WO2024005422A1 - B7-h6 variants with improved binding affinity for nkp30 - Google Patents

Abstract

The present invention relates to B7-H6 variants with improved binding affinity for NKp30. The B7-H6 variants of the present invention can increase the activation of natural killer (NK) cells due to significantly increased binding affinity for NKp30, which is an activating receptor of NK cells, compared to wild type, are easy to penetrate the tumor microenvironment due to having a much smaller size than antibodies, and are easily produced, and thus can be effectively used alone or in combination with various immunotherapeutic agents to treat cancer or infectious diseases.

Description

B7-H6 variant with improved NKP30 binding affinity

The present invention relates to B7-H6 variants with improved binding to NKp30.

Medicines for cancer treatment are largely divided into small molecule medicines and high molecule medicines. Compared to low molecule medicines, which have relatively greater side effects due to lack of specificity, high molecule medicines with specificity are receiving more attention as therapeutic agents. Cancer cells express immune checkpoint proteins on the cell surface, which are used by normal cells to suppress immune cell activation in order to avoid the killing mechanism of immune cells. Recently, immunotherapy has been used as a method to treat cancer. Research on checkpoint inhibitor proteins is actively underway. Patients who respond to immune checkpoint inhibitors have fewer side effects during the treatment process, and their use is increasing explosively as superior treatment effects are reported compared to existing anticancer drugs for various cancer types. However, more than half of patients still receive clinically approved immunizations. It does not respond to checkpoint inhibitors, and some early responders have also been observed to show resistance, with the cancer progressing again after treatment. Since the cause of resistance to immune checkpoint inhibitors varies depending on the tumor immunological characteristics of each patient, new biomarkers that can select patients suitable for treatment are needed to provide effective treatment and reduce medical costs, and immunostimulatory drugs are used in many preclinical and clinical research cases. It has been reported that combination therapy using , improves the patient's response rate, so effective immunostimulating treatments are also urgently needed. However, since antibodies are macromolecular proteins with a molecular weight of 150,000, they have the disadvantage of being difficult to penetrate into cancer tissue. Accordingly, for more effective treatment, there is a need for protein therapeutics that are much smaller than antibodies, can easily penetrate into cancer tissue, and target new immune checkpoint proteins to overcome low response rates.

Immune cells express immune checkpoint proteins on the cell surface that are used to suppress and activate themselves. Among various immune checkpoint receptors, it was confirmed that B7-H6 binds to NKp30, an activation receptor expressed on the surface of NK cells (Natural killer cells), thereby activating NK cells. NK cells are innate immune cells that detect and eliminate cancer cells and virus-infected cells without specific antigens, regulate immune and inflammatory responses by inducing the activity of other immune cells, and suppress proliferation, recurrence, and metastasis of cancer cells. Wild-type B7-H6, which activates it, binds to NKp30 with very low affinity (equilibrium dissociation constant = ~1.0 μM), making its use difficult. Therefore, in order to overcome the low response rate of existing immune checkpoint inhibitor treatments and induce effective immune checkpoint activation of NK cells that recognize and eliminate cancer cells and virus-infected cells without specific antigens, the binding force with the activation receptor, NKp30, is increased. There is a great need to discover the B7-H6 variant.

Meanwhile, cell therapy is a medicine that induces regeneration using living cells to restore damaged or diseased cells/tissues, using physical, chemical, and biological methods such as culturing, proliferating, or selecting autologous, allogeneic, or xenogeneic cells in vitro. It refers to medicines manufactured by manipulating. Among them, immunomodulatory cell therapy refers to medicines used to treat diseases by activating the body's immune response using immune cells such as dendritic cells, natural killer cells, and T cells. . Currently, immunomodulatory cell therapies are mainly being developed for cancer treatment indications. Since they achieve therapeutic effects by activating immune function by administering immune cells directly to the patient, they are used in existing cancer treatments such as surgery, anticancer drugs, or radiation therapy. It is a field that is expected to occupy a major part of new bio drugs in the future due to its differentiated treatment mechanism and efficacy. Depending on the type of immunomodulatory cell therapy, the physical and chemical characteristics of the antigen introduced into cells are different, and when a foreign gene is introduced into immune cells using a viral vector, it has the characteristics of both a cell therapy and a gene therapy. Immunomodulatory cell therapy activates various immune cells, such as PBMC (pheripheral blood mononuclear cells), T cells, and natural killer cells (NK cells), isolated from patients through apheresis, with various antibodies and cytokines, and then administers them in vitro. This is done by proliferating them in a cell and injecting them back into the patient, or by injecting immune cells into which genes such as TCR (T-Cell Receptor) or CAR (Chimeric Antigen Receptor) are introduced. In particular, they can be mass-produced and frozen. Immunotherapy using natural killer cells is being researched. Natural killer cells are lymphoid cells that make up about 15% of peripheral blood lymphocytes and play an important role in the innate immune response. Natural killer cells activate dendritic cells and induce cytotoxic T lymphocytes (CTL) to respond specifically to tumors to eliminate tumor cells. Natural killer cells directly kill malignant tumors such as sarcoma, myeloma, carcinoma, lymphomas, and leukemia. However, most natural killer cells present in the body of normal people exist in an inactive state, and activated natural killer cells are needed to remove tumors. In addition, in the case of natural killer cells present in the body of cancer patients, functional defects in natural killer cells exist due to the immune evasion mechanism of cancer cells. Therefore, in order to use natural killer cells as a therapeutic agent, it is very important to activate natural killer cells. Additionally, since the number of natural killer cells present in the body is limited, it is essential to develop technology to multiply and freeze natural killer cells in the blood of normal people or patients. The in vitro expansion method is used to proliferate natural killer cells in large quantities, using peripheral blood lymphocytes (PBMC), cord blood (CB), or human-induced pluripotent stem cells. Research is being conducted on mass culturing methods of natural killer cells using cells as raw materials. For the in vitro expansion culture of natural killer cells, PBMC, CD3 ^- cells, CD3-CD56 ⁺ cells, and CD56 ⁺ cells are used as raw material cells, and natural killer cell growth factors include IL-2, IL-12, IL-15, and IL. Cytokines such as -21, LPS (Goodier et al., J. Immunol. 165(1):139-147, 2000), and OKT-3 antibody that stimulates CD3 (Condiotti et al., Experimental Hematol. 29( 1):104-113, 2001), but the proliferation rate is such that it is difficult to commercialize natural killer cells as a treatment.

The purpose of the present invention is to provide a B7-H6 variant with increased binding ability to natural killer cells.

Additionally, an object of the present invention is to provide a natural killer cell activator.

Additionally, an object of the present invention is to provide bispecific or multispecific antibodies.

Additionally, an object of the present invention is to provide a pharmaceutical composition for treating or preventing cancer.

Additionally, an object of the present invention is to provide a method for in vitro proliferation of activated natural killer cells.

Additionally, an object of the present invention is to provide a use of the B7-H6 variant or a bispecific or multispecific antibody containing it for the prevention or treatment of cancer.

Additionally, an object of the present invention is to provide a method for treating cancer.

To achieve the above object, the present invention provides a B7-H6 variant with increased binding affinity to NKp30 (Natural cytotoxicity triggering receptor 3).

Additionally, the present invention provides a natural killer cell activator comprising the B7-H6 variant.

Additionally, the present invention provides a bispecific or multispecific antibody comprising a portion that binds to the B7-H6 variant and a target antigen.

Additionally, the present invention provides a pharmaceutical composition for the treatment or prevention of cancer comprising the B7-H6 variant or a bispecific or multispecific antibody.

Additionally, the present invention provides a method for in vitro proliferation of activated natural killer cells.

Additionally, the present invention provides a use of the B7-H6 variant, or bispecific or multispecific antibody, for preventing or treating cancer.

In addition, the present invention provides a method of treating cancer comprising administering the B7-H6 variant, or bispecific or multispecific antibody, in a pharmaceutically effective amount to an individual suffering from cancer.

The B7-H6 variant of the present invention has a significantly increased binding force to NKp30, an activation receptor for natural killer cells (NK cells), compared to the wild type, which can increase natural killer cell activation, and its size is significantly smaller than that of the antibody, so it can be used in the tumor microenvironment. Since it is easy to penetrate and produce, it can be usefully used alone or in combination with various immunotherapeutics to treat cancer or infectious diseases.

Figure 1 is a diagram showing an SDS-PAGE gel photograph of an expression vector for NKp30-streptavidin protein and purified protein.

Figure 2 is a diagram showing the results of flow cytometry for selecting the B7-H6 display method.

Figure 3 is a diagram showing the results of amino acid sequence analysis of the constructed initial library.

Figure 4 is a diagram showing the results of enrichment verification of clones with improved NKp30 binding ability according to the screening process using flow cytometry.

Figure 5 is a diagram showing the results of analysis of the binding affinity of B7-H6 variants with NKp30 obtained through flow cytometry.

Figure 6 is a diagram showing an SDS-PAGE gel photograph of purified B7-H6 variant-Fc fusion proteins.

Figure 7 is a diagram showing the results of ELISA analysis of the NKp30 binding capacity of B7-H6 variant-Fc fusion proteins.

Figure 8 is a diagram showing the results of amino acid sequence analysis of the constructed B7-H6 focused library.

Figure 9 is a diagram showing the results of verifying the amplification of B7-H6 variant clones with high NKp30 affinity according to the screening process using flow cytometry.

Figure 10 is a diagram showing the results of analysis of the binding affinity of B7-H6 variants to NKp30 using flow cytometry.

Figure 11 is a diagram showing an SDS-PAGE gel photograph of purified B7-H6 variant-Fc fusion proteins.

Figure 12 is a diagram showing the results of ELISA analysis of the NKp30 binding ability of the B7-H6 variant-Fc fusion proteins of the present invention.

Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following embodiments are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.

In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

All technical terms used in the present invention, unless otherwise defined, are used with the same meaning as commonly understood by a person skilled in the art in the field related to the present invention. In addition, preferred methods and samples are described in this specification, but similar or equivalent methods are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are hereby incorporated by reference.

