RU2780537C2 - Cd3-specific antibodies and their use - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: antibody (options), which specifically binds to CD3, a bispecific antibody, which specifically binds to CD3 and also specifically binds to a tumor antigen, are presented. A pharmaceutical composition for the treatment of cancer; a method for modulation of a T-cell-mediated immune response in a patient; a method for inhibition of tumor cell growth in an individual; polynucleotide for the production of an antibody or a bispecific antibody; and the use of an antibody or a bispecific antibody, or a pharmaceutical composition are presented.

EFFECT: claimed anti-CD3 antibodies can be useful for therapeutic purposes, including activation of T-cells.

20 cl, 21 tbl, 22 dwg, 11 ex

Description

This application is filed electronically via EFS-Web and includes sequence listings submitted electronically in .txt format. The .txt file created on May 13, 2019 and named "PC72375-PRV2_SequenceListing_ST25_05132019.txt" contains sequence listings and is 100 KB in size. The sequence listings contained in this .txt file are part of the specification and are incorporated herein by reference in their entirety.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention relates to antibodies and antigen-binding fragments specific for CD3, and methods for their use. The present invention also relates to polyspecific antibodies that bind to CD3 and a target antigen, and methods of using them.

BACKGROUND OF THE INVENTION

Cluster of differentiation 3 (CD3) is a homodimeric or heterodimeric antigen expressed on T cells in association with the T cell receptor complex (TCR) and is essential for T cell activation. T cell activation is a complex phenomenon that depends on the involvement of multiple cell surface molecules expressed in a population of reactive T cells. Functional CD3 results from the dimeric association of two of four distinct chains: epsilon, zeta, delta, and gamma. For example, the antigen-specific T-cell receptor (TCR) consists of a disulfide-linked heterodimer containing two clonally distributed chains of integral membrane glycoproteins: alpha and beta (α and β) or gamma and delta (γ and δ), non-covalently associated with a complex of small molecular weight invariant proteins commonly referred to as CD3. Dimeric structures of CD3 also include gamma/epsilon, delta/epsilon and zeta/zeta.

Anti-CD3 antibodies have been shown to cluster CD3 on T cells, thereby inducing T cell activation in a manner similar to TCR binding to peptide-loaded MHC molecules. Thus, anti-CD3 antibodies may be useful for therapeutic purposes, including T cell activation.

SUMMARY OF THE INVENTION

The present invention relates to antibodies that bind CD3, as well as compositions, such as pharmaceutical compositions, containing one or more anti-CD3 antibodies. The antibodies of this aspect of the invention are useful, among other things, for targeting CD3-expressing T cells and for stimulating T cell activation, for example in circumstances where T cell-mediated killing is useful or desirable. The anti-CD3 antibodies of the invention can be formulated into a multispecific antibody, such as a bispecific antibody, that directs CD3-mediated T cell activation to certain cell types, such as tumor cells or infectious agents.

In one aspect, the present invention provides an antibody that specifically binds to CD3, said antibody comprising: (a) a heavy chain variable region (VH) comprising a VH complementarity determining region one (VH CDR1), a VH complementarity determining region two (VH CDR2) and complementarity determining region three VH (VH CDR3) VH sequence with SEQ ID NO: 2, 4, 5, 7, 10, 12 or 35; and/or (b) a light chain variable region (VL) comprising a VL complementarity determining region one (VL CDR1), a VL complementarity determining region two (VL CDR2), and a VL complementarity determining region three (VL CDR3) of the VL sequence c SEQ ID NO : 1, 3, 6, 8, 9, 11, 34, 87 or 89.

In one embodiment of this aspect, the anti-CD3 antibody comprises: (a) a VH region containing (i) a VH CDR1 comprising the sequence of SEQ ID NO: 13, 14, or 15; (ii) VH CDR2 containing the sequence of SEQ ID NO: 16, 17, 19, 20, 21, 22, 23 or 24; and (iii) a VH CDR3 containing the sequence of SEQ ID NO: 18 or 25; and/or (b) a VL region containing (i) a VL CDR1 containing the sequence of SEQ ID NO: 26, 29, 30, 32, 91 or 92; (ii) VL CDR2 containing the sequence of SEQ ID NO: 27 or 31; and (iii) a VL CDR3 containing the sequence of SEQ ID NO: 28 or 33.

In some such embodiments, the invention relates to an antibody in which: (a) the VH region contains the sequence of SEQ ID NO: 2, 4, 5, 7, 10, 12, or 35; and/or (b) the VL region contains the sequence SEQ ID NO: SEQ ID NO: 1, 3, 6, 8, 9, 11, 34, 87, or 89.

In other embodiments, the antibody comprises a VL/VH amino acid sequence pair selected from the group consisting of: SEQ ID NOs: 1 and 2; SEQ ID NO: 3 and 2; SEQ ID NOs: 3 and 4; SEQ ID NOs: 3 and 5; SEQ ID NOs: 6 and 7; SEQ ID NOs: 8 and 7; SEQ ID NOs: 6 and 4; SEQ ID NOs: 8 and 4; SEQ ID NOs: 9 and 10; SEQ ID NOs: 9 and 7; SEQ ID NOs: 11 and 12; SEQ ID NOs: 34 and 35; SEQ ID NOs: 9 and 4; SEQ ID NO: 87 and 4; and SEQ ID NOs: 89 and 4.

In some embodiments of each of the above aspects, the antibody is selected from the group consisting of Fab, Fab fragment, F(ab) ₂ fragment, Fv fragment, single chain Fv fragment, disulfide stabilized Fv fragment, single chain antibody, monoclonal antibody, chimeric antibody, a bispecific antibody, a trispecific antibody, a polyspecific antibody, a bispecific heterodimeric diabody, a bispecific heterodimeric IgG, a polyclonal antibody, a labeled antibody, a humanized antibody, a human antibody, and fragments thereof.

In a specific embodiment, the antibody is a bispecific antibody. In some such embodiments, the bispecific antibody specifically binds to a tumor antigen.

In some embodiments, an antibody of the invention further comprises a human or humanized VH scaffold and a human or humanized VL scaffold. In some such embodiments, the antibody is a humanized antibody.

The present invention also relates to a pharmaceutical composition comprising an anti-CD3 antibody of the invention described hereinafter and a pharmaceutically acceptable carrier.

In another aspect, the present invention relates to a method for modulating a T cell-mediated immune response in a subject in need thereof, comprising administering to the subject an effective amount of an antibody or pharmaceutical composition disclosed herein to modulate the immune response in the subject.

In one aspect, the present invention relates to a method for suppressing the growth of tumor cells in a subject, comprising administering to the subject a bispecific antibody, a trispecific antibody, a multispecific antibody, a bispecific heterodimeric diabody, or a bispecific heterodimeric IgG disclosed herein, in an effective amount to suppress the growth of tumor cells.

In some such embodiments, the tumor cells are cancer cells. In specific embodiments, the cancer is selected from the group consisting of breast, ovarian, thyroid, prostate, cervical, lung, bladder, endometrial, head and neck, testicular, glioblastoma, and digestive system cancers.

In one embodiment, cancers of the digestive system are selected from the group consisting of cancers of the esophagus, stomach, small intestine, colon, rectum, anus, liver, gallbladder, appendix, bile ducts, and pancreas.

In one embodiment, the invention relates to an antibody, or a pharmaceutical composition containing such an antibody, for use in therapy.

In another embodiment, the invention relates to the use of an antibody of the invention in the manufacture of a medicament for use in therapy. In some such embodiments, the implementation of therapy includes the activation of the cytolytic T-cell response.

In one aspect, the present invention relates to a polynucleotide containing a nucleotide sequence encoding an antibody disclosed in the present description. In another aspect, the invention relates to a vector containing such a polynucleotide.

In another aspect, the invention relates to a host cell containing the vectors disclosed in the present description. In some such embodiments, the host cell recombinantly produces the antibody disclosed herein. In specific embodiments, the host cell is selected from the group consisting of bacterial cell lines, mammalian cell lines, insect cell lines, yeast cell lines. In a specific embodiment, the mammalian cell line is a CHO cell line.

In one aspect, the present invention provides a method for producing an antibody disclosed herein, comprising culturing a host cell under conditions that result in the production of an antibody disclosed herein and purifying the antibody from the culture supernatant.

In another aspect, the present invention relates to the use of an anti-CD3 antibody, polynucleotide, vector or host cell as disclosed herein in the manufacture of a medicament for the treatment of a disorder.

In one aspect, the invention relates to an antibody or pharmaceutical composition for use in a method for modulating a T-cell mediated immune response in a subject in need thereof. In specific embodiments, the invention relates to an antibody or pharmaceutical composition for use in a method for suppressing the growth of tumor cells in a subject.

In another aspect, the invention relates to an antibody or pharmaceutical composition for use in the treatment of cancer, optionally the cancer being selected from the group consisting of breast, ovarian, thyroid, prostate, cervix, lung, bladder, endometrial, head and neck, testicular , glioblastoma and cancer of the digestive system.

In another aspect, the invention relates to an antibody or pharmaceutical composition for use in the treatment of cancer, which modulates a T-cell mediated immune response or inhibits the growth of tumor cells.

Other embodiments will become apparent from the detailed description. Where aspects or embodiments of the invention are described in terms of the Markush group or other group of alternatives, the present disclosure covers not only the entire group listed as a whole, but each member of the group individually and all possible subgroups of the main group, as well as the main group, where one or more group members are missing. The present invention also provides for the explicit exclusion of one or more members of the group in the claimed invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a structural diagram of a bispecific antibody having a first domain providing a heterodimer and a second domain providing a heterodimer containing an Fc fragment chain (Fc chain) optimized for ridge-to-trough binding.

In FIG. 2 shows the results of differential scanning calorimetry thermograms showing the heat capacity of the GUCY2c-H2B4 and GUCY2c-2B5 bispecific antibodies as a function of temperature.

In FIG. 3 shows the binding of bispecific antibodies GUCY2c-H2B4 (GUCY2c-0247) and GUCY2c-2B5 (GUCY2c-0250) to human naive T cells by flow cytometry analysis.

In FIG. 4 shows the results of in vitro cytotoxicity mediated by the bispecific antibodies GUCY2c-H2B4 (GUCY2c-0247) and GUCY2c-2B5 (GUCY2c-0250) using cell viability data.

In FIG. 5 shows the potential energy surface (PES) of the Fv region of H2B4 obtained from the crystal structure obtained by X-ray diffraction. It includes 3 views which show VH area, VH/VL interface and VL area. CDRs with excess positive charge are shown in the drawing for reference. Dark black means positive charge, while white means negative.

In FIG. 6 shows the Fv surface of the H2B4 region with spatial aggregation propensity (SAP). Regions with concentrated hydrophobic residues are marked.

In FIG. 7 are capillary gel electrophoresis electropherograms showing the heterogeneity of bispecific antibodies containing the anti-CD3 binding domains of antibodies H2B4, 2B5 and 2B5v6.

In FIG. 8 is a differential scanning calorimetry (DSC) thermogram showing the heat capacity of the GUCY2c-1608 bispecific antibody (GUCY2c-2B5v6) as a function of temperature.

In FIG. 9 shows the binding of the bispecific antibody GUCY2c-1608 (GUCY2c-2B5v6) to naive T cells.

10 shows cell viability data showing in vitro cytotoxicity mediated by the GUCY2c-1608 bispecific antibody (GUCY2c-2B5v6).

In FIG. 11A-11F show measurements of bispecific GUCY2c-1608 (GUCY2c-2B5v6) mediated cytokine release in vitro. The bispecific antibody recruits naive human T cells to GUCY2c-expressing T84 cells to induce cytokine release in vitro. Luminex-based analysis showed increased expression of human IFN-gamma (Fig. 11A), IL10 (Fig. 11B), IL2 (Fig. 11C), IL4 (Fig. 11D), IL6 (Fig. 11E), and TNF-alpha (Fig. 11E). .11F).

In FIG. 12 shows the dose-dependent tumor growth suppression of the GUCY2c-1608 bispecific antibody (GUCY2c-2B5v6) in a PDX-CRX-11201 xenograft from a colorectal cancer patient in an adoptive transfer system.

In FIG. 13 shows dose-dependent tumor growth inhibition by the GUCY2C-1608 bispecific antibody (GUCY2C-2B5v6) in the LS1034 colorectal cancer xenograft cell line, in an adoptive transfer system.

In FIG. 14A shows the results of cell viability data demonstrating in vitro cytotoxicity mediated by the FLT3-2B5v6 bispecific antibody at various effector:target (E:T) ratios using Flt3-expressing MV4-11 cells. In FIG. 14B shows the results of cell viability data demonstrating in vitro cytotoxicity mediated by the FLT3-2B5v6 bispecific antibody at various effector:target (E:T) ratios using Flt3-expressing EOL-1 cells.

In FIG. 15 shows the binding of low affinity 2B5v6 variants to Jurkat cells expressing CD3.

In FIG. 16 is a graph summarizing the results of the study showing bispecific anti-GUCY2C antibodies dependent T-cell mediated cytotoxicity with anti-CD3 variants.

DETAILED DESCRIPTION OF THE INVENTION

The invention disclosed herein relates to antibodies that specifically bind to CD3 (eg, human CD3), including full-length antibodies or antigen-binding fragments thereof. The invention also relates to polynucleotides encoding these antibodies, compositions containing these antibodies, and methods for making and using these antibodies. The invention also relates to methods for treating a disorder using the antibodies disclosed herein.

General Methods

In the practice of the present invention, unless otherwise indicated, conventional methods of molecular biology (including recombinant methods), microbiology, cell biology, biochemistry and immunology are used, which are within the competence of specialists in this field. Such methods are fully described in the literature, for example, Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995). meanings are defined herein for clarity and/or merely by reference, and the inclusion of such definitions herein should not necessarily be construed as meaning that such definitions differ materially from their usual understanding in the art.

The invention is described in more detail below with reference to the following definitions and examples. All patents and publications, including all sequences disclosed in such patents and publications referred to in this specification, are expressly incorporated by reference.

Definitions

As a rule, unless otherwise specified, all terms in the art, designations and other scientific terms or terminology used in the present description have the meanings commonly understood by specialists in the art to which the present invention pertains. For example, the term "and/or" used in a phrase such as "A and/or B" includes "A and B", "A or B", "A" (only) and "B" (only) . Similarly, the term "and/or" used in a phrase such as "A, B and/or C" includes each of the following: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (only); B (only); and C (only).

As used herein, the singular forms include appropriate references to the plural, unless the context clearly dictates otherwise.

Used in the present description, the numerical ranges include numbers that define this range.

The terms "about", "approximately", etc., preceding a list of numerical values or a range, refer to each individual value in the list or range independently, as if the term preceded each individual value in the list or range. These terms mean that the meanings to which the above mentioned refer are exactly the same, close to or similar to them. For example, in some embodiments, about or approximately a particular value may indicate a value of 99%, 95%, or 90% of the specified value. For example, the expression "about 100" includes 99 and 101 and everything in between (eg, 99.1, 99.2, 99.3, 99.4, 99.5, etc.). As another example, when the temperature is 70°C, "about" or "about" 70°C may be 69°C, 66°C or 63°C. It should be understood that these are just examples.

In the context of the present description, nucleic acids are presented from left to right in the direction from the 5' to the 3' end; amino acid sequences are presented from left to right in the direction from the amino-terminus to the carboxy-terminus, respectively. Definitions and terms used in the art can be found in particular in Sambrook et al., 1989 and Ausubel FM et al., 1993. It should be understood that the present invention is not limited to the specific methods, protocols, and reagents described, as they can be changed.

The terms "polypeptide", "oligopeptide", "peptide", and "protein" are used interchangeably herein to refer to amino acid chains of any length, preferably relatively short (eg, 10-100 amino acids). The chain may be linear or branched, may contain modified amino acids, and/or may be interrupted by non-amino acids. The terms also cover an amino acid chain that has been naturally or tampered with; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification such as conjugation to a labeling component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids, etc.), as well as other modifications known in this field. It is understood that the polypeptides may exist as single chains or linked chains. Preferred for use are mammalian polypeptides (polypeptides that were originally obtained from the body of a mammal), more preferably polypeptides secreted directly into the environment.

An "antibody" is an immunoglobulin molecule capable of specifically binding to a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a bispecific antibody, a dual specificity antibody, a bifunctional antibody, a trispecific antibody, a polyspecific antibody, a bispecific heterodimeric diabody, a bispecific heterodimeric IgG, a labeled antibody, a humanized antibody, a human antibody, and fragments thereof (such as Fab, Fab', F(ab') ₂ , Fv), single chain (ScFv) and domain antibodies (including, for example, shark and camel antibodies), antibody-containing fusion proteins, and any other modified molecular configuration an immunoglobulin that includes an antigen recognition site. An antibody includes an antibody of any class such as IgG, IgA or IgM (or a subclass thereof) and the antibody need not be of any particular class. Depending on the amino acid sequence of the constant region of the heavy chains of the antibody, immunoglobulins can be classified into different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant regions that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known. The invention also includes "analogue(s) of the antibody(s)", scaffolds based on other proteins that are not antibody molecules, for example, fusion proteins and/or immunoconjugates that use CDRs to provide specific antigen binding. The antibodies of the invention may be from any species, including, but not limited to, mice, humans, camels, llamas, fish, sharks, goats, rabbits, chickens, horses, and cattle.

The term "antibody" further includes immunoglobulin molecules containing four polypeptide chains, two heavy (H) chains and two light (L) chains connected by disulfide bonds, as well as their multimers (eg, IgM). Each heavy chain contains a heavy chain variable region (abbreviated herein as VR HC or VH) and a heavy chain constant region. The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. The heavy chain constant region contains three domains, CH1, CH2 and CH3. The CH1 and CH2 domains are connected by a hinge region. Each light chain contains a light chain variable region (abbreviated herein as VR LC or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) that alternate with more conserved regions called framework regions (FRs). Each of the VH and VL consists of three CDRs and four FRs arranged in the amino-terminal to carboxy-terminal direction in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments, the FR of the anti-CD3 antibody (or antigen-binding portion thereof) may be identical to human germline sequences, or may be naturally or artificially modified. The amino acid consensus sequence can be determined by direct comparison of two or more CDRs. In each chain, the CDRs are held together in close proximity by the FR and, together with the CDRs of the other chain, contribute to the formation of the antigen-binding site of the antibodies.

The terms "antigen-binding fragment", "antibody fragment" or "antigen-binding portion" in the context of the present description refers to one or more antibody fragments that retain the ability to selectively bind to an antigen. Examples of binding fragments encompassed by the term "antigen binding fragment" of an antibody include (i) an antibody heavy chain variable domain (VH) and/or a light chain variable domain (VL) of an antibody, or a VH/VL pair derived from full-length antibodies or antibody fragments, such as a VH domain and/or a VL domain; (ii) Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (iii) a Fab' fragment that is essentially a Fab with part of the hinge region (eg, Fundamental Immunology, Paul ed., 3.sup.rd ed.1993); (iv) F(ab') ₂ fragment, divalent fragment containing two Fab fragment connected by a disulfide bridge in the hinge region; (v) an Fd fragment consisting of VH and CH1 domains; (vi) an Fv fragment consisting of the VL and VH domains of a single antibody arm; (vii) a single chain Fv fragment (scFv) which is a single protein chain in which the VL and VH regions are paired to form monovalent molecules (e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. ( 1988) Proc Natl Acad Sci USA 85:5879-5883); (viii) a disulfide-stabilized Fv fragment (dsFv), i.e. Fv with an engineered intermolecular disulfide bond to stabilize the VH-VL pair; (ix) an antibody fragment with a single variable domain (sdAb or dAb) (eg, Ward et al., (1989) Nature 341:544-546) that consists of a heavy chain variable domain and lacks a light chain; (x) complementarity determining region (CDR); and any of their derivatives.

As used herein, the term "antigen-binding fragment" of an antibody may contain at least one variable domain. The variable domain can be of any size or any amino acid composition, and typically contains at least one CDR that is adjacent to or in-frame with one or more framework sequences. In the context of the present description, the term "antigen-binding fragment of an antibody" may include a homodimer or heterodimer (or other multimer) of any configuration from the configurations consisting of the variable region and constant domain listed below, non-covalently associated with each other and / or with one or more monomeric regions VH or VL (eg via disulfide(s) bond(s)). For example, the variable region may be dimeric and contain VH-VH, VH-VL, or VL-VL dimers. Variable and constant domain configurations that can be found in an antigen-binding fragment of an antibody of the present invention include: VH-CH1; VH-CH2; VH-CH3; VH-CH1-CH2; VH-CH1-CH2-CH3; VH-CH2-CH3; VH-VL-CL, VH-VL-CH1, VH-VL-CH2; VH-CL; VL-CH1; VL-CH2; VL-CH3; VL-CH1-CH2; VL-CH1-CH2-CH3; VL-CH2-CH3; and VL-CL. Variable regions and constant domains may either be directly linked to each other or may be linked through a full or partial hinge or linker region. A hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible bond between adjacent variable regions and/or constant domains in a single polypeptide molecule.

As used herein, the term "binding domain" includes any region of a polypeptide (eg, antibody) that is responsible for selective binding to a molecule of interest (eg, antigen, ligand, receptor, substrate, or inhibitor). Examples of binding domains include an antibody variable region, a receptor binding domain, a ligand binding domain, and an enzymatic domain.

As used herein, the term “human acceptor framework” is a framework containing the amino acid sequence of a light chain variable region (VL) framework or a heavy chain variable region (VH) framework derived from a human immunoglobulin framework or a human consensus framework. , as defined below. A human acceptor framework "derived from" a human immunoglobulin framework or human consensus framework may contain the same amino acid sequence or may contain amino acid sequence modifications. In some embodiments, the number of amino acid modifications is 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some embodiments, the sequence of the human VL acceptor framework is identical to the sequence of the human immunoglobulin VL framework or the human framework consensus sequence.

As used herein, an "affinity matured antibody" refers to an antibody with one or more modifications in one or more variable regions (which include the CDR and FR) relative to the parent antibody that does not have such modifications, such modifications resulting in an improvement in the antibody's affinity for antigen.

In the context of the present description, the terms "Fc region", "Fc domain", "Fc chain" or similar terms are used to define the C-terminal region of the IgG heavy chain. The Fc region of IgG includes two constant domains, CH2 and CH3. The CH2 domain of the human IgG Fc region typically extends from amino acid 231 to amino acid 340 in the EU numbering system, or from amino acid 244 to amino acid 360 in the Kabat numbering system. The CH3 domain of the human IgG Fc region typically extends from amino acid 341 to 447 in the EU numbering system, or from amino acid 361 to amino acid 478 in the Kabat numbering system. The CH2 Fc domain of the human IgG region (also referred to as the "Cγ 2" domain) is unique in that it is not closely coupled to another domain. In contrast, two N-linked branched carbohydrate chains are located between the two CH2 domains of intact natural IgG. The Fc region may contain native or altered Fc sequences. Although the sequence boundaries of the immunoglobulin heavy chain Fc region may vary, the sequence of the human IgG heavy chain Fc region is generally defined as extending from an amino acid residue at about Cys226 or about Pro230 to the carboxyl terminus of the Fc region sequence. Unless otherwise indicated herein, the numbering of amino acid residues in an Fc region or constant region follows the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

In some embodiments, the Fc chain begins in the hinge region just before the papain cleavage site and ends at the C-terminus of the antibody. Accordingly, a complete Fc chain contains at least a hinge domain, a CH2 domain, and a CH3 domain. In some embodiments, the Fc chain contains at least one of: a hinge domain (eg, an upper, middle, and/or lower hinge), a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In some embodiments, the Fc domain contains a complete Fc chain (ie, a hinge domain, a CH2 domain, and a CH3 domain). In some embodiments, the Fc chain contains a hinge domain (or part thereof) fused to a CH3 domain (or part thereof). In some embodiments, the Fc chain contains a CH2 domain (or part thereof) fused to a CH3 domain (or part thereof). In some embodiments, the Fc chain consists of a CH3 domain or a portion thereof. In some embodiments, the Fc chain consists of a hinge domain (or part thereof) and a CH3 domain (or part thereof). In some embodiments, the Fc chain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In some embodiments, the Fc chain consists of a hinge domain (or part thereof) and a CH2 domain (or part thereof). In some embodiments, at least a portion of the CH2 domain is missing from the Fc chain (eg, all or part of the CH2 domain). As used herein, an Fc chain generally refers to a polypeptide containing all or part of an immunoglobulin heavy chain Fc chain. It includes, without limitation, polypeptides containing an entire CH1 domain, a hinge domain, CH2 and/or CH3 domains, as well as fragments of such peptides containing only, for example, a hinge domain, a CH2 domain, and a CH3 domain. The Fc chain can be derived from any immunoglobulin species and/or any subtype, including, without limitation, human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. The Fc domain includes native Fc molecules and Fc variants. As with Fc variants and native Fcs, the term "Fc chain" includes molecules in monomeric or polymeric form, whether cleaved from the whole antibody or prepared by other means. In some embodiments, the Fc chain contains the carboxy-terminal portions of both heavy chains held together by disulfide bonds. In some embodiments, the Fc chain consists of a CH2 domain and a CH3 domain.

As used in the art, the terms "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Moreover, a preferred FcR is a receptor that binds to an IgG antibody (gamma receptor) and includes receptors of the FcγRI, FcγRII and FcγRIII subclasses, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences that differ mainly in cytoplasmic domains. An overview of FcR species can be found in Ravetch and Kinet, Ann. Rev. Immunol. 9:457-92, 1991; Capel et al., Immunomethods, 4:25-34, 1994; and de Haas et al., J. Lab. Clin. Med., 126:330-41, 1995. "FcR" also includes the neonatal FcRn receptor, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol., 117:587, 1976; and Kim et al. , J. Immunol., 24:249, 1994).

A "native sequence Fc region" or "wild type Fc region" contains an amino acid sequence identical to the amino acid sequence of a naturally occurring Fc region. By "wild-type" human IgG Fc is meant an amino acid sequence that occurs naturally in the human population. Of course, just as Fc sequences may vary slightly from individual to individual, wild-type sequences may contain one or more changes, but still remain within the scope of the invention. For example, the Fc region may contain additional changes that are not relevant to the present invention, such as a mutation at the glycosylation site, the inclusion of a non-natural amino acid, or a bump-in-trough mutation.

An "Fc region variant" or "chain Fc variant" comprises an amino acid sequence that differs from the native Fc region sequence by at least one amino acid modification, but retains at least one effector function of the native sequence Fc region. In some embodiments, the Fc chain variant has at least one amino acid substitution compared to the native sequence Fc chain or the Fc region of the parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in Fc chains in the native sequence or in the Fc chain of the parent polypeptide. As used herein, a variant Fc chain may preferably have at least about 80% sequence identity with the native sequence Fc chain and/or with the Fc chain of the parent polypeptide, and most preferably at least about 90% sequence identity, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity.

In the context of the present description, the term "effector functions" refers to those biological activities that are inherent in the Fc chain (native sequence Fc chains or amino acid sequence Fc chain variants) of an antibody, and vary depending on the isotype of the antibody. Examples of antibody effector functions include: C1q binding and complement dependent cytotoxicity; binding to the Fc receptor; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (eg, B-cell receptor); and B cell activation. Such effector functions typically require association of an Fc chain with a binding domain (eg, the variable region of an antibody), and can be assessed using various assays known in the art to evaluate such antibody effector functions. Effector function can be measured, for example, through binding to Fcγ3 and/or C1q.

The effector functions of antibodies are determined by sequences in the Fc chain; this chain is also the part recognized by Fc receptors (FcRs) found on certain cell types.

In some embodiments, the Fc polypeptide contains part or all of the wild-type hinge sequence (typically at its N-terminus). In some embodiments, the Fc polypeptide does not contain a wild-type functional or hinge sequence.

The terms "hinge region", "hinge sequence", and variations thereof, as used herein, include the meaning known in the art as illustrated, for example, in Janeway et al., ImmunoBiology: the immune system in health and disease, Elsevier Science Ltd., NY (4th ed., 1999); Bloom et al., Protein Science, 6:407-415, 1997; and Humphreys et al., J. Immunol. Methods, 209:193-202, 1997.

The term "immunoglobulin-like hinge region", "immunoglobulin-like hinge sequence", or variants thereof, as used herein, refers to the hinge region and the hinge region sequence of an immunoglobulin-like or antibody-like molecule (eg, immunoadhesins). In some embodiments, the immunoglobulin-like hinge region may be derived from or derived from any IgG1, IgG2, IgG3, or IgG4 subtype, or from IgA, IgE, IgD, or IgM, including their chimeric forms, e.g., the chimeric IgG1/2 hinge region.

In some embodiments, the hinge region may be of the human IgG1 subtype, extending from amino acid 216 to amino acid 230 according to the EU numbering system, or from amino acid 226 to amino acid 243 according to the Kabat numbering system. In some embodiments, the sequence may be EPKSCDKTHTCPPCP (SEQ ID NO: 63). Those skilled in the art may define the exact amino acid boundaries corresponding to different domains of the IgG molecule in different ways. Thus, the N-terminus or C-terminus of the domains described above can be lengthened or shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9 or even 10 amino acids.

In some embodiments, the hinge region may be mutated in one or more amino acids. In some embodiments, the implementation of the hinge region may be shortened and contain only part of the full hinge region. In some embodiments, the implementation of the hinge region may contain only the last 5 amino acids of the hinge region, referred to in the present description "lower hinge" region. In some embodiments, the lower hinge region may be composed of CPPCP amino acids (SEQ ID NO: 64) or amino acid 226 to amino acid 230 according to the EU index numbering system, or amino acid 239 to amino acid 243 according to the Kabat numbering system.

As used herein, the term "modification" refers to an amino acid substitution, insertion and/or deletion in a polypeptide sequence, a change in a moiety chemically linked to a protein, or a modification in the function of a protein, such as an antibody. For example, the modification may be an altered function of an antibody, or an altered carbohydrate structure attached to a protein. In the context of the present description, the term "amino acid modification" refers to a mutation (substitution), insertion (addition) or deletion of one or more amino acid residues in an antibody. The term "amino acid mutation" means the replacement of at least one existing amino acid residue with another different amino acid residue (eg, a replacement amino acid residue). The term "deletion of an amino acid" means the removal of at least one amino acid residue at a predetermined position in the amino acid sequence. For example, the L234A mutation means that the lysine amino acid residue at position 234 in the Fc region of the antibody is replaced by an alanine amino acid residue (lysine to alanine substitution) (numbering according to the EU index numbering system). "Naturally occurring amino acid residue" means an amino acid residue from the group consisting of alanine (three letter code: Ala, one letter code: A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine (Cys, C), glutamine (Gin, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (He, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y) and valine (Val, V).

The term "agent" is used herein to refer to a biological macromolecule, an extract obtained from biological materials, a mixture of biological macromolecules, a chemical compound, a mixture of chemical compounds, and/or a mixture of chemical compounds and biological macromolecules. The term "therapeutic agent" refers to an agent with biological activity.

In the context of the present description, "monoclonal antibody" refers to an antibody derived from a population of essentially homogeneous antibodies, i.e. the individual antibodies that make up a population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Moreover, unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the nature of the antibody derived from a substantially homogeneous population of antibodies and should not be construed as requiring the production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be prepared by the hybridoma method first described by Kohler and Milstein, Nature 256:495, 1975, or may be prepared by recombinant DNA methods, such as those described in US Pat. No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries generated using the methods described in, for example, McCafferty et al., Nature 348:552-554, 1990.

The antibodies of the present invention may be "humanized antibodies". As used herein, the term "humanized" antibody refers to non-human (e.g., murine, rat, rabbit, non-human primate, or other mammalian) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab ', F(ab') ₂ or other antigen-binding antibody subsequences) that contain one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are usually derived from an "import" variable domain. The import residue, sequence or antibody has the required affinity and/or specificity or other desired antibody biological activity as discussed herein.

Preferably, the humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the complementarity determining region (CDR) of the recipient are replaced with residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of human Fv immunoglobulin are replaced by corresponding non-human residues. In addition, the humanized antibody may contain residues that are not present in either the recipient antibody or the imported CDR or framework sequences, but which are included to further improve and optimize the performance of the antibody. Typically, a humanized antibody contains substantially all, at least one, and typically two, variable regions in which all or substantially all of the CDRs correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions that are consensus sequence regions. human immunoglobulin. Optimally, the humanized antibody may include at least a portion of the constant region or domain (Fc) of an immunoglobulin, typically a human immunoglobulin. Preferred are antibodies having Fc chains modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) that are altered from the parent antibody, also referred to as one or more CDRs "derived from" one or more than the CDR of the parent antibody. The term "humanized" is intended to include deimmunized antibodies.

In the context of the present description, "human antibody" means an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or obtained using any method for producing human antibodies known to those skilled in the art or disclosed herein. Accordingly, the term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., random mutations or mutations resulting from in vitro site-directed mutagenesis or somatic mutations in vivo), for example, in CDRs and, in particular, in CDR3. This definition of a human antibody includes antibodies containing at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody containing mouse light chain and human heavy chain polypeptides. Human antibodies can be obtained by various methods known in this field. In one embodiment, the human antibody is selected from a phage library where the phage library expresses human antibodies (Vaughan et al., Nature Biotechnology, 14:309-314, 1996; Sheets et al., Proc. Natl. Acad. Sci. (USA ) 95:6157-6162, 1998; Hoogenboom and Winter, J. Mol. Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991). Human antibodies can also be obtained by immunizing animals that have been transgenically introduced with human immunoglobulin loci instead of endogenous loci, such as mice in which endogenous immunoglobulin genes are partially or completely inactivated. This approach is described in US Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5633425; and 5,661,016. Alternatively, a human antibody can be obtained by immortalizing human B-lymphocytes that produce an antibody directed against the target antigen (such B-lymphocytes can be obtained from an individual or obtained by cDNA cloning from a single cell, or by in vitro immunization). See, for example, Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985; Boerner et al., J. Immunol., 147(1):86-95, 1991; and US Patent No. 5,750,373.

The human antibodies of the present invention can exist in at least two forms that are associated with hinge region heterogeneity. For example, an immunoglobulin molecule contains a stable four chain construct of approximately 150-160 kDa, in which the dimers are held together by interchain heavy chain disulfide bonds. Alternatively, the dimers are not linked by interchain disulfide bonds, and a molecule of about 75-80 kDa is formed, consisting of covalently linked light and heavy chains (half-antibody).

As used herein, the term "recombinant human antibody" includes all human antibodies that are made, expressed, generated, or isolated by recombinant means, such as antibodies expressed with a recombinant expression vector transfected into a host cell (described further below), antibodies isolated from a recombinant combinatorial library of human antibodies (described further below), antibodies isolated from an animal (eg, mouse) that is transgenic for human immunoglobulin genes (see, for example, Taylor et al. (1992) Nucl. Acids Res 20:6287-6295) or antibodies produced, expressed, constructed or isolated by any other means which involve splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies undergo in vitro mutagenesis (or, when an animal transgenic for human Ig sequences, in vivo somatic mutagenesis is used) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist in the in vivo human germline antibody repertoire.

Antibodies of the invention may be "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, and the remainder of the chain(s) is identical or homologous corresponding sequences in antibodies derived from a different species or belonging to a different class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit the required biological activity (US patent 4816567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855, 1984). Chimeric antibodies of interest include primatized antibodies comprising variable region antigen-binding sequences derived from non-human primates (eg, Old World monkeys, monkeys, etc.) and human constant region sequences.

A "monovalent antibody" contains one antigen-binding site per molecule (eg, IgG or Fab). In some cases, a monovalent antibody may have more than one antigen-binding site, but these binding sites are derived from different antigens.

"Monospecific antibody" refers to an antibody or antibody preparation that contains two identical antigen-binding sites per molecule (eg, IgG), for example, two antigen-binding sites that are identical to an epitope on an antigen. Thus, they compete with each other for binding to the same antigen molecule. The term includes "monoclonal antibody" or "monoclonal antibody composition". Most natural antibodies are monospecific. In some cases, the monospecific antibody may also be a monovalent antibody (eg, Fab).

The terms "mutation burden", "mutation burden", "mutation burden" or "mutation burden" are used interchangeably herein. Tumor mutation load is a measure of the number of mutations in a tumor genome, defined as the total number of mutations per coding region of the tumor genome. The burden of mutation among tumor types varies greatly, from a few mutations to 1000 (Alexandrov LB et al., Nature 2013; 500(7463):415-421; Lawrence MS et al., Nature 2013;499:214-218; Vogelstein B et al., Science, 2013;339:1546-1558).

An "original amino acid residue" is a residue that is replaced by an "imported amino acid residue" with a side chain smaller or larger than that of the original residue. The imported amino acid residue may be a naturally occurring or non-naturally occurring amino acid residue, but is preferably the former. "Naturally occurring" amino acid residues are residues encoded by the genetic code. By "non-naturally occurring" amino acid residue is meant a residue that is not encoded by the genetic code, but which is capable of covalently binding to adjacent amino acid residue(s) in the polypeptide chain. Examples of non-naturally occurring amino acid residues are norleucine, ornithine, norvaline, homoserine, and other analogs of amino acid residues such as those described, for example, in Ellman et al., Meth. enzyme. 202:301-336 (1991). In some embodiments, the implementation of the method of the present invention includes the replacement of at least one original amino acid residue, but can be replaced by more than one original residue. The number of original amino acid residues that can be replaced usually does not exceed the total number of residues on the contact surface of the first or second polypeptide.

The term "compete" as used herein with respect to an antibody means that the first antibody, or an antigen-binding fragment (or portion) thereof, binds to an epitope in a manner substantially analogous to the binding of a second antibody, or antigen-binding portion thereof, resulting in the binding of the first antibody to its related epitope is markedly reduced in the presence of the second antibody relative to the level of binding of the first antibody in the absence of the second antibody. Alternatively, the binding of the second antibody to its epitope may also be markedly reduced in the presence of the first antibody, but this need not be. In other words, the first antibody can inhibit the binding of the second antibody to the epitope, while the second antibody does not inhibit the binding of the first antibody to its corresponding epitope. However, if each antibody detectably inhibits the binding of another antibody to its cognate epitope or ligand to the same, greater or lesser extent, the antibodies are said to "cross-compete" with each other for binding to the corresponding epitope(s). The present invention encompasses both competing and cross-competing antibodies. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or a portion thereof), the skilled artisan will recognize from the aspects presented herein that such competing and/or cross-competing antibodies are within the scope of the invention and may be useful for the methods disclosed in the present description.

In the context of the present description, the term "antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing Fc (FcR) (for example, natural killer (NK) cells, neutrophils and macrophages) , recognize the bound antibody on the target cell, and then cause lysis of the target cell. The ADCC activity of a molecule of interest can be assessed using an in vitro ADCC assay, such as that described in US Pat. No. 5,500,362 or 5,821,337. Suitable effector cells for such assays include peripheral blood mononuclear cells (PBMC) and NK cells. Alternatively or additionally, the ADCC activity of a molecule of interest can be assessed in vivo, for example, in an animal model such as that described in Clynes et al., PNAS (USA), 95:652-656, 1998.

