dead (PI ⁺ ) target cells. All values were determined using FlowJo software. E:T means the ratio of effector and target; IVT transcribed in vitro; mRNA, messenger RNA; TL, T-lymphocyte.

FIGURE 27. Human cytotoxic T cells can serve as a recipient of IVT-mRNA bi-scFv producer cells

Human cytotoxic T cells were isolated from fresh PBMCs by CD8 positive selection and then transiently transfected by electroporation with 80 or 240 μg/ml 1BiMAB IVT-mRNA. Transfected effector cells were co-incubated with NugC4 target cells endogenously expressing CLDN18.2 at a 5:1 effector to target ratio in 6-well plates in duplicate. As reference samples, untreated human T cells were grown with target cells. As a negative control, human T cells transfected with 80 or 240 μg/ml eGFP control IVT-mRNA were co-incubated with NugC4 target cells. After 48 hours, cells were harvested and labeled with anti-CD3-FITC, anti-CD25-PE, anti-CD69-APC, and propidium iodide (PI) to stain live and dead cells, and analyzed by flow cytometry. (A) shows the percentage of positively stained cytotoxic human T cells. (B) shows the percentage of dead (PI ⁺ ) target cells normalized to the reference sample. All values were determined using FlowJo software. contr means control; IVT; transcribed in vitro; mRNA, messenger RNA; TL, T lymphocyte.

FIGURE 28. CLDN18.2-negative target cells transfected with 1BiMAB IVT-mRNA are not lysed T cells

CLDN18.2-negative PA-1 cell line stably expressing luciferase served as the target cell line. 5×10 ⁶ PA-l/luc cells were transfected by electroporation of IVT-mRNA at a total of 40 μg/ml. As a positive control, 4 and 40 μg/ml IVT-mRNA 1BiMAB or 6RHU3, targeting endogenously expressed CLDN6, was transfected. As a bi-scFv negative control, 40 μg/ml of bi-scFv IVT-mRNA targeting unexpressed TAA (- counter) was transfected. This IVT-mRNA also served as filling RNA in 4 μg/ml IVT-mRNA samples (4 μg/ml IVT-mRNA 1BiMAB, 4 μg/ml IVT-mRNA 6RHU3). Protein controls containing 1BiMAB and 6PHU3 in combination with a bi-scFv negative control transfected with PA-l/luc and effector cells are included.

The transfected target cells were seeded with human cytotoxic T cells (pan T cells) in a 5:1 effector to target ratio. All samples were seeded in triplicate in a 96-well format and co-incubated for 72 hours. As a control for minimal lysis (L _min ), each individual sample of transfected target cells was seeded without effector cells. Maximum lysis (L _max ) to normalize to the number of spontaneous luminescence pulses was achieved by adding Triton X-100 to control wells containing effector cells and untreated target cells (L _max1 ) or only untreated target cells (L _max2 ) before adding luciferin. 30 min after the addition of the luciferin solution, luminescence was measured on an Infinite M200 Tecan microplate reader. Specific lysis of target cells was calculated by the formula: % specific lysis = [1 - (luminescence _{test sample} - L _{max 1} / (L _min _ _{test sample} - L _{max 2} )] × 100. counter means control; IVT; in vitro transcribed; mRNA , messenger RNA.

FIGURE 29. Confirmation of 1BiMAB production by mammalian cells transfected with bi-scFv IVT-mRNA or -replicon RNA

(A) 5×10 ⁶ BH 21 cells were transiently transfected by electroporation with 40 μg/ml of IVT-mRNA 1BiMAB or - replicon RNA. As a mock control, cells were electroporated without RNA. 18 hours after transfection, the supernatant and cells were collected. Cells were lysed and supernatants were subjected to ~50-fold concentration. Raw and concentrated supernatants were analyzed by ELISA using Ni-NTA columns, anti-chCLDN18.2ab idiotypic mAB and secondary AP-conjugated antibody. Purified protein 1BiMAB in a range of dilutions from 2.3 to 37.5 ng/ml with 2 steps was used as a standard. (B) Concentrated supernatant, cell lysates (A) and 0.1 μg of purified 1BiMAB protein as a positive control were separated by SDS-PAGE. Western blot analysis was performed with a primary anti-His monoclonal and a peroxidase-conjugated secondary anti-mouse antibody. (C) 5×10 ⁶ BHK21 cells were transiently transfected by electroporation with 40 μg/ml 1BiMAB or IVT-mRNA no.25. As a mock control, cells were subjected to electroporation without RNA. 48 hours after transfection, the supernatant was collected and subjected to 40-fold concentration. SN and 0.1 μg of purified 1BiMAB protein as a positive control were separated by SDS-PAGE. Western blot analysis was performed with a primary anti-His monoclonal and a peroxidase-conjugated secondary anti-mouse antibody. Counter means control; mAB, monoclonal antibody; SN, supernatant; WB, Western blotting.

FIGURE 30. Injection of 1BiMAB bi-scFv IVT mRNA or - replicon RNA results in in vivo production and detectable 1BiMAB bi-scFv molecules in mice

10 μg of 1BiMAB IVT mRNA with or without EBK IVT mRNA or 10 μg of 1BiMAB IVT replicon were injected intramuscularly (IM) into NSG mice. Serum from blood collected on days 2, 4 and 7 post-injection was used in an in vitro cytotoxicity assay. CLDN18.2 and NugC4-LVT-CLDN18.2/luc stably expressing luciferase target cells were co-incubated with human T cells at an E:T ratio of 30:1 with 20 µl of serum sample for 48 h. Standard control protein 1BiMAB, L _min and L _max contained 20 μl of NSG serum. EBK means mixture of proteins of vaccinia virus E3, Bl 8R, K3; IM, intramuscular.

FIGURE 31. Schematic illustration of IVT-RNA molecules encoding bi-scFv antibodies targeting CLDN6 TAA.

In vitro scheme of transcribed RNA sequences encoding bi-scFv anti-CLDN6 antibodies. (A) IVT mRNA at the 5' and 3' positions relative to the anti-TAA variable regions. (B) Alphavirus IVT replicon at the 3' position relative to the anti-TAA variable regions. The V _H and V _L regions of anti-CLDN6 were generated based on the sequence of the CLDN6 monoclonal antibody (mCLDN6ab). "Cap" is equally used for ARCA, beta-S-ARCA (Dl) or beta-S-ARCA (D2). The V _H and V _L regions of anti-CD3 were generated based on the sequence of the CD3 monoclonal antibody TR66. A means adenine; bi-scFv, bispecific single chain variable fragment; hAg, human alpha globin 5'-UTR; hBg, human beta-globin 3'-UTR; His, a histidine tag containing six histidine residues; IVT transcribed in vitro; LL, long linker (15-18 amino acids); nsPl-4, non-structural proteins 1-4; Sec, secretion signal; sgP, subgenomic promoter; SL, short linker (5-6 amino acids); TAA, tumor-associated antigen; UTR, untranslated region; V, heavy (H) and light (L) chain variable region of an antibody.

FIGURE 32. Co-incubation of bi-scFv anti-CLDN6 IVT-mRNA transfected target cells with human T cells results in the formation of T cell clusters.

PA-1 cells endogenously expressing CLDN6 were transiently transfected by electroporation with 20 μg/ml of 6RHU5 or 6RHU3 IVT-mRNA and co-incubated with human T cells (pan T cells) at a 5:1 effector to target ratio in 6-well tubes. tablets. As a negative control, PA-1 target cells transfected with bi-scFv IVT mRNA targeting unexpressed TAA (-contra) co-incubated with human T cells were used (upper row, left photo). Middle row shows untreated PA-1 cells and human T cells without protein as a negative control (mock control, photo left), or with 50 μg/mL purified anti-CLDN6 proteins 6PHU5 (middle) or 6PHU3 (right) bi -scFv as positive controls. The bottom row shows untreated PA-1 cells (left) and only human T cells (right). After 24 hours of co-incubation, samples were taken using a Nikon Eclipse Ti microscope at 200x magnification. White arrows indicate T-cell clusters on target cells. Bi-scFv is a bispecific single chain variable fragment; counter, control; hu, human; IVT transcribed in vitro; mRNA, messenger RNA; TL, T-lymphocyte.

FIGURE 33. Effect of domain targeting on efficiency: transfection of anti-CLDN6 target cells with 6RHU3 bi-scFv results in a higher percentage of activated T cells compared to 6RHU5.

PA-1 cells endogenously expressing CLDN6 were transiently transfected with two bi-scFv variants 6RHU5 and 6RHU3 directed against CLDN6 and CD3 to compare their activity in a T cell activation assay. For each variant, 5×10 ⁶ PA-1 cells were electroporated with 20 μg/ml IVT-mRNA. Transfected target cells were re-counted, 1×10 ⁵ cells were seeded in each 6-well plate and incubated with human cytotoxic T cells (CD8 ⁺ T cells selected) at an E:T ratio of 5:1. Untreated target cells (hu TL + untreated PA-1) and target cells transfected with IVT-mRNA of bi-scFv directed to unexpressed TAA (hu TL + PA-1 - counter) were selected as negative controls. Protein 6PHU5 served as a positive control at a concentration of 50 ng/ml (hu TL + PA-1 protein control). In addition, T cells were seeded without target cells with and without 6PHU5 protein as a reference background activation. Each sample was seeded in duplicate. Analysis was performed at 24h and 48h: T cells were harvested and labeled with anti-CD3-FITC, anti-CD25-PE, anti-CD69-APC and 7-AAD to stain live and dead cells and analyzed by flow cytometry. TAA-dependent bi-scFv-mediated T cell activation was observed in both anti-CLDN6 bi-scFv variants after 24 h (A) and 48 h (B) co-incubation. Transfection with 6RHU3 bi-scFv caused approximately 20% higher T cell activation at both time points. Bi-scFv is a bispecific single chain variable fragment; Counter, control; hu, human; IVT transcribed in vitro; mRNA, messenger RNA; TL, T lymphocyte.

FIGURE 34. Secretion of 6RHU3 mediates T-cell activation in a concentration dependent manner.

PA-1 cells endogenously expressing CLDN6 were transiently transfected by electroporation of IVT mRNA at a total of 20 μg/mL, comprising 0.2-20 μg/mL of 6RHU3 IVT mRNA plus appropriate amounts of bi-scFv targeting IVT mRNA. unexpressed TAA. Transfection with 20 µg/ml bi-scFv IVT mRNA targeting unexpressed TAA (0.0 µg/ml 6RHU3 IVT mRNA) served as a specificity control. Trasfected target cells were co-incubated with human cytotoxic T cells (pan T cells) at a 5:1 effector to target ratio in 6-well plates in duplicate. As a reference T cell activation, only human T cells were grown without 6PHU5 protein (hu TL -) or with 6PHU5 protein (hu TL protein counter). As a negative control, T cells were co-incubated with untreated PA-1 target cells (hu TL + PA-1 - Contr). Protein 6PHU5 served as a positive control at a concentration of 50 ng/ml (hu TL + PA-1 protein control). After 48 hours, T cells were harvested and labeled with anti-CD3-FITC, anti-CD25-PE and anti-CD69-APC and analyzed by flow cytometry. The graphs show the percentage of positively stained human T cells determined using the FlowJo software. Bi-scFv is a bispecific single chain variable fragment; counter, control; hu, human; IVT transcribed in vitro; mRNA, messenger RNA; TL, T lymphocyte.

FIGURE 35. The EC50 value of 6RHU3 for specific target cell lysis after 48 hours was approximately 200 ng/mL.

PA-1 cells endogenously expressing CLDN6, which stably express luciferase, were transiently transfected by electroporation of bi-scFv IVT-mRNA at a total concentration of 13.3 μg/ml, including 0.004 - 13.3 μg/ml of 6RHU3 and the corresponding amount of IVT-mRNA bi-scFv targeting unexpressed TAA. Transfected target cells were plated with human T cells at a 5:1 effector to target ratio in triplicate in a 96-well format. As a control for minimal lysis (L _min ), each individual sample of transfected target cells was seeded without effector cells. Maximum lysis (L _max ) to normalize with respect to the number of spontaneous luminescence pulses was achieved by adding Triton X-100 to control wells containing effector cells and untreated target cells immediately prior to the addition of luciferin. 30 min after the addition of _the luciferin solution, luminescence was measured on an Infinite M200 Tecan microplate _reader at 24 h and 48 _h . _{of the tested sample} - L _max )] × 100. The values were plotted against the logl0 concentration of 6RHU3. EC ₅₀ values mean half of the maximum effective concentration; L, lysis.

FIGURE 36. T cell proliferation is specifically induced in response to 6RHU3 secretion by target cells in the presence of CLDN6.

Human T cells were stained with CFSE for analysis. T cells were grown without target cells (T cells) in combination with 5 μg/ml OKT3 and 2 μg/ml αCD28 as a positive activation control (+cont), with 5 ng/ml non-targeted control bi-scFv (-cont. protein) or with 5 ng/ml of 6PHU3 protein (6PHU3 protein). T cells and PA-1 target cells endogenously expressing CLDN6 were incubated together (T cells + CLDN6 positive target cells) without anything (mimic) or with 5 ng/ml of 6PHU3 protein (6PHU3 protein). For IVT mRNA testing, PA-1 cells were transfected with 20 μg/ml of 6RHU3 IVT mRNA (6RHU3 mRNA) or bi-scFv IVT mRNA targeting unexpressed TAA (mRNA-counter) and incubated with T cells. In addition, PA-1 cells transfected with bi-scFv IVT mRNA targeting unexpressed TAA were combined with 5 ng/ml 6PHU3 protein (mRNA-contr + 6PHU3 protein). As an additional specificity control, samples with a CLDN6 non-expressing target cell line MDA-MB-231 along with T cells (T cells + CLDN6 negative target cells) were included. MDA-MB-231 was used untreated and incubated without anything (mock control), with 5 ng/ml bi-scFv control protein (-control protein) or 5 ng/ml 6PHU3 protein (6PHU3 protein), or MDA-MB- 231 were transfected with 20 μg/ml 6RHU3 IVT-mRNA (6RHU3 mRNA) or bi-scFv IVT-mRNA targeting unexpressed TAA (-counter mRNA). The assay was performed at a 5:1 effector to target ratio in a 96-well plate, with each sample in triplicate and an incubation time of 72 h. The decrease in CFSE signal indicative of T cell proliferation was analyzed by flow cytometry, calculated using software FlowJo and plotted as % proliferating T cells. CFSE is an ester of succinimidyl and carboxyfluorescein; IVT transcribed in vitro; mRNA, messenger RNA.

FIGURE 37. Confirmation of 6RHU3 translation by mammalian cells transfected with bi-scFv IVT-mRNA or -replicon RNA

(A) 5 x 10 ⁶ BHK21 cells were transiently transfected by electroporation with 40 μg/ml of 6RHU3 IVT-mRNA or β-replicon RNA. Transfection of 40 μg/ml IVT-mRNA no.25 was included as an additional sample. As a mock control, cells were subjected to electroporation without RNA. 18 hours after transfection, the supernatant and cells were collected. Cells were lysed and supernatants were subjected to 50-fold concentration. Raw and concentrated supernatants were analyzed by ELISA using Ni-NTA columns, anti-mCLDN6ab idiotypic mAB and secondary AP-conjugated antibody. Purified 6PHU3 protein diluted from 2.3 to 150 ng/ml in 2 steps was used as a standard. (B) Concentrated supernatant and cell lysates (A) and 0.1 μg of purified 6PHU3 protein as a positive control were separated by SDS-PAGE. Western blot analysis was performed with a primary anti-His monoclonal and a peroxidase-conjugated secondary anti-mouse antibody. (C) 5×10 ⁶ BHK21 cells were transiently transfected by electroporation with 40 μg/ml 6RHU3 or IVT-mRNA no.25. As a mock control, cells were subjected to electroporation without RNA. 48 hours after transfection, the supernatant was collected and subjected to 40-fold concentration. SN and 0.1 μg of purified 6PHU3 protein as a positive control were separated by SDS-PAGE. Western blot analysis was performed with a primary anti-His monoclonal and a peroxidase-conjugated secondary anti-mouse antibody. Contr means control; mAB, monoclonal antibody; SN, supernatant; WB, Western blotting.

FIGURE 38. Injection of 6RHU3 IVT-mRNA bi-scFv or - replicon RNA results in in vivo translation and detectable bi-scFv molecules in mice

10 μg of 6RHU3 IVT mRNA with or without EBK IVT mRNA or 10 μg of 6RHU3 IVT replicon were injected IM into NSG mice. Serum collected 7 days after injection was used in an in vitro cytotoxicity assay. PA-l/luc target cells endogenously expressing CLDN6 and stably expressing luciferase were co-incubated with human T cells at an E:T ratio of 30:1 with 20 µl of serum sample for 48 h. Standard control protein 6PHU3, L _min and L _max containing 20 μl of NSG mimic serum. EBK is a mixture of vaccinia E3, B-18R, K3 proteins; IM, intramuscular.

FIGURE 39. Cytotoxicity analysis results of anti-CLDN18.2 bi-scFv proteins containing the scFv anti-CD3 binding domain at the C-terminus of the protein.

Bi-scFv variants directed against CLDN18.2 and CD3 were transiently expressed in CHO cells and purified with Protein-L polymer to compare their potency in a cytotoxicity assay. NugC4 cells endogenously expressing CLDN18.2, which stably express luciferase, were used as target cells. Human T cells and target cells were incubated at a 5:1 E:T ratio with 5000, 1000, 200 and 40 ng/ml of each of the bi-scFv proteins in a 96-well format. Each test sample was seeded in triplicate, the control sample for L _min was seeded in triplicate. The duration of co-incubation before analysis was 24 h and 48 h. After adding the luciferin solution at the given time points, the luminescence was measured on an Infinite M200 TECAN reader. Specific target cell lysis was calculated for each concentration and recorded.

a. The anti-CD3 variable domains are arranged in VH-VL domain order and are separated by the LL4 peptide linker.

b. The anti-CD3 variable domains are arranged in VH-VL domain order and are separated by the LL4 peptide linker. The anti-CD3 scFv contains a disulfide link between the VH and VL domains.

c. The anti-CD3 variable domains are arranged in VL-VH order and are separated by the LL5 peptide linker.

d. The anti-CD3 variable domains are arranged in VL - VH order and are separated by the LL5 peptide linker. The anti-CD3 scFv contains a disulfide link between the VL and VH domains.

FIGURE 40. Intra-assay comparison of EC50 values obtained in a luciferase-based cytotoxicity assay using bi-scFv anti-CLDN6 proteins.

Luciferase based cytotoxicity assays were performed using three different donors for the T cell preparation. EC50 values calculated using the 6 bi-scFv anti-CLDN6 proteins tested after 24 h and 48 h of incubation are presented for each independent assay (A, B and C). PA-1 cells endogenously expressing CLDN6 were incubated for 24 h and 48 h with increasing concentrations (0.025-50000 ng/ml for A and B, 0.0025-5000 ng/ml for C) of bi-scFv anti-CLDN6 proteins. and human T cells in a 5:1 effector to target ratio in triplicate in a 96-well format. As a control for minimal lysis (L _min ), effector and target cells were seeded without bi-scFv proteins. Maximum lysis (L _max ) to normalize with respect to the number of spontaneous luminescence pulses was achieved by adding Triton X-100 to control wells containing effector and target cells in the absence of bi-scFv immediately prior to the addition of luciferin. Thirty minutes after the addition of the luciferin solution, luminescence was measured on an Infinite M200 Tecan microplate reader after 24 and 48 hours of target and effector cell incubation. Specific lysis of target cells was calculated by the formula: % specific lysis = [1 - (luminescence _{of the test sample} - L _max ) / (L _min - L _max )] × 100. EC ₅₀ means half the maximum effective concentration; L, lysis; NA, no data.

Detailed description of the invention

Although the present invention is described in detail below, it should be understood that this invention is not limited to the specific methods, protocols, and reagents described herein, as these methods, protocols, and reagents may vary. Also, it should be understood that the terminology used herein is only intended to describe specific embodiments and is not intended to limit the scope of the present invention, which may be limited only by the appended claims. Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art.

Next, the elements of the present invention will be described. These elements are listed along with certain embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the scope of the present invention to the specifically described embodiments. It is to be understood that this description is intended to support and cover embodiments that combine the clearly described embodiments with any number of disclosed and/or preferred elements. In addition, any permutations and combinations of all described elements in this application will be considered disclosed using the description of this application, unless the context indicates otherwise.

Preferably, the terms used here are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

Unless otherwise indicated, the practice of the present invention will involve the use of standard techniques in chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, ^2nd Edition, J. Sambrook et al.eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

Throughout this specification and the appended claims, unless the context otherwise requires, the word "comprise" and variations thereof such as "comprises" and "comprising" will be understood to include the specified element, integer, or step, or groups of elements, integers numbers or steps, but not to the exclusion of any other element, integer, or step, or groups of elements, integers, or steps, although in some embodiments, such other element, integer, or step, or groups of elements, integers, or steps may be excluded, that is, the object of the invention is to include the specified element, integer or stage, or groups of elements, integers or stages. The use of the singular in the context of the description of the invention (especially in the context of the appended claims) is to be construed to include both the singular and the plural, unless otherwise specified herein or clearly contrary to the context. The listing of value ranges in this document is only intended to serve as a shortcut for specifying each individual value that falls within the range. Unless otherwise noted, each individual value is included in the datasheet as if it were listed separately in this document. All of the methods described herein may be performed in any proper order unless otherwise noted herein or explicitly denied by the context. The use of any or all of the examples, or exemplary language (eg, "such as") provided herein, is intended only to better explain the invention, and does not impose any limitation on the scope of the invention, unless otherwise stated. No language in this specification should be construed as indicating any element not claimed in the claims as being essential to carrying out the invention.

In this description, some documents are cited. Each of the documents cited here (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), above or below, are incorporated here by reference in their entirety. Nothing here should be construed as an admission that the inventor is not entitled to predate the invention on account of the earlier date of the invention (antedate).

Claudins are a family of proteins that are the most important components of tight junctions, where they establish a paracellular barrier that controls the flow of molecules in the intercellular space between epithelial cells. Claudins are transmembrane proteins that cross the cell membrane four times, with both ends, N-terminus and C-terminus, located in the cytoplasm. The first extracellular loop, called ECl or ECL1, has an average of 53 amino acids, and the second extracellular loop, called EC2 or ECL2, has about 24 amino acids. Cell surface proteins of the claudin family, such as CLDN6 and CLDN18.2, are expressed in tumors of various origins, and are particularly suitable as target structures in antibody-mediated cancer immunotherapy due to their selective expression (expression is absent in normal tissues, significant in terms of toxicity). ) and localization in the plasma membrane.

In the context of the present invention, the preferred claudins are CLDN6 and CLDN18.2. CLDN6 and CLDN18.2 have been identified as being differentially expressed in tumor tissues, with the only normal tissue expressing CLDN18.2 being the stomach and the only normal tissue expressing CLDN6 being the placenta.

CLDN18.2 is selectively expressed in normal tissues in differentiated epithelial cells of the gastric mucosa. CLDN18.2 is expressed in cancers of various origins such as pancreatic carcinoma, esophageal carcinoma, gastric carcinoma, bronchial carcinoma, breast carcinoma, and ENT tumors. CLDN18.2 is a valuable target for the prevention and/or treatment of primary tumors such as gastric cancer, esophageal cancer, pancreatic cancer, lung cancer such as non-small cell lung cancer (NSCLC), ovarian cancer, colon cancer, liver cancer, cancer head and neck, and gallbladder cancer, and their metastases, in particular gastric cancer metastases such as Krukenberg tumors, peritoneal metastases and lymph node metastases.

CLDN6 has been found to be expressed, for example, in ovarian cancer, lung cancer, stomach cancer, breast cancer, liver cancer, pancreatic cancer, skin cancer, melonoma, head and neck cancer, sarcoma, bile duct cancer, renal cell carcinoma and bladder cancer. CLDN6 is a particularly preferred target for the prevention and/or treatment of ovarian cancer, in particular ovarian adenocarcinoma and ovarian teratocarcinoma; lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), in particular squamous cell lung cancer and adenocarcinoma; stomach cancer; breast cancer; liver cancer; pancreatic cancer; skin cancer, in particular basal cell carcinoma and squamous cell carcinoma; malignant melanoma; head and neck cancer, in particular malignant pleomorphic adenoma; sarcomas, in particular synovial sarcomas and carcinosarcomas; bile duct cancer; bladder cancer, in particular transitional cell carcinoma and papillary cancer; kidney cancer, in particular squamous cell carcinoma, including clear cell carcinoma of the kidney and papillary carcinoma of the kidney; colon cancer; cancer of the small intestine, including cancer of the ileum, in particular adenocarcinoma of the small intestine and adenocarcinoma of the ileum; embryonic testicular carcinoma; placental choriocarcinoma; cervical cancer; testicular cancer, in particular testicular seminoma, testicular teratoma and fetal testicular cancer; uterine cancer; germinomas such as teratocarcinoma or embryonic carcinoma, in particular testicular embryonic cell tumors and their metastatic forms. In one embodiment, cancers associated with CLDN6 expression are selected from the group consisting of ovarian cancer, lung cancer, metastatic ovarian cancer, and metastatic lung cancer. Preferably, the ovarian cancer is a carcinoma or adenocarcinoma. Preferably, the lung cancer is a carcinoma or adenocarcinoma, and preferably is a bronchiolar cancer such as bronchiolar carcinoma or bronchiolar adenocarcinoma.

The term "CLDN" as used herein means claudin and includes CLDN18.2 and CLDN6. Preferably, the claudin is human claudin.

The term "CLDN18" refers to claudin 18 and includes any variants including claudin 18 spliced variant 1 (claudin 18.1 (CLDN18.1)) and claudin 18 spliced variant 2 (claudin 18.2 (CLDN 18.2)).

The term "CLDN18.2" preferably refers to human CLDN18.2 and in particular to a protein comprising, preferably consisting of, the amino acid sequence according to SEQ ID NO: 1 of the sequence listing or a variant of said amino acid sequence. The first extracellular loop of CLDN18.2 preferably contains amino acids 27 to 81, more preferably amino acids 29 to 78 of the amino acid sequence shown in SEQ ID NO: 1. The second extracellular loop of CLDN18.2 preferably contains amino acids 140 to 180 of the amino acid sequence shown in SEQ ID NO: 1. Said first and second extracellular loops preferably form the extracellular portion of CLDN18.2.

The term "CLDN6" preferably refers to human CLDN6 and in particular to a protein containing, preferably consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 of the sequence listing or a variant of said amino acid sequence. The first extracellular loop of CLDN6 preferably contains amino acids 28 to 80, more preferably amino acids 28 to 76 of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 3. The second extracellular loop of CLDN6 preferably contains amino acids 138 to 160, preferably amino acids 141 to 159, more preferably amino acids 145 to 157 of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 3. Said first and second extracellular loops preferably form the extracellular portion of CLDN6.

The term "variant" according to the invention refers in particular to mutants, spliced variants, conformations, isoforms, allelic variants, variants of varieties and homologues of varieties, in particular those that are naturally present. An allelic variant refers to an alteration in the normal sequence of a gene, the meaning of which is often unclear. Whole gene sequencing often allows the identification of multiple allelic variants of a given gene. A species homologue is a nucleic acid or amino acid sequence with varieties having an origin different from that of a given nucleic acid or amino acid sequence. The term "variant" encompasses any post-translationally modified variants and conformational variants.

The second target molecule for binding agents described here is the cluster of cellular differentiation CD3 (Cluster of Differentiation 3). The CD3 complex denotes an antigen that is expressed on mature human T cells, thymocytes, and a subpopulation of natural killer cells as part of the T cell receptor (TCR) multimolecular complex. The T-cell co-receptor is a protein complex and consists of four different chains. In mammals, the complex contains a CD3γ chain, a CD35δ chain, and two CD3ε chains. These chains are coupled to a molecule known as the T cell receptor (TCR) and the ζ chain to generate an activation signal in T lymphocytes. Together, the TCR, ζ chain, and CD3 molecules form the TCR complex.

The amino acid sequence of human CD3 epsilon is disclosed in GenBank accession number NM_000733 and contains SEQ ID NO: 4. The amino acid sequence of human CD3 gamma is disclosed in GenBank accession number NM 000073. The amino acid sequence of human CD3 delta is disclosed in GenBank accession number NM_000732. CD3 is responsible for TCR signal transduction. As described in Lin and Weiss, Journal of Cell Science 114, 243-244 (2001), activation of the TCR complex by binding to MHC-presented specific antigen epitopes results in phosphorylation of immunoreceptor tyrosine activating motifs (ITAMs) by Src family kinases, triggering the recruitment of other kinases. , which leads to T-cell activation, including the release of Ca ²⁺ . Clustering of CD3 on T cells, for example by immobilization of anti-CD3 antibodies, results in T cell activation similar to that of the T cell receptor, but independent of the specificity typical of that clone.

The term "CD3" as used herein includes human CD3 and refers to an antigen that is expressed on human T cells as part of the multimolecular T cell receptor complex.

With respect to CD3, the binding agent of the invention preferably recognizes the CD3 epsilon chain, in particular it recognizes an epitope that corresponds to the first 27 N-terminal amino acids of the CD3 epsilon or functional fragments of this 27 amino acid extension.

In accordance with the invention, the term "claudin-positive cancer", or similar terms, means a cancer in which cancer cells expressing claudin are involved, preferably on the surface of said cancer cells.

"Cell surface" is used in accordance with its usual meaning in this field and, thus, includes the outer part of the cell, which is available for binding by proteins and other molecules.

Claudin is expressed on the cell surface if it is located on the surface of said cells and is available for binding by claudin-specific antibodies added to the cells.

The term "extracellular portion" in the context of the present invention refers to the portion of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from the outside of said cell, for example, to antigen-binding molecules, such as antibodies localized outside the cell. Preferably, the term refers to one or more extracellular loops or domains, or fragments thereof.

The terms "part" or "fragment" are used here interchangeably and refer to a continuous element. For example, a part of a structure, such as an amino acid sequence or a protein, refers to a contiguous element of said structure. The site, part or fragment of the structure preferably has one or more functional properties of the specified structure. For example, a region, portion, or fragment of an epitope or peptide is preferably immunologically equivalent to the epitope or peptide from which it is derived. Part or fragment of the protein sequence preferably contains a sequence consisting of at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30 , at least 50 or at least 100 consecutive amino acids of the protein sequence.

In accordance with the invention, CLDN18.2 is not significantly expressed in a cell if the expression level is lower compared to expression in gastric cells or gastric tissue. Preferably, the expression level is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05%, or even lower, of expression in stomach cells or tissue. stomach. Preferably, CLDN18.2 is essentially not expressed in a cell if the expression level exceeds the expression level in non-cancerous tissue other than the stomach by no more than 2 times, preferably 1.5 times, and preferably does not exceed the expression level in the specified non-cancerous tissue. Preferably, CLDN18.2 is essentially not expressed in the cell if the expression level is below the detection limit and/or if the expression level is too low to allow binding of CLDN18.2-specific antibodies added to the cells.

According to the invention, CLDN18.2 is expressed in a cell if the expression level exceeds the expression level in non-cancerous tissue other than the stomach, preferably more than 2 times, preferably 10 times, 100 times, 1000 times. Preferably, CLDN18.2 is expressed in a cell if the expression level is above the detection limit and/or if the expression level is high enough to allow binding of CLDN18.2-specific antibodies added to the cells. Preferably, CLDN18.2 expressed in a cell is expressed or displayed on the surface of said cell.

According to the invention, CLDN6 is generally not expressed in a cell if the level of expression is lower compared to expression in cells of the placenta or tissue of the placenta. Preferably, the expression level is less than 10%, preferably less than 5%, 3%, 2%, 1%, 0.5%, 0.1% or 0.05%, or even lower, of expression in placental or tissue cells. placenta. Preferably, CLDN6 is essentially not expressed in a cell if the expression level is no more than 2-fold, preferably 1.5-fold, and preferably not greater than the expression level in said non-cancerous tissue other than the placenta. . Preferably, CLDN6 is essentially not expressed in the cell if the expression level is below the detection limit and/or the expression level is too low to allow binding by CLDN6-specific antibodies added to the cells.

According to the invention, CLDN6 is expressed in a cell if the expression level exceeds the expression level in non-cancerous tissue other than the placenta, preferably more than 2 times, preferably 10 times, 100 times, 1000 times or 10000 times. Preferably, CLDN6 is expressed in a cell if the expression level is above the detection limit and/or if the expression level is high enough to allow binding by CLDN6-specific antibodies added to the cells. Preferably, CLDN6 expressed in a cell is expressed or displayed on the surface of said cell.

In accordance with the invention, the term "disease" refers to any pathological condition, including cancer, in particular, such forms of cancer as described herein. Any reference herein to cancer or specific forms of cancer also includes metastases of said cancer. In a preferred embodiment, the disease to be treated according to the present invention comprises claudin (CLDN) expressing cells such as CLDN18.2 and/or CLDN6.

"Disease associated with cells expressing CLDN" or similar expressions means in accordance with the invention that CLDN is expressed in cells of a diseased tissue or organ. In one embodiment, the expression of CLDN in cells of a diseased tissue or organ is increased compared to the state in a healthy tissue or organ. Increase refers to an increase of at least 10%, in particular at least 20%, at least 50%, at least 100%, at least 200%, at least 500% , at least 1000%, at least 10000%, or even more. In one embodiment, expression is found only in diseased tissue, while expression in healthy tissue is downregulated. In accordance with the invention, diseases associated with cells expressing CLDN include cancers. In addition, in accordance with the invention, cancers are preferably those diseases in which cancer cells express CLDN.

As used herein, the term "cancer" or "cancer" includes a disease characterized by aberrantly regulated cell growth, proliferation, differentiation, adhesion, and/or migration. The term "cancer cell" refers to an abnormal cell that grows by rapid uncontrolled cell proliferation and continues to grow after a stimulus that initiated a new growth arrest. Preferably, the "cancer disease" is characterized by cells expressing CLDN and cancer cells expressing CLDN. The CLDN expressing cell is preferably a cancer cell, preferably of the cancer types described herein.

The term "cancer" according to the invention includes leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer , cervical cancer, gastrointestinal cancer, liver cancer, colon cancer, stomach cancer, colon cancer, head and neck cancer, gastrointestinal cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and larynx (ENT), breast cancer, prostate cancer, uterine cancer, ovarian cancer and lung cancer and their metastases. Examples of said cancer are lung carcinoma, breast carcinoma, prostate carcinoma, colon carcinoma, renal cell carcinoma, cervical carcinoma, or metastases of the cancer or tumors described above. The term "cancer" in accordance with the invention also includes cancer metastases.

According to the invention, a "carcinoma" is a malignant tumor derived from epithelial cells. This group represents the most common cancers, including common breast, prostate, lung, and colon cancers.

"Adenocarcinoma" is a cancer that originates from glandular tissue. This tissue is also part of a larger category of tissue known as epithelial tissue. Epithelial tissue includes the skin, glands, and a variety of other tissues that line the body's cavities and organs. Epithelial tissue is derived from the germ layers, ectoderm, endoderm, and mesoderm. To classify a cancer as an adenocarcinoma, the cells do not have to be part of a gland, as they have secretory properties. This form of carcinoma can occur in some higher mammals, including humans. Well-differentiated adenocarcinomas resemble the glandular tissue from which they originate, while moderately differentiated adenocarcinomas do not. By staining the cells obtained from the biopsy, the pathologist determines whether the tumor is an adenocarcinoma or some other type of cancer. Adenocarcinoma can occur in many body tissues due to the ubiquitous nature of glands in the body. Although each gland may not secrete the same substance, since there is an exocrine function of the cell, the cancer is considered glandular and its malignant form is thus called adenocarcinoma. Malignant adenocarcinoma invades other tissues and often spreads metastases, provided it is given sufficient time. Adenocarcinoma of the ovary is the most common type of ovarian carcinoma. It includes serous and mucinous adenocarcinomas, clear cell adenocarcinoma, and endometrioid adenocarcinoma.

The term "metastasis" means the spread of cancer cells from their site of origin to another part of the body. The formation of metastases is a very complex process and depends on the detachment of malignant cells from the primary tumor, invasion of the extracellular matrix, penetration through the basement membranes of the endothelium to enter the body cavity and vessels, and then, after blood transfer, infiltration of the target organs. In conclusion, new tumor growth at the target site is dependent on angiogenesis. Tumor metastasis often occurs even after removal of the primary tumor, as tumor cells or components may remain and develop metastatic potential. In one embodiment, the term "metastasis" according to the invention refers to "distant metastases", which refers to metastases remote from the primary tumor and the regional lymph node system. In one embodiment, the term "metastases" in accordance with the invention refers to metastases to the lymph nodes. One particular form of metastasis that is treatable using the therapy of the invention is metastasis originating from gastric cancer as the primary site. In a preferred embodiment, such gastric cancer metastases are Krukenberg tumor, peritoneal metastases and/or lymph node metastases.

Krukenberg's tumor is a rare metastatic ovarian tumor, accounting for 1% to 2% of all ovarian tumors. The prognosis for Krukenberg tumors is still very poor and there is no approved treatment for Krukenberg tumors. Krukenberg tumor is a metastatic ring cell adenocarcinoma of the ovary. The stomach is the primary site for most cases of Krukenberg tumor (70%). Colon, appendicitis, and breast carcinomas (mostly invasive lobular carcinoma) are the next most common primary sites. Rare cases of Krukenberg tumor originating from carcinoma of the gallbladder, bile duct, pancreas, small intestine, nipple of Vater, cervix, and bladder/urinary duct have been reported.

The term "treat" means administering a compound or composition, or a combination of compounds or compositions, to a subject to prevent or eliminate a disease, including reducing the size of a tumor or the number of tumors in the subject; arresting or slowing the development of the disease in the subject; inhibiting or slowing down the development of a new disease in a subject; reducing the frequency or severity of symptoms and/or re-occurrence in a subject who currently has or previously had a disease, and/or prolongation, i.e., an increase in the subject's lifespan.

In particular, the term "treating a disease" includes curing, shortening, ameliorating, preventing, slowing or inhibiting the progression or worsening of, or preventing or delaying the onset of a disease or its symptoms.

In the context of the present invention, terms such as "protect", "prevent", "preventive" or "protective" refer to the prevention or treatment, or both, of the occurrence and/or spread of a disease in a subject and, in particular, to minimizing the likelihood that the subject will develop the disease, or delaying the development of the disease. For example, a person at risk of developing cancer would be a candidate for cancer treatment or prevention.

"At risk" means a subject who is identified as having a higher than normal likelihood of developing a disease, in particular cancer, compared to the general population. In addition, a subject who has had or currently has a disease, in particular cancer, is a subject who has an increased risk of developing the disease, eg, the subject may continue to develop the disease. Subjects who currently have or have had cancer also have an increased risk of cancer metastasis.

The term "patient" in accordance with the invention means a subject to be treated, in particular a sick subject, including humans, non-human primates or other animals, in particular mammals such as cows, horses, pigs, goats, dogs, cats or rodents, such like mice and rats. In a particularly preferred embodiment, the patient is a human.

"Target cell" means any unwanted cell, such as a cancer cell. In preferred embodiments, the target cell expresses CLDN.

The term "antigen" refers to an agent, such as a protein or peptide, containing an epitope against which the immune response is directed and/or to be directed. In a preferred embodiment, the antigen is a tumor-associated antigen such as CLDN18.2 or CLDN6, i.e. a component of cancer cells that can be derived from the cytoplasm, cell surface and cell nuclei, in particular such antigens that are produced, preferably in a large quantity, intracellularly or as surface antigens on cancer cells.

In the context of the present invention, the term "tumor-associated antigen" preferably refers to proteins that under normal conditions are specifically expressed in a limited number of tissues and/or organs, or at specific stages in the developmental process, and are expressed or aberrantly expressed in one or more tumors or cancer tissues. In the context of the present invention, the tumor-associated antigen is preferably associated with the cell surface of the cancer cell and is preferably not or only rarely expressed in normal tissues.

The term "epitope" refers to an antigenic determinant in a molecule, that is, a portion of a molecule that is recognized by the immune system, for example, that is recognized by an antibody. For example, epitopes are discrete, three-dimensional regions on an antigen that are recognized by the immune system. Epitopes usually consist of reactive groups located on the surface of molecules, such as side chains of amino acids or sugars, and usually have specific three-dimensional structure characteristics as well as specific charge characteristics. Conformational and non-conformational epitopes differ in that binding to the former, in contrast to binding to the latter, is lost in the presence of denaturing solvents. A protein epitope preferably contains a continuous or discontinuous portion of said protein and is preferably 5 to 100, preferably 5 to 50, more preferably 8 to 30, most preferably 10 to 25 amino acids in length, e.g., an epitope may preferably contain 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length.

The term "antibody" refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and chimeric antibodies. Each heavy chain is composed of a heavy chain variable region (abbreviated here as VH) and a heavy chain constant region. Each light chain is composed of a light chain variable region (abbreviated here as VL) and a light chain constant region. The VH and VL regions can be further subdivided into areas of hypervariability called complementarity determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells), and the first component (C1q) of the classical complement system.

The term "monoclonal antibody" as used herein refers to a preparation of antibody molecules of a single molecular composition. A monoclonal antibody exhibits one binding specificity and affinity. In one embodiment, the monoclonal antibodies are produced by a hybridoma that includes a B cell derived from a non-human animal, such as a mouse, fused to an immortalized cell.

The term "recombinant antibody" as used herein is intended to include all antibodies that are produced, expressed, generated, or isolated by recombinant methods, such as (a) antibodies isolated from an animal (e.g., mouse) that is transgenic or transchromosomal for immunoglobulin genes, or from a hybridoma derived therefrom, (b) antibodies isolated from a host cell transformed to express the antibody, e.g. from a transfectoma, (c) antibodies isolated from a library of recombinant combinatorial antibodies, and (d) antibodies obtained, expressed, created or isolated by any other means, which involve splicing immunoglobulin gene sequences with other DNA sequences.

The term "human antibody" as used herein is meant to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-directed mutagenesis in vitro, or by somatic mutation in vivo).

The term "humanized antibody" refers to a molecule having an antigen binding site that is essentially derived from an immunoglobulin from a non-human species and the rest of the immunoglobulin structure of that molecule based on the structure and/or sequence of a human immunoglobulin. This antigen binding site may either contain complete variable domains fused to constant domains or only complementarity determining regions (CDRs) grafted onto suitable framework regions in the variable domains. Antigen binding sites may be wild-type or modified with one or more amino acid substitutions, such as modified to more closely resemble human immunoglobulin. Some forms of humanized antibodies retain all CDR sequences (eg, a humanized mouse antibody that contains all six CDRs from a mouse antibody). Other forms have one or more CDRs that are altered from the parent antibody.

The term "chimeric antibody" refers to those antibodies in which one portion of each of the amino acid sequences of the heavy and light chains is homologous to the corresponding sequences of antibodies derived from certain species or belonging to a particular class, while the remainder of the chain segment is homologous to corresponding sequences from other species. Typically, the variable region of both the light and heavy chains mimics the variable regions of antibodies derived from one mammalian species, while the constant regions are homologous to sequences in antibodies derived from other species. One distinct advantage of such chimeric forms is that the variable region can be readily generated from currently known sources using readily available B cells or hybridomas of non-human hosts in combination with constant regions derived from, for example , from human cell preparations. While the variable region has the advantage of being easy to obtain and the specificity is not affected by its source, the constant region, if human, is less likely to elicit an immune response in a human subject when injected with antibodies than when the constant region is derived from source other than humans. However, the definition is not limited to such a specific example.

Antibodies can be obtained from various species, including, but not limited to, mice, rats, rabbits, guinea pigs, and humans.

The antibodies described herein include IgA antibodies such as IgAl or IgA2, IgGl, IgG2, IgG3, IgG4, IgE, IgM and IgD. In various embodiments, the antibody is an IgGl antibody, particularly an IgGl isotype kappa or lambda IgGl (i.e., IgGl, κ, λ), an IgG2a antibody (e.g., IgG2a, K, λ), an IgG2b antibody (e.g., IgG2b, κ , λ), an IgG3 antibody (eg, IgG3, κ, λ), or an IgG4 antibody (eg, IgG4, κ, λ).

As used herein, the term "heterologous antibody" is defined in relation to a transgenic organism that produces the antibody. The term refers to an antibody having an amino acid sequence or nucleic acid coding sequence corresponding to sequences found in an organism that is not a transgenic animal, and usually isolated from species other than the transgenic animal.

As used herein, the term "heterohybrid antibody" refers to an antibody having light and heavy chains from organisms of different origins. For example, an antibody having a human heavy chain associated with a mouse light chain is a heterohybrid antibody.

The antibodies described herein are preferably isolated. The term "isolated antibody" as used herein refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to CLDN18.2 contains substantially no antibodies that specifically bind to antigens other than from CLDN18.2). An isolated antibody that specifically binds to an epitope, isoform, or variant of human CLDN18.2 may, however, be cross-reactive with other related antigens, eg, from other species (eg, homologues of CLDN18.2 species). In addition, the isolated antibody may be substantially free of other cellular material and/or chemicals. In one embodiment, a combination of "isolated" monoclonal antibodies refers to antibodies having different specificities and combined into a well-characterized composition or mixture.

The terms "antigen-binding region" of an antibody (or simply "binding region") or "antigen-binding fragment" of an antibody (or simply "binding fragment"), or similar terms, refers to one or more antibody fragments that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of the entire sequence. Examples of binding fragments included in the term "antigen binding region" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH domains; (ii) F(ab') ₂ fragments, bivalent fragments consisting of two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragments consisting of VH and CH domains; (iv) Fv fragments consisting of the VL and VH domains of a single antibody region; (v) dAb fragments (Ward et al., (1989) Nature 341: 544-546) that consist of a VH domain; (vi) isolated complementarity determining regions (CDRs); and (vii) combinations of two or more isolated CDRs, which may optionally be connected by a synthetic linker. Moreover, despite the fact that the two domains in the Fv fragment, VL and VH, are encoded by different genes, they can be combined using recombinant technologies through a synthetic linker, which allows them to be built into a single protein chain in which the VL and VH regions are linked. to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85 : 5879-5883). Such single chain antibodies are also intended to be encompassed by the term "antigen binding fragment" of an antibody. A further example are binding domain-immunoglobulin fusion proteins comprising (i) a binding domain polypeptide that is fused to an immunoglobulin hinge polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge, and (iii) a heavy chain CH3 constant region. immunoglobulin fused to the CH2 constant region. The binding domain polypeptide may be a heavy chain variable region or a light chain variable region. Domain binding-immunoglobulin fusion proteins are further disclosed in US 2003/01 18592 and US 2003/0133939. Such antibody fragments are obtained using conventional methods known to those skilled in the art, and these fragments are screened in the same manner as intact antibodies.