Throughout this specification, the usual one- and three-letter codes for naturally occurring amino acids are used, as well as generally acceptable codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. A three-character code is used. Additionally, amino acids referred to by abbreviations in the present invention are described according to the IUPAC-IUB nomenclature as follows:

Alanine: A, Arginine: R, Asparagine: N, Aspartic Acid: D, Cysteine: C, Glutamic Acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine : M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y and valine: V.

In one aspect, the present invention relates to the 31st, 32nd, 37th, 40th, 51st, 53rd, 57th, 60th, One or more amino acids selected from the group consisting of the 67th, 86th, 101st, 102nd, 108th, 114th, 129th, 136th, 142nd and 143rd amino acids are substituted with a sequence different from the wild type amino acid. , relates to a B7-H6 variant or fragment thereof with increased binding to NKp30 (Natural cytotoxicity triggering receptor 3).

In one embodiment, the amino acid of wild-type B7-H6 may include the amino acid sequence of SEQ ID NO: 1, and the amino acid position may be based on the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the B7-H6 variant of the invention is M31I, A32T, I37T, I37F, L40Q, F51I, F51S, F51L, F51Y, F51T, F51H, F51Q, F51K, F51R, S53G, N57D, S60I, S60Y, S60T , S60H, S60L, W67R, Q86L, K101E, S102C, S102R, R108M, L114M, L129M, Q136R, S142N and P143S. It may include any one or more amino acid substitutions selected from the group consisting of.

In one embodiment, the B7-H6 variant of the present invention may be a variant comprising an amino acid mutation in the ectodomain region (SEQ ID NO: 2), which is a region exposed to the outside of the cell of all amino acids of wild-type B7-H6. .

In one embodiment, the B7-H6 variant of the present invention may be B5 containing the amino acid substitutions F51S and S60I, and may include an extracellular domain region (SEQ ID NO: 3) containing the amino acid substitutions, which is SEQ ID NO: It can be encoded as a nucleic acid molecule containing the nucleotide sequence of 4.

In one embodiment, the B7-H6 variant of the present invention may be B6 containing the amino acid substitution N57D, and may include an extracellular domain region (SEQ ID NO: 5) containing the amino acid substitution, which is of SEQ ID NO: 6. It can be encoded as a nucleic acid molecule containing a base sequence.

In one embodiment, the B7-H6 variant of the present invention may be B7 containing the amino acid substitutions K101E and S102R, and may include an extracellular domain region (SEQ ID NO: 7) containing the amino acid substitutions, which is SEQ ID NO: It can be encoded as a nucleic acid molecule containing the nucleotide sequence of 8.

In one embodiment, the B7-H6 variant of the present invention may be B8 containing the amino acid substitutions A32T and S60I, and may include an extracellular domain region (SEQ ID NO: 9) containing the amino acid substitutions, which is SEQ ID NO: It can be encoded as a nucleic acid molecule containing 10 base sequences.

In one embodiment, the B7-H6 variant of the present invention may be B9 containing amino acid substitutions A32T, L40Q and S60I, and may include an extracellular domain region (SEQ ID NO: 11) containing the amino acid substitutions, which It can be encoded as a nucleic acid molecule containing the base sequence of SEQ ID NO: 12.

In one embodiment, the B7-H6 variant of the present invention may be B14 containing the amino acid substitutions F51L and S60I, and may include an extracellular domain region (SEQ ID NO: 13) containing the amino acid substitutions, which is SEQ ID NO: It can be encoded as a nucleic acid molecule containing 14 base sequences.

In one embodiment, the B7-H6 variant of the present invention may be B16 containing the amino acid substitution K101E, and may include an extracellular domain region (SEQ ID NO: 15) containing the amino acid substitution, which is of SEQ ID NO: 16 It can be encoded as a nucleic acid molecule containing a base sequence.

In one embodiment, the B7-H6 variant of the present invention may be B19 comprising amino acid substitutions A32T, W67R, Q86L, K101E and L129M, and may include an extracellular domain region (SEQ ID NO: 17) comprising the amino acid substitutions. It can be encoded with a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 18.

In one embodiment, the B7-H6 variant of the present invention may be B23 containing amino acid substitutions S60I, L114M and S142N, and may include an extracellular domain region (SEQ ID NO: 19) containing the amino acid substitutions, which It can be encoded as a nucleic acid molecule containing the base sequence of SEQ ID NO: 20.

In one embodiment, the B7-H6 variant of the present invention may be B29 containing amino acid substitutions M31I, A32T, F51I, S60I, S102C and R108M, and the extracellular domain region (SEQ ID NO: 21) containing the amino acid substitutions. It may be encoded by a nucleic acid molecule containing the nucleotide sequence of SEQ ID NO: 22.

In one embodiment, the B7-H6 variant of the present invention may be B35 containing amino acid substitutions A32T, S60I, K101E and P143S, and may include an extracellular domain region (SEQ ID NO: 23) containing the amino acid substitutions, , which can be encoded as a nucleic acid molecule containing the base sequence of SEQ ID NO: 24.

In one embodiment, the B7-H6 variant of the present invention may be B40 containing the amino acid substitutions I37T, Q86L and K101E, and may include an extracellular domain region (SEQ ID NO: 25) containing the amino acid substitutions, which It can be encoded as a nucleic acid molecule containing the base sequence of SEQ ID NO: 26.

In one embodiment, the B7-H6 variant of the present invention may be B41 containing amino acid substitutions A32T, S53G, S60I and Q136R, and may include an extracellular domain region (SEQ ID NO: 27) containing the amino acid substitutions, , which can be encoded as a nucleic acid molecule containing the base sequence of SEQ ID NO: 28.

In one embodiment, the B7-H6 variant of the present invention may be B47 containing the amino acid substitutions F51Y and S60I, and may include an extracellular domain region (SEQ ID NO: 29) containing the amino acid substitutions, which is SEQ ID NO: It can be encoded as a nucleic acid molecule containing 30 base sequences.

In one embodiment, the B7-H6 variant of the present invention may be B52 containing the amino acid substitutions S60I and K101E, and may include an extracellular domain region (SEQ ID NO: 31) containing the amino acid substitutions, which is SEQ ID NO: It can be encoded as a nucleic acid molecule containing 32 base sequences.

In one embodiment, the B7-H6 variant of the present invention may be B53 containing the amino acid substitutions A32T and K101E, and may include an extracellular domain region (SEQ ID NO: 33) containing the amino acid substitutions, which is SEQ ID NO: It can be encoded as a nucleic acid molecule containing 34 base sequences.

In one embodiment, the B7-H6 variant of the present invention may be B54 containing amino acid substitutions A32T, S60I and K101E, and may include an extracellular domain region (SEQ ID NO: 35) containing the amino acid substitutions, which It can be encoded as a nucleic acid molecule containing the base sequence of SEQ ID NO: 36.

In one embodiment, the B7-H6 variant of the present invention may be BF2 containing the amino acid substitutions F51H and S60I, and may include an extracellular domain region (SEQ ID NO: 37) containing the amino acid substitutions, which is SEQ ID NO: It can be encoded as a nucleic acid molecule containing 38 base sequences.

In one embodiment, the B7-H6 variant of the present invention may be BF3 containing amino acid substitutions F51I and S60Y, and may include an extracellular domain region (SEQ ID NO: 39) containing the amino acid substitutions, which is SEQ ID NO: It can be encoded as a nucleic acid molecule containing 40 base sequences.

In one embodiment, the B7-H6 variant of the present invention may be BF5 containing amino acid substitutions I37F, F51L and S60T, and may include an extracellular domain region (SEQ ID NO: 41) containing the amino acid substitutions, which It can be encoded as a nucleic acid molecule containing the base sequence of SEQ ID NO: 42.

In one embodiment, the B7-H6 variant of the present invention may be BF8 containing amino acid substitutions F51T and S60T, and may include an extracellular domain region (SEQ ID NO: 43) containing the amino acid substitutions, which is SEQ ID NO: It can be encoded as a nucleic acid molecule containing 44 base sequences.

In one embodiment, the B7-H6 variant of the present invention may be BF11 containing amino acid substitutions F51L and S60H, and may include an extracellular domain region (SEQ ID NO: 45) containing the amino acid substitutions, which is SEQ ID NO: It can be encoded as a nucleic acid molecule containing 46 base sequences.

In one embodiment, the B7-H6 variant of the present invention may be BF19 containing the amino acid substitutions F51T and S60Y, and may include an extracellular domain region (SEQ ID NO: 47) containing the amino acid substitutions, which is SEQ ID NO: It can be encoded as a nucleic acid molecule containing 48 base sequences.

In one embodiment, the B7-H6 variant of the present invention may be BF25 containing the amino acid substitutions F51Q and S60H, and may include an extracellular domain region (SEQ ID NO: 49) containing the amino acid substitutions, which is SEQ ID NO: It can be encoded as a nucleic acid molecule containing 50 base sequences.

In one embodiment, the B7-H6 variant of the present invention may be BF39 containing amino acid substitutions F51K and S60L, and may include an extracellular domain region (SEQ ID NO: 51) containing the amino acid substitutions, which is SEQ ID NO: It can be encoded as a nucleic acid molecule containing 52 base sequences.

In one embodiment, the B7-H6 variant of the present invention may be BF46 containing amino acid substitutions F51R and S60T, and may include an extracellular domain region (SEQ ID NO: 53) containing the amino acid substitutions, which is SEQ ID NO: It can be encoded as a nucleic acid molecule containing 54 base sequences.

In the present invention, the amino acid substitution position is described based on the total amino acids of wild-type B7-H6 (SEQ ID NO: 1), but the actual mutation position is located in the extracellular domain region (SEQ ID NO: 2), so it is based on the total amino acids of wild-type B7-H6. The position described may be different from the position in the extracellular domain region containing the amino acid substitution. For example, the F51S amino acid substitution of the B5 variant is F27I based on the amino acid of SEQ ID NO: 3.