As used herein, the term "complement dependent cytotoxicity" or "CDC" refers to target lysis in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (eg, antibody) in complex with a related antigen. To evaluate complement activation, a CDC assay can be performed, such as that described in Gazzano-Santoro et al., J. Immunol. Methods, 202:163, 1996.

As used herein, the terms "immunospecifically binds", "immunospecifically recognizes", "specifically binds", "specifically recognizes", and similar terms refer to molecules, e.g., binding domains, that specifically bind to an antigen (e.g., epitope or immune complex) and do not bind specifically to another molecule. A molecule that specifically binds to an antigen may bind to other peptides or polypeptides with lower affinity, as determined by assays known in the art, such as immunoassays, BIACORE™, or other assays. Preferably, molecules that specifically bind to an antigen do not cross-react with other proteins.

Suitable "moderately stringent conditions" include a pre-wash in a solution of 5x SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization at 50°C-65°C, 5x SSC overnight; followed by washing twice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2× SSC containing 0.1% SDS.

In the context of the present description, "very stringent conditions" or "high stringency conditions" means conditions under which: (1) low ionic strength and high temperature are used for washing, for example, a mixture of 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1 % sodium dodecyl sulfate at 50°C; (2) a denaturing agent such as formamide, e.g. 50% (v/v) formamide, is used during hybridization with a mixture of 0.1% bovine serum albumin/0.1% ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) 50% formamide, 5× SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt's solution, sonicated Salmon sperm DNA (50 µg/mL), 0.1% SDS and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2× SSC (sodium chloride/sodium citrate) and 50% formamide at 55 °C, followed by a high stringency wash consisting of 0.1×SSC containing EDTA at 55°C. The skilled artisan will know how to adjust the temperature, ionic strength, etc., as needed, taking into account factors such as probe length, and the like.

Binding proteins, binding domains, CDRs, or antibodies (broadly defined) can be identified according to the Kabat, Chothia, both Kabat and Chothia accumulation definitions, the AbM definitions, the contact definition, the North definition, and /or conformational determination or using any method for determining the CDR, well known in this field. See, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed. (hypervariable regions); Chothia et al., Nature 342:877-883, 1989 (structural loop structures). The identity of the amino acid residues in a particular antibody that make up the CDR can be determined using methods well known in the art. The AbM CDR determination is a compromise between the Kabat and Chothia determinations, which uses Oxford Molecular's AbM antibody modeling software (Accelrys®). The definition of "contact" for CDRs is based on the observed contacts with antigens described in MacCallum et al., J. Mol. Biol., 262:732-745, 1996. The definition of "conformational" in relation to CDRs is based on residues that contribute to the enthalpy of antigen binding (see, for example, Makabe et al., J. Biol. Chem., 283: 1156-1166, 2008). North identified canonical CDR conformations using another preferred set of CDR definitions (North et al., J. Mol. Biol. 406:228-256, 2011). In another approach, referred to herein as "conformational determination" of CDRs, CDR positions can be identified as residues that contribute to the enthalpy of antigen binding (Makabe et al., J Biol. Chem. 283:1156-1166, 2008). However, other definitions of CDR boundaries may not strictly follow one of the above approaches, but will nonetheless overlap with at least part of the Kabat CDR, although they may be shorter or longer in light of predictions or experimental results, according to in which specific residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. As used herein, the term CDR may refer to CDRs determined using any approach known in the art, including a combination of approaches. The methods used herein may use CDRs defined in accordance with any of these approaches. For any given embodiment containing more than one CDR, the CDR (or other antibody residue) may be determined according to any of the Kabat, Chothia, extended (combination of Kabat and Chothia) systems, North, extended definitions, AbM definitions, contact and/or conformational definition.

Residues in the variable domain are numbered according to the Kabat system, which is the numbering system used for heavy chain variable regions or light chain variable regions in antibody formulation. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Using this numbering system, the actual linear amino acid sequence may include fewer or additional amino acid residues corresponding to a shortening or insertion in the FR or CDR of the variable region. For example, a heavy chain variable region may include one amino acid insertion (Kabat residue 52a) after the H2 residue at position 52 and inserted residues (e.g., Kabat residues 82a, 82b and 82c) after the residue at position 82 in heavy chain FR. The Kabat numbering of residues can be determined for a given antibody by aligning regions of antibody sequence homology to a "standard" Kabat numbered sequence. Various Kabat numbering algorithms are available. The present description uses the algorithm implemented in version 2.3.3 of Abysis (www.abysis.org) to number the variable regions VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2 and VH CDR3 according to Kabat. The positions of specific amino acid residues in an antibody can also be numbered according to Kabat.

Used in the present description, the term "library phage display" refers to a population of bacteriophages, each of which contains a foreign cDNA recombinantly fused in frame with a surface protein. The phage displays on its surface a foreign protein encoded by cDNA. After replication in a bacterial host, typically E. coli, the phage containing the foreign cDNA of interest is selected by expression of the foreign protein on the surface of the phage.

The term "epitope" refers to that part of the molecule that can be recognized, come into contact with and/or bind to the antibody in one or more antigen-binding regions of the antibody, known as the paratope. One antigen may have more than one epitope. Thus, different antibodies can bind to different regions of the antigen and have different biological effects. Epitopes often consist of reactive surface groups of molecules, such as amino acids or sugar side chains, and have certain three-dimensional structural characteristics as well as specific charge characteristics. In the context of the present description, epitopes can be either conformational or linear. A conformational epitope is formed by spatially arranged amino acids from different segments of a linear polypeptide chain. A linear epitope is an epitope formed by adjacent amino acid residues in a polypeptide chain. In some embodiments, the implementation of the epitope may include fragments of saccharides, phosphoryl groups or sulfonyl groups on the antigen.

The term "antigenic epitope" as used herein is defined as the portion of a polypeptide to which an antibody can specifically bind, as determined by any method well known in the art, such as conventional immunoassays. A "non-linear epitope" or "conformational epitope" comprises non-contiguous polypeptides (or amino acids) in an antigenic protein to which an antibody specific for the epitope binds. Once a desired epitope has been determined on an antigen, antibodies to that epitope can be generated, for example, using the methods disclosed herein.

The terms "specific binding" or "specifically binding", when used in relation to the interaction of an antibody with a protein or peptide, refer to an interaction that depends on the presence of a particular structure (ie, antigenic determinant or epitope) on the protein; in other words, the antibody recognizes and binds to a particular protein structure, not to the protein as a whole. For example, if the antibody is specific for an "A" epitope, the presence of a protein containing the A epitope (or free unlabeled A) in a reaction containing the labeled "A" and the antibody reduces the amount of labeled A bound to the antibody.

In some embodiments, "specifically binds" means, for example, that the antibody binds to a protein with a KD of about 0.1 nM or less, but more often less than about 1 μM. In some embodiments, "specifically binds" means that the antibody binds to a target in some cases with a KD of at least about 0.1 μM or less, in other cases at least about 0.01 μM or less, and in other cases at at least about 1 nM or less. Due to sequence identities between homologous proteins in different species, specific binding may involve an antibody that recognizes a protein from more than one species (eg, a human protein or a mouse protein). Similarly, due to homology in certain regions of the polypeptide sequences of different proteins, specific binding may include an antibody that recognizes more than one protein. It is understood that in some embodiments, an antibody or binding fragment that specifically binds to a first target may or may not specifically bind to a second target. As such, "specific binding" does not necessarily require (although it may include) exclusive binding, i. binding to a single target. Thus, in some embodiments, an antibody may specifically bind to more than one target. In some embodiments, multiple targets may bind to the same antigen-binding site on an antibody. For example, in some cases, an antibody may contain two identical antigen-binding sites, each of which specifically binds to the same epitope on two or more proteins. In some alternative embodiments, the antibody may be multispecific and contain at least two antigen-binding sites with different specificities. As a non-limiting example, a bispecific antibody may comprise one antigen-binding site that recognizes an epitope on one protein (e.g., human CD3) and may additionally contain a second, different antigen-binding site that recognizes a different epitope on a second protein. Usually, but not necessarily, a binding reference means a specific binding.

An antibody that specifically binds to an antigen may bind to other peptides or polypeptides with lower affinity as determined by assays known in the art, such as immunoassays, BIACORE™, or other assays. Preferably, an antibody that specifically binds to an antigen does not cross-react with other proteins.

The terms “non-specific binding” or “background binding”, when used in relation to the interaction of an antibody and a protein or peptide, refer to an interaction that is independent of the presence of a particular structure (i.e., the antibody binds to proteins in general and not to a particular structure). , such as an epitope).

The term "k _on " or "k _a " in the context of the present description refers to the rate constant of association of an antibody with an antigen. In particular, rate constants (k _on /k _a and k _ff /k _d ) and equilibrium dissociation constants are measured using whole antibodies (ie, divalent) and monomeric CD3 proteins.

As used herein, the term "k _off " or "k _d " refers to the dissociation rate constant of an antibody from an antibody/antigen complex.

As used herein, the term "K _D " refers to the equilibrium dissociation constant of an antibody-antigen interaction.

As used herein, the term "binding affinity" generally refers to the strength of the overall net non-covalent interactions between one binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to true binding affinity, which reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (K _D ). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (K _D ). For example, the Kd may be about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM or more. Affinity can be measured by conventional methods known in the art, including those disclosed herein. Low affinity antibodies generally bind antigen slowly and tend to dissociate easily, while high affinity antibodies generally bind antigen faster and tend to stay bound longer. Various methods for measuring binding affinity are known in the art, any of which can be used for the purposes of the present invention. In particular, the term "binding affinity" refers to the rate of dissociation of a particular antigen-antibody interaction. K _D is the ratio of the rate of dissociation, also referred to as "(k _off )", to the rate of association, or "(k _on )". Thus, K _D is equal to k _off /k _on and is expressed as a molar concentration (M). It follows that the smaller the K _D value, the stronger the binding affinity. Therefore, a K _D of 1 μM indicates poor binding affinity compared to a K _D of 1 nM. Antibody K _D values can be determined using methods well known in the art. One method for determining the K _D of an antibody is to use surface plasmon resonance (SPR), typically performed in a biosensor system such as the BIACORE ^™ system. The kinetic analysis of BIACORE™ includes the analysis of antigen binding and dissociation from chips with molecules immobilized on their surface (for example, molecules containing epitope-binding domains). Another method for determining the K _D of an antibody is biolayer interferometry, usually performed with the OCTET® system (Octet QKe system, ForteBio).

"Biologically active", "biological activity" and "biological characteristics" in relation to an antibody of the present invention, for example, an antibody, fragment or derivative thereof, means having the ability to bind to a biological molecule, unless otherwise indicated.

As used herein, the terms "nucleic acids" and "nucleotide sequences" include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), combinations of DNA and RNA molecules, or hybrid DNA/RNA molecules, and analogues of DNA molecules or RNA. Such analogs can be prepared using, for example, nucleotide analogs, which include, without limitation, inosine or tritylated bases. Such analogs may also include DNA or RNA molecules containing modified backbones that confer useful properties to the molecules, such as, for example, nuclease resistance or increased ability to permeate cell membranes. Nucleic acids or nucleotide sequences may be single stranded, double stranded, may contain both single and double stranded portions, and may contain triple stranded portions, but are preferably double stranded DNA.

The present invention also includes polynucleotides that encode the antibodies of the invention, including polypeptides and antibody binding regions. Polynucleotides encoding the molecules of the present invention can be obtained and the nucleotide sequence of the polynucleotides can be determined by any method known in the art.

Polynucleotides that encode antibodies of the present invention may include the following: a coding sequence for a variant alone, a coding sequence for a variant, and additional coding sequences such as a functional polypeptide or a signal or secretory sequence, or a proprotein sequence; a coding sequence for an antibody; and a non-coding sequence such as introns or a 5' and/or 3' non-coding sequence of an antibody coding sequence. The term "antibody-encoding polynucleotide" encompasses a polynucleotide that includes an additional coding sequence for a variant, as well as a polynucleotide that includes an additional coding and/or non-coding sequence. It is known in the art that a polynucleotide sequence optimized for a particular host cell/expression system can be easily derived from the amino acid sequence of the desired protein (see GENEART® AG, Regensburg, Germany).

The antibodies of the present invention may have additional conservative or non-essential amino acid substitutions that do not significantly affect the function of the polypeptide. Will a particular replacement be carried over, i.e. whether it will adversely affect desired biological properties, such as binding activity, can be determined as described by Bowie, JU et al. Science 247:1306-1310, 1990 or Padlan et al. FASEB J. 9:133-139, 1995.

In the context of the present description, the term "conservative amino acid substitution" means the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid), uncharged polar side chains (eg, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (eg, glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (eg, threonine, valine, isoleucine), and aromatic side chains (eg, tyrosine, phenylalanine , tryptophan, histidine).

As used herein, a "non-essential" amino acid residue is a residue that can be altered relative to the sequence of a wild-type binding agent, such as an antibody, without abolishing or substantially altering biological activity, while an "essential" amino acid residue leads to this change. In an antibody, the essential amino acid residue may be a specificity determining residue (SDR).

Used in the present description, the term "isolated" refers to material that is removed from its original environment (for example, from the natural environment, if it occurs in nature). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide that is separated from some or all of the materials coexisting with it in the natural system is isolated. Such a polynucleotide may be part of a vector and/or such a polynucleotide or polypeptide may be part of a composition, such as a mixture, solution or suspension, or containing an isolated cell or a cultured cell that contains the polynucleotide or polypeptide, and it remains isolated in the sense that that the vector or composition is not part of its natural environment. For example, a polypeptide that is chemically synthesized or synthesized in a cellular system other than the cell from which it naturally originates will be "separated" from its naturally associated components. A protein can also be made substantially free of its naturally associated components using protein purification techniques well known in the art.

An "isolated" or "purified" antibody is substantially free of cellular material or other contaminating proteins from the cell or tissue or environment from which the protein was derived, or substantially free of chemical precursors or other chemicals from chemical synthesis. The phrase "substantially free of cellular material" includes antibody preparations in which the polypeptide/protein is separated from the cellular components of the cells from which it is isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes antibody preparations containing less than about 50%, 40%, 30%, 20%, 10%, 5%, 2.5%, or 1% (by dry weight) of a contaminant. squirrel. If the antibody is produced recombinantly, it is also preferably substantially free of culture medium, ie. the culture medium is less than about 50%, 40%, 30%, 20%, 10%, 5%, 2.5%, or 1% of the volume of the protein preparation. If the antibody is made by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals and reagents, i.e. the antibody of interest is separated from chemical precursors or other chemicals that are involved in protein synthesis. Accordingly, such antibody preparations contain less than about 50%, 40%, 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors or compounds other than the antibody of interest. In a preferred embodiment of the present invention, the antibodies are isolated or purified. An antibody that is separated or removed from at least one component of an organism or tissue or cell in which the antibody exists or is naturally produced is an "isolated antibody" for the purposes of the present invention.

As used herein, the term "recovery" refers to a process that renders a chemical, such as a polypeptide, substantially free of its naturally associated components by isolation, for example, by protein purification methods well known in the art.

As used herein, the term "replicon" refers to any genetic element, such as a plasmid, chromosome, or virus, that behaves as an autonomous unit of polynucleotide replication within a cell.

Used in the present description, the term "operably linked" refers to a situation where the described components are in a relationship that allows them to function in the intended way. For example, a control sequence "operably linked" to a coding sequence is ligated such that the coding sequence is expressed under conditions appropriate or compatible with the control sequence. Generally, "operably linked" means that the DNA sequences to be linked are contiguous, and in the case of a secretory leader, they are contiguous and in read phase. However, enhancers do not need to be contiguous. Linking is accomplished by ligation of convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used in accordance with common practice.

As used herein, the term "vector" means a construct that is capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, without limitation, viral vectors, naked DNA or RNA expression vectors, plasmid vectors, cosmid or phage vectors, DNA or RNA based expression vectors associated with cationic condensing agents, DNA or RNA based expression vectors encapsulated in liposomes. and some eukaryotic cells such as producer cells.

As used herein, the term "expression control sequence" or "control sequence" refers to a polynucleotide sequence that is necessary to affect the expression of the coding sequence to which it is ligated. The nature of such control sequences varies depending on the host organism. For example, in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and terminators and, in some cases, enhancers. Thus, the term "control sequence" is intended to include at least all components that are required for expression, and may also include additional components that are advantageously present, such as leader sequences.

"Host cell" includes a single cell or cell culture that can be or has been a recipient of the vector(s) intended to include polynucleotide inserts. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or genomic DNA complementarity) to the original parent cell due to natural, accidental or intentional mutation. The host cell includes cells transfected in vivo with the polynucleotide(s) of the present invention.

Used in the present description, the term "mammalian cells" includes reference to cells derived from mammals, including humans, rats, mice, guinea pigs, chimpanzees or macaques. Cells can be cultured in vivo or in vitro.

As used herein, the term "purified product" refers to a preparation of a product that has been isolated from cellular components with which the product is normally associated and/or from other cell types that may be present in the sample of interest.

As used herein, the term "substantially pure" refers to a material that is at least 50% pure (i.e. free of impurities), more preferably at least 90% pure, more preferably at least 95% pure, even more preferably at least 98% pure, and most preferably at least 99% pure.

Used in the present description, the term "treat", "treating" or "treatment" is an approach to obtain useful or desired clinical results. For the purposes of the present invention, treatment is defined as administering an anti-CD3 antibody molecule (e.g., monoclonal antibody, bispecific antibody) to a subject, e.g., a patient, or administering, for example, by application to an isolated tissue or cell obtained from the subject, which is then returned to that subject. The anti-CD3 antibody molecule can be administered alone or in combination with one or more agents. Treatment may include recovery, healing, alleviation, alteration, repair, alleviation, alleviation, mitigation, relief, or effect on a disorder, the symptoms of a disorder, or a predisposition to a disorder, such as cancer.

As used herein, the term "subject" includes any animal (eg, mammal), including but not limited to humans, non-human primates, rodents, and the like, that is to be the recipient of a particular treatment. For example, the subject may be a patient (eg, a human or veterinary patient) suffering from cancer. Typically, the terms "subject", "individual" and "patient" are used interchangeably herein with respect to a human.

According to the present invention, the term "non-human animals" includes all non-human vertebrates, such as non-human mammals and non-mammals such as non-human primates, sheep, dogs, cows, chickens, amphibians, reptiles, mice, rat, rabbit or goat, etc., unless otherwise indicated.

As used herein, the term "pharmaceutically acceptable" refers to a product or compound approved (or warranted) by a federal or state government regulatory agency, or listed in the USP or other generally accepted pharmacopoeia for use in animals, including humans.

As used herein, the terms “pharmaceutically acceptable excipient, carrier, or adjuvant” or “pharmaceutically acceptable carrier” refers to an excipient, carrier, or adjuvant that can be administered to a subject in conjunction with at least one antibody of the present invention and that does not interfere with the activity of the antibody. . The excipient, carrier, or adjuvant must be non-toxic when administered with the antibody at doses sufficient to produce a therapeutic effect.

Used in the present description, the term "attenuation" means the reduction or relief of one or more symptoms compared to when the antibody molecules of the invention are not administered. "Relief" also includes a reduction or reduction in the duration of a symptom.

As used herein, the terms "prevent", "preventive" and "prophylaxis" refer to preventing the recurrence or occurrence of one or more symptoms of a disorder in a subject by administering a prophylactic or therapeutic agent.

As used herein, "effective amount", "therapeutically effective amount", "therapeutically sufficient amount", or "effective dose" refers to that amount of a therapeutic agent (e.g., anti-CD3 antibody, alone or as part of a multispecific antibody (e.g., bispecific) ) that is effective or sufficient when administered single or multiple doses to a subject to prevent, cure, alleviate, ameliorate, or relieve a disease, disorder, or side effect, or to reduce the rate of progression of a disease or disorder, or to prolong a state of recovery, alleviate, alleviate, or arrest a disorder disclosed herein in a subject beyond what would be expected in the absence of such treatment. The term also includes an amount effective to improve normal physiological function. An effective amount can be considered in the context of administering one or more therapeutic agents, and one agent can be considered to be administered in an effective amount if, when administered in combination with one or more other agents, the desired result can be or is achieved.

As used herein, an effective agent is capable of targeting effector T cells to induce T cell-mediated cytotoxicity of cells associated with a disease, such as cancer, an autoimmune disease, or an infectious disease. The therapeutic agent of the present invention induces a T cell mediated immune response by modulating the CD3 antigen on the surface of T cells, in particular CD3, more specifically CD3 epsilon.

With regard to cancer, a therapeutically effective amount refers to an amount of a therapeutic agent that inhibits the growth of a tumor or cancer by slowing, interrupting, arresting or stopping the growth of a malignant tumor and/or metastases.

Efficacy is a measure of the potency of a therapeutic agent, expressed as the amount necessary to produce an effect of a given intensity. A potent agent elicits a stronger response at low concentrations compared to an agent of lower potency, which elicits a less potent response at low concentrations. Potency is a function of affinity and potency. Efficiency is understood as the ability of a therapeutic agent to induce a biological response when bound to a target ligand and the quantitative magnitude of this response. Used in the present description, the term "half the maximum effective concentration (EC ₅₀ )" refers to the concentration of a therapeutic agent that causes a response equal to half between the initial level and the maximum achieved after a certain exposure time. The therapeutic agent may cause inhibition or stimulation. The EC ₅₀ value is commonly used, and is also used herein, as a measure of effectiveness.

Used in the present description, the term "combination therapy" or the introduction of "in combination with" refers to the use of more than one prophylactic and/or therapeutic agent. The use of the terms "combination therapy" or "in combination" does not limit the order in which prophylactic and/or therapeutic agents are administered to a subject with a disorder. In other words, combination therapy can be carried out as a separate, sequential or simultaneous treatment with therapeutic agents. In the case of "sequential administration", the first administered agent may have some physiological effect on the subject or become more active in the subject's body when the second agent is administered.

The term "simultaneous administration", as used herein in relation to the administration of a prophylactic and/or therapeutic agent, refers to the administration of the agents so that each of the agents is present in the subject's body at the same time. Simultaneous introduction can be carried out using molecules that are in the same composition or in separate compositions, administered at the same or similar time. Sequential administration may be in any order as needed.

The term "differentiation cluster 3" or "CD3" refers to a multimeric protein complex, historically known as the T3 complex, consisting of four distinct polypeptide chains; epsilon (ε), gamma (γ), delta (δ), and zeta (ζ), which assemble and function as three pairs of dimers (εγ, εδ, ζζ). The CD3 complex serves as a T cell co-receptor that binds non-covalently to the T cell receptor (TCR) (Smith-Garvin et al. 2009) and generates an activation signal in T lymphocytes. The CD3 protein complex is a defining feature of the T cell line, so anti-CD3 antibodies can be effectively used as T cell markers (Chetty and Gatter 1994). It is well known that anti-CD3 antibodies induce the formation of cytotoxic T cells through activation of endogenous lymphokine production and are able to selectively destroy tumor targets (Yun et al., Cancer Research, 49:4770-4774 (1989)).

T cells play a central role in cellular immunity in humans and animals. Recognition and binding of a particular antigen is mediated by TCRs expressed on the surface of T cells. TCR T cells are able to interact with immunogenic peptides (epitopes) associated with major histocompatibility complex (MHC) molecules and presented on the surface of target cells. Specific binding of the TCR triggers a signaling cascade within the T cell leading to proliferation and differentiation into a mature effector T cell. More specifically, T cells express TCR complexes that are capable of inducing antigen-specific immune responses (Smith Garvin et al, 2009). Antigens are peptides expressed by tumor cells and virus-infected cells that are capable of stimulating immune responses. Intracellularly expressed antigens bind to major histocompatibility class I (MHC class I) molecules and are transported to the surface where they are exposed to T cells. If the binding affinity of the TCR to MHC class I in complex with the antigen is sufficient, immune synapse formation will be initiated. Signaling across the immune synapse occurs through CD3 coreceptors, which form epsilon/delta, delta/gamma, and zeta/zeta dimers. These dimers bind to the TCR and generate an activation signal in T lymphocytes. This signaling cascade directs T-cell mediated killing of antigen-expressing cells. Cytotoxicity is mediated by the release and transfer of granzyme B and perforin from the T cell to the target cell.

In the context of the present description, the term "activating T-cell antigen" refers to an antigenic determinant expressed on the surface of a T-lymphocyte, in particular a cytotoxic T-lymphocyte, which is capable of inducing T-cell activation upon interaction with an antigen-binding molecule. In particular, interaction of an antigen-binding molecule with an activating T cell antigen can induce T cell activation by triggering the signaling cascade of the T cell receptor complex. In a specific embodiment, the activating T cell antigen is CD3, specifically the epsilon subunit of CD3 (see UniProt # P07766 (version 130), NCBI RefSeq # NP_000724.1, SEQ ID NO: 66 for the human sequence). Typically, a natural allelic variant has an amino acid sequence that is at least 95%, 97%, or 99% identical to the protein described in GenBank accession number: BAB71849.1.

"T cell activation" as used herein refers to one or more cellular responses of a T lymphocyte, in particular a cytotoxic T lymphocyte, selected from: proliferation, differentiation, secretion of cytokines, release of a cytotoxic effector molecule, cytotoxic activity, and expression of activation markers. The T cell activating bispecific antigen binding molecules of the invention are capable of inducing T cell activation. Suitable assays for measuring T cell activation are known in the art disclosed herein.

As used herein, "antibody that binds CD3", "antibody that recognizes CD3", "anti-CD3 antibody", or "CD3 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize one CD3 subunit (e.g., epsilon, delta , gamma or zeta), as well as antibodies and their antigen-binding fragments that specifically recognize the dimeric complex consisting of two CD3 subunits (for example, the gamma/epsilon, delta/epsilon and zeta/zeta CD3 dimers). Human CD3 epsilon is listed in GenBank accession number NM_000733. The antibodies of the present invention can bind soluble CD3 and/or cell surface expressed CD3. Soluble CD3 includes native CD3 proteins as well as recombinant CD3 protein variants such as, for example, CD3 monomeric and dimeric constructs that lack a transmembrane domain or are not otherwise associated with the cell membrane.

Antibodies directed against CD3 are able to generate an activation signal in T-lymphocytes. Other T cell activation ligands may also be used, including, but not limited to, CD28, CD134, CD137, and CD27.

The present invention includes CD3-binding single-arm antibodies. As used herein, the term "one-armed antibody" means an antigen-binding molecule comprising one antibody heavy chain and one antibody light chain. The single-armed antibodies of the present invention may contain any of the VL/VH or CDR amino acid sequences listed herein in Tables 1, 2, and 3.

The CD3 antibodies disclosed herein may contain one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from public antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof that are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids in one or more framework regions and/or CDR regions are mutated to the corresponding germline sequence residue(s), from which the antibody was generated, or to the corresponding residue(s) of another human germline sequence, or subjected to a conservative amino acid substitution of the corresponding germline residue(s) (such sequence changes are collectively referred to herein as "germline mutations"). From the heavy and light chain variable region sequences disclosed herein, numerous antibodies and antigen-binding fragments can readily be prepared by one skilled in the art that contain one or more individual germline mutations, or combinations thereof. In some embodiments, all framework and/or CDR residues in the VH and/or VL domains are mutated back to residues found in the original germline sequence from which the antibody originated. In some embodiments, only certain residues are mutated back to the original germline sequence, for example, only mutated residues present in the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues present in CDR1, CDR2, or CDR3. In some additional embodiments, one or more framework and/or CDR residues are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived).

In addition, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework region and/or CDR regions, for example, in which certain individual residues are mutated to the corresponding residue of a particular germline sequence, while some others residues that differ from the original germline sequence are retained or mutated to the corresponding residue of a different germline sequence. Once generated, antibodies that contain one or more germline mutations can be readily tested for one or more desirable properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as applicable), reduced immunogenicity, etc. Antibodies obtained in this conventional manner are within the scope of the present invention.

The present invention also includes anti-CD3 antibodies containing variants of any of the VH, VL and/or CDR amino acid sequences described herein having one or more conservative substitutions. For example, the present invention includes anti-CD3 antibodies having VH, VL, and/or CDR amino acid sequences, e.g., 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions relative to any of the VH, VL and/or CDR amino acid sequences presented herein in Table 1.

In the context of the present description, the term "complex" or "in a complex" refers to the association of two or more molecules that interact with each other through bonds and/or forces (for example, van der Waals, hydrophobic, hydrophilic forces) that are not peptide bonds. In one embodiment, the complex is heteromultimeric. The term "protein complex" or "polypeptide complex" as used herein is to be understood to include complexes that have a non-protein moiety conjugated to a protein in the protein complex (for example, including but not limited to chemical molecules such as a toxin or agent discovery).

In the context of the present description, the term "CD3 antibody" includes monovalent antibodies with a single specificity, as well as "bispecific antibody", "dual specificity antibody", "trispecific antibody", "bifunctional antibody", "heteromultimer", "heteromultimeric complex", " a bispecific heterodimeric diabody", "heteromultimeric polypeptide", or bispecific heterodimeric IgGs. In some embodiments, the CD3 antibodies of the invention are human or humanized antibodies.

In the context of the present description, the term "bispecific antibody" is a molecule containing at least a first polypeptide and a second polypeptide, and the amino acid sequence of the second polypeptide differs from the amino acid sequence of the first polypeptide by at least one amino acid residue. In some cases, the bispecific antibody is an artificial hybrid antibody having two different heavy chain and light chain regions. Preferably, the bispecific antibody has binding specificity for at least two different ligands, antigens, or binding sites. Accordingly, bispecific antibodies can simultaneously bind two different antigens. The two antigen-binding sites of a bispecific antibody bind to two different epitopes, which may be on the same or different protein targets, such as a tumor target.

In the context of the present description, the first polypeptide chain and the second polypeptide chain of a "bispecific antibody" include at least one VL region of an antibody and one VH region of an antibody or a fragment thereof, both antibody binding domains being contained in the same polypeptide chain, and wherein the VL and VH regions are in each polypeptide chain originate from different antibodies.

Bispecific antibodies can be derived from full length antibodies or antibody fragments (eg F(ab') ₂ bispecific antibodies). Diabodies are described more fully, for example, in EP 404097; WO93/11161; and Hollinger et al., Proc. Natl. Acad. sci. USA 90:6444-6448, 1993. Bispecific antibodies are heterodimers of two "crossover" sFv fragments in which the VH and VL regions of the two antibodies are present on different polypeptide chains.

In the context of the present description, the terms "linked", "fused", "fusion", "covalently linked" and "genetically fused" are used interchangeably. These terms refer to combining two more elements or components together by any means, including chemical conjugation or recombinant methods. Used in the present description, the term "covalently linked" means that these fragments are either directly covalently linked to each other, or indirectly covalently linked to each other through an intermediate fragment or fragments, such as a binding peptide or fragment. In a preferred embodiment, the fragments are covalently fused. One type of covalent bond is a peptide bond. Methods for chemical conjugation (eg, using heterobifunctional crosslinkers) are known in the art. Fusion fragments can also be genetically fused. In the context of the present description, the term "genetically linked" or "genetically fused" refers to the covalent linkage or attachment in one line of two or more proteins, polypeptides or fragments through their individual peptide backbones, through the genetic expression of a single polynucleotide molecule encoding these proteins, polypeptides or fragments. This genetic fusion results in the expression of one continuous genetic sequence. Preferred genetic fusions are in reading frame, ie. two or more open reading frames (ORFs) merge to form a continuous longer reading frame while maintaining the correct reading frame of the original ORFs. Thus, the resulting recombinant fusion protein is a single polypeptide containing two or more protein segments that correspond to the polypeptides encoded by the original ORFs (these segments are not usually connected in this way in nature). In this case, a single polypeptide is cleaved during processing to form dimeric molecules containing two polypeptide chains.

As used herein, the term "linked" or "bonds" refers to either a compound consisting of a direct peptide bond between the first and second amino acid sequences, or a compound that includes a third amino acid sequence linked by a peptide bond to the first and second amino acid sequences and located between them. For example, a linker peptide linked to the C-terminal end of one amino acid sequence and to the N-terminal end of another amino acid sequence.

As used herein, the term "linker" refers to an amino acid sequence of two or more amino acids in length. The linker may be composed of neutral polar or non-polar amino acids. The linker may be eg 2 to 100 amino acids in length, eg 2 to 50 amino acids, eg 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. The linker may be "cleavable", for example, by auto-cleavage or enzymatic or chemical cleavage. Cleavage sites in amino acid sequences and the enzymes and chemicals that cleave such sites are well known in the art and are also disclosed herein.

In the context of the present description, the term "disulfide bond" or "cysteine-cysteine disulfide bond" refers to a covalent interaction between two cysteines, in which the sulfur atoms of the cysteines are oxidized to form a disulfide bond. The average bond energy in a disulfide bond is about 60 kcal/mol compared to 1-2 kcal/mol in the case of a hydrogen bond. In the context of the present invention, cysteines that form a disulfide bond are found in the framework regions of a single chain antibody and serve to stabilize the conformation of the antibody. Cysteine residues can be introduced, for example, by site-directed mutagenesis, so that stabilizing disulfide bonds can be formed within the molecule.

The designation "ledge-to-trough" is similar to the designation "bulge and cavity" and can be used synonymously.

A "bulge" or "protrusion" refers to at least one amino acid side chain that protrudes at the contact surface (interface) of the first polypeptide and, therefore, can be located in the compensatory cavity on the adjacent contact surface (i.e., on the contact surface of the second polypeptide) providing stabilization of the heterodimer and thus promoting the formation of a heterodimer rather than a homodimer, for example. The bulge may be on the original contact surface, or may be introduced synthetically (eg, by altering the nucleic acid encoding the contact surface). Typically, the nucleic acid encoding the contact surface of the first polypeptide is modified to encode a bulge. To achieve this, the nucleic acid encoding at least one "original" amino acid residue on the contact surface of the first polypeptide is replaced with a nucleic acid encoding at least one "imported" amino acid residue having a larger side chain compared to the original amino acid residue. The upper limit of the number of initial residues to be replaced is equal to the total number of residues on the contact surface of the first polypeptide. Some imported bulge residues are usually naturally occurring amino acid residues, and are preferably selected from arginine (R), phenylalanine (F), tyrosine (Y) and tryptophan (W).

The bulge or protrusion is "placeable" in a cavity or cavity, which means that the spatial arrangement of the bulge and cavity at the contact surface of the first polypeptide and the second polypeptide, respectively, and the dimensions of the bulge and cavity are such that the bulge can be located inside the cavity without disturbing the normal association of the first and second polypeptides on the contact surface. Since bulges such as phenylalanine (F), tyrosine (Y), and tryptophan (W) are not normally perpendicular to the axis of the contact surface, alignment of the bulge with the corresponding cavity is based on modeling the bulge/cavity pair from a three-dimensional structure, such as obtained with using X-ray crystallography or nuclear magnetic resonance (NMR). This can be achieved using methods commonly available in the art.

"Cavity" or "trough" refers to at least one amino acid side chain that is recessed relative to the interface of the second polypeptide and therefore accommodates a corresponding protrusion on the adjacent interface of the first polypeptide. The cavity may be on the original contact surface or may be introduced synthetically (eg, by altering the nucleic acid encoding the contact surface). Typically, the nucleic acid encoding the contact surface of the second polypeptide is changed to encode the cavity. To achieve this, the nucleic acid encoding at least one "original" amino acid residue at the contact surface of the second polypeptide is replaced with DNA encoding at least one "imported" amino acid residue with a smaller side chain compared to the original amino acid residue. The upper limit of the number of original residues to be replaced is equal to the total number of residues on the contact surface of the second polypeptide. Some of the imported cavity residues are usually naturally occurring amino acid residues and are preferably selected from alanine (A), serine (S), threonine (T), and valine (V).

“Target antigen (target antigen)”, “target cell antigen”, “tumor antigen”, or “tumor-specific antigen”, as used herein, refers to an antigenic determinant present on the surface of a target cell, such as a cell in a tumor such as a cancer cell or tumor stroma cell.

In the context of the present description, the term "cancerous" or "malignant" refers to or describes a physiological condition in mammals, which is usually characterized by unregulated cell growth, neoplasm or tumor resulting from abnormal uncontrolled cell growth. In some aspects, cancer refers to a malignant primary tumor without metastases that remains localized. In other aspects, cancer refers to a malignant tumor that has invaded and destroyed adjacent body structures and spread to distant sites. In some aspects, cancer is associated with a specific cancer antigen. Examples of cancers include, but are not limited to, cancers of the mouth and pharynx, digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, gallbladder, bile ducts, and pancreas), respiratory system (e.g., larynx, lungs and bronchi, including non-small cell lung cancer), bones and joints (eg, bone metastases), soft tissues, skin (eg, melanoma), breasts, genitals (eg, cervix, corpus uteri, ovarian vulva, vagina, prostate) and testis), urinary system (eg, bladder, kidney, renal pelvis, and ureter), eye and orbit, brain and nervous system (eg, glioma), head and neck, or endocrine system (eg, thyroid gland). The cancer may also be lymphoma (eg, Hodgkin's disease and non-Hodgkin's lymphoma), multiple myeloma, or leukemia (eg, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, and the like). In some embodiments, the cancer of the digestive system is cancer of the esophagus, stomach, small intestine, colon, rectum, liver, gallbladder, bile duct, and pancreas.

With respect to cancer, treatment is useful in any one or more of the following: (a) treating, preventing, or alleviating one or more symptoms of a condition associated with malignant cells in a subject (e.g., cancer of the gastrointestinal tract, such as colorectal cancer (CRC )); (b) inhibition of growth or progression of a tumor in a subject having a cancer expressing a tumor antigen; (c) inhibition of metastasis of cancer (malignant) cells expressing a tumor antigen in a subject containing one or more malignant cells expressing a tumor antigen; (d) inducing regression (eg, long-term regression) of the tumor expressing the tumor antigen; (e) the manifestation of cytotoxic activity in malignant cells expressing the tumor antigen; (f) increasing the survival of a subject suffering from a tumor antigen-associated disorder without disease progression; (g) increasing the overall survival of a subject suffering from a disorder associated with a tumor antigen; (h) reducing the use of additional chemotherapeutic or cytotoxic agents by a subject suffering from a disorder associated with a tumor antigen; (i) reducing tumor burden in a subject suffering from a tumor antigen associated disorder; or (j) blocking the interaction of the tumor antigen with other as yet unidentified factors. Without wishing to be bound by theory, it is believed that treatment results in inhibition of growth, destruction, or killing of a cell in vitro or in vivo, or otherwise reduces the ability of a cell, including an aberrant cell, to mediate a disorder, such as a disorder as disclosed herein, such as cancer. .

As used herein, the term "cancer cell" or "cancer" refers to tumors or tumor cells that are invasive and/or capable of metastasizing, i. to the cancer cell.

While any materials and methods similar or equivalent to those disclosed herein may be used in the practice or testing or testing of the present invention, the following is a description of the preferred materials and methods.

Materials and methods

Various methods for producing antibodies have been described, which include the traditional hybridoma method for producing monoclonal antibodies, recombinant methods for producing antibodies (including chimeric antibodies, such as humanized antibodies), the production of antibodies in transgenic animals, and the recently described phage display technology for producing "fully human" antibodies. These methods are briefly described below.