The term "binding domain" is characterized in the context of the present invention by a structure, such as an antibody, that binds/interacts with a given target structure/antigen/epitope. Thus, the binding domain in accordance with the invention, means "site of interaction of the antigen".

All antibodies and antibody derivatives, such as antibody fragments described herein, are designated by the term "antibody" for the purposes of the invention. The term "antibody derivatives" refers to any modified form of an antibody, such as an antibody conjugate and another agent or antibody or antibody fragment. In addition, antibodies and antibody derivatives as described herein are useful in the preparation of binding agents of the invention, such as antibody fragments.

Antibodies of natural origin, as a rule, are monospecific, that is, they bind to one antigen. The present invention provides binding agents that bind to cytotoxic cells (by binding to the CD3 receptor) and cancer cells (by binding to CLDN). The binding agents of the present invention are at least bispecific or multispecific, such as trispecific, tetraspecific, etc.

The binding agent of the invention may be in the form of an antibody molecule or an antibody-like molecule, or a protein backbone with antibody-like properties, or a cyclic peptide with at least two binding specificities. Thus, the binding agent may contain one or more of the antibodies described here, or fragments thereof.

According to the invention, a bispecific molecule, in particular a bispecific protein such as a bispecific antibody, is a molecule that contains two different binding specificities and thus can bind to two different types of antigen, such as CLDN and CD3. In particular, the term "bispecific antibody" as used herein refers to an antibody comprising two antigen-binding sites, a first binding site having an affinity for a first antigen or epitope, and a second binding site having a binding affinity for a second antigen or epitope other than from the first. In particular, a bispecific antibody is an artificial protein that consists of fragments of two different antibodies (said fragments of two different antibodies form two binding domains) and thus binds to two different types of antigen. The bispecific antibody of the invention is designed to simultaneously bind to an immune system cell such as an immune effector cell, in particular a T cell such as a cytotoxic cell (by binding to CD3) and a target cell such as a cancer cell (by binding to the tumor-associated CLDN antigen) to be destroyed.

The term "bispecific antibody" also includes diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the use of a linker that is too short to allow pairing between two domains on the same chain causes these domains to pair with the complementary domains of the other chain. and creates two antigen binding sites (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121 - 1123).

"Multi-specific binding agents" are molecules that contain more than two different binding specificities.

Particularly preferred according to the invention are bispecific antibodies, including bispecific antibody fragments, in particular bispecific single chain antibodies, including bispecific single chain antibody fragments. The term "bispecific single chain antibody" means a single polypeptide chain containing two binding domains. In particular, the term "bispecific single chain antibody" or "single chain bispecific antibody", or related terms in accordance with the present invention, preferably means antibody constructs resulting from the combination of at least two antibody variable regions in a single polypeptide chain lacking constant and/or Fc portion(s) present in complete immunoglobulins.

For example, a bispecific single chain antibody may be a construct containing only two antibody variable regions, such as two VH regions, with each region capable of specific binding to a separate antigen and connected to each other via a short polypeptide spacer such that the two the variable regions of an antibody, with their included spacer, exist as one continuous polypeptide chain. Another example of a bispecific single chain antibody would be a single polypeptide chain with three antibody variable regions. In this case, two antibody variable regions, e.g. one VH and one VL, may constitute a scFv, wherein the two antibody variable regions are connected to each other via a synthetic polypeptide linker, said linker being genetically engineered to be minimally immunogenic, with while remaining as resistant to proteolysis as possible. Such an scFv is capable of specific binding to a particular antigen and is linked to a variable region of another antibody, eg a VH region capable of binding to an antigen other than the antigen bound by the scFv. Another example of a bispecific single chain antibody would be a single polypeptide chain with four antibody variable regions. In this case, the first two variable regions of an antibody, e.g., the VH region and the VL region, may form one scFv capable of binding to one antigen, while the second VH region and the VL region may form a second scFv capable of binding with a different antigen. Within a single contiguous polypeptide chain, individual antibody variable regions of the same specificity can be successfully separated by a synthetic polypeptide linker, while corresponding scFvs can be successfully separated by a short polypeptide spacer as described above.

In accordance with one embodiment of the invention, the first binding domain of the bispecific antibody comprises one antibody variable domain, preferably a VHH domain. According to one embodiment of the invention, the first binding domain of the bispecific antibody comprises two antibody variable domains, preferably scFv, ie VH-VL or VL-VH. In accordance with one embodiment of the invention, the second binding domain of the bispecific antibody comprises one antibody variable domain, preferably a VHH domain. In accordance with one embodiment of the invention, the second binding domain of the bispecific antibody comprises two antibody variable domains, preferably scFv, ie VH-VL or VL-VH. In its minimal form, the total number of antibody variable regions in a specific antibody according to the invention is thus only two. For example, such an antibody may contain two VH domains or two VHH domains.

In accordance with one embodiment of the invention, the first binding domain and the second binding domain of the bispecific antibody each contain one antibody variable domain, preferably a VHH domain. In accordance with one embodiment of the invention, the first binding domain and the second binding domain of the bispecific antibody each contain two antibody variable domains, preferably scFv, ie VH-VL or VL-VH. In this embodiment, the binding agent of the invention preferably comprises (i) an anti-CLDN antibody heavy chain variable domain (VH), (ii) an anti-CLDN antibody light chain variable domain (VL), (iii) an anti-CLDN antibody heavy chain variable domain (VH). CD3, and (iv) the light chain variable domain (VL) of an anti-CD3 antibody.

Full length bispecific antibodies can be generated by covalently linking two monoclonal antibodies or by standard cross-species hybridoma techniques. Covalent binding of two monoclonal antibodies is described in Anderson, Blood 80 (1992), 2826-34. In the context of this invention, one of the antibodies is specific for CLDN and the other for CD3.

In one embodiment, the bispecific binding agent is in an antibody-like format, wherein the heavy chain contains two consecutive N-terminal variable domains with different specificities and the light chain contains two consecutive variable domains with different specificities, resulting in four binding domains with two different specificities (Wu et al., Nat. Biotechnology, 2007, 25(11)), with one specificity being CD3 and the other specificity being CLDN.

In a preferred embodiment, the bispecific binding agent of the invention is in antibody fragment format.

In one embodiment, the bispecific molecules of the invention comprise two Fab regions, one directed against CLDN and the other directed against CD3. In one embodiment, the molecule of the invention is a complex of antigen-binding fragments (Fab)2. The Fab2 complex consists of two Fab fragments, with one Fab fragment containing an Fv domain, ie VH and VL domains specific for the CD3 antigen, and the other Fab fragment containing an Fv domain specific for CLDN. Each of the Fab fragments can consist of two single circuits, a VL-CL module and a VH-CH module. Alternatively, each of the individual Fab fragments may be single chained, preferably VL-CL-CH-VH, and the individual variable and constant domains may be connected via a peptide linker. In general, the individual single chain Fab fragments can be connected via disulfide bonds, adhesive domains, linked chemically and/or via a peptide linker. A bispecific molecule may also contain more than two Fab fragments, in particular, the molecule may be a Fab3, Fab4 or multimeric Fab complex with specificity for 2, 3, 4 or more different antigens. The invention also includes chemically linked Fabs.

In one embodiment, a binding agent according to the invention comprises various types of bivalent and trivalent single chain variable fragments (scFv), fusion proteins that mimic the variable domains of two antibodies. A single chain variable fragment (scFv) is a fusion protein consisting of immunoglobulin heavy (VH) and light chain (VL) variable regions joined by a short linker peptide of about 10-25 amino acids. The linker is usually rich in glycine for flexibility, and serine or threonine for solubility, and may connect either the N-terminus of the VH to the C-terminus of the VL, or vice versa. Divalent (bivalent) single chain variable fragments (di-scFv, bi-scFv) can be constructed by linking two scFv. This can be done by making a single peptide chain with two VH and two VL regions to produce tandem scFvs. The invention also includes multispecific molecules containing more than two scFv-binding domains. This ensures that the molecule has multiple antigen specificities and is trispecific, tetraspecific or multispecific, or the molecule is a bispecific molecule containing more than one scFv binding domain with specificity for the same antigen. In particular, the molecule of the invention may be a multispecific single chain Fv molecule.

Another possibility is to design scFvs with linker peptides that are too short to link two variable regions (about five amino acids), initiating scFv dimerization. This type is known as a diabody. Even shorter linkers (one or two amino acids) lead to the formation of trimers, the so-called triabodies or tribodies. Tetrabodies have also been received. They exhibit even higher affinity for their targets than diabodies.

A particularly preferred example of a bispecific antibody fragment is a diabody (Kipriyanov, Int. J. Cancer 77 (1998), 763-772), which is a small fragment of a bivalent and bispecific antibody. Diabodies contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL) by a peptide linker that is too short to allow pairing between the two domains on the same polypeptide chain. the same chain. This causes pairing with complementary domains of the other strand and facilitates the assembly of a dimeric molecule with two functional antigen-binding sites. To construct the bispecific diabodies of the invention, the V domains of an anti-CD3 antibody and an anti-CLDN antibody can be fused to create two chains VH(CD3)-VL(CLDN), VH(CLDN)-VL(CD3). Each strand is incapable of binding to the corresponding antigen by itself, but recreates functional anti-CD3 antibody and anti-CLDN antibody antigen-binding sites when paired with the other strand. In this case, a peptide linker is used that is too short to allow pairing between two domains on the same chain. Two scFv molecules with a linker between the heavy chain variable domain and the light chain variable domain that is too short for intramolecular dimerization co-express and self-assemble to form bispecific molecules with two binding sites at opposite ends.

In one embodiment, a multispecific molecule in accordance with the invention contains the variable (VH, VL) and constant (C) domains of immunoglobulins. In one embodiment, the bispecific molecule is a minibody, preferably a minibody, containing two VH-VL-C single chains that are linked to each other via the constant domains (C) of each chain. According to this aspect, the respective heavy chain variable regions (VH), the respective light chain variable regions (VL), and the constant domains (C) are located N-terminally to C-terminally in the order VH(CLDN)-VL(CLDN)-( C) and VH(CD3)-VL(CD3)-C, where C is preferably a CH3 domain. Pairing of the constant domains results in the formation of a minibody.

In another particularly preferred aspect, the bespecifically binding agent of the invention is in the format of a bispecific single chain antibody construct, said construct comprising or consisting of at least two binding domains, one of said domains binding to CLDN and the other domain binds to CD3. Such molecules, also referred to as "bispecific T cell engagers" (BiTE; the term BiTE refers only to bispecific molecules whose one arm is specific for CD3), consist of two scFv molecules connected via a linker peptide.

As used herein, the term "bispecific single chain antibody" means a single polypeptide chain containing two binding domains. Each binding domain contains one variable region from the antibody heavy chain ("VH region"), with the VH region of the first binding domain specifically binding to CLDN and the VH region of the second binding domain specifically binding to CD3. The two binding domains are optionally linked to each other by a short polypeptide spacer. A non-limiting example of a polypeptide spacer is Gly-Gly-Gly-Gly-Ser (G-G-G-G-S) and repeats thereof. Each binding domain may further comprise one antibody light chain variable region ("VL region"), wherein the VH region and the VL region within each of the first and second binding domains are linked to each other via a polypeptide linker of sufficient length to to allow pairing of the VH region and the VL region of the first binding domain with the VH region and the VL region of the second binding domain.

In accordance with this aspect, the respective heavy chain variable regions (VH) and the respective light chain variable regions (VL) are located N-terminally to C-terminally in the order VH(CLDN)-VL(CLDN)-VH(CD3)- VL(CD3), VH(CD3)-VL(CD3)-VH(CLDN)-VL(CLDN) or VH(CD3)-VL(CD3)-VL(CLDN)-VH(CLDN). However, the bispecific single chain antibodies of the invention are expected to contain other domain arrangements such as VL(CLDN)-VH(CLDN)-VH(CD3)-VL(CD3), VL(CLDN)-VH(CLDN)-VL(CD3) -VH(CD3), VH(CLDN)-VL(CLDN)-VL(CD3)-VH(CD3), VL(CD3)-VH(CD3)-VH(CLDN)-VL(CLDN), VL(CD3) -VH(CD3)-VL(CLDN)-VH(CLDN).

A long linker typically joins the respective heavy chain variable regions (VL) and respective light chain variable regions (VL) to form an scFv binding domain, while a short linker generally joins two scFv binding domains. The linker is typically designed to be flexible and resistant to protease and preferably the linker contains glycine and/or serine amino acid residues. Short peptide linker sequences may be 12 or fewer, such as 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acids, and preferably 5 or 6 amino acids. Short peptide linkers preferably contain the amino acid sequences SGGGGS or GGGGS. Long peptide linkers may be 12 or more, such as 15-25 or 15-20 or 15-18 amino acids. Long peptide linkers preferably contain the amino acid sequences (GGGGS)3 or VE(GGSGGS)2GGVD. Additional long peptide linkers may contain the amino acid sequences (GGGGS)4, (GGGGS)5 or GGGGS(GGS)3GGGS.

Binding agents according to the invention may also contain an amino acid sequence to assist in the secretion of the molecule, for example an N-terminal secretion signal, and/or one or more epitope tags to assist in binding, purification or detection of the molecule.

Preferably, the secretion signal is a signal sequence (eg, selected from any of SEQ ID NOs: 51, 52, 53, 54, 55) that allows sufficient passage through the secretory pathway and/or secretion of the binding agent into the extracellular space. Preferably, the secretion signal sequence is cleavable and removed from the mature binding agent. The secretion signal sequence is preferably selected according to the cell or organism in which the binding agent is being produced.

The amino acid sequence of the epitope tag may be inserted at any position within the amino acid sequence of the binding agent, and may take the form of a loop within the encoded protein structure, or may be fused at the N-terminus or C-terminus to the binding agent. Preferably, the epitope tag is C-terminally fused to a binding agent. The epitope label may contain a cleavage site that allows the label to be removed from the binding agent. Said epitope tag may be any kind of epitope tag that is functional under native and/or denaturing conditions, preferably a histidine tag, most preferably a tag containing six histidine residues.

The bispecific binding agent of the invention may contain, in addition to said first and second binding domains, an additional binding domain which serves, for example, to increase selectivity for tumor cells. This can be achieved, for example, by providing binding domains that bind to other antigens expressed on tumor cells.

In the context of the present invention, the created binding agents are preferably capable of inducing the immune effector functions described here. Preferably, said immune effector functions are directed against cells bearing the tumor-associated CLDN antigen on their surface.

The term "immune effector functions" in the context of the present invention includes any functions mediated by components of the immune system that result in, for example, inhibition of tumor growth and/or inhibition of tumor development, including inhibition of tumor spread and metastasis. Preferably, immune effector functions lead to the death of tumor cells. Such functions include complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), induction of apoptosis in cells bearing a tumor-associated antigen, cytolysis of cells bearing a tumor-associated antigen , and/or inhibition of proliferation of cells bearing a tumor-associated antigen. Binding agents may also exert their effect simply by binding to tumor-associated antigens on the surface of the cancer cell. For example, antibodies can block the function of a tumor-associated antigen or induce apoptosis only by binding to a tumor-associated antigen on the surface of a cancer cell.

The binding agents described herein may be conjugated to a therapeutic moiety or agent, such as a cytotoxin, a drug (eg, an immunosuppressant), or a radioactive isotope. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells and specifically kills cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tatracaine , lidocaine, propranolol and puromycin and analogues or homologues thereof. Suitable therapeutic agents for forming conjugates include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine), alkylating agents (eg, mechlorethamine, thiope chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomanitol, streptozotocin, mitomycin C and platinum(II) cis-dichlorodiamine (DDP) cisplatin), anthracyclines (eg daunorubicin (original name daunomycin) and doxorubicin), antibiotics (eg , dactinomycin (original name actinomycin), bleomycin, mithramycin and anthramycin (AMC), and antimitotic agents (e.g. vincristine and vinblastine).In a preferred embodiment, the therapeutic agent is a cytotoxic agent or a radiotoxic agent.In another embodiment, the therapeutic agent is immunosuppressant In yet another embodiment, the therapeutic agent is GM-CSF. In a preferred embodiment, the therapeutic agent is doxorubicin, cisplatin, bleomycin, sulfate, carmustine, chlorambucil, cycloforamide, or ricin A.

Also, binding agents can be conjugated with radioactive isotopes such as iodine-131, yttrium-90 or indium-111 to create cytotoxic radiopharmaceuticals.

Techniques for conjugating such therapeutic fragments to antibodies are well known, see, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy" in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery" in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review" in Monoclonal Antibodies '84: Biological And Clinical Applications, Pincheraet al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy" in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev. 62: 119-58 (1982).

The term "binding" in accordance with the invention preferably refers to specific binding.

In accordance with the present invention, an agent, such as an antibody, is capable of binding to a predefined target if it has significant affinity for said predefined target and binds to said predefined target in standard assays. "Affinity" or "binding affinity" is often measured by the dissociation constant (K _D ). Preferably, the term "significant affinity" refers to binding to a predetermined target with a dissociation constant (K _D ) of 10 ^-5 M or less, 10 ^-6 M or less, 10 ^-7 M or less, 10 ^-8 M or less , 10 ^-9 M or less, 10 ^-10 M or less, 10 ^-11 M or less, or 10 ^-12 M or less.

An agent is not able to (significantly) bind to a target unless it has significant affinity for said target and binds to a significant extent, in particular, does not bind in a detectable manner to said target in standard assays. Preferably, the agent does not detectably bind to said target when present at a concentration of up to 2, preferably 10, more preferably 20, especially 50 or 100 mg/ml or higher. Preferably, the agent does not have significant affinity for the target if it binds to said target with a K _D that is at least 10 times, 100 times, 10 ³ times, 10 ⁴ times, 10 ⁵ times, or 10 ⁶ times higher than K _D for binding to a predetermined target with which the agent is able to bind. For example, if the K _D for binding an agent to a target for which the agent is able to bind is 10 ^-7 M, the K _D for binding to a target for which the agent has no significant affinity would be at least 10 ^-6 M , 10 ^-5 M, 10 ^-4 M, 10 ^-3 M, 10 ^-2 M or 10 ^-1 M.

An agent, such as an antibody, is specific for a predefined target if it is able to bind to said predefined target without being able to bind to other targets, i.e. it does not have significant affinity for other targets and does not significantly bind to others. targets in standard assays. According to the invention, an agent is specific for CLDN if it is able to bind to CLDN but not (significantly) able to bind to other targets. Preferably, the agent is specific for CLDN if the affinity for such other targets and binding to such other targets does not significantly exceed affinity for unrelated CLDN proteins or binding to unrelated CLDN proteins such as bovine serum albumin (BSA), casein, human serum albumin (HSA), or non-claudin transmembrane proteins such as MHC molecules or transferrin receptor, or any other polypeptide specified. Preferably, an agent is specific for a predetermined target if it binds to said target with a K _D that is at least 10 times, 100 times, 10 ³ times, 10 ⁴ times, 10 ⁵ times, or 10 ⁶ times lower than the K _D for binding to a target for which it is not specific. For example, if the K _D for binding an agent to a target for which it is specific is 10 ^-7 M, the K _D for binding to a target for which it is not specific would be at least 10 ^-6 M , 10 ^-5 M, 10 ^-4 M, 10 ^-3 M, 10 ^-2 M or 10 ^-1 M.

The binding of an agent to a target can be determined experimentally using any suitable method; see, for example, Berzofsky et al., "Antibody-Antigen Interactions" in Fundamental Immunology, Paul, WE, Ed., Raven Press New York, NY (1984), Kuby, Janis Immunology, WH Freeman and Company New York, NY ( 1992), and the methods described herein. Affinity can be easily determined by standard methods such as equilibrium dialysis; analysis using the BIAcore 2000 device in accordance with the general procedures described by the manufacturer; radioimmunoassay using a radioactively labeled target antigen; or by other method known to those skilled in the art. Affinity data can be analyzed, for example, by the method of Scatchard et al., Ann NY Acad. ScL, 51:660 (1949). The measured affinity of a particular antibody-antigen interaction may vary when measured under different conditions, eg salt concentration, pH. Thus, measurements of affinity and other antigen-binding parameters, eg K _D , IC ₅₀ , are preferably carried out in standardized antibody and antigen solutions and standardized buffer.

The term "isotype" as used herein refers to the class of antibodies (eg, IgM or IgGl) encoded by heavy chain constant region genes.

The term "isotype switch" as used herein refers to the phenomenon whereby an antibody class or isotype changes from one class to one of the other Ig classes.

The term "natural", as used here, is applied to an object to denote the fact that this object can be found in natural conditions. For example, a polypeptide or polynucleotide sequence that is present in the body (including viruses) that can be isolated from a natural source and that has not been intentionally modified by humans in the laboratory is naturally occurring.

The term "rearranged" as used herein refers to the configuration of an immunoglobulin heavy chain or light chain locus where the position of the V segment is in close proximity to the D-J or J segment in a conformation encoding essentially a complete VH or VL domain, respectively. The rearranged immunoglobulin (antibody) gene locus can be identified by comparison with germline DNA; the rearranged locus will contain at least one recombinant heptameric/non-meric homologous element.

The term "unrearranged" or "germline configuration" as used herein in relation to a V segment refers to a configuration in which the V segment does not undergo recombination such that it would be in close proximity to the D or J segment.

In one embodiment, the binding agent of the invention has the ability to bind to CLDN18.2, i.e. the ability to bind to an epitope present in CLDN18.2, preferably an epitope located within the extracellular domains of CLDN18.2, especially the first extracellular loop, preferably , at amino acid positions 29 to 78 of CLDN18.2. In specific embodiments, an agent with the ability to bind to CLDN18.2 binds to an epitope on CLDN18.2 that is not present on CLDN18.1.

An agent with the ability to bind to CLDN18.2 preferentially binds to CLDN18.2 but not to CLDN18.1. Preferably, the CLDN18.2 binding agent is specific for CLDN18.2. Preferably, the CLDN18.2 binding agent binds to CLDN1 18.2 expressed on the cell surface. In particularly preferred embodiments, the CLDN18.2 binding agent binds to native CLDN18.2 epitopes present on the surface of living cells.

In a preferred embodiment, the CLDN18.2 binding agent comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, 8, 9, 10 and their fragments.

In a preferred embodiment, the CLDN18.2 binding agent comprises a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 1 , 12, 13, 14, 15, 16, 17, 18, 19 and their fragments.

In certain preferred embodiments, the CLDN18.2 binding agent comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following options (i) through (ix):

(i) VH contains the amino acid sequence shown in SEQ ID NO: 5, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 12, or a fragment thereof;

(ii) VH contains the amino acid sequence shown in SEQ ID NO: 6, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 11, or a fragment thereof;

(iii) VH contains the amino acid sequence shown in SEQ ID NO: 7, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 13, or a fragment thereof;

(iv) VH contains the amino acid sequence shown in SEQ ID NO: 9, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 16, or a fragment thereof;

(v) VH contains the amino acid sequence shown in SEQ ID NO: 8, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 15, or a fragment thereof;

(vi) VH contains the amino acid sequence shown in SEQ ID NO: 10, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 14, or a fragment thereof;

(vii) VH contains the amino acid sequence shown in SEQ ID NO: 10, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 17, or a fragment thereof;

(viii) VH contains the amino acid sequence shown in SEQ ID NO: 10, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 18, or a fragment thereof;

(ix) VH contains the amino acid sequence shown in SEQ ID NO: 10, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 19, or a fragment thereof.

In a particularly preferred embodiment, the CLDN18.2 binding agent comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL):

VH contains the amino acid sequence shown in SEQ ID NO: 8, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 15, or a fragment thereof.

In a further particularly preferred embodiment, the CLDN18.2 binding agent comprises the following combination of a heavy chain variable region (VH) and a light chain variable region (VL):

VH contains the amino acid sequence shown in SEQ ID NO: 6, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 11, or a fragment thereof.

The term "fragment" refers in particular to one or more complementarity determining regions (CDRs), preferably at least a CDR3 variable region, a heavy chain variable region (VH) and/or a light chain variable region (VL). In one embodiment, said one or more complementarity-determining regions (CDRs) are selected from the group of complementarity-determining regions CDR1, CDR2, and CDR3. In a particularly preferred embodiment, the term "fragment" refers to the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and/or the light chain variable region (VL).

In one embodiment, a binding agent containing one or more CDRs, a set of CDRs, or a combination of sets of CDRs, as described herein, contains these CDRs along with the framework regions located between them. Preferably, the portion will also contain at least about 50% of one or both of two or both of the first and fourth framework regions, with 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. The construction of linking agents by recombinant DNA methods may result in the introduction of N- or C-terminal residues into the variable regions encoded by linkers introduced to facilitate cloning or other processing steps, including the introduction of linkers to connect the variable regions according to of the invention with additional protein sequences, including immunoglobulin heavy chains, other variable domains (eg, in the production of diabodies), or protein tags.

In one embodiment, a binding agent containing one or more CDRs, a set of CDRs, or a combination of sets of CDRs, as described herein, contains said CDRs in a human antibody framework region.

In one embodiment, the binding agent of the invention has the ability to bind to CLDN6, i.e. the ability to bind to an epitope present in CLDN6, preferably an epitope located within the extracellular domains of CLDN6, in particular in the first extracellular loop, preferably at amino acid positions 28 to 76 CLDN6, or in a second extracellular loop, preferably at amino acid positions 141 to 159 of CLDN6. In specific embodiments, an agent with the ability to bind to CLDN6 binds to an epitope on CLDN6 that is not present on CLDN9. Preferably, the CLDN6 binding agent binds to an epitope on CLDN6 that is not present on CLDN4 and/or CLDN3. Most preferably, the CLDN6 binding agent binds to an epitope on CLDN6 that is not present on a CLDN protein other than CLDN6.

An agent having the ability to bind to CLDN6 preferably binds to CLDN6 but not to CLDN9, and preferably does not bind to CLDN4 and/or CLDN3. Preferably, the CLDN6 binding agent is specific for CLDN6. Preferably, the CLDN6 binding agent binds to CLDN6 expressed on the cell surface. In particularly preferred embodiments, the CLDN6 binding agent binds to native CLDN6 epitopes present on the surface of living cells.

In a preferred embodiment, the CLDN6 binding agent comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20, 22, 24, 26 and fragments thereof.

In a preferred embodiment, the CLDN6 binding agent comprises a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 23, 25, 27, 28, 29, 97, 98 , 99, 100 and their fragments.

In certain preferred embodiments, the CLDN6 binding agent comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following options (i) to (xi):

(i) VH contains the amino acid sequence shown in SEQ ID NO: 20, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 21, or a fragment thereof;

(ii) VH contains the amino acid sequence shown in SEQ ID NO: 22, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 23, or a fragment thereof;

(iii) VH contains the amino acid sequence shown in SEQ ID NO: 24, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 25, or a fragment thereof;

(iv) VH contains the amino acid sequence shown in SEQ ID NO: 26, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 27, or a fragment thereof;

(v) VH contains the amino acid sequence shown in SEQ ID NO: 22, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 21, or a fragment thereof;

(vi) VH contains the amino acid sequence shown in SEQ ID NO: 22, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 28, or a fragment thereof;

(vii) VH contains the amino acid sequence shown in SEQ ID NO: 22, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 29, or a fragment thereof;

(viii) VH contains the amino acid sequence shown in SEQ ID NO: 22, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 97, or a fragment thereof;

(ix) VH contains the amino acid sequence shown in SEQ ID NO: 22, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 98, or a fragment thereof;

(x) VH contains the amino acid sequence shown in SEQ ID NO: 22, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 99, or a fragment thereof;

(xi) VH contains the amino acid sequence shown in SEQ ID NO: 22, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 100, or a fragment thereof.

In a particularly preferred embodiment, the CLDN6 binding agent comprises the following combination of a heavy chain variable region (VH) and a light chain variable region (VL):

VH contains the amino acid sequence represented by SEQ ID NO: 22 or a fragment thereof and VL contains the amino acid sequence represented by SEQ ID NO: 23 or a fragment thereof.

In a further particularly preferred embodiment, the CLDN6 binding agent comprises the following combination of a heavy chain variable region (VH) and a light chain variable region (VL):

VH contains the amino acid sequence shown in SEQ ID NO: 22, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 97, or a fragment thereof.

In a further particularly preferred embodiment, the CLDN6 binding agent comprises the following combination of a heavy chain variable region (VH) and a light chain variable region (VL):

VH contains the amino acid sequence shown in SEQ ID NO: 22, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 98, or a fragment thereof.

In a further particularly preferred embodiment, the CLDN6 binding agent comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL):

VH contains the amino acid sequence shown in SEQ ID NO: 22, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 99, or a fragment thereof.

In a further particularly preferred embodiment, the CLDN6 binding agent comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL):

VH contains the amino acid sequence shown in SEQ ID NO: 22, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 100, or a fragment thereof.

The term "fragment" refers in particular to one or more complementarity determining regions (CDRs), preferably at least a CDR3 variable region, a heavy chain variable region (VH) and/or a light chain variable region (VL). In one embodiment, said one or more complementarity-determining regions (CDRs) are selected from a set of complementarity-determining regions CDR1, CDR2, and CDR3. In a particularly preferred embodiment, the term "fragment" refers to the complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and/or the light chain variable region (VL).

In one embodiment, a binding agent containing one or more CDRs, a set of CDRs, or a combination of sets of CDRs, as described herein, contains these CDRs along with the framework regions located between them. Preferably, the portion will also contain at least about 50% of one or both of two or both of the first and fourth framework regions, with 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. The construction of linking agents by recombinant DNA methods may result in the introduction of N- or C-terminal residues into the variable regions encoded by linkers introduced to facilitate cloning or other processing steps, including the introduction of linkers to connect the variable regions according to of the invention with additional protein sequences, including immunoglobulin heavy chains, other variable domains (for example, in the production of diabodies) or protein tags..

In one embodiment, a binding agent containing one or more CDRs, a set of CDRs, or a combination of sets of CDRs, as described herein, contains said CDRs in a human antibody framework region.

Anti-CD3 antibodies that are suitable for providing binding agents according to the invention include, but are not limited to, UCHT1-HS (humanized mAB), UCHT1-MM (mouse mAB), CLB-T3, TR66, 145-2C11.

UCHT1 is a monoclonal IgG1 anti-CD3 monoclonal antibody that detects CD3 in humans and primates. CLB-T3 is a mouse monoclonal anti-CD3 antibody that is directed against the CD3 antigen and interacts with 80-90% of human peripheral T lymphocytes and medullary thymocytes. TR66 is a mouse IgG1 monoclonal anti-CD3 antibody that recognizes the human CD3 epsilon chain. 145-2C11 is a gray hamster anti-mouse CD3 monoclonal antibody.

Preferably, the VH and VL regions of the CD3 binding domain are derived from antibody/antibody and antibody-like molecules that are capable of specifically recognizing human CD3 in the context of other TCR subunits that are present on activated primary human T cells expressing TCR in their native configuration. VH and VL regions derived from an antibody specific for the CD3 epsilon chain are most preferred, and said (parental) antibodies should be able to specifically bind epitopes reflecting native or near-native structure, or a conformational human CD3 epitope as shown in context of the TCR complex. In a preferred embodiment, the VH and VL regions of the CD3 binding domain are derived from a CD3-specific antibody selected from the group consisting of UCHT1-HS, UCHT1-MM, CLB-T3 and TR66, preferably TR66.

In a preferred embodiment, the CD3 binding agent comprises a heavy chain variable region (VH) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 32, 34, 36, 94, 95 and fragments thereof.

In a preferred embodiment, the CD3 binding agent comprises a light chain variable region (VL) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31, 33, 35, 37, 96 and fragments thereof.

In certain preferred embodiments, the CD3 binding agent comprises a combination of a heavy chain variable region (VH) and a light chain variable region (VL) selected from the following options (i) through (ix):

(i) VH contains the amino acid sequence shown in SEQ ID NO: 30, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 31, or a fragment thereof;

(ii) VH contains the amino acid sequence shown in SEQ ID NO: 32, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 33, or a fragment thereof;

(iii) VH contains the amino acid sequence shown in SEQ ID NO: 34, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 34, or a fragment thereof;

(iv) VH contains the amino acid sequence shown in SEQ ID NO: 36, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 37, or a fragment thereof;

(v) VH contains the amino acid sequence shown in SEQ ID NO: 94, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 37, or a fragment thereof;

(vi) VH contains the amino acid sequence shown in SEQ ID NO: 95, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 37, or a fragment thereof;

(vii) VH contains the amino acid sequence shown in SEQ ID NO: 36, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 96, or a fragment thereof;

(viii) VH contains the amino acid sequence shown in SEQ ID NO: 94, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 96, or a fragment thereof;

(ix) VH contains the amino acid sequence shown in SEQ ID NO: 95, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 96, or a fragment thereof.

In a particularly preferred embodiment, the CD3 binding agent comprises the following combination of heavy chain variable region (VH) and light chain variable region (VL):

VH contains the amino acid sequence shown in SEQ ID NO: 36 or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 37 or a fragment thereof.

In a further particularly preferred embodiment, the CD3 binding agent comprises the following combination of a heavy chain variable region (VH) and a light chain variable region (VL):

VH contains the amino acid sequence shown in SEQ ID NO: 94 or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 37 or a fragment thereof.

In a further particularly preferred embodiment, the CD3 binding agent comprises the following combination of a heavy chain variable region (VH) and a light chain variable region (VL):

VH contains the amino acid sequence shown in SEQ ID NO: 95, or a fragment thereof, and VL contains the amino acid sequence shown in SEQ ID NO: 96, or a fragment thereof.

The term "fragment" refers in particular to one or more complementarity determining regions (CDRs), preferably at least a CDR3 variable region, a heavy chain variable region (VH) and/or a light chain variable region (VL). In one embodiment, said one or more of the complementarity-determining regions (CDRs) are selected from a set of complementarity-determining regions CDR1, CDR2, and CDR3. In a particularly preferred embodiment, the term "fragment" refers to the complementarity-determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region (VH) and/or the light chain variable region (VL).

In one embodiment, a binding agent containing one or more CDRs, a set of CDRs, or a combination of sets of CDRs, as described herein, contains these CDRs along with the framework regions located between them. Preferably, the portion will also contain at least about 50% of one or both of two or both of the first and fourth framework regions, with 50% being the C-terminal 50% of the first framework region and the N-terminal 50% of the fourth framework region. The construction of linking agents by recombinant DNA methods may result in the introduction of N- or C-terminal residues into the variable regions encoded by linkers introduced to facilitate cloning or other processing steps, including the introduction of linkers to connect the variable regions according to of the invention with additional protein sequences, including immunoglobulin heavy chains, other variable domains (eg, in the production of diabodies), or protein tags.

In one embodiment, a binding agent containing one or more CDRs, a set of CDRs, or a combination of sets of CDRs as described herein contains said CDRs in a human framework.

According to the invention, a preferred binding agent targeting CLDN18.2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 38, 39, 40 and 41 or variants thereof.

According to the invention, a further preferred binding agent targeting CLDN18.2 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 103, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92 and 93, or fragments or variants thereof. In one embodiment, said amino acid sequence is devoid of secretion signals, such as N-terminal secretion signals, in particular the sequence according to SEQ ID NO: 51, and/or is devoid of His tags, such as C-terminal His tags, in particular sequence Gly-Gly-Ser-(His) ₆ or (His) ₆ if present.

According to the invention, a preferred CLDN6 targeting binding agent comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 42, 43, 44 and 45, or variants thereof.

According to the invention, a further preferred CLDN6 targeting binding agent comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 101, 102, 60, 61, 62, 63, 64 and 65, or fragments or variants thereof. In one embodiment, said amino acid sequence is devoid of secretion signals, such as N-terminal secretion signals, in particular the sequence according to SEQ ID NO: 51, and/or is devoid of His tags, such as C-terminal His tags, in particular sequence Gly-Gly-Ser-(His) ₆ or (His) ₆ if present.

It should be understood that the binding agents described herein can be delivered to a patient by administering a nucleic acid, such as an RNA coding agent, and/or by administering a host cell containing a nucleic acid, such as an RNA coding agent. Thus, the nucleic acid encoding the binding agent, when administered to a patient, may be present in free form or in a suitable delivery vehicle, eg in the form of liposomes or viral particles, or within the host cell. The provided nucleic acid can produce the agent for extended periods of time in a sustained manner, suppressing the volatility, at least in part, seen with therapeutic antibodies, especially bispecific antibodies. Nucleic acids to be delivered to a patient can be obtained using recombinant methods. If the nucleic acid is administered to a patient when present in a host cell, it is preferably taken up by the patient's cells to express the binding agent encoded by the nucleic acid. If the nucleic acid is administered to a patient when present in a host cell, it is preferably expressed by the host cell in the patient's body so as to produce the binding agent encoded by the nucleic acid.

The term "recombinant" in the context of the present invention means "genetically engineered". Preferably, a "recombinant object", such as a recombinant nucleic acid, in the context of the present invention is not of natural origin.

The term "natural" as used here refers to the fact that an object can be found in natural conditions. For example, a peptide or nucleic acid that is present in the body (including viruses) and that can be isolated from a natural source and has not been deliberately modified by humans in the laboratory is naturally occurring.

The term "nucleic acid" as used herein means the inclusion of DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. The nucleic acid can be single stranded or double stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA.

Nucleic acids may be included in the vector. The term "vector" as used herein includes any vectors known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus or baculovirus vectors, or vectors based on artificial chromosomes, such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or PI-based artificial chromosomes (PAC). These vectors include expression as well as cloning vectors. Expression vectors contain plasmids as well as viral vectors, and typically contain the desired coding sequence and corresponding DNA sequences necessary for expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or expression systems. in vitro. Cloning vectors are typically used to create and amplify certain desired DNA fragments and may lack the functional sequences required to express the desired DNA fragments.

In the context of the present invention, the term "RNA" refers to a molecule that contains ribonucleotide residues and preferably consists entirely or essentially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide containing a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. The term includes double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, essentially pure RNA, synthetic RNA obtained by recombinant RNA methods, as well as modified RNA that differs from natural RNA by addition, deletion, substitution and/or changes in one or more nucleotides. Such alterations may include the addition of material of a non-nucleotide nature, such as additives contained at one or both ends of the RNA molecule or introduced into the molecule, for example, located at one or more nucleotides in the RNA. Nucleotides within RNA molecules may also include non-standard nucleotides such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs can be considered as analogs or analogs of natural RNAs.

In accordance with the present invention, the term "RNA" includes and preferably refers to "mRNA", which means "messenger RNA" and refers to a "transcript" that can be obtained using DNA as a template and encodes a peptide or protein. mRNA typically contains a 5' untranslated region (5'-UTR), a protein or peptide coding region, and a 3' untranslated region (3'-UTR). mRNA has a limited half-life in cells and in vitro. Preferably, mRNA is obtained by in vitro transcription using a DNA template. In one embodiment, the RNA is obtained by in vitro transcription or chemical synthesis. The in vitro transcription method is known to those skilled in the art. For example, there are various commercially available vitro transcription kits.

In one embodiment of the present invention, the RNA is a self-replicating RNA, such as a self-replicating single-stranded RNA. In one embodiment, the self-replicating RNA is a single stranded positive polar RNA. In one embodiment, the self-replicating RNA is viral RNA or RNA derived from viral RNA. In one embodiment, the self-replicating RNA is alphavirus genomic RNA or RNA derived from alphavirus genomes. In one embodiment, the self-replicating RNA is an expression vector based on viral genes. In one embodiment, the virus is a Semliki forest virus. In one embodiment, the self-replicating RNA contains one or more transgenes, wherein at least one of these transgenes encodes a binding agent described here. In one embodiment, if the RNA is or derived from viral RNA, the transgenes may partially or completely replace viral sequences, such as viral sequences encoding structural proteins. In one embodiment, the self-replicating RNA is an in vitro transcribed RNA.

The alphavirus genome is a single-stranded positive polar RNA (ssRNA(+)) that encodes two open reading frames (ORFs) for large polyproteins. ORF at the 5' end of the genome encodes non-structural proteins nSP1 to nSP4 (nsPl-4), which are translated and processed into RNA-dependent RNA polymerase (replicase); ORF at the 3'-end encodes structural proteins - capsid and glycoproteins. Both ORFs are separated by the so-called subgenomic promoter (SGP), which drives the transcription of the structural ORF. When used as gene vectors, the structural proteins upstream of the subgene promoter (SGP) are usually replaced by transgenes. To package such vectors into viral particles, structural proteins are usually expressed in trans from helper constructs. Replication of alphaviruses occurs in the cytoplasm of infected cells, exclusively at the RNA level. After infection with the ssRNA(+) genome, it acts as mRNA for translation of the nsP1234 polyprotein precursor, which is located in the early stages of the viral life cycle, autoproteolytically processed into nsP123 and nsP4 fragments. Fragments nsP123 and nsP4 form a (-) strand replicase complex that transcribes a (-) strand RNA from a genomic RNA template. In later stages, the nsP1234 polyprotein is completely cleaved into single proteins that assemble into a (+)strand replicase complex that synthesizes new (+)strand genomes as well as subgenomic transcripts that encode structural proteins or transgenes. The subgenomic RNA, as well as the new genomic RNA, is capped and polyadenylated and thus recognized as mRNA upon infection of target cells. Only the new genomic RNA contains a packaging signal that provides specific packaging of the genomic RNA into budding virions. The attractiveness of alphavirus replicons for vectorology is based on the positive orientation of the capped and polyadenylated RNA genome. A translated replicon RNA can be easily synthesized in vitro, capping can be achieved with a cap analog added to the in vitro transcription reaction, and poly(A) tails can be encoded as traces of polytimylic acid (poly-T) on plasmid matrices. In vitro transcribed (IVT) replicons are transfected by conventional transfection methods and even low amounts of the original IVT RNA multiply rapidly. Within a few hours of transfer, the transgenes that are placed downstream of the SGP were transcribed into high copy numbers, approximately 40,000 to 200,000 subgenomic RNA copies per cell, so it is not surprising that expression of the recombinant proteins is high. Depending on the specific target, IVT replicons can be transfected directly into target cells and packaged into alphavirus particles with helper vectors that provide structural genes in trans. Transfer to skin or muscle results in high and sustained local expression, along with strong induction of humoral and cellular immune responses.

To increase the expression and/or stability of the RNA used in accordance with the present invention, the RNA can be modified, preferably without changing the sequence of the expressed peptide or protein.

The term "modification" in the context of RNA, as used in accordance with the present invention, includes any modification of the RNA, which is not naturally present in the specified RNA.

In one embodiment of the invention, the RNA used in accordance with the invention does not contain uncapped 5'-triphosphates. Removal of such uncapped 5'-triphosphates can be achieved by treating the RNA with phosphatase.

RNA according to the invention can be modified with naturally occurring or synthetic ribonucleotides to increase its stability and/or reduce cytotoxicity. For example, in one embodiment, in the RNA used in accordance with the invention, cytidine was replaced partially or completely, preferably completely, with 5-methylcytidine. Alternatively or additionally, in one embodiment, in the RNA used in accordance with the invention, the uridine has been replaced partially or completely, preferably completely, with pseudouridine.

In one embodiment, the term "modification" refers to providing an RNA with a 5'-cap or 5'-cap analog. The term "5' cap" refers to the cap structure found at the 5' end of the mRNA molecule and typically consists of a guanosine nucleotide linked to the mRNA via an unusual 5'-5' triphosphate bond. In one embodiment, this guanosine is 7-methylated. The term "typical" "5'-cap" refers to the 5'-cap of naturally occurring RNA, preferably the 7-methylguanosine cap (m7G). In the context of the present invention, the term "5'-cap" includes a 5'-cap analog that resembles the RNA cap structure and is modified to be capable of stabilizing RNA when attached to it, preferably in vivo and/or in the cell.

Providing the RNA with a 5'-cap or 5'-cap analog can be achieved by in vitro transcription of the template DNA in the presence of said 5'-cap or 5'-cap analog, said 5'-cap being co-transcriptionally inserted into the generated RNA strand. , or the RNA can be formed, for example, by in vitro transcription, and the 5'-cap can be attached to the RNA post-transcriptionally using capping enzymes, for example, vaccinia virus capping enzymes.

The RNA may contain additional modifications. For example, additional modification of the RNA used in the present invention may be an extension or shortening of the naturally occurring poly(A) tail, or alteration of the 5' or 3' untranslated regions (UTRs), such as the introduction of a UTR that is not related to the coding region of said RNA, for example by inserting one or more, preferably two copies of the 3'UTR derived from a globin gene such as alpha2-globin, alpha1-globin, beta-globin, preferably beta-globin, more preferably human beta-globin.

Thus, in order to increase the stability and/or expression of the RNA used in accordance with the present invention, the RNA can be modified to be present in association with a poly-A sequence, preferably having a length of 10 to 500, more preferably 30 to 300, even more preferably 65 to 200, and particularly preferably 100 to 150 adenosine residues. In a particularly preferred embodiment, the poly-A sequence is approximately 120 adenosine residues in length. In addition, insertion of two or more 3' untranslated regions (UTRs) into the 3' untranslated region of an RNA molecule can result in increased translation efficiency. In one specific embodiment, the 3'-UTR is derived from the human β-globin gene.

Preferably, the RNA, when delivered, ie transfected into a cell, in particular a cell present in vivo, expresses the protein, peptide or antigen it encodes.

The term "transfection" refers to the introduction of nucleic acids, in particular RNA, into a cell. For the purposes of the present invention, the term "transfection" also includes the introduction of a nucleic acid into a cell or the capture of a nucleic acid by such a cell, while the cell may be present in a subject, such as a patient. Thus, in accordance with the present invention, the cell intended for transfection of the nucleic acid described herein may be present in vitro or in vivo, for example, the cell may form part of an organ, tissue and/or body of a patient. In accordance with the invention, transfection may be transient or stable. For some applications of transfection, it is sufficient that the transfected genetic material is only transiently expressed. Since the nucleic acid introduced during the transfection process is usually not integrated into the nuclear genome, the foreign nucleic acid will be diluted by mitosis or degraded. Cells providing episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remain in the cell's genome and its daughter cells, stable transfection must occur. The RNA can be transfected into cells for transient expression of its encoded protein.

The term "stability" of RNA refers to the "half-life" of RNA. "Half-life" refers to the period of time it takes to eliminate half of the activity, amount, or number of molecules. In the context of the present invention, the RNA half-life is an indication of the stability of said RNA. The half-life of RNA can influence the "length of expression" of the RNA. It can be assumed that RNA having a long half-life will be expressed over a longer period of time.

In the context of the present invention, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA. Accordingly, RNA can be translated into protein. In accordance with the present invention, the term "transcription" includes "in vitro transcription", while the term "in vitro transcription" refers to the process in which RNA, in particular mRNA, is synthesized in vitro in a cell-free system, preferably using appropriate cellular extracts. Preferably, cloning vectors are used to generate transcripts. These cloning vectors are generally designed as transcription vectors and are encompassed by the term "vector" in accordance with the present invention.