The B7-H6 variant of the present invention refers to a substitution of some amino acid sequences in the wild-type B7-H6 protein (or peptide), and as used in the present invention, the term "variant" refers to at least one amino acid difference ( refers to the corresponding amino acid sequence including substitutions, insertions or deletions). In certain embodiments, a “variant” has high amino acid sequence homology and/or conservative amino acid substitutions, deletions and/or insertions when compared to a reference sequence. Additionally, as used in the present invention, the term “B7-H6 variant” refers to a B7-H6 variant protein mutated in one or more amino acids to regulate its binding activity with NKp30.

Specifically, the B7-H6 variant of the invention can be prepared by standard synthetic methods, recombinant expression systems, or any other art method. Accordingly, peptides according to the invention can be synthesized by a number of methods, including, for example, methods including:

(a) synthesizing peptides stepwise or by fragment assembly by means of solid phase or liquid phase methods, and isolating and purifying the final peptide product; or

(b) a method of expressing a nucleic acid construct encoding a peptide in a host cell and recovering the expression product from the host cell culture; or

(c) a method of performing cell-free in vitro expression of a nucleic acid construct encoding a peptide and recovering the expression product; or

A method of obtaining fragments of a peptide using any combination of (a), (b), and (c), then linking the fragments to obtain a peptide, and recovering the peptide.

As a more specific example, a gene encoding the B7-H6 variant of the present invention can be prepared through genetic manipulation, transformed into a host cell, and then expressed to produce the B7-H6 variant of the present invention.

In one aspect, the present invention relates to a nucleic acid molecule encoding the B7-H6 variant of the present invention, a vector containing the same, and a host cell containing the vector.

The term “nucleic acid molecule” used in the present invention refers to deoxyribonucleotides or ribonucleotides that exist in single-stranded or double-stranded form, and includes natural nucleic acid analogues unless specifically stated otherwise (Scheit, Nucleotide Analogs, John Wiley , New York (1980); Uhlman and Peyman, Chemical Reviews, 90:543-584 (1990)).

As used herein, the term “vector” refers to any nucleic acid containing a competent nucleotide sequence that is inserted into a host cell and recombines with and integrates into the host cell genome, or replicates spontaneously as an episome. These vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, etc.

The term “host cell” used in the present invention refers to a eukaryotic or prokaryotic cell into which one or more DNA or vectors are introduced, and should be understood to refer not only to a specific target cell but also to its descendants or potential descendants. In fact, the progeny are not identical to the parent cell since certain modifications may occur in subsequent generations due to mutations or environmental influences, but are still included within the scope of the term as used herein.

In one aspect, the present invention relates to a natural killer cell (NK cell) activator comprising the B7-H6 variant of the present invention or a fragment thereof.

In one embodiment, the fragment may be the ectodomain region of the B7-H6 variant of the present invention.

In one aspect, the present invention relates to a composition for detecting natural killer cells, comprising the B7-H6 variant of the present invention.

In one embodiment, the composition can detect and quantify NKp30 protein expressed on the surface of natural killer cells.

In one embodiment, the B7-H6 variant may be labeled with one selected from the group consisting of a chromogenic enzyme, a radioactive isotope, a chromophore, a luminescent substance, and a fluorescent substance, and the fluorescent substance is a Cy (cyanine) series, It may be a Rhodamine series, Alexa series, BODIPY series or ROX series fluorescent substance, such as Nile Red, BODIPY, 4,4-difluoro-4-bora-3a,4a -diaza-s-indacene), cyanine, fluorescein, rhodamine, coumarine, or Alexa.

In one aspect, the present invention relates to a bispecific or multispecific antibody comprising the B7-H6 variant of the present invention and a portion that binds to a target antigen.

In one embodiment, the bispecific antibody may be a bispecific NK cell engager.

In one embodiment, the portion that binds to the target antigen may include an antibody or an immunologically active fragment thereof, and the immunologically active fragment may include Fab, Fd, Fab', dAb, F(ab'), F (ab') ₂ , may be any one selected from the group consisting of scFv, Fv, single chain antibody, Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody and diabody, and is scFv or Fab It is more desirable.

In one embodiment, the target antigen is 17-1A antigen, GD3 ganglioside R24, EGFRvIII, PSMA, PSCA, HLA-DR, EpCAM, MUC1 core protein, aberrant glycosylation MUC1, a fibronectin isoform containing an ED-B domain, HER2/neu, carcinoembryonic antigen (CEA), gastrin-releasing peptide (GRP) receptor antigen, mucine antigen, epidermal growth factor receptor (EGF-R), HER3, HER4, MAGE antigen, SART antigen, MUC1 antigen. , c-erb-2 antigen, TAG 72, carbonic anhydrase IX, alpha-fetoprotein, A3, antigen specific for A33 antibody, Ba 733, BrE3-antigen, CA125, CDl, CD1a, CD3, CD5, CDl5, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD79a, CD80, CD138, Colon-specific antigen- p(CSAp), CSAp, EGP-1, EGP-2, Ep-CAM, FIt-1, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and its Subunits, hypoxia-inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, insulin growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KSl- 4, Le-Y, macrophage inhibitory factor (MIF), MAGE, MUCl, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibodies, placental growth factor, p53, prostatic acid phosphatase acid phosphatase, PSA, RS5, SlOO, TAC, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin ), angiogenesis marker, oncogene marker, or oncogene product may be any one or more selected from the group consisting of, and the oncogene is an apoptosis-related gene, a transcription factor gene. , it may be a metastasis-related gene, an angiogenesis-related gene, or a tyrosine-kinase gene.

In one embodiment, the apoptosis-related genes include ABL1, AKT1, AKT2, BARD1, BAX, BCL11B, BCL2, BCL2A1, BCL2L1, BCL2L12, BCL3, BCL6, BIRC2, BIRC3, BIRC5, BRAF, CARD11, CAV1, CBL, CDC25A, CDKN1A, CFLAR, CNR2, CTNNB1, CUL4A, DAXX, DDIT3, E2F1, E2F3, E2F5, ESPL1, FOXO1, HDAC1, HSPA5, IGF1R, IGF2, JUN, JUNB, JUND, MALT1, MAP3K7, MCL1, MDM2, MDM4, MYB, may be MYC, NFKB2, NPM1, NTRK1, PAK1, PAX3, PML, PRKCA, PRKCE, PTK2B, RAF1, RHOA, TGFB1, TNFRSF1B, TP73, TRAF6, YWHAG, YWHAQ or YWHAZ; The transcription factor genes are AR, ARID3A, ASCL1, ATF1, ATF3, BCL11A, BCL11B, BCL3, BCL6, CDC5L, CDX2, CREB1, CUX1, DDIT3, DLX5, E2F1, E2F3, E2F5, ELF4, ELK1, ELK3, EN2, ERG. , ETS1, ETS2, ETV1, ETV3, ETV4, ETV6, FEV, FEZF1, FLI1, FOS, FOSL1, FOXA1, FOXG1, FOXM1, FOXO1, FOXP1, FOXQ1, GATA1, GATA6, GFI1, GFI1B, GLI1, GLI2, GLI3, HES6 , HHEX, HLF, HMGA1, HMGA2, HOXA1, HOXA9, HOXD13, HOXD9, ID1, ID2, IKZF1, IRF2, IRF4, JUN, JUNB, JUND, KAT6A, KDM2A, KDM5B, KLF2, KLF4, KLF5, KLF6, KLF8, KMT2A , LEF1, LHX1, LMX1B, MAF, MAFA, MAFB, MBD1, MECOM, MEF2C, MEIS1, MITF, MYB, MYC, MYCL, MYCN, NANOG, NCOA3, NFIB, NFKB2, NKX2-1, OTX2, PATZ1, PAX2, PAX3 , PAX4, PAX8, PBX1, PBX2, PITX2, PLAG1, PLAGL2, PPARG, PPP1R13L, PRDM10, PRDM13, PRDM14, PRDM15, PRDM16, PRDM6, PRDM8, PRDM9, RARA, REL, RERE, RUNX1, RUNX3, SALL4, SATB1, SFPQ , SIX1, SNAI1, SOX2, SOX4, SPI1, SREBF1, STAT3, TAF1, TAL1, TAL2, TBX2, TBX3, TCF3, TFCP2, TFE3, THRA, TLX1, TP63, TP73, TWIST1, WT1, YBX1, YY1, ZBTB16, ZBTB7A , ZIC2, ZNF217 or ZNF268; The metastasis-related genes are AKT1, AKT2, AR, CBL, CDH1, CRK, CSF1, CTNNB1, CTTN, CXCR4, EGFR, FGFR1, FLT3, FYN, GLI1, ILK, ITGA3, JAK2, MET, PDGFRB, PLXNB1, PRKCI, PTCH1 , PTPN11, RAC1, RHOA, RHOC, ROCK1, SMO, SNAI1, SRC, TCF3 or WT1; The angiogenesis-related gene may be BRAF, CAV1, CTGF, EGFR, ERBB2, ETS1, FGF4, FGF6, FGFR1, FGFR3, FGFR4, ID1, NRAS, PDGFB, PDGFRA, PDGFRB or SPARC; The tyrosine-kinase genes are ABL1, ABL2, ALK, AXL, BLK, EGFR, EPHA2, ERBB2, ERBB3, ERBB4, FES, FGFR1, FGFR2, FGFR3, FGFR4, FGR, FLT3, FYN, ITK, JAK1, JAK2, KIT, It may be LCK, MERTK, MET, MST1R, NTRK1, NTRK3, PDGFRA, PDGFRB, PTK2B, PTK7, RET, ROS1, SRC, SYK, TEC or YES1.