Polyclonal antibodies to an antigen of interest can usually be obtained from animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of antigen and adjuvant. The antigen (or fragment containing the target amino acid sequence) can be conjugated to a protein that is immunogenic to the species to be immunized, such as serum albumin, bovine thyroglobulin, or a soybean trypsin inhibitor, using a bifunctional or derivatizing agent, such as a maleimidobenzoylsulfosuccinimide ester (conjugation via cysteine residues) or N-hydroxysuccinimide (via lysine residues). Animals are immunized against the immunogenic conjugates or derivatives and after a few weeks the animals are boosted by subcutaneous injection at several sites. After 7-14 days, blood is taken from the animals, and the serum is analyzed for antibody titer. Animals are given a booster injection until a titer plateau is reached. Preferably, animals are given a booster injection of a conjugate of the same antigen but conjugated to a different protein and/or with a different crosslinker. Conjugates can also be produced in recombinant cell culture as fusion proteins. Aggregating agents such as alum are also used to enhance the immune response.

Monoclonal antibodies can be obtained from a population of essentially homogeneous antibodies using the hybridoma method first described by Kohler & Milstein, Nature 256:495, 1975, or can be obtained by recombinant DNA methods (Cabilly et al., US patent No. 4816567). In the hybridoma method, a mouse or other suitable host animal is immunized as described above to obtain lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusion agent such as polyethylene glycol to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). The hybridoma cells thus obtained are seeded and grown in a suitable culture medium, which preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. In addition, hybridoma cells can be grown in vivo as ascitic tumors in animals. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascitic fluid or serum by conventional immunoglobulin purification procedures such as, for example, protein A-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

Expression of the antibody, antigen-binding fragments of the antibody, or any antibody construct may be carried out in appropriate prokaryotic or eukaryotic host cells, such as CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, PER.C6 cells, yeast cells, or E cells. .coli and secreted antibody are isolated and harvested from cells (cell culture supernatant, conditioned cell culture supernatant, cell lysate, or clarified mass). General methods for producing antibodies are well known in the art and are described, for example, in the review articles Makrides, S.C., Protein Expr. Purif. 17:183-202, 1999; Geisse, S., et al., Protein Expr. Purif. 8:271-282, 1986; Kaufman, R.J., MoI. Biotechnol. 16:151-160, 2000; Werner, R.G., Drug Res. 48:870-880, 1998. In a particular embodiment, the cell culture is a mammalian cell culture, such as a Chinese Hamster Ovary (CHO) cell culture.

In one embodiment, the antibody may be isolated or isolated from a source in which it is naturally produced. In some such embodiments, isolated or isolated antibodies may be subjected to additional purification steps using conventional chromatographic techniques known in the art. In particular, purification methods are contemplated to include, without limitation, affinity chromatography (e.g., protein A affinity chromatography), ion exchange chromatography (e.g., anion exchange chromatography or cation exchange chromatography), hydrophobic interaction chromatography, hydroxyapatite chromatography, size exclusion chromatography, and/ or dialysis. Among these, the preferred purification method is protein A chromatography. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.

Other antibody purification methods are also known in the art, such as ion exchange column fractionation, ethanol precipitation, reverse phase high pressure column chromatography, ethanol precipitation, reverse phase HPLC, silica chromatography, heparin-sepharose™ chromatography, chromatography on an anionic or cation exchange resin (for example, on a column of polyaspartic acid), chromatography, SDS-PAGE electrophoresis and precipitation with ammonium sulfate. The above list of cleaning methods is illustrative only and is not intended to be an exhaustive listing.

Alternatively, it is possible to obtain transgenic animals (eg mice) that are capable of producing the full repertoire of human antibodies after immunization without producing endogenous immunoglobulins. For example, homozygous deletion of the antibody heavy chain junction region gene (J.sub.H) in chimeric and mutant germline mice has been described as resulting in complete suppression of endogenous antibody production. Transfer of the human germline immunoglobulin gene array to such germline mutant mice will result in the production of human antibodies upon challenge with the antigen. See, for example, Jakobovits et al., Proc. Natl. Acad. sci. USA 90:2551-255; 1993 and Jakobovits et al., Nature 362:255-258, 1993.

In some embodiments, the antibodies of the present invention can be humanized while retaining high antigen affinity and other beneficial biological properties. Methods for humanizing non-human antibodies are well known in the art. Essentially humanization can be performed following the method of Winter et al. (Jones et al., Nature 321:522-525, 1986; Riechmann et al., Nature 332:323-327, 1988; Verhoeyen et al., Science 239:1534 -1536, 1988) by replacing the CDR or rodent CDR sequences with the corresponding human antibody sequences. Accordingly, such humanized antibodies are chimeric antibodies (Cabilly, supra) in which a substantially smaller portion of a relatively intact human variable domain has been replaced with a corresponding sequence derived from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are replaced by residues from similar regions in rodent antibodies. It is important that antibodies be humanized while retaining high antigen affinity and other favorable biological properties. To achieve this goal, according to the preferred method, humanized antibodies are obtained by analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are familiar to those skilled in the art. Computer programs are available that illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. The study of these images allows us to analyze the likely role of the residues in the functioning of the immunoglobulin candidate sequence, i.e. analyze residues that affect the ability of a candidate immunoglobulin to bind to its antigen. Thus, FR residues can be selected from the consensus and import sequences and combined such that the desired antibody characteristic, such as increased affinity for the target antigen(s), is obtained. For more information, see WO92/22653 published December 23, 1992.

The antibodies of the present invention can also be obtained using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them. In a specific aspect, such a phage can be used to display antigen-binding domains such as Fab and Fv, or disulfide-stabilized Fv expressed from a repertoire or library of combinatorial antibodies (eg, human or mouse). A phage expressing an antigen-binding domain that binds to an antigen of interest can be selected or identified by the antigen, such as a labeled antigen or an antigen bound or captured on a solid surface or bead. The phage used in these methods is usually filamentous phage, including fd and M13. The antigen binding domains are expressed as a recombinant fusion protein with either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to produce immunoglobulins or fragments thereof of the present invention include those disclosed in Brinkmann et al. "Phage Display Of Disulfide-Stabilized Fv Fragments," J. Immunol. Methods, 182:41-50, 1995.

The functional characteristics of several IgG isotypes and their domains are well known in the art. The amino acid sequences of IgG1, IgG2, IgG3 and IgG4 are known in the art. The selection and/or combination of two or more domains from particular IgG isotypes for use in the methods of the invention can be made based on any known parental isotype parameter, including affinity for FcγR. For example, the use of regions or domains from IgG isotypes that exhibit limited or no such binding to FcγRIIB, such as IgG2 or IgG4, may be of particular use in cases where the engineered bispecific antibody is required to maximize binding to an activating receptor and minimal binding to an inhibitory receptor. . Similarly, the use of Fc chains or domains from IgG isotypes known to preferentially bind to C1q or FcγRIIIA, such as IgG3, can be combined with Fc amino acid modifications known in the art to enhance antibody-dependent cell-mediated cytotoxicity (ADCC) and /or complement-dependent cytotoxicity (CDC) to create a bispecific antibody that demonstrates the maximum activity of the effector function, such as complement activation or ADCC. Likewise, mutations can be made in Fc chains or IgG isotype domains that minimize or abolish the effector function of the Fc chain.

During the discovery process, the creation and characterization of antibodies can shed light on information about desired epitopes. Based on this information, antibodies can then be competitively screened for binding to the same epitope. One approach to achieve this goal is to conduct competitive and cross-competitive analysis to look for antibodies that compete or cross-compete with each other, for example, antibodies that compete for binding to an antigen. One method is to identify the epitope to which the antibodies bind, or "epitope mapping". There are several methods known in the art for mapping and describing the position of epitopes on proteins, including crystal structure determination of the antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. As a further example, epitope mapping can be used to determine the sequence to which an antibody binds. Epitope mapping is commercially available from various sources such as Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). As another example, antigen-binding domain mutagenesis, domain swap experiments, and alanine scan mutagenesis can be used to identify residues required, sufficient, and/or necessary for epitope binding. The binding affinity and interaction rate of the antigen-binding domain can be determined using competitive binding assays. One example of a competitive binding assay is radioimmunoassay, which involves incubating a labeled antigen and detecting a molecule bound to the labeled antigen. The affinity of a molecule of the present invention for an antigen and dissociation rate for binding can be determined from saturation data using the Scatchard analysis.

The affinity and binding properties of the antibodies of the present invention for an antigen can be initially determined using in vitro assays known in the art (biochemical or immunological assays) for the antigen binding domain, including, but not limited to, ELISA assay, surface plasmon resonance assay ( SPR), biolayer interferometry or immunoprecipitation tests. Molecules of the present invention may have binding properties in in vivo models (eg, those described and disclosed herein) similar to those obtained from in vitro assays. However, the present invention does not exclude molecules of the invention that do not show the desired phenotype in in vitro assays but do show the desired phenotype in vivo.

Once the nucleic acid sequence encoding the molecules of the invention (ie binding domains) has been obtained, a vector for producing the molecules can be obtained by recombinant DNA technology using techniques well known in the art.

Polynucleotides encoding antibody binding domains of the present invention may include an expression control polynucleotide sequence operably linked to antibody coding sequences, including naturally associated or heterologous promoter regions known in the art. Expression control sequences can be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but prokaryotic host control sequences can also be used. After incorporation of the vector into an appropriate host cell line, the host cell is propagated under conditions suitable for the expression of the nucleotide sequences and, if necessary, for the collection and purification of antibodies. Eukaryotic cell lines include CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines, transformed B cells, or human embryonic kidney cell lines.

In one embodiment, the DNA encoding the antibodies of the invention is isolated and sequenced using conventional procedures (eg, with oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of mouse antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells, such as monkey COS cells, CHO cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to allow monoclonal antibody synthesis in recombinant host cells. The DNA can also be modified, for example by replacing the coding sequence of the human heavy and light chain constant domains in place of homologous murine sequences, Morrison et al., Proc. Nat. Acad. sci. 81:6851, 1984. In this way, chimeric antibodies are obtained that exhibit antigen binding specificity characteristic of the monoclonal antibodies disclosed herein.

CD3 antibodies

In one aspect, the present invention relates to agents that specifically bind to CD3 (eg, the full-length subunit of human CD3 epsilon (eg, accession number NP_000724 or SEQ ID NO: 66)).

In some embodiments, the anti-CD3 antibody specifically binds to human CD3.

Typical CD3 antibodies of the present invention are listed in the description in tables 1, 2 and 3. Table 1 provides the amino acid sequence identifiers of the VH regions and VL regions. In some embodiments, an antibody is provided having any of the partial light chain sequences listed in Table 1 and/or any of the partial heavy chain sequences listed in Table 1.

Table 1

Option light chain heavy chain 2B5-0001
(2B5 antibody) DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSRKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIK
(SEQ ID NO: 1) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNRARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 2) 2B5-0002 DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIK
(SEQ ID NO: 3) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNRARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 2) 2B5-0006
(2B5v6) DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIK
(SEQ ID NO: 3) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 4) 2B5-0009 DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIK
(SEQ ID NO: 3) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNRAQGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 5) 2B5-0517 DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSGKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIK
(SEQ ID NO: 6) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHQPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 7) 2B5-0522 DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSQKNYLAWYQQKPGKAPKLLIYWASDRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIK
(SEQ ID NO: 8) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHQPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 7) 2B5-0533 DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSGKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIK
(SEQ ID NO: 6) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 4) 2B5-0538 DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSQKNYLAWYQQKPGKAPKLLIYWASDRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIK
(SEQ ID NO: 8) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 4) 2B5-0598 DIQMTQSPSSLSASVGDRVTITCTSDQSLFNVRSGKNYLAWYQQKPGKAPKLLIYWASDRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIK
(SEQ ID NO: 9) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQDRGYTSDHQPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 10) 2B5-0623 DIQMTQSPSSLSASVGDRVTITCTSDQSLFNVRSGKNYLAWYQQKPGKAPKLLIYWASDRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIK
(SEQ ID NO: 9) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHQPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 7) 2B5-0707 DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYYLFTFGGGTKVEIK
(SEQ ID NO: 11) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRHSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 12) 2B5-0003 DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSRKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIK
(SEQ ID NO: 34) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 35) H2B4 DIVMTQSPDSLAVSLGERATINCKSSQSLFNVRSRKNYLAWYQQKPGQPPKLLISWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYDLFTFGSGTKLEIK
(SEQ ID NO: 85) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNRARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 2) 2B5-1038 DIQMTQSPSSLSASVGDRVTITCTSDQSLFNVRSGKNYLAWYQQKPGKAPKLLIYWASDRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIK
(SEQ ID NO: 9) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 4) 2B5-1039 DIQMTQSPSSLSASVGDRVTITCTSSESLFNVRSGKNYLAWYQQKPGKAPKLLIYWASDRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIK
(SEQ ID NO: 87) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 4) 2B5-1040 DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSGKNYLAWYQQKPGKAPKLLIYWASDRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIK
(SEQ ID NO: 89) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 4)

In Table 1, the underlined sequences are the CDR sequences by Kabat and those in bold by Chothia.

The invention provides CDR regions of anti-CD3 antibodies (including CDRs according to Chothia, Kabat and CDR contact regions). Methods and techniques for identifying CDRs in VH and VL amino acid sequences are well known in the art and can be used to identify CDRs in specific VH and/or VL amino acid sequences disclosed herein. Exemplary conventions that can be used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, for example, Kabat, "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et ai, J. Mol. Biol. 273:927-948 (1997); and Martin et ai, Proc. Natl. Acad. sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences in an antibody. It is understood that, in some embodiments, the CDRs may be a combination of Kabat and Chothia CDRs (also referred to as "combined CDRs" or "extended CDRs"). In some embodiments, the CDRs are Kabat CDRs. In some additional embodiments, the CDRs are Chothia CDRs. In other words, in embodiments with more than one CDR, these CDRs may be any of the Kabat, Chothia, combined CDRs, or combinations thereof. Tables 2 and 3 provide examples of CDR sequences of the invention.

In one embodiment, the present invention relates to antibodies containing the amino acid sequence shown in Table 1, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

In some such embodiments, the present invention relates to antibodies containing a VL region containing the amino acid sequence of any of the amino acid sequences of the VL regions listed in Table 1, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98% identity or at least 99% sequence identity.

In some other such embodiments, the present invention provides antibodies comprising a VH region comprising an amino acid sequence of any of the amino acid sequences of the VH regions listed in Table 1, or a substantially similar sequence thereof, having at least 90%, at least 95% , at least 98% identity or at least 99% sequence identity.

In one embodiment, an antibody is provided that specifically binds to CD3, said antibody comprising: (a) a heavy chain variable region (VH) comprising a VH complementarity determining region one (VH CDR1), a VH complementarity determining region two (VH CDR2) and complementarity determining region three VH (VH CDR3) sequence VH with SEQ ID NO: 2, 4, 5, 7, 10, 12 or 35; and/or (b) a light chain variable region (VL) comprising a VL complementarity determining region one (VL CDR1), a VL complementarity determining region two (VL CDR2), and a VL complementarity determining region three (VL CDR3) of the VL sequence c SEQ ID NO : 1, 3, 6, 8, 9, 11, 34, 87 or 89.

In one embodiment, the present invention provides an antibody comprising a set of six CDRs (i.e., VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, VH CDR3) contained in a VL/VH amino acid sequence pair as defined for any of the typical anti-CD3 antibodies listed in Table 1.

In specific embodiments, the present invention provides antibodies comprising a set of VL CDR1, VL CDR2, VL CDR3, VH CDR1, VH CDR2, or VH CDR3 amino acid sequences contained in a VL/VH amino acid sequence pair selected from the group consisting of SEQ ID NO : 1 and 2 (for example, 2B5-0001 (2B5)); SEQ ID NOs: 3 and 2 (2B5-0002); SEQ ID NOs: 3 and 4 (eg 2B5-0006 (2B5v6)); SEQ ID NOs: 3 and 5 (eg 2B5-0009); SEQ ID NOs: 6 and 7 (eg 2B5-0517); SEQ ID NOs: 8 and 7 (eg 2B5-0522); SEQ ID NOs: 6 and 4 (eg 2B5-0533); SEQ ID NOs: 8 and 4 (eg 2B5-0538); SEQ ID NOs: 9 and 10 (eg 2B5-0598); SEQ ID NOs: 9 and 7 (eg 2B5-0623); SEQ ID NOs: 11 and 12 (eg 2B5-0707); SEQ ID NOs: 34 and 35 (eg 2B5-0003); SEQ ID NO: 85 and 2 (eg H2B4); SEQ ID NOs: 9 and 4 (eg 2B5-1038); SEQ ID NOs: 87 and 4 (eg 2B5-1039); and SEQ ID NOs: 89 and 4 (eg 2B5-1040). Table 2 shows the VH CDR1, VH CDR2, and VH CDR3 regions of exemplary anti-CD3 antibodies.

table 2

Heavy chain variable region mAb VH CDR1 VH CDR2 VH CDR3 2B5-0001
(2B5) DYYMT
(SEQ ID NO: 13) (Kabat)
GFTFSDY (SEQ ID NO: 14) (Chothia)
GFTFSDYYMT
(SEQ ID NO: 15) (extended) FIRNRARGYTSDHNPSVKG (SEQ ID NO: 16) (Kabat)
RNRARGYT (SEQ ID NO: 17) (Chothia) DRPSYYVLDY (SEQ ID NO: 18) 2B5-0002 DYYMT
(SEQ ID NO: 13)
(Kabat)
GFTFSDY
(SEQ ID NO: 14) (Chothia)
GFTFSDYYMT
(SEQ ID NO: 15) FIRNRARGYTSDHNPSVKG (SEQ ID NO: 16) (Kabat)
RNRARGYT (SEQ ID NO: 17) (Chothia) DRPSYYVLDY
(SEQ ID NO: 18) 2B5-0006
(2B5v6) DYYMT
(SEQ ID NO: 13) (Kabat)
GFTFSDY
(SEQ ID NO: 14) (Chothia)
GFTFSDYYMT
(SEQ ID NO: 15) (extended) FIRNQARGYTSDHNPSVKG (SEQ ID NO: 19) (Kabat)
RNQARGYT
(SEQ ID NO: 20) (Chothia); DRPSYYVLDY
(SEQ ID NO: 18) 2B5-0009 DYYMT
(SEQ ID NO: 13) (Kabat)
GFTFSDY (SEQ ID NO: 14) (Chothia)
GFTFSDYYMT (SEQ ID NO: 15) (extended) FIRNRAQGYTSDHNPSVKG (SEQ ID NO: 21) (Kabat)
RNRAQGYT (SEQ ID NO: 22) (Chothia) DRPSYYVLDY
(SEQ ID NO: 18) 2B5-0517 DYYMT
(SEQ ID NO: 13) (Kabat)
GFTFS DY (SEQ ID NO: 14) (Chothia)
GFTFSDYYMT (SEQ ID NO: 15) (extended) FIRNQARGYTSDHQPSVKG (SEQ ID NO: 23) (Kabat)
RNQARGYT
(SEQ ID NO: 20) (Chothia) DRPSYYVLDY
(SEQ ID NO: 18) 2B5-0522 DYYMT
(SEQ ID NO: 13) (Kabat)
GFTFSDY
(SEQ ID NO: 14) (Chothia)
GFTFSDYYMT (SEQ ID NO: 15) (extended) FIRNQARGYTSDHQPSVKG (SEQ ID NO:23) (Kabat)
RNQARGYT (SEQ ID NO: 20) (Chothia) DRPSYYVLDY (SEQ ID NO: 18) 2B5-0533 DYYMT (SEQ ID NO: 13) (Kabat)
GFTFSDY (SEQ ID NO: 14) (Chothia)
GFTFSDYYMT
(SEQ ID NO: 15) (extended) FIRNQARGYTSDHNPSVKG
(SEQ ID NO: 19) (Kabat)
RNQARGYT
(SEQ ID NO: 20) (Chothia) DRPSYYVLDY
(SEQ ID NO: 18) 2B5-0538 DYYMT
(SEQ ID NO: 13) (Kabat)
GFTFS DY
(SEQ ID NO: 14) (Chothia)
GFTFSDYYMT
(SEQ ID NO: 15) (extended) FIRNQARGYTSDHNPSVKG
(SEQ ID NO: 19) (Kabat)
RNQARGYT
(SEQ ID NO: 20) (Chothia) DRPSYYVLDY
(SEQ ID NO: 18) 2B5-0598 DYYMT
(SEQ ID NO: 13) (Kabat)
GFTFSDY
(SEQ ID NO: 14) (Chothia)
GFTFSDYYMT
(SEQ ID NO: 15) (extended) FIRNQDRGYTSDHQPSVKG (SEQ ID NO: 24) (Kabat)
RNQARGYT (SEQ ID NO: 20) (Chothia) DRPSYYVLDY
(SEQ ID NO: 18) 2B5-0623 DYYMT (SEQ ID NO: 13) (Kabat)
GFTFS DY (SEQ ID NO: 14) (Chothia)
GFTFSDYYMT
(SEQ ID NO: 15) (extended) FIRNQARGYTSDHQPSVKG (SEQ ID NO: 23) (Kabat)
RNQARGYT (SEQ ID NO: 20) (Chothia) DRPSYYVLDY
(SEQ ID NO: 18) 2B5-0707 DYYMT
(SEQ ID NO: 13) (Kabat)
GFTFSDY
(SEQ ID NO: 14) (Chothia)
GFTFSDYYMT
(SEQ ID NO: 15) (extended) FIRNQARGYTSDHNPSVKG (SEQ ID NO: 19) (Kabat)
RNQARGYT
(SEQ ID NO: 20) (Chothia) DRHSYYVLDY
(SEQ ID NO: 25) 2B5-0003 DYYMT
(SEQ ID NO: 13) (Kabat)
GFTFSDY
(SEQ ID NO: 14) (Chothia)
GFTFSDYYMT
(SEQ ID NO: 15) (extended) FIRNQARGYTSDHNPSVKG
(SEQ ID NO: 19) (Kabat)
RNQARGYT
(SEQ ID NO: 20) (Chothia) DRPSYYVLDY
(SEQ ID NO: 18) H2B4 DYYMT
(SEQ ID NO: 13) (Kabat)
GFTFSDY (SEQ ID NO: 14) (Chothia)
GFTFSDYYMT
(SEQ ID NO: 15) (extended) FIRNRARGYTSDHNPSVKG (SEQ ID NO: 16) (Kabat)
RNRARGYT (SEQ ID NO: 17) (Chothia) DRPSYYVLDY
(SEQ ID NO: 18) 2B5-1038 DYYMT
(SEQ ID NO: 13) (Kabat)
GFTFSDY (SEQ ID NO: 14) (Chothia)
GFTFSDYYMT
(SEQ ID NO: 15) (extended) FIRNQARGYTSDHNPSVKG
(SEQ ID NO: 19) (Kabat)
RNQARGYT
(SEQ ID NO: 20) (Chothia) DRPSYYVLDY
(SEQ ID NO: 18) 2B5-1039 DYYMT
(SEQ ID NO: 13) (Kabat)
GFTFSDY (SEQ ID NO: 14) (Chothia)
GFTFSDYYMT
(SEQ ID NO: 15) (extended) FIRNQARGYTSDHNPSVKG
(SEQ ID NO: 19) (Kabat)
RNQARGYT
(SEQ ID NO: 20) (Chothia) DRPSYYVLDY
(SEQ ID NO: 18) 2B5-1040 DYYMT
(SEQ ID NO: 13) (Kabat)
GFTFSDY (SEQ ID NO: 14) (Chothia)
GFTFSDYYMT
(SEQ ID NO: 15) (extended) FIRNQARGYTSDHNPSVKG
(SEQ ID NO: 19) (Kabat)
RNQARGYT
(SEQ ID NO: 20) (Chothia) DRPSYYVLDY
(SEQ ID NO: 18)

In one embodiment, the present invention relates to antibodies containing a VH CDR1 containing any amino acid sequence from the VH CDR1 amino acid sequences listed in Table 2, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

In another embodiment, the present invention relates to antibodies containing a VH CDR2 containing any amino acid sequence from the VH CDR2 amino acid sequences listed in Table 2, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

In yet another embodiment, the present invention relates to antibodies containing a VH CDR3 containing any amino acid sequence of the amino acid sequences of the VH CDR3 listed in Table 2, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

Table 3 shows VL CDR1, VL CDR2, and VL CDR3 of typical anti-CD3 antibodies.

Table 3

Light chain variable region mAb CDR 1 CDR 2 CDR 3 2B5-0001
(2B5) TSSQSLFNVRSRKNYLA
(SEQ ID NO: 26) WASTRES
(SEQ ID NO: 27) KQSYDLFT
(SEQ ID NO: 28) 2B5-0002 TSSQSLFNVRSQKNYLA
(SEQ ID NO: 29) WASTRES
(SEQ ID NO: 27) KQSYDLFT
(SEQ ID NO: 28) 2B5-0006
(2B5v6) TSSQSLFNVRSQKNYLA
(SEQ ID NO: 29) WASTRES
(SEQ ID NO: 27) KQSYDLFT
(SEQ ID NO: 28) 2B5-0009 TSSQSLFNVRSQKNYLA
(SEQ ID NO: 29) WASTRES
(SEQ ID NO: 27) KQSYDLFT
(SEQ ID NO: 28) 2B5-0517 TSSQSLFNVRSGKNYLA
(SEQ ID NO: 30) WASTRES
(SEQ ID NO: 27) KQSYDLFT
(SEQ ID NO: 28) 2B5-0522 TSSQSLFNVRSQKNYLA
(SEQ ID NO: 29) WASDRES
(SEQ ID NO: 31) KQSYDLFT
(SEQ ID NO: 28) 2B5-0533 TSSQSLFNVRSGKNYLA
(SEQ ID NO: 30) WASTRES
(SEQ ID NO: 27) KQSYDLFT
(SEQ ID NO: 28) 2B5-0538 TSSQSLFNVRSQKNYLA
(SEQ ID NO: 29) WASDRES
(SEQ ID NO: 31) KQSYDLFT
(SEQ ID NO: 28) 2B5-0598 TSDQSLFNVRSGKNYLA
(SEQ ID NO: 32) WASDRES
(SEQ ID NO: 31) KQSYDLFT
(SEQ ID NO: 28) 2B5-0623 TSDQSLFNVRSGKNYLA
(SEQ ID NO: 32) WASDRES
(SEQ ID NO: 31) KQSYDLFT
(SEQ ID NO: 28) 2B5-0707 TSSQSLFNVRSQKNYLA
(SEQ ID NO: 29) WASTRES
(SEQ ID NO: 27) KQSYYLFT
(SEQ ID NO: 33) 2B5-0003 TSSQSLFNVRSRKNYLA
(SEQ ID NO: 26) WASTRES
(SEQ ID NO: 27) KQSYDLFT
(SEQ ID NO: 28) H2B4 TSSQSLFNVRSRKNYLA
(SEQ ID NO: 26) WASTRES
(SEQ ID NO: 27) KQSYDLFT
(SEQ ID NO: 28) 2B5-1038 TSDQSLFNVRSGKNYLA
(SEQ ID NO: 32) WASDRES
(SEQ ID NO: 31) KQSYDLFT
(SEQ ID NO: 28) 2B5-1039 TSSESLFNVRSGKNYLA (SEQ ID NO: 91) WASDRES
(SEQ ID NO: 31) KQSYDLFT
(SEQ ID NO: 28) 2B5-1040 TSSQSLFNVRSGKNYLA (SEQ ID NO: 92) WASDRES
(SEQ ID NO: 31) KQSYDLFT
(SEQ ID NO: 28)

In one embodiment, the present invention relates to antibodies containing a VL CDR1 containing any amino acid sequence from the VL CDR1 amino acid sequences listed in Table 3, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

In another embodiment, the present invention relates to antibodies containing a VL CDR2 containing any amino acid sequence from the VL CDR2 amino acid sequences listed in Table 3, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

In yet another embodiment, the present invention relates to an antibody containing a VL CDR3 containing any amino acid sequence from the VL CDR3 amino acid sequences listed in Table 3, or a sequence substantially similar to it, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

In one aspect, the present invention provides an antibody that specifically binds to CD3, said antibody comprising: (a) a heavy chain (HC) variable region (VH) comprising a VH complementarity determining region one (CDR1), a VH complementarity determining region two (VH CDR2) and complementarity determining region three VH (VH CDR3) of the VH sequence having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 5, 7, 10, 12 and 35; and/or (b) a light chain variable (VL) region (LC) comprising a VL complementary region defining one (VL CDR1), a VL complementary region defining two (VL CDR2) and a VL complementary region defining three (VL CDR3) of the VL sequence, having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 8, 9, 11, 34, 87 and 89.

In specific embodiments, the antibody comprises: (a) a VH region containing (i) a VH CDR1 containing the sequence of SEQ ID NO: 13, 14, or 15; (ii) VH CDR2 containing the sequence of SEQ ID NO: 16, 17, 19, 20, 21, 22, 23 or 24; and (iii) a VH CDR3 containing the sequence of SEQ ID NO: 18 or 25; and/or (b) a VL region containing (i) a VL CDR1 containing the sequence of SEQ ID NO: 26, 29, 30, 32, 91 or 92; (ii) VL CDR2 containing the sequence of SEQ ID NO: 27 or 31; and (iii) a VL CDR3 containing the sequence of SEQ ID NO: 28 or 33.

In some embodiments, the present invention provides an antibody that binds to CD3 and competes with an antibody disclosed herein, including 2B5-0001 (2B5), 2B5-0002, 2B5-0006 (2B5v6), 2B5-0009, 2B5-0517 , 2B5-0522, 2B5-0533, 2B5-0538, 2B5-0598, 2B5-0623, 2B5-0707, 2B5-0003, 2B5-1038, 2B5-1039 and 2B5-1040

In some embodiments, the invention also relates to anti-CD3 antibody CDRs based on CDR contact regions. CDR contact regions are regions of an antibody that confer antigen specificity to the antibody. Typically, CDR contact regions include residue positions in CDRs and Vernier zones that are restricted in order to maintain the correct loop structure of an antibody that binds to a particular antigen. See, for example, Makabe et al., J. Biol. Chem., 283:1156-1166, 2007. The definition of CDR contact regions is well known to those skilled in the art.

Some of the anti-CD3 antibodies described herein may have more than one name. For example, 2B5-0001 may be referred to as 2B5 or h2B5; and 2B5-0006 may be referred to as 2B5v6 or h2B5v6. Table 4 shows the antibody format and anti-CD3 antibody region present in the antibodies of the present invention.

Table 4

Subsequence Description Format Anti-CD3 antibody region 2B5-0001 (2B5) CD3 antibody IgG 2B5-0001 2B5-0002 CD3 antibody IgG 2B5-0002 2B5-0003 CD3 antibody IgG 2B5-0003 2B5-0006
(2B5v6) CD3 antibody IgG 2B5-0006 2B5-0009 CD3 antibody IgG 2B5-0009 2B5-0517 CD3 antibody IgG 2B5-0517 2B5-0522 CD3 antibody IgG 2B5-0522 2B5-0533 CD3 antibody IgG 2B5-0533 2B5-0538 CD3 antibody IgG 2B5-0538 2B5-0598 CD3 antibody IgG 2B5-0598 2B5-0623 CD3 antibody IgG 2B5-0623 2B5-0707 CD3 antibody IgG 2B5-0707 2B5-1038 CD3 antibody IgG 2B5-1038 2B5-1039 CD3 antibody IgG 2B5-1039 2B5-1040 CD3 antibody IgG 2B5-1040 H2B4 CD3 antibody IgG H2B4 GUCY2C-H2B4 CD3-GUCY2c bispecific antibody CD3 bispecific H2B4 GUCY2C-2B5 CD3-GUCY2c bispecific antibody CD3 bispecific 2B5-0001 GUCY2C-2B5v6 CD3-GUCY2c bispecific antibody CD3 bispecific 2B5-0006 xFLT3-2B5v6 CD3-FLT3 bispecific antibody CD3 bispecific 2B5-0006

The binding affinity (K _D ) of anti-CD3 antibodies disclosed herein (eg, human CD3 epsilon (eg, SEQ ID NO: 40)) can be from about 0.001 nM to about 6500 nM. In some embodiments, the binding affinity may have any of the following values, equal to about 6500 nM, 6000 nM, 5500 nM, 4500 nM, 4000 nM, 3500 nM, 3000 nM, 2500 nM, 2000 nM, 1500 nM, 1000 nM, 750 nM , 500nM, 400nM, 300nM, 250nM, 200nM, 150nM, 100nM, 75nM, 50nM, 45nM, 40nM, 35nM, 30nM, 25nM, 20nM, 19nM, 17 nM, 16 nM, 15 nM, 10 nM, 8 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.3 nM, 0.1 nM, 0.01 nM, 0.002 nM, or 0.001 nM. In some embodiments, the binding affinity may be less than any of the following values, equal to about 6500 nM, 6000 nM, 5500 nM, 5000 nM, 4000 nM, 3000 nM, 2000 nM, 1000 nM, 900 nM, 800 nM, 500 nM, 250 nM, 200 nM, 100 nM, 50 nM, 30 nM, 20 nM, 10 nM, 7.5 nM, 7 nM, 6.5 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or less than nM. In some embodiments, the antibody has a K _D of about 80 to about 200 pM. In a specific embodiment, the antibody has a K _D of about 10 to 2000 nm.

In one aspect, the invention relates to an antibody or pharmaceutical composition for use in a method for modulating a T-cell mediated immune response in a subject in need thereof. In specific embodiments, the invention relates to an antibody or pharmaceutical composition for use in a method for suppressing the growth of tumor cells in a subject.

In another aspect, the invention relates to an antibody or pharmaceutical composition for use in the treatment of cancer, optionally the cancer being selected from the group consisting of breast, ovarian, thyroid, prostate, cervix, lung, bladder, endometrial, head and neck, testicular , glioblastoma and cancer of the digestive system.

In yet another aspect, the invention provides an antibody or pharmaceutical composition for use in the treatment of cancer that modulates a T-cell mediated immune response or inhibits the growth of tumor cells.

The present invention also relates to nucleic acid molecules encoding anti-CD3 antibodies or parts thereof. For example, the present invention relates to nucleic acid molecules encoding any of the VH or VL amino acid sequences listed in Table 1, or a sequence substantially similar to them, containing at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the VH CDR1, VH CDR2 or VH CDR3 amino acid sequences listed in Table 2, or a sequence substantially similar to them, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding any of the VL CDR1, VL CDR2 or VL CDR3 amino acid sequences listed in Table 3, or a sequence substantially similar to them, having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

The present invention also relates to nucleic acid molecules encoding VR HC, where VH contains a set of three CDRs (i.e. VH CDR1, VH CDR2 or VH CDR3), and the set of amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 is as defined for any typical anti-CD3 antibody listed in Table 2.

The present invention also relates to nucleic acid molecules encoding VR LC, where VL contains a set of three CDRs (i.e. VL CDR1, VL CDR2 or VL CDR3), and the set of amino acid sequences of VL CDR1, VL CDR2 and VL CDR3 is as determined for any typical anti-CD3 antibody listed in Table 3.

The present invention also relates to nucleic acid molecules encoding both VH and VL, where VH contains any amino acid sequence from the VH amino acid sequences listed in Table 1, and where VL contains any amino acid sequence from the VL amino acid sequences shown in Table 1.

The present invention also relates to recombinant expression vectors capable of expressing a polypeptide containing the variable region of the heavy or light chains of an anti-CD3 antibody. For example, the present invention includes recombinant expression vectors containing any of the nucleic acid molecules mentioned above, ie. nucleic acid molecules encoding any of the VH and VL listed in Table 1, and/or the CDR sequences listed in Tables 2 or 3. The scope of the present invention also includes host cells into which such vectors have been introduced, as well as methods for obtaining antibodies or parts thereof by culturing the host cells under conditions allowing the production of antibodies or antibody fragments and recovering the antibodies and antibody fragments thus obtained.

In some embodiments, the polynucleotide contains a sequence encoding the heavy chain and/or light chain variable regions of the antibody 2B5-0001 (2B5), 2B5-0002, 2B5-0006 (2B5v6), 2B5-0009, 2B5-0517, 2B5-0522, 2B5 -0533, 2B5-0538, 2B5-0598, 2B5-0623, 2B5-0707, 2B5-0003, 2B5-1038, 2B5-1039, or 2B5-1040 listed in Table 4.

The sequence encoding the antibody of interest can be stored in a vector in a host cell, and then the host cell can be expanded and frozen for future use. Vectors (including expression vectors) and host cells are further disclosed herein.

Polynucleotides complementary to any such sequences are also included within the scope of the present invention. In one aspect, the invention relates to a method for obtaining any of the polynucleotides disclosed in the present description. Polynucleotides can be single stranded (coding or antisense) or double stranded and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and one-to-one correspondence with a DNA molecule, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may optionally be present in the polynucleotide of the present invention, and the polynucleotide may optionally be linked to other carrier molecules and/or materials.

Polynucleotides may contain a native sequence (ie, an endogenous sequence encoding an antibody or portion thereof) or may contain a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions and/or insertions such that the encoded polypeptide is not immunoreactive compared to the native immunoreactive molecule. The impact on the immunoreactivity of the encoded polypeptide can usually be assessed by the methods disclosed in the present description. The variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity, even more preferably at least about 90% identity, and most preferably at least about 95% identity with a polynucleotide sequence that encodes a native antibody or a portion thereof.

The polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods or PCR method. Methods for the chemical synthesis of polynucleotides are well known in the art and need not be described in detail. One skilled in the art can use the sequences provided herein and a commercial DNA synthesizer to obtain the desired DNA sequence.

To obtain polynucleotides using recombinant techniques, a polynucleotide containing the desired sequence can be inserted into a suitable vector, and the vector can in turn be introduced into a suitable host cell for replication and amplification, as further discussed herein. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct capture, endocytosis, transfection, F-pairing, or electroporation. Once introduced, the exogenous polynucleotide can be stored in the cell as a non-integrated vector (such as a plasmid) or integrated into the genome of the host cell. The thus amplified polynucleotide can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.

Alternatively, PCR allows DNA sequences to be reproduced. PCR technology is well known in the art and is described in US Pat. Nos. 4,683,195; 4,800,159; eds., Birkauswer Press, Boston, 1994.

RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. Once the cell has replicated and the DNA has been transcribed into RNA, this RNA can then be isolated using methods well known to those skilled in the art, such as those described in Sambrook et al., 1989, supra.

Suitable cloning vectors may be designed according to standard techniques or may be selected from a wide variety of cloning vectors available in the art. Although the cloning vector chosen may vary depending on the host cell to be used, useful cloning vectors are generally self-replicating, may have a single target for a particular restriction endonuclease, and/or may carry marker genes that can be used when selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g. pUC18, pUC19, Bluescript (e.g. pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and shuttle vectors such as pSA3 and pAT28 . These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors are typically replicable polynucleotide constructs that contain a polynucleotide of the present invention. The expression vector is intended to replicate in host cells either as an episome or as an integral part of chromosomal DNA. Suitable expression vectors include, without limitation, plasmids, viral vectors including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector(s) disclosed in PCT Publication No. WO 87/04462. Vector components typically may include, without limitation, one or more of the following: a signal sequence; replication source; one or more marker genes; suitable transcription control elements (such as promoters, enhancers and terminators). Expression (ie, translation) typically also requires one or more translation control elements, such as ribosome binding sites, translation initiation sites, and stop codons.