The term "translation" according to the invention refers to the process in the ribosomes of a cell by which a strand of an RNA messenger directs the assembly of an amino acid sequence to produce a peptide or protein.

The term "expression" is used in accordance with the invention in the most general sense and includes the production of RNA and/or peptides or proteins, for example, by transcription and/or translation. In relation to RNA, the term "expression" or "translation" refers in particular to the production of peptides or proteins. This term also includes partial expression of nucleic acids. Moreover, expression can be transient or stable. According to the invention, the term "expression" also includes "aberrant expression" or "abnormal expression".

"Aberrant expression" or "abnormal expression" means in accordance with the invention that the expression is altered, preferably increased, compared to reference expression, for example, the condition of a subject who does not have a disease associated with aberrant or abnormal expression of a particular protein, for example, tumor antigen. An increase in expression refers to an increase of at least 10%, in particular at least 20%, at least 50% or at least 100% or more. In one embodiment, expression is found only in diseased tissue, while expression in healthy tissue is suppressed.

The term "specifically expressed" means that the protein is predominantly expressed only in a specific tissue or organ. For example, a tumor antigen specifically expressed in the gastric mucosa means that said protein is predominantly expressed in the gastric mucosa and is not expressed in other tissues, or is not expressed to a significant extent in other types of tissues or organs. Thus, a protein that is exclusively expressed in gastric mucosal cells and to a much lesser extent in any other tissue, such as the testis, is specifically expressed in gastric mucosal cells. In some embodiments, the tumor antigen can also be specifically expressed under normal conditions in more than one tissue or organ type, such as 2 or 3 tissue or organ types, but preferably no more than 3 different tissue or organ types. In this case, the tumor antigen is then specifically expressed in these organs. For example, if a tumor antigen is expressed under normal conditions, preferably approximately equally in the lung and stomach, then said tumor antigen is specifically expressed in the lung and stomach.

According to the invention, the term "coding RNA" means that the RNA, when present in an appropriate environment, preferably within a cell, can be expressed to produce the protein or peptide it encodes.

Some aspects of the invention are based on the adoptive transfer of host cells that are transfected in vitro with a nucleic acid, such as an RNA encoding a binding agent described herein, and transferred by recipients, such as patients, preferably after ex vivo expansion from a low copy number of the precursor to clinically significant number of cells. The host cells used for treatment in accordance with the invention may be autologous, allogeneic or syngeneic in relation to the treated recipient.

The term "autologous" is used to describe something that is derived from the same subject. For example, "autologous transplant" refers to tissue or organs obtained from the same subject. Such procedures are preferred because they overcome the immunological barrier, which otherwise leads to rejection.

The term "allogeneic" is used to describe something that is obtained from different individuals of the same species. Two or more individuals are considered allogeneic to each other when the genes at one or more loci are not identical.

The term "syngeneic" is used to describe anything that is derived from individuals or tissues having identical genotypes, i.e. identical twins or animals of the same inbred strain, or tissues thereof.

The term "heterologous" is used to describe something that is made up of many different elements. For example, the transfer of the bone marrow of one individual to another individual represents a heterologous graft. A heterologous gene is a gene obtained from a source other than the subject.

The term "peptide" according to the invention includes oligo- and polypeptides, and refers to substances containing two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 9 or more, preferably 10 or more, preferably 13 or more, preferably 16 or more, preferably 21 or more, and up to preferably 8, 10, 20, 30, 40 or 50, in particular 100 amino acids connected covalently by peptide bonds. The term "protein" refers to large peptides, preferably peptides containing more than 100 amino acid residues, but, as a rule, the terms "peptides" and "proteins" are synonymous and are used interchangeably here.

Data provided herein with respect to specific amino acid sequences, such as those listed in a sequence listing, should be considered as also referring to variants of said specific sequences resulting in sequences that are functionally equivalent to said specific sequences, such as amino acid sequences exhibiting properties, identical or similar to the properties of specific amino acid sequences. One important property is the retention of target binding or effector functions. Preferably, a sequence that is a variant of a particular sequence when it replaces a particular sequence in an antibody retains the binding of said antibody to CLDN and/or CD3, and preferably the functions of said antibody as described herein, e.g., CDC-mediated lysis or ADCC-mediated lysis.

For example, the sequences presented in the sequence listing can be modified to remove one or more, preferably all, free cysteine residues, in particular by replacing cysteine residues with amino acids other than cysteine, preferably serine, alanine, threonine, glycine, tyrosine, leucine or methionine, most preferably alanine or serine. For example, a cysteine at position 103 of the sequence shown in SEQ ID NO: 36 of the sequence listing, or a corresponding cysteine in a sequence containing said sequence, may thus be modified. Other cysteines that can be modified in this manner are the cysteines at position 178 of SEQ ID NO: 42, at position 197 of SEQ ID NO: 43, at position 427 of SEQ ID NO: 44, or at position 446 of SEQ ID NO: :45.

Those skilled in the art will appreciate that the hypervariable and variable regions of the CDR sequence, in particular, can be modified without losing the ability to bind to CLDN and/or CD3. For example, the CDR regions will be identical or highly homologous to the regions of the antibodies indicated here. By "highly homologous" it is meant that 1 to 5, preferably 1 to 4, eg 1 to 3, or 1 or 2 substitutions can be made in the CDR. In addition, hypervariable and variable regions can be modified such that they show significant homology to the antibody regions specifically disclosed here.

For purposes of the present invention, amino acid sequence "variants" include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and/or amino acid substitution variants. Amino acid deletion variants that contain a deletion at the N-terminus and/or C-terminus of the protein are also referred to as N-terminal and/or C-terminal truncated variants.

Amino acid insertion variants include inserting one or two or more amino acids into a particular amino acid sequence. In the case of amino acid sequence variants having an insert, one or more amino acid residues are inserted at a specific site in the amino acid sequence, although random insertion by appropriate screening of the finished product is also possible.

Amino acid addition options include amino and/or carboxy terminal fusions of one or more amino acids, eg 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

Variants of amino acid deletions are characterized by the removal of one or more amino acids from a sequence, for example by removing 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. Deletions can be in any position of the protein.

Amino acid substitution variants are characterized in that at least one residue in the sequence is deleted and another residue is inserted in its place. Preference is given to modifications at positions in the amino acid sequence that are not conservative between homologous proteins or peptides, and/or replacement of amino acids with other amino acids having similar properties. Preferably, amino acid changes in protein variants are conservative amino acid changes, ie, substitutions of similarly charged or uncharged amino acids. A conservative amino acid change involves replacing one amino acid from a family of amino acids that are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.

Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence will be at least about 60%, 65%, 70%, 80%, 81%, 82%, 83 %, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. The degree of similarity or identity is preferably presented for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the total length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is presented, preferably for at least 20, at least 40, at least 60, at least 80, at least 100, at least 120, at least 140, at least 160, at least 180 or about 200 amino acids, preferably continuous amino acids. In preferred embodiments, the degree of similarity or identity is for the full length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be done using tools known in the art, preferably using optimal sequence alignment, e.g. using Align, using standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

"Sequence similarity" indicates the percentage of amino acids that are identical or that represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between sequences.

The term "percent identity" is intended to mean the percentage of identical amino acid residues between both compared sequences, obtained after optimal alignment, while this percentage is purely statistical and the differences between both sequences are randomly distributed and along their entire length. Comparison of two amino acid sequences is traditionally carried out by comparing these sequences after aligning them in an optimal way, this comparison can be carried out by comparing segments or using a "comparison window" to identify and compare local areas of sequence similarity. Optimal sequence alignment for comparison can also be done manually using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, using the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443 using a similar search method Pearson and Lipman, 1988, Proc. Natl Acad. sci. USA 85, 2444, or using software packages using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.) .

Percent identity is calculated by determining the number of identical positions common to two compared sequences and dividing this number by the total number of positions in the comparison window and multiplying the result by 100 to obtain the percentage identity between both these sequences.

Binding agents of the invention can be produced intracellularly (eg, in the cytosol, periplasm, or inclusion bodies) and then isolated from host cells and optionally further purified; or they can be obtained extracellularly (eg, in the medium in which the host cells are grown) and then isolated from the culture medium and optionally further purified. Methods and reagents used to produce recombinant polypeptides, such as specific suitable expression vectors, transformation or transfection methods, selectable markers, methods for inducing protein expression, growth conditions, and the like, are known in the art. Likewise, isolation and purification technologies are well known to those skilled in the art.

The term "cell" or "host cell" preferably refers to an intact cell, that is, a cell with an intact membrane that does not release its normal intracellular components such as enzymes, organelles, or genetic material. The intact cell is preferably a viable cell, ie a living cell capable of performing its normal metabolic functions. Preferably, said term refers according to the invention to any cell that can be transfected with an exogenous nucleic acid. Preferably, the cell, when transfected with an exogenous nucleic acid and transferred to a recipient, can express the nucleic acid in the recipient. The term "cell" includes a bacterial cell; other suitable cells are yeast cells, fungal cells or mammalian cells. Suitable bacterial cells include cells from Gram-negative bacterial strains such as Escherichia coli, Proteus and Pseudomonas strains and Gram-positive bacterial strains such as Bacillus, Streptomyces, Staphylococcus and Lactococcus strains. Suitable fungal cells include cells from Trichoderma, Neurospora and Aspergillus species. Suitable yeast cells include cells from Saccharomyces species (eg Saccharomyces cerevisiae), Schizosaccharomyces (eg Schizo saccharomyces pombe), Pichia (eg Pichia pastoris and Pichia methanolicd) and Hansenula. Suitable mammalian cells include, for example, CHO cells, BHK cells, HeLa cells, COS cells, 293 HEK, and the like. However, amphibian cells, insect cells, plant cells, and any other cells used in the art to express heterologous proteins can also be used. Mammalian cells are particularly preferred for adoptive transfer, such as cells from humans, mice, hamsters, pigs, sheep, and primates. Cells can be derived from a variety of tissue types and include primary cells and cell lines such as cells of the immune system, in particular antigen presenting cells such as dendritic cells and T cells, stem cells such as hematopoietic stem cells and mesenchymal stem cells, and other types of cells. An antigen presenting cell is a cell that presents an antigen by binding to the major histocompatibility complex on its surface. T cells can recognize this complex using their T cell receptor (TCR).

The term "reduce", "reduce" or "inhibit" as used herein means an overall reduction or the ability to cause an overall reduction, preferably 5% or more, 10% or more, 20% or more, more preferably 50% or more , and most preferably by 75% or more of the expression level or cell proliferation level.

Terms such as "increases" or "enhances" preferably refer to an increase or enhancement of at least about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or even more.

Antibody-dependent cell-mediated cytotoxicity

ADCC describes the ability of effector cells, in particular lymphocytes, to kill cells, which preferably requires labeling the target cell with an antibody.

ADCC preferably occurs when antibodies bind to antigens on tumor cells and antibody Fc domains bind to Fc receptors (FcRs) on the surface of immune effector cells. Several families of Fc receptors have been identified, and specific cell populations characteristically express certain Fc receptors. ADCC can be seen as a mechanism to directly induce a variable degree of immediate tumor destruction, leading to antigen presentation and induction of tumor-targeted T cell responses. Preferably, in vivo induction of ADCC will result in tumor-targeted T-cell responses and humoral host immune responses.

Complement dependent cytotoxicity

Complement dependent cytotoxicity (CDC) is another way of killing cells that can be directed by antibodies. IgM is the most effective isotype for complement activation. Both IgG1 and IgG3 are also effective in directing CDC along the classical complement activation pathway. Preferably, in this cascade, the formation of antigen-antibody complexes results in multiple C1q binding sites in close proximity on the _CH 2 domains of participating antibody molecules, such as IgG molecules (C1q is one of the three complement subcomponents of C1). Preferably, these C1q binding sites translate previously low affinity C1q-IgG interactions into high avidity interactions that trigger a cascade of events involving a series of other complement proteins and result in proteolytic release of C3a and C5a chemotactic/activating agents. Preferably, the complement cascade ends with the formation of a membrane attack complex that creates pores in the cell membrane to allow free passage of water and solutes into and out of the cell.

The antibodies described herein, for example, to provide the VL and VH regions, can be made using a variety of known technologies such as the standard somatic cell hybridization technology of Kohler and Milstein, Nature 256: 495 (1975). Although somatic cell hybridization procedures are preferred, other techniques for producing monoclonal antibodies can in principle be used, for example, transformation of B-lymphocytes with viruses or oncogenes, or phage display techniques using antibody gene libraries.

The preferred animal system for producing hybridomas that produce monoclonal antibodies is the murine system. Obtaining hybridoma using mice is well known in this field. Protocols and techniques for isolating immunized splenocytes for fusion are known in the art. Fusion partners (eg murine myeloma cells) and fusion methods are also known.

Other preferred animal systems for producing hybridomas that produce monoclonal antibodies are rat and rabbit systems (for example, those described in Spieker-Polet et al., Proc. Natl. Acad. Sci. U.S.A. 92:9348 (1995), see also Rossi et al. al., Am J Clin Pathol 124: 295 (2005)).

In yet another preferred embodiment, human monoclonal antibodies can be generated using transgenic or transchromosomal mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include the mice known as HuMAb mice and KM mice, respectively, and are collectively referred to herein as "transgenic mice". The production of human antibodies in such transgenic mice can be performed as described in detail for CD20 in WO2004 035607.

Another strategy for creating monoclonal antibodies is the direct isolation of genes encoding antibodies from lymphocytes that produce antibodies of a certain specificity, for example, see Babcock et al., 1996; a new strategy for the creation of monoclonal antibodies from single isolated lymphocytes that produce antibodies of certain specificities. For more information on recombinant antibody construction, see also Welschof and Kraus, Recombinant antibodes for cancer therapy ISBN-0-89603-918-8 and Benny K.C. Lo Antibody Engineering ISBN 1-58829-092-1.

To generate antibodies, mice can be immunized with carrier-conjugated peptides derived from the sequence of the antigen, i.e., the sequence against which antibodies are to be directed, enriched in a preparation of recombinantly expressed antigen or fragments thereof, and/or cells expressing the antigen, as described. Alternatively, mice can be immunized with DNA encoding the antigen or fragments thereof. In the event that immunizations using a purified or enriched antigen preparation do not produce antibodies, mice can also be immunized with antigen-expressing cells, such as a cell line, to stimulate immune responses.

The immune response can be monitored during the immunization protocol using plasma and serum samples obtained from blood sampling from the tail vein or retroorbital vein. Mice with sufficient immunoglobulin titers can be used for fusions. Mice are boosted by intraperitoneal or intravenous administration of antigen-expressing cells 3 days prior to sacrifice and removal of the spleen to increase the level of specific antibody-secreting hybridomas.

To obtain monoclonal antibody-producing hybridomas, splenocytes and lymph node cells from an immunized mouse can be isolated and fused to a suitable immortalized cell line, such as a mouse myeloma cell line. Then the resulting hybridomas are screened for the production of antigen-specific antibodies. Individual wells are then screened by ELISA for antibody-secreting hybridomas. By immunofluorescence and FACS analysis using antigen expressing cells, antibodies with specificity for the antigen can be identified. Antibody-secreting hybridomas can be replated, screened again and, if they remain positive for monoclonal antibodies, subcloned by limiting dilution. The stable subclones are then cultured in vitro in tissue culture medium to obtain an antibody for characterization.

Antibodies can also be generated in the transfectome of a host cell using, for example, a combination of recombinant DNA technologies and gene transfection techniques, as is well known in the art (Morrison, S. (1985) Science 229: 1202).

For example, in one embodiment, the gene(s) of interest, e.g., antibody genes, can be ligated into an expression vector, such as a eukaryotic expression plasmid, such as used in the GS gene expression system described in WO 87/04462, WO 89 /01036 and EP 338 841, or other expression systems well known in the art. The purified plasmid with the cloned antibody genes is introduced into eukaryotic host cells such as CHO cells, NS/0 cells, HEK 293T cells or HEK293 cells, or alternatively other eukaryotic cells such as cells isolated from plants, fungi or yeasts. The method used to introduce these genes may be by methods described in the art, such as electroporation, the use of lipofectamine, or other methods. Once these antibody genes are introduced into host cells, cells expressing the antibody can be identified and selected. These cells are transfectomes which can then be expanded based on expression levels and scaled up to generate antibodies. From the supernatants and/or cells of these cultures, recombinant antibodies can be isolated and purified.

Alternatively, these cloned antibody genes can be expressed in other expression systems, including prokaryotic cells such as microorganisms such as E. coli. In addition, antibodies can be obtained using transgenic non-human animals, for example, in the milk of sheep and rats or chicken eggs, or in transgenic plants; see, for example, Verma, R., et al. (1998) J. Immunol. Meth. 216:165-181; Pollock, et al. (1999) J. Immunol. Meth. 231:147-157; and Fischer, R., et al. (1999) Biol. Chem. 380: 825-839.

Chimerization

Unlabeled mouse antibodies are highly immunogenic in humans with repeated use, resulting in reduced therapeutic effect. The main immunogenicity is mediated by heavy chain constant regions. The immunogenicity of mouse antibodies in humans can be reduced or completely eliminated if the corresponding antibodies are chimerized or humanized. Chimeric antibodies are antibodies whose different parts are derived from different animal species, such as where the variable region is a mouse antibody and the constant region is a human immunoglobulin. Antibody chimerization is achieved by combining the heavy and light chain variable regions of a mouse antibody with a human heavy and light chain constant region (e.g., as described by Kraus et al., in Methods in Molecular Biology series, Recombinant antibodies for cancer therapy ISBN-0-89603-918 -8). In a preferred embodiment, the chimeric antibodies are generated by fusing a human kappa light chain constant region with a mouse light chain variable region. In a further preferred embodiment, chimeric antibodies can be generated by combining a human lambda light chain constant region with a murine light chain variable region. Preferred heavy chain constant regions for generating chimeric antibodies are IgG1, IgG3 and IgG4. Other preferred heavy chain constant regions for generating chimeric antibodies are IgG2, IgA, IgD and IgM.

humanization

Antibodies interact with target antigens primarily through amino acid residues that are found in the six hypervariable regions (CDRs) of the light and heavy chains. For this reason, the amino acid sequences within the CDR are more diverse among individual antibodies than those outside the CDR. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of specific natural antibodies by constructing expression vectors that include CDR sequences from a specific natural antibody grafted onto framework sequences from another antibody with different properties (see e.g. , Riechmann, L. et al (1998) Nature 332: 323-327; Jones, P. et al. (1986) Nature 321: 522-525; and Queen, C. et al. (1989) Proc. Natl. Acad. Sci. U. S. A. 86: 10029-10033). These framework sequences can be obtained from public DNA databases that include germline antibody gene sequences. These germline sequences will differ from mature antibody gene sequences because they will not include the fully assembled variable genes that are formed by V(D)J binding during B cell maturation. Germline gene sequences will also differ from high affinity antibody sequences from the secondary repertoire by a characteristic uniform arrangement in the variable region.

The ability of antibodies and other binding agents to bind to an antigen can be determined using standard binding assays (eg, ELISA, Western blot, immunofluorescence, and flow cytometry analysis).

For antibody purification, selected manufacturer-derived cell lines can be grown in 2 liter roller flasks to purify recombinant antibody. Alternatively, antibodies can be obtained using dialysis in bioreactors. Supernatants may be filtered and, if necessary, concentrated prior to affinity chromatography using L-sepharose protein. Eluted IgG can be checked by gel electrophoresis and high performance liquid chromatography to ensure purity. After replacing the buffer solution with PBS, the concentration can be determined from the OD280 using the appropriate instinct coefficient. Recombinant antibodies can be divided into aliquots and can be stored at -80°C.

Flow cytometry can be used to demonstrate the binding of monoclonal antibodies to living cells expressing the antigen. Cell lines naturally or transfectively expressing antigen and negative controls lacking antigen expression (grown under standard growth conditions) can be mixed with various concentrations of monoclonal antibodies in hybridoma supernatants or in PBS containing 1% FBS and incubated at 4°C. within 30 min. After washing, an anti-IgG antibody labeled with APC or Alexa647 is reacted with an antigen-bound monoclonal antibody under the same conditions as for staining the primary antibody. Samples can be analyzed by flow cytometry using a FACS device using the light and side scatter characteristics of the discrimination window for individual cells. To distinguish antigen-specific monoclonal antibodies from non-specific binders in one dimension, a co-transfection method can be used. Cells transiently transfected with antigen-encoding plasmids and fluorescent markers are stained as described above. Transfected cells can be detected in fluorescence channels other than antibody-stained cells. Since most of the transfected cells express both transgenes, antigen-specific monoclonal antibodies bind preferentially to cells expressing the fluorescent marker, while non-specific antibodies bind in a comparable ratio to non-transfected cells. In addition to or instead of flow cytometric analysis, an alternative method of analysis using fluorescence microscopy can be used. Cells are stained as described above and examined by fluorescence microscopy.

To demonstrate the binding of monoclonal antibodies to living cells expressing the antigen, analysis by immunofluorescence microscopy can be used. For example, cell lines expressing either spontaneously or after transfection the antigen and negative controls lacking antigen expression are grown on glass slides under standard growth conditions in DMEM/F12 medium supplemented with 10% fetal bovine serum (FCS), 2 mM L-glutamine , 100 U/ml penicillin and 100 µg/ml streptomycin. The cells are then fixed with methanol or paraformaldehyde, or left untreated. Then the cells are reacted with monoclonal antibodies against the antigen for 30 min at 25°C. After washing, the cells were reacted with an Alexa555 labeled secondary anti-mouse IgG antibody (Molecular Probes) under the same conditions. The cells are then examined using fluorescence microscopy.

Cellular extracts of cells expressing the antigen and corresponding negative controls are prepared and subjected to sodium dodecyl sulfate polyacrylamide (SDS) gel electrophoresis. After electrophoresis, the separated antigens are transferred to nitrocellulose membranes, blocked and probed with the monoclonal antibodies to be tested. IgG binding can be detected using peroxidase-conjugated anti-mouse IgG and displayed with ECL substrate.

Antibodies can be further tested for antigen interaction by immunohistochemistry methods well known to those skilled in the art, for example, using paraformaldehyde- or acetone-fixed cryosections, or paraformaldehyde-fixed paraffin-embedded tissue sections obtained from non-cancerous tissue or cancerous tissue samples from a patient during during routine surgical procedures, or from mice bearing grafted tumors inoculated with cell lines expressing spontaneously or after transfection the antigen. For immunostaining, antigen-reactive antibodies can be incubated with goat anti-mouse or goat anti-rabbit antibodies conjugated with horseradish peroxidase (DAKO) according to the manufacturer's instructions.

Preclinical studies

The binding agents described herein can also be tested in an in vivo model, eg, in an immunodeficient mouse bearing xenograft tumors inoculated with CLDN expressing cell lines, to determine their effectiveness in controlling the growth of CLDN expressing tumor cells.

In vivo studies following xenotransplantation of CLDN-expressing tumor cells into immunocompromised mice or other animals can be performed using the binding agents described herein. Binding agents can be administered to tumor-free mice followed by injection of tumor cells to measure the effects of the binding agents in preventing tumor formation or tumor-related symptoms. Binding agents can be administered to tumor-bearing mice to determine the therapeutic efficacy of the respective binders in reducing tumor growth, metastases, or tumor-related symptoms. The use of binding agents can be combined with the use of other agents such as cytotoxic drugs, growth factor inhibitors, cell cycle blockers, angiogenesis inhibitors or antibodies to determine the synergistic efficacy and potential toxicity of the combinations. For analysis of binding agent-mediated toxic side effects, animals can be inoculated with binding agents or control reagents and closely examined for symptoms possibly associated with CLDN binding agent therapy.

Mapping of epitopes that bind agents recognize can be done as detailed in "Epitope Mapping Protocols" (Methods in Molecular Biology) by Glenn E. Morris ISBN-089603-375-9 and in "Epitope Mapping: A Practical Approach" Practical Approach Series, 248 by Olwyn M. R. Westwood, Frank C. Hay.

The compounds and agents described herein may be administered in the form of any suitable pharmaceutical composition.

Pharmaceutical compositions of the invention are preferably sterile and contain an effective amount of the binding agents described herein, and optionally additional agents described herein, to achieve the desired reaction or desired effect.

Pharmaceutical compositions are usually presented in unit dosage form and can be prepared by methods known in the art. The pharmaceutical composition may, for example, be in the form of a solution or suspension.

The pharmaceutical composition may contain salts, buffers, preservatives, carriers, diluents and/or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" refers to the non-toxicity of the material, which does not interfere with the action of the active component of the pharmaceutical composition.

Salts that are not pharmaceutically acceptable can be used to prepare pharmaceutically acceptable salts and are included in the invention. Pharmaceutically acceptable salts of this kind include, but are not limited to, salts derived from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic acids, and the like. Pharmaceutically acceptable salts may also be prepared as alkali metal salts or alkaline earth metal salts such as sodium salts, potassium salts or calcium salts.

Buffers suitable for use in the pharmaceutical composition include acetic acid as a salt, citric acid as a salt, boric acid as a salt, and phosphoric acid as a salt.

Preservatives suitable for use in the pharmaceutical composition include benzalkonium chloride, chlorobutanol, paraben and thimerosal.

The injectable dosage form may contain a pharmaceutically acceptable excipient such as Ringer's lactate.

The term "carrier" refers to an organic or inorganic component of natural or synthetic origin, with which the active component is combined to promote, enhance or enable application. In accordance with the invention, the term "carrier" also includes one or more compatible solid or liquid excipients, diluents, or encapsulating agents that are suitable for administration to a patient.

Possible carrier materials for parenteral administration are, for example, sterile water, Ringer's solution, Ringer's lactate, sterile sodium chloride solution, polyalkylene glycols, hydrogenated naphthalenes and in particular biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxypropylene copolymers.

The term "excipient" as used herein means all substances that may be present in a pharmaceutical composition and are not active ingredients, such as, for example, carriers, binders, lubricants, thickeners, surfactants, preservatives, emulsifiers, buffers, flavoring agents. agents and dyes.

The agents and compositions described herein can be administered by any conventional route, such as parenteral administration, including injection or infusion. Preferably, the administration is parenteral, eg intravenous, intra-arterial, subcutaneous, intradermal or intramuscular.

Compositions suitable for parenteral administration generally contain a sterile aqueous or non-aqueous preparation of the active compound, which is preferably isotonic with the recipient's blood. Examples of compatible vehicles and solvents are Ringer's solution and isotonic sodium chloride solution. In addition, usually sterile fixed oils are used as a solution or suspension medium.

The agents and compositions described herein are administered in effective amounts. "Effective amount" refers to the amount that achieves the desired response or the desired effect, alone or together with additional doses. In the treatment of a particular disease or condition, the desired response preferably refers to inhibition of the course of the disease. This includes slowing the progression of the disease and, in particular, interrupting or reversing the course of the disease. The desired response in the treatment of a disease or condition may also be a delay or prevention of the onset of said disease or said condition.

The effective amount of an agent or composition described herein will depend on the condition being treated, the severity of the disease, individual patient parameters including age, physiology, size and weight, duration of treatment, type of concomitant therapy (if present), specific route of administration, and similar factors. Accordingly, the doses of the administered agents described herein may depend on these various parameters. In the event that the patient's response is insufficient after the initial dose, higher doses may be administered (or, in fact, higher doses are achieved by a different, more localized route of administration).

The agents and compositions described herein may be administered to patients, for example, in vivo, for the treatment or prevention of various disorders such as those described herein. Preferred patients include human patients with disorders that can be corrected or attenuated by the administration of the agents and compositions described herein. This includes disorders that involve cells characterized by an altered expression pattern of CLDN, such as CLDN18.2 and/or CLDN6.

For example, in one embodiment, the agents described herein can be used to treat a patient with a cancer, such as a cancer described here, characterized by the presence of cancer cells expressing CLDN.

Pharmaceutical compositions and methods of treatment described in accordance with the invention can also be used for immunization or vaccination to prevent the disease described here.

The pharmaceutical composition of the invention may be administered together with additional immune enhancing substances such as one or more adjuvants, and may contain one or more immune enhancing substances to further enhance their effectiveness, preferably to achieve a synergistic immunostimulatory effect. The term "adjuvant" refers to compounds that prolong or enhance or accelerate the immune response. Different mechanisms are possible in this regard depending on the different types of adjuvants. For example, compounds that allow DC maturation, such as lipopolysaccharides or CD40 ligand, form a first class of suitable adjuvants. Generally, any agent that has a "danger signal" effect on the immune system (LPS, GP96, dsRNA, etc.) or cytokines such as GM-CSF can be used as an adjuvant that provides the ability to enhance immune response and/or influencing the immune response in a controlled manner. Oligodeoxynucleotides can optionally be used in this case, although their side effects, which occur under certain circumstances, as indicated above, are subject to specification. Particularly preferred adjuvants are cytokines such as monokines, lymphokines, interleukins or chemokines, for example IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8 , IL-9, IL-10, IL-12, INFα, INF-γ, GM-CSF, LT-α, or growth factors such as hGH. The following known adjuvants are aluminum hydroxide, Freund's adjuvant or an oil such as Montanide®, most preferably Montanide® ISA51. Lipopeptides such as Pam3Cys are also suitable for use as adjuvants in the pharmaceutical composition of the present invention.

The agents and compositions provided herein may be used alone or in combination with conventional forms of treatment such as surgery, radiation, chemotherapy, and/or bone marrow transplantation (autologous, syngeneic, allogeneic, or unrelated).

Cancer treatment represents an area in which combination strategies are particularly desirable because often the combined action of two, three, four or even more anti-cancer drugs/therapies creates synergistic effects that are more potent than those of a monotherapeutic approach. Thus, in another embodiment of the present invention, cancer treatment using immunity- or vaccination-based mechanisms, such as the methods and pharmaceutical compositions of the present invention, can be effectively combined with various other drugs and/or methods having a similar or different directional mechanism. These include, for example, combinations with conventional tumor therapies, multi-epitope strategies, complementary immunotherapy, and treatment approaches that target angiogenesis or apoptosis (for a review, see e.g. Andersen et al., 2008: Cancer treatment: the combination of vaccination with other therapies Cancer Immunology Immunotherapy, 57(11): 1735-1743.). Sequential administration of different agents may inhibit the growth of cancer cells at different time points, while other agents may, for example, inhibit neoangiogenesis, malignant cell survival or metastasis, potentially converting the cancer into a chronic disease. The following list represents some non-limiting examples of anti-cancer drugs and therapies that may be used in combination with the present invention:

1. Chemotherapy

Chemotherapy is the standard of care for many types of cancer. The most common chemotherapy agents work by killing cells that are rapidly dividing, which is one of the main features of cancer cells. Thus, combination with established chemotherapeutic drugs, such as, for example, alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents that affect cell division or DNA synthesis, can significantly improve the therapeutic effects of the present invention by clearing suppressor cells, restoring the immune system by rendering tumor cells more susceptible to immune-mediated killing, or by further activating immune system cells. The synergistic anticancer effects of chemotherapy and vaccine-based immunotherapeutic drugs have been demonstrated in many studies (see e.g. Quoix et al. 2011: Therapeutic vaccination with TG4010 and first-line chemotherapy in advanced non-small-cell lung cancer: a controlled phase 2B trial Lancet Oncol 12(12): 1125-33 see also Liseth et al 2010: Combination of intensive chemotherapy and anticancer vaccines in the treatment of human malignancies: the hematological experience J Biomed Biotechnol 2010: 6920979 see See also Hirooka et al 2009: A combination therapy of gemcitabine with immunotherapy for patients with inoperable locally advanced pancreatic cancer Pancreas 38(3): e69-74). There are hundreds of chemotherapeutic agents available that are mostly suitable for combination therapy. Some (non-limiting) examples of chemotherapy drugs that can be used in combination with the present invention are carboplatin (paraplatin), cisplatin (platinol, platinol-AQ), cyclophosphamide (cytoxan, neozar), docetaxel (taxotere), doxorubicin (adriamycin), erlotinib (Tartceva), etoposide (Vepezid), fluorouracil (5-FU), gemcitabine (Gemzar), imatinib mesylate (Gleevec), irinotecan (Camptosar), methotrexate (Folex, Mexat, Amethopterin), paclitaxel (Taxol, Abraxane), sorafinib (Nexavar), sunitinib (Sutent), topotecan (hycamtin), vincristine (Oncovin, Vincazar PFS), and vinblastine (Velban).

2. Surgery

Cancer surgery - surgery to remove a tumor - remains the mainstay of cancer treatment. Surgery may be combined with other cancer therapies to remove any remaining tumor cells. Combining surgical techniques with subsequent immunotherapy is a promising approach that has been repeatedly demonstrated.

3. Irradiation

Radiation therapy remains an important component in cancer treatment, with approximately 50% of all cancer patients receiving radiation therapy during the course of their disease. The main goal of radiation therapy is to deprive cancer cells of their potential to reproduce (cell division). The types of irradiation used to treat cancer are photon irradiation (X-rays and gamma rays) and particle irradiation (electron, photon and neutron beams). There are two ways to deliver radiation to the site of a malignant tumor. External beam irradiation is delivered from sources located outside the body by targeting high-energy beams (photons, protons or particle irradiation) to the site of the tumor. Internal radiation or brachytherapy is delivered from radioactive sources located outside the body, placed in sealed catheters, or injected directly into the tumor site. The radiotherapy methods that can be used in combination with the present invention are fractionation (radiotherapy sessions in fractionated mode, for example, daily fractions of 1.5 to 3 Gy for several weeks), 3D conformal radiotherapy (3DCRT; delivery radiation into the macroscopic volume of the tumor), intensity-modulated radiation therapy (IMRT; computerized beam intensity modulation), image-guided radiation therapy (IGRT; a method that creates images at short intervals during a radiation therapy session, which allows for correction ), and stereotactic radiotherapy (SRBT, delivers very high doses of radiation in just a few fractions). For a review of radiotherapy, see Baskar et al. 2012: Cancer and radiation therapy: current advances and future directions. Int. J Med Sci. 9(3): 193-199.

4. Antibodies

Antibodies (preferably monoclonal antibodies) achieve their therapeutic effect against cancer cells through various mechanisms. Antibodies may have a direct effect on apoptosis or programmed cell death. Antibodies can block components of signal transduction pathways, such as, for example, growth factor receptors, effectively inhibiting tumor cell proliferation. In cells that express monoclonal antibodies, they can promote the formation of anti-idiotypic antibodies. Indirect effects include the recruitment of cells that are cytotoxic, such as monocytes and macrophages. This type of antibody-mediated cell death is called antibody-dependent cell-mediated cytotoxicity (ADCC). The antibodies also bind complement, resulting in a direct cellular toxicity known as complement-dependent cytotoxicity (CDC). Combining surgical techniques with immunotherapeutic drugs and techniques is a successful approach, as demonstrated, for example, in Gadri et al. 2009: Synergistic effect of dendritic cell vaccination and anti-CD20 antibody treatment in the therapy of murine lymphoma. J Immunother. 32(4): 333-40. The following list provides some non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that can be used in combination with the present invention: abagovomab (CA-125), abciximab (CD41), adecatumumab (EpCAM), afutuzumab (CD20), alacizumab pegol (VEGFR2), altumomab pentetate (CEA), amatuximab (MORAb-009), anatumomab mafenatox (TAG-72), apolizumab (HLA-DR), arcitumomab (CEA), bavituximab (phosphatidylserine), bektumomab (CD22), belimumab (BAFF) ), bevacizumab (VEGF-A), bivatuzumab mertansine (CD44 v6), blinatumomab (CD19), brentuximab vedotin (CD30 TNFRSF8), cantuzumab mertansine (CanAg mucin), cantuzumab ravtansine (MUC1), capromab pendetide (prostate carcinoma cells), carlumab (CNT0888), catumaxomab (EpCAM, CD3), cetuximab (EGFR), citutuzumab richox (EpCAM), cixutumumab (IGF-1 receptor), claudiximab (Claudin), clivatuzumab tetraketane (MUC1), conatumumab (TRAIL-R2), dacetuzumab (CD40), dolatuzumab (insulin-like growth factor I receptor), denosumab (RANKL), detumomab (B-lymphoma cells), drozitumab (DR5), ecromeximab (ganglioside GD3), edrecolomab (EpCAM), elotuzumab (SLAMF7), enavatuzumab (PDL192) ), ensituximab (NPC-1 C), epratuzumab (CD22), ertumaxomab (HER2/neu, CD3), etaracizumab (ανβ3 integrin), farletuzumab (folate receptor 1), FBTA05 (CD20), ficlatuzumab (SCH 900105), figitumumab ( IGF-1 receptor), flanvotumab (glycoprotein 75), fresolimumab (TGF-β), galiximab (CD80), ganitumab (IGF-I), gemtuzumab ozogamicin (CD33), gevokizumab (IL-1β), girentuximab (carbonic anhydrase 9 (CA -IX)), glembatumumab vedotin (GPNMB), ibritumumab tiuxetan (CD20), ikrucumab (VEGFR-1), igovoma (CA-125), indatuximab ravtansine (SDC1), intetumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab ( CD 152), iratumumab (CD30), labetuzumab (CEA), lexatumumab (TRAIL-R2), libivirumab (hepatitis B surface antigen), lintuzumab (CD33), lorvotuzumab mertansine (CD56), lucatumumab (CD40), lumiliximab (CD23), mapatumumab (TRAIL-R1) (5T4), narnatumab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumab (PDGF-R α), onartuzumab (human hepatocyte growth factor receptor kinase), oportuzumab monatox ( EpCAM), oregovomab (CA-125), oxelumab (OX-40), panitumumab (EGFR), patritumab (HER3), pemtumoma (MUC1), pertuzumab (HER2/neu), pintumumab (adenocarcinoma antigen), pritumumab (vimentin), pacotumomab (N-glycolylneuraminic acid), radretumab (fibronectin extra domain B), rafivirumab (rabies virus glycoprotein), ramucipumab (VEGFR2), rilotumumab (HGF), rituximab (CD20), robatumumab (IGF-1 receptor), samalizumab (CD200) , sibrotuzumab (FAP), siltuximab (IL-6), tabalumab (BAFF), takatuzumab tetraxetane (alpha-fetoprotein), taplitumomab paptox (CD 19), tenatumomab (tenascin C), teprotumumab (CD221), ticilimumab (CTLA-4) , tigatuzumab (TRAIL-R2), TNX-650 (IL-13), tositumomab (CD20), trastuzumab (HER2/neu), TRBS07 (GD2), tremelimumab (CTLA-4), tucotuzumab celloleukin (EpCAM), ublituximab (MS4A1) ), urelumab (4-IBB), volociximab (α5β1 integrin), votumumab (CTAA16.88 tumor antigen), zalutumumab (EGFR), zanolimumab (CD4).

5. Cytokines, chemokines, costimulatory molecules, fusion proteins

The combined use of the antigen-encoding pharmaceutical compositions of the present invention with cytokines, chemokines, co-stimulatory molecules and/or fusion proteins thereof to elicit beneficial immune modulating or tumor inhibitory effects is another embodiment of the present invention. Various chemokines with C, CC, CXC, and CX3C structures can be used to increase immune cell infiltration into tumors and promote movement of antigen-presenting cells to tumor-draining lymph nodes. Some of the most promising chemokines are, for example, CCR7 and its ligands CCL19 and CCL21, as well as CCL2, CCL3, CCL5 and CCL16. Other examples are CXCR4, CXCR7 and CXCL12. In addition, co-stimulatory or regulatory molecules such as, for example, B7 ligands (B7.1 and B7.2) can be used. Other cytokines may also be used, such as, for example, interleukins (eg, IL-1 to IL17), interferons (eg, IFNalphal to IFNalpha8, IFNalpha10, IFNalpha13, IFNalpha14, IFNalpha16, IFNalpha17, IFNalpha21, IFNbeta1, IFNW, IFNE1, and IFNK), hematopoietic factors, TGF (e.g., TGF-α, TGF-β, and other members of the TGF family), and finally the tumor necrosis factor family receptor and its ligands, as well as other stimulatory molecules, including, but not limited to, 4- 1BB, 4-1BB-L, CD 137, CD137L, CTLA-4GITR, GITRL, Fas, Fas-L, TNFR1, TRAIL-R1 , TRAIL-R2, p75NGF-R, DR6, LT.beta.R, RANK, EDAR1 , XEDAR, Fnl l4, Troy/Trade, TAJ, TNFRII, HVEM, CD27, CD30, CD40, 4-1BB, OX40, GITR, GITRL, TACI, BAFF-R, BCMA, RELT and CD95 (Fas/APO-1) , glucocorticoid-induced TNFR-associated proteins, TNF receptor-associated apoptosis-mediating protein (TRAMP) and cell death receptor-6 (DR6). T-cell survival and proliferation. For a review, see Lechner et al. 2011: Chemokines, costimulatory molecules and fusion proteins for the immunotherapy of solid tumors. Immunotherapy 3(11), 1317-1340.

6. Bacterial therapies

The researchers used anaerobic bacteria such as Clostridium novyi to engulf the interior of oxygen-poor tumors. The bacteria are then killed on contact with the oxygenated sides of the tumor, which means they will not have a harmful effect on the rest of the body. Another strategy is to use anaerobic bacteria that have been transformed with an enzyme that can convert a non-toxic prodrug into a toxic drug. During the proliferation of bacteria in necrotic and hypoxic areas of the tumor, the enzyme is expressed only in the tumor. Thus, a systemically administered prodrug is only metabolized to a toxic drug in the tumor. It has been shown to be effective with non-pathogenic Clostridium anaerobic sporogens.

7. Kinase inhibitors

Another large group of potential targets for complementary cancer therapy includes kinase inhibitors, since the growth and survival of cancer cells is closely related to the deregulation of kinase activity. A wide variety of inhibitors have been used to restore normal kinase activity and hence reduce tumor growth. The group of target kinases includes receptor tyrosine kinases, e.g. , FGFR4, CSF1R, c-Met, RON, c-Ret, ALK, cytoplasmic tyrosine kinases, e.g. c-SRC, c-YES, Abl, JAK-2, serine/threonine kinases, e.g. ATM, Aurora A & B, CDKs , mTOR, PKCi, PLKs, b-Raf, S6K, STK1 1/LKB1 and lipid kinases eg PI3K, SK1. Low molecular weight inhibitors of kinaz are, for example, PHA-739358, Nilotinib, Dazatinib and PD166326, NSC 743411, Lapatinib (GW-572016), CAVERTINIB (CI-1033), Semaxinib (SU5416), VATALANIB (PTK787/ZK22/ZK22 2584), ration (SU1 1248), sorafenib (BAY 43-9006), and leflunomide (SU101). For more information, see, for example, Zhang et al. 2009: Targeting cancer with small molecule kinase inhibitors. Nature Reviews Cancer 9, 28-39.

8. Toll-like receptors

Members of the Toll-like receptor (TLRs) family are the key junction between innate and adaptive immunity, and the effect of many adjuvants is based on TLR activation. A large number of cancer vaccines being developed include ligands for TLR8 to induce vaccine responses. In addition to TLR2, TLR3, TLR4, especially TLR7 and TLR 8 have been investigated for cancer therapy in passive immunotherapeutic approaches. The closely related TLR7 and TLR8 promote antitumor responses by affecting immune system cells, tumor cells, and the tumor microenvironment, and can be activated by nucleoside analog structures. All TLRs are useful as standalone immunotherapeutics or adjuvants in cancer vaccines and can be combined synergistically with the dosage forms and methods of the present invention. For more details see van Duin et al. 2005: Triggering TLR signaling in vaccination. Trends in Immunology, 27(1):49-55.

9. Angiogenesis inhibitors

In addition to therapies that target immune modulatory receptors affected by tumor-mediated escape and immune suppression mechanisms, there are therapies that target the tumor environment. Angiogenesis inhibitors prevent the overgrowth of blood vessels (angiogenesis) that tumors require to survive. Angiogenesis, stimulated by tumor cells to meet their growing demands for nutrients and oxygen, can be blocked by targeting various molecules. Non-limiting examples of angiogenesis mediating molecules or angiogenesis inhibitors that can be combined with the present invention include soluble VEGF (VEGF, VEGF121 and VEGF165 isoforms, VEGFR1 receptors, VEGFR2, and neuropilin-1 and neuropilin-2 co-receptors) 1 and NRP-1, angiopoietin 2, TSP-1 and TSP-2, angiostatin and related molecules, endostatin, vasostatin, calreticulin, platelet factor-4, TIMP and CDAI, Meth-1 and Meth-2, IFN-α, -β and -γ, CXCL10, IL-4, -12 and -18, prothrombin (kringle domain 2), antithrombin III fragment, prolactin, VEGI, SPARC, osteopontin, maspin, canstatin, proliferin-related protein, restin, and drugs such as, for example, bevacizumab , itraconazole, carboxyamidotriazole, TNP-470, CM101 , IFN-α platelet factor-4, suramin, SU5416, thrombospondin, VEGFR antagonists, angiostatic steroids + heparin, cartilage angiogenesis inhibitory factors, matrix metalloproteinase inhibitors, 2-methoxyestradiol, tec ogalan, tetrathiomolybdate, thalidomide, thrombospondin, ανβ3 prolactin inhibitors, linomide, tasquinimod. For a review see Schoenfeld and Dranoff 2011: Anti-angiogenesis immunotherapy. Hum Vaccines. (9):976-81.

10. Small molecule drugs for targeted therapy

In general, small molecule targeted therapy drugs are inhibitors of enzyme domains on mutated, overexpressed, or otherwise important proteins in a cancer cell. Notable and non-limiting examples are the tyrosine kinase inhibitors imatinib (Gleevec/Glivec) and gefininib (Iressa). The use of small molecules such as sunitinib malate and/or sorafenib tosylate targeting certain kinases in combination with vaccines for cancer therapy is also described in earlier patent application US2009004213.

11. Virus based vaccines

There are a number of virus-based cancer vaccines available or under development that can be used in a combination therapeutic approach along with the dosage forms of the present invention. One advantage of using such viral vectors is their innate ability to initiate immune responses, with the inflammatory responses resulting from viral infection creating the danger signal required for immune activation. The ideal viral vector should be safe and should not lead to an anti-vector immune response in order to allow booster immunization of anti-tumor specific responses. Recombinant viruses such as vaccinia, herpes simplex viruses, adenoviruses, adeno-associated viruses, retroviruses, and viruses of the genus Avipox have been used in animal tumor models and human clinical trials have been initiated based on their encouraging results. Virus-based vaccines of particular importance are virus-like particles (VLPs), small particles that contain specific proteins from the outer envelope of the virus. Virus-like particles do not contain any viral genetic material and cannot cause infection, but they can be engineered to present tumor antigens on their envelope. VLPs can be derived from various viruses such as, for example, hepatitis B virus or other families of viruses including Parvoviridae (eg, adeno-associated virus), Retroviridae (eg, HIV), and Flaviviridae (eg, hepatitis C virus). For a general overview, see Sorensen and Thompsen 2007: Virus-based immunotherapy of cancer: what do we know and where are we going? APMIS 1 15(11):1 177-93; virus-like particles against cancer are described in Buonaguro et al. 2011: Developments in virus-like particle-based vaccines for infectious diseases and cancer. Expert Rev Vaccines 10(1 1): 1569-83; and in Guillen et al. 2010: Virus-like particles as vaccine antigens and adjuvants: application to chronic disease, cancer immunotherapy and infectious disease preventive strategies. Procedia in Vaccinology 2(2), 128-133.