In one embodiment, the oncogene is SEPTIN9, ACOD1, ACTN4, ADAM28, ADAM9, ADGRF1, ADRBK2, AFF1, AFF3, AGAP2, AGFG1, AGRN, AHCYL1, AHI1, AIMP2, AKAP13, AKAP9, AKIRIN2, AKTIP, ALDH1A1, ALL1, ANIB1, ANP32C, ANP32D, AQP1, ARAF, ARHGEF1, ARHGEF2, ARHGEF5, ASPSCR1, AURKA, BAALC, BAIAP2L1, BANP, BCAR4, BCKDHB, BCL9, BCL9L, BCR, BMI1, BMP7, BOC, BRD4, BRF2, CABIN1, CAMK1D, CAPG, CBFB, CBLB, CBLL1, CBX7, CBX8, CCDC28A, CCDC6, CCNB1, CCNB2, CCND1, CCNE1, CCNL1, CD24, CDC25C, CDC6, CDH17, CDK1, CDK14, CDK4, CDK5R2, CDK6, CDK8, CDKN1B, CDKN3, CDON, CEACAM6, CENPW, CHD1L, CHIC1, CHL1, CKS1B, CMC4, CNTN2, COPS3, COPS5, CRKL, CRLF2, CROT, CRTC1, CRYAB, CSF1R, CSF3, CSF3R, CSNK2A1, CSNK2A2, CT45A1, CTBP2, CTNND2, CTSZ, CUL7, CXCL1, CXCL2, CXCL3, CYGB, CYP24A1, DCD, DCUN1D1DDB2, DDHD2, DDX6, DEK, DIS3, DNPH1, DPPA2, DPPA4, DSG3, DUSP12, DUSP26, ECHS1, ECT2, EEF1A1, EEF1A2, EEF1D, EIF3E, EIF3I, EIF4E, EIF5A2, ELAVL1, ELL, EML4, EMSY, ENTPD5, EPCAM, EPS8, ERAS, ERGIC1, ERVW-1, EVI2A, EVI5, EWSR1, EZH2, FAM189B, FAM72A, FAM83D, FASN, FDPS, FGF10, FGF3, FGF5, FGF8, FR1OP, FHL2, FIP1L1, FNDC3B, FRAT1, FUBP1, FUS, FZD2, GAB2, GAEC1, GALNT10, GALR2, GLO1, GMNN, GNA12, GNA13, GNAI2, GNAQ, GNAS, GOLPH3, GOPC, GPAT4, GPM6A, GPM6B, GPR132, GREM1, GRM1, GSK3A, GSM1, H19, HAS1, HAX1, HDGFRP2, HMGN5, HNRNPA1, HOTAIR, HOTTIP, HOXA-AS2, HRAS, HSPA1A, HSPA4, HSPB1, HULC, IDH1, IFNG, IGF2BP1, IKBKE, IL7R, INPPL1, INTS1, INTS2, INTS3, INTS4, INTS5, INTS7, INTS8, IRS2, IST1, JUP, KDM4C, KIAA0101, KIAA1524, KIF14, KRAS, KSR2, LAMTOR5, LAPTM4B, LCN2, LDHB, LETMD1, LIN28A, LIN28B, LMO1, LMO2, LMO3, LMO4, LSM1, LUADT1, MACC1, MACROD1, MAGEA11, MALAT1, MAML2, MAP3K8, MAPRE1, MAS1, MCC, MCF2, MCF2L, MCTS1, MEFV, MFHAS1, MFNG, MIEN1, MINA, MKL2, MLANA, MLLT1, MLLT11, MLLT3, MLLT4, MMP12, MMS22L, MN1, MNAT1, MOS, MPL, MPST, MRAS, MRE11A, MSI1, MTCP1, MTDH, MTOR, MUC1, MUC4, MUM1, MYD88, NAAA, NANOGP8, NBPF12, NCOA4, NEAT1, NECTIN4, NEDD4, NEDD9, NET1, NINL, NME1, NOTCH1, NOTCH4, NOV, NSD1, NUAK2, NUP214, NUP98, NUTM1, OLR1, PA2G4, PADI2, PAK7, PARK7, PARM1, PBK, PCAT1, PCAT5, PDGFA, PDZK1IP1, PELP1, PFN1P3, PIGU, PIK3CA, PIK3R1, PIM1, PIM2, PIM3, PIR, PIWIL1, PLAC8, PLK1, PPM1D, PPP1R10, PPP1R14A, PPP2R1A, PRAME, PRDM12, PRMT5, PSIP1, PSMD10, PTCH2, PTMA, PTP4A1, PTP4A2, PTP4A3, PTTG1, PTTG1IP, PTTG2, PVT1, RAB11A, RAB18, RAB22A, RAB23, RAB8A, RALGDS, RAP1A, RASSF1, RBM14, RBM15, RBM3, RBMY1A1, RFC3, RGL4, RGR, RHO, RING1, RINT1, RIT1, RNF43, RPL23, RRAS, RRAS2, RSF1, RUNX1T1, S100A4, S100A7, S100A8, SAG, SART3, SBSN, SEA, SEC62, SERTAD1, SERTAD2, SERTAD3, SET, SETBP1, SETDB1, SGK1, SIRT1, SIRT6, SKI, SKIL, SKP2, SLC12A5, SLC3A2, SMR3B, SMURF1, SNCG, SNORA59A, SNORA80E, SPAG9, SPATA4, SPRY2, SQSTM1, SRSF1, SRSF2, SRSF3, SRSF6, SS18, SSX1, SSX2, SSX2B, STIL, STMN1, STRA6, STYK1, SUZ12, SWAP70, SYT1, TAC1, TACSTD2, TAF15, TALDO1, TAZ, TBC1D1, TBC1D15, TBC1D3, TBC1D3C, TBC1D7, TCL1A, TCL1B, TCL6, TCP1, TFG, TGM3, TINCR, TKTL1, TLE1, TMEM140, TMPOP2, TMPRSS2, TNS4, TPD52, TPR, TRE17, TREH, TRIB1, TRIB2, TRIM28, TRIM32, TRIM8, TRIO, TRIP6, TSPAN1, TSPY1, TXN, TYMS, TYRP1, UBE2C, UBE3C, UCA1, UCHL1, UHRF1, URI1, USP22, USP4, USP6, VAV1, VAV2, VAV3, VIM, WAPL, WHSC1, WHSC1L1, WISP1, WNT1, WNT10A, WNT10B, WNT2, WNT3, WNT5A, WWTR1, XCL1, It could be ZNHIT6.

In one embodiment, the target antigen may be a cell surface antigen or an autoantigen, and the cell surface antigen may be CEA, ED-B fibronectin, CD20, CD22, CDl9, EGFR, IGFlR, VEFGRl/Flt-1, VEGFR2/KDR, VEGRF3 /Flt-4, HER2/neu, CD30, CD33, CD3, CD16, CD64, CD89, CD2, adenovirus fiber knob, PfMSP-1, HN/NDV, EpCAM/17-lA, hTR, IL-2R/Tac, It may be any one or more selected from the group consisting of CAl9-9, MUCl, HLA class II, GD2, G250, TAG-72, PSMA, CEACAM6, HMWMAA, CD40, Ml3 coat protein, and GPIIb/IIIa.

In one embodiment, the immunologically active fragment of the present invention is Fab, Fd, Fab', dAb, F(ab'), F(ab') ₂ , scFv (single chain fragment variable), Fv, single chain antibody. , Fv dimer, complementarity determining region fragment, humanized antibody, chimeric antibody, and diabody.

The antibodies are in whole antibody form as well as functional fragments of the antibody molecule. A full antibody has a structure of two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. A functional fragment of an antibody molecule refers to a fragment that possesses an antigen-binding function. Examples of antibody fragments include (i) the variable region (VL) of the light chain, the variable region (VH) of the heavy chain, the constant region (CL) of the light chain, and Fab fragment consisting of the first constant region (CH1) of the heavy chain; (ii) Fd fragment consisting of VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 341:544-546 (1989)); (v) an isolated CDR region; (vi) a bivalent fragment comprising two linked Fab fragments. F(ab')2 fragment; (vii) single chain Fv molecule (scFv) joined by a peptide linker that joins the VH domain and VL domain to form an antigen binding site; (viii) bispecific single chain Fv dimer (PCT/US92/09965) and (ix) diabody WO94/13804, which is a multivalent or multispecific fragment produced by gene fusion.

The antibody or immunologically active fragment thereof of the present invention may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and immunologically active fragments thereof. The antibody may be recombinantly or synthetically produced.

Animal-derived antibodies produced by immunizing an immunized animal with a desired antigen can generally cause immune rejection when administered to humans for therapeutic purposes, and chimeric antibodies have been developed to suppress such immune rejection. A chimeric antibody is one in which the constant region of an animal-derived antibody, which causes an anti-isotype reaction, is replaced with the constant region of a human antibody using genetic engineering methods. Chimeric antibodies have significantly improved anti-isotype responses compared to animal-derived antibodies, but animal-derived amino acids still exist in the variable region, resulting in potential side effects on anti-idiotypic responses. I'm doing it. Humanized antibodies were developed to improve these side effects. It is produced by transplanting the CDR (complementary determining regions) region, which plays an important role in antigen binding, among the variable regions of a chimeric antibody, into the human antibody framework.

The most important thing in CDR grafting technology to produce humanized antibodies is to select an optimized human antibody that can best accept the CDR region of an animal-derived antibody. To this end, use of an antibody database and crystal structure (crystal structure) Structure analysis, molecular modeling technology, etc. are used. However, even when the CDR region of an animal-derived antibody is transplanted onto an optimized human antibody framework, there are cases where amino acids that affect antigen binding are present in the framework of the animal-derived antibody, so antigen-binding ability is not preserved in many cases. Therefore, the application of additional antibody engineering technology to restore antigen binding ability can be said to be essential.

The antibody or fragment thereof with immunological activity may be isolated from a living body (not present in the living body) or non-naturally occurring, for example, synthetically or recombinantly produced. You can.