The vectors containing the polynucleotides of interest can be introduced into the host cell by any of several suitable methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microparticle bombardment; lipofection; and infection (eg, when the vector is an infectious agent such as vaccinia virus). The choice of vectors to introduce or polynucleotides often depends on the characteristics of the host cell.

The invention also relates to host cells containing any of the polynucleotides disclosed in the present description. Any host cell capable of overexpressing heterologous DNA can be used to isolate genes encoding an antibody, polypeptide, or protein of interest. Non-limiting examples of mammalian host cells include, without limitation, COS, HeLa, and CHO cells. See also PCT Publication No. WO 87/04462. Suitable non-mammalian host cells include prokaryotes (such as E. coli or B. subtillis) and yeast (such as S. cerevisae, S. pombe; or K. lactis). Preferably, the host cells express cDNA at a level greater than about 5-fold, more preferably 10-fold, even more preferably 20-fold, the level of the corresponding endogenous antibody or CD3 protein or CD3 domain (e.g., domains 1-4) determined by immunoassay. or FACS. A cell overexpressing an antibody or protein of interest can be identified.

The invention also includes scFv antibodies of the present invention. Single chain variable region fragments are generated by linking the light and/or heavy chain variable region with a short binding peptide (Bird et al., Science 242:423-426, 1988). An example of a binding peptide includes the sequence of SEQ ID NO: 65. In one embodiment, the binding peptide is a bridge, approximately 3.5 nM in size, between the carboxy terminus of one variable region and the amino terminus of another variable region. Linkers having other sequences have been developed and are being used (Bird et al., Science 242:423-426, 1988). Linkers should be short, flexible polypeptides and preferably less than about 20 amino acid residues. The linkers, in turn, can be modified to perform additional functions such as drug attachment or attachment to a solid support. Single chain variants can be produced either recombinantly or synthetically. An automated synthesizer can be used to synthesize scFv. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding scFv can be introduced into a suitable host cell, either eukaryotic, such as a yeast, plant, insect, or mammalian cell, or prokaryotic, such as an E. coli cell. Polynucleotides encoding the scFv of interest can be obtained by conventional manipulations such as polynucleotide ligation. The resulting scFv can be isolated by standard protein purification methods known in the art.

In one aspect, a polynucleotide sequence is provided encoding any one of a light chain partial sequence and/or any one of a heavy chain partial sequence disclosed herein. Exemplary CD3 polynucleotides encoding the VH and VL regions of the antibodies of the present invention are shown in Table 5.

Table 5

Option light chain heavy chain 2B5-0001
(2B5) GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGCACAAGCTCACAGTCACTGTTTAATGTCCGCAGCCGGAAAAACTATCTTGCGTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTGGGCCAGTACACGAGAATCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCAAACAGTCTTACGACCTTTTCACTTTTGGCGGCGGAACAAAGGTGGAGATCAAG
(SEQ ID NO: 68) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATAGGGCACGCGGGTACACTTCAGACCACAATCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACCATCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCTCT
(SEQ ID NO: 69) 2B5-0002 GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGCACAAGCTCACAGTCACTGTTTAATGTCCGCAGCCAGAAAAACTATCTTGCGTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTGGGCCAGTACACGAGAATCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCAAACAGTCTTACGACCTTTTCACTTTTGGCGGCGGAACAAAGGTGGAGATCAAG
(SEQ ID NO: 70) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATAGGGCACGCGGGTACACTTCAGACCACAATCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACCATCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCTCT
(SEQ ID NO: 69) 2B5-0006
(2B5v6) GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGCACAAGCTCACAGTCACTGTTTAATGTCCGCAGCCAGAAAAACTATCTTGCGTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTGGGCCAGTACACGAGAATCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCAAACAGTCTTACGACCTTTTCACTTTTGGCGGCGGAACAAAGGTGGAGATCAAG
(SEQ ID NO: 70) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATCAGGCACGCGGGTACACTTCAGACCACAATCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACCATCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCTCT
(SEQ ID NO: 71) 2B5-0009 GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGCACAAGCTCACAGTCACTGTTTAATGTCCGCAGCCAGAAAAACTATCTTGCGTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTGGGCCAGTACACGAGAATCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCAAACAGTCTTACGACCTTTTCACTTTTGGCGGCGGAACAAAGGTGGAGATCAAG
(SEQ ID NO: 70) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATAGGGCACAGGGGTACACTTCAGACCACAATCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACCATCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCTCT
(SEQ ID NO: 72) 2B5-0517 GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGCACAAGCTCACAGTCACTGTTTAATGTCCGCAGCGGAAAAAACTATCTTGCGTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTGGGCCAGTACACGAGAATCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCAAACAGTCTTACGACCTTTTCACTTTTGGCGGCGGAACAAAGGTGGAGATCAAG
(SEQ ID NO: 83) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATCAGGCACGCGGGTACACTTCAGACCACCAGCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACCATCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCTCT
(SEQ ID NO: 73) 2B5-0522 GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGCACAAGCTCACAGTCACTGTTTAATGTCCGCAGCCAGAAAAACTATCTTGCGTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTGGGCCAGTGATCGAGAATCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCAAACAGTCTTACGACCTTTTCACTTTTGGCGGCGGAACAAAGGTGGAGATCAAG
(SEQ ID NO: 74) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATCAGGCACGCGGGTACACTTCAGACCACCAGCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACCATCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCTCT
(SEQ ID NO: 73) 2B5-0533 GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGCACAAGCTCACAGTCACTGTTTAATGTCCGCAGCGGAAAAAACTATCTTGCGTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTGGGCCAGTACACGAGAATCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCAAACAGTCTTACGACCTTTTCACTTTTGGCGGCGGAACAAAGGTGGAGATCAAG
(SEQ ID NO: 83) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATCAGGCACGCGGGTACACTTCAGACCACAATCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACCATCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCTCT
(SEQ ID NO: 71) 2B5-0538 GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGCACAAGCTCACAGTCACTGTTTAATGTCCGCAGCCAGAAAAACTATCTTGCGTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTGGGCCAGTGATCGAGAATCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCAAACAGTCTTACGACCTTTTCACTTTTGGCGGCGGAACAAAGGTGGAGATCAAG
(SEQ ID NO: 74) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATCAGGCACGCGGGTACACTTCAGACCACAATCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACCATCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCTCT
(SEQ ID NO: 71) 2B5-0598 GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGCACAAGCGACCAGTCACTGTTTAATGTCCGCAGCGGCAAAAACTATCTTGCGTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTGGGCCAGTGACCGAGAATCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCAAACAGTCTTACGACCTTTTCACTTTTGGCGGCGGAACAAAGGTGGAGATCaag
(SEQ ID NO: 75) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATCAGGACCGCGGGTACACTTCAGACCACCAGCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACCATCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCtct
(SEQ ID NO: 76) 2B5-0623 GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGCACAAGCGACCAGTCACTGTTTAATGTCCGCAGCGGCAAAAACTATCTTGCGTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTGGGCCAGTGACCGAGAATCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCAAACAGTCTTACGACCTTTTCACTTTTGGCGGCGGAACAAAGGTGGAGATCaag
(SEQ ID NO: 75) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATCAGGCACGCGGGTACACTTCAGACCACCAGCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACCATCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCTCT
(SEQ ID NO: 73) 2B5-0707 GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGCACAAGCTCACAGTCACTGTTTAATGTCCGCAGCCAGAAAAACTATCTTGCGTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTGGGCCAGTACACGAGAATCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCAAACAGTCTTACTACCTTTTCACTTTTGGCGGCGGAACAAAGGTGGAGATCAAG
(SEQ ID NO: 77) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATCAGGCACGCGGGTACACTTCAGACCACAATCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACACTCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCTCT
(SEQ ID NO: 84) 2B5-0003 GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGCACAAGCTCACAGTCACTGTTTAATGTCCGCAGCCGGAAAAACTATCTTGCGTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTGGGCCAGTACACGAGAATCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCAAACAGTCTTACGACCTTTTCACTTTTGGCGGCGGAACAAAGGTGGAGATCAAG
(SEQ ID NO: 68) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATCAGGCACGCGGGTACACTTCAGACCACAATCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACCATCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCTCT
(SEQ ID NO: 71) H2B4 GACATCGTGATGACCCAGAGCCCCGATAGCCTGGCCGTGTCTCTGGGAGAGAGAGCCACCATCAACTGCAAGAGCAGCCAGAGCCTGTTCAACGTGAGAAGCCGGAAGAACTACCTGGCCTGGTATCAGCAGAAACCCGGCCAGCCCCCCAAGCTGCTGATCAGCTGGGCCAGCACCAGAGAAAGCGGCGTGCCCGATAGATTCAGCGGCAGCGGAAGCGGCACCGACTTCACCCTGACAATCAGCTCCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCAAGCAGAGCTACGACCTGTTCACCTTCGGCAGCGGCACCAAGCTGGAAATCAAA
(SEQ ID NO: 86) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATAGGGCACGCGGGTACACTTCAGACCACAATCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACCATCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCTCT
(SEQ ID NO: 69) 2B5-1038 GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGCACAAGCGACCAGTCACTGTTTAATGTCCGCAGCGGCAAAAACTATCTTGCGTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTGGGCCAGTGACCGAGAATCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCAAACAGTCTTACGACCTTTTCACTTTTGGCGGCGGAACAAAGGTGGAGATCaag
(SEQ ID NO: 75) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATCAGGCACGCGGGTACACTTCAGACCACAATCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACCATCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCTCT
(SEQ ID NO: 71) 2B5-1039 GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGCACAAGCTCAGAGTCACTGTTTAATGTCCGCAGCGGCAAAAACTATCTTGCGTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTGGGCCAGTGACCGAGAATCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCAAACAGTCTTACGACCTTTTCACTTTTGGCGGCGGAACAAAGGTGGAGATCAAG
(SEQ ID NO: 88) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATCAGGCACGCGGGTACACTTCAGACCACAATCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACCATCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCTCT
(SEQ ID NO: 71) 2B5-1040 GACATCCAGATGACCCAGTCCCCCTCTTCTCTGTCTGCCTCTGTGGGCGACAGAGTGACCATCACCTGCACAAGCTCACAGTCACTGTTTAATGTCCGCAGCGGCAAAAACTATCTTGCGTGGTATCAGCAGAAGCCTGGCAAGGCTCCCAAGCTGCTGATCTACTGGGCCAGTGACCGAGAATCCGGCGTGCCTTCCAGATTCTCCGGCTCTGGCTCTGGCACCGATTTCACCCTGACCATCTCCTCCCTCCAGCCTGAGGATTTCGCCACCTACTACTGCAAACAGTCTTACGACCTTTTCACTTTTGGCGGCGGAACAAAGGTGGAGATCAAG
(SEQ ID NO: 90) GAAGTGCAGCTTGTTGAATCTGGCGGCGGTTTGGTTCAGCCCGGTGGATCACTGCGACTCAGTTGCGCAGCTAGCGGCTTCACCTTTTCTGATTACTACATGACATGGGTACGACAGGCGCCAGGCAAGGGTTTGGAATGGGTAGCATTCATACGCAATCAGGCACGCGGGTACACTTCAGACCACAATCCCTCAGTAAAAGGAAGATTTACCATCTCAAGAGACAATGCCAAAAATTCACTCTACCTGCAAATGAACTCACTTCGCGCCGAGGATACCGCCGTGTATTACTGTGCCAGAGACAGACCATCTTATTACGTGCTGGACTATTGGGGACAGGGCACTACAGTCACCGTCAGCTCT
(SEQ ID NO: 71)

In one aspect, the invention relates to compositions (such as a pharmaceutical composition) containing any of the polynucleotides of the invention. In some embodiments, the composition may contain any of the polynucleotides shown in Table 5. In specific embodiments, the composition contains an expression vector containing a polynucleotide encoding any of the antibodies disclosed herein, for example, any of the polynucleotides shown in Table 5.

Polynucleotides complementary to any such sequences are also included in the present invention. Polynucleotides can be single stranded (coding or antisense) or double stranded and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include mature and immature mRNAs such as progenitor mRNAs (pre-mRNAs) or heterogeneous nuclear mRNAs (hnRNAs) and mature mRNAs. Additional coding or non-coding sequences may optionally be present in the polynucleotide of the present invention, and the polynucleotide may optionally be linked to other carrier molecules and/or materials.

Polynucleotides may contain a native sequence (ie, an endogenous sequence encoding an antibody or portion thereof) or may contain a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions, and/or insertions that do not reduce the immunoreactivity of the encoded polypeptide relative to the native immunoreactive molecule. The impact on the immunoreactivity of the encoded polypeptide can usually be assessed by the methods disclosed in the present description. The variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity, even more preferably at least about 90% identity, and most preferably at least about 95% identity. with a polynucleotide sequence that encodes a native antibody or part thereof.

Two polynucleotide or polypeptide sequences are said to be "identical" if two nucleotide or amino acid sequences are the same when aligned for maximum matching, as described below. Comparison of two sequences is typically performed by comparing sequences within a comparison window to identify and compare local areas of sequence similarity. "Comparison window" as used herein refers to a segment of at least about 20 contiguous positions, typically 30 to about 75 or 40 to about 50, in which a sequence can be compared to a reference sequence with the same number of contiguous positions after optimal alignment of the two sequences.

Optimal sequence alignment for comparison can be performed using the Megalign program from the Lasergene Bioinformatics Software Suite (DNASTAR, Inc., Madison, WI) using the default parameters. Preferably, the "percent sequence identity" is determined by comparing two optimally aligned sequences in a comparison window of at least 20 positions, and for optimal alignment of the two sequences, a portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e. gaps) of 20 percent or less, typically 5 to 15 percent or 10 to 12 percent, relative to reference sequences (which contain no additions or deletions). The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences to obtain the number of matching positions by dividing the number of matching positions by the total number of positions in the reference sequence (i.e. window size) and multiplying the results by 100 to give percent sequence identity.

Variants may also or alternatively be substantially homologous to a native gene, or a portion thereof, or a complementary sequence. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to the native DNA sequence encoding the native antibody (or complementary sequence).

Those skilled in the art will appreciate that, as a result of the degeneracy of the genetic code, there are multiple nucleotide sequences that encode the polypeptide disclosed herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any natural gene. However, polynucleotides that differ due to differences in codon usage are specifically noted in the present invention. In addition, alleles of genes containing the polynucleotide sequences presented herein are within the scope of the present invention. Alleles are endogenous genes that are changed by one or more mutations such as deletions, additions and/or substitutions of nucleotides. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles can be identified by standard methods (such as hybridization, amplification and/or database sequence comparison).

In one aspect, the invention relates to a method for obtaining any of the polynucleotides disclosed in the present description. For example, the polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods or PCR. Methods for the chemical synthesis of polynucleotides are well known in the art and need not be described in detail. One skilled in the art can use the sequences provided herein and a commercial DNA synthesizer to obtain the desired DNA sequence.

To obtain polynucleotides using recombinant techniques, a polynucleotide containing the desired sequence can be inserted into a suitable vector, and the vector can in turn be introduced into a suitable host cell for replication and amplification, as further discussed herein. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide via direct capture, endocytosis, transfection, F-pairing, or electroporation. Once introduced, the exogenous polynucleotide can be stored in the cell as a non-integrated vector (such as a plasmid) or integrated into the genome of the host cell. The thus amplified polynucleotide can be isolated from the host cell by methods well known in the art (eg, Sambrook et al., 1989).

Alternatively, PCR allows DNA sequences to be reproduced. PCR technology is well known in the art and is described in US Pat. Nos. 4,683,195; 4,800,159; eds., Birkauswer Press, Boston, 1994.

RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. Once the cell has replicated and the DNA has been transcribed into RNA, this RNA can then be isolated using techniques well known to those skilled in the art, such as described in Sambrook et al., 1989, supra.

Suitable cloning vectors may be designed according to standard techniques or may be selected from a wide variety of cloning vectors available in the art. Although the cloning vector chosen may vary depending on the host cell to be used, useful cloning vectors are generally self-replicating, may have a single target for a particular restriction endonuclease, and/or may carry marker genes that can be used when selecting clones containing the vector. Suitable examples include plasmids and bacterial viruses, e.g. pUC18, pUC19, Bluescript (e.g. pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and shuttle vectors such as pSA3 and pAT28 . These and many other cloning vectors are available from commercial vendors such as BioRad, Strategene, and Invitrogen.

Expression vectors are typically replicable polynucleotide constructs that contain a polynucleotide of the present invention. The expression vector is intended to replicate in host cells either as an episome or as an integral part of chromosomal DNA. Suitable expression vectors include, without limitation, plasmids, viral vectors including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector(s) disclosed in PCT Publication No. WO87/04462. Vector components typically may include, without limitation, one or more of the following: a signal sequence; replication source; one or more marker genes; suitable transcription control elements (such as promoters, enhancers and terminators). Expression (ie, translation) typically also requires one or more translation control elements, such as ribosome binding sites, translation initiation sites, and stop codons.

The vectors containing the polynucleotides of interest can be introduced into the host cell by any of several suitable methods, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microparticle bombardment; lipofection; and infection (eg, when the vector is an infectious agent such as vaccinia virus). The choice of vectors to introduce or polynucleotides often depends on the characteristics of the host cell.

Any host cell capable of overexpressing heterologous DNA can be used to isolate genes encoding an antibody, polypeptide, or protein of interest. Non-limiting examples of mammalian host cells include, without limitation, COS, HeLa, and CHO cells. See also PCT Publication No. WO87/04462. Suitable non-mammalian host cells include prokaryotic cells (such as E. coli or B. subtillis) and yeast cells (such as S. cerevisae, S. pombe; or K. lactis). Preferably, the host cells express cDNA at a level greater than about 5-fold, more preferably 10-fold, even more preferably 20-fold, the level of the corresponding endogenous antibody or CD3 protein or CD3 domain (e.g., domains 1-4) determined by immunoassay. or FACS. A cell overexpressing an antibody or protein of interest can be identified.

In one aspect, the CD3 antibody molecule may have an affinity for CD3, eg, as measured by direct binding or competitive binding assays in the picomolar-micromolar range of binding affinity, preferably in the picomolar-low nanomolar range.

Bispecific antibodies can be generated using the sequences disclosed herein. Methods for making bispecific antibodies are known in the art (see, for example, Suresh et al., Methods in Enzymology 121:210, 1986). Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy chain-light chain pairs, the two heavy chains having different specificities (Millstein and Cuello, Nature 305, 537-539, 1983).

In another embodiment, an antibody disclosed herein comprises a full-length human antibody, wherein the antibody variable region of the heterodimeric protein is capable of recruiting the activity of a human effector immune cell by specifically binding to an effector antigen (e.g., CD3 antigen) located on a human immune effector cell, and wherein the second variable region of the antibody of the heterodimeric protein is capable of specifically binding to the target antigen. In some embodiments, the human antibody is an IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the heterodimeric protein contains an immunologically inert Fc chain.

The human effector immune cell can be any of a variety of immune effector cells known in the art. For example, the effector immune cell may be a member of a human lymphoid cell line, including, but not limited to, a T cell (eg, a cytotoxic T cell), a B cell, and a natural killer (NK) cell. The immune effector cell may also be, for example, without limitation, a member of a human myeloid lineage, including, but not limited to, a monocyte, a neutrophilic granulocyte, and a dendritic cell. Such immune effector cells can exert either a cytotoxic or apoptotic effect on the target cell, or another desired effect when activated through effector antigen binding.

An effector antigen is an antigen (eg, protein or polypeptide) that is expressed on a human effector immune cell. Examples of effector antigens that can bind to a heterodimeric protein (eg, heterodimeric antibody or bispecific antibody) include, without limitation, human CD3, CD16, NKG2D, NKp46, CD2, CD28, CD25, CD64, and CD89.

The target cell may be native or foreign to humans. In the case of a native target cell, the cell may have transformed into a malignant cell or may have been pathologically modified (eg, a native target cell infected with a virus, plasmodium, or bacterium). In the case of the target foreign cell, the cell is an invading pathogen such as a bacterium, plasmodium, or virus.

In some embodiments, an antibody used in the present invention is a Fab, Fab fragment, F(ab) ₂ fragment, Fv fragment, single chain Fv fragment, disulfide stabilized Fv fragment, single chain antibody, monoclonal antibody, chimeric antibody, bispecific antibody, a trispecific antibody, a polyspecific antibody, a bispecific heterodimeric diabody, a bispecific heterodimeric IgG, a polyclonal antibody, a labeled antibody, a humanized antibody, a human antibody, or fragments thereof.

In some embodiments, the anti-CD3 antibody disclosed herein is a monoclonal antibody. For example, the anti-CD3 antibody is a humanized monoclonal antibody or a chimeric monoclonal antibody.

The present invention includes an antibody containing a chain or Fc domain, or portions thereof. In some embodiments, the Fc chain, or portion(s) thereof, comprises one or more IgG1, IgG2, IgG3, or IgG4 Fc chain constant domains (eg, a CH2 or CH3 domain). In another embodiment, the invention encompasses molecules comprising an Fc chain, or portion thereof, wherein the Fc chain, or portion thereof, contains at least one amino acid modification (eg, substitution) relative to a comparable wild-type Fc chain, or portion thereof. Variant Fc regions are well known in the art and are primarily used to alter the phenotype of an antibody containing a variant Fc region as assessed by any binding activity or effector function assay well known in the art, such as ELISA, SPR assay, or ADCC. Such altered Fc chains or portions thereof can increase the plasma half-life and stability exhibited by a bispecific antibody of the invention comprising an Fc chain or portion thereof. In another embodiment, the invention includes the use of any Fc variant known in the art.

In one embodiment, one or more amino acid modifications of the Fc chain are made to reduce the affinity and avidity of the Fc chain, and thus the bispecific antibody of the invention, for one or more FcγR receptors. In a particular embodiment, the invention includes bispecific antibodies comprising a Fc chain variant or a portion thereof, wherein the Fc chain variant contains at least one amino acid modification relative to the wild-type Fc chain, and wherein the altered Fc region binds to only one FcγR, wherein the FcγR is FcγRIIIA . In another specific embodiment, the invention includes bispecific antibodies comprising a Fc chain variant or a portion thereof, wherein the Fc chain variant contains at least one amino acid modification relative to a wild-type (WT) Fc chain, and wherein the altered Fc region binds to only one FcγR, wherein FcγR is FcγRIIA. In another specific embodiment, the invention includes bispecific antibodies comprising a Fc chain variant, or a portion thereof, wherein the Fc chain variant contains at least one amino acid modification relative to a wild-type Fc chain, and wherein the Fc chain variant binds to only one FcγR, wherein the FcγR is is FcγRIIB. In another embodiment, the invention encompasses molecules containing an Fc chain variant, where the variant provides or mediates reduced ADCC activity (or other effector function) and/or increased binding to FcγRIIB (CD32B) compared to a molecule that does not contain an Fc chain or contains an Fc chain wild type as measured by methods known to those skilled in the art and disclosed herein.

The invention also includes the use of an Fc region containing domains or regions from two or more IgG isotypes. As is known in the art, amino acid modification of an Fc region can have a major impact on Fc-mediated effector function and/or binding activity. However, these functional changes can be further modified and/or controlled if implemented in the context of selected IgG isotypes. Similarly, the natural characteristics of the Fc isotype can be altered with one or more amino acid modifications. Numerous IgG isotypes (eg, IgG1, IgG2, IgG3, and IgG4) exhibit different physical and functional properties, including serum half-life, complement fixation, FcγR binding affinity, and activity of effector functions (eg, ADCC, CDC), due to differences in their amino acid sequences of the hinge and/or Fc regions.

In one embodiment, the amino acid modification and Fc region of the IgG are independently selected based on their respective binding activities and/or effector function, evaluated separately, to design a bispecific antibody with the desired characteristics. In a specific embodiment, amino acid modifications and IgG hinge/Fc regions are analyzed separately for binding activity and/or effector function, as disclosed herein or known in the art in the context of IgG1. In one embodiment, the amino acid modification and the IgG hinge/Fc region have similar functionality, such as reduced ADCC activity (or other effector function) and/or increased binding to FcγRIIB in the context of a bispecific antibody or other Fc containing molecule (e.g., and immunoglobulin) . In another embodiment, the invention encompasses variant Fc regions containing combinations of amino acid modifications known in the art and selected IgG regions exhibiting novel properties that were not detected by independent analysis of the modifications and/or regions disclosed herein.

One way to determine the binding affinity of an antibody to CD3 is to measure the binding affinity of monofunctional Fab fragments of an antibody. To obtain monofunctional Fab fragments, an antibody (eg, IgG) can be digested with papain or expressed recombinantly. The Fab affinity of an antibody fragment for CD3 can be determined by surface plasmon resonance (SPR system Biacore™ 3000™, Biacore™, INC, Piscataway NJ) using pre-immobilized streptavidin (SA) or anti-mouse Fc sensor chips or anti-human Fc using HBS-EP working buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v surfactant P20). Biotinylated or Fc-fused human CD3 can be diluted in HBS-EP buffer to less than 0.5 µg/mL and injected through individual channels of the chip using variable contact time to achieve two antigen density ranges: either 50-200 response units (RU ) for detailed study of kinetic characteristics, or 800-1000 RU for screening analyses. Regeneration studies have shown that 25 mM NaOH at 25% v/v. ethanol effectively removes bound Fab while maintaining CD3 activity on the chip for over 200 injections. Typically, serial dilutions (covering concentrations of 0.1-10x estimated K _D ) of purified Fab samples are administered over 1 min at 100 μl/min and allowed to dissociate for 2 hours. The concentrations of Fab proteins are determined by ELISA and/or SDS-PAGE electrophoresis using Fab at a known concentration (determined by amino acid analysis) as a standard. Kinetic rates of association (k _on ) and rates of dissociation (k _off ) are obtained simultaneously by fitting the data as a whole to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99-110) using the BIAevaluation program. The values of the equilibrium dissociation constant (K _D ) are calculated by the formula: k _off /k _on . This protocol is suitable for determining the binding affinity of an antibody for any CD3, including human CD3, other mammalian CD3 (eg, mouse CD3, rat CD3, or primate CD3), and various forms of CD3 (eg, glycosylated CD3). The binding affinity of an antibody is usually measured at 25°C, but it can also be measured at 37°C.

The antibodies disclosed herein may be prepared by any method known in the art. To obtain hybridoma cell lines, the route and schedule for immunizing the host animal generally follows established and conventional methods for stimulating and producing antibodies, as further disclosed herein. General methods for obtaining human and mouse antibodies are known in the art and/or disclosed in the present description.

It is contemplated that any mammalian subject, including a human or an antibody-producing cell, can be used to serve as a basis for the production of mammalian, including human, hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and/or intradermally with a specified amount of the immunogen, including the methods disclosed herein.

Hybridomas can be generated from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization method proposed by Kohler, B. and Milstein, C., Nature 256:495-497, 1975 or modified by Buck, DW, et al., In Vitro, 18: 377-381, 1982. Available myeloma lines can be used for hybridization, including but not limited to X63-Ag8.653 and lines from the Salk Institute, Cell Distribution Center, San Diego, CA, USA. Typically, the method involves the fusion of myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art. After fusion, the cells are separated from the confluence medium and grown in a selective growth medium such as hypoxanthine-aminopterin-thymidine (HAT) medium to remove unhybridized parental cells. Any of the media disclosed herein, supplemented with or without serum, can be used to culture hybridomas that secrete monoclonal antibodies. As another alternative, immortalized EBV B cells can be used in the cell fusion method to generate the monoclonal antibodies of the present invention. If desired, the hybridomas are expanded and subcloned, and the supernatants are assayed for anti-immunogenic activity using conventional immunoassay procedures (eg, radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay).

Hybridomas that can be used as a source of antibodies include all derivatives, progeny cells of parental hybridomas that produce monoclonal antibodies specific for CD3, or parts thereof.

Hybridomas that produce such antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from culture media or body fluids using conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if necessary. Unwanted activity, if present, can be eliminated, for example, by passing the preparation through adsorbents consisting of the immunogen attached to the solid phase and eluting or releasing the desired antibodies from the immunogen. Immunization of a host animal with human CD3 or a fragment containing a target amino acid sequence conjugated to a protein that is immunogenic to the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional agent or a derivatizing agent, e.g. maleimidobenzoylsulfosuccinimide ester (conjugation via cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl ₂ or R ¹ N=C=NR, where R and R ¹ are different alkyl groups, can give a population antibodies (eg monoclonal antibodies).

If desired, the antibody of interest can be sequenced and then the polynucleotide sequence can be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be stored in a vector in a host cell, and then the host cell can be expanded and frozen for future use. Production of recombinant monoclonal antibodies in cell culture can be accomplished by cloning antibody genes from B cells by methods known in the art. See, for example, Tiller et al., J. Immunol. Methods, 329, 112, 2008; U.S. Patent No. 7,314,622.

Alternatively, the polynucleotide sequence can be used for genetic manipulation to humanize the antibody or improve the affinity or other characteristics of the antibody. For example, the constant region can be designed to more closely resemble human constant regions to avoid initiating an immune response when the antibody is used in clinical trials and in human therapy. It may be necessary to genetically alter the sequence of the antibody to obtain higher affinity for CD3 and higher therapeutic efficacy.

In some embodiments, the antibody has a modified constant region that removes or reduces binding to the Fc gamma receptor. For example, the Fc may be a human IgG2 containing the D265 mutation, with amino acid residues numbered with reference to the sequence of the wild-type IgG2. Accordingly, in some embodiments, the constant region has a modified constant region having the sequence shown in SEQ ID NO: 80. In some embodiments, the antibody has a modified constant region having the sequence shown in SEQ ID NO: 81.

There are four main steps in the humanization of monoclonal antibodies. These include: (1) determining the nucleotide and predicted amino acid sequences of the variable regions of the light and heavy chains of the original antibody (2) developing a humanized antibody, i.e. deciding which antibody framework to use in the humanization process (3) practical humanization techniques/methods; and (4) transfection and expression of the humanized antibody. See, for example, US Pat. Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370.

Several humanized antibody molecules are described containing an antigen-binding site derived from a non-human immunoglobulin, including chimeric antibodies having rodent V-regions or modified rodent V-regions and associated CDRs fused to human constant regions. See, for example, Winter et al. Nature 349:293-299, 1991, Lobuglio et al. Proc. Nat. Acad. sci. USA 86:4220-4224, 1989, Shaw et al. J Immunol. 138:4534-4538, 1987, and Brown et al. Cancer Res. 47:3577-3583, 1987. Other references describe rodent CDRs grafted into a human supportive framework region (FR) prior to fusion with a suitable human antibody constant region. See, for example, Riechmann et al. Nature 332:323-327, 1988, Verhoeyen et al. Science 239:1534-1536, 1988, and Jones et al. Nature 321:522-525, 1986. Rodent CDRs supported by recombinantly engineered rodent framework regions are described elsewhere. See, for example, European Publication No. EP0519596. These "humanized" antibodies are designed to minimize the undesirable immunological response to rodent anti-human antibody molecules, which limits the duration and effectiveness of the therapeutic use of these fragments in human recipients. For example, an antibody constant region can be designed to be immunologically inert (eg, not trigger complement lysis). See, for example, PCT Application No. PCT/GB99/01441; UK Patent Application No. 9809951.8. Other methods for humanizing antibodies that can also be used are disclosed in Daugherty et al., Nucl. Acids Res. 19: 2471-2476, 1991, and US Pat. Nos. 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; and 6,350,861; and in PCT Publication No. WO01/27160.

The general principles relating to humanized antibodies discussed above also apply to adapting antibodies for use in, e.g., other mammals (e.g., human, non-human primates, mouse, donkey, sheep, rabbit, goat, guinea pig, camel). , horses or chickens). In addition, one or more aspects of the humanization of an antibody disclosed herein may be combined, such as CDR grafting, framework mutation, and CDR mutation.

In one embodiment, fully human antibodies can be generated using commercially available mice that have been engineered to express human-specific immunoglobulin proteins. Transgenic animals that are designed to produce a more preferable (eg, fully human antibody) or more robust immune response can also be used to generate humanized or human antibodies. Examples of such a method are Xenomouse™ from Abgenix, Inc. (Fremont, CA) and HuMAb-Mouse® and TC Mouse™ from Medarex, Inc. (Princeton, NJ).

Alternatively, antibodies can be generated recombinantly and expressed using any method known in the art. In another alternative, antibodies can be generated recombinantly using phage display techniques. See, for example, US Pat. Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., Annu. Rev. Immunol. 12:433-455, 1994. Alternatively, phage display technology (McCafferty et al., Nature 348:552-553, 1990) can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable (V) gene repertoires from unimmunized donors. In this method, antibody V domain genes are cloned in frame into the major or minor coat protein gene of a filamentous bacteriophage such as M13 or fd and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a copy of the single-stranded DNA of the phage genome, selection based on the functional properties of the antibody also results in the selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of the B cell. The phage display can be made in various formats; for a review, see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571, 1993. Several sources of V gene segments can be used for phage display. Clackson et al., Nature 352:624-628, 1991, isolated a set of different antioxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V-genes derived from non-immunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated essentially following the methods described by Mark et al., J. Mol. Biol. 222:581-597, 1991, or Griffith et al., EMBO J. 12:725-734, 1993. In a natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the changes made will provide higher affinity, and B cells displaying high affinity surface immunoglobulin preferentially replicate and differentiate during subsequent antigen challenge. This natural process can be mimicked using a technique known as "chain shuffling" (Marks et al., Bio/Technol. 10:779-783, 1992). In this method, the affinity of "primary" human antibodies obtained by phage display can be improved by sequentially replacing the genes of the heavy and light chain V regions with repertoires of naturally occurring variants (repertoires) of the V domain genes obtained from non-immunized donors. This method makes it possible to obtain antibodies and antibody fragments with an affinity in the pM-nM range. A strategy for generating very large repertoires of phage antibodies (also known as "mother libraries") is described by Waterhouse et al., Nucl. Acids Res. 21:2265-2266, 1993. Gene shuffling can also be used to generate human antibodies from rodent antibodies, where the human antibody has similar affinity and specificity to the parent rodent antibody. In this method, also called "epitope imprinting", a rodent antibody heavy or light chain V domain gene obtained by phage display is replaced with a repertoire of human V domain genes, creating rodent-human chimeras. Antigen selection results in the isolation of human variable regions capable of restoring a functional antigen-binding site, i.e. the epitope governs (imprints) mate choice. When the process is repeated to replace the remaining rodent V domain, a human antibody is obtained (see PCT Publication No. WO93/06213). In contrast to the traditional humanization of rodent antibodies by CDR transplantation, this method produces fully human antibodies that do not have residues of rodent framework regions or CDRs.

Antibodies can be produced recombinantly by first isolating antibodies and antibody-producing cells from host animals, obtaining a gene sequence, and using that gene sequence to recombinantly express the antibody in host cells (eg, CHO cells). Another method that can be used is the expression of the antibody sequence in plants (eg tobacco) or transgenic milk. Disclosed are methods for recombinant expression of antibodies in plants or milk. See, for example, Peeters, et al. Vaccine 19:2756, 2001; Lonberg, N. and D. Huszar Int. Rev. Immunol 13:65, 1995; and Pollock, et al., J Immunol Methods 231:147, 1999. Methods for derivatizing antibodies, eg, humanized, single chain, etc., are known in the art.

Immunoassays and flow cytometry sorting methods such as fluorescence activated cell sorting (FACS) can also be used to isolate CD3-specific antibodies or antigens of interest.

Antibodies disclosed herein may be associated with many different solid phase supports or carriers. Such carriers may be active and/or inert. Well-known supports include polypropylene, polystyrene, polyethylene, dextran, nylon, amylases, glass, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The support may be inherently soluble or insoluble for the purposes of the invention. Those skilled in the art are aware of other suitable antibody-binding supports or are able to determine them using routine experimentation. In some embodiments, the implementation of the substrate contains a fragment that targets the myocardium.

DNA encoding monoclonal antibodies can be easily isolated and sequenced using conventional procedures (eg, using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of monoclonal antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors (such as the expression vectors disclosed in PCT Publication No. WO87/04462) which are then transfected into host cells such as E. coli cells, monkey COS cells, CHO cells, or myeloma cells, which do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in recombinant host cells. See, for example, PCT Publication No. WO87/04462. The DNA can also be modified, for example by replacing the coding sequence of the human heavy and light chain constant regions in place of homologous mouse sequences, Morrison et al., Proc. Nat. Acad. sci. 81:6851, 1984, or by covalently attaching all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Thus, “chimeric” or “hybrid” antibodies are obtained that have the binding specificity of the monoclonal antibody disclosed in the present description.

Antibodies to CD3 or other antigens disclosed herein can be identified or characterized using methods known in the art that detect and/or measure antigen expression levels. In some embodiments, an anti-CD3 antibody is identified by incubating the candidate agent with CD3 and monitoring binding and/or concomitant reduction in CD3 expression levels. The binding assay can be performed on purified CD3 polypeptide(s) or on cells naturally expressing or transfected to express CD3 polypeptide(s). In one embodiment, the binding assay is a competitive binding assay that evaluates the ability of a candidate antibody to compete with a known anti-CD3 antibody for binding to CD3. The assay can be performed in various formats, including the ELISA format.

After initial identification, the activity of a CD3 candidate or other antigenic antibody can be further confirmed and refined using bioassays known to test biological targets of interest. Alternatively, bioassays can be used to directly select candidates. Some methods for identifying and characterizing antibodies are described in detail in the examples.

CD3 or other antigenic antibodies can be characterized using methods well known in the art. For example, one method is to identify the epitope to which it binds, or to map the epitope. There are several techniques known in the art for mapping and characterizing the location of epitopes on proteins, including crystal structure determination of the antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In a further example, epitope mapping can be used to determine the sequence to which an antibody binds. Epitope mapping is commercially available from various sources such as Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitope may be a linear epitope, i.e. contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids, which may optionally be contained in a single stretch. Peptides of various lengths (eg, at least 4-6 amino acids long) can be isolated or synthesized (eg, recombinantly) and used for anti-CD3 antibody or other antigen binding assays. In another example, an epitope that an anti-CD3 antibody or other antigen binds to can be determined by systematic screening using overlapping peptides derived from a CD3 or other antigen sequence and determining binding to an anti-CD3 antibody or other antigen. According to gene fragment expression assays, an open reading frame encoding CD3 or other antigen is fragmented either randomly or using specific genetic constructs, and the reactivity of the expressed CD3 or other antigen fragments with the test antibody is determined. Gene fragments can, for example, be obtained by PCR and then transcribed and translated into protein in vitro in the presence of radioactive amino acids. Binding of the antibody to radiolabeled CD3 or other antigen fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a specific library of overlapping peptide fragments can be tested for binding to a test antibody in simple binding assays. In a further example, antigen binding domain mutagenesis, domain switch experiments, and alanine scan mutagenesis can be performed to identify residues required, sufficient, and/or necessary for epitope binding. For example, domain swap experiments can be performed using a mutant CD3 or other antigen, wherein various fragments of CD3 or other antigenic protein are replaced (swapped) with sequences from an antigen of a different species (e.g., mouse) or a closely related but antigen-different protein. (for example, Trop-1). By evaluating the binding of an antibody to a mutant CD3 or other antigen, the importance of a particular CD3 or other antigen fragment for antibody binding can be assessed.