12. Multi-epitope strategies

The use of multi-epitopes has shown promising results in relation to vaccination. Rapid sequencing technologies combined with smart algorithm systems allow the use of tumor mutant and can provide multi-epitopes for individual vaccines that can be combined with the present invention. For more information see 2007: Vaccination of metastatic colorectal cancer patients with matured dendritic cells loaded with multiple major histocompatibility complex class I peptides. J Immunother 30: 762-772; additionally, Castle et al. 2012: Exploiting the mutanome for tumor vaccination. Cancer Res 72(5): 1081-91.

13. Adoptive T cell transfer

For example, the combination of tumor antigen vaccination and T cell transfer is described in: Rapoport et al. 2011: Combination immunotherapy using adoptive T-cell transfer and tumor antigen vaccination on the basis of hTERT and survivin after ASCT for myeloma. Blood 1 17(3):788-97.

14. Targeted peptide therapies

Peptides can bind to cell surface receptors or act on the extracellular matrix surrounding the tumor. Radionuclides that are attached to these peptides (eg RGD) end up killing cancer cells if the nuclide is destroyed near the cell. Oligo- or multimers of these binding motifs are of particular interest as they may lead to increased tumor specificity and avidity. For a review of non-limiting examples, see Yamada 2011: Peptide-based cancer vaccine therapy for prostate cancer, bladder cancer, and malignant glioma. Nihon Rinsho 69(9): 1657-61.

15. Other therapies

There are a number of other cancer therapies that can be combined with the dosage forms and methods of the present invention to create synergistic effects. Non-limiting examples are therapies targeting apoptosis, hyperthermia, hormone therapy, telomerase therapy, insulin potentiated therapy, gene therapy, and photodynamic therapy.

The present invention is further illustrated by the following examples, which should not be construed as limiting the scope of the invention.

Examples

Example 1: Design and Testing of Bispecific Binding Agents Targeting CLDN18.2 and CD3

a. Sequence origin, design of bi-scFv constructs, and cloning into expression vectors

A tandem bispecific single chain antibody (bi-scFv) construct containing binding domains specific for the CD3 component of the human T-cell receptor and human tumor-associated antigens (TAA) was prepared. The respective heavy chain variable regions (V _H ) and the respective light chain variable regions (V _L ) for each construct are specifically located in the N- to C-terminal direction in the following order:

N - V _H ^αCLDN18.2 - V _L ^αCLDNl8.2 - V _H ^αCD3 - V _L ^αCD3 -C (1BiMAB, 18PHU5, no.11-15)

N - V _H ^αCD3 - V _L ^αCD3 - V _H ^αCLDN18.2 - V _L ^αCLDN18.2 -C (18PHU3, no.16-20)

Table 1 lists all bi-scFv constructs specific for TAA CLDN18.2 and PLAC1 that have been generated within the scope of the invention. The bi-scFv constructs were generated by gene synthesis from GeneArt AG (GeneArt/Life Technologies GmbH, Regensburg, Germany) using the V _H and V _L sequences of the respective antibodies. Codon optimizations such as Homo sapiens (HS), Mus musculus (MM) or Chinese Hamster Ovary (CHO) cells were implemented using GeneArt's GeneOptimizer® software and are listed in Table 1. Information on specificity, sequence origin from monoclonal antibodies (mAB), codon usage, additional sequence distinguishing features, and references to all domains used are summarized in Table 2. The origin of the variable domain sequences of the respective CD3 antibodies is shown in Table 2. Due to the high homology between human and murine TAAs, the same V _H and The V _L anti-TAA can be used to generate bi-scFv constructs for testing in mice, but in combination with the V _H , V _L sequences of mouse specific anti-CD3 antibody clone 145-2C1.

DNA cloning and expression vector construction were performed according to standard procedures (Green/Sambrook, Molecular Cloning, 2012) well known to those skilled in the art. Briefly, the bi-scFv core DNA sequences contained 5' HindIII and 3' XhoI (HindIII and XbaI in the case of 1BiMAB bi-scFv) restriction sites for cloning into expression plasmids. The secretion signal sequence was introduced at the 5' end (upper) of the bi-scFv sequence to secrete the protein from the cell cytoplasm into the culture medium. A sequence encoding a flexible glycine-serine peptide linker of 15-18 amino acids was inserted to connect the V _H and V _L domains for the composition of single chain variable antibody fragments (scFv), one of which binds to CD3 and the other to TAA. To form a bispecific single chain antibody, the sequences of the two scFv domains were joined by a sequence encoding a short peptide linker (GGGGS). Along with this linker sequence, a BamHI restriction site was introduced for scFv domain exchanges to clone subsequent bi-scF _V constructs. In particular, 5'scFv domains can be replaced with HindIII and BamHI restriction sites and 3'scFv domains with BamHI and XhoI restriction sites. See also figure 1 for the design diagram.

All bi-scFv antibody constructs used were cloned into the standard mammalian expression vector pcDNA™3.1/myc-His (+) (Invitrogen/Life Technologies GmbH, Darmstadt, Germany). The C-terminal 6xHis tag can be used for protein metal affinity purification and detection assays. All constructs were verified by sequencing through the MWG single read sequence service (Eurofins MWG Operon, Ebersberg, Germany).

Table 1. Brief description of bispecific single chain antibody constructs specific for TAA and CD3

Internal name TAA Specificity 5'- _VH - _VL ° 3'- _VH - _VL ° Codon usage 1BiMAB CLDN18.2 Human mCLDN18.2ab TR66 HS no.11 CLDN18.2 mouse mCLDN18.2ab 145-2C11 CHO no.12 CLDN18.2 Human mCLDN18.2ab UCHT1-HU CHO no.13 CLDN18.2 Human mCLDN18.2ab UCHT1 CHO no.14 CLDN18.2 Human mCLDN18.2ab CLB-T3 CHO no.15 CLDN18.2 Human mCLDN18.2ab TR66 CHO no.16 CLDN18.2 mouse 145-2C11 mCLDN18.2ab CHO no.17 CLDN18.2 Human UCHT1-HU mCLDN18.2ab CHO no.18 CLDN18.2 Human UCHT1 mCLDN18.2ab CHO no.19 CLDN18.2 Human CLB-T3 mCLDN18.2ab CHO no.20 CLDN18.2 Human TR66 mCLDN18.2ab CHO 18PHU5 CLDN18.2 Human mCLDN18.2ab TR66 HS 18PHU3 CLDN18.2 Human TR66 mCLDN18.2ab HS 18PMU5 CLDN18.2 mouse mCLDN18.2ab 145-2C11 MM 18PMU3 CLDN18.2 mouse 145-2C11 mCLDN18.2ab MM control bi-scFv no.35 PLAC1 Human 78H11 TR66 CHO

CHO, Chinese hamster ovary cells; HS, Homo sapiens; HU, humanized; MM, Mus.

Table 2. Summary of bi-scFv constructs

binding fragment of CD3 TAA binding fragment Int. name Origin mAB Reactive variant. TAA Origin of mAB. Reactive variety 5'- _VH - _VL 3'- _VH - _VL Short linker 1BiMAB TR66 Human CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab TR66 GGGGS no.11 145-2C11 mouse CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab 145-2C11 SGGGGS no.12 UCHT1-HU Human CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab UCHT1-HU SGGGGS no.13 UCHT1 Human CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab UCHT1 SGGGGS no.14 CLB-T3 Human CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab CLB-T3 SGGGGS no.15 TR66 Human CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab TR66 SGGGGS no.16 145-2C11 mouse CLDN18.2 mCLDN18.2ab man, mouse 145-2C11 mCLDN18.2ab SGGGGS no.17 UCHT1-HU Human CLDN18.2 mCLDN18.2ab man, mouse UCHT1-HU mCLDN18.2ab SGGGGS no.18 UCHT1 Human CLDN18.2 mCLDN18.2ab man, mouse UCHT1 mCLDN18.2ab SGGGGS no.19 CLB-T3 Human CLDN18.2 mCLDN18.2ab man, mouse CLB-T3 mCLDN18.2ab SGGGGS no.20 TR66 Human CLDN18.2 mCLDN18.2ab man, mouse TR66 mCLDN18.2ab SGGGGS 18PHU5 TR66 Human CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab TR66 SGGGGS 18PHU3 TR66 Human CLDN18.2 mCLDN18.2ab man, mouse TR66 mCLDN18.2ab SGGGGS 18PMU5 145-2C11 mouse CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab 145-2C11 SGGGGS 18PMU3 145-2C11 mouse CLDN18.2 mCLDN18.2ab man, mouse 145-2C11 mCLDN18.2ab SGGGGS no.35 TR66 Human PLAC1 78H11 man, mouse 78H11 TR66 SGGGGS

table 2

Continuation

Int. names. 5' long linker 3'-long linker secretion signal Use codons Anti-CD3
mAB
links 1BiMAB (GGGGS) ₃ VE(GGSGGS) ₂ GGVD MGWSCIILFLVATATGVHS HS Lanzavecchia & Scheidegger, Eur J Immunol 1987 no.11 (GGGGS) ₃ (GGGGS) ₃ MGWSCIILFLVATATGVHS CHO Leo et al., Proc Natl Acad Sci, 1987 no.12 (GGGGS) ₃ (GGGGS) ₃ MGWSCIILFLVATATGVHS CHO Shalaby et al. J Exp Med 1992 no.13 (GGGGS) ₃ (GGGGS) ₃ MGWSCIILFLVATATGVHS CHO Beverley et al., Eur J Immunol 1981 no.14 (GGGGS) ₃ (GGGGS) ₃ MGWSCIILFLVATATGVHS CHO Van Lier et al. Immunology 1989 no.15 (GGGGS) ₃ (GGGGS) ₃ MGWSCIILFLVATATGVHS CHO Lanzavecchia & Scheidegger, Eur J Immunol 1987 no.16 (GGGGS) ₃ (GGGGS) ₃ MNSGLQLVFFVLTLKGIQG CHO Leo et al., Proc Natl Acad Sci, 1987 no.17 (GGGGS) ₃ (GGGGS) ₃ MGWSCIILFLVATATGVHS CHO Shalaby et al. J Exp Med 1992 no.18 (GGGGS) ₃ (GGGGS) ₃ MNSGLQLVFFVLTLKGIQG CHO Beverley et al., Eur J Immunol 1981 no.19 (GGGGS) ₃ (GGGGS) ₃ MNFGLSLIFLALILKGVQC CHO Van Lier et al. Immunology 1989 no.20 (GGGGS) ₃ (GGGGS) ₃ MEWSWIFLFLLSVTTGVHS CHO Lanzavecchia & Scheidegger, Eur J Immunol 1987 18PHU5 (GGGGS) ₃ VE(GGSGGS) ₂ GGVD MGWSCIILFLVATATGVHS HS Lanzavecchia & Scheidegger, Eur J Immunol 1987 18PHU3 VE(GGSGGS) ₂ GGVD (GGGGS) ₃ MGWSCIILFLVATATGVHS HS Lanzavecchia & Scheidegger, Eur J Immunol 1987 18PMU5 (GGGGS) ₃ VE(GGSGGS) ₂ GGVD MGWSCIILFLVATATGVHS MM Leo et al., Proc Natl Acad Sci, 1987 18PMU3 VE(GGSGGS) ₂ GGVD (GGGGS) ₃ MNSGLQLVFFVLTLKGIQG MM Leo et al., Proc Natl Acad Sci, 1987 no.35 (GGGGS) ₃ (GGGGS) ₃ MGWLWNLLFLMAAAQSAQA CHO Lanzavecchia & Scheidegger, Eur J Immunol 1987

CHO stands for Chinese Hamster Ovary Cells; HS, Homo sapiens; mAB, monoclonal antibody; MM, Mus musculus; TAA, tumor-associated antigen.

b. Generation of stable cell lines-producers

To generate stable clones of cells producing CLDN18.2-specific bi-scFv proteins, the human embryonic kidney cell line HEK293 (ATCC CRL-1573) and the Chinese hamster ovary cell line CHO-K1 (ATCC CCL-61) were used.

HEK293 transfection

1×10 ⁷ HEK293 cells were seeded two days prior to transfection onto 14.5 cm Petri dishes in 20 ml of complete DMEM medium (DMEM/F-12 GlutaMax supplemented with 10% heat-inactivated FBS and 0.5% penicillin-streptomycin; all reagents from Gibco/Life Technologies GmbH, Darmstadt, Germany). Prior to transfection, cells were washed with Dulbecco's phosphate-buffered saline (DPBS) supplemented with 2 mM EDTA, then 20 ml of pure DMEM was added without addition of FBS or antibiotics. 20 μg of the linearized DNA constructs described in Example 1 were diluted in 0.5 ml of pure DMEM/F-12 medium. 75 μl of a 1 mg/ml linear PEI solution (polyethyleneimine; Polysciences Europe GmbH, Eppelheim, Germany) was added to the diluted DNA and vortexed vigorously. After 15 min incubation at room temperature (RT), DNA/PEI complexes were added dropwise to the cells, the cell culture dishes were gently swirled and then incubated at 37° C., 5% CO ₂ . The medium was changed 24 hours after transfection. Selection of transfected cells was started 48 h after transfection with G418 sulfate (Gibco/Life Technologies GmbH, Darmstadt, Germany) at a final concentration of 0.8 mg/mL. G418 was continuously added to the culture medium to grow the cells.

CHO-K1 transfection

1×10 ⁶ CHO-K1 cells were seeded the day before transfection in 6-well tissue culture plates in 2 ml of complete DMEM medium (DMEM/F-12 GlutaMax supplemented with 10% heat-inactivated FBS, no antibiotics; all reagents from Gibco/Life Technologies GmbH, Darmstadt, Germany). Prior to transfection, cells were washed with DPBS supplemented with 2 mM EDTA, then 1.5 ml of normal DMEM was added without addition of FCS or antibiotics. 4 µg of the linearized DNA constructs described in Example 1.a were diluted in 0.25 ml of normal DMEM/F-12 medium and mixed gently. In the second reaction tube, 2.5 μl of Lipofectamine 2000 (Invitrogen/Life Technologies GmbH, Darmstadt, Germany) was diluted in 0.25 ml of normal DMEM/F-12, mixed gently and incubated for 5 min at room temperature (RT). The DNA mixture and Lipofectamine mixture were combined in a 1:1 ratio, mixed gently and incubated for 20 minutes at room temperature (RT). DNA/Lipofectamine 2000 complexes were added dropwise to the cells, the cell culture dishes were gently swirled and then incubated at 37° C., 5% CO ₂ . 6 hours after transfection, the medium was replaced with complete DMEM/F-12 medium. Cells were split the next day at a ratio of 1:10. Selection of transfected cells was started 48 h after transfection with G418 sulfate (Gibco/Life Technologies GmbH, Darmstadt, Germany) at a final concentration of 0.5 mg/mL. G418 was continuously added to the culture medium to grow the cells.

c. Selection of HEK293 as producer cells

The expression of bi-scFv proteins by the stably transfected HEK293 and CHO-1 cell lines described in example lb was characterized by immunofluorescent staining to detect bi-scFv expression according to standard procedures (Current Protocols in Immunology, 2012). Briefly, 2×10 ⁵ cells were grown on glass slides for 24 h and then permeabilized with 2% PFA. DPBS supplemented with 5% BSA and 0.2% saponin was used as blocking buffer. After washing with DPBS and blocking with blocking buffer, the cells were incubated with primary antibody Anti-HIS Epitope-Tag (Dianova GmbH, Hamburg, Germany) at a 1:500 dilution in blocking buffer for 30 min at RT. After washing with blocking buffer, a secondary Cy3-conjugated goat anti-mouse IgG (H+L) antibody (Jackson ImmunoResearch Europe, Suffolk, England) at a 1:500 dilution in blocking buffer was added and incubated for 3 h at RT. After washing with blocking buffer and H ₂ O, cells were immersed in DAKO fixing medium (Dako, Carpinteria, CA, USA) supplemented with Hoechst 33342 dye (Pierce/Thermo Fisher Scientific, Rockford, IL, USA). Sections were examined and imaged using a Nikon-Eclipse Ti fluorescence microscope to detect the presence of bi-scFv positive cells (data not shown). HEK293 cells showed better overall expression of bi-scFv proteins compared to CHO-K1 cells and thus were selected as a producer cell line.

d. Production and detection of 1BiMAB bi-scFv protein using clone #28 HEK293

1BiMAB bi-scFv has been selected as the first bi-scFv protein to be generated, purified and used in various assays. For this purpose, clonal cell lines of all HEK293 cells stably expressing 1BiMAB (see Example l.b) were obtained by single cell sorting using a FACS Aria cell sorter (BD Biosciences, Heidelberg, Germany). After propagating about 40 clonal lines, the best producing clone was selected by immunofluorescence as described in Example 1.c.

Selected producer clone #28 was expanded and cultured in a 10-layer cell factory (Nunc, Roskilde, Denmark) in DMEM/F-12 GlutaMax supplemented with 10% FBS, 0.5% penicillin-streptomycin, and 0.8 mg/ml G418 (all reagents from Gibco/Life Technologies GmbH, Darmstadt, Germany) according to the manufacturer's instructions. At the confluent stage, cells were washed with DPBS and medium changed to DMEM/F-12 with antibiotics but no FBS. Cellular supenatant containing 1BiMAB bi-scFv protein was harvested every 3-5 days for 4 weeks. The supernatant was filtered with 500 ml Steritop Filter Units (Merck Millipore, Billerica, MA, USA) and stored at 4° C. until FPLC purification.

Before FPLC purification, the presence of bi-scFv in the cell culture supernatant was tested by polyacrylamide gel electrophoresis followed by Coomassie staining and Western blot analysis performed in a standard manner (Current Protocols in Protein Science, 2012). The supernatant was concentrated 5×-10× using Centricon Centrifugal Filter Devices -10K MWCO (Merck Millipore, Billerica, MA, USA) according to the manufacturer's protocol. Concentrated and unconcentrated supernatants were separated on NuPAGE Novex 4-12% Bis-Tris Gels (Invitrogen/Life Technologies GmbH, Darmstadt, Germany). The gels were then stained with Coomassie Brilliant Blue according to standard procedures for detecting the 50 to 60 kD bi-scFv 1BiMAB protein and other proteins contained in the cell culture supernatant. Western blot analysis was performed to specifically detect the 1BiMAB bi-scFc protein via its 6xHis tag. Briefly, after proteins were blotted onto a PVDF membrane and blocked with PBST/3% milk powder, the membrane was incubated for 1 h at 4°C with Primary Anti-HIS Epitope-Tag (Dianova GmbH, Hamburg, Germany) at a dilution of 1:500 in the blocking buffer. After washing with blocking buffer, the membranes were incubated with Fc-specific secondary goat-anti-mouse IgG peroxidase-conjugated antibody (Sigma Aldrich, Germany) at a dilution of 1 : 10000 in blocking buffer for 1 h at 4°C. After washing with blocking buffer, signals were visualized with a SuperSignal West Femto Chemiluminescent Substrate (Pierce/Thermo Fisher Scientific, Rockford, IL, USA) and recorded with an ImageQuant LAS 4000 Imager (GE Healthcare Life Sciences, Munich, Germany). Bi-scFv signals were detected in the range of 50 to 60 kD compared to internal molecular weight standards (see Figure 3 A and B).

e. Purification and quantification of 1BiMAB bi-scFv protein

The cell culture supernatant of clone #28 HEK293 containing the 1BiMAB bi-scFv protein (described in example ld) was subjected to immobilized metal affinity chromatography (IMAC) using standard procedures (Current Protocols in Protein Science, 2012). Briefly, the filtered cell culture supernatant was applied to a 5 ml His Trap FF column connected to an AKTA Purifier 10 FPLC system (both from GE Healthcare Life Sciences, Munich, Germany). The PBS wash buffer contained 10 mM imidazole, the PBS elution buffer contained 500 mM NaCl, 50 mM NaH ₂ PO ₄ and 250 mM imidazole, both buffers were adjusted to pH 7.4. Elution was performed using a stepped gradient. The eluted bi-scFv 1BiMAB protein was immediately dialyzed against lx PBS using a Slide-A-Lyzer G2 Dialysis Cassette 10K MWCO (Pierce/Thermo Fisher Scientific, Rockford, IL, USA). After dialysis against lx PBS, bi-scFv were dialyzed against 200 mM H ₂ O based arginine buffer (L-arginine monohydrochloride; Roth, Karlsruhe, Germany).

The bi-scFv concentration was determined by measuring at 280 nm with a NanoDrop 2000c, taking into account the extinction coefficient and molecular weight of the 1BiMAB bi-scFv protein determined using the ProtParam tool (http://web.expasy.org/protparam/). The purified protein was aliquoted and stored at -80°C for long term storage or at 4°C for immediate use.

Bi-scFv 1BiMAB protein quality and purity were tested by Coomassie staining and Western blot analysis as described in Example l.d (see also Figures 3A and B). A standard dilution of BSA was included in the Coomassie staining procedure to preliminarily confirm the concentration measured by NanoDrop (data not shown).

f. Conducting an ELISA assay

To quantify 1BiMAB in the cell culture supernatant of HEK 293 cells, a specific ELISA assay was performed. For this purpose, the supernatant from Example 1.d and the purified IBiMAB bi-scFv protein described in Example le were used. 6xHis-tags. All washing steps were performed three times with 200 µl l×PBS/0.05% Tween (wash buffer) per well and all steps were performed at room temperature. Purified 1BiMAB protein diluted in lx PBS in the range of 10-500 ng/mL was used as a standard. The supernatants were diluted 1:10 in lx PBS. 100 μl of diluted protein or supernatant was transferred to each well and incubated for one hour with shaking. After washing, the anti-idiotypic antibody against the V _H -V _L domains mCLDN18.2ab was diluted to a final concentration of 0.5 μg/ml in l×PBS/3% BSA. 100 μl of anti-mCLDN18.2ab solution was added to each well and incubated for one hour with shaking. After washing, AP-conjugated anti-mouse-Fc antibody (Jackson ImmunoResearch Europe, Suffolk, England) was diluted to a final concentration of 300 ng/ml in l×PBS/3% BSA. 100 μl of this secondary antibody solution was added to each well and incubated for an additional hour with shaking. Secondary antibody alone, 1BiMAB plus secondary antibody, and anti-mCLDN18.2ab plus secondary antibody were used as negative controls. In addition, HEK293 cell supernatant without bi-scFv protein was included in the analysis. Finally, after washing, 50 μl of AP substrate solution (1.5 mg pNPP per ml substrate buffer, AppliChem GmbH, Darmstadt, Germany) was added to each well. After 5, 15 and 30 min incubation in the dark, absorbance was measured at 405 nm with an emission wavelength of 492 nm using an Infinite M200 Tecan microplate reader (Tecan, Mannedorf, Switzerland). The concentration of bi-scFv protein in the supernatant was determined by calculation against a standard series (data not shown).

g. Transient transfection of CLDN18.2-specific bi-scFv proteins for comparative studies

To temporarily generate preferably high amounts of CLDN18.2-specific bi-scFv proteins, the human embryonic kidney cell line HEK293T (ATCC CRL-11268) was used for transfection.

1×10 ⁷ HEK293T cells were seeded two days prior to transfection onto 14.5 cm petri dishes in 20 ml of complete DMEM medium (DMEM/F-12 GlutaMax supplemented with 10% heat-inactivated FBS and 0.5% penicillin-streptomycin; all reagents from Gibco/Life Technologies GmbH, Darmstadt, Germany). Before transfection, cells were washed with DPBS supplemented with 2 mM EDTA, then 20 ml of normal DMEM without FBS or antibiotics was added. 20 µg of 1BiMAB circular DNA constructs, no. 11-20 and no.35 (described in Example lb) were diluted in 0.5 ml of regular DMEM/F-12 medium. 75 μl of a linear PEI (polyethyleneimine; Polysciences Europe GmbH, Eppelheim, Germany) solution was added to the diluted DNA at a concentration of 1 mg/ml and vortexed vigorously. After 15 min incubation at room temperature (RT), DNA/PEI complexes were added dropwise to the cells, the cell culture dishes were gently swirled and then incubated at 37°C, 5% CO ₂ for 24 h. DMEM/F-12 cells were incubated for an additional 48 h at 33°C, 5% CO ₂ . Cell supernatant was collected after incubation and sterile filtered using 0.2 µm Minisart syringe filters (Sigma-Aldrich, Germany). Bi-scFv proteins were then purified on a small scale from cell culture supernatants using Ni-NTA spin columns according to the manufacturer's protocol (Qiagen, Hilden, Gemany). Bi-scFv protein concentrations were assessed by ELISA as described in Example 1.f and confirmed by Western blot analysis as described in Example le (data not shown). Purified proteins were stored at 4°C for immediate use.

Example 2 Performance of functional assays to monitor specific T cell activation and target cell lysis with redirected T cells mediated by bi-scFv proteins.

The FPLC-purified 1BiMAB bi-scFv protein was used in in vitro assays to monitor the ability of the bi-scFv proteins to specifically redirect human effector cells to TAA-positive target cells. The aim is to visualize the effects and quantify the activation of human T cells and the specific lysis of target cells.

a. Microscopic analysis of T cells redirected to target cells by the bi-scFv protein

To visualize the functionality of the bi-scFv protein, an assay was performed to demonstrate redirection of effector cells to CLDN18.2-expressing target cells by bi-scFv proteins using a microscope. For this purpose, the NugC4 gastric carcinoma cell line, which endogenously expresses relatively high levels of human CLDN18.2, was used as a target cell line (Sahin U. et al., Clin Cancer Res. 2008 Dec l;14(23):7624-34 ).

Human effector cells were freshly isolated from human blood from healthy donors according to standard procedures (Current Protocols in Immunology, 2012): Briefly, blood was diluted with DPBS, layered on Ficoll-Paque Plus (GE Healthcare Life Sciences, Munich, Germany) and centrifuged. Peripheral blood mononuclear cells (PBMC) were harvested from interphase, washed with cold DPBS supplemented with 2 mM EDTA and counted. Human T cells were then separated by separation of magnetically activated cells (MACS) from PBMCs using the Pan T Cell Isolation Kit II (Miltenyi Biotec, Teterow, Germany) according to the manufacturer's protocol.

1×10 ⁵ NugC4 cells were seeded per well in tissue culture 6-well plates. Human cells were prepared as described above and added at an effector to target ratio (E:T) of 5:1. RPMI 1640 medium supplemented with 5% heat-inactivated human AB serum, 0.5% penicillin-streptomycin, l×NEAA, and 1 mM sodium pyruvate (Gibco/Life Technologies GmbH, Darmstadt, Germany) was used for all cells, and the final volume of each well adjusted to 2 ml per well. Control samples contained target cells or T cells with or without bi-scFv protein. Tissue culture plates were then incubated at 37°C, 5% CO ₂ . The analysis progress was monitored continuously on a Wilovert S inverted microscope (Hund, Wetzlar, Germany) in the range from 6 h to 48 h of co-incubation. Significant effects on T cell clustering on target cells, immunological synapse formation, and target cell killing in the presence of 1BiMAB bi-scFv protein were observed after 24 hours. After 48 hours, viable target cells could hardly be detected. Pictures were taken 24 h later using a Nikon Eclipse TS 100 inverted microscope (Nikon, Japan). See also figure 5.

This assay was subsequently used as a visual control in all cytotoxicity assays in various well formats.

b. Target dependent activation of T cells by 1BiMAB bi-scFv protein

To detect specific activation of human T cells by bi-scFv proteins, analysis was performed by flow cytometry. To detect T cell activation, the early activation marker CD69 and the late activation marker CD25 were selected for staining with antibodies conjugated to fluorescent labels. For detection of human T cells in a mixture of target cells and T cells, CD3 on T cells was stained.

The order of the analysis was chosen from the above example (Example 2.a). Briefly, NugC4 target cells were seeded with human T cells at a 5:1 E:T ratio in 2 ml of complete medium, and bi-scFv 1BiMAB protein was added at a concentration ranging from 0.001-1000 ng/ml. Control samples contained target cells or T cells with or without bi-scFv 1BiMAB protein. After 24 h and/or 48 h - depending on the result of visual control - all cells were collected by gentle scraping with Cell Scrapers (Sarstedt AG & Co, Niirmbrecht, Germany) and transferred to 5 ml round bottom tubes (BD Falcon, Heidelberg, Germany). Cells were centrifuged and washed with DPBS. Cells were stained with mouse anti-human CD3-FITC, mouse anti-human CD69-APC, and mouse anti-human CD25-PE antibodies (all from BD Biosciences, Heidelberg, Germany). Cell pellets were resuspended in 50 µl of FACS buffer (DPBS supplemented with 5% FBS) containing antibodies conjugated to fluorescent labels. After incubation for 20 min at 4°C in the dark, the samples were washed with 4 ml of DPBS and the cell pellet was resuspended in 200 µl of FACS buffer containing propidium iodide (PI) or 7-AAD (both from Sigma Aldrich, Germany) at the final dilution 1 : 1000 for the detection of dead cells. The samples were kept on ice and in the dark during the measurement. Analysis was performed with a FACSCalibur, subsequent measurements were performed with a FACSCanto II flow cytometer (both from BD Biosciences, Heidelberg, Germany). Analytical data were evaluated using FlowJo software (Tree Star, San Carlos, CA, USA).

As shown in Figures 6A and B, 1BiMAB-mediated T cell activation is not detected in the absence of target cells, confirming the strong dependence of bi-scFv functionality on the target. Significant T cell activation in the presence of target cells only occurred at 0.01 ng/mL 1BiMAB after 24 hours. Maximum efficacy was achieved using 100 ng/mL 1BiMAB.

In addition to investigating T cell activation, this assay also provides quantitative assays for bi-scFv mediated effects on target cell killing by gating the target cell population and assessing the percentage of PI- or 7-AAD-positive target cells (data not available) . All analyzes were performed using FlowJo software (Tree Star, San Carlos, CA, USA).

c. Cytotoxicity assay using luciferase

To determine the differences in the potential of bi-scFv proteins directed against CLDN18.2 and CD3 to kill target cells, a highly sensitive assay should be developed. The aim was to provide an assay that could quantitatively control the killing of target cells in a high throughput manner. To achieve this goal, a cytotoxic assay using luciferase was chosen. With this assay, measurement of luciferase expression by viable target cells allows indirect determination of target cell lysis mediated by cytotoxic effector cells in the presence of an antibody.

First, NugC4 cells (described above) were transduced with a lentiviral vector carrying firefly luciferase, an EGFP reporter gene, and an antibiotic selection marker. After antibiotic selection of transduced cells, cells expressing high levels of EGFP were sorted using a FACSAria cell sorter (BD Biosciences, Heidelberg, Germany), analyzed for high luciferase expression, and then expanded for further studies.

Human effector cells were prepared as described in Example 2.a. The assay was performed at a bi-scFv 1BiMAB protein concentration in the range of 1-100 ng/mL, with a concentration of 5 ng/mL found to provide highly efficient and reproducible results, and was subsequently used as the standard concentration. NugC4 cells stably expressing luciferase (described above) were used as target cells. In each well, 1×10 ⁴ target cells were seeded in white flat-bottomed 96-well plates. Human T cells (prepared as described in Example 2.a) were added in an E:T ratio of 5:1. The medium described above (Example 2.a) was used and the final volume of each well was adjusted to 100 µl. Test samples and controls were seeded in at least triplicate. Cell culture microplates were incubated for 24 h and 48 h at 37°C, 5% CO ₂ . For the assay, 50 μl of an aqueous solution containing 1 mg/ml luciferin (BD Monolight, BD Biosciences, Heidelberg, Germany) and 50 mM HEPES was added to each well and the plates were then incubated for 30 min in the dark at 37°C. The luminescence resulting from the oxidation of luciferin by luciferase expressing viable cells was measured on a microplate reader (Infinite M200, Tecan, Mannedorf, Switzerland). The percentage specific lysis of target cells was calculated using the following formula: % specific lysis = [1 - (luminescence _{of the test} sample - L _max ) / (L _min - L _max )] × 100, where "L" means lysis. L _min refers to the minimum lysis in the absence of bi-scFv, and L _max to the maximum lysis (equal to the number of spontaneous luminescence pulses) in the absence of bi-scFv, achieved by the addition of Triton X-100 (2% final concentration).

Potential direct effects of bi-scFv proteins on target cells, regardless of effector cells, were determined by seeding target cells without human T cells, including all controls such as L _min and L _max . This analysis was used for subsequent studies to study the specific lysis of target cells mediated by T cells. Modifications were made, for example, by changing the concentrations of bi-scFv, bi-scFv proteins, E:T ratios, or effector cells (CD8+, CD4+ T cells, PBMC).

Example 3: Selection of a leader candidate CLDN18.2-specific bi-scFv

Luciferase cytotoxicity assay with different CLDN18.2-specific bi-scFv proteins to select the most potent bi-scFv variant

All 10 codon-optimized CHO constructs (no. 11-20) specific for CLDN18.2 TAA were tested against the codon-optimized human 1BiMAB bi-scFv protein in a luciferase cytotoxicity assay with NugC4 target cells, which endogenously express CLDN18.2 and ectopically express luciferase (see also example 2.c). The characteristics of the bi-scFv proteins used are shown in Table 2. Bi-scFv no.35, specific for PLAC1 TAA, was used as an isotype control since PLAC1 is not expressed by NugC4 cells. CD3 binding activity on human T cells was confirmed in a FACS binding assay (data not shown). All bi-scFv proteins were generated as described in Example 1.g and used for cytotoxicity assay as described in Example 2.c.

All bi-scFv proteins were used at a final concentration of 5 ng/ml. To determine L _min , the bi-scFv no.35 control protein was seeded with target cells and T cells in nine replications, the test samples were seeded in six replications. At each time point, one plate was prepared for analysis.

Specific lysis at each analyzed time point (8 h, 16 h, 24 h) is shown depending on the bi-scFv proteins used. The 1BiMAB bi-scFv (SEQ ID NO: 39) and no.15 (SEQ ID NO: 41) proteins, which are designed in the same orientation and contain the same anti-CD3 (TR66) sequence, have been shown to differ only in their use codons at the nucleic acid level and at the linker sequences are the strongest antibodies in terms of mediating the lysis of target cells (see figure 2). Since 1BiMAB and no.15 are equivalent in performance, the best studied 1BiMAB bi-scFv protein to date was selected for all subsequent analyses. The 18PHU3 and 18PHU5 constructs (see Tables 1 and 2) were compared at a later time point with 1BiMAB. The potency of 18PHU5 was equivalent to 1BiMAB, 18PHU3 was less potent (data not shown).

Example 4 Binding capacity of 1BiMAB bi-scFv protein

Performing a FACS-Based Binding Assay

Flow cytometry analysis was used to evaluate the binding capacity of CLDN18.2 and bi-scFv protein fragments targeting CD3. CLDN18.2 endogenously expressed by NugC4 cells was used to study the anti-CLDN18.2 site and human T cells were used to study the anti-CD3 site.

To study the binding capacity of anti-CLDN18.2, NugC4 cells were trypsinated, washed with complete RPMI 1640 medium and then with DPBS. All washing steps were performed by centrifuging at 1200 rpm for 6 min at 4°C. 2.5 x l0 ⁵ NugC4 cells were transferred into 5 ml round bottom tubes and incubated with 50 μg/ml FPLC-purified 1BiMAB protein in FACS buffer for 30 min at 4°C. Cells were washed with 2 ml FACS buffer and then incubated with 3.3 μg/ml Anti-HIS Epitope-Tag monoclonal antibody (Dianova GmbH, Hamburg, Germany) for 30 min at 4°C. After washing with 2 ml of FACS buffer, the cell pellet was incubated with APC-conjugated goat-anti-mouse secondary antibody (Jackson ImmunoResearch Europe, Suffolk, England) at a 1:200 dilution in FACS buffer for 20 min at 4°C under conditions darkness. Cells were washed twice with 2 ml FACS buffer and finally resuspended in 150 μl FACS buffer supplemented with 1 μg/ml PI (Sigma Aldrich, Germany) to counterstain dead cells. Another stain was included using the same procedure with 50 μg/ml 1BiMAB and APC-conjugated goat-anti-mouse secondary antibody (1:200) but without Anti-HIS Epitope-Tag antibody. Negative controls included allophycocyanin (APC) conjugated goat anti-mouse secondary antibody, Anti-HIS Epitope-Tag monoclonal antibody plus APC conjugated goat anti-mouse secondary antibody. As a positive control, 10 μg/ml of the CLDN18.2-specific monoclonal antibody mCLDN18.2ab stained with an APC-conjugated secondary goat-anti-human antibody (Jackson ImmunoResearch Europe, Suffolk, England) and its secondary antibody was used as a positive control.

Samples were measured with a FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany) and analyzed with FlowJo Software (Tree Star, San Carlos, CA, USA). Strong signals were detected by sequential staining with 1BiMAB, Anti-HIS Epitope-Tag and goat-anti-mouse APC. Signal intensity was comparable to mCLDN18.2ab positive control with goat anti-human APC. A low level of direct binding of goat-anti-mouse APC to 1BiMAB was observed in the sample stained with 1BiMAB and goat-anti-mouse APC without Anti-HIS Epitope-Tag (see Figure 4A).

For all subsequent FACS binding assays to examine the binding capacity of bi-scFv proteins, a sequential protocol was used, staining with bi-scFv, Anti-HIS Epitope-Tag, and goat-anti-mouse APC (see Figure 4B, C and D). To rule out non-specific binding of 1BiMAB, target cells that do not express CLDN18.2, as confirmed by RT-PCR data (data not shown), were subjected to a FACS-based binding assay. No non-specific binding of 1BiMAB was detected, as shown in Figure 4D.

To study the binding capacity of the anti-CD3 arm of the 1BiMAB bi-scFv protein, human T cells were used, 1×10 ⁶ T cells obtained as described in example 2.a were transferred into 5 ml round bottom tubes and incubated with FPLC-purified protein 1BiMAB at a concentration in the range of 0.002 - 2 µg/ml in FACS buffer for 30 min at 4°C. The subsequent staining procedure is described above. Control samples included only secondary goat anti-mouse APC antibody and monoclonal antibody Anti-HIS Epitope-Tag plus secondary goat anti-mouse APC antibody. Measurements and analysis were performed as described above. A significant signal was obtained at 2 μg/ml 1BiMAB (see figure 4C).

Example 5 Study of Highly Specific, Target-Dependent Activation of T Cells by 1BiMAB bi-scFv Protein

Cancer cell lines that endogenously express high or low levels of CLDN 18.2, and cancer cell lines that do not express CLDN18.2, were selected to confirm strong bi-scFv 1BiMAB protein target dependence in an in vitro cytotoxicity assay. The cell lines selected were the two main types of carcinoma that express CLDN18.2: gastric (NugC4, MKN7, SNU-1) and pancreatic (DanG, KP-4) carcinomas. The MCF7 breast cancer cell line was used as a negative control.

a. RT-PCR analysis of CLDN18.2 cancer cell lines

Total RNA was extracted from the carcinoma cell lines above using the RNEasy Mini Kit according to the procedure according to the manufacturer's protocol (Quiagen, Hilden, Germany). 5 µg of RNA was used for cDNA synthesis using Superscript II reverse transcriptase (Life Technologies GmbH, Darmstadt, Germany).

RT-PCR analyzes were performed using the ABI Prism 7300 Real Time PCR System (Applied Biosystems/Life Technologies GmbH, Darmstadt, Germany) using Sybr Green dye and the following primers:

CLDN18.2: for TGGCTCTGTGTCGACACTGTG; rev GTGTACATGTTAGCTGTGGAC HPRT: for TGACACTGGCAAAACAATGCA; rev GGTCCTTTTCACCAAGCT

The delta Ct value was calculated by subtracting the Ct value of the HPRT housekeeping gene from the Ct value of CLDN18.2 (results are shown in Figure 7A).

b. Exclusive T cell activation in the presence of CLDN18.2

Cytotoxicity analysis was performed as described in example 2.a. Carcinoma cell lines tested for CLDN18.2 transcripts according to Example 5.a by quantitative RT-PCR were used as target cells. The 1BiMAB bi-scFv protein concentration in this assay was 5 ng/mL. Target cells were seeded with human T cells and 1BiMAB in duplicate for analysis of T cell activation. To control for any potential alloreativity of T cells against target cells regardless of the bi-scF 1BiMAB protein, target cells and T cells were seeded without 1BiMAB in duplicate. Cells were continuously observed under a microscope for T cell cluster formation and target cell binding. In the case of the NugC4 cell line, highly expressing CLDN18.2, a significant effect occurred after 2 hours; after 48 h, viable target cells were barely visible. In the case of the low-expressing CLDN18.2 cell line DanG, the first effects were observed after 96 hours and significant effects after 120 hours. In the case of CLDN18.2-negative cell lines, there were no effects indicating any activation of T cells even after 144 hours. -cells of all samples were analyzed after 144 h co-incubation with target cells by flow cytometry as described in example 2. for dead cells. Surprisingly, up to 100% of T cells co-incubated with NugC4 and 1BiMAB were CD25 positive but CD69 negative, indicating continued T cell activation when CD69 downregulation had already occurred. Approximately 75% of T cells co-incubated with DanG and 1BiMAB were activated, of which approximately 40% co-expressed CD25 and CD69, indicating that T cell activation is still ongoing. T cells co-incubated with CLDN18.2-negative cell lines did not show any sign of induction of T cell activation: CD69 and CD25 expression was not significantly increased compared to expression levels of samples without 1BiMAB (see also Figure 7B).

Example 6 Study of Bi-scFv 1BiMAB Protein-Induced T Cell Function

a. Induction of T cell proliferation

T cell proliferation is a sign of T cell activation. Flow cytometry analysis was used to demonstrate T cell proliferation in response to bi-scFv 1BiMAB protein in the presence of CLDN18.2 positive target cells. Briefly, 1×10 ⁶ human T cells were isolated as described in Example 2.a, stained in the dark at 37°C for 10 min with 0.5 μM carboxyfluorescein diacetate succinimidyl ester (CellTrace CFSE, Invitrogen/Life Technologies GmbH, Germany) dissolved in DPBS. Staining was stopped by adding 5 volumes of cold complete RPMI 1640 medium. Cells were stored on ice for 5 min and washed 3 times with complete RPMI medium (5% heat-inactivated human AB serum, 0.5% penicillin-streptomycin, 1x NEAA and 1 mM sodium pyruvate ) and then resuspended to 1×10 ⁵ cells per ml. The cytotoxicity assay described in Example 2.b was performed with CLDN18.2 endogenously expressing NugC4 cells and human T cells as effector cells. 50 units of IL-2 per ml of medium were added to the cells. Samples included T cells only, T cells with 1 ng/ml 1BiMAB, T cells and NugC4 cells, and T cells with 1 ng/ml 1BiMAB and NugC4 cells. After 120 h of co-incubation, T cells were collected, collected in 5 ml round bottom tubes, washed, and stained with a 1:1000 dilution of 7-AAD DPBS to counterstain dead cells for 15 min at 4°C. After washing with DPBS, cells were resuspended in FACS buffer and analyzed with FACSCanto II (BD Biosciences, Heidelberg, Germany).

T cell proliferation was detected by a decrease in the CFSE signal only in the presence of target cells and bi-scFv 1BiMAB protein (see also Figure 8A).

b. Granzyme B serine protease induction

To demonstrate the upregulation of proteolytic molecules following bi-scFv 1BiMAB protein-mediated T cell activation in the presence of CLDN18.2 positive target cells, detection of Granzyme B serine protease by flow cytometry analysis was chosen. The cytotoxicity assay described in Example 2.b was performed with CLDN18.2 endogenously expressing NugC4 cells and human T cells as effector cells. Samples included only T cells, T cells with 5 ng/ml 1BiMAB, T cells and NugC4 cells, and T cells with 5 ng/ml 1BiMAB and NugC4 cells. After 96 h of co-incubation, T cells were collected, collected in 5-mL round bottom tubes, washed, and stained with a 1 : 1000 dilution of 7-AAD DPBS for counterstaining dead cells for 15 min at 4°C. After washing with DPBS, cells were fixed with 100 μl of Cytoperm/Cytofix solution for 20 min at room temperature (RT). Cells were washed with lx Perm/Wash and then stained with PE-conjugated mouse anti-human Granzyme B antibody for 20 min at RT. After washing, cells were resuspended in FACS buffer and analyzed on a FACSCanto II (all reagents and FACS machine BD Biosciences, Heidelberg, Germany).

Upregulation of Granzyme B in T cells was detected only in the presence of target cells and bi-scFv 1BiMAB protein (see also Figure 8B).

Example 7 Determination of the EC50 of the 1BiMAB bi-scFv protein in an in vitro cytotoxicity assay

Cytotoxicity assay using luciferase

To determine half the maximum effective dose of bi-scFv 1BiMAB protein, a series of 1BiMAB titrations were tested in an in vitro assay for cytotoxicity using luciferase, essentially as described in Example 2.c.

The luciferase-stably expressing NugC4 cells described in Example 2.c were inoculated with human T cells and 1BiMAB bi-scFv protein at concentrations ranging from 1 pg/mL to 1 μg/mL (10 steps) and without 1BiMAB to determine L values. _min . Viable cell luminescence was measured on an Infinite M200 Tecan plate reader 24 and 48 hours after the start of the assay. Specific lysis of target cells was calculated using the formula given in example 2.c.

Maximum lysis was achieved after 48 hours using 1-10 ng/ml 1BiMAB. The determined EC50 value at 48 h in this assay was 10 pg/ml (see also figure 9). The result of this assay depends to a large extent on the activity of human T cells, which differs depending on the immune status of the donor, as described in other works (for example, Lutterbuese, R et al., Proc. Natl. Acad. Sci. USA. 2010 Jul 13; 107(28): 12605-10). In addition, the NugC4 target cell line used shows altered expression of CLDN18.2, also influencing the result. Thus, during the course of the invention, deviations in the EC ₅₀ values of the 1BiMAB bi-scFv protein were observed in the range of 10-300 pg/ml.