In the present invention, “antibody” refers to a substance produced by stimulation of an antigen within the immune system, the type of which is not particularly limited, and can be obtained naturally or unnaturally (e.g., synthetically or recombinantly). You can. Antibodies are very stable not only in vitro but also in vivo and have a long half-life, making them advantageous for mass expression and production. In addition, antibodies inherently have a dimer structure, so their adhesion ability (avidity) is very high. A complete antibody has a structure of two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. The constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and subclasses. It has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

As used herein, the term "heavy chain" refers to a variable region domain V _H and three constant region domains C _H1 , C _H2 and C _H3 comprising an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen. It is interpreted to include both the full-length heavy chain including the hinge and fragments thereof. Additionally, the term "light chain" refers to both a full-length light chain and fragments thereof comprising a variable region domain V _L and a constant region domain C _L comprising an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen. It is interpreted to mean inclusive.

In the present invention, the term "variable region or variable domain" refers to a portion of an antibody molecule that performs the function of specifically binding to an antigen and exhibits many variations in sequence, and the variable region has complementary There are crystalline regions CDR1, CDR2 and CDR3. A framework region (FR) exists between the CDRs and serves to support the CDR ring. The “complementarity determining region” is a ring-shaped region involved in antigen recognition, and as the sequence of this region changes, the specificity of the antibody to the antigen is determined.

The term "scFv (single chain fragment variable)" used in the present invention refers to a single chain antibody made by expressing only the variable region of an antibody through genetic recombination, and is a single chain form in which the VH region and VL region of the antibody are connected by a short peptide chain. refers to the antibodies of The term “scFv” is intended to include scFv fragments, including antigen-binding fragments, unless otherwise specified or understood from context. This is self-evident to those skilled in the art.

In the present invention, the term “complementarity determining region (CDR)” refers to the amino acid sequence of the hypervariable region of the heavy and light chains of immunoglobulin. The heavy and light chains may each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDR may provide key contact residues for the antibody to bind to the antigen or epitope.

In the present invention, the term "specifically binds" or "specifically recognizes" has the same meaning commonly known to those skilled in the art, and means that an antigen and an antibody specifically interact to produce an immunological reaction. .

In the present invention, the term “antigen-binding fragment” refers to a fragment of the entire structure of an immunoglobulin and a portion of a polypeptide containing a portion to which an antigen can bind. For example, it may be scFv, (scFv) 2, scFv-Fc, Fab, Fab' or F(ab') 2, but is not limited thereto. Among the antigen-binding fragments, Fab has one antigen-binding site with a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (C _H1 ) of the heavy chain. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain C _H1 domain. The F(ab') 2 antibody is produced when the cysteine residue in the hinge region of Fab' forms a disulfide bond. Fv is a minimal antibody fragment containing only the heavy chain variable region and the light chain variable region, and recombinant techniques for producing Fv fragments are widely known in the art. A two-chain Fv (two-chain Fv) is a non-covalent bond in which the heavy chain variable region and a light chain variable region are connected, while a single-chain Fv (single-chain Fv) is generally shared between the heavy chain variable region and the short chain variable region through a peptide linker. They can be connected by a bond or directly connected at the C-terminus to form a dimer-like structure, such as double-chain Fv. The linker may be a peptide linker consisting of 1 to 100 or 2 to 50 amino acids, and suitable sequences are known in the art. The antigen-binding fragment can be obtained using a proteolytic enzyme (for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F(ab') 2 fragment can be obtained by digestion with pepsin), It can be produced through genetic recombination technology.

In the present invention, the term "hinge region" refers to a region contained in the heavy chain of an antibody, which exists between the C _H1 and C _H2 regions and functions to provide flexibility of the antigen binding site in the antibody. It means area. For example, the hinge may be derived from a human antibody, specifically, IgA, IgE, or IgG, such as IgG1, IgG2, IgG 3, or IgG4.

In one aspect, the invention relates to an isolated nucleic acid molecule encoding the B7-H6 variant or fragment thereof, or bispecific or multispecific antibody of the invention, a vector containing the same, and a host cell transformed therewith. .

Nucleic acid molecules of the invention may be isolated or recombinant and include single- and double-stranded forms of DNA and RNA as well as corresponding complementary sequences. An isolated nucleic acid, in the case of a nucleic acid isolated from a naturally occurring source, is a nucleic acid that has been separated from the surrounding genetic sequence present in the genome of the individual from which the nucleic acid was isolated. In the case of nucleic acids synthesized enzymatically or chemically from a template, such as PCR products, cDNA molecules, or oligonucleotides, the nucleic acids resulting from these procedures may be understood as isolated nucleic acid molecules. Isolated nucleic acid molecules refer to nucleic acid molecules either in the form of separate fragments or as components of larger nucleic acid constructs. A nucleic acid is operably linked when placed in a functional relationship with another nucleic acid sequence. For example, the DNA of the presequence or secretion leader is operably linked to the DNA of the polypeptide when the polypeptide is expressed as a preprotein in a form before secretion, and the promoter or enhancer is a polypeptide sequence. is operably linked to the coding sequence when it affects transcription, or the ribosome binding site is operably linked to the coding sequence when configured to facilitate translation. In general, operably linked means that the DNA sequences to be linked are located adjacent to each other, and in the case of a secretory leader, it means that they are adjacent and exist within the same reading frame. However, enhancers do not need to be located adjacently. Linking is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

Isolated nucleic acid molecules encoding the antibodies of the present invention or immunologically active fragments thereof, or the bispecific or multispecific antibodies of the present invention, may be used due to codon degeneracy or for the purpose of expressing the antibodies. Considering the codons preferred in organisms, various modifications can be made to the coding region within the range that does not change the amino acid sequence of the antibody expressed from the coding region, and does not affect gene expression in parts other than the coding region. A person skilled in the art will understand that various modifications or modifications can be made within the gene, and that such modified genes are also included within the scope of the present invention. That is, as long as the nucleic acid molecule of the present invention encodes a protein with equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included within the scope of the present invention. The sequence of these nucleic acid molecules may be single or double stranded, and may be DNA molecules or RNA (mRNA) molecules.

An isolated nucleic acid molecule encoding an antibody of the present invention or a fragment having immunological activity thereof, or a bispecific or multispecific antibody of the present invention may be inserted into an expression vector for protein expression. Expression vectors typically contain proteins operably linked, i.e., placed in a functional relationship, with regulatory or control sequences, selectable markers, optional fusion partners, and/or additional elements. Under appropriate conditions, a host cell transformed with a nucleic acid, preferably containing an antibody of the invention or an immunologically active fragment thereof, or an isolated nucleic acid molecule encoding a bispecific or multispecific antibody of the invention, is expressed. An antibody of the present invention, a fragment having immunological activity thereof, or a bispecific or multispecific antibody of the present invention can be produced by culturing a vector to induce protein expression. A variety of suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are known in the art and will vary depending on the host cell used. Preferably, E. coli, which has low production cost and thus has high industrial value, can be produced as a host cell.

Vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, etc. Suitable vectors include expression control elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. The signal sequence includes the PhoA signal sequence and OmpA signal sequence when the host is Escherichia sp., and the α-amylase signal sequence and subtilisin signal when the host is Bacillus sp. For sequences, etc., if the host is yeast, the MFα signal sequence, SUC2 signal sequence, etc. can be used, and if the host is an animal cell, the insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used. It is not limited to this. Additionally, the vector may include a selection marker for selecting host cells containing the vector, and if it is a replicable expression vector, it will include an origin of replication.

In the present invention, the term “vector” refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence. Nucleic acid sequences may be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids, and viruses (eg, bacteriophages). Those skilled in the art can construct vectors by standard recombination techniques (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994, etc.).

In one embodiment, when constructing the vector, expression control sequences such as promoters, terminators, enhancers, etc., depending on the type of host cell for producing the antibody, membrane targeting or secretion Sequences, etc. can be appropriately selected and combined in various ways depending on the purpose.

In the present invention, the term “expression vector” refers to a vector containing a nucleic acid sequence encoding at least a portion of the gene product to be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors may contain various control sequences. In addition to regulatory sequences that regulate transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that also serve other functions.

In the present invention, the term “host cell” includes eukaryotes and prokaryotes and refers to any transformable organism capable of replicating the vector or expressing the gene encoded by the vector. The host cell may be transfected or transformed by the vector, which refers to a process in which an exogenous nucleic acid molecule is transferred or introduced into the host cell.

In one embodiment, the host cells may be bacteria or animal cells, the animal cell line may be CHO cells, HEK cells, or NSO cells, and the bacteria may be Escherichia coli.

In one aspect, the present invention relates to a pharmaceutical composition for the treatment or prevention of cancer, comprising the B7-H6 variant or fragment thereof, or a bispecific or multispecific antibody of the present invention.

In one embodiment, the cancer is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, and colon cancer. , small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer. , soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, renal or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma. and pituitary adenoma.

In the present invention, the term “prevention” refers to all actions that inhibit or delay the occurrence, spread, and recurrence of a disease or condition by administering the composition according to the present invention.

The term "treatment" used in the present invention refers to any action that improves or beneficially changes the symptoms of a disease or condition and complications resulting therefrom by administering the composition according to the present invention. Anyone with ordinary knowledge in the technical field to which the present invention pertains can refer to the data presented by the Korean Medical Association, etc. to know the exact criteria for diseases for which our composition is effective and to determine the degree of improvement, improvement, and treatment. will be.

The term "therapeutically effective amount" used in combination with an active ingredient in the present invention refers to an amount effective in preventing or treating a disease or disorder, and the therapeutically effective amount of the composition of the present invention is determined by several factors, such as administration. It may vary depending on the method, target area, patient condition, etc. Therefore, when used in the human body, the dosage must be determined as appropriate by considering both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. These considerations in determining an effective amount include, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceutically effective amount" refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's Factors including health status, type of disease or condition, severity of disease or condition, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, treatment period, drugs combined or used simultaneously, and other factors. It can be determined based on factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The pharmaceutical composition of the present invention may contain a carrier, diluent, excipient, or a combination of two or more commonly used in biological products. As used in the present invention, the term “pharmaceutically acceptable” means that the composition exhibits non-toxic properties to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compounds described in, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients can be mixed and used, and if necessary, other ingredients such as antioxidants, buffers, and bacteriostatic agents. Normal additives can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group including oral formulations, topical formulations, suppositories, sterile injectable solutions, and sprays, with oral or injectable formulations being more preferable.