Another method that can be used to characterize an antibody to CD3 or another antigen is to use competitive assays with other antibodies known to bind to the same antigen, ie. with different fragments on CD3 or another antigen to determine if an anti-CD3 antibody or an antibody against another antigen binds to the same epitope as other antibodies, respectively. Competitive assays are well known to those skilled in the art.

An expression vector can be used to drive the expression of an anti-CD3 antibody or other antigen. One skilled in the art is familiar with the introduction of expression vectors to obtain expression of an exogenous protein in vivo. See, for example, US Pat. Nos. 6,436,908; 6,413,942; and 6,376,471. Administration of expression vectors includes topical or systemic administration, including injection, oral administration, gunshot or catheterization, and topical administration. In another embodiment, the expression vector is injected directly into the sympathetic trunk or ganglion, or into the coronary artery, atrium, ventricle, or pericardium.

You can also use the targeted delivery of therapeutic compositions containing the expression vector or subgenomic polynucleotides. Methods for receptor-mediated DNA delivery are described, for example, in Findeis et al., Trends Biotechnol., 11:202, 1993; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer, J.A. Wolff, ed., 1994; Wu et al., J. Biol. Chem. 263:621, 1988; Wu et al., J. Biol. Chem. 269:542, 1994; Zenke et al., Proc. Natl. Acad. sci. USA 87:3655, 1990; and Wu et al., J. Biol. Chem., 266:338, 1991. Therapeutic compositions containing a polynucleotide are administered in the range of about 100 ng to about 200 mg of DNA for topical administration according to a gene therapy protocol. Concentration ranges from about 500 ng to about 50 mg, from about 1 µg to about 2 mg, from about 5 µg to about 500 µg, and from about 20 µg to about 100 µg of DNA can also be used according to the gene therapy protocol. Therapeutic polynucleotides and polypeptides can be delivered using gene delivery vehicles. The gene delivery vehicle may be of viral or non-viral origin (for review, see Jolly, Cancer Gene Therapy, 1:51, 1994; Kimura, Human Gene Therapy, 5:845, 1994; Connelly, Human Gene Therapy, 1:185, 1995 ; and Kaplitt, Nature Genetics, 6:148, 1994). Expression of such coding sequences can be induced using endogenous mammalian promoters or heterologous promoters. Expression of a coding sequence may be constitutive or regulated.

Virus-based vectors for delivery of the desired polynucleotide and expression in the desired cell are well known in the art. Examples of virus-based carriers include, without limitation, recombinant retroviruses (see, for example, PCT Publication Nos. WO90/07936; WO94/03622; WO93/25698; WO93/25234; WO93/11230; US Nos. 5,219,740 and 4,777,127; GB Patent No. 2200651 and EP Patent No. EP0345242), alpha virus vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus ( ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)) and adeno-associated viral (AAV) vectors (see, for example, publications PCT Nos. WO94/12649, WO93/03769; WO93/19191; WO94/28938; WO95/11984 and WO95/00655). You can also enter the DNA associated with killed adenovirus, as described in Curiel, Hum. Gene Ther. 3:147, 1992.

Non-viral carriers and delivery methods can also be used, including, but not limited to, polycationic condensed DNA associated or not associated only with killed adenovirus (see, for example, Curiel, Hum. Gene Ther., 3:147, 1992); ligand-linked DNA (see, for example, Wu, J. Biol. Chem., 264:16985, 1989); carrier cells for delivery to eukaryotic cells (see, for example, US Patent No. 5,814,482; PCT Publication Nos. WO95/07994; WO96/17072; WO95/30763; and WO97/42338) and nucleic acid charge neutralization or fusion with cell membranes. Naked DNA can also be used. Exemplary methods for introducing naked DNA are described in PCT Publication No. WO90/11092 and US Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in US Pat. No. 5,422,120; PCT Publication No. WO95/13796; WO94/23697; WO91/14445; and European publication EP 0524968. Additional approaches are described in Philip, Mol. Cell Biol., 14:2411, 1994 and Woffendin, Proc. Natl. Acad. Sci. 91:1581, 1994.

In some embodiments, the invention includes compositions, including pharmaceutical compositions, containing the antibodies of the invention disclosed herein or obtained by methods and having the characteristics disclosed herein. In the context of the present description, pharmaceutical compositions may contain one or more antibodies that bind to a tumor antigen, one or more bispecific antibodies that bind to CD3 and a tumor antigen, and/or one or more polynucleotides containing sequences encoding one or more of these antibodies. . These compositions may additionally contain suitable excipients, such as pharmaceutically acceptable excipients, including buffers, which are well known in the art.

In one embodiment, the present invention relates to a composition that is a combination of an anti-CD3 antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is any agent that is advantageously combined with an anti-CD3 antibody. Examples of agents that can be advantageously combined with an anti-CD3 antibody include, without limitation, other agents that bind and/or activate CD3 signaling (including other antibodies or antigen-binding fragments thereof, etc.) and/or agents which do not bind directly to CD3, but nevertheless activate or stimulate the activation of immune cells. The present invention further includes additional combination therapies and co-pharmaceutical formulations comprising the anti-CD3 antibodies of the present invention. The invention also relates to methods for producing any of these antibodies. The antibodies of the present invention can be produced by methods known in the art, including de novo protein synthesis and recombinant expression of nucleic acids encoding binding proteins. Desired nucleic acid sequences can be obtained by recombinant methods (eg, PCR mutagenesis of a previously obtained variant of the desired polynucleotide) or solid phase DNA synthesis. Recombinant expression methods are commonly used. In one aspect, the invention relates to a polynucleotide that contains a sequence encoding VH and/or VL CD3. Due to the degeneracy of the genetic code, multiple nucleic acid sequences encode the same immunoglobulin amino acid sequence, and the present invention includes all nucleic acids encoding the binding proteins disclosed herein.

Chimeric or hybrid antibodies can also be produced in vitro using known methods of chemical protein synthesis, including methods involving cross-linking agents. For example, immunotoxins can be constructed through a disulfide exchange reaction or through the formation of a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl 4-mercaptobutyrimidate.

In recombinant humanized antibodies, the Fcγ portion can be modified to avoid interaction with the Fcγ receptor, the complement system, and the immune system. Methods for making such antibodies are described in PCT Publication WO99/58572. For example, the constant region can be designed to more closely resemble human constant regions in order to avoid triggering an immune response when the antibody is used in clinical trials and in human therapy. See, for example, U.S. Patent Nos. 5,997,867 and 5,866,692.

The CD3 antibodies disclosed herein may contain one or more amino acid substitutions, insertions and/or deletions in the framework and/or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be readily identified by comparing the amino acid sequences disclosed herein with germline sequences available, for example, from publicly available antibody sequence databases. The present invention includes antibodies and antigen-binding fragments thereof that are derived from any amino acid sequence disclosed herein, wherein one or more amino acids in one or more framework regions and/or CDR regions are mutated to the corresponding residue(s) of the germline sequence from which an antibody has been generated, either against the corresponding residue(s) of another human germline sequence, or by conservative amino acid substitution of the corresponding germline residue(s) (such sequence substitutions are collectively referred to herein as "germline mutations"). One of skill in the art can readily generate numerous antibodies and antigen-binding fragments that contain one or more individual germline mutations, or combinations thereof, from the heavy and light chain variable region sequences disclosed herein. In some embodiments, all framework and/or CDR residues in the VH and/or VL domains are mutated back to residues occurring in the original germline sequence from which the antibody is derived. In some embodiments, only certain residues are mutated back to the original germline sequence, for example, only mutated residues contained within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only mutated residues contained within CDR1, CDR2, or CDR3. In some additional embodiments, one or more framework and/or CDR residues are mutated to the corresponding residue(s) of a different germline sequence (i.e., a germline sequence that differs from the germline sequence from which the antibody was originally derived).

In addition, the antibodies of the present invention may contain any combination of two or more germline mutations within the framework region and/or CDR regions, for example, in which certain individual residues are mutated to the corresponding residue of a particular germline sequence, while some others residues that differ from the original germline sequence are retained or mutated to the corresponding residue of a different germline sequence. Once generated, antibodies and antigen-binding fragments that contain one or more germline mutations can be readily tested for one or more of the desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (depending on circumstances), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained by such a general method are covered by the present invention.

The present invention also includes anti-CD3 antibodies containing variants of any of the HC VR, LC VR and/or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present invention includes anti-CD3 antibodies having the amino acid sequences HC VR, LC VR and/or CDRs containing, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions relative to any of the HC VR, LC VR and/or CDR amino acid sequences disclosed herein.

Accordingly, the invention encompasses modifications of the antibodies and polypeptides of the embodiments of the invention disclosed herein, including functionally equivalent antibodies that do not significantly affect their properties, and variants that have increased or decreased activity and/or affinity. For example, the amino acid sequence can be mutated to produce an antibody with the desired binding affinity for the tumor antigen and/or CD3. Modification of polypeptides is a common practice in this field and does not require detailed description. Examples of modified polypeptides include polypeptides with conservative amino acid residue substitutions, one or more deletions or additions of amino acids that do not result in a significant change in functional activity, or that allow maturation (increase) in the affinity of the polypeptide for its ligand, or the use of chemical analogues.

Amino acid sequence insertions include amino-terminal and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as insertions of one or more amino acid residues within the sequence. Examples of end inserts include an antibody with an N-terminal methionyl residue or an antibody fused to an epitope tag. Other insertion variants of the antibody molecule include the fusion of the N- or C-terminus of the antibody with an enzyme or polypeptide that can increase the circulating half-life of the antibody.

Substitution variants have at least one amino acid residue removed from the antibody molecule and another residue inserted in its place. Regions of greatest interest for displacement mutagenesis include hypervariable regions, but changes in FR are also considered. Conservative substitutions are shown in Table 6 in the column headed "conservative substitutions". If such substitutions result in a change in biological activity, then more significant changes, referred to in Table 6 as "illustrative substitutions" or further described below with reference to amino acid classes, can be made and the products can be screened.

Table 6

original balance
(naturally occurring amino acid) Conservative substitutions Illustrative substitutions Ala (A) Val Val; Leu; ile Arg(R) Lys Lys; gln; Asn Asn(N) Gln gln; His; Asp, Lys; Arg Asp(D) Glu Glu; Asn Cys(C) Ser Ser; Ala Gln(Q) Asn asn; Glu Glu(E) asp asp; Gln Gly (G) Ala Ala His(H) Arg asn; gln; Lys; Arg Ile (I) Leu Leu; Val; met; Ala; Ph; Norleucine Leu(L) ile norleucine; Ile; Val; met; Ala; Phe Lys (K) Arg Arg; gln; Asn Met(M) Leu Leu; Ph; ile Phe(F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser TRP(W) Tyr Tyr; Phe Tyr (Y) Phe trp; Ph; Thr; Ser Val(V) Leu Ile; Leu; met; Ph; Ala; norleucine

Substantial modifications to the biological properties of an antibody are achieved by selecting substitutions that differ greatly in their effect on maintaining (a) the structure of the polypeptide backbone at the site of the substitution, e.g., in a sheet or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain. Naturally occurring amino acid residues are divided into groups based on common side chain properties:

(1) non-polar: norleucine, Met, Ala, Val, Leu, Ile;

(2) polar without charge: Cys, Ser, Thr, Asn, Gln;

(3) acidic (negatively charged): Asp, Glu;

(4) basic (positively charged): Lys, Arg;

(5) residues that affect chain orientation: Gly, Pro; and

(6) aromatic: Trp, Tyr, Pheb His.

Non-conservative substitutions are made by replacing a member of one of these classes with a member from another class.

Any cysteine residue not involved in maintaining the correct conformation of the antibody can also be replaced, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to an antibody to improve its stability, especially when the antibody is an antibody fragment, such as an Fv fragment.

The number of amino acid modifications can range from changing or modifying one or more amino acids to completely rearranging a region, such as a variable region. Changes in the variable region can change the binding affinity and/or specificity. In some embodiments, no more than one to five conservative amino acid substitutions are made in the CDR domain. In some embodiments, no more than one to three conservative amino acid substitutions are made in the CDR domain. In other embodiments, the CDR domain is a VH CDR3 and/or a VL CDR3.

Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications such as, for example, glycosylation with various sugars, acetylation, and phosphorylation. Antibodies are glycosylated at conserved positions in constant regions (Jefferis and Lund, Chem. Immunol. 65:111-128, 1997; Wright and Morrison, TibTECH 15:26-32, 1997). The oligosaccharide side chains of immunoglobulins influence protein function (Boyd et al., Mol. Immunol. 32:1311-1318, 1996; Wittwe and Howard, Biochem. conformation and are represented by the three-dimensional surface of the glycoprotein (Jefferis and Lund, supra; Wyss and Wagner, Current Opin. Biotech. 7:409-416, 1996). Oligosaccharides can also serve to target a given glycoprotein to specific molecules based on specific recognition patterns. There are also reports that antibody glycosylation affects antibody-dependent cellular cytotoxicity (ADCC). In particular, there are reports that CHO cells with tetracycline-regulated expression of (1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyses the formation of bisector GlcNAc, have improved ADCC activity (Umana et al., Mature Biotech. 17 :176-180, 1999).

Glycosylation of antibodies is usually either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine and asparagine-X-cysteine, where X is any amino acid other than proline, are recognition sequences for enzymatic attachment of a carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the addition of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to an antibody is typically accomplished by changing the amino acid sequence to contain one or more of the tripeptide sequences described above (for N-linked glycosylation sites). The change can also be made by adding or replacing one or more serine or threonine residues in the sequence of the original antibody (for O-linked glycosylation sites).

The glycosylation pattern of antibodies can also be changed without changing the underlying nucleotide sequence. Glycosylation is highly dependent on the host cell used to express the antibody. Since the cell type used to express recombinant glycoproteins, such as antibodies, as potential therapeutic agents is rarely native cell, a change in the glycosylation pattern of an antibody can be expected (see, for example, Hse et al., J. Biol. Chem. 272:9062 -9070, 1997). In addition to host cell selection, factors that influence glycosylation during recombinant antibody production include growth mode, medium composition, culture density, oxygenation, pH, purification schemes, and the like. Various methods have been proposed to alter the pattern of glycosylation achieved in a particular host organism, including the introduction or overexpression of certain enzymes involved in the production of oligosaccharides (US Pat. Nos. 5,047,335; 5,510,261 and 5,278,299). Glycosylation or certain types of glycosylation can be removed from the glycoprotein enzymatically, for example, using endoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. In addition, the recombinant host cell may be genetically engineered to be incapable of processing certain types of polysaccharides. These and similar methods are well known in the art.

Other modification methods include the use of coupling methods known in the art, including, without limitation, enzymatic agents, oxidative displacement, and chelation. Modifications can be used, for example, to attach labels for immunoassays. Modified polypeptides are prepared using art-established procedures and can be screened using standard assays known in the art, some of which are described below and in the examples.

In some embodiments, the antibody contains a modified constant region, such as a constant region that has increased affinity for the human Fc gamma receptor, is immunologically inert or partially inert, e.g., does not trigger complement-mediated lysis, does not stimulate antibody-dependent cellular cytotoxicity (ADCC) or does not activate macrophages; or has reduced activity (compared to unmodified antibody) in one or more of the following: triggering complement-mediated lysis, stimulation of antibody-dependent cellular cytotoxicity (ADCC), or microglial activation. To achieve the optimal level and/or combination of effector functions, various modifications of the constant region can be used. See, for example, Morgan et al., Immunology 86:319-324, 1995; Lund et al., J. Immunology 157:4963-9 157:4963-4969, 1996; Idusogie et al., J. Immunology 164:4178-4184, 2000; Tao et al., J. Immunology 143:2595-2601, 1989; and Jefferis et al., Immunological Reviews 163:59-76, 1998. In some embodiments, the constant region is modified as described by Eur. J. Immunol., 29:2613-2624, 1999; in PCT Application No. PCT/GB99/01441; and/or UK Patent Application No. 9809951.8. In other embodiments, the constant region is aglycosylated for N-linked glycosylation. In some embodiments, the constant region is aglycosylated for N-linked glycosylation by mutating a glycosylated amino acid residue or flanking residues that are part of the N-glycosylation recognition sequence in the constant region. For example, the N-glycosylation site N297 can be mutated to A, Q, K, or H. See, Tao et al., J. Immunology 143:2595-2601, 1989; and Jefferis et al., Immunological Reviews 163:59-76, 1998. In some embodiments, the constant region is aglycosylated for N-linked glycosylation. The constant region can be aglycosylated for N-linked glycosylation enzymatically (eg, by removal of carbohydrates by the PNGase enzyme) or by expression in a glycosylation-deficient host cell.

Other antibody modifications include antibodies that are modified as described in PCT Publication No. WO99/58572. These antibodies contain, in addition to a binding domain directed to the target molecule, an effector domain having an amino acid sequence substantially homologous to all or part of the human immunoglobulin heavy chain constant region. These antibodies are able to bind the target molecule without triggering significant complement-dependent lysis or cell-mediated destruction of the target. In some embodiments, the effector domain is capable of specifically binding to FcRn and/or FcγRIIb. They are typically based on chimeric domains derived from two or more human immunoglobulin heavy chain CH2 domains. Antibodies thus modified are particularly suitable for long-term antibody therapy, avoiding the inflammatory and other adverse reactions associated with conventional antibody therapy.

The invention includes affinity matured embodiments. For example, affinity matured antibodies can be prepared using procedures known in the art (Marks et al., Bio/Technology, 10:779-783, 1992; Barbas et al., Proc Nat. Acad. Sci, USA 91:3809 -3813, 1994; Schier et al., Gene, 169:147-155, 1995; Yelton et al., J. Immunol., 155:1994-2004, 1995; Jackson et al., J. Immunol., 154 ( 7):3310-9, 1995, Hawkins et al., J. Mol. Biol., 226:889-896, 1992; and PCT Publication No. WO2004/058184).

The methods described below can be used to adjust the affinity of an antibody and to characterize CDRs. One method of characterizing the CDR of an antibody and/or altering (eg, improving) the binding affinity of a polypeptide, such as an antibody, is referred to as "library scanning mutagenesis". Typically, library-scanning mutagenesis works as follows. One or more amino acid positions in the CDR are replaced by two or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids using methods recognized in the art. Thus, small libraries of clones are formed (in some embodiments, one for each amino acid position analyzed), each with a complexity of two or more members (if two or more amino acids are changed at each position). Typically, the library also includes a clone containing the native (unsubstituted) amino acid. A small number of clones, e.g., about 20-80 clones (depending on the complexity of the library), from each library are screened for binding affinity for the target polypeptide (or other binding target), and candidates with increased, equal, decreased binding, or no are identified. with binding ability. Methods for determining binding affinity are well known in the art. Binding affinity can be determined using the BIACORE™ Surface Plasmon Resonance assay, which detects 2-fold or more differences in binding affinity approximately. BIACORE™ is particularly useful when the parent antibody already binds with a relatively high affinity, such as a K _D of about 10 nM or less. Screening using surface plasmon resonance BIACORE™ disclosed in the present description in the examples.

Binding affinity can be determined using the Kinexa biosensor, proximity scintillation assay, ELISA, ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, and/or yeast display. Binding affinity screening is also possible with a suitable bioassay.

In some embodiments, each amino acid position in the CDR is replaced (in some embodiments, one at a time) with all 20 naturally occurring amino acids using art-recognized mutagenesis techniques (some of which are disclosed herein). This generates small libraries of clones (in some embodiments, one is analyzed for each amino acid position), each with a complexity of 20 members (if all 20 amino acids are changed at each position).

In some embodiments, the library to be screened contains substitutions at two or more positions, which may be in the same CDR or in two or more CDRs. Thus, a library may contain substitutions at two or more positions in a single CDR. A library may contain a substitution at two or more positions in two or more CDRs. The library may contain substitutions in 3, 4, 5 or more positions, these positions being in two, three, four, five or six CDRs. The substitution can be made using codons with low redundancy. See, for example, Table 2 in Balint et al., Gene 137(1):109-18, 1993.

Candidates with improved binding can be sequenced, thereby identifying a mutant with a CDR substitution that provides improved affinity (also referred to as an "improved" substitution). Candidates that bind can also be sequenced, thereby identifying a CDR substitution that retains binding.

Several rounds of screening are possible. For example, candidates (each containing an amino acid substitution at one or more positions of one or more CDRs) with improved binding are also useful for generating a second library containing at least the original and substituted amino acid at each improved CDR position (i.e., amino acid position in the CDR in which the substitution mutant showed improved binding). The preparation, screening, or selection of this library is discussed below.

Library scanning mutagenesis also provides a means to characterize CDRs, as the frequency of clones with improved binding, same binding, reduced binding, or no binding also provides information regarding the importance of each amino acid position to the stability of the antibody-antigen complex. For example, if a CDR position retains binding in the event of a change in all 20 amino acids, that position is identified as a position that is unlikely to be required for antigen binding. Conversely, if a CDR position retains binding in only a small percentage of substitutions, that position is identified as a position that is important to CDR function. Thus, library scanning mutagenesis methods provide information about positions in the CDR that can be replaced by multiple amino acids (including all 20 amino acids) and about positions in the CDR that cannot be replaced or that can only be replaced by a few amino acids.

Affinity improved candidates can be combined into a second library that includes the improved amino acid, the original amino acid at that position, and may optionally include additional substitutions at that position, depending on the complexity of the library, which is preferred or acceptable for the desired screening or selection method to be used. In addition, if necessary, the position of an adjacent amino acid may be randomized for at least two or more amino acids. Randomization of adjacent amino acids may provide additional conformational flexibility to the mutant CDR, which in turn may permit or facilitate the introduction of more enhancing mutations. The library may also contain substitutions at positions that did not show improved affinity in the first round of screening.

The second library is screened or selected for library members with improved and/or altered binding affinity using any method known in the art, including screening with a BIACORE™ Surface Plasmon Resonance assay and selection using any method known in the art for selection, including phage display, yeast display, and ribosome display.

This is followed by methods for isolating antibodies and obtaining antibodies. However, it should be appreciated that antibodies may be formed from other polypeptides or other polypeptides may be incorporated into them using methods known in the art. For example, a nucleic acid encoding a polypeptide of interest (eg, a ligand, receptor, or enzyme) can be isolated from a cDNA library derived from tissue that is expected to contain and express the mRNA of the polypeptide at a detectable level. Libraries are checked with probes (such as antibodies or oligonucleotides of about 20-80 bases) designed to identify the gene of interest or the protein it encodes. Screening of a cDNA or genomic library with a chosen probe can be performed using the standard procedures described in chapters 10-12 of Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989).

Anti-CD3 antibodies for the treatment of an autoimmune disorder

In one aspect, a method for treating an autoimmune disorder in a subject is provided, comprising administering to a subject in need thereof an effective amount of a composition comprising the antibodies of the present invention.

As used herein, autoimmune disorders include, but are not limited to, inflammatory bowel disease, systemic lupus erythematosus, rheumatoid arthritis, diabetes (type I), multiple sclerosis, Addison's disease, celiac disease, dermatomyositis, Graves' disease, Hashimoto's thyroiditis, Hashimoto's encephalopathy, myasthenia gravis, reactive артрит, синдром Шегрена, атопическую аллергию, атопический дерматит, аутоиммунную энтеропатию, аутоиммунный гепатит, аутоиммунный лимфопролиферативный синдром, аутоиммунную периферическую нейропатию, аутоиммунный панкреатит, аутоиммунный полиэндемический синдром, аутоиммунный прогестероновый дерматит, аутоиммунную крапивницу, аутоиммунный увеит, болезнь Беше, болезнь Кастлемана, болезнь холодовых agglutinins, Crohn's disease, dermatomyositis, eosinophilic fasciitis, gastrointestinal pemphigoid, Goodpasture's syndrome, Guillain-Barré syndrome, suppurative hidradenitis, narcolepsy, pemphigus vulgaris, polymyositis, relapsing polychondritis, rheumatic fever, poper chronic myelitis, ulcerative colitis, undifferentiated connective tissue disease, vasculitis, and Wegener's granulomatosis.

Polyspecific antibodies and their uses

The anti-CD3 antibodies of the present invention may be specific for different epitopes of a single target polypeptide, or may contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244. The anti-CD3 antibodies of the present invention may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, an antibody or antibody fragment can be operably linked (e.g., by chemical linkage, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce polyspecific antibodies with a second binding specificity. .

In one aspect, the antibody of the present invention is a polyspecific antibody. In a specific embodiment, the anti-CD3 antibody is a bispecific antibody that specifically binds to human CD3. In some such embodiments, the bispecific antibody further specifically binds to a tumor antigen. In another such embodiment, the bispecific antibodies of the present invention target both T cells (CD3) and tumor cells and successfully direct and activate T cell cytotoxicity against tumor cells expressing a tumor antigen.

In additional embodiments of each of the above aspects, the CD3-binding bispecific antibody is characterized by one or more of the following: (a) treating, preventing, alleviating one or more symptoms of a condition associated with cancer cells expressing a specific tumor antigen in a subject (eg, B-cell associated cancer such as multiple myeloma); (b) suppression of tumor growth or progression in a subject (having a cancer expressing a specific tumor antigen); (c) inhibition of metastasis of cancerous (malignant) cells expressing a specific tumor antigen in a subject (having one or more malignant cells expressing a tumor antigen); (d) inducing regression (eg, long-term regression) of the tumor expressing the tumor antigen; (e) the manifestation of cytotoxic activity in malignant cells expressing the tumor antigen; (f) increasing the survival of a subject with a tumor-associated disorder without disease progression; (g) increasing the overall survival of a subject with a tumor-associated disorder; (h) reducing the use of additional chemotherapeutic or cytotoxic agents in a subject with a tumor associated disorder; (i) reducing tumor burden in a subject with a tumor associated disorder; or (j) blocking the interaction of the tumor antigen with other as yet unidentified factors.

In the context of the present description, a bispecific antibody of the present invention refers to a complex consisting of two or more polypeptide chains, each of which contains at least one VL region of an antibody and one VH region of an antibody, or a fragment thereof, and the VL and VH regions in each polypeptide chain obtained from different antibodies. In specific aspects, the bispecific antibody includes dimers or tetramers of polypeptide chains containing both a VL region and a VH region. Individual polypeptide chains containing multimeric proteins can be covalently linked to at least one other multimeric peptide via interchain disulfide bonds.

In some such embodiments, the bispecific antibodies of the present invention comprise a first heterodimer providing domain on the first polypeptide chain and a second heterodimer providing domain on the second polypeptide chain (FIG. 1). Together, the first and second heterodimer-providing domains drive heterodimerization and/or stabilize the bispecific antibody (e.g., via ridge-to-trough interaction on the heterodimer-providing domains of the complementary strand) and/or serve to stabilize the bispecific antibody. In some embodiments, both the first heterodimer providing domain and the second heterodimer providing domain comprise a CH2 domain and a CH3 domain, wherein the amino acid sequence of each of the CH2 domains and/or each of the CH3 domains is modified to control heterodimerization and/or stabilization of the bispecific antibodies.

In some embodiments, the first heterodimer-providing domain may comprise an Fc chain having a CH2 and/or CH3 domain modified to contain either a ridge (bulge) or a pit (cavity). In some such embodiments, the amino acid sequence of the CH2 domain and/or CH3 domain comprises at least one amino acid modification in which: (a) the CH3 domain of the first domain providing the heterodimer forms an overhang; and (b) the CH3 domain of the second domain providing the heterodimer forms a pit. In another such embodiment, the CH3 domain of the first heterodimer-providing domain contains the Y349C and/or T366W mutations; and the CH3 domain of the second domain providing the heterodimer contains mutations S354C, T366S, L368A and/or Y407V (numbering according to the EU index).

In one embodiment, the first heterodimer-providing domain may contain a CH2 and/or CH3 domain modified to contain a bulge containing the sequence of SEQ ID NO: 78 if the second heterodimer-providing domain contains a CH2 domain and/or CH3 modified to contain a cavity. In another embodiment, the first heterodimer-providing domain may contain a cavity (cavity) containing the sequence of SEQ ID NO: 79 if the second heterodimer-providing domain contains a CH2 and/or CH3 domain modified to contain a bulge (bulge ).

Each polypeptide chain of a bispecific antibody includes a VL region and a VH region that can be covalently linked by a glycine-serine linker containing glycine and serine residues (linker 1 or linker 2) that prevents self-assembly of the antibody domains. In addition, each polypeptide chain includes a heterodimerization domain that promotes heterodimerization and/or stabilization of multiple polypeptide chains and reduces the likelihood of homodimerization of different polypeptide chains. The heterodimerization domain may be located at the N-terminus of the polypeptide chain or at the C-terminus. The heterodimerization domain may contain a cysteine linker (linker 3) that is 1, 2, 3, 4, 5, 6 or more amino acid residues in length. The interaction of two polypeptide chains can lead to VL/VH pairing to form two epitope-binding domains, i.e. divalent molecule. Neither the VH region nor the VL region is limited to any position in the polypeptide chain, i.e. are not limited by the amino or carboxy terminus, nor are any of these regions restricted by their position relative to other regions, i.e. the VL region may be N-terminal to the VH region and vice versa. The only limitation is the availability of the complementary polypeptide chain for the formation of a functional bispecific antibody. If the VL and VH regions are derived from antibodies specific for different antigens, the formation of a functional bispecific antibody requires the interaction of two different polypeptide chains, i.e. formation of a heterodimer. In contrast, if two different polypeptide chains can interact freely, for example, in a recombinant expression system in which one chain contains VLA and VHB (A is the first epitope and B is the second epitope) and the other chain contains VLB and VHB, then two different binding sites can form: VLA-VHA and VLB-VHB. Any of the polypeptide chain pairs of the bispecific antibodies may have a misalignment or misalignment of the two chains, such as an interaction between the VL-VL or VH-VH regions. However, the purification of functional bispecific antibodies based on the immunospecificity of a correctly dimerized binding site can easily be selected using any affinity-based method known in the art or exemplary herein, for example, affinity chromatography.

In one embodiment, the polypeptide chains of the bispecific antibody may contain various linkers and peptides. Linkers and peptides may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or more amino acids.

In some embodiments, domain 1 is covalently linked to the first heterodimer-providing domain via a cysteine linker, and domain 2 is covalently linked to the second heterodimer-providing domain via a cysteine linker. Each cysteine linker includes at least one cysteine residue, allowing the formation of intramolecular disulfide bonds. In additional embodiments of each of the above aspects, the cysteine linker (linker 3) contains at least five amino acids.

In some embodiments, the first polypeptide chain is covalently linked to the second polypeptide chain by at least one disulfide bond. In some such embodiments, at least one disulfide bond is formed between linker 3 of the first polypeptide chain and linker 3 of the second polypeptide chain. In another such embodiment, at least one disulfide bond is formed between the first heterodimer-providing domain and the second heterodimer-providing domain. In specific embodiments, each disulfide bond is formed by two cysteine residues. In one aspect of the present invention, a bispecific antibody, as shown in FIG. 1 contains a first polypeptide chain and a second polypeptide chain. In some such embodiments, linker 3 may comprise a truncated human IgG1 lower hinge having the sequence of SEQ ID NO: 64 with at least one glycine residue preceding the lower hinge.

The bispecific antibodies of the invention can simultaneously bind to two separate and distinct epitopes. In some embodiments, at least one epitope binding site is specific for a CD3 determinant expressed on an immune effector cell, eg, expressed on T lymphocytes. In one embodiment, the bispecific antibody molecule binds to an effector cell determinant and also activates the effector cell.

In one aspect of the invention, a first polypeptide chain and a second polypeptide chain are provided. In one embodiment, the first polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 36. In another embodiment, the second polypeptide chain contains the amino acid sequence shown in one or more of SEQ ID NO: 37 or 38 (Table 7). In a preferred embodiment, the first polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 36; and the second polypeptide chain contains the amino acid sequence shown in SEQ ID NO: 37 or 38. In one embodiment, the second polypeptide chain is any polypeptide capable of association with the first polypeptide chain through the contact surface. Table 7 shows the sequences of the first polypeptide chain and the second polypeptide chain for the bispecific antibodies GUCY2c-H2B4 and GUCY2c-2B5.

Table 7

Bispecific antibody First polypeptide chain Second polypeptide chain GUCY2c-H2B4 DIQLTQSPSSLSASVGDRVTITCRASESVDYYGTSLMQWYQQKPGKPPKLLIYAASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTRKVYTFGQGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNRARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(SEQ IN NO: 36) DIVMTQSPDSLAVSLGERATINCKSSQSLFNVRSRKNYLAWYQQKPGQPPKLLISWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYDLFTFGSGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEIKPSNGLTNYIEKFKNRFTISVDKAKNSAYLQMNSLRAEDTAVYYCTRTITTTEGYWFFDVWGQGTLVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(SEQ IN NO: 37) GUCY2c-2B5 DIQLTQSPSSLSASVGDRVTITCRASESVDYYGTSLMQWYQQKPGKPPKLLIYAASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTRKVYTFGQGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNRARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(SEQ ID NO: 36) DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSRKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWMHWVRQAPGKGLEWIGEIKPSNGLTNYIEKFKNRFTISVDKAKNSAYLQMNSLRAEDTAVYYCTRTITTTEGYWFFDVWGQGTLVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(SEQ ID NO: 38)

In specific embodiments, the bispecific antibody of the present invention (a) binds to the extracellular domain of a human tumor antigen; (b) demonstrates increased serum and tumor half-life from 30 minutes to 100 days; and/or (c) exhibits a lower EC ₅₀ value of 0.0001 to 100 nM in the presence of increased levels of tumor expression or increased levels of receptor density.

In one embodiment, the epitope-binding domain is capable of binding to a tumor-associated antigen associated with breast, ovarian, thyroid, prostate, cervical, lung (including, but not limited to, non-small cell lung cancer and small cell lung cancer), urinary bladder, endometrium, head and neck, testis, glioblastoma and cancer of the digestive system. Cancer of the digestive system includes, without limitation, cancer of the esophagus, stomach, small intestine, colon, rectum, anus, liver, gallbladder, appendix, bile duct, and pancreas. In specific embodiments, the implementation of therapy activates a cytolytic T-cell response.

Null effector function mutations in CH2-CH3 of human IgG1

The human IgG1 Fc chain is modified by introducing mutations L234A, L235A and G237A (SEQ ID NO: 82, numbered according to the EU index) using standard site-directed mutagenesis by PCR using primers carrying the mutation to reduce effector function by binding to FcγRIII, providing a null effector function phenotype (Canfield et al., J. Exp. Med (1991) 173:1483-1491; Shields et al., J. Biol. Chem. (2001) 276: 6591-604) .

Lump-in-trough mutations in CH2-CH3 of human IgG1

"Help-to-trough" is an efficient design strategy known in the art for designing antibody heavy chain homodimers for heterodimerization. In this approach, the 'overhang' variant was generated by replacing a small amino acid with a larger one in one of the IgG1 Fc chains, eg Y349C and T366W (EU numbering). The "ledge" was chosen to fit into a "trough" in the CH3 domain of the complementary Fc chain, created by replacing a large residue with a smaller one, for example, S354C, T366S, L368A and Y407V (numbering according to the EU index).

In some embodiments, complementary mutations are introduced to heterodimerize the resulting Fc strands such that each Fc strand carries one set of mutations, Y349C and T366W for the overhang (or bulge) in the Fc strand (SEQ ID NO: 78), or S354C, T366S, L368A and Y407V for a recessed (or hollow) Fc chain (SEQ ID NO: 79) as shown in Table 8. When co-transfected into a suitable mammalian host, DNA encoding amino acid sequences, e.g., SEQ ID NO: 78 and 79, produces an Fc domain, which is predominantly a bispecific antibody having a single ridge (or bulge) Fc chain linked to a single pit (or cavity) Fc chain.

Table 8

Fc chain with a "ledge" mutation
(SEQ ID NO: 78) APEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVHYTVXVMQHE Fc chain with "trough" mutation
(SEQ ID NO: 79) APEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQLSGNFSCS

The anti-CD3 tumor antigen bispecific antibodies of the present invention are aggregation stable as measured by thermostability studies and are effective bispecific antibody-Fc fusion proteins targeting both human CD3 and tumor antigen. The Fc domain with the tang-to-trough mutation provides improved expression in CHO cells and improved purification, resulting in high purity of the desired heterodimer. Mutations engineered in the Fc domain abolish FcR binding, thus avoiding ADCC-mediated T cell depletion. In addition, the inclusion of an Fc domain in a bispecific antibody enhances the stability of the molecule, as shown by differential scanning calorimetry (DSC).

A bispecific antibody can be designed to contain at least one cysteine residue that can interact with a similar cysteine residue on another polypeptide chain of the invention to form an interchain disulfide bond. Interchain disulfide bonds can serve to stabilize the bispecific antibody, improve expression and recovery in recombinant systems, which provides a stable and constant composition, and also improves the stability of the isolated and/or purified product in vivo. The cysteine residue or residues can be introduced as a single amino acid or as part of a larger amino acid sequence, such as the hinge region, at any part of the polypeptide chain. In a specific aspect, at least one cysteine residue is introduced that is located at the C-terminus of the polypeptide chain.

The invention includes methods and compositions for treating, preventing, or treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of an antibody constructed in accordance with the invention, the molecule of which further binds to a cancer antigen. The antibodies of the present invention may be particularly useful in preventing, inhibiting, reducing the growth and/or regression of primary tumors and cancer cell metastasis. Without being limited to any particular mechanism of action, the antibodies of the invention may mediate an effector function that may result in tumor disappearance, tumor reduction, or a combination thereof.

In one aspect, the present invention relates to methods of therapeutic treatment aimed at stimulating T cell activation with an anti-CD3 antibody of the invention, the therapeutic methods comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition containing an antibody of the invention. A treatable disorder is any disease or condition that is relieved, alleviated, suppressed, or prevented by stimulation of CD3 activity or signaling. In specific embodiments, the present invention relates to bispecific antigen-binding molecules, eg, bispecific antibodies, that bind to CD3 and a target antigen.

In one aspect, the invention relates to methods for suppressing tumor growth, comprising contacting the tumor with an effective amount of an antibody that binds to CD3, including each of the antibodies disclosed herein.

In another aspect, the invention relates to a method for suppressing tumor growth in a subject, comprising administering to the subject a therapeutically effective amount of an antibody that binds to CD3, including any antibody disclosed herein.

In another aspect, the invention relates to a method for modulating angiogenesis in a subject, comprising administering to the subject a therapeutically effective amount of an antibody that binds CD3, including any antibody disclosed herein.

In another aspect, the invention relates to a method for reducing the tumorigenicity of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of an antibody that binds CD3, including any antibody disclosed herein.

In one aspect, the invention relates to a method of treating a condition associated with tumor antigen expression in a subject by constructing a bispecific antibody that immunospecifically recognizes tumor antigen and CD3 antigen on T cells. The bispecific antibodies which are constructed in accordance with the invention are useful in the prevention or treatment of cancer because they have cytotoxic activity against anti-CD3 antibody induced activated killer T cells.