Example 8 Efficacy in a Mouse Xenograft Model

The mouse strain NOD.Cg-Prkd ^scid IL2rg ^tmlwjl /SzJ or short NSG (Jackson laboratory, Bar Harbor, ME, USA) was chosen to study the therapeutic potential of the 1BiMAB bi-scFv protein in vivo. For the study described, inoculation of human effector cells and human T-lymphocytes in mice is required to investigate the effects of bi-scFv T-cell uptake in vivo. Due to the complete absence of B, T and NK cells, the NSG mouse strain is suitable for this type of xenograft research. A mouse model inoculated with predominantly human T cells following PBMC injection forms part of the invention.

a. Treatment of Late-Onset, Late-Stage Tumors Highly Expressing CLDN18.2 in Mice with Bi-scFv 1BiMAB Protein

In the described study, 40 female NSG mice at 8 weeks of age were subcutaneously inoculated with 1 x 10 ⁷ HEK293 cells stably expressing high levels of human CLDN18.2 (HEK293-CLDN18.2). 5 days after tumor cell inoculation, mice were divided according to the volume of their tumors into treatment groups, and mice that did not grow tumors were excluded. On the same day, peripheral blood mononuclear cells (PBMCs) were isolated from human blood of healthy donors by ficoll density gradient centrifugation as described in Example 2.a and used as effector cells in vivo. 2×10 ⁷ PBMC diluted in 300 μl DPBS were injected intraperitoneally on the day of release to the experimental treatment groups, designated "PBMC". Treatment groups, designated "PBS", received 300 μl of plain DPBS ip and served as controls without human effector cells. PBS controls can be used to investigate the potential effect on tumor growth of 1BiMAB itself, or any potential side effects that are induced by 1BiMAB or vehicle, but not human effector cells, against mouse tissue (i.e., graft-versus-host disease induced by human effector cells, against mouse tissue). The PBS/vehicle group included 4 mice (n=4), PBS/1BiMAB 5 mice (n=5), PBMC/vehicle 13 mice (n=13), and PBMC/1BiMAB 15 mice (n =15). Therapy was started 1 day after DPBS or PBMC application: the "PBS/1BiMAB" and "PBMC/1BiMAB" groups received intraperitoneally 5 μg of bi-scFv purified 1BiMAB protein diluted in 200 μl of DPBS per animal. The PBS/vehicle and PBMC/vehicle groups received intraperitoneally 200 μl of vehicle buffer (200 mM L-arginine monohydrochloride dissolved in H ₂ O, sterile filtered) diluted in DPBS. Treatment groups are summarized in Table 3. Therapy was administered on a daily basis for 22 days. Tumor size was measured twice a week with a digital calibrated caliper, and tumor volume was calculated by the formula mm ³ = length × width × (width/2). Figures 10A and B show tumor growth inhibition and tumor halving in PBMC/1BiMAB mice by antibody alone in the presence of human effector cells. Mice were sacrificed by dislocation of the cervical vertebrae when the tumor volume exceeded 500 mm ³ or in case of severe clinical manifestations (some mice showed symptoms of graft versus host reaction).

Table 3. Treatment groups

Treatment group (G) Number of mice
(n) Effector
cells bi-scFv protein µg protein bi-scFv/mouse G1 4 - - - G2 5 - 1BiMAB 5 G3 13 PBMC - - G4 15 PBMC 1BiMAB 5

b. Determination of the effect of therapy on body weight

The body weight of each mouse was measured twice a week using a laboratory balance. None of the mice in either group showed a decrease in body weight during the treatment period (data not shown). Some mice in both PBMC groups showed symptoms of graft-versus-host disease 4 weeks after PBMC injection and a few days after completion of treatment. No effects of 1BiMAB itself on body weight or other health related side effects were observed in mice.

c. Tissue preservation and splenocyte isolation

After the mice were sacrificed, the tumors were removed and the tissue was immediately fixed in 10 ml of 4% Roti-Histofix (Carl Roth, Karlsruhe, Germany) for immunohistochemical analysis. In addition, spleens were removed to detect engraftment of human cells by flow cytometry. Isolation of splenocytes was performed immediately after removal of the spleens by forcing them through a 70 µm cell filter placed in a 50 ml reaction tube with a sterile 3-5 ml syringe plunger, and repeated washing of the cell filter with warm DPBS. The isolated splenocytes were centrifuged, the DPBS was decanted and the splenocyte pellet was resuspended in 1 ml heat-inactivated fetal bovine serum supplemented with 10% DMSO. Samples were immediately frozen at -80° C. and stored until splenocyte samples were obtained from all mice.

d. Engraftment assay of human T-lymphocytes in mouse spleens

Splenocytes from all mice were collected and frozen as described in Example 8.c. A complete collection of splenocyte samples were thawed simultaneously, all cells were washed twice with warm DPBS, and 1×10 ⁶ splenocytes per sample were incubated with antibodies conjugated to fluorescent labels for 20 min at 4°C in the dark to detect engraftment of human cells by staining with anti -CD45 and percentage of human cells by anti-CD3, anti-CD4 and anti-CD8 staining. Flow cytometry analysis was performed on a FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany). Engraftment of human T cells in both PBMC groups can be confirmed by a high percentage of double CD45-CD3 positive splenocytes, as shown in Figure 10D.

Example 9 Generation and Testing of Bispecific Binding Agents Targeting CLDN6 and CD3

a. Sequence origin, development of bi-scFv constructs and cloning into expression vectors

The bispecific tandem single chain antibody (bi-scFv) constructs contained binding domains specific for the CD3 component of the human T-cell receptor and human tumor-associated antigens (TAA). The respective heavy chain variable regions (V _H ) and the respective light chain variable regions (V _L ) are arranged for each construct specifically in the N- to C-terminal direction in the following order:

NV _H ^αCLDN6 - V _L ^αCLDN6 - V _H ^αCD3 - V _L ^αCD3 - C (6PHU5; SEQ ID NO: 43)

N- V _H ^αCD3 - V _L ^αCD3 -V _H ^αCLDN6 - V _L ^αCLDN6 C (6PHU5; SEQ ID NO:45)

Table 4 summarizes all bi-scFv constructs specific for TAA CLDN6 that have been generated within the scope of the invention. The CLDN18.2-specific 1BiMAB bi-scFv construct was used as a control antibody. The bi-scFv constructs were generated by gene synthesis from GeneArt AG (GeneArt/Life Technologies GmbH, Regensburg, Germany) using the V _H and V _L sequences of the respective antibodies. Codon optimizations such as Homo sapiens (HS) or Mus musculus (MM) were implemented using GeneArt's GeneOptimizer® software, and are listed in Table 5. Information on specificity, sequence origin from monoclonal antibodies (mAB), codon usage, additional distinguishing sequence indications and references to all applicable domains are summarized in Table 5. The origin of the variable domain sequences of the respective CD3 antibodies are listed in Table 5. Due to the high homology between human and murine TAAs, the V _H and V _L sequences of the same anti-TAA can be used. to generate bi-scFv constructs for mouse assays, but in combination with the V _H , V _L sequences of the mouse specific anti-CD3 antibody clone 145-2C11.

DNA cloning and construction of expression vectors were performed according to standard procedures (Sambrook, 1989) well known to those skilled in the art. Briefly, the bi-scFv DNA sequences contained a 5' HindIII and 3' BamHI restriction for cloning into expression plasmids. The secretion signal sequence was introduced at the 5' end (upper) of the bi-scFv sequence to secrete the protein from the cell cytoplasm into the culture medium. A sequence encoding a flexible glycine-serine peptide linker of 15-18 amino acids was inserted to connect the V _H and V _L domains for the composition of single chain variable antibody fragments (scFv), one of which binds to CD3 and the other to TAA. To form a bispecific single chain antibody, the sequences of the two scFv domains were joined by a sequence encoding a short peptide linker (GGGGS). Along with this linker sequence, a BamHI restriction site was introduced for scFv domain exchanges to clone subsequent bi-scF _V constructs. In particular, 5'scFv domains can be replaced with HindIII and BamHI restriction sites and 3'scFv domains with BamHI and XhoI restriction sites. See also figure 1 for the design diagram.

All bi-scFv antibody constructs used were cloned into the standard mammalian expression vector pcDNA™3.1/myc-His (+) (Invitrogen/Life Technologies GmbH, Darmstadt, Germany). The C-terminal 6xHis tag was used for protein metal affinity purification and detection assays. All constructs were verified by sequencing through the MWG single read sequence service (Eurofins MWG Operon, Ebersberg, Germany).

Table 4. Brief description of bispecific single chain antibody constructs specific for TAA and CD3

Internal name TAA Specificity 5'- _VH - _VL 3'-V _H -V Usage
codons 1BiMAB CLDN18.2 Human mCLDN18.2ab TR66 HS 6PHU5 CLDN6 Human mCLDN6ab TR66 HS 6PHU3 CLDN6 Human TR66 mCLDN6ab HS 6PMU5 CLDN6 mouse mCLDN6ab 145-2C11 MM 6PMU3 CLDN6 mouse 145-2C11 mCLDN6ab MM

HS, Homo sapiens; MM, Mus musculus; TAA, tumor-associated antigen.

Table 5. Brief information on the bi-scFv construct

CD3 binding fragment TAA binding fragment Int. name Origin mAB Reactive variety TAA Origin mAB Reactive variety 5'- _VH - _VL 3'-V _Η -V _L Short linker 1BiMAB TR66 Human CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab TR66 GGGGS 6PHU5 TR66 Human CLDN6 mCLDN6ab man, mouse mCLDN6ab TR66 SGGGGS 6PHU3 TR66 Human CLDN6 mCLDN6ab man, mouse TR66 mCLDN6ab SGGGGS 6PMU5 145-2C11 mouse CLDN6 mCLDN6ab man, mouse mCLDN6ab 145-2C11 SGGGGS 6PMU3 145-2C11 mouse CLDN6 mCLDN6ab man, mouse 145-2C11 mCLDN6ab SGGGGS

Table 5

Continuation

Int. names 5 - long linker 3 - long linker secretion signal Use
codons Anti-CD3
mAB link 1BiMAB (GGGGS) ₃ VE(GGSGGS) ₂ GGVD MGWSCIILFLVATATGVHS HS Lanzavecchia & Scheidegger, Eur J Immunol 1987 6PHU5 (GGGGS) ₃ VE(GGSGGS) ₂ GGVD MGWSCIILFLVATATGVHS HS Lanzavecchia & Scheidegger, Eur J Immunol 1987 6PHU3 VE(GGSGGS) ₂ GGVD (GGGGS) ₃ MGWSCIILFLVATATGVHS HS Lanzavecchia & Scheidegger, Eur J Immunol 1987 6PMU5 (GGGGS) ₃ VE(GGSGGS) ₂ GGVD MGWSCIILFLVATATGVHS MM Leo et al., Proc Natl Acad Sci, 1987 6PMU3 VE(GGSGGS) ₂ GGVD (GGGGS) ₃ MNSGLQLVFFVLTLKGIQG MM Leo et al., Proc Natl Acad Sci, 1987

HS, Homo sapiens; mAB, monoclonal antibody; MM, Mus musculus; TAA, tumor-associated antigen.

b. Generation of stable cell lines-producers

The human embryonic kidney cell line HEK293 (ATCC CRL-1573) was used to generate stable clones of cells producing CLDN6-specific bi-scFv proteins.

1×10 ⁷ HEK293 cells were seeded two days prior to transfection onto 14.5 cm Petri dishes in 20 ml of complete DMEM medium (DMEM/F-12 GlutaMax supplemented with 10% heat-inactivated FBS and 0.5% penicillin-streptomycin; all reagents from Gibco/Life Technologies GmbH, Darmstadt, Germany). Before transfection, cells were washed with DPBS supplemented with 2 mM EDTA, then 20 ml of normal DMEM was added without addition of FBS or antibiotics. 20 μg of the linearized DNA constructs pcDNA3.1/6PHU5 and pcDNA3.1/6PHU3 (described in Example 9.a) were diluted in 0.5 ml of normal DMEM/F-12 medium. 75 μl of a 1 mg/ml linear PEI solution (polyethyleneimine; Polysciences Europe GmbH, Eppelheim, Germany) was added to the diluted DNA and vortexed vigorously. After 15 min incubation at room temperature (RT), DNA/PEI complexes were added dropwise to the cells, the cell culture dishes were gently swirled and then incubated at 37° C., 5% CO ₂ . The medium was changed 24 hours after transfection. Selection of transfected cells was started 48 h after transfection with G418 sulfate (Gibco/Life Technologies GmbH, Darmstadt, Germany) at a final concentration of 0.8 mg/mL. G418 was continuously added to the culture medium to grow the cells.

With. Small scale production of 6PHU5 and 6PHU3 bi-scFv proteins using HEK293 polyclonal cells

The 6PHU5 and 6PHU3 bi-scFv proteins were generated on a small scale and purified from HEK293 polyclonal cell supernatants for in vitro comparison.

Briefly, the confluent FBS-free supernatant was collected from the polyclonal cell lines described in Example 9.b and filtered through a 0.2 μm Minisart filter head (Sigma-Aldrich, Germany) using a syringe. Bi-scFv proteins were then purified on a small scale from cell culture supernatants on Ni-NTA spin columns according to the manufacturer's protocol (Qiagen, Hilden, Germsny). Bi-scFv protein concentrations were determined by absorbance measurement at 280 nm using a NanoDrop 2000c, taking into account the extinction coefficient and molecular weight - determined by the ProtParam tool (http://web.expasy.org/protparam/) - of the 6PHU5 and 6PHU3 bi- scFv. Purified proteins were stored at 4°C for immediate use.

Bi-scFv proteins were tested by polyacrylamide gel electrophoresis followed by Coomassie staining and Western blot analysis performed using standard procedures (Current Protocols in Protein science, 2012). Small scale purified proteins were resolved on NuPAGE Novex 4-12% Bis-Tris Gels (Invitrogen/Life Technologies GmbH, Darmstadt, Germany). The gels were then stained with Coomassie Brilliant Blue according to standard procedures (Current Protocols in Protein Science, 2012) to detect the 6PHU5 and 6PHU3 bi-scFv proteins and other proteins contained in the cell culture supernatant. Western blot analysis was performed to specifically detect the 6PHU5 and 6PHU3 bi-scFv proteins through their 6xHis tag. Briefly, after proteins were blotted onto a PVDF membrane and blocked with PBST/3% milk powder, the membranes were incubated for 1 h at 4°C with Primary Anti-HIS Epitope-Tag (Dianova GmbH, Hamburg, Germany) diluted 1:500 in the blocking buffer. After washing with blocking buffer, membranes were incubated with Fc-specific secondary peroxidase-conjugated goat-anti-mouse IgG antibody (Sigma Aldrich, Germany) at a dilution of 1:10,000 in blocking buffer for 1 h at 4°C. After washing again with blocking buffer, signals were visualized with SuperSignal West Femto Chemiluminescent Substrate (Pierce/Thermo Fisher Scientific, Rockford, IL, USA) and recorded on an ImageQuant LAS 4000 Imager (GE Healthcare life sciences, Munich, Germany). Bi-scFv protein signals were detected between 50 and 60 kD, obtained by comparison with an internal molecular weight standard.

d. Large scale production of 6PHU3 bi-scFv proteins using HEK293 polyclonal cells

The polyclonal producer cell line was grown in a 10-layer cell factory (Nunc, Roskilde, Denmark) in DMEM/F-12 GlutaMax supplemented with 10% FBS and 0.5% penicillin-streptomycin and 0.8 mg/ml G418 (all reagents from Gibco/Life Technologies GmbH, Darmstadt, Germany) in accordance with the manufacturer's instructions. At the confluent stage, cells were washed with DPBS and medium changed to DMEM/F-12 with antibiotics but no FBS. Cell supernatant containing 6PHU3 bi-scFv protein was collected every 3-5 days up to 3 weeks. The supernatant was filtered with 500 ml Steritop Filter Units (Merck Millipore, Billerica, MA, USA) and stored at 4° C. until FPLC filtered.

Prior to FPLC filtration, the presence of bi-scFv in the cell culture supernatant was tested by polyacrylamide gel electrophoresis followed by Coomassie staining and Western blot analysis performed using standard procedures as summarized in Example 9.c.

e. Purification and quantification of 6PHU3 bi-scFv protein

Cell culture supernatant of polyclonal HEK293 cells containing the 6PHU3 bi-scFv protein (described in Example 9.b) was subjected to immobilized metal affinity chromatography (IMAC) using a standard procedure (Current Protocol in Protein Science, 2012). Briefly, the cell culture supernatant was applied to a 5 ml His Trap FF column connected to an AKTA Purifier 10 FPLC system (both from GE Healthcare Life Science, Munich, Germany). The PBS wash buffer contained 10 mM imidazole, the PBS elution buffer contained 500 mM NaCl, 50 mM NaH ₂ PO ₄ and 250 mM imidazole, both buffers were adjusted to pH 7.4. Elution was carried out using a stepped gradient. The eluted 6PHU3 bi-scFv protein was immediately dialyzed against 1x PBS using a Slide-A-Lyzer G2 Dialysis Cassette 10K MWCO (Pierce/Thermo Fisher Scientific, Rockford, IL, USA). After PBS dialysis, bi-scFv were dialyzed against 200 mM arginine buffer (L-arginine monohydrochloride; Roth, Karlsruhe, Germany) based on H ₂ O.

The concentration of bi-scFv was determined by measuring the absorbance at 280 nm using the NanoDrop 2000c, taking into account the extinction coefficient and the molecular weight of the 6PHU3bi-scFv protein. The purified protein was aliquoted and stored at -80°C for long term storage or at 4°C for immediate use.

The quality and purity of the bi-scFv 6PHU3 protein was tested by Coomassie staining and Western blot analysis as described in Example 9.c. BSA at standard dilution was included in the Coomassie staining procedure to roughly confirm the concentration measured with the Nano Drop (data not shown).

Example 10: Efficacy of 6PHU5 and 6PHU3 bi-scFv Candidates Targeting CLDN6

A. Microscopic analysis of T cells redirected to target cells by 6PHU5 and 6PHU3 bi-scFv proteins

To visualize the redirection of effector cells to CLDN6-expressing target cells by 6PHU5 and 6PHU3 bi-scFv proteins by microscopic analysis, an in vitro cytotoxicity assay was performed. The 6PHU3 and 6PHU5 bi-scFv proteins purified on a NiNTA column (see Example 9.c) were used to compare the two variants according to their performance. The target cell line used was the ovarian teratocarcinoma cell line PA-1, which endogenously expresses high levels of human CLDN6.

Human effector cells were isolated from freshly drawn human blood from healthy donors according to standard procedures (Current Protocols in Protein Science, 2012): in brief, blood was diluted with DPBS, layered on Ficoll-Paque Plus (GE Healthcare Life Sciences, Munich, Germany) and centrifuged. Peripheral blood mononuclear cells (PBMC) were harvested from interphase, washed with cold DPBS supplemented with 2 mM EDTA, and counted. Human T cells were then separated from PBMCs by magnetically activated cell separation (MACS) using the Pan T Cell Isolation Kit II (Miltenyi Biotec, Teterow, Germany) according to the manufacturer's guidelines.

1×10 ⁵ PA-1 cells per well were seeded in tissue culture 6-well plates. Human cells were prepared as described above and added at an effector to target ratio (E:T) of 5:1. MEM medium supplemented with 10% heat-inactivated FBS, 5% penicillin-streptomycin, 1x NEAA, 1 mM sodium bicarbonate and 1 mM sodium pyruvate (Gibco/Life Technologies GmbH, Darmstadt, Germany) was used for all cells and the final volume of each well was adjusted to 2 ml. The bi-scFv protein concentration used in this assay was 50 ng/mL. Control samples contained target cells or only T cells without bi-scFv protein. Tissue culture plates were then incubated at 37°C, 5% CO ₂ . During the analysis, continuous observation was carried out on a Wilovert S inverted microscope (Hund, Wetzlar, Germany) in the range from 6 h to 24 h of co-incubation. Significant effects on T cell clustering on target cells, immunological synapse formation, and target cell death in the presence of 6PHU5 and 6PHU3 bi-scFv proteins were observed after 24 h and imaged using a Nikon Eclipse TS100 inverted microscope (Nikon, Japan) . Both bi-scFv proteins showed strong T cell aggregation and target cell death as shown in Figure 12.

A. T cell activation mediated by 6PHU5 and 6PHU3 bi-scFv proteins

A FACS-based T cell activation assay was used to detect T cell activation and determine the difference in potency between the two CLDN6 specific bi-scFv variants. The early activation marker CD69 and the late activation marker CD25 were chosen to stain antibodies conjugated with fluorescent labels. To detect human T cells in a mixture of target cells and T cells, CD3 staining was performed on T cells.

In general, the analysis conditions indicated above were chosen (Example 10.a). Briefly, PA-1 target cells endogenously expressing CLDN6 were seeded with human T cells at an E:T ratio of 5:1 in 2 ml of complete medium, and 6PHU5 and 6PHU3 bi-scFv proteins were added at a concentration in the range of 5- 200 ng/ml. Control samples contained only target cells or T cells with or without bi-scFv proteins. After 24 hours and 48 hours, T cells were collected by rinsing and transferred to 5 ml round bottom tubes (BD Falcon, Heidelberg, Germany). Cells were centrifuged and washed with DPBS. Cells were stained with mouse anti-human CD3-FITC, mouse anti-human CD69-APC, and mouse anti-human CD25-PE antibodies (all from BD Biosciences, Heidelberg, Germany). The cell pellet was resuspended in 50 µl of FACS buffer (DPBS supplemented with 5% FBS) containing antibodies conjugated to fluorescent labels and 2 µl of 7-AAD (BD Biosciences, Heidelberg, Germany). After incubation for 20 min at 4° C. in the dark, the samples were washed with 4 ml of DPBS and the cell pellet was resuspended in 200 μl of FACS buffer. Samples were kept on ice and in the dark while measured with a FACSCanto II flow cytometer (both from BD Biosciences, Heidelberg, Germany). The results of the analysis were evaluated using FlowJo software (Tree Star, San Carlos, CA, USA).

Both variants of CDLN6-specific bi-scFv resulted in effective T-cell activation up to 60%. The 6PHU3 variant (bi-scFv CD3 x CLDN6) was more potent in the low concentration range of 5-10 ng/mL (see also Figure 13) and was therefore chosen for further studies.

Example 11: Binding capacity of 6PHU3bi-scFv

FACS Binding Assay

Flow cytometric analysis was used to assess the binding capacity of the 6PHU3 bi-scFv protein fragments targeting CLDN6 and CD3. PA-1 and OV-90 cells endogenously expressing CLDN6 were used to study the anti-CLDN6 site, and human T cells were used to study the anti-CD3 site. CLDN6-negative NugC4 cells were used as control cells.

To study the binding capacity of anti-CLDN6, CLDN6 positive cells (PA-1, OV-90) and CLDN6 negative cells (NugC4) were trypsinized, washed with complete medium and then with DPBS. All washing steps were performed by centrifugation at 1200 rpm for 6 min at 4°C. 1×10 ⁵ cells were transferred into 5 ml round bottom tubes and incubated with 0.01-10 μg/ml of FPLC-purified 6PHU3 protein or bi-scFv control 1BiMAB protein in FACS buffer for 30 min at 4°C. Cells were washed with 2 ml FACS buffer and then incubated with 3.3 μg/ml Anti-HIS Epitope-Tag monoclonal antibody (Dianova GmbH, Hamburg, Germany) for 30 min at 4°C. After washing with 2 ml of FACS buffer, the cell pellet was incubated with APC-conjugated goat-anti-mouse secondary antibody (Jackson ImmunoResearch Europe, Suffolk, England) at a 1:200 dilution in FACS buffer for 20 min at 4°C in the dark . Cells were washed twice with 2 ml FACS buffer and finally resuspended in 150 μl FACS buffer supplemented with 1 μg/ml PI (Sigma Aldrich, Germany) to counterstain dead cells. Negative controls included only secondary goat-anti-mouse APC antibody. As a positive control, 10 μg/ml of the CLDN6-specific monoclonal antibody mCLDNab was stained with a goat-anti-human APC secondary antibody (Jackson Immunoresearch Europe, Suffolk, England) and an appropriate control secondary antibody.

Samples were measured with a FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany) and analyzed with FlowJo Software (Tree Star, San Carlos, CA, USA). The signal intensity of 10 μg/ml 6PHU3 was 4-9 times lower than the mCLDN6ab positive control (see Figure 15A). Non-specific binding of 6PHU3 to the CLDN6-negative NugC4 cell line was not detected (Figure 15C).

Human T cells were used to study the binding capacity of the anti-CD3 arm of the bi-scFv 6PHU3 protein. 5×10 ⁵ T cells were transferred to 5 ml round bottom tubes and incubated with FPLC-purified 6PHU3 protein at a concentration in the range of 100 ng/ml - 10 μg/ml in FACS buffer for 30 min at 4°C. The subsequent staining procedure was the same as described above. Control samples included only secondary goat anti-mouse APC antibody and monoclonal antibody Anti-HIS Epitope-Tag plus secondary goat anti-mouse PE antibody. Measurement and analysis were performed as described above. A significant signal was obtained at 100 ng/ml 6PHU3 (see also Figure 15B).

Example 12 Target-Dependent T Cell Activation Assay with bi-scFv 6PHU3

Cytotoxicity assay was performed as described in Example 10.a and b. Briefly, PA-1 target cells endogenously expressing CLDN6 were seeded with human T cells at an E:T ratio of 5:1 in 2 ml of complete medium, and bi-scFv 6PHU3 protein was added at a concentration ranging from 0.001-1000 ng/ml. For target-dependence analysis of bi-scFv mediated T cell activation, T cells were seeded without target cells but incubated with the same concentrations of bi-scFv 6PHU3 as target cell samples with T cells. After 24 hours and 48 hours, T cells were harvested and transferred to 5 ml round bottom tubes (BD Falcon, Heidelberg, Germany). Cell staining and analysis was performed as described in Example 10.b.

As shown in Figures 16A and B, 6PHU3 mediated T cell activation was not detected in the absence of target cells, indicating a direct strong dependence of bi-scFv functionality on the target. Significant T cell activation only occurred with 0.1 ng/mL 6PHU3 after 48 h.

Example 13 Determination of the EC50 of 6PHU3 bi-scFv in an in vitro cytotoxicity assay

Cytotoxicity assay using luciferase

To determine half the maximum effective dose of bi-scFv 6PHU3 protein, a series of 6PHU3 titrations were tested in an in vitro luciferase cytotoxicity assay.

Luciferase-stably-expressing PA-1 cells and human T cells at a 5:1 E:T ratio were incubated with bi-scFv 6PHU3 protein concentrations ranging from 1 pg/mL to 1 μg/mL (10 steps) or without 6PHU3 to determine the values of L _min .

Cell culture microplates were incubated for 24 h and 48 h at 37°C, 5% CO ₂ . For the assay, 50 µl of an aqueous solution containing 1 mg/ml luciferin (BD Monolight, BD Biosciences, Heidelberg, Germany) and 50 mM HEPES was added to each well and the plates were then incubated for 30 min in the dark at 37°C. The luminescence resulting from the oxidation of luciferin by luciferase expressing viable cells was measured using an Inifinite M200 Tecan microplate reader (Tecan, Mannedorf, Switzerland). The percentage specific lysis of target cells was calculated by the following formula: % specific lysis = [1-(luminescence _{test sample} - L _max ) / (L _min - L _max )] × 100, where "L" means lysis. L _min refers to the minimum lysis in the absence of bi-scFv, and L _max to the maximum lysis (equal to the spontaneous luminescence value) in the absence of bi-scFv, achieved by the addition of Triton X-100 (at a final concentration of 2%).

Maximum lysis was achieved after 48 hours with 1-10 ng/ml 6PHU3, the determined EC ₅₀ after 48 hours was approximately 10 pg/ml (see also figure 17). The result of this assay is highly dependent on the strength of human T cells, which differs according to the immune status of the donor, which has also been described elsewhere (see e.g. Lutterbuese, R et al., 2010, Proc. Natl. Acad. Sci USA, 2010 Jul 13;107(28):12605-10). Thus, the deviation of the EC ₅₀ value of the 6PHU3 bi-scFv protein by factor 3 was observed during the course of the invention.

Example 14 Efficacy in a Mouse Xenograft Model

The murine strain NOD.Cg-Prkd ^scid IL2rg ^tmlwjl /SzJ or short NSG (Jackson laboratory, Bar Harbour, ME, USA) was chosen to study the therapeutic potential of the 6PHU3 bi-scFv protein in vivo. For the study described, inoculation of human effector cells and human T-lymphocytes in mice is required to investigate the effects of bi-scFv T-cell uptake in vivo. Due to the complete absence of B, T and NK cells, the NSG mouse strain is suitable for this type of xenograft research. A mouse model inoculated with predominantly human T cells following PBMC injection forms part of the invention.

A. Treatment of delayed-onset, late-stage, high-expressing CLDN6 tumors in mice with 6PHU3bi-scFv protein

In the described study, 25 female and 25 male NSG mice aged 8-11 were subcutaneously inoculated with 1×10 ⁷ PA-1 cells endogenously expressing high levels of human CLDN6. 5 days after tumor cell inoculation, mice were divided according to the volume of their tumors into treatment groups, and mice that did not grow tumors were excluded. On the same day, peripheral blood mononuclear cells (PBMC) were isolated from human blood of healthy donors by ficoll density gradient centrifugation and used as effector cells in vivo. 2×10 ⁷ PBMC diluted in 200 μl DPBS were injected intraperitoneally on the day of release to the experimental treatment groups, designated "PBMC". Treatment groups, designated "PBS", received 200 μl of conventional DPBS ip and served as controls without human effector cells. PBS controls can be used to investigate the potential effect on tumor growth of 6PUH3 itself or any potential side effects induced in 25 female and 25 male NSG mice aged 8-11 or vehicle, but not human effector cells, against mouse tissue. (i.e. graft-versus-host reaction induced by human effector cells against mouse tissue). The "PBS/vehicle" group included 8 mice (n=8), "PBS/6PUH3" 8 mice (n=8), "PBMC/vehicle" 7 mice (n=7), "PBMC/6PUH3" 7 mice (n =7) and "PBMC/1BiMAB" 8 mice (n=8). Therapy was initiated 7 days after DPBS or PBMC: PBS/6PHU3, PBMC/6PHU3, and PBMC/1BiMAB groups received ip 5 μg of purified 6PHU3 protein bi-scFv or 1BiMAB diluted in 200 μl of DPBS per animal. The PBS/vehicle and PBMC/vehicle groups received intraperitoneally 200 μl of vehicle buffer (200 mM L-arginine monohydrochloride dissolved in H ₂ O, sterile filtered) diluted in DPBS. Treatment groups are summarized in Table 3. Therapy was administered on a daily basis for 26 days. Tumor size was measured twice a week with a digital calibrated caliper and tumor volume was calculated as mm ³ = length x width x (width/2). Figures 18A and B show the inhibition of tumor growth in all PBMC/6PHU3 mice by antibody in the presence of human effector cells. Mice were sacrificed by dislocation of the cervical vertebrae when the tumor volume reached 1500 mm ³ or in case of severe clinical manifestations (some mice showed symptoms of graft versus host reaction).

Table 6. Treatment groups

Treatment group (G) Number of mice effector cells bi-scFv protein µg protein bi-scFv/mouse G1 8 - - - G2 8 - 6PHU3 5 G3 7 PBMC - - G4 7 PBMC 6PHU3 5 G5 8 PBMC 1BiMAB 5

b. Determination of the effect of therapy on body weight

The body weight of each mouse was measured twice a week using a laboratory balance. None of the mice in either group showed weight loss during treatment (data not shown).

With. Tissue preservation and splenocyte isolation

After the mice were sacrificed, the tumors were removed and the tissue was immediately fixed in 10 ml of 4% Roti-Histofix (Carl Roth, Karlsruhe, Germany) for immunohistochemical analysis. In addition, spleens were removed to detect engraftment of human cells by flow cytometry. Isolation of splenocytes was performed immediately after removal of the spleens by forcing them through a 70 µm cell filter placed in a 50 ml reaction tube with a sterile 3-5 ml syringe plunger, and re-washing the cell filter with warm DPBS. The isolated splenocytes were centrifuged, the DPBS was decanted and the splenocyte pellet was resuspended in 1 ml heat-inactivated fetal bovine serum supplemented with 10% DMSO. Samples were immediately frozen at -80° C. and stored until splenocyte samples were obtained from all mice.

d. Engraftment assay of human T-lymphocytes in mouse spleens

Splenocytes from all mice were collected and frozen as described in Example 14.c. A complete collection of splenocyte samples were thawed simultaneously, all cells were washed twice with warm DPBS, and 1×10 ⁶ splenocytes per sample were incubated with antibodies conjugated to fluorescent labels for 20 min at 4°C in the dark to detect engraftment of human cells by staining with anti -CD45 and percentage of human cells by anti-CD3, anti-CD4 and anti-CD8 staining. Flow cytometry analysis was performed on a FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany). Engraftment of human T cells in both PBMC groups can be confirmed by a high percentage of double CD45-CD3 positive splenocytes, as shown in Figure 18D.

e. Immunohistochemistry to determine target cell expression and T-cell infiltration

Tumors were fixed after removal using 4% formaldehyde buffer solution (Roti-Histofix, Carl Roth, Karlsruhe, Germany) for 48 h at 4°C. Fixed tumors were divided into two parts and transferred to an ASP200 automated vacuum tissue processor for dehydrogenation (Leica Microsystems GmbH, Wetzlar, Germany) followed by paraffin embedding (Paraplast, Carl Roth, Karlsruhe, Germany) using an MPS/C paraffin dispenser station (Slee Medical GmbH, Mainz, Germany). For immunohistochemical staining, 3 μm thick sections of formalin-fixed and paraffin-embedded tissues were obtained using a rotary microtome RM2255 (Leica Microsystems GmbH, Wetzlar, Germany). Deparaffinization and rehydrogenation were performed in a StainMate Max bilinear stainer (Thermo Ficher Scientific, Rockford, IL, USA) followed by heat-induced epitope extraction in 10 mM lemon buffer, pH 6, with 0.05% Tween 20 for 10 min at 120°C. WITH. Endogenous peroxidase activity was quenched by immersion in 0.1% H ₂ O ₂ in PBS for 15 min (Carl Roth) followed by incubation with 10% goat serum in PBS (PAA Laboratories GmbH/GE Healthcare, Pasching, Austria) for 30 min to block non-specific antibody binding sites. TAA claudin 6 was detected by incubation with the polyclonal primary antibody Anti-Mouse Claudin 6 (C) Rabbit (IBL-America, Minneapolis, MN, USA) at 4° C. overnight; T cells were detected on serial sections using polyclonal anti-CD3 AB (Abcam, Cambridge, UK) at 4°C overnight followed by incubation with anti-rabbit secondary antibody conjugated to Bright Vision HRP polymer (ImmunoLogic, Duiven, Netherlands). ). Binding reactions were visualized using the Vector NovaRED kit (Vector Laboratories Ltd., Peterborough, UK) according to the manufacturer's instructions, followed by hematoxylin counterstaining (Carl Roth), dehydrogenation, and media incorporation. Analyzes and registration were performed using an Axio Imager M2 imaging unit or a Mirax scanner (both from Carl Zeiss Microscopy GmbH, Goettingen, Germany).

As shown in Figure 19, the highest T cell infiltration was detected in tumors of the "PBMC/6PHU3" group by CD-3 staining, especially in the border regions of CLDN6 expression. The pattern of heterogeneous expression of TAA CLDN6 in the control groups (figures 19A, B, C and D) changed into more compact areas of CLDN6 expression in tumors of the "PBMC/6PHU3" group as a result of therapy (figure 19D).

Example 15: Design and Testing of Bispecific Binding Agents Targeting CLDN18.2 and CD3

A. Initial sequences, design of bi-scFv constructs and vector matrix cloning

Bispecific tandem single chain antibody (bi-scFv) constructs were prepared containing binding domains specific for the CD3-epsilon component of the human T-cell receptor and tumor-associated antigens (TAA). The respective heavy chain variable regions (V _H ) and the respective light chain variable regions (V _L ) for each construct were specifically arranged in the 5' to 3' direction in the following order:

pST1-hAgKozak-V _H ^αCLDN18.2 -V _L ^αCLDN18.2 -V _H ^αCD3 - V _L ^αCD3 -His-2hBgUTR-A120

(1BiMAB, 18RHU5, no. 1-5)

pST1-hAgKozak-V _H ^αCD3 - V _L ^αCD3 - V _H ^αCLDN18.2 -V _L ^αCLDN18.2 - His-2hBgUTR-A120

(18RHU3, no. 6-10)

Table 7 summarizes all bi-scFv constructs specific for TAA CLDN18.2 and PLAC1 that have been generated within the scope of the invention. The bi-scFv constructs were generated by gene synthesis from GeneArt AG (GeneArt/Life Technologies GmbH, Regensburg, Germany) using the V _H and V _L sequences of the respective antibodies. Codon optimizations such as Homo sapiens (HS) or Mus musculus (MM) or Chinese Hamster Ovary (CHO) cells were implemented using GeneArt's GeneOptimizer® software, and are listed in Table 7. Information on specificity, sequence origin from monoclonal antibodies (mAB), codon usage, additional sequence distinguishing features, and references to all applicable domains are summarized in Table 8. The origin of the variable domain sequences of the respective CD3 antibodies are listed in Table 8. Due to the high homology between human and murine TAAs, V _H and V _L of the same anti-TAA to generate bi-scFv constructs for mouse assays, but in combination with V _H , V _L sequences of the mouse specific clone 145-2C11 anti-CD3 antibody.

DNA cloning and construction of expression vectors were performed according to standard procedures (Sambrook, 1989) well known to those skilled in the art. Briefly, the bi-scFv core DNA sequences contained a 5'-BsmBI and 3'-XhoI restriction site for cloning into pST1 plasmids. The secretion signal sequence was introduced at the 5' end (upper) of the bi-scFv sequence for protein secretion. A sequence encoding a flexible glycine-serine peptide linker of 15-18 amino acids was inserted to connect the V _H and V _L domains for the composition of single chain variable antibody fragments (scFv), one of which binds to CD3 and the other to TAA. To form a bispecific single chain antibody, the sequences of the two scFv domains were joined by a sequence encoding a short peptide linker (GGGGS). Along with this linker sequence, a BamHI restriction site was introduced for scFv domain exchanges to clone subsequent bi-scF _V constructs. Briefly, 5'scFv domains were replaced with BsmBI and XhoI restriction and 3'scFv domains with BamHI and XhoI restriction. The C-terminal 6xHis tag was used for assays for the detection of translated protein. The untranslated regions of human alpha globin 5' and human beta globin 3' of the bi-scFv sequence were present in the pST1 vector (see more details in WO2007/036366A2: Waggoner, S. et al. (2003) Exp. Biol. Med. (Maywood ) 228 (4), pp. 387-395).

To obtain the 1BiMAB replicon vector, the entire 1BiMAB sequence, including the secretion signal and the 6xHis tag, was subcloned 3' from the subgenomic promoter of the Semliki Forest Virus (pSFV) replicon vector, courtesy of K. Lundstrom (Lundstrom, K. et al. (2001) ) Histochem Cell Biol 115(1), pp. 83-91; Ehrengruber, M.U. et al.(1999) Proc. Natl. Acad. Sci. U.S.A. 96(12), pp. 7041-7046).

All constructs were verified by sequencing through the MWG single read sequence service (Eurofins MWG Operon, Ebersberg, Germany) and only constructs with the correct sequence and a poly(A) tail consisting of more than 100 adenines were used for in vitro transcription of RNA.

Structural diagrams are also shown in Figure 20A.

Table 7. Brief description of mRNA template constructs for a bispecific single chain antibody specific for TAA and CD3

Internal name TAA Specificity 5'- _VH - _VL 3'- _VH - _VL Usage
codons 1BiMAB CLDN18.2 Human mCLDN18.2ab TR66 HS no.1 CLDN18.2 mouse mCLDN18.2ab 145-2C11 CHO no.2 CLDN18.2 Human mCLDN18.2ab UCHT1-HU CHO no.3 CLDN18.2 Human mCLDN18.2ab UCHT1 CHO no.4 CLDN18.2 Human mCLDN18.2ab CLB-T3 CHO no.5 CLDN18.2 Human mCLDN18.2ab TR66 CHO no.6 CLDN18.2 mouse 145-2C11 mCLDN18.2ab CHO no.7 CLDN18.2 Human UCHT1-HU mCLDN18.2ab CHO no.8 CLDN18.2 Human UCHT1 mCLDN18.2ab CHO no.9 CLDN18.2 Human CLB-T3 mCLDN18.2ab CHO no.10 CLDN18.2 Human TR66 mCLDN18.2ab CHO 18RHU5 CLDN18.2 Human mCLDN18.2ab TR66 HS 18RHU3 CLDN18.2 Human TR66 mCLDN18.2ab HS 18RMU5 CLDN18.2 mouse mCLDN18.2ab 145-2C11 MM 18RMU3 CLDN18.2 mouse 145-2C11 mCLDN18.2ab MM control bi-scFv no.25 PLAC1 Human 78H11 TR66 CHO

bi-scFv is a bispecific single chain variable fragment; CHO, Chinese hamster ovary cells; HS, Homo sapiens; HU, humanized; MM, Mus musculus; TAA, tumor-associated antigen; V _H , heavy chain variable domain, V _L , light chain variable domain.

Table 8. Brief information on the construction of the bi-scFv mRNA template

CD3 binding fragment TAA binding fragment . ker Internal name Origin of mAB. Reactive variety TAA Origin mAB Reactive variant 5'- _VH - _VL 3' V _Η -V _L short ling 1BiMAB TR66 Human CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab TR66 GGGGS no.1 145-2C11 mouse CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab 145-2C11 SGGGGS no.2 UCHT1-HU Human CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab UCHT1-HU SGGGGS no.3 UCHT1 Human CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab UCHT1 SGGGGS no.4 CLB-T3 Human CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab CLB-T3 SGGGGS no.5 TR66 Human CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab TR66 SGGGGS no.6 145-2C11 mouse CLDN18.2 mCLDN18.2ab man, mouse 145-2C11 mCLDN18.2ab SGGGGS no.7 UCHT1-HU Human CLDN18.2 mCLDN18.2ab man, mouse UCHT1-HU mCLDN18.2ab SGGGGS no.8 UCHT1 Human CLDN18.2 mCLDN18.2ab man, mouse UCHT1 mCLDN18.2ab SGGGGS no.9 CLB-T3 Human CLDN18.2 mCLDN18.2ab man, mouse CLB-T3 mCLDN18.2ab SGGGGS no.10 TR66 Human CLDN18.2 mCLDN18.2ab man, mouse TR66 mCLDN18.2ab SGGGGS 18RHU5 TR66 Human CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab TR66 SGGGGS 18RHU3 TR66 Human CLDN18.2 mCLDN18.2ab man, mouse TR66 mCLDN18.2ab SGGGGS 18RMU5 145-2C11 mouse CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab 145-2C11 SGGGGS 18RMU3 145-2C11 mouse CLDN18.2 mCLDN18.2ab man, mouse 145-2C11 mCLDN18.2ab SGGGGS no.25 TR66 Human PLAC1 78H11 man, mouse 78H11 TR66 SGGGGS

Table 8

Continuation

Int. names. 5'-long linker 3'-long linker secretion signal Use
codons Anti-CD3
mAB
links 1BiMAB (GGGGS) ₃ VE(GGSGGS) ₂ GGVD MGWSCIILFLVATATGVHS HS Lanzavecchia & Scheidegger, Eur J Immunol 1987 no.1 (GGGGS) ₃ (GGGGS) ₃ MGWSCIILFLVATATGVHS CHO Leo et al., Proc Natl Acad Sci, 1987 no.2 (GGGGS) ₃ (GGGGS) ₃ MGWSCIILFLVATATGVHS CHO Shalaby et al. J Exp Med 1992 no.3 (GGGGS) ₃ (GGGGS) ₃ MGWSCIILFLVATATGVHS CHO Beverley et al., Eur J Immunol 1981 no.4 (GGGGS) ₃ (GGGGS) ₃ MGWSCIILFLVATATGVHS CHO Van Lier et al. Immunology 1989 no.5 (GGGGS) ₃ (GGGGS) ₃ MGWSCIILFLVATATGVHS CHO Lanzavecchia & Scheidegger, Eur J Immunol 1987 no.6 (GGGGS) ₃ (GGGGS) ₃ MNSGLQLVFFVLTLKGIQG CHO Leo et al., Proc Natl Acad Sci, 1987 no.7 (GGGGS) ₃ (GGGGS) ₃ MGWSCIILFLVATATGVHS CHO Shalaby et al. J Exp Med 1992 no.8 (GGGGS) ₃ (GGGGS) ₃ MNSGLQLVFFVLTLKGIQG CHO Beverley et al., Eur J Immunol 1981 no.9 (GGGGS) ₃ (GGGGS) ₃ MNFGLSLIFLALILKGVQC CHO Van Lier et al. Immunology 1989 no.10 (GGGGS) ₃ (GGGGS) ₃ MEWSWIFLFLLSVTTGVHS CHO Lanzavecchia & Scheidegger, Eur J Immunol 1987 18RHU5 (GGGGS) ₃ VE(GGSGGS) ₂ GGVD MGWSCIILFLVATATGVHS HS Lanzavecchia & Scheidegger, Eur J Immunol 1987 18RHU3 VE(GGSGGS) ₂ GGVD (GGGGS) ₃ MGWSCIILFLVATATGVHS HS Lanzavecchia & Scheidegger, Eur J Immunol 1987 1RPMU5 (GGGGS) ₃ VE(GGSGGS) ₂ GGVD MGWSCIILFLVATATGVHS MM Leo et al., Proc Natl Acad Sci, 1987 18RMU3 VE(GGSGGS) ₂ GGVD (GGGGS) ₃ MNSGLQLVFFVLTLKGIQG MM Leo et al., Proc Natl Acad Sci, 1987 no.25 (GGGGS) ₃ (GGGGS) ₃ MGWLWNLLFLMAAAQSAQA CHO Lanzavecchia & Scheidegger, Eur J Immunol 1987

CHO stands for Chinese Hamster Ovary Cells; HS, Homo sapiens; mAB, monoclonal antibody; MM, Mus musculus; TAA, tumor-associated antigen.

b. Synthesis of IVT-RNA

To generate anti-CLDN18.2-specific bi-scFv IVT templates, plasmids were linearized downstream of the poly(A) tail using class IIs endonuclease. The linearized DNA template was purified by phenol/chloroform extraction and precipitated with sodium acetate as described in the literature (Holtkamp, S. et al. (2006) Blood 108 (13), pp. 4009-4017).