As used in the present invention, the term "administration" means providing a predetermined substance to an individual or patient by any appropriate method, and is administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally) according to the desired method. Alternatively, it can be applied topically as an injection formulation) or orally administered, and the dosage range varies depending on the patient's weight, age, gender, health status, diet, administration time, administration method, excretion rate, and severity of the disease. Liquid preparations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and in addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are used. etc. may be included together. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc. The pharmaceutical composition of the present invention may be administered by any device capable of transporting the active agent to target cells. Preferred administration methods and formulations include intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, and drip injection. Injections include aqueous solvents such as physiological saline solution and Ringer's solution, non-aqueous solvents such as vegetable oil, higher fatty acid esters (e.g., ethyl oleate, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). It can be manufactured using stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulphite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH adjustment, and agents to prevent microbial growth. It may contain pharmaceutical carriers such as preservatives (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

As used in the present invention, the term "individual" refers to monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, bats, including humans that have or may develop the disease or condition. It means any animal, including camel, rat, rabbit or guinea pig, and “specimen” may be droplets, sputum, whole blood, plasma, serum, urine or saliva isolated therefrom.

The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and calcium hydrogen phosphate. , lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, white sugar, dextrose, sorbitol, and talc can be used. The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

In one aspect, the present invention includes the steps of isolating natural killer cells; and culturing the isolated natural killer cells in the presence of the B7-H6 variant or fragment thereof of the present invention.

In one aspect, the present invention relates to the use of the B7-H6 variant of the present invention or a bispecific or multispecific antibody comprising the same for the prevention or treatment of cancer.

In one aspect, the present invention relates to a method of treating cancer comprising administering the B7-H6 variant of the present invention or a bispecific or multispecific antibody containing the same in a pharmaceutically effective amount to an individual suffering from cancer. will be.

The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.

Example 1. Cloning of tetrameric human NKp30 for detection of B7-H6 variants

In order to be used in the search for B7-H6 variants with improved binding to NKp30, the avidity effect was induced through tetramerization of the NKp30 protein. Specifically, streptavidin was expressed in the C-terminal part of NKp30 to induce tetramerization, and glycine and serine linkers were inserted between NKp30 and streptavidin to ensure the fluidity of each protein. The NKp30 and streptavidin genes were each amplified using primers and Vent polymerase (New England Biolab), and then assembly PCR was performed using Vent polymerase. The created gene was subjected to restriction enzyme treatment using Bss HII and Xba I (New England Biolab). The restriction enzyme-treated NKp30-Streptavdin gene was ligated into pMAZ vector, a vector for animal cells, treated with the same restriction enzyme. After transforming the ligated plasmid into E. coli Jude1, a single clone was obtained and sequenced to confirm that NKp30-streptavidin was successfully inserted into the pMAZ vector (Figure 1).

Example 2. Expression, purification and labeling of tetrameric human NKp30

One day after subculturing Expi293F cells in 300 ml at a density of 2x10 ⁶ cells/ml, PEI (Polyehylenimine, Polyscience, 23966) and NKp30 prepared in Example 1 were added to 30 ml of Freestyle 293 expression culture medium (Gibco. 12338-018). -Streptavidin-His tag expression vector was mixed at a ratio of 1:4, left at room temperature for 20 minutes, and then transfected into the Expi293F animal cells. The cells were cultured in a CO ₂ shaking incubator at 37°C, 125 rpm, and 8% CO ₂ for 7 days, then centrifuged, and only the supernatant was collected. Afterwards, the mixture was equilibrated using 25x PBS and filtered through a 0.2 μm filter (Merck Millipore) using a bottle top filter. 1 ml of Ni-NTA resin was added to the filtered culture medium, stirred at 4°C for 16 hours, and then passed through a column to recover the resin and washed with 10 ml of PBS. The washed resin was sequentially washed with 10 ml each of 10mM imidazole buffer and 20mM imidazole buffer, and then eluted with 4ml of 250mM imidazole buffer. After exchange with PBS buffer using centrifugal filter units 3K (Merck Millipore), the purified NKp30-streptavidin tetramer protein was fluorescently labeled using an Alexa-488 labeling kit.

Example 3. Screening Method Selection

3-1. B7-H6 cloning for selection of yeast surface display method

For efficient screening, we decided to determine the yeast surface anchoring motif and used a system that anchors the N-terminus of B7-H6 to Aga2 (pCTCON-Aga2-B7-H6-FLAG) and a system that anchors the C-terminus to Aga2 (pCTCON). -B7-H6-Aga2-FLAG) was cloned to compare B7-H6. Specifically, the gene was amplified using primers designed by synthesizing the wild-type B7-H6 gene and Vent Polymerase (New England Biolab). The amplified wild-type B7-H6 gene was subjected to restriction enzyme treatment using Sfi I (New England Biolab), and the restriction enzyme-treated gene was ligated into the pCTCON vector treated with the same restriction enzyme. After transforming the ligated plasmid into Jude1 E. coli, a single clone was obtained and sequenced, and two plasmids, pCTCON-Aga2-B7-H6_WT-FLAG and pCTCON-B7-H6_WT-Aga2-FLAG, were successfully cloned. was confirmed.

3-2. Selection of display method through verification of cohesion

The display method was selected by verifying the binding affinity between B7-H6 expressed on the yeast surface and the probe NKp30-streptavidin using a flow cytometer. Specifically, the two plasmids prepared in Example 3-1 were transformed into the AWY101 (Trp-) strain, and 50 μg/ml of Kanamycin and 40 μg/ml of Chloramphenicol were added. 30 in 5 ml of medium containing SDCAA (20 g/L Glucose, 6.7 g/L Yeast nitrogen base without amino acids, 5 g/L casamino acids, 5.4 g/L Na ₂ HPO ₄ and 8.56 g/L NaH ₂ PO ₄ ). Cultured for 16 hours at ℃ and 225 rpm. 5x10 ⁷ cultured cells were obtained by centrifugation (2,500g, 5 minutes, 4°C), and then incubated with SGCAA (20 g/L Galactose, 6.7 g) to which 50 μg/ml kanamycin and 40 μg/ml chloramphenicol were added. /L Yeast nitrogen base without amino acids, 5 g/L casamino acids, 5.4 g/L Na ₂ HPO ₄ and 8.56 g/L NaH ₂ PO ₄ ) Induction in 5 ml of medium at 20℃ and 225 rpm for 24 hours did. After induction, 2x10 ⁷ cells were centrifuged (14,000g, 30 seconds, 4°C) and recovered in an e-tube. Add 1 ml of PBSB (0.1% BSA in PBS) to each e-tube from which the cells were recovered, resuspend them, collect the cells again through centrifugation (14,000 g, 30 seconds, 4°C), and then add 0.5 ml of PBSB and resuspend. It was cloudy, making 4x10 ⁷ cells/ml. After transferring 25 μl of cells to a new e-tube, 25 μl of PBSB, NKp30-Streptavidin-Alexa488 (200 nM), and Anti-FLAG-iFluor647 (1000:1) probes were respectively added and incubated for 30 minutes at room temperature. Then, the cells were labeled with a fluorescent probe. Afterwards, centrifugation (14,000g, 30 seconds, 4°C) discarded the supernatant, resuspended in 200 μl of PBSB, washed, and centrifuged again (14,000g, 1 minute, 4°C) and resuspended in 200 μl of PBSB. A cloudy sample was prepared. The binding affinity of wild-type B7-H6 and NKp30 was indirectly analyzed by measuring the fluorescence signal values of samples prepared using FACSLyric (BD Biosciences) equipment.

As a result of the analysis, it was confirmed that the fluorescently labeled tetrameric NKp30 was active when tetrameric NKp30 was used through the signal of wild-type B7-H6. In addition, all expressions were good, but compared to the wild-type B7-H6 displayed in yeast with its N-terminal part anchored to Aga2, B7-H6 displayed in yeast with its C-terminal part anchored to Aga2 had significantly lower binding affinity to NKp30. After confirming the increase, it was decided to proceed with screening in the form of anchoring the C-terminus (Figure 2).

Example 4. Construction of a large B7-H6 error-prone library for using ultra-fast screening techniques

In order to quickly search for B7-H6 variants with increased binding affinity to NKp30, pCTCON-B7-H6_WT-Aga2-FLAG contains Sfi I sites on both sides to allow random mutations to all regions of B7-H6. A primer was designed. DNA was first amplified using the Error-Prone PCR technique using the designed primers, Taq Polymerase (TAKARA), dNTPs (Invitrogen), MgCl ₂ and MnCl ₂ (SIGMA). The amplified gene was prepared by secondary amplification using Vent polymerase (24 μg), and the vector was prepared by treatment with Sfi I restriction enzyme (8 μg). The two prepared genes were transformed into the AWY101 strain to construct a library through homologous recombination. The constructed library had a size of ^5.1 This was confirmed (Figure 3).