In a specific embodiment, the present invention relates to a method of treating cancer. In specific embodiments, the cancer is breast, ovarian, thyroid, prostate, cervical, lung (including but not limited to non-small cell lung cancer and small cell lung cancer), bladder, endometrial, head and neck, testicular, glioblastoma or cancer of the digestive system. In some embodiments, the digestive system cancer is selected from the group consisting of the esophagus, stomach, small intestine, colon, rectum, anus, liver, gallbladder, appendix, bile ducts, and pancreas.

In one aspect, the present invention relates to an antibody, bispecific antibody or pharmaceutical composition, as disclosed in the present description, for use in therapy. In a specific embodiment, the invention also provides an anti-CD3 bispecific antibody for use in the method of treating cancer as defined herein. In specific embodiments, the implementation of therapy provides activation of the cytolytic T-cell response.

The present invention also relates to an antibody or bispecific antibody as disclosed herein for use in the manufacture of a medicament for use in therapy. In some embodiments, the therapy is a cancer treatment. In some embodiments, the cancer is selected from the group consisting of breast, ovarian, thyroid, prostate, cervix, lung (including, but not limited to, non-small cell lung cancer and small cell lung cancer), bladder, endometrial, head and neck, testicular, glioblastoma and cancer of the digestive system. In some embodiments, the digestive system cancer is selected from the group consisting of the esophagus, stomach, small intestine, colon, rectum, anus, liver, gallbladder, appendix, bile ducts, and pancreas.

In one aspect, the present invention relates to a polynucleotide that encodes an antibody or bispecific antibody disclosed in the present description. In another embodiment, the invention relates to a vector containing the polynucleotides disclosed in the present description. In yet another embodiment, the invention relates to a host cell containing the vectors disclosed in the present description. In some such embodiments, the host cell recombinantly produces an antibody or a bispecific antibody disclosed herein. In specific embodiments, the host cell is selected from the group consisting of bacterial cell lines, mammalian cell lines, insect cell lines, yeast cell lines, and in vitro cell-free protein synthesis systems. In a specific embodiment, the mammalian cell line is a CHO cell line.

In one aspect, a method for treating cancer in a subject in need thereof is provided, comprising: a) administering a bispecific antibody of the present invention, and b) administering said bispecific antibody to said subject.

In some embodiments, a method is provided for inhibiting tumor growth or progression in a subject having cancer cells expressing a tumor antigen, comprising administering to a subject in need thereof an effective amount of a composition comprising antibodies of the present invention. In some embodiments, a method is provided for inhibiting metastatic cells expressing a tumor antigen in a subject, comprising administering to a subject in need thereof an effective amount of a composition comprising the antibodies of the present invention. In some embodiments, a method for inducing tumor regression in malignant cells in a subject is provided, comprising administering to a subject in need thereof an effective amount of a composition comprising the antibodies of the present invention.

In a specific aspect, the antibodies of the invention inhibit or reduce the growth of cancer cells by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% relative to the growth of cancer cells in the absence of an antibody or bispecific antibody of the invention.

In a specific aspect, antibodies kill cells or inhibit or reduce cancer cell growth by at least 5%, at least 10%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least at least 90%, at least 95%, or at least 100% more effective than in the absence of the antibody or bispecific antibodies of the invention.

In one aspect, the invention provides an effective amount of a composition (eg, pharmaceutical composition) comprising a bispecific antibody of the present invention for treating a condition (eg, cancer) associated with tumor antigen expression in a subject in need thereof.

In another aspect, the invention provides antibodies provided herein for use in the treatment of a condition (eg, cancer) associated with tumor antigen expression in a subject in need thereof. In some embodiments, the antibodies provided herein are provided for inhibiting the growth or progression of a tumor in a subject having cancer cells expressing a tumor antigen. In some embodiments, the antibodies provided herein are provided for inhibiting the metastasis of cancer cells expressing a tumor antigen in a subject in need thereof. In some embodiments, antibodies provided herein are provided for inducing tumor regression in a subject having cancer cells expressing a tumor antigen.

In another aspect, the invention relates to the use of the antibodies described herein in the manufacture of a medicament for the treatment of a condition (eg, cancer) associated with the expression of a tumor antigen. In some embodiments, the use of the antibodies provided herein is contemplated in the manufacture of a medicament for inhibiting tumor growth or progression. In some embodiments, the use of the antibodies provided herein in the manufacture of a medicament for inhibiting the metastasis of cancer cells expressing a tumor antigen is contemplated. In some embodiments, the use of the antibodies provided herein is contemplated in the manufacture of a medicament for inducing tumor regression.

In some embodiments, the methods disclosed herein further comprise the step of treating the subject with an additional form of therapy. In some embodiments, the additional form of therapy is an additional anti-cancer therapy, including, but not limited to, chemotherapy, radiation therapy, surgery, hormonal therapy, and/or additional immunotherapy.

The invention also includes administering the molecules of the invention in combination with other cancer treatment or prevention methods known to those skilled in the art, including, without limitation, current standard and experimental chemotherapy, biological therapies, immunotherapy, radiation therapy, or surgery. In some aspects, molecules of the invention may be administered in combination with a therapeutically or prophylactically effective amount of one or more agents, therapeutic antibodies, or other agents known to those of skill in the art for the treatment and/or prevention of cancer.

Accordingly, methods of treating cancer comprise administering to a subject in need thereof an effective amount of a polyspecific antibody (eg, a bispecific antibody) of the present invention in combination with a chemotherapeutic agent. Such combined treatment can be administered separately, sequentially or simultaneously.

Disclosed in the present description, the number of doses and frequency of administration is covered by the terms therapeutically effective and prophylactically effective. The dosage and frequency may further vary depending on factors specific to each subject, depending on the particular therapeutic or prophylactic administered, the severity and type of cancer, the route of administration, as well as the age, body weight, response of the subject and medical history. One of skill in the art can select an appropriate treatment regimen considering such factors and following, for example, the dosages given in the literature and recommended in the Physician's Desk Reference (56th ed., 2002).

Accordingly, in addition to targeting T cells to tumor-specific antigens, T cell-interacting bispecific molecules can also be used to carry other diagnostic or therapeutic compounds to tumor-expressing cells on their surface. Thus, a bispecific T-cell interacting molecule can be attached directly or indirectly, eg via a linker, to a drug for delivery directly to tumor-bearing cells. Therapeutic agents include compounds such as nucleic acids, proteins, peptides, amino acids or derivatives, glycoproteins, radioisotopes, lipids, carbohydrates, or recombinant viruses. Therapeutic and diagnostic nucleic acid fragments include antisense nucleic acids, derivatized oligonucleotides for covalent linkage to single or double stranded DNA, and triplex forming oligonucleotides.

Pharmaceutical compositions

The present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of an antibody disclosed herein and a pharmaceutically acceptable carrier.

Antibodies of the present invention may be presented in the form of a pharmaceutical composition intended for administration, prepared in accordance with the chosen method of administration with a pharmaceutically acceptable diluent or excipients, such as buffers, surfactants, preservatives, solubilizing agents, isotonicity adjusting agents, stabilizing agents , carriers, etc. Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton PA, 18th ed., 1995, provides a compendium of methods used to prepare drug formulations that are generally known to practitioners.

These pharmaceutical compositions can be administered by any means known in the art that achieve the goal, which is usually the treatment of cancer. The route of administration may be parenteral, which as used herein refers to routes of administration including, but not limited to, intravenous, intramuscular, intraperitoneal, subcutaneous, and intraarticular injection and infusion. The route of administration and dosage of the molecules of the invention (e.g. antibodies, pharmaceutical composition) depend on the type of disease to be treated or the stage of the disease, the antigen to be monitored, the type of concomitant treatment, if any, the frequency of treatment, the nature of the desired effect , as well as body weight, age, health status, diet and gender of the patient. Thus, the actual dosage regimen used may vary widely and therefore may deviate from the dosage regimens disclosed herein.

Various antibody formulations containing the antibodies of the present invention may be used for administration. In some embodiments, antibodies can be administered in pure form. In some embodiments, the antibody and the pharmaceutically acceptable excipient may be in different formulations. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that facilitate the administration of a pharmacologically effective substance. For example, the excipient may impart shape or texture or act as a diluent. Suitable excipients include, without limitation, stabilizing agents, wetting and emulsifying agents, osmolarity salts, encapsulating agents, buffers, and skin penetration enhancers. Excipients and formulations for parenteral and non-parenteral drug delivery are disclosed in Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005.

In some embodiments, these agents are formulated for administration by injection (eg, intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, these agents may be combined with pharmaceutically acceptable carriers such as saline, Ringer's solution, dextrose solution, and the like. The specific dosing regimen, i.e. the dose, timing, and re-administration will depend on the individual and their medical history.

Antibodies disclosed herein can be administered by any suitable method, including injection (eg, intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). An antibody, such as a monoclonal antibody or a bispecific antibody, can also be administered by inhalation, as disclosed herein. Generally, the dose of an antibody of the present invention to be administered depends on the host being treated and the particular route of administration. In one embodiment, the dosage range for an antibody of the present invention may be from about 0.001 µg/kg body weight to about 20,000 µg/kg body weight. The term "body weight" is applied to the patient receiving treatment. When isolated cells are processed, "body weight" in the context of the present description refers to "total cellular body weight". The term "total body weight" can be used for both cell treatment treatment and patient treatment. All concentrations and treatment levels herein expressed as "body weight" or simply "kg" should also be considered to include similar concentrations of "total cell body mass" and "total body weight". However, those skilled in the art will appreciate the practical value of a variety of dose ranges, e.g. 03 mcg/kg body weight up to 10000 mcg/kg body weight, from 0.04 mcg/kg body weight to 5000 mcg/kg body weight, from 0.05 mcg/kg body weight to 2500 mcg/kg body weight, from 0.06 µg/kg body weight up to 1000 µg/kg body weight, from 0.07 µg/kg body weight to 500 µg/kg body weight, from 0.08 µg/kg body weight to 400 µg/kg body weight, from 0.09 µg/kg of body weight to 200 µg/kg of body weight or from 0.1 µg/kg of body weight to 100 µg/kg of body weight. In addition, those skilled in the art will appreciate that different dosage levels can be used, e.g. 0007 mcg/kg, 0.001 mcg/kg, 0.1 mcg/kg, 1.0 mcg/kg, 1.5 mcg/kg, 2.0 mcg/kg, 5.0 mcg/kg, 10.0 mcg/ kg, 15.0 µg/kg, 30.0 µg/kg, 50 µg/kg, 75 µg/kg, 80 µg/kg, 90 µg/kg, 100 µg/kg, 120 µg/kg, 140 µg/kg . . , 1 µg/kg, 5 µg/kg, 10 µg/kg, 12 µg/kg, 15 mg/kg, 20 mg/kg and/or 30 mg/kg. All of these dosages are exemplary, and it is expected that, according to the invention, any dosage between these points can be applied. For an antibody of the present invention, any of the above dose ranges or dose levels can be used. With repeated administrations for several days or longer, depending on the condition, treatment is continued until the desired level of symptom suppression or until sufficient therapeutic levels are achieved, for example, until tumor growth/progression is suppressed or retarded or cancer cells metastasize.

Other dosage regimens may also be useful, depending on the form of pharmacokinetic degradation desired by the practitioner. In one embodiment, an antibody of the present invention is administered at an initial dose followed by a higher dose and/or at a continuous, substantially constant dose. In some embodiments, the implementation provides for the introduction of a dose of one to four times a week. In some embodiments, the implementation provides for the introduction of a dose once a month, once every two months or every three months. The progress of this therapy is easily monitored using conventional methods and tests. Dosing regimen may change over time.

For the purposes of the present invention, the appropriate dose of an antibody will depend on the antibody or compositions used, the type and severity of the symptoms to be treated, whether the agent is being administered therapeutically, prior therapy, the patient's medical history and response to the agent, the rate of clearance of the administered agent from the patient's body and the discretion of the attending physician. Usually, the doctor prescribes the administration of the antibody until the desired result is achieved. The dose and/or frequency of dosing may vary depending on the course of treatment. The dose is usually determined based on empirical considerations such as half-life. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to prolong the half-life of an antibody and prevent attack by the host's immune system on the antibody. The frequency of administration can be determined and adjusted during therapy and is usually, but not necessarily, based on treatment and/or suppression and/or alleviation and/or delay of symptoms, e.g. inhibition or delay of tumor growth, etc. Alternatively, sustained sustained release antibody formulations may be suitable. Various formulations and devices are known in the art to achieve sustained release.

In one embodiment, doses of the antibody may be determined empirically in individuals receiving one or more administrations of the antibody. People are given increasing doses of the antibody. To evaluate the effectiveness, you can monitor the indicator of the disease.

In some embodiments, administration of the antibody results in at least one effect selected from the group consisting of: tumor growth inhibition, tumor regression, tumor size reduction, tumor cell reduction, tumor growth retardation, abscopal effect, tumor metastasis inhibition, tumor reduction metastatic lesions over time, reduced use of chemotherapeutic or cytotoxic agents, decreased tumor burden, increased progression-free survival, increased overall survival, complete response, partial response, and stable disease.

Administration of an antibody according to the method of the present invention may be continuous or intermittent, depending on, for example, the physiological state of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Administration of the antibody may be substantially continuous over a preselected period of time, or may be administered in multiple, spaced doses.

In some embodiments, more than one antibody may be present. At least one, at least two, at least three, at least four, at least five or more different antibodies may be present. Typically, these antibodies may have additional activities that do not adversely affect each other. For example, one or more of the following antibodies can be used: a first anti-CD3 antibody directed at one epitope on CD3 and a second anti-CD3 antibody directed at a different epitope on CD3.

In some embodiments, the antibodies of the invention are particularly useful for parenteral administration, such as intravenous administration or administration into a body cavity or lumen of an organ.

Therapeutic formulations of antibodies used in accordance with the present invention are prepared for storage by mixing the antibody of the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005) as lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed and may include buffers such as phosphate, citrate and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Liposomes containing the antibody are prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. sci. USA 82:3688, 1985; Hwang, et al., Proc. Natl Acad. sci. USA 77:4030, 1980; and US patent. Nos. 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed in US Pat. No. 5,013,556. Particularly useful liposomes can be obtained by reverse phase evaporation with a lipid composition containing phosphatidylcholine, cholesterol and a phosphatidylethanolamine derivative with PEG (PEG-PE). Liposomes are extruded through filters with a certain pore size to obtain liposomes of the desired diameter.

The active ingredients may also be encapsulated in microcapsules prepared, for example, by coacervation or interfacial polymerization methods, such as hydroxymethyl cellulose or gelatin microcapsules and polymethyl methacrylate microcapsules, respectively, in colloidal drug delivery systems (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Such methods are disclosed in Remington, The Science and Practice of Pharmacy 21st Ed. Mack Publishing, 2005.

Sustained release formulations may be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g. poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactides (US Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres consisting of a copolymer of lactic acid and glycolic acid and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

Formulations used for in vivo administration must be sterile. This is easily achieved, for example, by filtration through sterile filter membranes. Therapeutic antibody compositions (eg, anti-CD3 antibodies) are typically placed in a container having a sterile access port, such as an intravenous solution bag or a vial having a stopper that can be pierced with a hypodermic needle.

The compositions of the present invention may be in the form of unit dosage forms such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories for oral, parenteral or rectal administration or administration by inhalation or insufflation.

To prepare solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical carrier, for example, traditional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and others. pharmaceutical diluents, such as water, to form a solid precomposition containing a homogeneous mixture of a compound of the present invention or a non-toxic pharmaceutically acceptable salt thereof. When such pre-formulations are said to be homogeneous, it is meant that the active ingredient is uniformly dispersed throughout the composition so that the composition can be easily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then divided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Tablets or pills of the new composition may be coated or otherwise mixed to obtain a dosage form that provides the benefit of prolonged action. For example, a tablet or pill may contain an internal dose component and an external dose component, the latter being in the form of a shell covering the first component. The two components can be separated by an enteric layer which prevents the breakdown of the internal component in the stomach and allows it to enter the duodenum intact or delays its release. Various materials can be used for such enteric layers or coatings, including a variety of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol, and cellulose acetate.

Suitable surface active agents include, in particular, nonionic agents such as polyoxyethylene sorbitans (eg Tween™ 20, 40, 60, 80 or 85) and other sorbitans (eg Span™ 20, 40, 60, 80 or 85). Compositions with a surfactant typically contain 0.05 to 5% surfactant, eg 0.1 to 2.5%. It should be understood that other ingredients may be added as needed, such as mannitol or other pharmaceutically acceptable carriers.

The compositions of the invention include all compositions in which the antibody is present in an amount effective to achieve the desired medical effect in the treatment of cancer. Although individual needs may vary from one patient to another, it is within the skill of the ordinary physician to determine the optimal ranges for effective amounts of all components.

Examples of such compositions, as well as methods for their preparation, have also been described above and are further described below. In some embodiments, the implementation of the composition contains one or more antibodies. In some embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In some embodiments, the antibody contains a constant region that is capable of inducing the desired immune response, such as antibody-mediated lysis or ADCC. In some embodiments, the antibody contains a constant region that does not elicit an unwanted immune response, such as antibody-mediated lysis or ADCC.

It is understood that the compositions may contain more than one antibody, for example a mixture of anti-CD3 antibodies that recognize different CD3 or CD3 epitopes and a tumor antigen. Other illustrative compositions contain more than one antibody that recognizes the same epitope(s), or different types of antibodies, or that bind to different CD3 epitopes and tumor antigen (eg, human CD3).

In some embodiments, the antibody may be administered in combination with one or more additional therapeutic agents. This includes, without limitation, administering a biotherapeutic agent and/or a chemotherapeutic agent such as, but not limited to, a vaccine, CAR-T cell based therapy, radiation therapy, cytokine therapy, a bispecific anti-CD3 antibody, an inhibitor of other immunosuppressive pathways, an inhibitor angiogenesis inhibitor, T cell activator, metabolic pathway inhibitor, mTOR inhibitor, adenosine pathway inhibitor, tyrosine kinase inhibitor including but not limited to Inlyta, ALK inhibitors and sunitinib, BRAF inhibitor, epigenetic modifier, IDO1 inhibitor, JAK inhibitor, STAT inhibitor, cyclin inhibitor α-dependent kinase, biotherapeutic agent (including but not limited to antibodies to VEGF, VEGFR, EGFR, Her2/neu, other growth factor receptors, CD40, CD-40L, CTLA-4, OX-40, 4-1BB, TIGIT and ICOS ), an immunogenic agent (e.g., attenuated cancer cells, tumor antigens, antigen-presenting cells such as dendritic cells stimulated with tumor-derived antigen or nuclei inoic acids, immunostimulatory cytokines (eg, IL-2, IFNα2, GM-CSF), and cells transfected with genes encoding immunostimulatory cytokines such as GM-CSF, without limitation).

Examples of biotherapeutic agents include therapeutic antibodies, immunomodulatory agents, and therapeutic immune cells.

Therapeutic antibodies may have specificity for many different antigens. For example, therapeutic antibodies can be directed to a tumor-associated antigen such that binding of the antibody to the antigen results in the death of the cell expressing the antigen. In another example, therapeutic antibodies can be directed to an antigen (eg, PD-1) on an immune cell such that binding of the antibody prevents suppression of activity of the antigen-expressing cell (and thus activates the antigen-expressing cell). In some situations, a therapeutic antibody may function through several different mechanisms (for example, it may both i) promote the death of an antigen-expressing cell and ii) prevent antigen-mediated downregulation of immune cells in contact with an antigen-expressing cell).

Therapeutic antibodies can be directed to, for example, the antigens listed below. For some antigens, below (in parentheses/parentheses after the antigen) are also indicated typical antibodies directed to that antigen. The following antigens may also be referred to herein as "target antigens" or the like. As used herein, target antigens for therapeutic antibodies include, for example: 4-1BB (eg, utilumab); 5T4; A33; alpha folic acid receptor 1 (eg, mirvetuximab soravtansin); Alk-1; BCMA [for example, PF-06863135 (see US9969809)]; BTN1A1 (for example, see WO2018222689); CA-125 (eg, abagovomab); carbonic anhydrase IX; CCR2; CCR4 (for example, mogamulizumab); CCR5 (eg, leronlimab); CCR8; CD3 [eg, blinatumomab (CD3/CD19 bispecific), PF-06671008 (CD3/P-cadherin bispecific), PF-06863135 (CD3/BCMA bispecific), PF-07062119 (CD3/GUCY2c bispecific)] CD19 (eg, blinatumomab, MOR208); CD20 (eg, ibritumomab tiuxetan, obinutuzumab, ofatumumab, rituximab, ublituximab); CD22 (inotuzumab ozogamicin, moxetumomab pasudotox); CD25; CD28; CD30 (eg, brentuximab vedotin); CD33 (eg, gemtuzumab ozogamicin); CD38 (eg, daratumumab, isatuximab), CD40; CD-40L; CD44v6; CD47; CD52 (eg, alemtuzumab); CD63; CD79 (eg, Polatuzumab vedotin); CD80; CD123; CD276/B7-H3 (eg, omburtamab); CDH17; CEA; ClhCG; CTLA-4 (eg, ipilimumab, tremelimumab), CXCR4; desmoglein 4; DLL3 (eg, rovalpituzumab tezirine); DLL4; E-cadherin; EDA; EDB; EFNA4; EGFR (eg, cetuximab, depatuxizumab, mafodotin, necitumumab, panitumumab); EGFRvIII; Endosialin; EpCAM (eg, oportuzumab monatox); FAP; fetal acetylcholine receptor; FLT3 (for example, see WO2018/220584); GD2 (eg, dinutuximab, 3F8); GD3; GITR; GloboH; GM1; GM2; GUCY2C (for example, PF-07062119); HER2/neu [eg, margetuximab, pertuzumab, trastuzumab; ado-trastuzumab emtansine, trastuzumab duocarmazine, PF-06804103 (see US8828401)]; HER3; HER4; ICOS; IL-10; ITG-AvB6; LAG-3 (eg, relatlimab); Lewis-Y; LG; Ly-6; M-CSF [e.g. PD-0360324 (see US7326414)]; MCSP; mesothelin; MUC1; MUC2; MUC3; MUC4; MUC5AC; MUC5B; MUC7; MUC16; notch1; notch3; Nectin-4 (for example, enfortumab vedotin); OX40 [for example, PF-04518600 (see US7960515)]; P-cadherin [e.g. PF-06671008 (see WO2016/001810)]; PCDHB2; PD-1 [e.g., BCD-100, camrelizumab, cemiplimab, genolimzumab (CBT-501), MEDI0680, nivolumab, pembrolizumab, RN888 (see WO2016/092419), sintilimab, spartalizumab, STI-A1110, tisselizumab, TSR-0] ; PD-L1 (eg, atezolizumab, durvalumab, BMS-936559 (MDX-1105) or LY3300054); PDGFRA (eg, olaratumab); plasma cell antigen; PolySA; PSCA; PSMA; PTK7 [e.g. PF-06647020 (see US9409995)]; Ror1; S.A.S. SCRx6; SLAMF7 (eg elotuzumab); SHH; SIRPa (eg ED9, Effi-DEM); STEAP; TGF-beta; TIGIT; TIM-3; TMPRSS3; precursor TNF-alpha; TROP-2 (eg, scituzumab govitecan); TSPAN8; VEGF (eg, bevacizumab, brolucizumab); VEGFR1 (eg ranibizumab); VEGFR2 (eg ramucirumab, ranibizumab); wue-1.

Therapeutic antibodies may be in any suitable format. For example, therapeutic antibodies may be in any of the formats disclosed elsewhere herein. In some embodiments, the therapeutic antibody may be a naked antibody. In some embodiments, the therapeutic antibody may be linked to a drug/agent (also known as an "antibody-drug conjugate" (ADC)). In some embodiments, a therapeutic antibody to a particular antigen may be included in a polyspecific antibody (eg, a bispecific antibody).

In some embodiments, an antibody directed to an antigen may be conjugated to a drug/agent. Antibody-bound drug molecules are also referred to as "antibody drug conjugates" (ADCs). Drugs/agents can be linked to the antibody directly or indirectly via a linker. Most commonly, toxic drugs are bound to an antibody such that binding of the ADC to the corresponding antigen results in the killing of cells expressing the antigen. For example, ADCs that are associated with toxic drugs are particularly useful for targeting tumor-associated antigens to help kill tumor cells that express tumor-associated antigens. In other embodiments, agents that can be associated with an antibody can be, for example, an immunomodulatory agent (for example, to modulate the activity of immune cells in close proximity to the ADC), an imaging agent (for example, to facilitate visualization of the ADC in a subject or in a biological sample obtained from the subject), or an agent to increase the serum half-life of the antibody or increase bioactivity.

Methods for conjugating a cytotoxic agent or other therapeutic agents with antibodies are described in various publications. For example, antibodies can be chemically modified either through side chain lysine amines or through cysteine sulfhydryl groups activated by reduction of interchain disulfide bonds to cause a conjugation reaction. See, for example, Tanaka et al., FEBS Letters 579: 2092-2096, 2005, and Gentle et al., Bioconjugate Chem. 15:658-663, 2004. Reactive cysteine residues have also been described introduced into specific sites of antibodies to conjugate a specific drug with a specific stoichiometry. See, for example, Junutula et al., Nature Biotechnology, 26:925-932, 2008. Conjugation using a label containing glutamine (acyl donor) or endogenous glutamine brought into a reactive state (i.e., the ability to form a covalent bond as an acyl donor) by creating a polypeptide in the presence of transglutaminase and an amine (for example, a cytotoxic agent containing or attached to a reactive amine) is also described in international applications WO2012/059882 and WO2015015448. In some embodiments, the implementation of the ADC may have any of the functions or characteristics of the ADC presented in WO2016166629, which is incorporated herein by reference for all purposes.

Drugs/agents that can be linked to an antibody in ADC format may include, for example, cytotoxic agents, immunomodulators, imaging agents, therapeutic proteins, biopolymers, or oligonucleotides.

Typical cytotoxic agents that can be included in ADCs include anthracycline, auristatin, dolastatin, combretastatin, duocarmycin, pyrrolobenzodiazepine dimer, indolinobenzodiazepine dimer, enediine, geldanamycin, maytansine, pyromycin, taxane, vinca alkaloid, camptothecin, tubulisin, hemiasterlin, splicenolidine, pladienolidine and their stereoisomers, isosteres, analogs or derivatives.

Typical immunomodulatory agents that may be included in ADCs include ganciclovir, etanercept, tacrolimus, sirolimus, voclosporin, cyclosporine, rapamycin, cyclophosphamide, azathioprine, mycophenolate mofetil, methotrextrate, glucocorticoid and its analogs, cytokines, stem cell growth factors, lymphotoxins, factor tumor necrosis factor (TNF), hematopoietic factors, interleukins (eg, interleukin-1 (IL-1), IL-2, IL-3, IL-6, IL-10, IL-12, IL-18, and IL-21) , colony stimulating factors (eg, granulocyte colony stimulating factor (G-CSF) and granulocyte and macrophage colony stimulating factor (GM-CSF)), interferons (eg, interferons-alpha, -beta and -gamma), stem cell growth factor , designated as "factor S 1", erythropoietin and thrombopoietin, or a combination of both.

Examples of imaging agents that may be included in the ADC include fluorescein, rhodamine, lanthanide phosphors and their derivatives, or a chelating radioisotope. Examples of fluorophores include, without limitation, fluorescein isothiocyanate (FITC) (eg, 5-FITC), fluorescein amidite (FAM) (eg, 5-FAM), eosin, carboxyfluorescein, erythrosin, Alexa Fluor.RTM. (e.g. Alexa 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 647, 660, 680, 700 or 750), carboxytetramethylrhodamine (TAMRA) (e.g. 5- TAMRA), tetramethylrhodamine (TMR) and sulforodamine (SR) (eg SR101). Examples of chelators include, without limitation, 1,4,7,10-tetraazacyclododecane-N, N',N”,N”'-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), 1,4,7-triazacyclononane, 1-glutaric acid-4,7-acetic acid (deferoxamine), diethylenetriaminepentaacetic acid (DTPA) and 1,2-bis(o-aminophenoxy)ethane-N, N, N,N'-tetraacetic acid) (BAPTA).

Examples of therapeutic proteins that can be included in an ADC include a toxin, a hormone, an enzyme, and a growth factor.

Examples of biocompatible polymers that can be included in ADCs include water-soluble polymers such as polyethylene glycol (PEG) or derivatives thereof, and zwitterion-containing biocompatible polymers (eg, a polymer containing phosphorylcholine).

Examples of biocompatible polymers that can be included in ADCs include antisense oligonucleotides.

In some embodiments, an antibody directed to an antigen disclosed herein may be included in a bispecific antibody molecule. Bispecific antibodies are monoclonal antibodies that have binding specificity for at least two different antigens.

In some embodiments, the bispecific antibody comprises a first antibody variable domain and a second antibody variable domain, wherein the first antibody variable domain is capable of recruiting the activity of a human immune system effector cell by specifically binding to CD3, as provided herein, and wherein the second antibody variable domain is capable of specifically bind to the target antigen. In some embodiments, the antibody is IgG1, IgG2, IgG3, or IgG4 isotype. In some embodiments, the implementation of the antibody contains an immunologically inert Fc region. In some embodiments, the antibody is a human antibody or a humanized antibody.

The target antigen is usually expressed on the target cell in a disease state (eg, a cancer cell). Examples of target antigens of particular interest in the case of bispecific antibodies include, but are not limited to, BCMA, EpCAM (epithelial cell adhesion molecule), CCR5 (chemokine type 5 receptor), CD19, HER (human epidermal growth factor receptor)-2/neu, HER-3, HER-4, EGFR (epidermal growth factor receptor), PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, CIhCG, Lewis-Y, CD20, CD33, CD30 , ganglioside GD3, 9-O-acetyl-GD3, GM2, Globo H, fucosyl GM1, Poly SA, GD2, carbonic anhydrase IX (MN/CA IX), CD44v6, Shh (Sonic Hedgehog), Wue-1, plasma cell antigen, (membrane bound) IgE, MCSP (melanoma chondroitin sulfate proteoglycan), CCR8, TNF alpha precursor, STEAP, mesothelin, A33 antigen, PSCA (prostate stem cell antigen), Ly-6; desmoglein 4, E-cadherin neoepitope, fetal acetylcholine receptor, CD25, CA19-9 marker, CA-125 marker and type II MIS receptor, sTn (sialylated Tn antigen; TAG-72), FAP (fibroblast activation antigen ), endosialin, EGFRvIII, LG, SAS, PD-L1, CD47, SIRPa and CD63.

In some embodiments, the bispecific antibody comprises a full-length human antibody, wherein the first variable domain of the bispecific antibody is capable of recruiting the activity of a human immune system effector cell by specifically binding to CD3, and wherein the second variable domain of the antibody of the heterodimeric protein is capable of specifically binding to a target antigen (e.g., CD20, EpCAM or P-cadherin).

Methods for making bispecific antibodies are known in the art (see, for example, Suresh et al., Methods in Enzymology 121:210, 1986). Traditionally, recombinant production of bispecific antibodies has been based on the co-expression of two immunoglobulin heavy chain-light chain pairs, with the two heavy chains having different specificities (Millstein and Cuello, Nature 305, 537-539, 1983).

According to one approach to the production of bispecific antibodies, antibody variable domains with the desired binding specificity (antibody-antigen combining regions) are fused to immunoglobulin constant region sequences. The fusion is preferably performed with an immunoglobulin heavy chain constant region comprising at least a portion of the hinge, CH2 and CH3 regions. Preferably, at least one of the fusion partners has a first heavy chain constant region (CH1) containing the site required for light chain binding. The DNA encoding the immunoglobulin heavy chains to be fused and, optionally, the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. This allows the proportion of each of the polypeptide fragments to be controlled in embodiments where unequal ratios of the three polypeptide chains used in the construct provide optimal yields. However, it is possible to insert sequences encoding two or all three polypeptide chains into one expression vector when the expression of at least two polypeptide chains in equal ratios provides a high yield or when the ratios are not particularly important.

In one approach, the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm and a hybrid immunoglobulin heavy/light chain pair (providing a second binding specificity) on the other arm. This asymmetric structure with an immunoglobulin light chain in only one half of the bispecific molecule facilitates separation of the desired bispecific compound from unwanted combinations of immunoglobulin chains. This approach is described in PCT Publication No. WO 94/04690.

In another approach, bispecific antibodies consist of a modification of an amino acid in a first hinge region on one arm, wherein the substituted/substituted amino acid in the first hinge region has the opposite charge of the corresponding amino acid in the second hinge region on the other arm. This approach is described in International Patent Application No. PCT/US2011/036419 (WO2011/143545).

In another approach, production of the desired heteromultimeric or heterodimeric protein (eg, bispecific antibody) is increased by altering or creating a contact area between the first and second immunoglobulin-like Fc regions (eg, the hinge region and/or the CH3 region). In this approach, bispecific antibodies may consist of a CH3 region, wherein the CH3 region includes a first CH3 polypeptide and a second CH3 polypeptide that interact together to form a CH3 contact region, wherein one or more amino acids in that CH3 contact region destabilize homodimer formation and are not electrostatically unfavorable. to form homodimers. This approach is described in International Patent Application No. PCT/US2011/036419 (WO2011/143545).

In another approach, bispecific antibodies can be generated using a glutamine-containing peptide tag designed for an antibody directed to an epitope (e.g., BCMA) on one arm and another peptide tag (e.g., a Lys-containing peptide tag or reactive endogenous Lys), constructed on the second antibody directed to the second epitope on the other arm in the presence of transglutaminase. This approach is described in International Patent Application No. PCT/IB2011/054899 (WO2012/059882).

In some embodiments, the first and second variable domains of the bispecific antibody contain amino acid modifications at positions where the first and second variable domains of the bispecific antibody contain amino acid modifications at positions 223, 225, and 228 (e.g., (C223E or C223R), (E225R) and ( P228E or P228R)) in the hinge region and at position 409 or 368 (eg K409R or L368E (EUC numbering scheme)) in the CH3 region of human IgG2.

In some embodiments, the first and second variable domains of the bispecific antibody comprise amino acid modifications at positions 221 and 228 (e.g., (D221R or D221E) and (P228R or P228E)) in the hinge region and at position 409 or 368 (e.g., K409R or L368E ( EU numbering scheme)) in the CH3 region of human IgG1.

In some embodiments, the first and second variable domains of the bispecific antibody comprise amino acid modifications at positions 228 (e.g., (P228E or P228R)) in the hinge region and at position 409 or 368 (e.g., R409 or L368E (EU numbering scheme)) in the CH3 region. human IgG4.

In some embodiments, the implementation of the bispecific antibody may have any features or characteristics of any of the bispecific antibodies presented in patent application WO2016166629, incorporated by reference for all purposes.

Immunomodulatory agents include many different types of molecules that can stimulate an immune response in a subject, such as pattern recognition receptor (PRR) agonists, immunostimulatory cytokines, and cancer vaccines.

Pattern recognition receptors (PRRs) are receptors that are expressed by cells of the immune system and recognize a variety of molecules associated with pathogens and/or cell damage or death. PRRs are involved in both the innate immune response and the adaptive immune response. PRR agonists can be used to stimulate an immune response in a subject. There are several classes of PRR molecules, including toll-like receptors (TLRs), RIG-I-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), C-type lectin receptors (CLRs), and protein interferon gene stimulator (STING).

The terms "TLR" and "toll-like receptor" refer to any toll-like receptor. Toll-like receptors are receptors involved in the activation of immune responses. TLRs recognize, for example, pathogen-associated molecular patterns (PAMPs) expressed in microbes as well as endogenous damage-associated molecular patterns (DAMPs) that are released from dead or dying cells.

Molecules that activate TLRs (and thereby activate immune responses) are referred to herein as "TLR agonists". TLR agonists may include, for example, small molecules (eg, an organic molecule with a molecular weight of less than about 1000 Daltons) as well as large molecules (eg, oligonucleotides and proteins). Some TLR agonists are specific to one TLR type (eg TLR3 or TLR9), while some TLR agonists activate two or more TLR types (eg both TLR7 and TLR8).

Examples of TLR agonists provided herein include TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, and TLR9 agonists.

Examples of small molecular weight TLR agonists include those disclosed in, for example, US Pat. Nos. 4,689,338; 4,929,624; 5,266,575; 5,268,376; 5,346,905; 5,352,784; 5,389,640; 5,446,153; 5,482,936; 5,756,747; 6,110,929; 6,194,425; 6,331,539; 6,376,669; 6,451,810; 6,525,064; 6,541,485; 6,545,016; 6,545,017; 6,573,273; 6,656,938; 6,660,735; 6,660,747; 6,664,260; 6,664,264; 6,664,265; 6,667,312; 6,670,372; 6,677,347; 6,677,348; 6,677,349; 6,683,088; 6,756,382; 6,797,718; 6,818,650; and 7.7091.214; US Patent Publication Nos. 2004/0091491, 2004/0176367 and 2006/0100229; and international publications Nos. WO 2005/18551, WO 2005/18556, WO 2005/20999, WO 2005/032484, WO 2005/048933, WO 2005/048945, WO 2005/051317, WO9 2005/051304/WO0 2616 Wo 2005/066170, Wo 2005/066172, Wo 2005/076783, Wo 2005/079195, Wo 2005/094531, Wo 2005/123079, Wo 2005/123080, Wo 2006/009826, Wo 2006/009832, Wo 2006/026660, Wo 2006/028451, Wo 2006/028545, Wo 2006/028962, Wo 2006/029115, Wo 2006/038923, Wo 2006/065280, Wo 2006/074003, Wo 2006/083440, Wo 2006/086449, Wo 2006/091394, Wo 2006/091394, WO 2006/086633, WO 2006/086634, WO 2006/091567, WO 2006/091568, WO 2006/091647, WO 2006/093514 and WO 2006/098852.

Additional examples of small molecule TLR agonists include certain purine derivatives (e.g., described in US Pat. Nos. 6,376,501 and 6,028,076), some imidazoquinoline amide derivatives (e.g., described in US Pat. No. 6,069,149), some imidazopyridine derivatives (e.g., described in US Pat. No. 6,518,265 ), some benzimidazole derivatives (for example, described in US patent No. 6,387,938), some derivatives of 4-aminopyrimidine fused to a five-membered nitrogen-containing heterocyclic ring (for example, adenine derivatives described in US patents No. 6,376,501; 6,028,076 and 6,329,381; and in WO 02 /08905), and some 3-β-D-ribofuranosylthiazolo[4,5-d]pyrimidine derivatives (for example, those described in US Publication No. 2003/0199461), and some small molecular weight immunostimulatory compounds, such as those described, for example, in patent publication US No. 2005/0136065.