Linearized DNA templates were subjected to in vitro transcription using MEGAscript Kits (Ambion/Life Technologies, Darmstadt, Germany) according to the manufacturer's instructions: pST1 templates were transcribed using the MEGAscript T7 Kit and pSFV template using the MEGAscript SP6 Kit. For cap analog reactions, the GTP concentration was reduced to 1.5 mM and 6 mM ARCA, beta-S-ARCA(D1) or beta-S-ARCA(D2) synthesized as described in the literature was added to the reaction (Kowalska, J et al (2008) RNA 14 (6), pp. 1119-1131 Grudzien, E. et al (2004) RNA 10 (9), pp. 1479-1487 Stepinski, J. et al (2001 ) RNA 7 (10), pp. 1486-1495). Purification of IVT-mRNA was performed using the MEGAclear Kit (Ambion/Life Technologies, Darmstadt, Germany) according to the manual. The concentration and quality of IVT-RNA was assessed by spectrophotometry and analyzed on a bioanalyzer 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

Example 16 T cell activation in response to bi-scFv secretion by target cells transfected with various CLDN18.2-specific IVT mRNAs

To investigate the functionality of the CLDN18.2 IVT-mRNA-specific bi-scFv gastric carcinoma cell line NugC4, which endogenously expresses relatively high levels of human CLDN18.2 (Sahin, U. et al. (2008) Clin. Cancer Res. 14 (23) , pp. 7624-7634) was used as the target cell line.

NugC4 target cells - which were tested in the usual manner for TAA expression by FACS analysis before each assay - were washed twice with ice-cold X-Vivo 15 medium (LONZA, Basel, Switzerland) and resuspended to a density of 2×10 ⁷ cells/ml. 250 μl of the cell suspension was transferred into pre-chilled 0.4 cm Gene Pulser/MicroPulser Cuvettes (Bio-Rad, Dreieich, Germany) and 20 μg/ml IVT-mRNA was added. The following IVT mRNAs were used: 1BiMAB, no. 2, no. 3, no. 4, no. 5, no. 7, no. 8, no. 9 and no. 10. For more details on bi-scFv variants, see Tables 7 and 8. After gentle mixing, cells were transfected with a BTX ECM 830 electroporator (Harvard Apparatus, Holliston, MA, USA) using the following conditions: 250 V, 2 pulses, duration pulse 12 ms, pause between pulses 40 ms. Immediately after electroporation, the cuvettes were placed on ice for a short period of time, and then the cell suspensions were transferred to a room temperature assay medium (RPMI 1640 medium supplemented with 5% heat-inactivated human AB serum, 0.5% penicillin-streptomycin, 1x NEAA and 1 mM sodium pyruvate (Gibco/Life Technologies GmbH, Darmstadt, Germany)) in 15 ml tubes Transfected target cells were counted and adjusted to 1 x 10 ⁵ cells per ml.

Human effector cells were isolated from fresh blood of healthy human donors according to standard procedures (Current Protocols in Immunology, 2012): in brief, blood was diluted with DPBS, applied to Ficoll-Paque Plus (GE Healthcare Life Sciences, Munich, Germany) and centrifuged. Peripheral blood mononuclear cells (PBMC) were harvested from interphase, washed with cold DPBS supplemented with 2 mM EDTA and counted. Human cytotoxic T cells were isolated by magnetically activated cell sorting (MACS) from PBMCs using the CD8+ T cell Isolation Kit, human (Miltenyi Biotec, Teterow, Germany) according to the manufacturer's instructions. The separated effector cells were tested in the usual way for successful isolation of T cells by FACS analysis (CD4, CD8 staining). T cells were adjusted to 5×10 ⁵ cells per ml in assay medium.

1×10 ⁵ target cells were seeded per well of a 6-well plate and human cytotoxic T cells were added to an E:T ratio of 5:1. The final volume per well was 2 ml. Control samples containing effector cells and target cells contained target cells secreting no.25, chCLDN18.2ab parent mAB IgG and protein 1BiMAB as a positive control at a final concentration of 5 ng/ml. Controls included only electroporated target cells or only T cells with and without 1BiMAB protein. After 48 hours, T cells and target cells were harvested, labeled and analyzed by flow cytometry. Briefly, all cells were harvested by gentle scraping with Cell scrapers (Sarstedt AG & Co, Nurmbrecht, Germany) and transferred to 5 ml round bottom tubes (BD Falcon, Heidelberg, Germany). Cells were centrifuged and washed with DPBS. Cells were stained with mouse anti-human CD3-FITC, mouse anti-human CD69-APC, and mouse anti-human CD25-PE (all antibodies from BD Biosciences, Heidelberg, Germany). Cell pellets were resuspended in 50 µl of FACS buffer (DPBS supplemented with 5% FBS) containing antibodies conjugated to fluorescent labels. After incubation for 20 min at 4°C in the dark, the samples were washed with 4 ml of DPBS and the cell pellets were resuspended in 200 µl of FACS buffer containing propidium iodide (PI) (Sigma Aldrich, Germany) at a final dilution of 1:1000 to detect dead cells. Samples were kept on ice and in the dark until measured. Analysis was performed using a FACSCalibur instrument (BD Biosciences, Heidelberg, Germany). The results of the analysis were evaluated using FlowJo software (Tree Star, San carlos, CA, USA).

As shown in Figure 21A, each CLDN18.2-specific IVT mRNA caused secretion of the bi-scFv protein, as seen by efficient T cell activation. The strongest variants with only minor differences in T-cell activation were no.5 > no.8 > no.10 > no.3 > 1BiMAB in descending order. All of these variants resulted in an overall T cell activation of greater than 55%, while options no.2, no.4, no.7, and no.9 resulted in an overall T cell activation of 40 to 50%.

Specific lysis of target cells was calculated by the formula: % lysis = (% PI ⁺ target cells _sample - % PI ⁺ target cells _{EP reference} ), where "sample" means co-incubated effector cells and target cells, and "EP reference" only the electroporated target cells of each individual IVT-mRNA electroporation. As shown in Figure 21B, target cell lysis above 65% was achieved by 1BiMAB > no.5 > no.8 > no.3 variants in descending order. Other variants mediated the lysis of target cells, amounting to 55-64%. The most potent CLDN18.2-specific bi-scFv contains the V _H and V _L domains of TR66 (1BiMAB, no.5, no.10) or UCHT1 (no.3, no.8). With respect to domain orientations, no significant difference was observed in contrast to bi-scFv protein variants (see example 3). In accordance with protein studies, the IVT mRNA encoding 1BiMAB was selected for further studies.

The 18RHU5 and 18RHU3 constructs (see Tables 7 and 8) were compared at a later time point with 1BiMAB. The potency of 18RHU5 was equivalent to 1BiMAB, 18RHU3 was less potent (data not shown).

Example 17 Microscopic analysis of T cells redirected to target cells secreting CLDN18.2-specific 1BiMAB bi-scFv

The procedure for conducting the analysis was essentially the same as described in example 2.a.

Human cytotoxic T cells were separated using MACS from PBMC isolated from fresh blood using the CD8+ T Cell Isolation Kit, human (Miltenyi Biotec, Teterow, Germany) according to the manufacturer's instructions.

NugC4 target cells were prepared and transfected as described in Example 16, except that 80 μg/ml 1BiMAB IVT-mRNA or 80 μg/ml no.25 IVT-mRNA was used. Transfected target cells were counted and adjusted to 2×10 ⁵ cells per ml. 1×10 ⁴ target cells were seeded in each well of a 96-well plate and human cytotoxic T cells were added at an E:T ratio of 5:1. The final volume per well was 100 μl. Control samples contained only transfected target cells to confirm therapeutic properties after electroporation and control transfected bi-scFv target cells with effector cells. Tissue culture plates were then incubated at 37°C, 5% CO ₂ . Significant effects on T cell clustering on target cells, immunological synapse formation, and target cell death in samples containing 1BiMAB-transfected target cells were observed after 24 h and recorded using a Nikon Eclipse TS100 inverted microscope (Nikon, Japan) . No T cell clustering or target cell lysis was observed in the control sample with no.25 transfected target cells, indicating a strong dependence on TAA expression to induce T cell activation. See also figure 22.

Example 18: Flow Cytometry Analysis of Concentration-Dependent Activation of CLDN18.2 T Cells by Bi-scFv-Specific 1BiMAB

To study the concentration-dependent bi-scFv activation of T cells in the presence of TAA-expressing target cells, a three-fold dilution series was used for the transfection process.

NugC4 target cells were prepared and transfected as described in Example 16, but with a final concentration of IVT mRNA of 40 μg/ml. The concentrations of 1BiMAB IVT-mRNA varied from 0.4 to 40 μg/ml and were supplemented with appropriate amounts of luciferase IVT-mRNA in order to expose all cells to the same level of stress in relation to the amounts of IVT-mRNA.

1×10 ⁵ transfected target cells and human cytotoxic T cells (isolated as described in Example 16) were seeded at an E:T ratio of 10:1 in 2 ml assay medium in a 6-well format. Control samples containing target cells were transfected with 40 μg/ml of luciferase IVT-mRNA, but not 1BiMAB IVT-mRNA. After 24 hours and 48 hours of co-incubation of target and effector cells, cells were harvested, stained and analyzed as described in Example 16.

As shown in Figures 23 A and B, significant T cell activation was observed in samples containing target cells transfected with 4 μg/ml IVT-mRNA 1BiMAB. Maximal T cell activation in this assay was achieved with 40 μg/ml IVT-mRNA 1BiMAB. The expression of CD69 and CD25 differed depending on the time of incubation (figure 23 from A to B) and the concentration of IVT-mRNA. Higher amounts of 1BiMAB IVT-mRNA (12 and 40 μg/ml) resulted in more rapid initiation of the T cell activation mechanism, observed, for example, with increased CD25 expression compared to CD69 expression in Figure 23B. The total activation of T cells did not exceed ~40%.

Example 19: Flow Cytometry Analysis of Concentration-Dependent, T Cell-Mediated Target Cell Lysis with CLDN18.2-Specific 1BiMAB bi-scFv

The method described in Example 18 was used to study bi-scFv concentration-dependent T cell-mediated target cell lysis. . The latter served as reference samples ("EP Comparison") to subtract target cells killed by the electroporation process from target cells lysed with T cells.

Harvesting and staining were performed according to Example 18. Target cells were finally analyzed by injecting them with propidium iodide using a FACSCalibur (BD Biosciences, Heidelberg, Germany).

The percentage specific lysis of target cells was determined by two-step subtraction of the initial dead cells:

1. % T cell mediated lysis = (% PI ⁺ target cells _sample - % PI ⁺ target cells _{EP comparison} )

2. % specific lysis = (% T cell mediated lysis _sample - % T cell mediated lysis _control )

"T cell-mediated lysis _control " is subtracted from the difference in PI ⁺ target cells of controls (40 µg/ml luciferase IVT-mRNA only) with and without effector cells.

Using this calculation, a maximum specific lysis of 55.5% +/- 5.6% is achieved with 12 μg/ml IVT-mRNA 1BiMAB in this experiment. Due to the sensitivity of NugC4 target cells to electroporation-induced stress and the associated inevitable death of the original target cells that are subtracted, the specific target cell lysis noted in the graph may be lower under these experimental conditions than the actual percentage lysis.

Example 20: T cell proliferation in response to 1BiMAB IVT-mRNA transfection

T cell proliferation is a sign of T cell activation. Flow cytometry analysis was used to demonstrate T cell proliferation of bi-scFv encoding 1BiMAB IVT mRNA in the presence of CLDN18.2 positive target cells. Briefly, 1×10 ⁷ human T cells isolated as described in Example 16 were stained with 1 μM carboxyfluorescein diacetate succinimidyl ester (CellTrace CFSE, Invitrogen/Life Technologies GmbH, Germany) dissolved in DPBS under dark conditions at room temperature within 5 min.

Cells were washed twice with DPBS/5%FCS and resuspended in assay medium to 2×10 ⁶ cells per ml. Target cells were NugC4 cells lentivirally transduced with human CLDN18.2 and, for specificity testing, the CLDN18.2-negative breast cancer cell line MDA-MB-231. 20 μg/ml 1BiMAB IVT-mRNA or a non-targeted control was used for electroporation. Electroporation of NugC4 was performed as described in Example 16. Electroporation of MDA-MB-231 was performed using the following conditions: 400 V, pulse duration 3 ms, 1 pulse, pulse interval 400 ms in 0.4 cm Gene Pulser/MicroPulser Cuvettes ( Bio-Rad, dreieich, Germany). The cytotoxicity assay described in Example 16 was performed with transfected target cells and human CFSE-labeled T cells as effector cells. Only T cells and untransfected or control transfected target cells plus T cells were used as negative controls. Only T cells stimulated with 5 μg/ml OKT3 (Bio X Cell, West Lebanon, NH, USA) and 2 μg/ml anti-CD28 (BioLegend, Fell, Germany) served as positive controls. Protein 1BiMAB at a concentration of 5 ng/ml was applied to non-transfected target cells plus T cells to confirm the validity of the analysis. Samples fused with bi-scFv non-targeted protein 6PHU3 were included as a specificity control. All samples were prepared in triplicate in a total volume of 0.2 ml of assay medium in 96 wells. After 72 h of co-incubation, T cells were harvested, transferred to 5 ml round bottom tubes, washed and stained at 4°C for 30 min with 2 μl of anti-CD45-APC to differentiate human T cells from tumor cells, and 0. 25 µl eFluor506 (BD Biosciences, Heidelberg, Germany) to counterstain dead cells in 200 µl DPBS. After washing with DPBS, cells were resuspended in FACS buffer and analyzed with FACSCanto II (BD Biosciences, Heidelberg, Germany).

T cell proliferation was detected by a decrease in CFSE signal only in the presence of CLDN18.2 positive target cells and 1BiMAB bi-scFv (see also Figure 25). In addition to the positive control, T cell proliferation of 42-48% could be observed in the presence of CLDN18.2 positive target cells in combination with 1BiMAB protein. Target positive cells transfected with 1BiMAB IVT-mRNA resulted in 22% +/- 3%. The lower proliferation in response to IVT-mRNA than to protein may be due to the low transfection efficiency achieved by NugC4 target cells. T cells incubated with CLDN18.2-negative MDA-MB-231 target cells showed no significant proliferation under any condition, and T cells without target cells except for the positive control.

Example 21 Titration of Effector to Target Ratios to Determine Effective Ratio

To determine the appropriate E:T ratio for in vitro cytotoxicity analysis based on FACS analysis, E:T ratios ranging from 0.3:1 to 10:1 were selected in 3 steps.

NugC4 target cells were prepared and transfected as described in Example 16 with 40 μg/ml IVT mRNA. One preparation of transfected IVT-mRNA 1BiMAB cells was used for all tested samples. Transfection with 40 μg/ml luciferase IVT-mRNA was chosen as a negative control.

Human cytotoxic T cells were separated from PBMC isolated from fresh blood as described in Example 16 and served as effector cells. 1×10 ⁵ transfected target cells were co-incubated with cytotoxic T cells in 6-well plates in duplicate at the following ratios of effector to target: 0.3:1 - 1:1 - 3:1 - 10:1. In addition, human cytotoxic T cells were grown in the absence of target cells to determine initial T cell activation. Target cells transfected with control IVT mRNA or IVT mRNA 1BiMAB were also grown in the absence of effector cells to determine the original dead cells by subjecting to electroporation stress. Luciferase negative controls were seeded only at the maximum E:T ratio of 10:1. After 48 hours of co-incubation, cells were harvested, labeled and analyzed as described in Example 16.

Figure 26A shows the specific activation of cytotoxic T cells in response to 1BiMAB secretion by NugC4 target cells. Regardless of the number of T cells in the samples, an overall activation of 50-60% was found. In addition, the distribution of CD25 and CD69 expression in the samples is highly comparable. This indicates that there is a fixed percentage of T cells in the cytotoxic T cell population that can be activated.

In Figure 26B, the dependence of effective target cell lysis on the number of cytotoxic T cells becomes apparent. Even if target cells are lysed at an E:T ratio of only 0.3:1, active lysis begins at a ratio of 3:1.

Example 22: T Cell Activation and Target Cell Lysis Assay in a FACS Based Assay Using IVT-mRNA Transfected 1BiMAB Effector Cells

In this experiment, the aim was to test the ability of human effector cells to produce and secrete 1BiMAB after transfection with IVT mRNA. The rationale was a hypothetical patient scenario where the patient's own T cells could be transfected with bi-scFv and then retransferred into the patient's body.

Human cytotoxic T cells - isolated as described in Example 16 - were washed twice with X-Vivo 15 medium (LONZA, Basel, Switzerland) and resuspended to a density of 2×10 ⁷ cells/ml. 250 μl of the cell suspension was transferred into pre-chilled 0.4 cm Gene Pulser/MicroPulser Cuvettes (Bio-Rad, dreieich, Germany) and 80 or 240 μg/ml IVT-mRNA was added. The IVT mRNAs used were 1BiMAB and eGFP as controls. After gentle agitation, cells were electroporated using a BTX ECM 830 electroporator (Harvard Apparatus, Holliston, MA, USA) using the following conditions: 500 V, 1 pulse, pulse duration 3 ms, pulse interval 400 ms. Immediately after electroporation, the cuvettes were placed on ice for a short period of time and then the cell suspensions were transferred to the assay medium (RPMI 1640 medium supplemented with 5% heat-inactivated human AB serum, 0.5% penicillin-streptomycin, 1x NEAA, and 1 mM sodium pyruvate (Gibco /Life Nechnologies GmbH, Darmstadt, Germany) containing 10 U/ml IL-2. Transfected effector cells were grown overnight at 37°C, 5% CO ₂ . The next day, transfection efficiency was analyzed by FACS, showing transfection efficiency above 70% for both concentrations of eGFP IVT-mRNA. Each sample of effector cells was counted and adjusted to 5×10 ⁵ cells per ml. NugC4 target cells were harvested for analysis by trypsinization, washed with assay medium, counted and adjusted to 1 x 10 ⁵ cells per ml. Effector and target cells were mixed and plated in duplicate in 6-well plates in a final E:T ratio of 5:1 and a final volume of 2 ml. Raw T cells were seeded without target cells to determine the original activation signals. The analysis was carried out as described in example 16, after 48 h of co-incubation at 37°C, 5% CO ₂ .

Significant activation of cytotoxic T cells transfected with 1BiMAB IVT-mRNA and co-incubated with target cells was achieved as shown in Figure 27A. Lysis of target cells transfected with IVT-mRNA 1BiMAB T-cells was more than 60%, as shown in figure 27B. The effects of 80 μg/ml IVT-mRNA 1BiMAB could not be increased with higher amounts of RNA.

As a result of this experiment, it can be concluded that effector cells can theoretically be used as bi-scFv producing and secreting recipient cells.

Example 23: 1BiMAB IVT-mRNA target specificity study in a luciferase-based cytotoxicity assay using CLDN18.2-negative target cells.

Strict target specificity is an important issue to avoid unwanted side effects in patients. In this primary study, a CLDN18.2-negative cell line, the PA-1 teratocarcinoma cell line (ATCC CRL-1572), was used to investigate the non-specific cytolytic potential of 1BiMAB administered as IVT mRNA.

The PA-1 cell line used was stably transduced with a luciferase-based lentiviral vector and thus could be used in a luciferase-based cytotoxicity assay. PA-1/luc target cells were prepared for electroporation as described in Example 16. A total of 40 μg/ml IVT mRNA per sample was transfected. The following IVT mRNAs were used: 1BiMAB, no. 25 and 6RHU3. no. 25 targets non-expressed PLAC-1 TAA, and 6RHU3 targets highly expressed target CLDN6 in PA-1/luc cells. See Tables 7 and 8 for more details regarding bi-scFv variants. 25 was used as an addition to IVT-mRNA in samples for electroporation of 4 μg/ml IVT-mRNA to ensure the same level of stress caused by RNA transfection for all target cell samples. After gentle mixing, cells were transfected with a BTX ECM 830 electroporator (Harvard Apparatus, Holliston, MA, USA) using the following conditions for a 0.4 cm cuvette: 200 V, 2 pulses, pulse duration 12 ms, pulse interval 400 ms. Immediately after electroporation, the cuvettes were placed on ice for a short period of time, and then the cell suspensions were transferred to room temperature PA-1 assay medium (MEM medium supplemented with 10% heat-inactivated FCS, 0.5% penicillin-streptomycin, 1x NEAA, 1 .5 g/l sodium bicarbonate and 1 mM sodium pyruvate (Gibco/Life Technologies GmbH, Darmstadt, Germany)) in 15 ml tubes. Transfected target cells were counted and adjusted to 1 x 10 ⁵ cells per ml.

Human effector cells were isolated as described in Example 16. Human cytotoxic T cells were isolated by magnetically activated cell sorting (MACS) from PBMC using the Pan T Cell Isolation Kit II, human (Miltenyi Biotec, Tererow, Germany) according to the manufacturer's instructions . T cells were adjusted to 5×10 ⁵ cells per ml in PA-1 assay medium.

1×10 ⁴ target cells were seeded into each well of a 96-well plate and human cytotoxic T cells were added to an E:T ratio of 5:1. The final volume of each well was 100 μl. Control samples containing effector cells and target cells contained target cells secreting no. 25, as a positive control, the 6RHU3 or 6PHU3 protein, and the 1BiMAB protein. Protein concentrations were adjusted to a final concentration of 100 ng/ml and combined with no. 25-transfected target cells to ensure the same conditions for the target cells used. Minimal lysis controls (L _min ) included only each sample of electroporated target cells. Spontaneous lysis controls (L _max ) consisted of untreated target cells and effector cells (L _max ) to subtract from L _{test sample} , or untreated target cells only (L _max2 ) to subtract from L _min . Each sample was seeded in triplicate. The analysis was carried out after 72 h of incubation at 37°C, 5% CO ₂ . Spontaneous lysis controls (L _max ) were treated with Triton X-100 at a final concentration of 2%.

For the assay, 50 µl of an aqueous solution containing 1 mg/ml luciferin (BD Monolight, BD, Biosciences, Heidelberg, Germany) and 50 mM HEPES was added to each well and the plates were then incubated for 30 min in the dark at 37°C . The luminescence resulting from the oxidation of luciferin by luciferase expressing viable cells was measured on a microplate reader (Infinite M200, Tecan, Mannedorf, Switzerland). The percentage specific lysis of target cells was calculated using the following formula: % specific lysis = [1 - (luminescence _{of the test sample} - L _max ) / (L _min - L _max2 )] × 100.

The figure 28 shows the percentage of specific lysis of target cells. CLDN18.2 negative PA-1/luc cells transfected with negative control no. 25 or 1BiMAB did not show significant lysis even after 72 h incubation. Also, 100 ng/mL of 1BiMAB protein did not result in significant lysis, whereas lysis caused by bi-scFv secretion of 6RHU3 targeting TAA or 100 ng /ml protein 6RHU3, amounted to 85-93%. The analysis was also performed after 24 h and 48 h, which showed equivalent results (data not shown).

Example 24 Quantification of 1BiMAB Protein Production Following Transfection of Mammalian Cells with IVT-mRNA

To study the translation of RNA into protein in mammalian cells, the BHK21 cell line (ATCC CRL-13001) was chosen as the expression system. 2x10 ⁷ BHK21 cells per ml were transfected by electroporation as described in Example 16, with the difference that all steps were performed at room temperature. 250 μl of the cell suspension was transferred into 0.4 cm Gene Pulser/MicroPulser Cuvettes (Bio-rad, Dreiech, Germany) and 40 μg/ml IVT-mRNA 1BiMAB or IVT-replicon RNA was added. The electroporation conditions were as follows: 300 V, pulse duration 16 ms, 1 pulse, interval between pulses 400 ms.

Electroporated cells were resuspended in culture medium at room temperature (RPMI 1640, 10% FCS) and transferred to 15 cm culture dishes. 5 hours after inoculation, the medium containing FCS was replaced with medium not containing FCS. In Examples 24 a and b, the cell culture supernatant and cells were collected for analysis separately 18 hours after electroporation. Cell pellets were lysed in 1x LDS sample buffer (catalog number NP0008; Life technologies, Darmstadt, Germany) and heated at 72°C for 15 min. The unconcentrated and approximately 50-fold concentrated supernatant was used for ELISA analysis. Concentration was performed using an Amicon ultra-15 centrifuge filter (Merck Millipore, Billerica, MA, USA) according to the manufacturer's protocol. In the case of example 24c (IVT mRNA only), the supernatant was collected 48 hours after transfection and subjected to 40-fold concentration as described above.

A. ELISA using supernatant

For the ELISA assay, nickel coated plates (Thermo Fisher Scientific, Bonn, Germany) were used to capture the analyte through its His-tag. First, the plate was washed three times with 200 μl wash buffer (0.01% Tween-20 in 1x PBS) per well. As a standard, the purified 1BiMAB protein was diluted in 1.75% Na-Casein in 1 x PBS (= diluent). The dilution series varied from 2.34 to 37.50 ng/mL in increments of 2. 100 μl per standard dilution was transferred to the wells in triplicate of each concentration. Accordingly, 100 μl of samples were transferred in triplicate. The tablet was sealed with an adhesive film and incubated at 37°C for 30 minutes. Thereafter, the plates were washed three times with 200 μl wash buffer per well. For the detection of 1BiMAB, the anti-idiotypic monoclonal Ig 8B1F3 was used, which specifically binds to V _H -V _L of CLDN18.2ab and therefore also to 1BiMAB. 8B1F3 was mixed with diluent to a final concentration of 1 µg/mL. 100 μl of anti-idiotypic antibody solution was transferred to each well, followed by incubation for 30 min at 37°C. The plate was then washed 3 times with 200 µl of wash buffer per well, and 100 µl of AP-conjugated anti-mouse detection antibody (Jackson Immuno Research Laboratories, West Grove, PA, USA) - at a 1:500 dilution in diluent - was added to each well followed by incubation for 30 min at 37°C. After a final wash step (3 x 200 µl wash buffer), 1.5 mg/ml pNPP substrate in the appropriate substrate buffer (1M diethanolamine, 0.5 mM MgCl ₂ , 0.01% Na-azide, pH 9, 8) followed by incubation for 30 min at room temperature in the dark. To stop the enzymatic reaction, 100 μl of 3M KOH was used per well. Absorbance was measured using a microplate reader (Infinite M200, Tecan, Mannedorf, Switzerland). For two wavelength analysis, 405 nm was set as the measurement wavelength and 492 nm as the reference wavelength. Absorption values were calculated by subtracting the reference wavelength from the measurement wavelength.

FIG. 29A plots average absorbances at 405 nm including standard deviations. The concentrated supernatant from cells transfected with IVT mRNA 1BiMAB and the IVT replicon elicited a significant signal, confirming translation of the bi-scFv encoding the IVT RNA. The concentrated supernatant from the mock-transfected cells did not produce any signal. Actual protein concentrations cannot be suggested due to the differing toxicity of the IVT mRNA and IVT replicon constructs and the slightly different x-fold concentrates. The determined approximate protein concentration in the non-concentrated supernatant was in the range of 1.5 ng/ml for IVT-replicon samples and 2.4 ng/ml for IVT-mRNA samples.

b. Western blot analysis of supernatant and cell lysate (IVT mRNA and IVT replicon samples)

For Western blot analysis, concentrated supernatants and cell lysates were separated on NuPAGE Novex 4-12% Bis-tris Gels (Invitrogen/Life Technologies GmbH, Darmstadt, Germany). The supernatant and cell lysate of BHK21 cells transfected with 1BiMAB IVT-mRNA, IVT-replicon RNA, or untreated cells and positive control was loaded with 0.1 μg of purified 1BiMAB protein. Western blot analysis was performed using standard procedures (Current Protocols in Protein Science, 2012). Briefly, after proteins were blotted onto a PVDF membrane and blocked with PBST/3% milk powder, the membrane was incubated for 1 h at 4°C with Primary Anti-HIS Epitope-Tag (Dianova GmbH, Hamburg, Germany) at a dilution of 1: 500 in blocking buffer. After repeated washing with blocking buffer, the membranes were incubated with a peroxidase-conjugated Fc-specific secondary goat-anti-mouse IgG antibody (Sigma Aldrich, Germany) at a dilution of 1:10,000 in blocking buffer for 1 h at 4°C. After washing again with blocking buffer, signals were visualized with SuperSignal West Femto Chemiluminescent Substrate (Pierce/Thermo Fisher Scientific, Rockford, IL, USA) and recorded with an ImageQuant LAS 4000 Imager (GE Healthcare Life Sciences, Munich, Germany). 1BiMAB signals were detected in the range of 50 to 60 kDa compared to the molecular weight of the internal standard.

As shown in Figure 29B, weak signals were detected in the supernatant of cells transfected with IVT mRNA (lane 2) and IVT replicon RNA (lane 3), while no signal was detected in lane 4 (supernatant from untreated cells). Strong signals can be generated by cell lysates of cells transfected with IVT mRNA (lane 5) and IVT replicon RNA (lane 6). The signal was again absent in the cell lysate of untreated cells (lane 7). All signals occurred at the same height as the purified 1BiMAB control protein (lane 8). The weak signal in the supernatant and the weak secretion of 1BiMAB may be due to the relatively short incubation time after transfection. Due to the toxic effect of replicon RNA, longer incubation periods cannot be tested.

Both assays are qualitative and cannot be used to determine protein concentration.

With. Western blot analysis of the supernatant (IVT-mRNA samples)

The supernatant collected 48 hours after transfection was separated by SDS-PAGE followed by Western blot analysis as described in example 24b. As shown in Figure 29C, no.25 and 1BiMAB translated from IVT-mRNA and secreted into the supernatant were detected by their His-tag. In this case, the production and secretion of 1BiMAB, as well as no.25, which was used as a bi-scFv specificity control, can be confirmed.

Example 25: In vivo detection of translated and functional 1BiMAB protein after intramuscular RNA injection

Female and male NSG mice aged 8-16 weeks were selected and divided into 4 groups of 5 mice. Each mouse was injected with 40 μl of RNA solution into the thigh muscle. 40 µl RNA solution contained 1x PBS, 5 µg D2-capped IVT-mRNA 1BiMAB or replicon, 2 µg D1-capped IVT-mRNA luciferase, and 0 or 15 µg D1-capped IVT-mRNA EBK. EBK IVT mRNA encoding vaccinia virus E3L, B18R and K3L (EBK) proteins was co-injected to inhibit the IFN response and counteract PKR activation to enhance RNA translation (patent application PCT/EP2012/04673). The luciferase signal was observed 24 hours after Xenogen IVIS 2000 injection to exclude mice without signal from the sample collection.

Blood was collected 2, 4 and 7 days after injection. Serum was collected and then frozen at -80°C. Mice with a strong luminescent signal were excised and histochemically fixed for immunohistochemical analysis (IHC) or frozen by shock freezing for Western blot analysis 4 days after RNA injection.

Cytotoxicity assay

Serum from NSG mice was analyzed in an in vitro cytotoxicity assay. NugC4 target cells stably transfected with firefly luciferase and human CLDN18.2 for better target expression were seeded with human T cells (isolated as described in Example 16) at an E:T ratio of 30:1 for maximum sensitivity. The assay medium consisted of RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 0.5% penicillin-streptomycin, 1x NEAA, and 1 mM sodium pyruvate (Gibco/Life Technologies GmbH, Darmstadt, Germany). 20 μl of thawed test serum was added to each test sample well. Wells with standard control protein 1BiMAB, wells with L _min and L _max were filled with 20 μl of serum from untreated NSG mice. The final volume of each well was 100 μl. L _min was inoculated twelve times, L _max six times, and the test samples in triplicate. After 48 hours of incubation at 37° C. and 5% CO ₂ , wells with L _max were mixed with 10 μl of 2% Triton X-100 solution and incubated for 10 minutes. 10 μl of assay medium was added to all other wells. 50 μl of luciferin solution (see example 23) was added and the plates were measured - after a 30 min incubation step at 37° C. in the dark - on an Infinite M200 microplate reader (TECAN, Mannedorf, Switzerland). Calculation of specific lysis of target cells was performed as described in example 23.

The figure 30 plotted the percentage of specific lysis. Significant cytotoxic effects were detected in each group. Cytotoxic effects increased by a factor of 1.7 in wells containing sera from mice injected with EBK and IVT-mRNA 1BiMAB and were collected for analysis 2 days after injection. Significantly lower effects were observed in samples collected 4 or 7 days after injection. In the case of 1BiMAB replicon samples, serum collected at later time points also generated strong cytolytic effects.

These data support in vivo translation of 1BiMAB from IVT-mRNA or β-replicon and secretion into the circulation after intramuscular injection.

Example 26 Generation and Testing of Bispecific Binding Agents Targeting CLDN6 and CD3

A. Initial sequences, design of the bi-scFv construct, and cloning into a vector matrix

Bispecific tandem single chain antibody (bi-scFv) constructs were prepared containing binding domains specific for the CD3 component of the human T-cell receptor and human tumor-associated antigens (TAA). The respective heavy chain variable regions (V _H ) and the respective light chain variable regions (V _L ) for each construct are located specifically in the 5' to 3' direction in the following order:

pST1-5'AgKozak-V _H ^αCLDN6 -V _L ^αCLDN6 -V _H ^αCD3 - V _L ^αCD3 -His-2hBgUTR-A120 (6RHU5)

pST1-5'AgKozak-V _H ^αCD3 - V _L ^αCD3 - V _H ^αCLDN6 -V _L ^αCLDN6 - His-2hBgUTR-A120 (6RHU3)

Table 9 summarizes all bi-scFv constructs specific for TAA CLDN6 that have been generated within the scope of the invention. The CLDN18.2-specific 1BiMAB bi-scFv construct was used as a control antibody. The bi-scFv constructs were generated by gene synthesis from GeneArt AG (GeneArt/Life Technologies GmbH, Regensburg, Germany) using the V _H and V _L sequences of the respective antibodies. Codon optimizations such as Homo sapiens (HS) or Mus musculus (MM) were implemented using GeneArt's GeneOptimizer® software and are listed in Table 9. Information regarding specificity, sequence origin from monoclonal antibodies (mAB), codon usage, additional distinguishing sequence features and references to all _applicable domains are summarized in Table _10. The original sequences of the variable domains of the respective CD3 antibodies are listed in Table 10. bi-scFv constructs for murine assays, but in combination with the V _H , V _L sequences of the mouse specific anti-CD3 antibody clone 145-2C11.

DNA cloning and construction of expression vectors were performed according to standard procedures (Green/Sambrook, Molecular Cloning, 2012), which are well known to those skilled in the art. Briefly, the major DNA sequences of bi-scFv contained the restriction site 5'-BsmBI and 3'-XhoI for cloning into pST1 plasmids. The secretion signal sequence was introduced at the 5' end (upper) of the bi-scFv sequence for protein secretion. A sequence encoding a flexible glycine-serine peptide linker of 15-18 amino acids was inserted to connect the V _H and V _L domains for the composition of single chain variable antibody fragments (scFv), one of which binds to CD3 and the other to TAA. To form a bispecific single chain antibody, the sequences of the two scFv domains were joined by a sequence encoding a short peptide linker (GGGGS). Along with this linker sequence, a BamHI restriction site was introduced to exchange scFv domains for cloning subsequent bi-scF _V constructs. Briefly, the 5'scFv- domains were replaced by restriction with BsmBI and BamHI and the 3'scFv-domains by restriction with BamHI and XhoI. The C-terminal 6xHis tag was used for assays for the detection of translated protein. To obtain the 6RHU3 replicon vector, the entire 6RHU3 sequence, including the secretion signal and 6xHis tag, was subcloned 3' from the subgenomic promoter of the Semliki Forest Virus (pSFV) replicon vector, courtesy of K. Lundstrom (Lundstrom, K. et al. (2001) ) Histochem Cell Biol 115 (1), pp. 83-91; Ehrengruber, MU et al (1999) Proc. Natl Acad. Sci. USA 96 (12), pp. 7041-7046).

All constructs were verified by sequencing through the MWG single read sequence service (Eurofins MWG Operon, Ebersberg, Germany) and only constructs with the correct sequence and a poly(A) tail consisting of more than 100 adenines were used for in vitro transcription of RNA.

Structural diagrams are also shown in Figure 31A.

Table 9. Brief description of mRNA template constructs for a bispecific single chain antibody specific for TAA and CD3

Internal name TAA specific
ness 5'- _VH - _VL 3'- _VH - _VL Codon usage 1BiMAB CLDN18.2 Human mCLDN18.2ab TR66 HS 6RHU5 CLDN6 Human mCLDN6ab TR66 HS 6RHU3 CLDN6 Human TR66 mCLDN6ab HS 6RMU5 CLDN6 mouse mCLDN6ab 145-2C11 MM 6RMU3 CLDN6 mouse 145-2C11 mCLDN6ab MM

bi-scFv is a bispecific single chain variable fragment; HS, Homo sapiens; HU, humanized; MM, Mus musculus; TAA, tumor-associated antigen; V _H , heavy chain variable domain, V _L , light chain variable domain.

Table 10 Brief description of the bi-scFv mRNA template design

Binding fragment of CD3 TAA binding fragment Int. name Origin mAB Reactive view TAA Origin.mAB Reactive view 5'- _VH - _VL 3'- _VH - _VL Short linker 1BiMAB TR66 Human CLDN18.2 mCLDN18.2ab man, mouse mCLDN18.2ab TR66 GGGGS 6RHU5 TR66 Human CLDN6 mCLDN6ab man, mouse mCLDN6ab TR66 SGGGGS 6RHU3 TR66 Human CLDN6 mCLDN6ab man, mouse TR66 mCLDN6ab SGGGGS 6RMU5 145-2C11 mouse CLDN6 mCLDN6ab man, mouse mCLDN6ab 145-2C11 SGGGGS 6RMU3 145-2C11 mouse CLDN6 mCLDN6ab man, mouse 145-2C11 mCLDN6ab SGGGGS Continuation
Table 10 Int. name 5' long linker 3'-long linker secretion signal codon usage link on anti-CD3
mAB 1BiMAB (GGGGS) ₃ VE(GGSGGS) ₂ GGVD MGWSCIILFLVATATGVHS HS Lanzavecchia & Scheidegger, Eur J Immunol 1987 6RHU5 (GGGGS) ₃ VE(GGSGGS) ₂ GGVD MGWSCIILFLVATATGVHS HS Lanzavecchia & Scheidegger, Eur J Immunol 1987 6RHU3 VE(GGSGGS) ₂ GGVD (GGGGS) ₃ MGWSCIILFLVATATGVHS HS Lanzavecchia & Scheidegger, Eur J Immunol 1987 6RMU5 (GGGGS) ₃ VE(GGSGGS) ₂ GGVD MGWSCIILFLVATATGVHS MM Leo et al., Proc Natl Acad Sci, 1987 6RMU3 VE(GGSGGS) ₂ GGVD (GGGGS) ₃ MNSGLQLVFFVLTLKGIQG MM Leo et al., Proc Natl Acad Sci, 1987

HS, Homo sapience; mAB, monoclonal antibody; MM, Mus musculus; TAA, tumor-associated antigen.

b. Synthesis of IVT-RNA

To generate anti-CLDN6-specific bi-scFv IVT templates, plasmids were linearized downstream of the poly(A) tail using class IIs endonuclease. The linearized DNA template was purified by phenol/chloroform extraction and precipitated with sodium acetate as described in the literature (Holtkamp, S. et al. (2006) Blood 108 (13), pp. 4009-4017).

Linearized DNA templates were subjected to in vitro transcription using MEGAscript Kits (Ambion/Life Technologies, Darmstadt, Germany) according to the manufacturer's instructions: pST1 templates were transcribed using the MEGAscript T7 Kit and pSFV template using the MEGAscript SP6 Kit. For cap analog reactions, the GTP concentration was reduced to 1.5 mM and 6 mM ARCA, beta-S-ARCA(D1) or beta-S-ARCA(D2) synthesized as described in the literature was added to the reaction (Kowalska, J et al (2008) RNA 14 (6), pp. 1119-1131 Grudzien, E. et al (2004) RNA 10 (9), pp. 1479-1487 Stepinski, J. et al (2001 ) RNA 7 (10), pp. 1486-1495). Purification of IVT-mRNA was performed using the MEGAclear Kit (Ambion/Life Technologies, Darmstadt, Germany) according to the manual. The concentration and quality of IVT-RNA was assessed by spectrophotometry and analyzed on a bioanalyzer 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).

Example 27: Microscopic analysis of T cells redirected to target cells secreting CLDN6-specific bi-scFv 1BiMAB

The procedure for conducting the analysis was essentially the same as described in example 2.a.

The target cell line was a subclone of the ovarian teratocarcinoma cell line PA-1 (ATCC CRL-1572), which endogenously expresses high levels of human CLDN6. Human cytotoxic T cells were separated using MACS from PBMC isolated from fresh blood using the Pan T Cell Isolation Kit, human (Miltenyi Biotec, Teterow, Germany) according to the manufacturer's instructions.

PA-1 target cells - which were tested in the usual manner for TAA expression by FACS analysis before each assay - were washed twice with ice-cold X-Vivo 15 medium (LONZA, Basel, Switzerland) and resuspended to a density of 2×10 ⁷ cells/ml . 250 μl of the cell suspension was transferred into pre-chilled 0.4 cm Gene Pulser/MicroPulser Cuvettes (Bio-Rad, Dreieich, Germany) and 20 μg/ml IVT-mRNA was added. Conditions for a BTX ECM 830 electroporator (Harvard apparatus, Holliston, MA, USA) were as follows: 200 V, pulse length 12 ms, 2 pulses, pulse interval 400 ms. The following IVT mRNAs were used: 6RHU5, 6RHU3, no. 25 (see tables 9 and 10 for details). Transfected target cells were counted and adjusted to 1×10 ⁵ cells per ml. 1×10 ⁵ target cells were seeded in each well of a 6-well plate, and human cytotoxic T cells were added in accordance with the E:T ratio of 5:1. The final volume of each well was 2 ml. Control samples contained only transfected target cells to confirm therapeutic properties after electroporation and control transfected bi-scFv target cells with effector cells. As positive controls, the corresponding CLDN6-specific 6RHU5 and 6RHU3 bi-scFv proteins were prepared at a concentration of 50 μg/ml. Thus, untreated PA-1 cells with human T cells were used. The tissue culture plates were then incubated at 37° C., 5% CO ₂ . Significant effects in terms of T cell cluster formation on target cells, immunological synapse formation, and target cell death in samples containing 6RHU5 and 6RHU3 transfected target cells were observed after 24 h and recorded using a Nikon Eclipse TS100 inverted microscope (Nikon , Japan). As shown in Figure 32, no T cell clustering or target cell lysis was observed in the no. 25 transfected target cells, indicating a strong dependence on TAA expression to induce T cell activation. The mimic control without bi-scFv also shows no T-cell clustering. Control proteins, on the contrary, lead to the formation of T-cell clusters and the lysis of target cells.

Example 28 Activation of T Cells Induced by 6RHU5 and 6RHU3 Bi-scFv Candidates Targeting CLDN6

To detect T cell activation and determine the differences in potency between the two CLDN6-specific bi-scFv variants, a FACS-based T cell activation assay was performed. The early activation marker CD69 and the late activation marker CD25 were chosen for staining with antibodies conjugated to a fluorescent label. To detect human T cells in a mixture of target cells and T cells, they were stained with CD3, which is expressed by all T cells.

Target cells and effector cells were prepared as described above (example 27). Briefly, PA-1 target cells endogenously expressing CLDN6 were transfected by electroporation with 20 μg/ml of the following IVT mRNAs: 6RHU5, 6RHU3 and no. 25. No.25 targeting unexpressed TAA was used as a specificity control, untreated target cells were used as a mime control. 50 ng/ml 6RHU5 protein was used as a positive control. In addition, T cells were seeded without target cells with or without 6RHU5 protein as reference initial activations. Each sample was seeded in duplicate in 6-well plates and incubated at 37°C, 5% CO ₂ . After 24 hours and 48 hours, T cells were collected by scraping and transferred to 5 ml round bottom tubes (BD Falcon, Heidelberg, Germany). Cells were centrifuged and washed with DPBS. Cells were stained with mouse anti-human CD3-FITC, mouse anti-human CD69-APC, and mouse anti-human CD25-PE (all antibodies from BD Biosciences, Heidelberg, Germany). Cell pellets were resuspended in 50 µl of FACS buffer (DPBS supplemented with 5% FBS) containing antibodies conjugated to fluorescent labels and 2 µl of 7-AAD (BD Biosciences, Heidelberg, Germany). After incubation for 20 min at 4°C in the dark, the samples were washed with 4 ml of DPBS and the cell pellets were resuspended in 200 μl of FACS buffer. Samples were kept on ice and in the dark while measured with a FACSCanto II flow cytometer (both from BD Biosciences, Heidelberg, Germany). Results were evaluated using FlowJo software (Tree Star, San Carlos, CA, USA).

As shown in Figure 33A and B, both variants resulted in T cell activation, while none of the negative controls showed significant expression of the CD69 and CD25 markers of T cell activation.

Overall T cell activation in response to 6RHU3 was 1.53-fold higher at 24 h (A) and 1.35-fold higher at 48 h (B) of co-incubation compared to the response to 6RHU5. Based on these findings, all subsequent studies were performed with the 6RHU3 variant only.

Example 29: Concentration-Dependent Activation of T Cells by Co-Incubation with 6RHU3 Transfected Target Cells

To determine the lowest concentration of 6RHU3 IVT mRNA required to induce target-dependent T cell activation, a series of dilutions in the range of 0.2-20 μg/ml of IVT mRNA were transfected into PA-1 target cells. To ensure that all samples were subjected to the same level of RNA electroporation-induced stress, IVT-mRNA was transfected at a total concentration of 20 μg/ml. No.25 - targeting unexpressed TAA - was used as a filler for the IVT mRNA. Accordingly, 0 μg/ml 6RHU3 correlates with 20 μg/ml no.25. Electroporation was performed as described in Example 27. Human T cells were used as effector cells. PBMC isolation was performed according to the manufacturer's instructions (Pan T Cell Isolation Kit II, Miltenyi, Teterow, Germany). Effector cells and target cells were mixed in a ratio of 5:1. Untreated target cells with T cells served as a sham control. T cells were then seeded without target cells with or without 6RHU5 protein as reference initial activations. As a positive control, untreated target cells were mixed with T cells and 50 ng/ml 6RHU5 protein. All samples were prepared in duplicate in 6-well plates.

Target cells and effector cells were co-incubated for 48 h at 37°C, 5% CO ₂ . Samples were prepared for analysis by flow cytometry as described in Example 28.

Figure 34 plots the percentage of CD3 positive T cells that express activation markers. T cell activation was not observed in the controls. The onset of significant T cell activation was detected at a concentration of 0.7 μg/ml of 6RHU3 IVT-mRNA. Effects in response to 2.0 μg/ml 6RHU3 IVT mRNA were comparable to those mediated by 50 ng/ml control 6RHU5 protein. Thus, protein translation and secretion from transfected IVT mRNA appears to be an efficient process.