Example 5. B7-H6 variant screening

The initial library prepared by transformation into AWY101 in Example 4 was grown in SDCAA (20 g/L Glucose, 6.7 g/L Yeast nitrogen base without amino acids, 50 μg/ml kanamycin and 40 μg/ml chloramphenicol). Cultured in 500 ml of medium (5 g/L casamino acids, 5.4 g/L Na ₂ HPO ₄ and 8.56 g/L NaH ₂ PO ₄ ) at 30°C, 225 rpm for 16 hours, washed with SDCAA medium 100 to remove dead cells. After inoculating ml with OD ₄₅₀ = 0.7, the cells were further cultured at 30°C and 225 rpm for 16 hours. Afterwards, the cultured cells were inoculated into 100 ml of SGCAA medium at an OD of ₄₅₀ = 0.7 and cultured at 20°C and 225 rpm for 2 days for induction, followed by centrifugation of 1x10 ⁸ cells (14,000g, 30 seconds, 4°C). ) and recovered in an e-tube. Resuspend 1 ml of PBSB (0.1% BSA in PBS) in the e-tube from which the cells were recovered, collect the cells again through centrifugation (14,000 g, 30 seconds, 4°C), and then add Anti-FLAG-iFluor647 (1000 :1) and NKp30-Streptavidin-Alexa488 (200 nM) probes were resuspended in 1 ml of PBSB and incubated at room temperature for 1 hour for labeling. Afterwards, centrifugation (14,000g, 30 seconds, 4℃) was performed, the supernatant was discarded, resuspended in 1 ml of PBSB, washed, and centrifuged again (14,000g, 1 minute, 4℃) with 1 ml of PBSB. The sample was prepared by resuspending it. Yeasts with high binding affinity to NKp30 were recovered by measuring the fluorescence signal value of the prepared library sample using S3 sorter (Bio-Rad) equipment, and the recovered yeasts were cultured in 20 ml of SDCAA at 30°C and 225 rpm. The cultured cells were recovered the next day, cultured in 100 ml of SGCAA at 20°C and 225 rpm for 2-3 days, and then inducted, before proceeding with the next round. The above screening process was performed a total of four times while decreasing the concentration of the probe.

Example 6. Confirmation of amplification of B7-H6 variants with increased binding affinity to NKp30

SDCAA (20 g/L Glucose, 6.7 g/L Yeast nitrogen base without amino acids, 5 g/L casamino acids, 5.4 g/L Na ₂ HPO ₄ ) with 50 μg/ml kanamycin and 40 μg/ml chloramphenicol , 8.56 g/L NaH ₂ PO ₄ ) The initials, 1st round, 2nd round, 3rd round, and 4th round libraries of Example 5 were separately inoculated in 100 ml of medium and cultured at 30°C and 225 rpm for 16 hours. . To remove dead cells, the cultured cells were inoculated into 100 ml of SDCAA medium at an OD _{of 450} = 0.7, cultured at 30°C and 225 rpm for 16 hours, and then inoculated into 100 ml of SGCAA medium at an OD _{of 450} = 0.7. Cultured for 2 days at 20°C and 225 rpm. After induction, the libraries were collected in e-tubes by centrifugation (14,000g, 30 seconds, 4°C) in an amount equivalent to 2x10 ⁷ cells, and each e-tube was injected with PBSB (0.1% BSA in PBS) 1. ml was added and the cells were re-collected through centrifugation (14,000 g, 30 seconds, 4°C), then 0.5 ml of PBSB was added and resuspended to make 4x10 ⁷ cells/ml. After transferring 25 μl of cells to a new e-tube, add 25 μl of PBSB, NKp30-Streptavidin-Alexa488 (100 nM), and Anti-FLAG-iFluor647 (1000:1) probes, respectively, and incubate for 30 minutes at room temperature. Then, the cells were labeled with a fluorescent probe. Afterwards, centrifugation (14,000 g, 30 seconds, 4°C) was performed, the supernatant was discarded, resuspended with 200 μl of PBSB, washed, and centrifuged again (14,000 g, 1 minute, 4°C) and resuspended with 200 μl of PBSB. Samples were prepared by resuspension. The binding ability of each library to NKp30 was indirectly analyzed by measuring the fluorescence signal value of the samples prepared using FACSLyric (BD Biosciences) equipment. As a result, it was confirmed that variants with improved binding ability to NKp30 were amplified as the number of screening rounds increased (Figure 4).

Example 7. Amino acid sequence analysis of B7-H6 variants and securing of B7-H6 variants with increased binding affinity to NKp30

After securing the DNA of the 4th round library using Zymoprep Yeast Plasmid Miniprep kits (Zymo Research), nucleotide sequence analysis of 50 colonies obtained by transformation into Jude1 E. coli was performed. As a result, 26 variants were selected, and the plasmids of each of the 26 variants were transformed into the AWY101 (Trp-) strain to analyze their binding ability to NKp30. To confirm the binding ability of the mutants to NKp30 using flow cytometry, wild type B7-H6 and 26 mutants were incubated with SDCAA (20 g/L Glucose, 6.7 g) with 50 μg/ml kanamycin and 40 μg/ml chloramphenicol, respectively. /L Yeast nitrogen base without amino acids, 5 g/L casamino acids, 5.4 g/L Na ₂ HPO ₄ , 8.56 g/L NaH ₂ PO ₄ ) and cultured in 5 ml of medium at 30℃ and 225 rpm for 16 hours. 5x10 ⁷ cells were obtained by centrifugation (2,500g, 5 minutes, 4℃), and then incubated with SGCAA (20 g/L Galactose, 6.7 g/mL) to which 50 μg/ml kanamycin and 40 μg/ml chloramphenicol were added. L Yeast nitrogen base without amino acids, 5 g/L casamino acids, 5.4 g/L Na ₂ HPO ₄ , 8.56 g/L NaH ₂ PO ₄ ) was inducted in 5 ml of medium at 20°C and 225 rpm for 24 hours. After induction, 2x10 ⁷ cells were centrifuged (14,000g, 30 seconds, 4°C) and recovered in an e-tube. Add 1 ml of PBSB (0.1% BSA in PBS) to each e-tube from which the cells were recovered, resuspend them, collect the cells again through centrifugation (14,000 g, 30 seconds, 4°C), and then add 0.5 ml of PBSB and resuspend. It was cloudy, making 4x10 ⁷ cells/ml. After transferring 25 μl of cells to a new e-tube, add 25 μl of PBSB, NKp30-Streptavidin-Alexa488 (100 nM), and Anti-FLAG-iFluor647 (1000:1) probes, respectively, and incubate for 30 minutes at room temperature. Then, the cells were labeled with a fluorescent probe. Afterwards, centrifugation (14,000 g, 30 seconds, 4°C) was performed, the supernatant was discarded, resuspended with 200 μl of PBSB, washed, and centrifuged again (14,000 g, 1 minute, 4°C) and resuspended with 200 μl of PBSB. A resuspended sample was prepared. By measuring the fluorescence signal value of samples prepared with FACSLyric (BD Biosciences) equipment, the expression level and binding ability of each variant with NKp30 were indirectly analyzed, and a total of 17 variants with improved binding ability with NKp30 were identified (B5, B6, B7, B8, B9, B14, B16, B19, B23, B29, B35, B40, B41, B47, B52, B53, and B54) were selected (Figure 5 and Table 1).

B7-H6 variant	B7-H6 mutation location and substituted amino acid
B5	F51S/S60I
B6	N57D
B7	K101E/S102R
B8	A32T/S60I
B9	A32T/L40Q/S60I
B14	F51L/S60I
B16	K101E
B19	A32T/W67R/Q86L/K101E/L129M
B23	S60I/L114M/S142N
B29	M31I/A32T/F51I/S60I/S102C/R108M
B35	A32T/S60I/K101E/P143S
B40	I37T/Q86L/K101E
B41	A32T/S53G/S60I/Q136R
B47	F51Y/S60I
B52	S60I/K101E
B53	A32T/K101E
B54	A32T/S60I/K101E

Example 8. Expression and purification of B7-H6 variants with increased binding affinity to NKp30

To construct animal cell expression vectors for B7-H6 variants with increased binding affinity to NKp30, the wild-type B7-H6 and B7-H6 variants were used as controls to express the Fc domain of an IgG antibody to induce dimer formation. At this time, a GS linker composed of glycine and serine was added between the Fc and the B7-H6 variant to ensure the fluidity of each protein. Specifically, the wild-type B7-H6, the genes of three variants (B5, B7, and B14) selected from among the 17 B7-H6 variants selected in Example 7, the Fc domain, a designed primer, and Vent Polymerase ( After amplification using (New England Biolab), the amplified gene was subjected to Assembly PCR and then treated with Bss HII and Xba I restriction enzymes (New England Biolab). The genes of the restriction enzyme-treated B7-H6 mutants were ligated into the pMAZ vector, a vector for animal cells, treated with the same restriction enzyme. After transforming the ligated plasmid into E. coli Jude1, a single clone was obtained and sequence analysis was performed, confirming that the genes of the mutants were successfully inserted into the vector. The constructed B7-H6 mutant-Fc fusion protein expression vector was transfected into Expi293F animal cells, cultured in a CO ₂ shaking incubator at 37°C, 125 rpm, and 8% CO ₂ for 7 days, and then centrifuged to collect only the supernatant. separated. Afterwards, it was equilibrated using 25x PBS and filtered using a 0.2 μm syringe filter (Sartorius, S6634). 0.15 ml of Protein A resin was added to the filtered culture medium, stirred at room temperature for 1 hour to recover the resin, and then washed with PBS. Afterwards, it was eluted with 100 mM glycine buffer (pH 2.7), neutralized using 1 M Tris-HCl (pH 8.0), and buffered with 1×PBS (pH 7.4) using centrifugal filter units 10K (Merck Millipore). was exchanged. The expression of the wild-type B7-H6-Fc fusion protein expressed and purified in this way and the B7-H6 variant-Fc fusion protein, which includes each of the three variants (B5, B7, and B14) discovered in the present invention, was confirmed by SDS-PAGE. (Figure 6).

Example 9. Verification of NKp30 binding ability of B7-H6 variants

ELISA was performed to analyze the NKp30 binding ability of the B7-H6 variant-Fc fusion proteins purified in Example 8. Specifically, the B7-H6 variant-Fc fusion proteins diluted to 4 μg/ml in 0.05 M Na ₂ CO ₃ pH 9.6 were dispensed in 50 μl portions into Flat Bottom Polystyrene High Bind 96-well microplates (costar) and incubated for 16 minutes at 4°C. After immobilization for an hour, the cells were blocked with 100 μl of 4% skim milk (Biopure) (in PBS, pH 7.4) at room temperature for 1 hour. After washing 4 times with 180 μl of 0.05% PBST (pH 7.4), 50 μl of NKp30-GST serially diluted with 1% skim milk (Biopure) (in PBS, pH 7.4) was dispensed into each well and reacted at room temperature for 1 hour. I ordered it. After washing 4 times, 50 μl of anti-GST-HRP conjugate was added, reacted at room temperature for 1 hour, and washed 4 times. After that, 50 μl of 1-Step Ultra TMB-ELISA Substrate Solution (Thermo Fisher Scientific) was added to develop color, and then 2 MH ₂ SO ₄ was added at a time of 50 μl to terminate the reaction, and then analyzed using an Epoch Microplate Spectrophotometer (BioTek). analyzed.