Exemplary macromolecular TLR agonists include oligonucleotide sequences. Some TLR agonist oligonucleotide sequences contain cytosine guanine dinucleotides (CpG) and are described, for example, in US Pat. Nos. 6,194,388; 6,207,646; 6,239,116; 6,339,068; and 6,406,705. Some CpG-containing oligonucleotides may include synthetic immunomodulatory structural motifs such as those described, for example, in US Pat. Nos. 6,426,334 and 6,476,000. Other TLR agonist nucleotide sequences lack CpG sequences and are described, for example, in International Patent Publication No. WO 00/75304. Still other TLR agonist nucleotide sequences include guanosine and uridine rich single-stranded RNA (ssRNA) such as described, for example, in Heil et al., Science, vol. 303, pp. 1526-1529 Mar. 5, 2004.

Other TLR agonists include biological molecules such as aminoalkylglucosamine phosphates (AGPs) and are described, for example, in US Pat. Nos. 6,113,918; 6 303 347; 6,525,028; and 6,649,172.

TLR agonists also include inactivated pathogens, or fractions thereof, which can activate several different types of TLR receptors. Exemplary pathogen-derived TLR agonists include BCG, M. obuense mycobacterium extract, talimogen laherparepvec (T-Vec) (derived from HSV-1), and Pexa-Vec (derived from vaccine virus).

In some embodiments, a TLR agonist may be an agonist antibody that specifically binds to a TLR.

Below is a brief description of the various TLRs as well as TLR agonists. The list of TLR agonists below should not be interpreted in relation to a particular TLR as indicating that a given TLR agonist necessarily activates only that TLR (eg, some molecules may activate multiple types of TLRs or even multiple classes of PRRs). For example, some of the molecules shown below as an example of a TLR4 agonist may also be a TLR5 agonist.

The terms "TLR1" and "toll-like receptor 1" refer to any form of the TLR1 receptor, as well as variants, isoforms, and species homologues that retain at least some of the activity of TLR1. Unless otherwise indicated, for example by specific reference to human TLR1, TLR1 includes native TLR1 sequences from all mammalian species, eg human, monkey and mouse. One example of a human TLR1 is provided by UniProt under accession number Q15399.

"TLR1 agonist" as used herein means any molecule that, after binding to TLR1, (1) stimulates or activates TLR1, (2) enhances, enhances, stimulates, induces or prolongs the activity, function or presence of TLR1, or (3) enhances, increases, promotes or induces TLR1 expression. TLR1 agonists that can be used in any of the treatments, drugs and uses of the present invention include, for example, bacterial lipoproteins and derivatives thereof that bind TLR1.

Examples of TLR1 agonists that can be used in the treatments, drugs and uses of the present invention include, for example, bacterial lipoproteins and their derivatives such as SPM-105 (derived from autoclaved mycobacteria), OM-174 (a lipid A derivative), OmpS1 (porin from Salmonella typhi), OmpS1 (porin from Salmonella typhi), OspA (from Borrelia burgdorferi), MALP-2 (lipopeptide-2kD, activating mycoplasma macrophages), STF (soluble tuberculosis factor), CU-T12-9, diprovozim and lipopeptides derived from cell wall components such as PAM2CSK4, PAM3CSK4 and PAM3Cys.

TLR1 can form a heterodimer with TLR2 and, accordingly, many TLR1 agonists are also TLR2 agonists.

The terms "TLR2" and "toll-like receptor 2" refer to any form of the TLR2 receptor, as well as variants, isoforms, and species homologues that retain at least some of the activity of TLR2. Unless otherwise indicated, for example by specific reference to human TLR2, TLR2 includes native TLR2 sequences from all mammalian species, eg human, monkey and mouse. One example of a human TLR2 is available from UniProt under accession number Q60603.

"TLR2 agonist" as used herein means any molecule that, after binding to TLR2, (1) stimulates or activates TLR2, (2) enhances, enhances, stimulates, induces or prolongs the activity, function or presence of TLR2, or (3) enhances, increases, promotes or induces TLR2 expression. TLR2 agonists that can be used in any of the treatments, drugs and uses of the present invention include, for example, bacterial lipoproteins and derivatives thereof that bind TLR2.

Examples of TLR2 agonists that can be used in the treatments, drugs, and uses of the present invention include, for example, bacterial lipoproteins (e.g., diacylated lipoproteins) and derivatives thereof, such as SPM-105 (derived from autoclaved mycobacteria), OM-174 (lipid A derivative), OmpS1 (porin from Salmonella typhi), OmpS1 (porin from Salmonella typhi), OspA (from Borrelia burgdorferi), MALP-2 (lipopeptide-2kD, activating mycoplasma macrophages), STF (soluble tuberculosis factor), CU -T12-9, diprovocyme, amplivant and lipopeptides derived from cell wall components such as PAM2CSK4, PAM3CSK4 and PAM3Cys.

TLR2 can form a heterodimer with TLR1 or TLR6 and, accordingly, many TLR2 agonists are also TLR1 or TLR6 agonists.

The terms "TLR3" and "toll-like receptor 3" refer to any form of the TLR3 receptor, as well as variants, isoforms, and species homologues that retain at least some of the activity of TLR3. Unless otherwise indicated, for example by specific reference to human TLR3, TLR3 includes native TLR3 sequences from all mammalian species, eg human, monkey and mouse. One example of human TLR3 is available from UniProt under accession number Q15455.

"TLR3 agonist" as used herein means any molecule that, after binding to TLR3, (1) stimulates or activates TLR3, (2) enhances, increases, stimulates, induces or prolongs the activity, function or presence of TLR3, or (3) enhances, increases, promotes or induces TLR3 expression. TLR3 agonists that can be used in any of the treatments, drugs and uses of the present invention include, for example, nucleic acid ligands that bind TLR3.

Examples of TLR3 agonists that can be used in the treatments, drugs, and uses of the present invention include TLR3 ligands such as synthetic dsRNA, polyinosine polycytidylic acid ["poly(I:C)"] (available, for example, from InvivoGen in high molecular weight ( HMW) and small molecule (LMW) formulations), polyadenylic-polyuridylic acid ["poly(A:U)"] (available, for example, from InvivoGen), polyICLC (see Levy et al., Journal of Infectious Diseases, vol. 132 , No. 4, pp. 434-439, 1975), ampligen (see Jasani et al., Vaccine, vol. 27, no. 25-26, pp. 3401-3404, 2009), quintonol, rintatolimod and RGC100 ( see Naumann et al., Clinical and Developmental Immunology, vol 2013, article ID 283649).

The terms "TLR4" and "toll-like receptor 4" refer to any form of the TLR4 receptor, as well as variants, isoforms, and species homologues that retain at least some of the activity of TLR4. Unless otherwise indicated, for example by specific reference to human TLR4, TLR4 includes native TLR4 sequences from all mammalian species, eg human, monkey and mouse. One example of a human TLR4 is available from UniProt under accession number Q00206.

"TLR4 agonist" as used herein means any molecule that, after binding to TLR4, (1) stimulates or activates TLR4, (2) enhances, enhances, stimulates, induces or prolongs the activity, function or presence of TLR4, or (3) enhances, increases, promotes or induces TLR4 expression. TLR4 agonists that can be used in any of the treatments, drugs and uses of the present invention include, for example, bacterial lipopolysaccharides (LPS) and derivatives thereof that bind TLR4.

Examples of TLR4 agonists that can be used in the treatments, drugs and uses of the present invention include, for example, bacterial lipopolysaccharides (LPS) and derivatives thereof such as B:0111 (Sigma), monophosphoryl lipid A (MPLA), 3DMPL (3- O-deacylated MPL), GLA-AQ, G100, AS15, ASO2, GSK1572932A (GlaxoSmithKline, UK).

The terms "TLR5" and "toll-like receptor 5" refer to any form of the TLR5 receptor, as well as variants, isoforms, and species homologues that retain at least some of the activity of TLR5. Unless otherwise indicated, for example by specific reference to human TLR5, TLR5 includes native TLR5 sequences from all mammalian species, eg human, monkey and mouse. One example of human TLR5 is available from UniProt under accession number Q60602.

"TLR5 agonist" as used herein means any molecule that, after binding to TLR5, (1) stimulates or activates TLR5, (2) enhances, enhances, stimulates, induces or prolongs the activity, function or presence of TLR5, or (3) enhances, increases, promotes or induces TLR5 expression. TLR5 agonists that can be used in any of the treatments, drugs and uses of the present invention include, for example, bacterial flagellins and derivatives thereof that bind TLR5.

Examples of TLR5 agonists that can be used in the treatments, drugs and uses of the present invention include, for example, bacterial flagellin purified from B. subtilis, flagellin purified from P. aeruginosa, flagellin purified from S. typhimurium, and recombinant flagellin (all available from InvivoGen), entolimod (CBLB502; pharmacologically optimized flagellin derivative).

The terms "TLR6" and "toll-like receptor 6" refer to any form of the TLR6 receptor, as well as variants, isoforms, and species homologues that retain at least some of the activity of TLR6. Unless otherwise indicated, for example by specific reference to human TLR6, TLR6 includes native TLR6 sequences from all mammalian species, eg human, monkey and mouse. One example of human TLR6 is available from UniProt under accession number Q9Y2C9.

"TLR6 agonist" as used herein means any molecule that, after binding to TLR6, (1) stimulates or activates TLR6, (2) enhances, enhances, stimulates, induces or prolongs the activity, function or presence of TLR6, or (3) enhances, increases, promotes or induces TLR6 expression. TLR6 agonists that can be used in any of the treatments, drugs and uses of the present invention include, for example, bacterial lipopeptides and derivatives thereof that bind TLR6.

Examples of TLR6 agonists that can be used in the treatments, drugs, and uses of the present invention include, for example, many of the TLR2 agonists presented above, since TLR2 and TLR6 can form a heterodimer. TLR6 may also form a heterodimer with TLR4, and TLR6 agonists may include the various TLR4 agonists noted above.

The terms "TLR7" and "toll-like receptor 7" refer to any form of the TLR7 receptor, as well as variants, isoforms, and species homologues that retain at least some of the activity of TLR7. Unless otherwise indicated, for example by specific reference to human TLR7, TLR7 includes native TLR7 sequences from all mammalian species, eg human, monkey and mouse. One example of human TLR7 is available from UniProt under accession number Q9NYK1.

"TLR7 agonist" as used herein means any molecule that, after binding to TLR7, (1) stimulates or activates TLR7, (2) enhances, enhances, stimulates, induces or prolongs the activity, function or presence of TLR7, or (3) enhances, increases, promotes or induces TLR7 expression. TLR7 agonists that can be used in any of the treatments, drugs and uses of the present invention include, for example, nucleic acid ligands that bind TLR7.

Examples of TLR7 agonists that can be used in the treatments, drugs, and uses of the present invention include recombinant single-stranded ("ss") RNA, imidazoquinoline compounds such as imiquimod (R837), gardiquimod, and resiquimod (R848); loxoribin (7-allyl-7,8-dihydro-8-oxoguanosine) and related compounds; 7-thia-8-oxoguanosine, 7-deazaguanosine and related guanosine analogues; ANA975 (Anadys Pharmaceuticals) and related compounds; SM-360320 (Sumimoto); 3M-01, 3M-03, 3M-852 and 3M-S-34240 (3M Pharmaceuticals); GSK2245035 (GlaxoSmithKline; 8-oxoadenine molecule), AZD8848 (AstraZeneca; 8-oxoadenine molecule), MEDI9197 (Medimmune; formerly 3M-052), ssRNA40, and adenosine analogs such as UC-1V150 (Jin et al. Chem Lett (2006) 16:4559-4563, compound 4). Many TLR7 agonists are also TLR8 agonists.

The terms "TLR8" and "toll-like receptor 8" refer to any form of the TLR8 receptor, as well as variants, isoforms, and species homologues that retain at least some of the activity of TLR8. Unless otherwise indicated, for example by specific reference to human TLR8, TLR8 includes native TLR8 sequences from all mammalian species, eg human, monkey and mouse. One example of a human TLR8 is available from UniProt under accession number Q9NR97.

"TLR8 agonist" as used herein means any molecule that, after binding to TLR8, (1) stimulates or activates TLR8, (2) enhances, enhances, stimulates, induces or prolongs the activity, function or presence of TLR8, or (3) enhances, increases, promotes or induces TLR8 expression. TLR8 agonists that can be used in any of the treatments, drugs and uses of the present invention include, for example, nucleic acid ligands that bind TLR8.

Examples of TLR8 agonists that can be used in the treatments, drugs and applications of the present invention include recombinant single strand ssRNA, imiquimod (R837), gardiquimod, resiquimod (R848), 3M-01, 3M-03, 3M-852 and 3M-S -34240 (3M Pharmaceuticals); GSK2245035 (GlaxoSmithKline; 8-oxoadenine molecule), AZD8848 (AstraZeneca; 8-oxoadenine molecule), MEDI9197 (Medimmune; formerly 3M-052), poly-G10, motolimod, and the various TLR7 agonists above (as previously noted, many TLR7 agonists are also TLR8 agonists).

The terms "TLR9" and "toll-like receptor 9" refer to any form of the TLR9 receptor, as well as species variants, isoforms, and homologues that retain at least some of the activity of TLR9. Unless otherwise indicated, for example by specific reference to human TLR9, TLR9 includes native TLR9 sequences from all mammalian species, eg human, monkey and mouse. One example of a human TLR9 is available from UniProt under accession number Q9NR96.

"TLR9 agonist" as used herein means any molecule that, after binding to TLR9, (1) stimulates or activates TLR9, (2) enhances, enhances, stimulates, induces or prolongs the activity, function or presence of TLR9, or (3) enhances, increases, promotes or induces TLR9 expression. TLR9 agonists that can be used in any of the treatments, drugs and uses of the present invention include, for example, nucleic acid ligands that bind TLR9.

Examples of TLR9 agonists that can be used in the treatments, drugs, and uses of the present invention include unmethylated CpG-containing DNA, immunostimulatory oligodeoxynucleotides (ODNs), such as CpG-containing ODNs, such as CpG24555, CpG10103, CpG7909 (PF-3512676/agatolimod ), CpG1018, AZD1419, ODN2216, MGN1703, SD-101, 1018ISS and CMP-001. TLR9 agonists also include nucleotide sequences containing synthetic cytosine phosphate-2'-deoxy-7-deazaguanosine dinucleotide (CpR) (Hybridon, Inc.), dSLIM-30L1, and immunoglobulin-DNA complexes. Exemplary TLR9 agonists are disclosed in WO2003/015711, WO2004/016805, WO2009/022215, PCT/US95/01570, PCT/US97/19791 and US Pat. all purposes.

RLRs include various cytosolic PRRs that allow detection of, for example, dsRNA. Examples of RLRs include, for example, retinoic acid inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA-5), and genetics and physiology laboratory 2 (LGP2).

"RLR agonist" as used herein means any molecule that, after binding to the RLR, (1) stimulates or activates the RLR, (2) enhances, enhances, stimulates, induces or prolongs the activity, function or presence of the RLR, or (3) enhances, increases, promotes or induces RLR expression. RLR agonists that can be used in any of the treatments, drugs and uses of the present invention include, for example, nucleic acids and derivatives thereof that bind RLR and agonist monoclonal antibodies (mAbs) that specifically bind to RLR.

Examples of RLR agonists that can be used in the treatments, medicaments and uses of the present invention include, for example, uncapped 5'-triphosphate short double-stranded RNA (RIG-I agonist); poly I:C (MDA-5 agonist) and BO-112 (MDA-A agonist).

NLRs include various PRRs that allow the detection of, for example, damage-associated molecules (DAMPs). NLRs include the subfamilies NLRA-A, NLRB-B, NLRC-C, and NLRP-P. Examples of NLRs include, for example, NOD1, NOD2, NAIP, NLRC4, and NLRP3.

"NLR agonist" as used herein means any molecule that, after binding to an NLR, (1) stimulates or activates the NLR, (2) enhances, increases, stimulates, induces or prolongs the activity, function or presence of the NLR, or (3) enhances, increases, promotes or induces NLR expression. NLR agonists that can be used in any of the treatments, drugs and uses of the present invention include, for example, DAMPs and derivatives thereof that bind NLR and agonist monoclonal antibodies (mAbs) that specifically bind to NLR.

Examples of NLR agonists that can be used in the treatments, medicaments and uses of the present invention include, for example, liposomal muramyl tripeptide/mifamurtide (NOD2 agonist).

CLRs include various PRRs that allow detection of, for example, carbohydrates and glycoproteins. CLRs include both transmembrane and secreted CLRs. Examples of CLRs include, for example, DEC-205/CD205, macrophage mannose receptor (MMR), dectin-1, dectin-2, minkle, DC-SIGN, DNGR-1, and mannose-binding lectin (MBL).

"CLR agonist" as used herein means any molecule that, after binding to the CLR, (1) stimulates or activates the CLR, (2) enhances, increases, stimulates, induces or prolongs the activity, function or presence of the CLR, or (3) enhances, increases, promotes or induces CLR expression. CLR agonists that can be used in any of the treatments, drugs and uses of the present invention include, for example, carbohydrates and their derivatives that bind CLR and agonist monoclonal antibodies (mAbs) that specifically bind to CLR.

Examples of CLR agonists that can be used in the treatments, medicaments and uses of the present invention include, for example, MD fraction (purified extract of soluble beta-glucan from Grifola frondosa) and Imprime PGG (beta 1,3/1,6- yeast-derived PAMP glucan).

The STING protein acts as a cytosolic DNA sensor and as an adapter protein in type 1 interferon signaling. The terms "STING" and "interferon gene stimulator" refer to any form of the STING protein, as well as species variants, isoforms, and homologues that retain at least some STING activity. Unless otherwise indicated, for example, by specific reference to human STING, STING includes native STING sequences from all mammalian species, eg, human, monkey, and mouse. One example of a human TLR9 is available from UniProt under accession number Q86WV6. STING is also known as TMEM173.

"STING agonist" as used herein means any molecule that, after binding to TLR9, (1) stimulates or activates STING, (2) enhances, enhances, stimulates, induces or prolongs the activity, function, or presence of STING, or (3) enhances, increases, promotes or induces the expression of STING. STING agonists that can be used in any of the treatments, drugs and uses of the present invention include, for example, nucleic acid ligands that bind STING.

Examples of STING agonists that can be used in the treatments, drugs and uses of the present invention include various immunostimulatory nucleic acids such as synthetic double-stranded DNA, cyclic di-GMP, cyclic GMP-AMP (cGAMP), synthetic cyclic dinucleotides (CDN) , such as MK-1454 and ADU-S100 (MIW815), and small molecules such as P0-424.

Other PRRs include, for example, DNA-dependent IFN regulatory factor activator (DAI) and Absent in melanoma 2 (AIM2).

Immunostimulatory cytokines include various signaling proteins that stimulate the immune response, such as interferons, interleukins, and hematopoietic growth factors.

Examples of immunostimulatory cytokines include GM-CSF, G-CSF, IFN-alpha, IFN-gamma; IL-2 (eg, denileukin diphytox), IL-6, IL-7, IL-11, IL-12, IL-15, IL-18, IL-21, and TNF-alpha.

The immunostimulatory cytokines may be in any suitable format. In some embodiments, the immunostimulatory cytokine may be a recombinant version of the wild-type cytokine. In some embodiments, the immunostimulatory cytokine may be a mutein that has one or more amino acid changes compared to the corresponding wild-type cytokine. In some embodiments, the immunostimulatory cytokine may be incorporated into a chimeric protein containing the cytokine and at least one other functional protein (eg, antibody). In some embodiments, an immunostimulatory cytokine may be covalently linked to a drug/agent (eg, any drug/agent as disclosed elsewhere herein as a possible component of the ADC).

Cancer vaccines include various compositions that contain tumor-associated antigens (or that can be used to generate a tumor-associated antigen in a subject) and thus can be used to provoke an immune response in a subject that will be directed to tumor cells containing a tumor-associated antigen.

Examples of materials that can be included in a cancer vaccine include attenuated cancer cells, tumor antigens, antigen presenting cells such as dendritic cells stimulated with a tumor derived antigen, or nucleic acids encoding tumor associated antigens. In some embodiments, the cancer vaccine may be prepared from the patient's own cancer cells. In some embodiments, the cancer vaccine may be prepared from biological material that is not derived from the patient's own cancer cells.

Cancer vaccines include, for example, sipuleucel-T and talimogen lacherparepvec (T-VEC).

Immunocellular therapy involves treating a patient with immune cells that are capable of attacking cancer cells. Immunocellular therapy includes, for example, tumor infiltrating lymphocytes (TILs) and chimeric antigen receptor T cells (CAR-T cells).

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbochone, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamin; acetogenins (especially bullatacin and bullatacinone); camptothecin (including the synthetic analog of topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues of KW-2189 and CBI-TMI); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterin, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as enediyne antibiotics (e.g. calicheamycin, especially calicheamycin gamma 1I and calicheamycin phiI1, see e.g. Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994); dynemycin, including dynemycin A, bisphosphonates ; such as clodronate; esperamycin; as well as neocarzinostatin chromophore and chromophores of related chromoprotein enediyne antibiotics), aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, kaminomycin, carzinophilin, chromomycins, dactinomycin, detorubicin, detorubicin-5, -oxo-L-norleucine, doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), pegylated liposomal doxorubicin, epirubicin, esorubicin, idarubicin, makrellomycin, mitomycins such as mitomycin C, mycophenolic acid, olivomycin, olivomycin , potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubic n; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; and antibodies to the tissue of the adrenal cortex, such as aminoglutethimide, mitotane, trilostane; folic acid compensator such as frolinic acid; aceglatone; aldofosfamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; demecolcin; diaziquon; elformitin; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pyrarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anhidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids such as paclitaxel and doxetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantron; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included are antihormonal agents that regulate or inhibit the action of hormones in tumors, such as antiestrogen and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen, raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapriston, and toremifene ( Fareston); aromatase inhibitors which inhibit the aromatase enzyme that regulates adrenal estrogen production, such as, for example, 4(5)-imidazoles, aminoglutethimide, megestrol acetate, exemestane, formestane, fadrozole, vorozol, letrozole and anastrozole; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; KRAS inhibitors; MCT4 inhibitors; MAT2a inhibitors; tyrosine kinase inhibitors such as sunitinib, axitinib; alk/c-Met/ROS inhibitors such as crizotinib, lorlatinib; mTOR inhibitors such as temsirolimus, gedatolisib; src/abl inhibitors such as bosutinib; cyclin-dependent kinase (CDK) inhibitors such as palbociclib, PF-06873600; erb inhibitors such as dacomitinib; PARP inhibitors such as talazoparib; SMO inhibitors such as glasdegib, PF-5274857; EGFR T790M inhibitors such as PF-06747775; EZH2 inhibitors such as PF-06821497; PRMT5 inhibitors such as PF-06939999; TGFRβr1 inhibitors such as PF-06952229; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In specific embodiments, such additional therapeutic agent is bevacizumab, cetuximab, sirolimus, panitumumab, 5-fluorouracil (5-FU), capecitabine, tivozanib, irinotecan, oxaliplatin, cisplatin, trifluridine, tipiracil, leucovorin, gemcitabine, regorafinib, or erlotinib hydrochloride.

In some embodiments, the antibody is used in combination with one or more other therapeutic agents that target an immune checkpoint modulator or a costimulatory agent, such as, for example, without limitation, an agent that targets CTLA-4, LAG-3, B7-H3 , B7-H4, B7-DC (PD-L2), B7-H5, B7-H6, B7-H8, B7-H2, B7-1, B7-2, ICOS, ICOS-L, TIGIT, CD2, CD47, CD80, CD86, CD48, CD58, CD226, CD155, CD112, LAIR1, 2B4, BTLA, CD160, TIM1, TIM-3, TIM4, VISTA (PD-H1), OX40, OX40L, GITR, GITRL, CD70, CD27, 4 -1BB, 4-BBL, DR3, TL1A, CD40, CD40L, CD30, CD30L, LIGHT, HVEM, SLAM (SLAMF1, CD150), SLAMF2 (CD48), SLAMF3 (CD229), SLAMF4 (2B4, CD244), SLAMF5 (CD84) ), SLAMF6 (NTB-A), SLAMCF7 (CS1), SLAMF8 (BLAME), SLAMF9 (CD2F), CD28, CEACAM1 (CD66a), CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, CEACAM8, CEACAM1-3AS CEACAM3C2, CEACAM1- 15, PSG1-11, CEACAM1-4C1, CEACAM1-4S, CEACAM1-4L, IDO, TDO, CCR2, CD39-CD73-adenosine pathway (A2AR), BTK, TIK, CXCR2, CXCR4, CCR4, CCR8, CCR5, CSF- 1 or m modulator of the innate immune response.

In some embodiments, the antibody is used in combination with, for example, an anti-CTLA-4 antagonist antibody, such as, for example, ipilimumab; an anti-LAG-3 antagonist antibody such as BMS-986016 and IMP701; an anti-TIM-3 antagonist antibody; an anti-B7-H3 antagonist antibody such as, for example, MGA271; an anti-VISTA antagonist antibody; an anti-TIGIT antagonist antibody; antibody; anti-CD80 antibody; anti-CD86 antibody; an anti-B7-H4 antagonist antibody; an anti-ICOS agonist antibody; an anti-CD28 agonist antibody; an innate immune response modulator (eg, TLR, KIR, NKG2A); and an IDO inhibitor.

In some embodiments, the antibody is used in combination with an OX40 agonist, such as, for example, an anti-OX-40 agonist antibody. In some embodiments, the antibody is used in combination with a GITR agonist, such as, for example, an anti-GITR agonist antibody, such as, for example, without limitation, TRX518. In some embodiments, the antibody is used in combination with an IDO inhibitor. In some embodiments, the antibody is used in combination with a cytokine therapy such as, for example, without limitation, IL-15, CSF-1, MCSF-1, etc.

In some embodiments, the antibody is used in combination with one or more other therapeutic antibodies, such as, for example, without limitation, an antibody that targets CD19, CD22, CD40, CD52, or CCR4.

In some embodiments, the antibody composition comprises at least one additional agent such as bevacizumab, cetuximb, sirolimus, panitumumab, 5-fluorouracil (5-FU), capecitabine, tivozanib, irinotecan, oxaliplatin, cisplatin, trifluridine, tipiracil, leucovorin, gemcitabine and erlotinib hydrochloride.

In some embodiments of the invention, antibody therapy can be carried out together or sequentially before or after treatment with another agent at intervals of several minutes to several weeks. In embodiments in which other agents and/or proteins or polynucleotides are administered separately, it is generally necessary that the period of time between each delivery does not exceed the period of time during which the agent and composition of the present invention are still able to provide a beneficial combined effect on the subject. . In such cases, it is expected that both administrations should be carried out at intervals of about 12-24 hours, and more preferably at intervals of about 6-12 hours. However, in some situations, in which the time between the respective injections is from several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8), it may be it is desirable to significantly increase the time of administration.

In some embodiments, an antibody of the present invention is combined with a treatment regimen further comprising conventional therapy selected from the group consisting of: surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, hormonal therapy, angiogenesis inhibition, and palliative care.

The composition of the present invention may further comprise pharmaceutically acceptable carriers, adjuvants or stabilizers (Remington: The Science and Practice of Pharmacy 21st Ed., 2005, Lippincott Williams and Wilkins, Ed. KE Hoover) in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at certain doses and concentrations and may include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrans; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are listed below in the present description.

In one aspect, the present invention provides a method for producing an antibody of the present invention, comprising culturing a host cell under conditions that result in the production of an antibody of the present invention and purifying the antibody or bispecific antibody from the culture.

In another aspect, the present invention relates to the use of an antibody, pharmaceutical composition, polynucleotide, vector or host cell as disclosed herein in the manufacture of a medicament for the detection, diagnosis and/or treatment of a tumor antigen associated disorder.

Application in diagnostics

In one aspect, the anti-CD3 antibodies of the present invention can be used to detect and/or measure CD3 or cells expressing CD3 in a sample, for example, for diagnostic purposes. For example, an anti-CD3 antibody can be used to diagnose a condition or disease characterized by aberrant expression (eg, overexpression, underexpression, lack of expression, etc.) of CD3. Exemplary CD3 diagnostic assays may include, for example, contacting a sample obtained from a subject with an anti-CD3 antibody of the invention, wherein the CD3 antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-CD3 antibody can be used for diagnostic purposes in combination with a secondary antibody that is itself a detectable label. The detectable label or reporter molecule may be a radioisotope such as ¹⁴ C, ³ H, ¹²⁵ I, ³² P or ³⁵ S; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine; or an enzyme such as alkaline phosphatase, beta-galactosidase, horseradish peroxidase or luciferase. Specific exemplary assays that can be used to detect or measure CD3 in a sample include, without limitation, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorting (FACS), and the like. Samples that may be used in the CD3 diagnostic assays of the present invention include any tissue or fluid sample obtained from a patient that contains detectable amounts of CD3 protein or fragments thereof under normal or pathological conditions. Typically, CD3 levels in a specific sample from a healthy patient (eg, a patient not suffering from a disease or condition associated with abnormal CD3 levels or activity) is measured to determine a baseline or standard CD3 level. This baseline CD3 level can then be compared with CD3 levels measured in samples obtained from individuals suspected of having a disease or condition associated with CD3.

In another aspect, a method for detecting, diagnosing, and/or monitoring a condition associated with tumor antigen expression is provided. For example, polyspecific antibodies disclosed herein may be labeled with a detectable moiety, such as a visualizer and an enzyme-substrate tag. The antibodies of the present invention can also be used for in vivo diagnostic assays, such as in vivo imaging (eg, PET or SPECT), or with a staining reagent.

Sets

In a further aspect of the invention, a kit is provided comprising an antibody as described above and instructions for use in accordance with any of the methods of the invention disclosed herein. Typically, these instructions contain instructions for administering the antibody in the therapeutic procedures described above. This kit includes any pharmaceutical composition disclosed herein. The pharmaceutical compositions and other reagents may be present in the kits in any convenient form, such as a solution or powder.

In another aspect, the kit further comprises one or more other prophylactic or therapeutic agents useful in the treatment of cancer in one or more containers. In one embodiment, the other prophylactic or therapeutic agent is a chemotherapeutic agent. In other aspects, the prophylactic or therapeutic agent is a biological or hormonal therapeutic agent.

Certain aspects of pharmaceutical compositions, prophylactic or therapeutic agents of the invention are preferably tested in vitro for the desired therapeutic activity in a cell culture system and in a model animal, such as a rodent model system, prior to use in humans.

The toxicity and effectiveness of the prophylactic and/or therapeutic protocols of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD ₅₀ (dose lethal in 50% of the population) and ED ₅₀ (therapeutically effective dose in 50% of the population ). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD ₅₀ /ED ₅₀ . Prophylactic and/or therapeutic agents showing high therapeutic performance are preferred.

In addition, any assay known to those skilled in the art can be used to evaluate the prophylactic and/or therapeutic use of the treatments or combination treatments disclosed herein for the treatment or prevention of cancer.

Instructions regarding the use of a bispecific antibody disclosed herein typically include information on the dose, dosing regimen, and route of administration for the intended treatment. The containers may be unit dosage forms, bulk packs (eg, multi-dose packs), or divisible unit doses. The instructions provided in the kits of the invention are usually written instructions on a label or package insert (for example, a sheet of paper included in the kit), but machine-readable instructions (for example, instructions stored on a magnetic or optical storage device) are also acceptable.

The kits of the invention are in suitable packaging. Suitable packaging includes, but is not limited to, vials, ampoules, test tubes, bottles, jars, flexible packaging (eg sealed mylar or plastic bags), and the like. for each pharmaceutical composition and other included reagents, such as buffers, balanced salt solutions, etc., used when administering pharmaceutical compositions to subjects. Packages are also provided for use in combination with a particular device such as an inhaler, a nasal device (eg a nebulizer) or an infusion device such as a minipump. The kit may have a sterile inlet (for example, the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). The container may also have a sterile inlet (for example, the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an antibody. The container may further contain a second pharmaceutically active agent.

Typically, the kit comprises a container and a label or insert(s) on or associated with the container.

Deposition of biological material

Presented materials of the present invention were deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, USA, February 13, 2018. Strand A of vector GUCY2C-1608, (VH-Hole SEQ ID NO: 94 ), having ATCC accession number PTA-124943, contains a DNA insert encoding the VH chain containing the pit of the bispecific antibody GUCY2C-1608, which includes a polynucleotide (SEQ ID NO: 70) encoding the variable region of the anti-CD3 antibody light chain (SEQ ID NO: 3); and the B strand of the GUCY2C-1608 vector (VL-Knob, SEQ ID NO: 93) having the ATCC accession number PTA-124944 contains a DNA insert encoding the VL strand containing the overhang of the bispecific antibody GUCY2C-1608, which contains a polynucleotide (SEQ ID NO: 71) encoding the heavy chain variable region of an anti-CD3 antibody (SEQ ID NO: 4).

These deposits were received in accordance with the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure and the Establishment of Rules Based on that Treaty (the Budapest Treaty). This ensures that the culture of the deposit is kept viable for 30 years from the date of deposit. Deposits are available at ATCC subject to the terms of the Budapest Agreement and subject to an agreement between Pfizer Inc. and ATCC, which guarantees permanent and unrestricted user access to the progeny of the deposit culture after the publication of the relevant US patent or after the release of any US patent application or foreign patent application, whichever comes first, and which also guarantees access to the offspring of a party designated by the U.S. Patent and Trademark Commissioner as eligible under 35 USC 122 and related commissioner rules (including 37 CFR § 1.14 with special reference to 886 OG 638).

The owner of this application agrees that in case of death, loss or destruction of the culture of materials on deposit when cultivated under acceptable conditions, the materials will immediately, with notification, be replaced with other similar materials. Availability of deposited material should not be construed as a license to practice the invention in violation of rights guaranteed by any authority under local patent law.

The following examples of specific aspects of the implementation of the present invention are offered solely for the purpose of illustration and are in no way intended to limit the scope of the present invention.

Example 1

Creation of soluble CD3ed

A protein named CD3ed representing the extracellular domains of the epsilon and delta subunits of CD3 fused in tandem through a short linker (SEQ ID NO: 39) was generated by standard molecular biology, protein expression and purification methods. The gene carrying the plasmid encoding the amino acid shown in Table 9 (SEQ ID NO: 40) was transiently transfected into HEK293 cells in a 20 liter culture volume using PEI as the transfection agent. After incubation for 7 days at 37° C., 20 L of the crop was bound in portions with 50 ml of His60 resin (equilibrated with PBS) overnight. The bound resin was then washed first with PBS, then with 10 mM imidazole containing PBS buffers (10-15 CV). After washing, the protein was eluted with 200 mM imidazole containing phosphate buffer. After that, the eluate fractions were analyzed using SDS-gel. Fractions containing pure protein were pooled and concentrated using Amicon 3 kDa ultra centricons. During the process, the concentration of glycerol in the sample was maintained at 5% to maintain protein stability. The His60 pool was additionally loaded onto a Superdex200 SEC column. Fractions containing the epsilon/delta CD3 heterodimer were analyzed by SDS-PAGE, the appropriate fractions were pooled and filtered through a 0.22 μm filter. Table 9 shows the sequence of CD3 epsilon/delta subunits fused in tandem and used as antigen for all optimization work.

Table 9

SEQID NO: Protein Subsequence 40 CD3 epsilon/delta fusion protein MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARGGGGSGGGGSGGGGSPIEELEDRVFVNCNTSITWVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHHY

Example 2

Structure optimization of the anti-CD3 antibody H2B4

The lead anti-CD3e (CD3-epsilon) antibody H2B4 showed lower thermal stability when used in the bispecific diabody-Fc format. To increase stability in the light chain scaffold, VK4 was replaced with VK1 (DPK9) and the heavy chain scaffold was saved in VH3 (DP54) format. VL CDR1 (SEQ ID NO: 26), VL CDR2 (SEQ ID NO: 27), and VL CDR3 (SEQ ID NO: 28) were transferred to the DPK9 framework and expressed in combination with the heavy chain CDRs VH CDR1 (SEQ ID NO: 28). : 13), VH CDR2 (SEQ ID NO: 16) and VH CDR3 (SEQ ID NO: 18). In addition to the CDRs grafted onto the working scaffold, some backmutations were also tested. Backmutations introduced into both the heavy and light chain scaffolds are shown in Table 10 with the number of the residue at which the backmutation was performed.

cDNAs were synthesized containing the human acceptor framework, heavy chain VH3 and light chain VK1 with the appropriate donor CDR sequences. The synthesized cDNA products were subcloned and fused in frame with the human IgG1 heavy chain and human kappa light chain constant region in mammalian expression vectors. To be able to distinguish from the H2B4 antibody, these variants are referred to as 2B5, and all optimized molecules are referred to hereinafter as 2B5 and variants thereof. All 2B5 mutants were analyzed for competitive binding to Jurkat cells with endogenous CD3e expression.

Competitive FACS was performed to check if the humanized 2B5 retained its binding epitope on the cell surface. Chimeric cH2B4 was biotinylated prior to competitive FACS. The EC ₅₀ value for binding of biotinylated cH2B4 to Jurkat cells was determined by direct binding using FACS analysis, which was 0.8 nM. For competitive FACS, 3-fold serially diluted antibody variants were mixed with a constant amount of biotin-cH2B4 representing an EC ₅₀ concentration and 1.0+E05 Jurkat cells per well. The mixtures were incubated for 1 hour on ice, followed by washing three times with FACS buffer. The secondary antibody, streptavidin conjugated to PE (phycoerythrin) (PE-SA), was added to the cells and incubated for 30 minutes on ice. The mean fluorescence intensity (MFI) was then analyzed by FACS on a FACS CANTO™ analyzer (BD Biosciences). Y-axis calculation: % binding up to Max. = [MFI (wells with serially diluted Ab) - MFI (2nd PE-SA)]/[max. MFI (Jurkat cells with biotin-cH2B4 only) - MFI (2nd PE-SA)]. Table 10 shows the IC ₅₀ values resulting from competitive binding, with mutations introduced into the CD3 H2B4 antibody to optimize scaffolds. Based on these results, H2B4 1.0/1.0T showed binding characteristics similar to H2B4 and is designated as the binding domain of the anti-CD3 antibody 2B5.

Table 10

Antitheor heavy chain light chain IC ₅₀ competitive binding h2B4 1.0/1.0T (2B5) Grafted CDRs Grafted CDRs, R24T 1.27 h2B4 1.0/1.1 vH1.0 Q13V, R24T, Y49S, L78V 1.68 h2B4 1.0/1.2 vH1.0 Q13V, R24T, Y49S Not tested h2B4 1.0/1.3 vH1.0 R24T, Y49S 1.309 c2B4 (chimeric 2B4) mouse vH mouse vK 0.752

Example 3

Creation and characterization of a bispecific diabody-Fc molecule using the H2B4 and 2B5 sequences

Bispecific diantibody-Fc molecules were generated using the 2B5 or H2B4 anti-CD3 antibody sequences (Tables 2 and 3) and the GUCY2c antibody in one of the configurations shown in Figure 1 (schematic) using standard expression and purification methods known in the art. . The resulting bispecific antibodies (Table 7; SEQ.ID.NO.36-38) were tested for stability by differential scanning calorimetry.