Example 30: Definition of EC ₅₀ for 6RHU3

To determine half the maximum effective dose of bi-scfv encoding 6RHU3 IVT mRNA, a series of 6RHU3 titrations were tested in an in vitro luciferase-based cytotoxicity assay. Luciferase-stably-expressing PA-1 cells were transiently transfected by electroporation as described in Example 27. The concentrations of 6RHU3 IVT-mRNA used ranged from 0.004-13.3 μg/mL in 6-step dilutions. The total concentration of IVT-mRNA was constant and amounted to 13.3 µg/ml, no. 25 was used as filling IVT-mRNA. As controls for minimal lysis (L _min ), all samples of transfected target cells were seeded without effector cells. By this procedure, the initially dead cells of each individual electroporation sample are subtracted and only the effects of T-cell mediated lysis are obtained.

Transfected target cells were plated with human T cells at a 5:1 effector to target ratio in triplicate in a 96-well format and incubated at 37° C., 5% CO ₂ . Maximum lysis (Lmax) to normalize to spontaneous luminescence values was achieved by adding Triton X-100 to control wells containing effector cells and untreated target cells immediately prior to the addition of luciferin. After adding the luciferin solution - to each well 50 µl of an aqueous solution containing 1 mg/ml luciferin (BD Monolight, BD Biosciences, Heidelberg, Germany) and 50 mM HEPES - luminescence was measured on an Infinite M200 Tecan microplate reader after 24 h and 48 h. Specific lysis of target cells was calculated by the formula: % specific lysis = [1-(luminescence _{test sample} - L _max ) / (L _min _ _{test sample} - L _max )] × 100.

Figure 35 shows a concentration-dependence curve of specific target cell lysis in response to 6RHU3. Using the GraphPad Prism equation to calculate EC ₅₀ values "log(agonist) vs. response - variable slope" revealed EC ₅₀ (24 h) = 548.0 ng/mL and EC ₅₀ (48 h) = 194.5 ng/mL . The result of this assay depends to a large extent on the strength of human T cells, which varies according to the immune status of the donor, as also described elsewhere (see e.g. (Lutterbuese, R. et al. (2010) Proc. Natl. Acad. Sci USA 107 (28), pp. 12605-12610).

Example 31 T cell proliferation in response to 6RHU3 IVT-mRNA transfection

T cell proliferation is a sign of T cell activation. Flow cytometry analysis was used to demonstrate specific T cell proliferation in response to bi-scFv encoding IVT mRNA 1BiMAB in the presence of CLDN6 positive target cells. Briefly, 1×10 ⁷ human T cells isolated as described in Example 16 were stained with 1 μM carboxyfluorescein diacetate succinimidyl ester (CellTrace CFSE, Invitrogen/Life Technologies GmbH, Germany) dissolved in DPBS under dark conditions at room temperature within 5 min. Cells were washed twice with DPBS/5%FCS and resuspended in assay medium to 2×10 ⁶ cells per ml. PA-1 cells endogenously expressing CLDN6 and, for specificity testing, the CLDN6-negative breast cancer cell line MDA-MB-231 were selected as target cells. 20 μg/ml 6RHU3 IVT-mRNA or a non-targeted control was used for electroporation. PA-1 electroporation was performed as described in Example 27. MDA-MB-231 electroporation was performed using the following conditions: 400 V, 3 ms pulse duration, 1 pulse, 400 ms interval between pulses in 0.4 cm Gene Pulser/MicroPulser cuvettes Cuvettes (Bio-Rad, dreieich, Germany). The cytotoxicity assay described in Example 27 was performed with transfected target cells and human CFSE-labeled T cells as effector cells. Only T cells and untransfected or control transfected target cells plus T cells were used as negative controls. Only T cells stimulated with 5 μg/ml OKT3 (Bio X Cell, West Lebanon, NH, USA) and 2 μg/ml anti-CD28 (BioLegend, Fell, Germany) served as positive controls. Protein 6PHU3 at a concentration of 5 ng/ml was applied to non-transfected target cells plus T cells to confirm the validity of the analysis. Samples combined with the non-targeted 1BiMAB protein were included as a specificity control. All samples were prepared in triplicate in a total volume of 0.2 ml of assay medium in 96-well plates. After 72 h of co-incubation, T cells were harvested, transferred to 5 ml round bottom tubes, washed and stained at 4°C for 30 min with 2 μl of anti-CD45-APC to differentiate human T cells from tumor cells, and 0. 25 µl eFluor506 (BD Biosciences, Heidelberg, Germany) to counterstain dead cells in 200 µl DPBS. After washing with DPBS, cells were resuspended in FACS buffer and analyzed with FACSCanto II (BD Biosciences, Heidelberg, Germany).

T cell proliferation was detected by a decrease in CFSE signal only in the presence of CLDN6-positive target cells and anti-CLDN6-specific bi-scFv (see also Figure 36). In addition to the positive control, T cell proliferation of 49-61% could be observed in the presence of CLDN6 positive target cells in combination with the 6PHU3 protein. Target positive cells transfected with 6PHU3 IVT-mRNA resulted in 62% +/- 2%. T cells incubated with CLDN6-negative MDA-MB-231 target cells showed no significant proliferation under any conditions, and T cells without target cells except for the positive control.

Example 32 Quantification of protein production after transfection of 6PHU3 IVT-mRNA in mammalian cells

To study the translation of RNA into protein in mammalian cells, the BHK21 cell line (ATCC CRL-13001) was chosen as an expression system. 2×10 ⁷ BHK cells per ml were transfected by electroporation as described in example 16, with the difference that all steps were carried out at room temperature. 250 μl of the cell suspension was transferred into 0.4 cm Gene Pulser/MicroPulser Cuvettes (Bio-Rad, dreieich, Germany) and 40 μg/ml no.25 IVT mRNA, 6PHU3 IVT mRNA or 6PHU3 IVT replicon RNA was added. Electroporation was performed using the following conditions: 300 V, pulse duration 16 ms, 1 pulse, interval between pulses 400 ms.

Electroporated cells were resuspended in culture medium at room temperature (RPMI1640, 10% FCS) and transferred to 15 cm culture dishes. 5 hours after inoculation, the medium containing FCS was replaced with medium not containing FCS. For examples 32 a and b, cell culture supernatant and cells were collected separately 18 hours after electroporation. Cell pellets were lysed in 1x LDS sample buffer (catalog number NP0008; Life technologies, Darmstadt, Germany) and heated at 72°C for 15 min. The unconcentrated and approximately 50-fold concentrated supernatant was used for ELISA analysis. Concentration was performed using Amicon ultra-15 centrifuge filters (Merck Millipore, Billerica, MA, USA) according to the manufacturer's protocol. For example 32c (IVT mRNA only), the supernatant was collected 48 hours after transfection and subjected to 40-fold concentration as described above.

A. ELISA using supernatant

For the ELISA assay, nickel coated plates (Thermo Fisher Scientific, Bonn, Germany) were used to capture the analyte through its His-tag. First, the plate was washed three times with 200 μl wash buffer (0.01% Tween-20 in 1x PBS) per well. As a standard, the purified 6PHU3 protein was diluted in 1.75% Na-Casein in 1x PBS (= diluent). The dilution range varied from 2.34 to 150 ng/mL in steps of 2. 100 µl per standard dilution was transferred to wells in triplicate of each concentration. Accordingly, 100 μl of samples were transferred in triplicate. The tablet was sealed with an adhesive film and incubated at 37°C for 30 minutes. Thereafter, the plates were washed three times with 200 μl wash buffer per well. For the detection of 6PHU3/6RHU3, the anti-idiotypic monoclonal IgG 4F9 was used, which specifically binds to V _H -V _L of CLDN6ab and therefore also to 6PHU3/6RHU3. 4F9 was mixed with diluent to a final concentration of 2.5 μg/ml. 100 μl of anti-idiotypic antibody solution was transferred to each well, followed by incubation for 30 min at 37°C. The plate was then washed 3 times with 200 µl of wash buffer per well, and 100 µl of AP-conjugated anti-mouse detection antibody (Jackson Immuno Research Laboratories, West Grove, PA, USA) - at a 1:500 dilution in diluent - was added to each well followed by incubation for 30 min at 37°C. After a final wash step (3 x 200 µl wash buffer), 1.5 mg/ml pNPP substrate in the appropriate substrate buffer (1M diethanolamine, 0.5 mM MgCl ₂ , 0.01% Na-azide, pH 9, 8) followed by incubation for 30 min at room temperature in the dark. To stop the enzymatic reaction, 100 μl of 3M KOH was used per well. Absorbance was measured using a microplate reader (Infinite M200, Tecan, Mannedorf, Switzerland). For two wavelength analysis, 405 nm was set as the measurement wavelength and 492 nm as the reference wavelength. Absorption values were calculated by subtracting the reference wavelength from the measurement wavelength.

FIG. 37A plots average absorbances including standard deviations. The concentrated supernatant from cells transfected with 6RHU3 IVT mRNA and the IVT replicon elicited a significant signal, confirming translation of the bi-scFv-coding IVT RNA. The concentrated supernatant from mock-transfected cells and control no.25 (-con) did not produce any signal as expected. Actual protein concentrations cannot be predicted due to the differing toxicity of the IVT mRNA and IVT replicon constructs and the slightly different x-fold concentrates. The determined approximate protein concentration in the unconcentrated supernatant was in the range of 1.4 ng/ml for IVT replicon samples and 5.9 ng/ml for IVT mRNA samples.

b. Western blot analysis of supernatant and cell lysates (IVT-mRNA and IVT-replicon samples)

For Western blot analysis, concentrated supernatants and cell lysates were separated on NuPAGE Novex 4-12% Bis-tris Gels (Invitrogen/Life Technologies GmbH, Darmstadt, Germany). The supernatant and cell lysate of BHK21 cells transfected with 6RHU3 IVT-mRNA, IVT-replicon RNA, no.25 IVT-mRNA or untreated cells and positive control was loaded with 0.1 μg of purified 6RHU3 protein. Western blot analysis was performed using standard procedures (Current Protocols in Protein Science, 2012). Briefly, after proteins were blotted onto a PVDF membrane and blocked with PBST/3% milk powder, the membrane was incubated for 1 h at 4°C with Primary Anti-HIS Epitope-Tag (Dianova GmbH, Hamburg, Germany) at a dilution of 1: 500 in blocking buffer. After repeated washing with blocking buffer, the membranes were incubated with a peroxidase-conjugated Fc-specific secondary goat-anti-mouse IgG antibody (Sigma Aldrich, Germany) at a dilution of 1:10,000 in blocking buffer for 1 h at 4°C. After washing again with blocking buffer, signals were visualized with SuperSignal West Femto Chemiluminescent Substrate (Pierce/Thermo Fisher Scientific, Rockford, IL, USA) and recorded with an ImageQuant LAS 4000 Imager (GE Healthcare Life Sciences, Munich, Germany). 6RHU3 signals were detected in the range of 50 to 60 kDa compared to the molecular weight of the internal standard.

As shown in Figure 37B, signals were detected in supernatants of cells transfected with no.25 IVT mRNA (lane 1), 6RHU3 IVT mRNA (lane 4), and 6RHU3 IVT replicon RNA (lane 5), while no signal was present in lane 6 - supernatant from untreated cells. Strong signals can be generated by cell lysates of cells transfected with 6RHU3 IVT mRNA (lane 8) and IVT replicon RNA (lane 9). Cell lysate of cells transfected with no.25 resulted in a very weak signal (lane 2), untreated cells (lane 10) showed no signal. The purified 6RHU3 control protein was detectable (lane 11). The relatively weak signals in the supernatant and, at the same time, the weak secretion of 6RHU3 may be due to the relatively short incubation period after transfection. Due to the toxic effect of replicon RNA, longer incubation periods cannot be tested.

Both assays are qualitative and cannot be used to determine protein concentration.

With. Western blot analysis of the supernatant (IVT-mRNA samples)

Supernatant collected 48 hours after transfection was separated by SDS-PAGE followed by Western blot analysis as described in example 32b. As shown in Figure 27C, no.25 and 6RHU3 translated from IVT-mRNA and secreted into the supernatant were detected by their His-tag. This can be confirmed production and secretion of 6RHU3, as well as no.25, which was used as a control for the specificity of bi-scFv.

Example 33: In vivo detection of translated and functional CLDN6-specific bi-scFv protein after intramuscular RNA injection

Female and male NSG mice aged 8-16 weeks were selected and divided into 4 groups of 5 mice. Each mouse was injected with 40 μl of RNA solution into the thigh muscle. 40 µl RNA solution contained 1x PBS, 5 µg D2-capped 6RHU3 IVT-mRNA or replicon, 2 µg D1-capped luciferase IVT-mRNA, and 0 or 15 µg D1-capped EBK IVT-mRNA. EBK IVT mRNA encoding vaccinia virus E3L, B18R and K3L (EBK) proteins was co-injected to inhibit the IFN response and counteract PKR activation to enhance RNA translation (patent application PCT/EP2012/04673). The luciferase signal was observed 24 hours after Xenogen IVIS 2000 injection to exclude mice without signal from the sample collection.

Blood was collected 2, 4 and 7 days after injection. Serum was collected and then frozen at -80°C.

Cytotoxicity assay

Serum from NSG mice was analyzed in an in vitro cytotoxicity assay. PA-1 target cells stably transduced with firefly luciferase and endogenously expressed CLDN6 were seeded with human T cells (isolated as described in Example 16) at an E:T ratio of 30:1 for maximum sensitivity. The assay medium consisted of RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 0.5% penicillin-streptomycin, 1x NEAA, and 1 mM sodium pyruvate (Gibco/Life Technologies GmbH, Darmstadt, Germany). 20 μl of thawed test serum was added to each test sample well. Wells with standard control protein 6PHU3, wells with L _min and L _max were filled with 20 μl of serum from untreated NSG mice. The final volume per well was 100 μl. L _min was inoculated twelve times, L _max six times, and the test samples in triplicate. After 48 hours of incubation at 37° C. and 5% CO ₂ , L _max wells were mixed with 10 μl of 2% Triton X-100 solution and incubated for 10 minutes. 10 μl of assay medium was added to all other wells. 50 μl of luciferin solution (see example 23) was added and the plates were measured - after a 30 min incubation step at 37° C. in the dark - on an Infinite M200 microplate reader (TECAN, Mannedorf, Switzerland). Calculation of specific lysis of target cells was performed as described in example 23.

The figure 28 shows the percentage of specific lysis. Significant cytotoxic effects were detected in each group. The concentration of CLDN6-specific protein bi-scFv was significantly increased by co-injection with EBK in the case of 6RHU3 IVT mRNA.

These data support in vivo translation of 6RHU3 from IVT-mRNA or β-replicon and secretion into the circulation after intramuscular injection.

Example 34 Generation and Testing of Bispecific Binding Agents Targeting CLDN18.2 or CLDN6 and CD3

For further development of anti-CLDN18.2 and anti-CLDN6 specific bi-scFv antibody fragments, other aspects were considered to optimize the original protein. These aspects are mainly aimed at obtaining preparations with higher homogeneity in relation to the various types of folding, disulfide isomers and oligomers that can be formed during recombinant production of the protein. These modifications should not affect the functional activity of the bi-scFv proteins.

Substitution of an additional (unpaired) cysteine residue in the anti-CD3 binding domain of bi-scFv proteins.

Several synthetic constructs were generated to investigate the ability of unpaired cysteine residues arising within the primary sequence of the Ig domain to prevent the correct formation of intra- and/or inter-chain disulfide bonds that are important for proper folding and stability of the resulting antibody fragment. Such unpaired cysteines can adversely affect the potency, uniformity, productivity and stability of the final protein product and should therefore be avoided. In addition to the "standard" set of cysteines involved in disulfide bond pairing, free cysteine residues may be present in variable domains.

For example, in the VH domain of the OKT3 antibody, three conserved cysteine residues are present at position H92 before the onset of CDR-H3 and form a structural disulfide bond to position H22. But in this molecule at position H100A (CDR-H3), another cysteine (Cys) can provide misfolding, in which H100A instead of H92 is involved in the formation of a disulfide bond with H22, thus generating a misfolded, insoluble and non-functional product. To overcome this possible mismatch of cysteine residues, site-directed substitution of free cysteine was performed (Kipriyanov, Protein Engineering 10:445-453, 1997). With this single substitution, a significant increase in productivity and stability of OKT3-derived scFv was achieved while maintaining overall binding activity.

The VH domain of the anti-CD3 antibody TR66 (SEQ ID NO: 36) used in the present study contains such a free cysteine at position H103 of the primary sequence as shown in SEQ ID NO: 36. Sequence comparison of the VH domain of the anti-CD3 antibody TR66 (SEQ ID NO: 36) with the VH domain of the anti-CD3 antibody OKT3 showed a sequence homology of 96.6%.

According to these results, substitution of free cysteine with a serine residue within the CDR-H3 domain of the VH of the anti-CD3 antibody TR66 was performed for CD3-targeting bi-scFv proteins (SEQ ID NO: 94) and either for CLDN18.2 or CLDN6. The introduction of these substituents allowed the construction of bi-scFv proteins 1-BiMAB-S (SEQ ID NO: 103) and 6-PHU3-S (SEQ ID NO: 110) (see tables 11 and 12, respectively).

Substitution of additional (unpaired) cysteine residues in bi-scFv anti-CLDN6 proteins

The parental anti-CLDN6 antibody mCLDN6ab, whose variable domains were used to assemble the respective bi-scFv proteins 6PHU3 (SEQ ID NO: 45) and 6PHU5 (SEQ ID NO: 43), contains an unpaired cysteine residue within the flanked CDR-L2 region of the L2 domain in position 46 of the corresponding primary sequence. This corresponds to position 45 within SEQ ID NO: 23 where the first amino acid VL is omitted. Various substitutions were made to replace this free cysteine:

a serine residue similar to a substitution in the VH domain of an anti-CD3 antibody (TR66 (SEQ ID NO: 100);

- leucine residue, by comparison with the amino acid sequence of other anti-CLDN6 antibodies (SEQ ID NO: 97 and 98);

- tryptophan residue, by comparing the amino acid sequence with the germline database (SEQ ID NO: 99).

Estimation of linker length, V-domain order, and artificial linking disulfide bonds in anti-CLDN18.2 bi-scFv proteins

As another example, Arndt et al. (Biochemistry 37:12918-12926, 1998) describes the so-called domain change as a possible explanation for the appearance of non-covalently bound oligomers of scFv fragments. According to this model, the protein is in a state of possible thermodynamic equilibrium between the monomeric and dimeric/oligomeric form due to the constantly occurring intra- and intermolecular exchange of VL/VH boundary contacts. These oligomers may already be present in the cell culture supernatant and must be removed during the purification process. However, these molecular species may also form during storage of the purified monomeric species.

The preferred energy status is strongly influenced by the overall design (primary sequence, linker length, VL/VH orientation, etc.).

, и расстояние между С-концом VH и N-концом VL составляет 32-34

(Pluckthun et al., From PCR to fermentation. (J. McCafferty, H. R. Hoogenboom, & D. J. Chriswell, Eds.). In: (IRL Press., pp. 203-252, 1996). Для получения аналогичных молекулярных свойств линкер для ориентации VL-VH должен быть длиннее, чем линкер VH-VL. В публикации Pluckthun et al., From PCR to fermentation. (J. McCafferty, H. R. Hoogenboom, & D. J. Chriswell, Eds.). In: (IRL Press., pp. 203-252, 1996) рекомендовано использование линкеров длиной от 15 до 20 аминокислот в ориентации VH/VL и линкеров длиной 20-25 аминокислот в ориентации VL/VH.Worn and Pluckthun (JMB 305:989-1010, 1999) indicate that forms with a higher content of monomeric species can be obtained by using a linker of 20 or more residues. Desplancq et al., (Ptotein Eng. 7:1027-1033, 1994) indicate that variable domain orientation can also influence the formation of dimers and high molecular weight forms. In the same publication, Desplancq indicated that a linker consisting of 25 or 30 amino acids (aa) showed the best ratio of monomer compared to dimer for their particular antibody. The distance between the C-terminus of VL and the N-terminus of VH is about 39-43

, and the distance between the C-terminus of VH and the N-terminus of VL is 32-34

(Pluckthun et al., From PCR to fermentation. (J. McCafferty, HR Hoogenboom, & DJ Chriswell, Eds.). In: (IRL Press., pp. 203-252, 1996). The VL-VH orientation must be longer than the VH-VL linker In: (IRL Press., pp. 203-252, 1996) recommend the use of linkers 15 to 20 amino acids long in the VH/VL orientation and linkers 20 to 25 amino acids long in the VL/VH orientation.

Another possibility to direct the formation of monomers and stabilize the interaction of the VH/VL domains is to design a boundary disulfide bond in the boundary region of contact between the two domains. The introduction of a disulfide bridge at position H44-L100 (Kabat numbering) has been most commonly used in scFv with satisfactory results (Brinkmann et al., PNAS. 90:7538-7542, 1993; Worn and Pluckthun, Biochemistry 38: 8739-8750, 1999; Weatherill et al., PEDS 25:321-329, 2012). This strategy has been successfully used to stabilize IgG-like bispecific antibodies by combining scFv fused to full-length IgG (Michaelson et al., mAbs 1: 128-141, 2009; Schanzre et al., Antimicrob. Agents. Chemother. 55:2369- 2378, 2011).

Weatherill et al., (PEDS 25:321-239, 2012) stabilized human scFv (VH-(G4S) ₄ -VL and VL-(G4S) ₄ -VH) with a disulfide bond between V _H 44 and V _L -100 . Moreover, this publication highlights the problem of possibly changing the domain to a scFv that does not contain an interface disulfide bond by performing different SE-HPLC experiments at different loading volumes and concentrations. The assays showed different results depending on the sample loading conditions for the unstabilized scFv, but regardless of the conditions under which the disulfide stabilized molecules eluted similarly to the monomer were used. Zhao et al., (Int. J. Mol. Sci. 12:1-11, 2011) introduced the same mutation in scFv and observed higher stability of the stabilized molecule after storage for 20 hours at 37°C.

For a bispecific format using scFv fused to full length IgG, Schanzer et al., (Antimicrob. Agents Chemother, 55:2369-2378 2011) compared the effect of linker length and border disulfide bond. They fused the parent scFv or scdFv (VH-(G4S) ₃ -VL) at the C- or N-terminus of the heavy chain or light chains. For different linker lengths (20, 25 and 30 amino acids), they fused the parent scFv to the C-terminus of the heavy or light chains. The results obtained using various linker lengths identified the 30 aa peptide as the preferred linker for obtaining stable monomers. The level of aggregates after 7 days of storage at 40°C was 50% for scFv15, 18% for scFv20, 8% for scFv25 and 6% for scFv30, but scFv15 disulfide, stabilized by the border disulfide bond, was slightly superior to scFv30. The same approach was taken by Michaelson et al., (mAbs. 1:128-141 2009), and they improved their parental IgG-like bispecific antibody containing scFv with a 15aa linker in VH/VL orientation (generating 40% aggregates), by increasing the length of the linker to 20 aa scFv and introducing a boundary disulfide bond between the position of VH ₄₄ and V _L -100. The resulting molecule contained more than 98% monomers, which were stable after three months at 4°C. The authors decided to work on improving the scFv molecule before moving to a bispecific format.

In the case of anti-CLDN18.2-specific bi-scFv proteins, it is not known whether the formation of dimers and high molecular weight forms can occur and what the involvement of anti-Claudin and/or anti-CD3 scFv molecules is. To evaluate the overall optimal molecule for the anti-CLDN18.2-specific bi-scFv protein, the following modifications were evaluated for each individual scFv:

- domain orientation;

- linker length;

- introduction of a border disulfine bond;

- a combination of three modifications.

For the anti-Claudin 18.2 binding domain, in addition to the variable domains derived from mCLDN18.2ab (VH: SEQ ID NO: 8; VL: SEQ ID NO: 15), sequences derived from mCLDN18.2ab1 (VH: SEQ ID NO: 6; VL: SEQ ID NO: 11) was used to construct anti-CLDN18.2-specific bi-scFv proteins.

Table 11 in section A.a "Initial sequences, design of 32 anti-CLDN18.2-specific bi-scFv constructs and cloning into expression vectors" describes the various constructs.

A. Generation and testing of bispecific binding agents targeting CLDN18.2 and CD3

A. Initial sequences, design of 32 bi-scFv anti-CLDN18.2-specific constructs and cloning into expression vectors

The bi-scFv bispecific tandem single chain antibody constructs presented here contain two distinct binding domains, of which the first is specific for the human tumor-associated antigen (TAA), while the second is specific for the human T cell receptor ε chain ( CD3). Each of the two binding domains of the bispecific molecule contains two antibody variable domains arranged as an scFv fragment in the VH-VL or VL-VH orientation. The variable domains of an antibody are connected by a flexible glycine-serine peptide linker, consisting of four or five repeats of the G4S subunit, depending on orientation. Thus, scFv fragments located in the VH-VL orientation are connected by a 20 amino acid linker (named "LL4"), VL-VH are connected by a 25 amino acid linker (named "LL5"). The two scFv fragments, on the other hand, are connected via a long six amino acid SG ₄ S linker (named "SL"). Information on parent sequences derived from monoclonal antibodies (mAB) and domain organization is summarized in Table 11.

Options 5504, 5505, 5506, 5507, 5512, 5513, 5514, 5515, 5520, 5521, 5522, 5523, 5528, 5529, 5530, 5531, 5536, 5537, 5538, 5539, 5544, 5545, 5546, 5547, 5552 , 5553, 5554, 5555, 5560, 5561, 5562 and 5563 contain anti-CD3 VH of SEQ ID NO: 95 and anti-CD3 VL of SEQ ID NO: 96.

In the specific case of 1-BiMAB-S, only a substitution of free cysteine for serine in the anti-CD3 VH (SEQ ID NO: 94) was made compared to the amino acid sequence of the 1-BiMAB sequence (SEQ ID NO: 39). It should be noted that SEQ ID NO: 39 still contains the amino acid sequence of an N-terminal signal sequence that mediates secretion of the 1-BiMAB bi-scFv protein into the cell culture supernatant when expressed in a mammal. This signal sequence is not part of the secreted recombinant protein, as it is cleaved by a signal peptidase in the lumen of the endoplasmic reticulum.

The genes encoding the bi-scFv constructs were generated by GeneArf® gene synthesis (Life Technologies GmbH, Darmstadt, Germany) using the GeneOptimizer® software to optimize codon usage for expression in CHO cells. In addition to a common secretion signal, all DNA constructs contain the same Kozak sequence and a HindIII restriction site at the 5' end. At the 3'end, BsiWI and XhoI recognition sites are added to allow flexible subcloning into various expression vectors. Subcloning into the preferred expression vector pCEP4 (Life Technologies GmbH, Darmstadt, Germany) was performed by Life Technologies using HindIII and XhoI restriction sites.

Table 11. Summary of anti-CLDN18.2 bi-scFv constructs

Ident.
option Origin anti-CLDN18.2 Origin
anti-CD3 Replacement. to the floor H103 TR66 Domain order (from N- to C-terminus) Domain Organization*) SEQ ID NO; 1 BiMAB-S mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 see 1 BiMAB in example 1 103 5504 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VH-LL4-VL-SL-VH-LL4-VL 66 5505 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-(VH-LL4-VL)-SL-VH-LL4-VL 67 5506 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VH-LL4-VL-SL-VH-LL4-VL 68 5507 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-(VH-LL4-VL)-SL-VH-LL4-VL 69 5512 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VH-LL4-VL-SL-ds(VH-LL4-VL) 70 5513 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-VH-LL4-VL-SL-ds(VH-LL4-VL) 71 5514 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VH-LL4-VL-SL-ds(VH-LL4-VL) 72 5515 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-VH-LL4-VL-SL-ds(VH-LL4-VL) 73 5520 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VH-LL4-VL-SL-VL-LL5-VH 74 5521 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-(VH-LL4-VL)-SL-VL-LL5-VH 75 5522 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VH-LL4-VL-SL-VL-LL5-VH 76 5523 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-(VH-LL4-VL)-SL-VL-LL5-VH 77 5528 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VH-LL4-VL-SL-ds(VL-LL5-VH) 78 5529 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-VH-LL4-VL-SL-ds(VL-LL5-VH) 79 5530 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VH-LL4-VL-SL-ds(VL-LL5-VH) 80 5531 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-VH-LL4-VL-SL-ds(VL-LL5-VH) 81 5536 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VL-LL5-VH-SL-VH-LL4-VL 82 5537 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-(VL-LL5-VH)-SL-VH-LL4-VL 83 5538 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VL-LL5-VH-SL-VH-LL4-VL 84 5539 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-(VL-LL5-VH)-SL-VH-LL4-VL 85 5544 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VL-LL5-VH-SL-ds(VH-LL4-VL) 86 5545 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-VL-LL5-VH-SL-ds(VH-LL4-VL) 87 5546 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VL-LL5-VH-SL-ds(VH-LL4-VL) 88 5547 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-VL-LL5-VH-SL-ds(VH-LL4-VL) 89 5552 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VL-LL5-VH-SL-VL-LL5-VH -- 5553 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-(VL-LL5-VH)-SL-VL-LL5-VH -- 5554 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VL-LL5-VH-SL-VL-LL5-VH -- 5555 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-(VL-LL5-VH)-SL-VL-LL5-VH -- 5560 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VL-LL5-VH-SL-ds(VL-LL5-VH) 90 5561 mCLDN18.2ab1 TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-VL-LL5-VH-SL-ds(VL-LL5-VH) 91 5562 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 VL-LL5-VH-SL-ds(VL-LL5-VH) 92 5563 mCLDN18.2ab TR66 Serene scFv-anti-CLDN18.2-scFv anti-CD3 ds-VL-LL5-VH-SL-ds(VL-LL5-VH) 93

*) LL4, (G ₄ S) ₄ ; LL5; (G ₄ S) ₅ ; SL, SG ₄ S.

It should be noted that SEQ ID NOs: 66 to 93 still contain the amino acid sequence of the N-terminal signal sequence that mediates secretion of the 1-BiMAB bi-scFv protein into the cell culture supernatant when expressed in a mammal. This signal sequence is not part of the secreted recombinant protein, as it is cleaved by a signal peptidase in the lumen of the endoplasmic reticulum.

b. Generation and purification of 32 anti-CLDN18.2-specific bi-scFv proteins by transient transfection

Suspension-adapted CHO cells were subcultured in a serum-free medium in a humidified CO ₂ atmosphere on an orbital shaker. The day before transfection, cells were seeded in serum-free medium in shaker flasks. On the day of transfection, cells were centrifuged (5 min at 200×g) and resuspended in fresh DMEM (Invitrogen, 41965-039) in shaker flasks. DNA and transfection reagent were added to the cells and mixed gently by shaking. After incubation under standard conditions in a CO ₂ incubator, cells were diluted with serum-free growth medium and then grown for expression in an incubation shaker. Cells were fed with CHO CD EfficientFeed™ C (Invitrogen, A13275) according to nutritional requirements. Bi-scFv proteins were harvested after cell viability began to decline. The antibody constructs were purified with Capto L Sepharose. Protein concentration was determined by measuring absorbance at 280 nm.

With. Luciferase-based cytotoxicity assay using 32 bi-scFv anti-CLDN18.2-specific proteins

For a functional screen of 32 anti-CLDN18.2-specific bi-scFv proteins, four titrations (5000, 1000, 200 and 40 ng/mL) were tested in an in vitro luciferase-based cytotoxicity assay as described in Example 2.c.

The luciferase-stably-expressing NugC4 cells described in Example 2.c were incubated with human T cells and bi-scFv proteins or without bi-scFv protein to determine L _min values. Viable cell luminescence was measured using an Infinite M200 Tecan plate reader 24 and 48 hours after the start of the assay. The specific lysis of target cells was calculated using the formula given in example 2.c.

After quantifying the results of the cytotoxicity assay (Figures 39 a, b, c and d) based on the scFv anti-CD3 binding domains, the following conclusions can be drawn.

The most effective anti-CLDN18.2-specific bi-scFv proteins in a luciferase-based cytotoxicity assay contain an anti-CD3 fragment in the VH/VL domain orientation linked by an "LL4" linker with or without a border disulfide bridge (5504, 5505, 5506 , 5507, 5536, 5537, 5538, 5539, 5512, 5513, 5514, 5515, 5544, 5545, 5546, 5547; figures 39 a and b). Lower cytotoxic activity was obtained for variants containing an anti-CD3 fragment in the orientation of the VL/VH domains with the "LL5" peptide linker and containing a border disulfide bridge (5528, 5529, 5530, 5531, 5560, 5561, 5562, 5563; figure 39 d ). The lowest cytotoxic activity was obtained for variants containing an anti-CD3 fragment in the orientation of the VL/VH domains with a peptide linker "LL5" without a border disulfide bridge (5520, 5521, 5522, 5523, 5552, 5553, 5554, 5555; figure 39 c) .

B. Generation and testing of bispecific binding agents targeting CLDN6 and CD3

A. Sequence origin, design of bi-scFv constructs, and cloning of expression vectors

The bi-scFv bispecific tandem single chain antibody constructs presented here contain two distinct binding domains, one of which is specific for the human tumor-associated antigen (TAA) and the other is specific for the human T-cell receptor (CD3) ε chain. ). Each of the two domains of the bispecific molecule contains two antibody variable domains arranged as an scFv fragment in either VH-VL or VL-VH orientation. The variable domains of an antibody are connected via a flexible glycine-serine peptide linker. The scFv fragments targeting TAA are located in the VH-VL orientation and linked by a 15 amino acid linker (named "LL3") consisting of three identical repeats of the G ₄ S subunit. The scFv fragments targeting CD3 are also located in the VH orientation. -VL, but linked by an ₁₈ amino acid linker (named "LLv1") to the G ₄ S(G ₂ S) ₃ G ₃ S sequence. "SL"). Information on sequence derivation from monoclonal antibodies (mAB) and domain organization is summarized in Table 12. Variants 5454, 5456, 5458, 5460, 5462, and 5464 contain for the anti-CD3 binding domain an anti-CD3 VH with SEQ ID NO: 95 and an anti-CD3 VL. -CD3 of SEQ ID NO: 96. Variants 5454 and 5458 contain anti-CLDN6 VL of SEQ ID NO: 98; options 5457 and 5460 contain VL anti-CLDN6 with SEQ ID NO: 99; variants 5462 and 5464 contain the anti-CLDN6 VL of SEQ ID NO: 100.

In the specific case of 6PHU3-S (SEQ ID NO: 101), only a substitution of free cysteine for a serine residue in anti-CD3 VH (SEQ ID NO: 94) was made compared to the amino acid sequence of 6-PHU3 (SEQ ID NO: 45). It should be noted that SEQ ID NO: 45 still contains the amino acid sequence of an N-terminal signal sequence that mediates the secretion of the 6-PHU-3 bi-scFv protein into the cell culture supernatant when expressed in a mammal. This signal sequence is not part of the secreted recombinant protein, as it is cleaved by a signal peptidase in the lumen of the endoplasmic reticulum. Moreover, for 6PHU3-SL (SEQ ID NO: 102), a substitution of free cysteine for a leucine residue in the anti-CLDN VL (SEQ ID NO: 97) at position 46 of the corresponding primary sequence was made compared to the amino acid sequence of 6PHU3-S. This corresponds to position 45 within SEQ ID NO: 23 where the first amino acid VL is missing.

The genes encoding the bi-scFv constructs were generated by GeneArf® gene synthesis (Life Technologies GmbH, Darmstadt, Germany) using the GeneOptimizer® software to optimize codon usage for expression in CHO cells. In addition to a common secretion signal, all DNA constructs contain the same Kozak sequence and a HindIII restriction site at the 5' end. At the 3'end, BsiWI and XhoI recognition sites are added to allow flexible subcloning into various expression vectors. Subcloning into the preferred expression vector pEE12.4 (LONZA Group Ltd., Basel, Switzerland) was performed using standard techniques using HindIII and BsiWI restriction sites.

Table 12. Summary of anti-CLDN6 bi-scFv constructs

Ident.
option Origin
anti-CLDN6 Floor replacement. L46 mCLDN6ab Origin anti-CD3 Floor replacement. H103 TR66 Domain order (N- to C-terminal) Organization domains*) SEQID NO: 5454 mCLDN6ab Leucine TR66 Serene scFvanti-CLDN6-scFvanti-CD3 VH-LL3-VL-SL-VH-LLv1-VL 60 5456 mCLDN6ab tryptophan TR66 Serene scFvanti-CLDN6-scFvanti-CD3 VHLL3VL-SL-VH-LLv1-VL 61 5462 mCLDN6ab Serene TR66 Serene scFvanti-CLDN6-scFvanti-CD3 VH-LL3-VL-SL-VH-LLv1-VL 62 6PHU3-S mCLDN6ab No data TR66 Serene scFvanti-CD3-scFvanti-CLDN6 See 6PHU3 in Example 9 101 6PHU3-SL mCLDN6ab Leucine TR66 Serene scFvanti-CD3-scFvanti-CLDN6 See 6PHU3 in Example 9 102 5458 mCLDN6ab Leucine TR66 Serene scFvanti-CD3-scFvanti-CLDN6 VH-LLv1-VL-SL-VH-LL3-VL 63 5460 mCLDN6ab tryptophan TR66 Serene scFvanti-CD3-scFvanti-CLDN6 VH-LLv1-VL-SL-VH-LL3-VL 64 5464 mCLDN6ab Serene TR66 Serene scFvanti-CD3-scFvanti-CLDN6 VH-LLv1-VL-SL-VH-LL3-VL 65

*) LL3, (G ₄ S) ₃ ; LLv1, G ₄ S(G ₂ S) ₃ G ₃ S; SL; _SG4S .

b. Production and purification of 6 anti-CLDN6-specific bi-scFv proteins by transient transfection

Suspension-adapted CHO cells were subcultured in a serum-free medium in a humidified CO ₂ atmosphere on an orbital shaker. The day before transfection, cells were seeded in serum-free medium in shaker flasks. On the day of transfection, cells were centrifuged (5 min at 200×g) and resuspended in fresh DMEM (Invitrogen, 41965-039) in shake flasks. DNA and transfection reagent were added to the cells and mixed gently by shaking. After incubation under standard conditions in a CO ₂ incubator, cells were diluted with serum-free growth medium and then grown for expression in an incubation shaker. Cells were fed with CHO CD EfficientFeed™ C (Invitrogen, A13275) according to nutritional requirements. Bi-scFv proteins were harvested after cell viability began to decline. The antibody constructs were purified by FPLC using Capto L Sepharose. Protein concentration was determined by measuring absorbance at 280 nm.

With. Determination of EC ₅₀ 5 anti-CLDN6-specific bi-scFv proteins

To determine half the maximum effective dose of anti-CLDN6-specific bi-scFv proteins, a series of bi-scFv protein titrations were tested in an in vitro luciferase cytotoxicity assay as described in Example 13.

Luciferase-stably-expressing PA-1 cells and human T cells at a 5:1 E:T ratio were incubated with bi-scFv protein at a concentration ranging from 2.5 pg/mL to 5 µg/mL (10 steps) or without anti-CLDN6-specific proteins bi-scFv to determine the values of L _min .

Three independent analyzes of the human T cell preparation were performed with different human donors. The results are presented in figure 40.

After performing a quantitative comparison of the results of the cytotoxicity assay based on the substitution of a cysteine residue within the flanking region of CDR-L2 with serine, leucine or tryptophan, and based on the position of the anti-CLDN6 and anti-CD3 binding domains, the following conclusions can be drawn.

The most effective anti-CLDN6-specific bi-scFv proteins in the luciferase-based cytotoxicity assay contain a cysteine-tryptophan substitution for the anti-CLDN6 binding domain at the N-terminus of the bi-scFv protein (variant 5456). A slightly lower cytotoxic activity was obtained for variants containing anti-CLDN6 in the C-terminal part with the replacement of cysteine with tryptophan (variant 5460) or serine (variant 5464). For cysteine to leucine substitution variants containing anti-CLDN6 at the N-terminus (variant 5454) or C-terminus (variant 5458) of the bi-scFv proteins, lower cytotoxic activity was measured compared to previous variants. Surprisingly, it was found that the variant containing the substitution of cysteine for serine with anti-CLDN6 in the N-terminal portion (variant 5462) of the bi-scFv protein had the lowest cytotoxic activity.

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SEQUENCE LIST

<110> BioNTech AG et al.