As a result, the three selected mutants (B5, B7, and B14) showed significantly higher binding affinity than the wild type B7-H6 (Figure 7).

Example 10. Additional screening of B7-H6 variants

10-1. Production of a large B7-H6 focused library to use ultra-fast screening techniques

A library to further discover new B7-H6 variants with improved NKp30 binding by introducing mutations at two amino acid positions in the three variants (B5, B7, B14), excluding mutations matching wild-type B7-H6. was produced. For the library genes, a yeast surface display library was constructed in the same manner as in Example 4 above. The constructed library was 1.5 × 10 ⁷ in size, and through sequence analysis, it was confirmed that mutations were included at two locations (FIG. 8).

10-2. B7-H6 variant screening

Yeasts with high binding affinity to NKp30 were recovered by measuring the fluorescence signal value of the library sample in the same manner as in Example 5, and the screening process was performed once.

10-3. Confirmation of enrichment of B7-H6 variants with increased binding affinity to NKp30

The binding ability of the library to NKp30 was indirectly analyzed in the same manner as in Example 6 above. As a result, it was confirmed that variants with improved binding ability to NKp30 were amplified after one screening (Figure 9).

Example 11 Amino acid sequence analysis and selection of additional B7-H6 variants

The expression level of the variants and their binding ability to NKp30 were indirectly analyzed by measuring the fluorescence signal value in the same manner as in Example 7. Through this, a total of 9 variants (BF2, BF3, BF5, BF8, BF11, BF19, BF25, BF39, and BF46) with improved binding to NKp30 were selected (Table 2 and Figure 10).

B7-H6 variant	B7-H6 mutation location and substituted amino acid
B5	F51S/S60I
B6	N57D
B7	K101E/S102R
B8	A32T/S60I
B9	A32T/L40Q/S60I
B14	F51L/S60I
B16	K101E
B19	A32T/W67R/Q86L/K101E/L129M
B23	S60I/L114M/S142N
B29	M31I/A32T/F51I/S60I/S102C/R108M
B35	A32T/S60I/K101E/P143S
B40	I37T/Q86L/K101E
B41	A32T/S53G/S60I/Q136R
B47	F51Y/S60I
B52	S60I/K101E
B53	A32T/K101E
B54	A32T/S60I/K101E
BF2	F51H/S60I
BF3	F51I/S60Y
BF5	I37F/F51L/S60T
BF8	F51T/S60T
BF11	F51L/S60H
BF19	F51T/S60Y
BF25	F51Q/S60H
BF39	F51K/S60L
BF46	F51R/S60T

Example 12. Preparation, expression, and purification of animal cell expression vectors for additionally secured B7-H6 variants

Animal cell expression vectorial production, expression, and After purification, it was confirmed by SDS-PAGE that the wild type B7-H6 and all 9 additionally discovered mutants were well expressed (FIG. 11).

Example 13. Verification of NKp30 binding ability of B7-H6 variants of the present invention

ELISA was performed in the same manner as Example 9 above for the three initially discovered variants (B5, B7, and B14) and the three variants with the highest NKp30 binding affinity (BF2, BF8, and BF19) among the additionally discovered variants. As a result, all variants discovered in the present invention showed significantly higher binding affinity than wild-type B7-H6 (FIG. 12).

Claims (34)

1. 31st, 32nd, 37th, 40th, 51st, 53rd, 57th, 60th, 67th, 86th, 101 of the amino acid sequence of wild type B7-H6 (B7 homolog 6, NCR3LG1) NKp30 (Natural cytotoxicity triggering receptor) in which one or more amino acids selected from the group consisting of the 102nd, 102nd, 108th, 114th, 129th, 136th, 142nd and 143rd amino acids are substituted with a sequence different from the wild type amino acid. 3) B7-H6 variant with increased binding affinity.
2. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, wherein the amino acid of wild-type B7-H6 includes the amino acid sequence of SEQ ID NO: 1.
3. The method of claim 1, M31I, A32T, I37T, I37F, L40Q, F51I, F51S, F51L, F51Y, F51T, F51H, F51Q, F51K, F51R, S53G, N57D, S60I, S60Y, S60T, S60H, S60L, W67R, A B7-H6 variant with increased binding affinity to NKp30, comprising one or more amino acid substitutions selected from the group consisting of Q86L, K101E, S102C, S102R, R108M, L114M, L129M, Q136R, S142N and P143S.
4. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, comprising amino acid substitutions F51S and S60I.
5. The B7-H6 variant according to claim 1, which has increased binding affinity to NKp30, comprising amino acid substitutions K101E and S102R.
6. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, comprising amino acid substitutions A32T and S60I.
7. The B7-H6 variant according to claim 1, comprising amino acid substitutions A32T, L40Q, and S60I, with increased binding affinity to NKp30.
8. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, comprising amino acid substitutions F51L and S60I.
9. The B7-H6 variant according to claim 1, which has increased binding affinity to NKp30, comprising amino acid substitutions A32T, W67R, Q86L, K101E, and L129M.
10. The B7-H6 variant according to claim 1, which has increased binding affinity to NKp30, comprising amino acid substitutions S60I, L114M, and S142N.
11. The B7-H6 variant according to claim 1, which has increased binding affinity to NKp30, comprising amino acid substitutions M31I, A32T, F51I, S60I, S102C, and R108M.
12. The B7-H6 variant according to claim 1, which has increased binding affinity to NKp30, comprising amino acid substitutions A32T, S60I, K101E, and P143S.
13. The B7-H6 variant according to claim 1, which has increased binding affinity to NKp30, comprising amino acid substitutions I37T, Q86L, and K101E.
14. The B7-H6 variant according to claim 1, which has increased binding affinity to NKp30, comprising amino acid substitutions A32T, S53G, S60I, and Q136R.
15. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, comprising amino acid substitutions F51Y and S60I.
16. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, comprising amino acid substitutions S60I and K101E.
17. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, comprising amino acid substitutions A32T and K101E.
18. The B7-H6 variant according to claim 1, comprising amino acid substitutions A32T, S60I and K101E, with increased binding affinity to NKp30.
19. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, comprising amino acid substitutions F51H and S60I.
20. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, comprising amino acid substitutions F51I and S60Y.
21. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, comprising amino acid substitutions I37F, F51L, and S60T.
22. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, comprising amino acid substitutions F51T and S60T.
23. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, comprising amino acid substitutions F51L and S60H.
24. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, comprising amino acid substitutions F51T and S60Y.
25. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, comprising amino acid substitutions F51Q and S60H.
26. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, comprising amino acid substitutions F51K and S60L.
27. The B7-H6 variant with increased binding affinity to NKp30 according to claim 1, comprising amino acid substitutions F51R and S60T.
28. A natural killer cell (NK cell) activator comprising the B7-H6 variant of claim 1.
29. A bispecific or multispecific antibody comprising the B7-H6 variant of claim 1 and a portion that binds to a target antigen.
30. 30. The method of claim 29, wherein the target antigen is 17-1A antigen, GD3 ganglioside R24, EGFRvIII, PSMA, PSCA, HLA-DR, EpCAM, MUC1 core protein, aberrant glycosylated MUC1, fibronectin variant containing an ED-B domain. , HER2/neu, carcinoembryonic antigen (CEA), gastrin-releasing peptide (GRP) receptor antigen, mucine antigen, epidermal growth factor receptor (EGF-R), HER3, HER4, MAGE antigen, SART antigen, MUC1 Antigen, c-erb-2 antigen, TAG 72, carbonic anhydrase IX, alpha-fetoprotein, A3, antigen specific for A33 antibody, Ba 733, BrE3-antigen, CA125 , CDl, CD1a, CD3, CD5, CDl5, CD16, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD33, CD38, CD40, CD45, CD52, CD74, CD79a, CD80, CD138, colon-specific antigen -p(CSAp), CSAp, EGP-1, EGP-2, Ep-CAM, FIt-1, Flt-3, folate receptor, HLA-DR, human chorionic gonadotropin (HCG) and Its subunits, hypoxia-inducible factor (HIF-I), Ia, IL-2, IL-6, IL-8, insulin growth factor-1 (IGF-1), KC4-antigen, KS-1-antigen, KSl -4, Le-Y, macrophage inhibitory factor (MIF), MAGE, MUCl, MUC2, MUC3, MUC4, NCA66, NCA95, NCA90, antigen specific for PAM-4 antibodies, placental growth factor, p53, prostatic acid phosphatase ( prostatic acid phosphatase), PSA, RS5, SlOO, TAC, tenascin, TRAIL receptors, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, VEGF, ED-B fibronectin ( A bispecific or multispecific antibody that is at least one selected from the group consisting of fibronectin, angiogenesis marker, oncogene marker, or oncogene product.
31. A pharmaceutical composition for the treatment or prevention of cancer, comprising the B7-H6 variant of claim 1 or the bispecific or multispecific antibody of claim 29.
32. i) isolating natural killer cells; and

    ii) A method of in vitro proliferation of activated natural killer cells, comprising culturing the isolated natural killer cells in the presence of the B7-H6 variant or fragment thereof of claim 1.
33. Use of the B7-H6 variant of claim 1 or the bispecific or multispecific antibody of claim 29 for the prevention or treatment of cancer.
34. A method of treating cancer comprising administering the B7-H6 variant of claim 1 or the bispecific or multispecific antibody of claim 29 in a pharmaceutically effective amount to a subject suffering from cancer.

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