<120> AGENTS FOR TREATMENT OF CLAUDIN EXPRESSING CANCER DISEASES

<130> 674-86 PCT

<150> PCT/EP2012/004712

<151> 2012-11-13

<150> PCT/EP2013/002270

<151> 2013-07-30

<160> 108

<170>PatentIn version 3.5

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Gly Val Leu Thr Leu Ile Pro Val Cys Trp Thr Ala His Ala Ile Ile

130 135 140

Arg Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Gln Lys Arg Glu Leu

145 150 155 160

Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu

165 170 175

Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Ser Gly Gly Ser Gln Gly

180 185 190

Pro Ser His Tyr Met Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser

195 200 205

Arg Gly Pro Ser Glu Tyr Pro Thr Lys Asn Tyr Val

210 215 220

<210> 3

<211> 220

<212> PRT

<213> Homo sapiens

<400> 3

Met Ala Ser Ala Gly Met Gln Ile Leu Gly Val Val Leu Thr Leu Leu

1 5 10 15

Gly Trp Val Asn Gly Leu Val Ser Cys Ala Leu Pro Met Trp Lys Val

20 25 30

Thr Ala Phe Ile Gly Asn Ser Ile Val Val Ala Gln Val Val Trp Glu

35 40 45

Gly Leu Trp Met Ser Cys Val Val Gln Ser Thr Gly Gln Met Gln Cys

50 55 60

Lys Val Tyr Asp Ser Leu Leu Ala Leu Pro Gln Asp Leu Gln Ala Ala

65 70 75 80

Arg Ala Leu Cys Val Ile Ala Leu Leu Val Ala Leu Phe Gly Leu Leu

85 90 95

Val Tyr Leu Ala Gly Ala Lys Cys Thr Thr Cys Val Glu Glu Lys Asp

100 105 110

Ser Lys Ala Arg Leu Val Leu Thr Ser Gly Ile Val Phe Val Ile Ser

115 120 125

Gly Val Leu Thr Leu Ile Pro Val Cys Trp Thr Ala His Ala Val Ile

130 135 140

Arg Asp Phe Tyr Asn Pro Leu Val Ala Glu Ala Gln Lys Arg Glu Leu

145 150 155 160

Gly Ala Ser Leu Tyr Leu Gly Trp Ala Ala Ser Gly Leu Leu Leu Leu

165 170 175

Gly Gly Gly Leu Leu Cys Cys Thr Cys Pro Ser Gly Gly Ser Gln Gly

180 185 190

Pro Ser His Tyr Met Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser

195 200 205

Arg Gly Pro Ser Glu Tyr Pro Thr Lys Asn Tyr Val

210 215 220

<210> 4

<211> 207

<212> PRT

<213> Homo sapiens

<400> 4

Met Gln Ser Gly Thr His Trp Arg Val Leu Gly Leu Cys Leu Leu Ser

1 5 10 15

Val Gly Val Trp Gly Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr

20 25 30

Gln Thr Pro Tyr Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr

35 40 45

Cys Pro Gln Tyr Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys

50 55 60

Asn Ile Gly Gly Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp

65 70 75 80

His Leu Ser Leu Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr

85 90 95

Val Cys Tyr Pro Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu

100 105 110

Tyr Leu Arg Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp Val Met

115 120 125

Ser Val Ala Thr Ile Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu

130 135 140

Leu Leu Leu Val Tyr Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys

145 150 155 160

Pro Val Thr Arg Gly Ala Gly Ala Gly Gly Arg Gln Arg Gly Gln Asn

165 170 175

Lys Glu Arg Pro Pro Pro Val Pro Asn Pro Asp Tyr Glu Pro Ile Arg

180 185 190

Lys Gly Gln Arg Asp Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile

195 200 205

<210> 5

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 5

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr

20 25 30

Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Gly Ser Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Asp Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ala

115

<210> 6

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 6

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr

20 25 30

Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 7

<211> 116

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 7

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Tyr Ile Asn Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Ser Gly Asn Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Ser Tyr Gly Ala Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 8

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 8

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser

115

<210> 9

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 9

Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr

20 25 30

Val Ile Ser Trp Val Lys Gln Arg Thr Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Tyr Pro Gly Ser Gly Ser Thr Tyr Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Gly Val Leu Leu Arg Ala Met Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Ser Val Thr Val Ser Ser

115

<210> 10

<211> 120

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 10

Gln Val His Leu Gln Gln Ser Gly Ser Glu Leu Arg Ser Pro Gly Ser

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Asp Phe Asp Ser Glu Val Phe Pro Phe

20 25 30

Ala Tyr Met Ser Trp Ile Arg Gln Lys Pro Gly His Gly Phe Glu Trp

35 40 45

Ile Gly Asp Ile Leu Pro Ser Ile Gly Arg Thr Ile Tyr Gly Glu Lys

50 55 60

Phe Glu Asp Lys Ala Thr Leu Asp Ala Asp Thr Val Ser Asn Thr Ala

65 70 75 80

Tyr Leu Glu Leu Asn Ser Leu Thr Ser Glu Asp Ser Ala Ile Tyr Tyr

85 90 95

Cys Ala Arg Gly Glu Gly Tyr Gly Ala Trp Phe Ala Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ala

115 120

<210> 11

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 11

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 12

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 12

Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ser Tyr Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 13

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 13

Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly

1 5 10 15

Asp Arg Val Ser Ile Thr Cys Lys Ala Ser Gln Asn Val Arg Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile

35 40 45

Tyr Leu Ala Ser Asn Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser

65 70 75 80

Glu Asp Leu Ala Asp Tyr Phe Cys Leu Gln His Trp Asn Tyr Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 14

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 14

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 15

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 15

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 16

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 16

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asn Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 17

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 17

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr His Cys Gly Gln

85 90 95

Gly Tyr Ser Tyr Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 18

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 18

Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser

20 25 30

Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu

100 105 110

Lys

<210> 19

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 19

Asn Ile Val Met Thr Gln Ser Pro Lys Ser Met Ser Met Ser Val Gly

1 5 10 15

Glu Arg Val Thr Leu Thr Cys Lys Ala Ser Glu Asn Val Val Thr Tyr

20 25 30

Val Ser Trp Tyr Gln Gln Lys Pro Glu Gln Ser Pro Lys Leu Leu Ile

35 40 45

Tyr Gly Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly

50 55 60

Ser Gly Ser Ala Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Lys Ala

65 70 75 80

Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Ser Tyr Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 20

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 20

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ile Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Gly Tyr Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 21

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 21

Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu

1 5 10 15

Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Leu His

20 25 30

Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Val Tyr Ser

35 40 45

Thr Ser Asn Leu Pro Ser Gly Val Pro Ala Arg Phe Gly Gly Ser Gly

50 55 60

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu Asp

65 70 75 80

Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Ile Tyr Pro Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 22

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 22

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Gly Phe Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 23

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 23

Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly Glu

1 5 10 15

Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His

20 25 30

Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Cys Ile Tyr Ser

35 40 45

Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg Gly

50 55 60

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu Asp

65 70 75 80

Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 24

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 24

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Ile Ile Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Phe Tyr Cys

85 90 95

Ala Arg Asp Phe Gly Tyr Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 25

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 25

Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu

1 5 10 15

Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His

20 25 30

Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr Ser

35 40 45

Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly

50 55 60

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu Asp

65 70 75 80

Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Thr Tyr Pro Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 26

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 26

Glu Val Gln Leu Gln Gln Ser Arg Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Leu Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Ser Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Gly Tyr Val Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 27

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 27

Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu

1 5 10 15

Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met His

20 25 30

Trp Phe Gln Leu Lys Pro Gly Thr Ser Pro Lys Leu Leu Ile Tyr Ser

35 40 45

Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg Gly

50 55 60

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu Asp

65 70 75 80

Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Asn Asn Tyr Pro Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 28

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 28

Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly Glu

1 5 10 15

Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His

20 25 30

Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Gly Ile Tyr Ser

35 40 45

Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg Gly

50 55 60

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu Asp

65 70 75 80

Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 29

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 29

Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly Glu

1 5 10 15

Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His

20 25 30

Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Ser Ile Tyr Ser

35 40 45

Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg Gly

50 55 60

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu Asp

65 70 75 80

Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 30

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 30

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Ser Gly Tyr Thr Lys Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Trp Gln Asp Tyr Asp Val Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 31

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 31

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 32

<211> 122

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 32

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Met

35 40 45

Gly Leu Ile Asn Pro Tyr Lys Gly Val Ser Thr Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Phe Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Gly Tyr Tyr Gly Asp Ser Asp Trp Tyr Phe Asp Val Trp

100 105 110

Gly Ala Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 33

<211> 107

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 33

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Arg Asn Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp

85 90 95

Thr Phe Ala Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 34

<211> 119

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 34

Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Phe Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Arg Tyr Ser Arg Tyr Ile Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Arg Pro Leu Tyr Gly Ser Ser Pro Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 35

<211> 108

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 35

Asp Ile Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser

1 5 10 15

Pro Gly Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Thr

20 25 30

Tyr Val His Trp Tyr Gln Gln Lys Ser Asn Thr Ser Pro Lys Leu Trp

35 40 45

Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Gly Arg Val Ser

50 55 60

Gly Ser Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu

65 70 75 80

Ala Glu Asp Val Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro

85 90 95

Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Met Arg

100 105

<210> 36

<211> 117

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 36

Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val

1 5 10 15

Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met

20 25 30

His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr

35 40 45

Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp

50 55 60

Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln

65 70 75 80

Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg

85 90 95

Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 37

<211> 106

<212> PRT

<213> Artificial Sequence

<220>

<223> Antibody fragment

<400> 37

Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

35 40 45

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105

<210> 38

<211> 495

<212> PRT

<213> Artificial Sequence

<220>

<223> Bispecific molecule

<400> 38

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu

130 135 140

Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln

145 150 155 160

Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr

180 185 190

Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val

210 215 220

Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu

245 250 255

Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met

260 265 270

Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp

275 280 285

Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn

290 295 300

Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala

305 310 315 320

Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser

325 330 335

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr

340 345 350

Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

355 360 365

Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

370 375 380

Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met

385 390 395 400

Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser

405 410 415

Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro

420 425 430

Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr

435 440 445

Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser

450 455 460

Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser

465 470 475 480

Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

485 490 495

<210> 39

<211> 520

<212> PRT

<213> Artificial Sequence

<220>

<223> Bispecific molecule

<400> 39

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro

145 150 155 160

Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys

165 170 175

Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr

180 185 190

Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp

195 200 205

Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly

210 215 220

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp

225 230 235 240

Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe

245 250 255

Gly Ser Gly Thr Lys Leu Glu Ile Lys Ser Gly Gly Gly Gly Ser Asp

260 265 270

Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser

275 280 285

Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr

290 295 300

Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly

305 310 315 320

Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys

325 330 335

Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met

340 345 350

Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala

355 360 365

Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr

370 375 380

Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly Gly

385 390 395 400

Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser Pro

405 410 415

Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg

420 425 430

Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly

435 440 445

Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly

450 455 460

Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu

465 470 475 480

Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln

485 490 495

Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu

500 505 510

Leu Lys His His His His His His

515 520

<210> 40

<211> 490

<212> PRT

<213> Artificial Sequence

<220>

<223> Bispecific molecule

<400> 40

Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu

130 135 140

Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln

145 150 155 160

Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln

165 170 175

Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr

180 185 190

Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr

195 200 205

Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val

210 215 220

Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly

225 230 235 240

Thr Lys Leu Glu Ile Lys Ser Gly Gly Gly Gly Ser Lys Leu Gln Gln

245 250 255

Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys

260 265 270

Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys

275 280 285

Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser

290 295 300

Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu

305 310 315 320

Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu

325 330 335

Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp

340 345 350

His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

355 360 365

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

370 375 380

Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly

385 390 395 400

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met

405 410 415

Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr

420 425 430

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser

435 440 445

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu

450 455 460

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr

465 470 475 480

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

485 490

<210> 41

<211> 515

<212> PRT

<213> Artificial Sequence

<220>

<223> Bispecific molecule

<400> 41

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro

145 150 155 160

Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys

165 170 175

Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr

180 185 190

Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp

195 200 205

Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly

210 215 220

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp

225 230 235 240

Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe

245 250 255

Gly Ser Gly Thr Lys Leu Glu Ile Lys Ser Gly Gly Gly Gly Ser Lys

260 265 270

Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys

275 280 285

Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His

290 295 300

Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile

305 310 315 320

Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys

325 330 335

Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu

340 345 350

Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr

355 360 365

Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu

370 375 380

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

385 390 395 400

Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala

405 410 415

Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val

420 425 430

Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg

435 440 445

Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe

450 455 460

Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met

465 470 475 480

Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn

485 490 495

Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys His His His

500 505 510

His His His

515

<210> 42

<211> 487

<212> PRT

<213> Artificial Sequence

<220>

<223> Bispecific molecule

<400> 42

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Gly Phe Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser

130 135 140

Val Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser

145 150 155 160

Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys

165 170 175

Leu Cys Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg

180 185 190

Phe Ser Gly Arg Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg

195 200 205

Val Ala Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn

210 215 220

Tyr Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Ser

225 230 235 240

Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu

245 250 255

Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr

260 265 270

Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln

275 280 285

Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn

290 295 300

Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser

305 310 315 320

Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser

325 330 335

Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp

340 345 350

Tyr Trp Gly Glyn Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly

355 360 365

Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile

370 375 380

Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys

385 390 395 400

Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp

405 410 415

Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr

420 425 430

Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser

435 440 445

Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala

450 455 460

Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly

465 470 475 480

Ala Gly Thr Lys Leu Glu Leu

485

<210> 43

<211> 513

<212> PRT

<213> Artificial Sequence

<220>

<223> Bispecific molecule

<400> 43

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe

35 40 45

Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu

50 55 60

Glu Trp Ile Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ile Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asp Tyr Gly Phe Val Leu Asp Tyr Trp Gly Gln

115 120 125

Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Ser

145 150 155 160

Ile Met Ser Val Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala

165 170 175

Ser Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Thr

180 185 190

Ser Pro Lys Leu Cys Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val

195 200 205

Pro Ala Arg Phe Ser Gly Arg Gly Ser Gly Thr Ser Tyr Ser Leu Thr

210 215 220

Ile Ser Arg Val Ala Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

225 230 235 240

Arg Ser Asn Tyr Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu

245 250 255

Ile Lys Ser Gly Gly Gly Gly Ser Asp Ile Lys Leu Gln Gln Ser Gly

260 265 270

Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr

275 280 285

Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg

290 295 300

Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly

305 310 315 320

Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr

325 330 335

Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser

340 345 350

Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr

355 360 365

Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Val

370 375 380

Glu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val

385 390 395 400

Asp Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro

405 410 415

Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr

420 425 430

Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile

435 440 445

Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly

450 455 460

Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala

465 470 475 480

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu

485 490 495

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys His His His His His

500 505 510

His

<210> 44

<211> 488

<212> PRT

<213> Artificial Sequence

<220>

<223> Bispecific molecule

<400> 44

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly

115 120 125

Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser

130 135 140

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

145 150 155 160

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

165 170 175

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

180 185 190

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

195 200 205

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

210 215 220

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu

225 230 235 240

Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser

245 250 255

Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys Ile Ser Cys Lys

260 265 270

Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp Val Lys Gln

275 280 285

Ser His Gly Lys Asn Leu Glu Trp Ile Gly Leu Ile Asn Pro Tyr Asn

290 295 300

Gly Gly Thr Ile Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr

305 310 315 320

Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr

325 330 335

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Asp Tyr Gly Phe Val

340 345 350

Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

355 360 365

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

370 375 380

Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly Glu Lys Val

385 390 395 400

Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Phe

405 410 415

Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Cys Ile Tyr Ser Thr Ser

420 425 430

Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg Gly Ser Gly

435 440 445

Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu Asp Ala Ala

450 455 460

Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp Thr Phe Gly

465 470 475 480

Gly Gly Thr Lys Leu Glu Ile Lys

485

<210> 45

<211> 513

<212> PRT

<213> Artificial Sequence

<220>

<223> Bispecific molecule

<400> 45

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg

20 25 30

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe

35 40 45

Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly

130 135 140

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu

145 150 155 160

Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr

165 170 175

Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln

180 185 190

Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys

195 200 205

Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly

245 250 255

Thr Lys Leu Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

260 265 270

Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys Ile

275 280 285

Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp

290 295 300

Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile Gly Leu Ile Asn

305 310 315 320

Pro Tyr Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe Lys Gly Lys Ala

325 330 335

Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu

340 345 350

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Asp Tyr

355 360 365

Gly Phe Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

370 375 380

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

385 390 395 400

Asp Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly

405 410 415

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

420 425 430

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Cys Ile Tyr

435 440 445

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg

450 455 460

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu

465 470 475 480

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp

485 490 495

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys His His His His His

500 505 510

His

<210> 46

<211> 15

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker

<400> 46

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 47

<211> 16

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker

<400> 47

Val Glu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Val Asp

1 5 10 15

<210> 48

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker

<400> 48

Ser Gly Gly Gly Gly Ser

15

<210> 49

<211> 5

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker

<400> 49

Gly Gly Gly Gly Ser

15

<210> 50

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker

<400> 50

Val Glu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

1 5 10 15

Val Asp

<210> 51

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> Secretion signal

<400> 51

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser

<210> 52

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> Secretion signal

<400> 52

Met Asn Ser Gly Leu Gln Leu Val Phe Phe Val Leu Thr Leu Lys Gly

1 5 10 15

Ile Gln Gly

<210> 53

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> Secretion signal

<400> 53

Met Asn Phe Gly Leu Ser Leu Ile Phe Leu Ala Leu Ile Leu Lys Gly

1 5 10 15

Val Gln Cys

<210> 54

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> Secretion signal

<400> 54

Met Glu Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Val Thr Thr Gly

1 5 10 15

Val His Ser

<210> 55

<211> 19

<212> PRT

<213> Artificial Sequence

<220>

<223> Secretion signal

<400> 55

Met Gly Trp Leu Trp Asn Leu Leu Phe Leu Met Ala Ala Ala Gln Ser

1 5 10 15

Ala Gln Ala

<210> 56

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 56

tggctctgtg tcgacactgt g 21

<210> 57

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 57

gtgtacatgt tagctgtgga c 21

<210> 58

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 58

tgacactggc aaaacaatgc a 21

<210> 59

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Oligonucleotide

<400> 59

ggtccttttc accagcaagc t 21

<210> 60

<211> 507

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 60

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe

35 40 45

Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu

50 55 60

Glu Trp Ile Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ile Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asp Tyr Gly Phe Val Leu Asp Tyr Trp Gly Gln

115 120 125

Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ser

145 150 155 160

Ile Met Ser Val Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala

165 170 175

Ser Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Thr

180 185 190

Ser Pro Lys Leu Leu Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val

195 200 205

Pro Ala Arg Phe Ser Gly Arg Gly Ser Gly Thr Ser Tyr Ser Leu Thr

210 215 220

Ile Ser Arg Val Ala Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

225 230 235 240

Arg Ser Asn Tyr Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu

245 250 255

Ile Lys Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly

260 265 270

Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr

275 280 285

Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg

290 295 300

Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly

305 310 315 320

Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr

325 330 335

Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser

340 345 350

Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr

355 360 365

Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly

370 375 380

Gly Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly

385 390 395 400

Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro

405 410 415

Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr

420 425 430

Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile

435 440 445

Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly

450 455 460

Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala

465 470 475 480

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu

485 490 495

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

500 505

<210> 61

<211> 507

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 61

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe

35 40 45

Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu

50 55 60

Glu Trp Ile Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ile Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asp Tyr Gly Phe Val Leu Asp Tyr Trp Gly Gln

115 120 125

Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ser

145 150 155 160

Ile Met Ser Val Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala

165 170 175

Ser Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Thr

180 185 190

Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val

195 200 205

Pro Ala Arg Phe Ser Gly Arg Gly Ser Gly Thr Ser Tyr Ser Leu Thr

210 215 220

Ile Ser Arg Val Ala Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

225 230 235 240

Arg Ser Asn Tyr Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu

245 250 255

Ile Lys Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly

260 265 270

Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr

275 280 285

Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg

290 295 300

Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly

305 310 315 320

Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr

325 330 335

Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser

340 345 350

Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr

355 360 365

Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly

370 375 380

Gly Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly

385 390 395 400

Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro

405 410 415

Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr

420 425 430

Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile

435 440 445

Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly

450 455 460

Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala

465 470 475 480

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu

485 490 495

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

500 505

<210> 62

<211> 507

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 62

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe

35 40 45

Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu

50 55 60

Glu Trp Ile Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ile Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asp Tyr Gly Phe Val Leu Asp Tyr Trp Gly Gln

115 120 125

Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ser

145 150 155 160

Ile Met Ser Val Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala

165 170 175

Ser Ser Ser Val Ser Tyr Met His Trp Phe Gln Gln Lys Pro Gly Thr

180 185 190

Ser Pro Lys Leu Ser Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val

195 200 205

Pro Ala Arg Phe Ser Gly Arg Gly Ser Gly Thr Ser Tyr Ser Leu Thr

210 215 220

Ile Ser Arg Val Ala Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln

225 230 235 240

Arg Ser Asn Tyr Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu

245 250 255

Ile Lys Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly

260 265 270

Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr

275 280 285

Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg

290 295 300

Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly

305 310 315 320

Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr

325 330 335

Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser

340 345 350

Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr

355 360 365

Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly

370 375 380

Gly Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly

385 390 395 400

Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro

405 410 415

Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr

420 425 430

Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile

435 440 445

Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly

450 455 460

Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala

465 470 475 480

Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu

485 490 495

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

500 505

<210> 63

<211> 507

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 63

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg

20 25 30

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe

35 40 45

Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly Ser Gln Ile Val Leu

145 150 155 160

Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr

165 170 175

Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln

180 185 190

Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys

195 200 205

Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly

245 250 255

Thr Lys Leu Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

260 265 270

Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys Ile

275 280 285

Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp

290 295 300

Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile Gly Leu Ile Asn

305 310 315 320

Pro Tyr Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe Lys Gly Lys Ala

325 330 335

Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu

340 345 350

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Asp Tyr

355 360 365

Gly Phe Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

370 375 380

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

385 390 395 400

Gln Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly

405 410 415

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

420 425 430

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Leu Ile Tyr

435 440 445

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg

450 455 460

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu

465 470 475 480

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp

485 490 495

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

500 505

<210> 64

<211> 507

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 64

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg

20 25 30

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe

35 40 45

Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly Ser Gln Ile Val Leu

145 150 155 160

Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr

165 170 175

Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln

180 185 190

Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys

195 200 205

Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly

245 250 255

Thr Lys Leu Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

260 265 270

Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys Ile

275 280 285

Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp

290 295 300

Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile Gly Leu Ile Asn

305 310 315 320

Pro Tyr Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe Lys Gly Lys Ala

325 330 335

Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu

340 345 350

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Asp Tyr

355 360 365

Gly Phe Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

370 375 380

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

385 390 395 400

Gln Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly

405 410 415

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

420 425 430

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr

435 440 445

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg

450 455 460

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu

465 470 475 480

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp

485 490 495

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

500 505

<210> 65

<211> 507

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 65

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg

20 25 30

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe

35 40 45

Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Gly Ser Gln Ile Val Leu

145 150 155 160

Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr

165 170 175

Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln

180 185 190

Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys

195 200 205

Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly

245 250 255

Thr Lys Leu Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

260 265 270

Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys Ile

275 280 285

Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp

290 295 300

Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile Gly Leu Ile Asn

305 310 315 320

Pro Tyr Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe Lys Gly Lys Ala

325 330 335

Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu

340 345 350

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Asp Tyr

355 360 365

Gly Phe Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

370 375 380

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

385 390 395 400

Gln Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly

405 410 415

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

420 425 430

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Ser Ile Tyr

435 440 445

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg

450 455 460

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu

465 470 475 480

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp

485 490 495

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

500 505

<210> 66

<211> 530

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 66

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys

20 25 30

Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu

50 55 60

Lys Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala

65 70 75 80

Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser

85 90 95

Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr

100 105 110

Tyr Phe Cys Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala

275 280 285

Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr

290 295 300

Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly

305 310 315 320

Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr

325 330 335

Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser

340 345 350

Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

355 360 365

Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr

370 375 380

Trp Gly Glyn Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

405 410 415

Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu

420 425 430

Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn

435 440 445

Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp

450 455 460

Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly

465 470 475 480

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp

485 490 495

Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe

500 505 510

Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Ser His His His His

515 520 525

His His

530

<210> 67

<211> 530

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 67

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys

20 25 30

Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Cys Leu

50 55 60

Lys Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala

65 70 75 80

Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser

85 90 95

Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr

100 105 110

Tyr Phe Cys Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala

275 280 285

Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr

290 295 300

Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly

305 310 315 320

Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr

325 330 335

Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser

340 345 350

Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

355 360 365

Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr

370 375 380

Trp Gly Glyn Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

405 410 415

Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu

420 425 430

Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn

435 440 445

Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp

450 455 460

Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly

465 470 475 480

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp

485 490 495

Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe

500 505 510

Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Ser His His His His

515 520 525

His His

530

<210> 68

<211> 530

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 68

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala

275 280 285

Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr

290 295 300

Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly

305 310 315 320

Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr

325 330 335

Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser

340 345 350

Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

355 360 365

Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr

370 375 380

Trp Gly Glyn Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

405 410 415

Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu

420 425 430

Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn

435 440 445

Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp

450 455 460

Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly

465 470 475 480

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp

485 490 495

Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe

500 505 510

Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Ser His His His His

515 520 525

His His

530

<210> 69

<211> 530

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 69

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Cys Leu

50 55 60

Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Phe Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala

275 280 285

Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr

290 295 300

Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly

305 310 315 320

Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr

325 330 335

Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser

340 345 350

Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

355 360 365

Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr

370 375 380

Trp Gly Glyn Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

405 410 415

Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu

420 425 430

Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn

435 440 445

Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp

450 455 460

Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly

465 470 475 480

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp

485 490 495

Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe

500 505 510

Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Ser His His His His

515 520 525

His His

530

<210> 70

<211> 530

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 70

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys

20 25 30

Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu

50 55 60

Lys Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala

65 70 75 80

Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser

85 90 95

Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr

100 105 110

Tyr Phe Cys Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala

275 280 285

Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr

290 295 300

Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Cys

305 310 315 320

Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr

325 330 335

Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser

340 345 350

Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

355 360 365

Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr

370 375 380

Trp Gly Glyn Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

405 410 415

Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu

420 425 430

Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn

435 440 445

Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp

450 455 460

Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly

465 470 475 480

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp

485 490 495

Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe

500 505 510

Gly Cys Gly Thr Lys Leu Glu Leu Lys Gly Gly Ser His His His His

515 520 525

His His

530

<210> 71

<211> 530

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 71

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys

20 25 30

Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Cys Leu

50 55 60

Lys Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala

65 70 75 80

Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser

85 90 95

Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr

100 105 110

Tyr Phe Cys Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala

275 280 285

Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr

290 295 300

Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Cys

305 310 315 320

Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr

325 330 335

Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser

340 345 350

Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

355 360 365

Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr

370 375 380

Trp Gly Glyn Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

405 410 415

Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu

420 425 430

Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn

435 440 445

Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp

450 455 460

Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly

465 470 475 480

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp

485 490 495

Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe

500 505 510

Gly Cys Gly Thr Lys Leu Glu Leu Lys Gly Gly Ser His His His His

515 520 525

His His

530

<210> 72

<211> 530

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 72

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala

275 280 285

Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr

290 295 300

Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Cys

305 310 315 320

Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr

325 330 335

Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser

340 345 350

Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

355 360 365

Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr

370 375 380

Trp Gly Glyn Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

405 410 415

Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu

420 425 430

Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn

435 440 445

Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp

450 455 460

Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly

465 470 475 480

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp

485 490 495

Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe

500 505 510

Gly Cys Gly Thr Lys Leu Glu Leu Lys Gly Gly Ser His His His His

515 520 525

His His

530

<210> 73

<211> 530

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 73

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Cys Leu

50 55 60

Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Phe Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala

275 280 285

Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr

290 295 300

Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Cys

305 310 315 320

Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr

325 330 335

Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser

340 345 350

Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

355 360 365

Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr

370 375 380

Trp Gly Glyn Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln

405 410 415

Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu

420 425 430

Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn

435 440 445

Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp

450 455 460

Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly

465 470 475 480

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp

485 490 495

Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe

500 505 510

Gly Cys Gly Thr Lys Leu Glu Leu Lys Gly Gly Ser His His His His

515 520 525

His His

530

<210> 74

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 74

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys

20 25 30

Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu

50 55 60

Lys Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala

65 70 75 80

Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser

85 90 95

Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr

100 105 110

Tyr Phe Cys Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

275 280 285

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

290 295 300

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

305 310 315 320

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

325 330 335

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

340 345 350

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

355 360 365

Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly

370 375 380

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala

405 410 415

Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser

420 425 430

Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro

435 440 445

Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr

450 455 460

Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp

465 470 475 480

Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu

485 490 495

Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser

500 505 510

Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 75

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 75

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys

20 25 30

Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Cys Leu

50 55 60

Lys Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala

65 70 75 80

Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser

85 90 95

Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr

100 105 110

Tyr Phe Cys Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

275 280 285

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

290 295 300

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

305 310 315 320

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

325 330 335

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

340 345 350

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

355 360 365

Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly

370 375 380

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala

405 410 415

Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser

420 425 430

Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro

435 440 445

Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr

450 455 460

Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp

465 470 475 480

Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu

485 490 495

Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser

500 505 510

Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 76

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 76

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

275 280 285

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

290 295 300

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

305 310 315 320

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

325 330 335

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

340 345 350

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

355 360 365

Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly

370 375 380

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala

405 410 415

Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser

420 425 430

Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro

435 440 445

Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr

450 455 460

Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp

465 470 475 480

Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu

485 490 495

Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser

500 505 510

Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 77

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 77

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Cys Leu

50 55 60

Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Phe Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

275 280 285

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

290 295 300

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

305 310 315 320

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

325 330 335

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

340 345 350

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

355 360 365

Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly

370 375 380

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala

405 410 415

Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser

420 425 430

Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro

435 440 445

Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr

450 455 460

Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp

465 470 475 480

Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu

485 490 495

Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser

500 505 510

Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 78

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 78

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys

20 25 30

Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Gly Leu

50 55 60

Lys Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala

65 70 75 80

Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser

85 90 95

Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr

100 105 110

Tyr Phe Cys Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

275 280 285

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

290 295 300

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

305 310 315 320

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

325 330 335

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

340 345 350

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

355 360 365

Asn Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys Gly Gly

370 375 380

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala

405 410 415

Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser

420 425 430

Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro

435 440 445

Gly Gln Cys Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr

450 455 460

Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp

465 470 475 480

Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu

485 490 495

Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser

500 505 510

Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 79

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 79

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys

20 25 30

Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Asn Tyr Gly Met Asn Trp Val Lys Gln Ala Pro Gly Lys Cys Leu

50 55 60

Lys Trp Met Gly Trp Ile Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala

65 70 75 80

Glu Glu Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser

85 90 95

Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr

100 105 110

Tyr Phe Cys Ala Arg Leu Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

275 280 285

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

290 295 300

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

305 310 315 320

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

325 330 335

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

340 345 350

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

355 360 365

Asn Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys Gly Gly

370 375 380

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala

405 410 415

Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser

420 425 430

Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro

435 440 445

Gly Gln Cys Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr

450 455 460

Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp

465 470 475 480

Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu

485 490 495

Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser

500 505 510

Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 80

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 80

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

275 280 285

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

290 295 300

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

305 310 315 320

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

325 330 335

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

340 345 350

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

355 360 365

Asn Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys Gly Gly

370 375 380

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala

405 410 415

Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser

420 425 430

Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro

435 440 445

Gly Gln Cys Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr

450 455 460

Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp

465 470 475 480

Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu

485 490 495

Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser

500 505 510

Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 81

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 81

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Cys Leu

50 55 60

Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

145 150 155 160

Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val

165 170 175

Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln

180 185 190

Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys

195 200 205

Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg

210 215 220

Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser

225 230 235 240

Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser

245 250 255

Tyr Pro Phe Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Ser Gly

260 265 270

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

275 280 285

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

290 295 300

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

305 310 315 320

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

325 330 335

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

340 345 350

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

355 360 365

Asn Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys Gly Gly

370 375 380

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala

405 410 415

Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser

420 425 430

Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro

435 440 445

Gly Gln Cys Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr

450 455 460

Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp

465 470 475 480

Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu

485 490 495

Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser

500 505 510

Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 82

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 82

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

20 25 30

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys

115 120 125

Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Gln

145 150 155 160

Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys

165 170 175

Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn

180 185 190

Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile

195 200 205

Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe Lys Gly Arg

210 215 220

Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile

225 230 235 240

Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Leu

245 250 255

Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

260 265 270

Val Ser Ser Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser

275 280 285

Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys

290 295 300

Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln

305 310 315 320

Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg

325 330 335

Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr

340 345 350

Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

355 360 365

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His

370 375 380

Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

405 410 415

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

420 425 430

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

435 440 445

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

450 455 460

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

465 470 475 480

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

485 490 495

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

500 505 510

Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 83

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 83

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

20 25 30

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Cys Gly Thr Lys

115 120 125

Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Gln

145 150 155 160

Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys

165 170 175

Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn

180 185 190

Trp Val Lys Gln Ala Pro Gly Lys Cys Leu Lys Trp Met Gly Trp Ile

195 200 205

Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe Lys Gly Arg

210 215 220

Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile

225 230 235 240

Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Leu

245 250 255

Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

260 265 270

Val Ser Ser Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser

275 280 285

Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys

290 295 300

Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln

305 310 315 320

Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg

325 330 335

Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr

340 345 350

Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

355 360 365

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His

370 375 380

Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

405 410 415

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

420 425 430

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

435 440 445

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

450 455 460

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

465 470 475 480

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

485 490 495

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

500 505 510

Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 84

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 84

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

20 25 30

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

145 150 155 160

Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys

165 170 175

Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Asn

180 185 190

Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Asn Ile

195 200 205

Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys

210 215 220

Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu

225 230 235 240

Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Ser

245 250 255

Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

260 265 270

Val Ser Ser Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser

275 280 285

Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys

290 295 300

Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln

305 310 315 320

Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg

325 330 335

Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr

340 345 350

Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

355 360 365

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His

370 375 380

Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

405 410 415

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

420 425 430

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

435 440 445

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

450 455 460

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

465 470 475 480

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

485 490 495

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

500 505 510

Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 85

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 85

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

20 25 30

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Cys Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

145 150 155 160

Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys

165 170 175

Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Asn

180 185 190

Trp Val Lys Gln Arg Pro Gly Gln Cys Leu Glu Trp Ile Gly Asn Ile

195 200 205

Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys

210 215 220

Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu

225 230 235 240

Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Ser

245 250 255

Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

260 265 270

Val Ser Ser Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser

275 280 285

Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys

290 295 300

Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln

305 310 315 320

Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg

325 330 335

Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr

340 345 350

Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

355 360 365

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His

370 375 380

Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

405 410 415

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

420 425 430

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

435 440 445

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

450 455 460

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

465 470 475 480

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

485 490 495

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

500 505 510

Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 86

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 86

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

20 25 30

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys

115 120 125

Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Gln

145 150 155 160

Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys

165 170 175

Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn

180 185 190

Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile

195 200 205

Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe Lys Gly Arg

210 215 220

Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile

225 230 235 240

Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Leu

245 250 255

Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

260 265 270

Val Ser Ser Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser

275 280 285

Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys

290 295 300

Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln

305 310 315 320

Arg Pro Gly Gln Cys Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg

325 330 335

Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr

340 345 350

Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

355 360 365

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His

370 375 380

Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

405 410 415

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

420 425 430

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

435 440 445

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

450 455 460

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

465 470 475 480

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

485 490 495

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

500 505 510

Asn Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 87

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 87

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

20 25 30

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Cys Gly Thr Lys

115 120 125

Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Gln

145 150 155 160

Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys

165 170 175

Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn

180 185 190

Trp Val Lys Gln Ala Pro Gly Lys Cys Leu Lys Trp Met Gly Trp Ile

195 200 205

Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe Lys Gly Arg

210 215 220

Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile

225 230 235 240

Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Leu

245 250 255

Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

260 265 270

Val Ser Ser Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser

275 280 285

Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys

290 295 300

Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln

305 310 315 320

Arg Pro Gly Gln Cys Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg

325 330 335

Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr

340 345 350

Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

355 360 365

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His

370 375 380

Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

405 410 415

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

420 425 430

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

435 440 445

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

450 455 460

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

465 470 475 480

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

485 490 495

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

500 505 510

Asn Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 88

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 88

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

20 25 30

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

145 150 155 160

Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys

165 170 175

Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Asn

180 185 190

Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Asn Ile

195 200 205

Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys

210 215 220

Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu

225 230 235 240

Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Ser

245 250 255

Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

260 265 270

Val Ser Ser Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser

275 280 285

Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys

290 295 300

Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln

305 310 315 320

Arg Pro Gly Gln Cys Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg

325 330 335

Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr

340 345 350

Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

355 360 365

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His

370 375 380

Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

405 410 415

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

420 425 430

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

435 440 445

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

450 455 460

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

465 470 475 480

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

485 490 495

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

500 505 510

Asn Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 89

<211> 535

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 89

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

20 25 30

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Cys Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

145 150 155 160

Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys

165 170 175

Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Asn

180 185 190

Trp Val Lys Gln Arg Pro Gly Gln Cys Leu Glu Trp Ile Gly Asn Ile

195 200 205

Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys

210 215 220

Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu

225 230 235 240

Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Ser

245 250 255

Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

260 265 270

Val Ser Ser Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser

275 280 285

Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys Lys

290 295 300

Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys Gln

305 310 315 320

Arg Pro Gly Gln Cys Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg

325 330 335

Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr

340 345 350

Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr

355 360 365

Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His

370 375 380

Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser

385 390 395 400

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

405 410 415

Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser

420 425 430

Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser

435 440 445

Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys

450 455 460

Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg

465 470 475 480

Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser

485 490 495

Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser

500 505 510

Asn Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu Glu Leu Lys Gly Gly

515 520 525

Ser His His His His His His His

530 535

<210> 90

<211> 540

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 90

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

20 25 30

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys

115 120 125

Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Gln

145 150 155 160

Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys

165 170 175

Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn

180 185 190

Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp Ile

195 200 205

Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe Lys Gly Arg

210 215 220

Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile

225 230 235 240

Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Leu

245 250 255

Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

260 265 270

Val Ser Ser Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser

275 280 285

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

290 295 300

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

305 310 315 320

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

325 330 335

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

340 345 350

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

355 360 365

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu

370 375 380

Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

405 410 415

Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met

420 425 430

Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp

435 440 445

Val Lys Gln Arg Pro Gly Gln Cys Leu Glu Trp Ile Gly Tyr Ile Asn

450 455 460

Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala

465 470 475 480

Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser

485 490 495

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr

500 505 510

Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

515 520 525

Val Ser Ser Gly Gly Ser His His His His His His

530 535 540

<210> 91

<211> 540

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 91

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

20 25 30

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp Tyr Ser Tyr Pro Leu Thr Phe Gly Cys Gly Thr Lys

115 120 125

Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile Gln

145 150 155 160

Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val Lys

165 170 175

Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr Gly Met Asn

180 185 190

Trp Val Lys Gln Ala Pro Gly Lys Cys Leu Lys Trp Met Gly Trp Ile

195 200 205

Asn Thr Asn Thr Gly Glu Pro Thr Tyr Ala Glu Glu Phe Lys Gly Arg

210 215 220

Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln Ile

225 230 235 240

Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Leu

245 250 255

Gly Phe Gly Asn Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr

260 265 270

Val Ser Ser Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser

275 280 285

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

290 295 300

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

305 310 315 320

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

325 330 335

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

340 345 350

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

355 360 365

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu

370 375 380

Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

405 410 415

Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met

420 425 430

Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp

435 440 445

Val Lys Gln Arg Pro Gly Gln Cys Leu Glu Trp Ile Gly Tyr Ile Asn

450 455 460

Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala

465 470 475 480

Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser

485 490 495

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr

500 505 510

Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

515 520 525

Val Ser Ser Gly Gly Ser His His His His His His

530 535 540

<210> 92

<211> 540

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 92

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

20 25 30

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

145 150 155 160

Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys

165 170 175

Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Asn

180 185 190

Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Asn Ile

195 200 205

Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys

210 215 220

Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu

225 230 235 240

Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Ser

245 250 255

Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

260 265 270

Val Ser Ser Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser

275 280 285

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

290 295 300

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

305 310 315 320

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

325 330 335

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

340 345 350

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

355 360 365

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu

370 375 380

Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

405 410 415

Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met

420 425 430

Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp

435 440 445

Val Lys Gln Arg Pro Gly Gln Cys Leu Glu Trp Ile Gly Tyr Ile Asn

450 455 460

Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala

465 470 475 480

Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser

485 490 495

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr

500 505 510

Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

515 520 525

Val Ser Ser Gly Gly Ser His His His His His His

530 535 540

<210> 93

<211> 540

<212> PRT

<213> artificial

<220>

<223> artificial Antibody

<400> 93

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val

20 25 30

Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu

35 40 45

Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys

50 55 60

Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe

85 90 95

Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr

100 105 110

Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe Gly Cys Gly Thr Lys

115 120 125

Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln

145 150 155 160

Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys

165 170 175

Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Ile Asn

180 185 190

Trp Val Lys Gln Arg Pro Gly Gln Cys Leu Glu Trp Ile Gly Asn Ile

195 200 205

Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys

210 215 220

Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu

225 230 235 240

Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Thr Arg Ser

245 250 255

Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

260 265 270

Val Ser Ser Ser Gly Gly Gly Gly Ser Gln Ile Val Leu Thr Gln Ser

275 280 285

Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys

290 295 300

Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser

305 310 315 320

Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser

325 330 335

Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser

340 345 350

Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys

355 360 365

Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Cys Gly Thr Lys Leu

370 375 380

Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

385 390 395 400

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu

405 410 415

Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met

420 425 430

Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp

435 440 445

Val Lys Gln Arg Pro Gly Gln Cys Leu Glu Trp Ile Gly Tyr Ile Asn

450 455 460

Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala

465 470 475 480

Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser

485 490 495

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr

500 505 510

Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr

515 520 525

Val Ser Ser Gly Gly Ser His His His His His His

530 535 540

<210> 94

<211> 119

<212> PRT

<213> Mus musculus

<400> 94

Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr

20 25 30

Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe

50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Thr Leu Thr Val Ser Ser

115

<210> 95

<211> 116

<212> PRT

<213> Mus musculus

<400> 95

Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys

1 5 10 15

Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His

20 25 30

Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile

35 40 45

Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys

50 55 60

Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu

65 70 75 80

Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr

85 90 95

Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser

115

<210> 96

<211> 103

<212> PRT

<213> Mus musculus

<400> 96

Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val

1 5 10 15

Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr

20 25 30

Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser

35 40 45

Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly

50 55 60

Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala

65 70 75 80

Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala

85 90 95

Gly Thr Lys Leu Glu Leu Lys

100

<210> 97

<211> 107

<212> PRT

<213> Mus musculus

<400> 97

Asp Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Leu Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 98

<211> 106

<212> PRT

<213> Mus musculus

<400> 98

Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly Glu

1 5 10 15

Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His

20 25 30

Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Leu Ile Tyr Ser

35 40 45

Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg Gly

50 55 60

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu Asp

65 70 75 80

Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 99

<211> 106

<212> PRT

<213> Mus musculus

<400> 99

Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly Glu

1 5 10 15

Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His

20 25 30

Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Trp Ile Tyr Ser

35 40 45

Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg Gly

50 55 60

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu Asp

65 70 75 80

Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 100

<211> 106

<212> PRT

<213> Mus musculus

<400> 100

Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly Glu

1 5 10 15

Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met His

20 25 30

Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Ser Ile Tyr Ser

35 40 45

Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg Gly

50 55 60

Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu Asp

65 70 75 80

Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 101

<211> 513

<212> PRT

<213> artificial

<220>

<223> bispecific molecule 107

<400> 101

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg

20 25 30

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe

35 40 45

Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly

130 135 140

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu

145 150 155 160

Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr

165 170 175

Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln

180 185 190

Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys

195 200 205

Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly

245 250 255

Thr Lys Leu Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

260 265 270

Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys Ile

275 280 285

Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp

290 295 300

Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile Gly Leu Ile Asn

305 310 315 320

Pro Tyr Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe Lys Gly Lys Ala

325 330 335

Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu

340 345 350

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Asp Tyr

355 360 365

Gly Phe Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

370 375 380

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

385 390 395 400

Asp Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly

405 410 415

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

420 425 430

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Cys Ile Tyr

435 440 445

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg

450 455 460

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu

465 470 475 480

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp

485 490 495

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys His His His His His

500 505 510

His

<210> 102

<211> 513

<212> PRT

<213> artificial

<220>

<223> bispecific molecule 123

<400> 102

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg

20 25 30

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe

35 40 45

Thr Arg Tyr Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp

115 120 125

Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly

130 135 140

Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu

145 150 155 160

Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr

165 170 175

Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln

180 185 190

Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys

195 200 205

Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr

210 215 220

Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr

225 230 235 240

Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly

245 250 255

Thr Lys Leu Glu Leu Lys Ser Gly Gly Gly Gly Ser Glu Val Gln Leu

260 265 270

Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Met Lys Ile

275 280 285

Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Thr Met Asn Trp

290 295 300

Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile Gly Leu Ile Asn

305 310 315 320

Pro Tyr Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe Lys Gly Lys Ala

325 330 335

Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Leu

340 345 350

Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Asp Tyr

355 360 365

Gly Phe Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser

370 375 380

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

385 390 395 400

Asp Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly

405 410 415

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

420 425 430

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Leu Ile Tyr

435 440 445

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg

450 455 460

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu

465 470 475 480

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp

485 490 495

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys His His His His His

500 505 510

His

<210> 103

<211> 520

<212> PRT

<213> artificial

<220>

<223> bispecific molecule 124

<400> 103

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg

20 25 30

Pro Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Trp Ile Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Asn Ile Tyr Pro Ser Asp Ser Tyr Thr Asn Tyr Asn

65 70 75 80

Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Thr Arg Ser Trp Arg Gly Asn Ser Phe Asp Tyr Trp Gly

115 120 125

Gly Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

130 135 140

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro

145 150 155 160

Ser Ser Leu Thr Val Thr Ala Gly Glu Lys Val Thr Met Ser Cys Lys

165 170 175

Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr

180 185 190

Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp

195 200 205

Ala Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly

210 215 220

Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp

225 230 235 240

Leu Ala Val Tyr Tyr Cys Gln Asn Asp Tyr Ser Tyr Pro Phe Thr Phe

245 250 255

Gly Ser Gly Thr Lys Leu Glu Ile Lys Ser Gly Gly Gly Gly Ser Asp

260 265 270

Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser

275 280 285

Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr

290 295 300

Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly

305 310 315 320

Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys

325 330 335

Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met

340 345 350

Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala

355 360 365

Arg Tyr Tyr Asp Asp His Tyr Ser Leu Asp Tyr Trp Gly Gln Gly Thr

370 375 380

Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly Gly

385 390 395 400

Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr Gln Ser Pro

405 410 415

Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg

420 425 430

Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly

435 440 445

Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly

450 455 460

Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu

465 470 475 480

Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln

485 490 495

Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu

500 505 510

Leu Lys His His His His His His His

515 520

<210> 104

<211> 20

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker

<400> 104

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 105

<211> 25

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker

<400> 105

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<210> 106

<211> 18

<212> PRT

<213> Artificial Sequence

<220>

<223> Linker

<400> 106

Gly Gly Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly

1 5 10 15

Gly Ser

<210> 107

<211> 6

<212> PRT

<213> Artificial Sequence

<220>

<223> His Tag

<400> 107

His His His His His His His

15

<210> 108

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> His Tag

<400> 108

Gly Gly Ser His His His His His His

15

<---

Claims (32)

1. A bispecific claudin 6 (CLDN6) and CD3 binding agent containing a first binding domain that binds to CLDN6 and a second binding domain that binds to CD3, wherein the bispecific agent comprises an immunoglobulin heavy chain (VH) variable domain with specificity for claudin antigen (VH(CLDN6)), an immunoglobulin light chain variable domain (VL) with specificity for claudin antigen (VL(CLDN6)), a heavy chain variable domain an immunoglobulin (VH) with specificity for CD3 (VH(CD3)) and an immunoglobulin light chain (VL) variable domain with specificity for CD3 (VL(CD3)), characterized in that (i) said VH(CLDN6) contains an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 20, 22, 24 and 26, or a fragment thereof, or a variant of said amino acid sequence or said fragment, where the fragment contains complementarity-determining regions CDR1, CDR2 and CDR3 of the specified VH, and VL(CLDN6) contains an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 21, 23, 25 and 27-29, or a fragment thereof, or a variant of the specified amino acid sequence or specified fragment, where the fragment contains the complementarity-determining regions CDR1, CDR2 and CDR3 of the specified VL, and (ii) said VH(CD3) contains an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 30, 32, 34, 36 and 95, or a fragment thereof, or a variant of said amino acid sequence or said fragment, where the fragment contains the complementarity determining regions CDR1, CDR2 and CDR3 of the specified VH, and VL(CD3) contains an amino acid sequence selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 31, 33, 35, 37 and 96, or a fragment thereof, or a variant of the specified amino acid sequence or the specified fragment, where the fragment contains complementarity-determining sections CDR1, CDR2 and CDR3 of the specified VL, where option includes: (i) an amino acid sequence that is at least 98% identical to the specified amino acid sequence; (ii) one or two conservative amino acid residue substitutions. 2. The bispecific agent according to claim 1, wherein the binding agent is a bispecific molecule. 3. The bispecific agent of claim 2, wherein the bispecific molecule is a bispecific antibody. 4. The bispecific agent of claim 3, wherein the bispecific antibody is a bispecific single chain antibody. 5. Bispecific agent according to any one of paragraphs. 1-4, wherein binding of said bispecific agent to CD3 on T cells results in proliferation and/or activation of said T cells, wherein said activated T cells preferentially release cytotoxic factors such as perforins and granzymes and initiate cytolysis and apoptosis cancer cells. 6. Bispecific agent according to any one of paragraphs. 1-5, where the binding agent is in the format of a full-length antibody, antibody fragment, diabody, which contains a heavy chain variable domain connected to a light chain variable domain on the same polypeptide chain in such a way that the two domains do not form a pair. 7. The bispecific agent of claim 6 wherein the diabody contains two polypeptide chains, wherein one polypeptide contains VH(CLDN6) and VL(CD3) and the other polypeptide chain contains VH(CD3) and VL(CLDN6). 8. Bispecific agent according to any one of paragraphs. 1-6, where the bispecific agent is in the format of a bispecific single chain antibody that consists of two scFv molecules connected via a linker peptide. 9. The bispecific agent of claim 8 wherein the heavy chain variable regions (VH) and the corresponding light chain variable regions (VL) are N-terminal to C-terminal in the order VH(CLDN6)-VL(CLDN6)-VH (CD3)-VL(CD3), VH(CD3)-VL(CD3)-VH(CLDN6)-VL(CLDN6) or VH(CD3)-VL(CD3)-VL(CLDN6)-VH(CLDN6). 10. The bispecific agent of claim 9, wherein said heavy chain variable regions (VH) and corresponding light chain variable regions (VL) are connected via a long peptide linker, preferably a peptide linker comprising the amino acid sequence represented by SEQ ID NOs: 46, 47 or 104. 11. A bispecific agent according to claim 9 or 10, wherein said two VH-VL or VL-VH scFv units are connected via a short peptide linker, preferably a peptide linker containing the amino acid sequence represented by SEQ ID NO: 48 or 49. 12. Bispecific agent according to any one of paragraphs. 1-11, where the specified bispecific agent contains an amino acid sequence selected from the group consisting of SEQ ID NO: 42, 43, 44 and 45, or a fragment or variant. 13. Bispecific agent according to any one of paragraphs. 1-12, wherein the bispecific agent comprises an N-terminal secretion signal and/or a C-terminal epitope histidine tag, preferably an epitope tag containing six histidine residues. 14. Recombinant nucleic acid that encodes a bispecific agent according to any one of paragraphs. 1-13. 15. Recombinant nucleic acid according to claim 14, which is in the form of a vector or in the form of RNA. 16. A host cell for expressing a bispecific agent encoded by nucleic acids, containing a recombinant nucleic acid according to claim 14 or 15. 17. The use of a bispecific agent according to any one of paragraphs. 1-13, a recombinant nucleic acid according to claim 14 or 15, or a host cell according to claim 16 for the manufacture of a pharmaceutical composition for the treatment or prevention of cancer, characterized by cancer cells expressing CLDN6. 18. Pharmaceutical composition for the treatment or prevention of cancer, characterized by cancer cells expressing CLDN6, containing one of the following: i) a bispecific agent according to any one of paragraphs. 1-13, ii) a recombinant nucleic acid according to claim 14 or 15, iii) recombinant nucleic acids encoding a bispecific agent according to any one of paragraphs. 1-13, iv) a host cell according to claim 16 or v) a host cell containing recombinant nucleic acids encoding a bispecific agent according to any one of paragraphs. 1-13. 19. The pharmaceutical composition of claim 18, wherein the recombinant nucleic acids are in vector form or in RNA form. 20. A method for treating or preventing a cancer characterized by cancer cells expressing CLDN6, comprising administering to a patient a pharmaceutical composition according to claim 18 or 19. 21. The use of claim 17, the pharmaceutical composition of claim 18 or 19, or the method of claim 20, wherein said cancer is selected from the group consisting of bladder cancer; ovarian cancer, in particular ovarian adenocarcinoma and ovarian teracarcinoma; lung cancer, including small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC), in particular squamous cell lung cancer and adenocarcinoma; stomach cancer; breast cancer; liver cancer; pancreatic cancer; skin cancer, in particular basal cell carcinoma and squamous cell carcinoma; malignant melanoma; head and neck cancer, in particular malignant pleomorphic adenoma; sarcomas, in particular synovial sarcomas and carcinosarcomas; bile duct cancer; bladder cancer, in particular transitional cell carcinoma and papillary cancer; kidney cancer, in particular renal cell carcinoma, including clear cell carcinoma of the kidney and papillary carcinoma of the kidney; colon cancer; cancer of the small intestine, including cancer of the ileum, in particular adenocarcinoma of the small intestine and adenocarcinoma of the ileum; embryonic testicular carcinoma; placental choriocarcinoma; cervical cancer; testicular cancer, in particular testicular seminoma, testicular teratoma and fetal testicular cancer; uterine cancer; germinomas such as teratocarcinoma or embryonic carcinoma, in particular testicular germ cell tumors and their metastatic forms.

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