Example 4

Assessing the Stability of Bispecific Antibodies Using Differential Scanning Calorimetry

Proteins were diluted with phosphate buffered saline (PBS) to 0.6 mg/ml in a volume of 400 μl. PBS was used as a blank buffer in the control well. PBS contained 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na ₂ HPO ₄ and 1.47 mM KH ₂ PO ₄ , pH 7.2. Samples were placed in a MicroCal VP-Capillary DSC sample tray with autosampler (Malvern Instruments Ltd, Malvern, UK). Samples were equilibrated for 5 minutes at 10°C and then scanned to 110°C at a rate of 100°C per hour. The filtration period was chosen to be 16 seconds. Baseline data was corrected to baseline and protein concentration was normalized. Origin 7.0 software (OriginLab Corporation, Northampton, MA) was used to fit the data to the MN2-State model with the appropriate number of hops. As shown in FIG. 2, the bispecific GUCY2c-2B5 showed an improved first order phase transition by 5°C compared to the bispecific GUCY2c-H2B4. This indicates that bispecific antibodies containing 2B5 are more stable.

The two bispecific antibodies were tested for T cell binding by FACS analysis using standard procedures known in the art and as shown in FIG. 3, both bispecific antibodies showed approximately the same binding to T cells, indicating retention of binding when converting H2B4 to 2B5.

Cytotoxicity of both H2B4 and 2B5 bispecific antibodies was also assessed to evaluate T cell redirection activity. As shown in FIG. 4, both bispecific antibodies showed similar cytotoxicity, indicating that 2B5 activity remained the same as that of H2B4.

Example 5

Structural rational mutagenesis 2B5

The H2B4 binding domain of the anti-CD3 antibody showed polyreactivity as measured by DNA and insulin binding, a tendency to self-assemble as evidenced by high AC-SINS scores, and a tendency to clip at position R53 in CDR-H2 when expressed in cells- SNO owners. To reduce polyreactivity, self-assembly and truncation in CDRH2, mutations were introduced into CDR 2B5 to maintain binding affinity and stability. To this end, all predictions were made based on the X-ray diffraction crystal structure of the CD3 H2B4 antibody complexed with a peptide representing a binding epitope derived from the N-terminus of the CD3 epsilon subunit. It is well known in the art that excess positive charge and/or hydrophobicity can play an important role in polyreactivity. The Poisson-Boltzmann DelPhi calculator in Discovery Studio 4.01 was used to determine the electrostatic surface Fv of the H2B4 region based on the crystal structure. In FIG. 5 you can see spots of excess positive charge, which are not destroyed by negative charges. This suggests a correlation with high polyreactivity. In addition, the Spatial Aggregation Propensity (SAP) tool in Discovery Studio1 was used to determine excessive hydrophobic patches (FIG. 6). As shown in FIG. 6, the hydrophobic spot appears small and insignificant. Based on this observation, the focus has been on reducing positive charges by removing positive charges or adding negative charges to destroy spots.

In order to determine suitable positions for mutagenesis, mutations were calculated on the computer using the Discovery Studio and FoldX methods to change both affinity and stability. Mutations were classified as tolerant mutations, which, according to the predictions of both Discovery Studio and FoldX methods, did not give a ΔΔG value >1 kcal/mol. Based on these predictions, a set of residues were identified that were expected to have the least effect on binding and stability while still destroying charged spots. In addition, preference was given to hydrophilic mutations where possible so as not to increase the hydrophobic surface. A list of possible CDR mutations introduced into the 2B5 anti-CD3 antibody to optimize biophysical properties is shown in Table 11. This includes mutations that are expected to be tolerant at every position and reduce polyreactivity and the risk of truncation.

Table 11

Remainder # relative to 2B5-0001 (by Kabat) WT amino acid Amino acids mutated to heavy chain H52 R Q H52B R Q H53 R Q L E H65 K Q H96 R Q light chain L27F R T S L29 R Q N L30 K Q

In addition, a set of several mutations (in combination) for the heavy chain and light chain was developed. These include R52Q/R52bQ/R53Q, R52Q/R52bQ/R53Q/R96Q, R52bQ/R53Q/R96Q and R52Q/R52bQ/R96Q for the heavy chain and R29Q/K30Q/R54L/V27eE, R29Q_R54L_V27eEE30, R29Q_R54Q_R54Q

Example 6

Characteristics of mutants

All mutants were generated as IgG molecules using standard expression and purification methods well known in the art and tested for CD3ed binding, polyreactivity (DNA and insulin binding) and AC-SINS.

Binding assays to evaluate variants

An Octet assay was designed and performed to study association and dissociation rates of 10 μg/ml soluble human CD3ed antigen (SEQ ID NO: 40). Anti-human IgG tips were used to capture anti-CD3 antibodies at a concentration of 10 μg/ml for 120 seconds, followed by a baseline in PBS for 30 seconds, after which the tips were immersed in a concentration of 10 μg/ml of soluble human CD3 antigen on 120 seconds, and finally performed dissociation for 300 seconds. Sensograms were studied to determine if the antigen was bound to the antibody and Octet software was used to calculate kd (1/s) and kd error. Twenty-six rational mutations maintained binding to the hCD3ed antigen. The soluble CD3 binding kinetics of twenty-six CD3 antibody variants that still showed antigen binding were then studied by SPR using a Biacore assay. Anti-CD3 antibodies were captured with anti-human Fc and soluble CD3 antigen at concentrations of 0, 3.7, 11.1, 33.3 and 100 nM was injected over the captured antibodies to determine binding kinetics. Twenty-three anti-CD3 antibodies bound to the soluble CD3 antigen with the same affinity as the original CDR-grafted antibody.

DNA and insulin ELISA to measure polyreactivity

384-well ELISA plates (Nunc Maxisorp) were coated with DNA (10 μg/ml) (Sigma-Aldrich, D1626) and insulin (5 μg/ml) (Sigma-Aldrich, I9278-5 ml) in PBS, pH 7.5 overnight at 4°C. ELISA adapted from the assays described in Tiller et al (17) was performed on a PerkinElmer Janus automated liquid handling workstation. The wells were washed with water, blocked with 50 μl of polyreactive ELISA buffer (PEB; PBS containing 0.05% Tween-20, 1 mM EDTA) for 1 hour at room temperature and washed three times with water. Serially diluted mAbs in 25 μl were added in quadruplicate to the wells and incubated for 1 h at room temperature. The plates were washed three times with water and 25 μl of 10 ng/ml goat anti-human IgG (Face specific fragment) conjugated with horseradish peroxidase (Jackson ImmunoResearch, 109-035-008) was added to each well. The plates were incubated for 1 h at room temperature, washed three times with 80 μl of water, and 25 μl of TMB substrate (Sigma-Aldrich, T-0440) was added to each well. Reactions were stopped after approximately 7 minutes by adding 25 μl of 0.18 M phosphoric acid to each well and the absorbance was read at 450 nm. The amount of DNA and insulin binding was calculated as the ratio of the ELISA signal of the antibody at 10 μg/ml to the signal of the buffered well, not the primary antibody. Values below 8 were considered ideal and indicated low polyreactivity.

AC-SINS analysis to measure self-assembly propensity

The AC-SINS (Affinity Capture Nanoparticle Self Interaction Spectroscopy) analysis was standardized to a 384-well format for the Perkin-Elmer Janus Automated Fluid Handling System. 20 nm gold nanoparticles (Ted Pella, Inc., #15705) were coated with a mixture of 80% goat anti-human Fc (Jackson ImmunoResearch Laboratories, Inc. # 109-005-098) and 20% non-specific goat polyclonal antibodies (Jackson ImmunoResearch Laboratories, Inc. # 005-000-003), which were buffer replaced with 20 mM sodium acetate, pH 4.3, and diluted to 0.4 mg/ml. After one hour of incubation at room temperature, the areas not occupied by gold nanoparticles were blocked with thiolated polyethylene glycol (2 kDa). The coated nanoparticles were then concentrated 10-fold using a syringe filter and 10 μl was added to 100 μl of mAb at a concentration of 0.05 mg/ml in PBS pH 7.2. The coated nanoparticles were incubated with the antibody of interest for 2 hours in a 96-well polypropylene plate and then transferred to a 384-well polystyrene plate and read on a Tecan M1000 spectrophotometer. Absorbance was read in the range of 450-650 in 2 nm steps, and a Microsoft Excel macro was used to determine the maximum absorbance, smooth the data, and fit the data with a second order polynomial. The smoothed maximum absorbance of the average control sample (PBS buffer only) was subtracted from the smoothed maximum absorbance of the antibody sample to determine the AC-SINS score of the antibody. A result less than 8 was considered ideal and indicated a low degree of self-assembly.

Evaluation of Leading Variants of Anti-CD3 Antibody 2B5 in Diabody-Fc Bispecific Format

Comprehensive analysis of binding, AC-SINS, and polyreactivity data narrowed down the number of binding variants to five, which were reformatted into bispecific diabody-Fc molecules using antitumor sequences and standard cloning strategies well known in the art. These bispecific antibodies were transiently expressed and purified by standard methods well known in the art. The resulting bispecific antibodies were tested for binding to jurkat cells, in vitro cytotoxicity using T cell retargeting assay, polyreactivity, and thermal stability by DSC and AC-SINS. Of all five bispecific antibodies, only one bispecific variant, 2B5v6, carrying the anti-CD3 antibody 2B5, showed strong binding and, as a result, showed strong cytotoxicity. This variant also showed reduced polyreactivity as assessed by DNA and insulin binding and AC-SINS. The 2B5v6 bispecific antibody values were below 8, in contrast to the values observed for the H2B4 bispecific antibody, which are above 8. Based on these results, the 2B5v6 variant was selected as the optimized 2B5 variant for further analysis.

Example 7

Truncation evaluation in CDR-H2

Clipping in VH CDR2 (SEQ ID NO: 16) at position R53 was observed in anti-CD3 antibody (H2B4 and 2B5) when the expression of the bispecific antibody occurred in CHO cells and not in HEK293 cells. In order to reduce this risk and provide better product uniformity on an industrial scale, care was taken to select mutations in the truncation region along with other mutations to mitigate other risks as described in the previous examples. Briefly, to evaluate truncation, diatelo-Fc bispecific antibodies were generated using the variable domain sequence of the antibody as a negative control and anti-CD3 antibody 2B5 or anti-CD3 antibody H2B4 or anti-CD3 antibody 2B5v6. All bispecific diabody-Fc molecules were prepared in a similar manner using expression and purification methods well known in the art. CHO SSI cell lines were created using standard procedures. The two strands encoding the bispecific antibody were cloned into the pRY19-GA-Q mammalian expression vector containing a dual promoter and a recombination site for single site integration (SSI) (Zhang, L. et al. Biotechnology Progress. 2015; 31:1645-1656) into the genome of the CHO cell. The resulting plasmid was transfected into CHO-K1 SV 10e9 cells by electroporation with the pRY19 vector containing the gene of interest, followed by positive and negative selection pressure using hygromycin-B and ganciclovir. These CHO pools went through a three week recovery phase. When the observed viability exceeded 90%, cell banks were created for future work. The generated CHO pools were then subjected to 12 day fed-batch expression in CD-CHO media (ThermoFisher Scientific, Waltham, MA) at a volume of 1 L. Cultures were harvested on day 12, passed through a protein A column and eluted at low pH. The proA pool was immediately neutralized to pH 8.1. The resulting pure proteins were evaluated by capillary gel electrophoresis (cGE) following standard protocols well known in the art. As shown in Table 12 and in FIG. 7, the 2B5 bispecific antibody had a significantly lower truncation rate compared to H2B4, and no truncation was observed in 2B5v6, while H2B4 showed an increased truncation rate or increased fragmentation. Table 12 presents the results of capillary gel electrophoresis showing fragmentation of CD3-GUCY2c bispecific antibodies. %POI indicates the peak of interest.

Table 12

Recovered cGE sample %POI % fragmentation Negative control-H2B4 88.8 11.2 Negative control-2B5 98.9 1.1 Negative control-2B5v6 99.6 0.4

Example 8

GUCY2c-2B5v6 Bispecific Antibodies: Expression and Purification of Chimeric CD3-GUCY2c Bispecific Antibodies with CD3 2B5v6 Antibody

Complementary pairs of constructs (12.5 μg each strand) were co-transfected into 25 ml log-phase cultures containing 1 million cells/ml HEK 293 cells using the ExpiFectamine™ 293 transfection kit (Life Technologies). Twenty-four hours after transfection, the transfection enhancer ExpiFectamine was added and cells were grown for an additional 4-5 days before harvest. The depleted cultures were then harvested, centrifuged to remove cell debris, and passed through a 20 µm filter.

Thereafter, purified conditioned medium containing bispecific antibodies to GUCY2c T cells was purified by protein A affinity chromatography. Samples were loaded onto 0.45 ml microcolumns (Repligen) prepackaged with MabSelect SuRe resin and protein A (GE Healthcare), using a liquid manipulator (Tecan). Bound protein was washed with PBS, pH 7.2, then eluted with 20 mM citric acid, 150 mM sodium chloride, pH 3.5 and neutralized with 2M Tris, pH 8.0. The samples were then desalted in PBS pH 7.2 using G25 Sephadex packed columns (GE Healthcare) according to the manufacturer's methods. Purified proteins were analyzed for purity by analytical size exclusion chromatography on a Mab HTP (TOSOH) column on an Aglient 1200 HPLC system according to the manufacturer's protocols. Concentrations were determined by measuring at OD280 nm with a microspectrophotometer (Trinean).

Assessing the Stability of Bispecific Antibodies Using Differential Scanning Calorimetry

Proteins were diluted with phosphate buffered saline (PBS) to 0.6 mg/ml in a volume of 400 μl. PBS was used as a blank buffer in the control well. PBS contained 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na ₂ HPO ₄ and 1.47 mM KH ₂ PO ₄ , pH 7.2. Samples were placed in the sample tray of a MicroCal VP-Capillary DSC calorimeter with autosampler (Malvern Instruments Ltd, Malvern, UK). Samples were equilibrated for 5 minutes at 10°C and then scanned to 110°C at a rate of 100°C per hour. The filtration period was chosen to be 16 seconds. Baseline data were corrected for baseline and protein concentration was normalized. Origin 7.0 software (OriginLab Corporation, Northampton, MA) was used to fit the data to the MN2-State model with the appropriate number of hops. As shown in FIG. 8, the GUCY2c-2B5v6 bispecific antibody showed excellent thermal stability with T _m 1=70°C.

Binding to naive T cells

Purified bispecific anti-CD3 antibodies were titrated for cell surface binding to human CD3 on naïve human T cells purified from fresh human peripheral blood mononuclear cells (PBMC) using standard flow cytometry techniques known in the art on a BD analyzer. LSRII Fortessa. As shown in FIG. 9, the bispecific GUCY2c-2B5v6 binds very well to T cells with an EC ₅₀ binding of 35.44 nM.

Assessing the activity of bispecific T cells

Human PBMCs were isolated from the blood of healthy donors using Histopaque-177 (Sigma). Naïve T cells were isolated from PBMCs using the Stem Cell Technologies T cell enrichment kit (negative T cell selection). GUCY2c T84-expressing human tumor cells transfected with a luciferase expression construct were treated with serial dilutions of GUCY2c bispecific antibodies or CD3 bispecific antibodies serving as a negative control along with T cells. The ratio of effector cells to target cells (E:T) was 10:1 or 5:1. T cells and tumor cells were incubated at 37° C. for 48 hours, followed by measurement of the luciferase signal using the Neolite reagent (Perkin Elmer). EC ₅₀ values were calculated using the Graphpad PRISM program using a four-parameter non-linear regression analysis. HCT116 or HT29 negative target cells did not exhibit GUCY2c mediated bispecific cytotoxicity. As shown in FIG. 10, bispecific GUCY2c-2B5v6 showed strong cytotoxicity in a T cell retargeting assay.

T84 cells were treated with T cells (E:T ratio=5:1) and GUCY2c-1608 (GUCY2c-2B5v6) at different doses. Supernatants were collected at different time points (24 hours, 48 hours, 96 hours) and analyzed by Lumninex multiplex analysis according to the manufacturer's instructions and read on a Luminex 200 using xPONENT software. The cytokines measured were human IFN-gamma, IL10, IL2, IL4, IL6, TNF-alpha. In FIG. 11 shows increased expression of the cytokines (11A) IFN-gamma, (11B) IL10, (11C) IL2, (11D) IL4, (11E) IL6, (11F) TNF-alpha after activation of GUCY2c-1608 T cells.

In vivo assessment of activity mediated by bispecific GUCY2c-2B5v6

An in vivo efficacy study was performed on an adoptive transfer model. Tumor cell lines/xenograft fragments obtained from the patient were implanted in NSG mice and adjusted to a volume of approximately 200 mm ³ . Mice were intravenously injected with bispecific antibodies (unless otherwise indicated). Two million activated and expanded T cells (using T cell expansion and activation kits from Miltenyi Biotec) were administered intravenously 24 hours after administration of the bispecific compounds. Bispecific antibodies were administered weekly. As shown in FIG. 12 and FIG. 13, the GUCY2c-2B5v6 bispecific antibody (GUCY2c-1608) showed excellent robust tumor regression in both the CRX-11201 PDX model and the in vivo LS1034 cell line representing colorectal cancer.

Example 9

FLT3-CD3 bispecific antibodies and generation of FLT3-2B5v6 bispecific IgG using optimized anti-CD3 antibodies (2B5v6) and cytotoxicity

Human anti-FLT3 antibody (xFLT3) and human anti-CD3 antibody (2B5v6) were expressed separately as human IgG2dA_D265A engineered with EEEE on one arm and RRRR on the other arm for bispecific exchange at positions 223, 225 and 228 (e.g., (C223E or C223R), (E225E or E225R) and (P228E or P228R)) in the hinge region and at position 409 or 368 (e.g. K409R or L368E (EU numbering scheme)) in the CH3 region of human IgG2. The antibodies also had a D to A mutation at position 265 (EU numbering scheme).

Each antibody arm was purified separately using protein A resin. Then, one mg/ml of purified xFLT3 antibody was replaced with 1 mg/ml of purified 2B5v6 antibody in the presence of 2 mM reduced glutathione. The exchange reaction was carried out at 37°C for 16 hours. Then, 2 mM oxidized glutathione was added to the proteins and the mixture was incubated at 37° C. for 30 minutes. The mixture was then diluted 1:5 with 20 mM MES buffer pH 5.4 without NaCl. The diluted sample was loaded onto a MonoS ion exchange column and washed with 20 mM MES, pH 5.4, 25 mM NaCl buffer. The xFLT3-2B5v6 bispecific antibody was then eluted with a gradient of 0-500 mM NaCl in 20 mM MES buffer, pH 5.4. Finally, the bispecific antibody was dialyzed in phosphate buffered saline (PBS) for cell analysis. For cytotoxicity assays, unstimulated human T cells and target cells expressing luciferase (Eol-1 or MV411) were co-incubated at specific ratios in 96 U-bottom well plates in 200 μl RPMI+10% FBS. The bispecific antibodies xFLT3-2B5v6 were serially diluted in PBS and added to the wells. After 24 hours, 100 μl of the cell suspension was mixed with 100 μl of One-GLO reagent in 96-well white wall plates and luminescence was measured on an Envision luminometer. Cytotoxicity values were determined by the following formula:

% cytotoxicity = [1- (RLU sample)/RLU target cell)] × 100RLU: relative light units

As shown in FIG. 14A and 14B, the xFLT3-2B5v6 bispecific antibody containing an optimized anti-CD3 antibody (2B5v6) was found to be very effective in an alternative IgG bispecific format when tested in both cell types expressing higher copy number of FLT3 (EOL-1) and lower copies (MB4-11).

Table 13 shows the light and heavy chain sequences of FLT3 (EOL-1) and the light and heavy chains of the 2B5v6 bispecific antibodies.

Table 13

mAb light chain heavy chain xFLT3 EIVLTQSPATLSLSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYDTFTRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQYGSSPPTTFGQGTRLEIK
(SEQ ID NO: 41) EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMNWVRQAPGKGLEWVSAISGGGRSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSPSDVGWGYGFDIWGQGTLVTVSS
(SEQ ID NO: 42) 2B5v6 EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSS
(SEQ ID NO: 4) DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIK
(SEQ ID NO: 3)

Example 10

Reducing the risk associated with the presence of T-cell epitopes of the bispecific antibody 2B5v6

The optimized clone 2B5v6 was analyzed for potential T-cell epitopes with the desire to reduce the risk of immunogenicity. For this analysis, the Epivax Tool was used for in silico prediction of potential T-cell epitopes. This tool predicts the number of 9-mer backbone peptides binding to any of 8 different HLA alleles. Hits were defined as hits with Z-scores in the top 5% and strong hits in the top 1%. Hits on 4 or more alleles or a 1-strong hit were considered predicted T-cell epitopes. Based on these criteria, 3 predicted non-germline T-cell epitopes in CDRH2, 2 in CDRL1, and 1 predicted germline epitope in L2 were identified (Table 15). In addition to the scores for individual peptides, the overall risk for the sequence was determined by total hits or an adjusted Epivax T-regitop score, which was optimized for correlation with the incidence of clinical ADA. In this case, for these predicted values, a lower score meant a lower expected risk of developing ADA. In this case, the VH domain had 70 hits with a score of -42.82, and the VL domain had 60 hits with a score of -23.79.

To reduce the risk of immunogenicity, mutations were identified that would reduce the overall score of this clone while removing hits and, ideally, identify predicted T-cell epitopes. To identify single mutations that reduce hit counts, reduce the overall T regitop adjusted EpiMatrix score, and remove T cell epitopes, all possible single mutant changes in CDR-L1, CDR-L2, and CDR-H2 were considered. Because these potential mutations can also affect binding and stability, the predicted stability and affinity data described above were taken into account, and tolerant mutations were identified. Tables 14A, 14B and 14C show mutations identified as reducing the risk of 2B5v6 immunogenicity. In addition, it is assumed that the mutations shown in SEQ ID NO: 44-47, 51-57, 60 and 62, reduce the number of T-cell epitopes.

Table 14A

# remainder VH CDR2 in 2B5v6 WT remainder Mutated on Chothia Kabat H50 H50 F H H52A H52A N D H52B H52B Q D H52C H52C A D H55 H55 Y E H56 H56 T P H57 H57 S D H60 H60 N Q

Table 14B

# remainder VL CDR1 in 2B5v6 WT remainder Mutated on Chothia Kabat wt Mut L26 L26 S D L27 L27 Q E L28 L27A S D L30B L27E V E L30D L28 S T L30E L29 Q G L30F L30 K Q L31 L31 N E L34 L34 A N

Table 14C

# remainder of VL CDR2 in 2B5v6 WT remainder Mutated on Chothia Kabat wt Mut L51 L51 A T L53 L53 T D L54 L54 R D L54 L54 R Q L56 L56 S D germ line germ line WASTRES
(SEQ ID NO: 27) AASSLQS
(SEQ ID NO: 43)

Table 15 shows regions of the variable domain of an anti-CD3 antibody (2B5v6) with T-cell epitopes that pose a potential risk of developing immunogenicity.

Table 15

VH/VL Peptide Hits hard hits vh VQPGGSLRL
(SEQ ID NO: 44) 2 0 vh LRLSCAASG
(SEQ ID NO: 45) 7 four vh WVRQAPGKG
(SEQ ID NO: 46) four 3 vh VRQAPGKGL
(SEQ ID NO: 47) 3 0 vh WVAFIRNQA
(SEQ ID NO: 48) 2 one vh FIRNQARGY
(SEQ ID NO: 49) 5 2 vh YTSDHNPSV
(SEQ ID NO: 50) four 2 vh VKGRFTISR
(SEQ ID NO: 51) 2 0 vh FTISRDNAK
(SEQ ID NO: 52) 5 0 vh LYLQMNSLR
(SEQ ID NO: 53) 6 2 vh YLQMNSLRA
(SEQ ID NO: 54) eight 5 vh LQMNSLRAE
(SEQ ID NO: 55) 2 0 VL IQMTQSPSS
(SEQ ID NO: 56) 6 one VL MTQSPSSLS
(SEQ ID NO: 57) 7 0 VL ITCTSSQSL
(SEQ ID NO: 58) 3 one VL VRSQKNYLA
(SEQ ID NO: 59) eight 5 VL WYQQKPGKA
(SEQ ID NO: 60) 5 0 VL LLIYWASTR
(SEQ ID NO: 61) 2 one VL FTLTISSLQ
(SEQ ID NO: 62) 7 5

All mutants were generated as monovalent IgG antibodies using standard HEK293 transient expression followed by protein A column purification. The resulting clones were tested for binding to CD3-expressing Jurkat cells by FACS analysis and self-assembly potential was assessed by AC-SINS as described in Example 7 above. As shown in Table 16, five molecules with improved Epivax score and reduced T-cell epitopes and retained affinity for CD3 were selected for further testing and tested in diatelo-Fc format. The 2B5v6 variants with improved Epivax scores were compared to the parent 2B5v6. The results show comparable binding to Jurkat cells expressing CD3 and AC-SINS.

Table 16

Option EC50 (nM) Jurkat cells AC-SINS Epivax score 2B5v6-0453 11.1 0 -47.18 2B5v6-0517 9.9 one -42.92 2B5v6-0522 8.6 0 -46.6 2B5v6-0533 6.1 one -38.8 2B5v6-0538 9.2 0 -42.47 2B5v6-0453 11.1 0 -47.18

Among the mutants tested, several mutants showed lower binding to Jurkat cells compared to 2B5v6. Of these mutants, mutants with all T-cell epitopes removed and improved Epivax scores were used to generate diatelo-Fc bispecific molecules with the GUCY2c antibody as the other binding domain. Table 17 shows the Epivax scores and FIG. 15 shows reduced binding of these mutants to jurkat cells. Variants of the low affinity CD3 antibody 2B5v6 have improved Epivax scores.

Table 17

Anti-CD3 antibody variants Epivax score
(tReg-adapted) 2B5v6-0598 -65.15 2B5v6-0623 -56.74

Example 11

Three anti-CD3 variants show improved affinity and cytotoxicity in the form of GUCY2C-CD3 bispecific antibodies

Three anti-CD3 variants: 2B5-1038 (SEQ ID NOS: 4 and 9), 2B5-1039 (SEQ ID NOS: 4 and 87) and 2B5-1040 (SEQ ID NOS: 4 and 89), which were derived from 2B5v6, reformatted into bispecific diabody-Fc molecules fused with the antitumor antibody GUCY2c sequence in one of the configurations shown in FIG. 1 using the standard cloning, expression and purification methods described above. The resulting bispecific antibodies shown in Table 18 were tested for in vitro cytotoxicity by T cell retargeting assay and for binding affinity by surface plasmon resonance (SPR) assay and nonspecificity by AC-SIN. The risk of immunogenicity was assessed using the Epivax Tool for in silico prediction of potential T-cell epitopes. Raw mass spectroscopy data show that all bispecific antibodies are correctly paired and are 100% heterodimers. Table 18 shows that all three bispecific antibodies (1) are approximately 2-fold more potent, as measured by in vitro cytotoxicity analysis, compared to the control bispecific GUCY2C-1608, which is paired with anti-CD3 2B5v6 (FIG. 16); (2) have a binding affinity for recombinant human CD3 according to SPR results, which is consistent with cytotoxic activity; (3) have higher levels of immunogenicity compared to controls; and (4) AC-SIN scores remained the same as control bispecific GUCY2C-1608. These results demonstrate that anti-GUCY2c bispecific antibodies with various CD3 variants recruit naive human T cells to induce cell killing in T84 tumor cells (FIG. 16). The three anti-CD3 variants derived from 2B5v6 showed approximately 2-fold potent activity compared to the control bispecific GUCY2C-1608.

Table 18

Bispecific antibody Option
CD3 Epivax score (tReg-adapted) MassSpec (intact) score
AC-SIN In vitro cytotoxicity (nM) KD (nM, SPR) GUCY2C-1608 2B5-0542 -33.7 100% heterodimer 3 0.772 127.88±1.63 GUCY2C-1678 2B5-1038 -52.61 100% heterodimer 2 0.247 63.55±0.49 GUCY2C-1679 2B5-1040 -47.58 100% heterodimer 3 0.194 72.02±0.36 GUCY2C-1680 2B5-1039 -53.51 100% heterodimer 3 0.385 78.57±0.9

The sequences of the bispecific antibodies are shown in Table 19.

Bispecific antibody First polypeptide chain Second polypeptide chain GUCY2C-1608 DIQLTQSPSSLSASVGDRVTITCRASESVDYYGSSLLQWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTRKAYTFGQGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(SEQ ID NO: 93) DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIKGGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLEWIGEIKPSNELTNVHEKFKDRFTISVDKAKNSAYLQMNSLRAEDTAVYYCTRTITTTEGYWFFDVWGQGTLVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(SEQ ID NO: 94) GUCY2C-1678 DIQLTQSPSSLSASVGDRVTITCRASESVDYYGSSLLQWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTRKAYTFGQGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(SEQ ID NO: 95) DIQMTQSPSSLSASVGDRVTITCTSDQSLFNVRSGKNYLAWYQQKPGKAPKLLIYWASDRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIKGGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLEWIGEIKPSNELTNVHEKFKDRFTISVDKAKNSAYLQMNSLRAEDTAVYYCTRTITTTEGYWFFDVWGQGTLVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(SEQ ID NO: 96) GUCY2C-1679 DIQLTQSPSSLSASVGDRVTITCRASESVDYYGSSLLQWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTRKAYTFGQGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(SEQ ID NO: 97) DIQMTQSPSSLSASVGDRVTITCTSSQSLFNVRSGKNYLAWYQQKPGKAPKLLIYWASDRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIKGGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLEWIGEIKPSNELTNVHEKFKDRFTISVDKAKNSAYLQMNSLRAEDTAVYYCTRTITTTEGYWFFDVWGQGTLVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(SEQ ID NO: 98) GUCY2C-1680 DIQLTQSPSSLSASVGDRVTITCRASESVDYYGSSLLQWYQQKPGKAPKLLIYAASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTRKAYTFGQGTKLEIKGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSDYYMTWVRQAPGKGLEWVAFIRNQARGYTSDHNPSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRPSYYVLDYWGQGTTVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(SEQ ID NO: 99) DIQMTQSPSSLSASVGDRVTITCTSSESLFNVRSGKNYLAWYQQKPGKAPKLLIYWASDRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQSYDLFTFGGGTKVEIKGGGGSGGGGEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYWMHWVRQAPGKGLEWIGEIKPSNELTNVHEKFKDRFTISVDKAKNSAYLQMNSLRAEDTAVYYCTRTITTTEGYWFFDVWGQGTLVTVSSGCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
(SEQ ID NO: 100)

--->

SEQUENCE LIST

<110> Pfizer Inc.

Apgar, James

Jin, Fan

Katraghadda, Madan

Mathur, Divia

Chistyakova, Lyudmila Gennadievna

<120> CD3 SPECIFIC ANTIBODIES AND THEIR USES

<130> PC72375

<160> 100

<170>PatentIn version 3.5

<210> 1

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 1

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val

20 25 30

Arg Ser Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 2

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Asn Arg Ala Arg Gly Tyr Thr Ser Asp His Asn Pro

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr Val Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 3

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 3

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val

20 25 30

Arg Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 4

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 4

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr Thr Ser Asp His Asn Pro

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr Val Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 5

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 5

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Asn Arg Ala Gln Gly Tyr Thr Ser Asp His Asn Pro

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr Val Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 6

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 6

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val

20 25 30

Arg Ser Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 7

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr Thr Ser Asp His Gln Pro

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr Val Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 8

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 8

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val

20 25 30

Arg Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Asp Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 9

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 9

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Thr Ser Asp Gln Ser Leu Phe Asn Val

20 25 30

Arg Ser Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Asp Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 10

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 10

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Asn Gln Asp Arg Gly Tyr Thr Ser Asp His Gln Pro

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr Val Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 11

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 11

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val

20 25 30

Arg Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Tyr Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 12

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 12

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr Thr Ser Asp His Asn Pro

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg His Ser Tyr Tyr Val Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 13

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 13

Asp Tyr Tyr Met Thr

fifteen

<210> 14

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 14

Gly Phe Thr Phe Ser Asp Tyr

fifteen

<210> 15

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 15

Gly Phe Thr Phe Ser Asp Tyr Tyr Met Thr

1 5 10

<210> 16

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 16

Phe Ile Arg Asn Arg Ala Arg Gly Tyr Thr Ser Asp His Asn Pro Ser

1 5 10 15

Val Lys Gly

<210> 17

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 17

Arg Asn Arg Ala Arg Gly Tyr Thr

fifteen

<210> 18

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 18

Asp Arg Pro Ser Tyr Tyr Val Leu Asp Tyr

1 5 10

<210> 19

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 19

Phe Ile Arg Asn Gln Ala Arg Gly Tyr Thr Ser Asp His Asn Pro Ser

1 5 10 15

Val Lys Gly

<210> 20

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 20

Arg Asn Gln Ala Arg Gly Tyr Thr

fifteen

<210> 21

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 21

Phe Ile Arg Asn Arg Ala Gln Gly Tyr Thr Ser Asp His Asn Pro Ser

1 5 10 15

Val Lys Gly

<210> 22

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 22

Arg Asn Arg Ala Gln Gly Tyr Thr

fifteen

<210> 23

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 23

Phe Ile Arg Asn Gln Ala Arg Gly Tyr Thr Ser Asp His Gln Pro Ser

1 5 10 15

Val Lys Gly

<210> 24

<211> 19

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 24

Phe Ile Arg Asn Gln Asp Arg Gly Tyr Thr Ser Asp His Gln Pro Ser

1 5 10 15

Val Lys Gly

<210> 25

<211> 10

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 25

Asp Arg His Ser Tyr Tyr Val Leu Asp Tyr

1 5 10

<210> 26

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 26

Thr Ser Ser Gln Ser Leu Phe Asn Val Arg Ser Arg Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 27

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 27

Trp Ala Ser Thr Arg Glu Ser

fifteen

<210> 28

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 28

Lys Gln Ser Tyr Asp Leu Phe Thr

fifteen

<210> 29

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 29

Thr Ser Ser Gln Ser Leu Phe Asn Val Arg Ser Gln Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 30

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 30

Thr Ser Ser Gln Ser Leu Phe Asn Val Arg Ser Gly Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 31

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 31

Trp Ala Ser Asp Arg Glu Ser

fifteen

<210> 32

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 32

Thr Ser Asp Gln Ser Leu Phe Asn Val Arg Ser Gly Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 33

<211> 8

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 33

Lys Gln Ser Tyr Tyr Leu Phe Thr

fifteen

<210> 34

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 34

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val

20 25 30

Arg Ser Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 35

<211> 121

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 35

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr

20 25 30

Tyr Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr Thr Ser Asp His Asn Pro

50 55 60

Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr Val Leu Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 36

<211> 461

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 36

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Tyr Tyr

20 25 30

Gly Thr Ser Leu Met Gln Trp Tyr Gln Gln Lys Pro Gly Lys Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Val Glu Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Arg

85 90 95

Lys Val Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly

115 120 125

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

130 135 140

Phe Thr Phe Ser Asp Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly

145 150 155 160

Lys Gly Leu Glu Trp Val Ala Phe Ile Arg Asn Arg Ala Arg Gly Tyr

165 170 175

Thr Ser Asp His Asn Pro Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

180 185 190

Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala

195 200 205

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr

210 215 220

Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly

225 230 235 240

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe

245 250 255

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

260 265 270

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

275 280 285

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

290 295 300

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

305 310 315 320

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

325 330 335

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

340 345 350

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

355 360 365

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

370 375 380

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

385 390 395 400

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

405 410 415

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

420 425 430

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

435 440 445

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

450 455 460

<210> 37

<211> 464

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 37

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Asn Val

20 25 30

Arg Ser Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Leu Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly

115 120 125

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

130 135 140

Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met His Trp Val Arg Gln Ala

145 150 155 160

Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Lys Pro Ser Asn Gly

165 170 175

Leu Thr Asn Tyr Ile Glu Lys Phe Lys Asn Arg Phe Thr Ile Ser Val

180 185 190

Asp Lys Ala Lys Asn Ser Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala

195 200 205

Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Thr Ile Thr Thr Thr Glu

210 215 220

Gly Tyr Trp Phe Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Gly Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro

245 250 255

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

260 265 270

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

275 280 285

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

290 295 300

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

305 310 315 320

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

325 330 335

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

340 345 350

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

355 360 365

Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

370 375 380

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

385 390 395 400

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

405 410 415

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

420 425 430

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

435 440 445

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

450 455 460

<210> 38

<211> 464

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 38

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val

20 25 30

Arg Ser Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly

115 120 125

Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala

130 135 140

Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met His Trp Val Arg Gln Ala

145 150 155 160

Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Lys Pro Ser Asn Gly

165 170 175

Leu Thr Asn Tyr Ile Glu Lys Phe Lys Asn Arg Phe Thr Ile Ser Val

180 185 190

Asp Lys Ala Lys Asn Ser Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala

195 200 205

Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Thr Ile Thr Thr Thr Glu

210 215 220

Gly Tyr Trp Phe Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val

225 230 235 240

Ser Ser Gly Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro

245 250 255

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

260 265 270

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

275 280 285

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

290 295 300

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

305 310 315 320

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

325 330 335

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

340 345 350

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

355 360 365

Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser

370 375 380

Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

385 390 395 400

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

405 410 415

Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys

420 425 430

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

435 440 445

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

450 455 460

<210> 39

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 39

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 40

<211> 214

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 40

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Tyr Leu Arg Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

115 120 125

Gly Gly Gly Ser Pro Ile Glu Glu Leu Glu Asp Arg Val Phe Val Asn

130 135 140

Cys Asn Thr Ser Ile Thr Trp Val Glu Gly Thr Val Gly Thr Leu Leu

145 150 155 160

Ser Asp Ile Thr Arg Leu Asp Leu Gly Lys Arg Ile Leu Asp Pro Arg

165 170 175

Gly Ile Tyr Arg Cys Asn Gly Thr Asp Ile Tyr Lys Asp Lys Glu Ser

180 185 190

Thr Val Gln Val His Tyr Arg Met Cys Gln Ser Cys Val Glu Leu Asp

195 200 205

His His His His His His His

210

<210> 41

<211> 107

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 41

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Thr Phe Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys

100 105

<210> 42

<211> 124

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 42

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

Ser Ala Ile Ser Gly Gly Gly Arg Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

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

85 90 95

Ala Arg Asp Leu Ser Pro Ser Asp Val Gly Trp Gly Tyr Gly Phe Asp

100 105 110

Ile Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 43

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 43

Ala Ala Ser Ser Leu Gln Ser

fifteen

<210> 44

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 44

Val Gln Pro Gly Gly Ser Leu Arg Leu

fifteen

<210> 45

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 45

Leu Arg Leu Ser Cys Ala Ala Ser Gly

fifteen

<210> 46

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 46

Trp Val Arg Gln Ala Pro Gly Lys Gly

fifteen

<210> 47

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 47

Val Arg Gln Ala Pro Gly Lys Gly Leu

fifteen

<210> 48

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 48

Trp Val Ala Phe Ile Arg Asn Gln Ala

fifteen

<210> 49

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 49

Phe Ile Arg Asn Gln Ala Arg Gly Tyr

fifteen

<210> 50

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 50

Tyr Thr Ser Asp His Asn Pro Ser Val

fifteen

<210> 51

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 51

Val Lys Gly Arg Phe Thr Ile Ser Arg

fifteen

<210> 52

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 52

Phe Thr Ile Ser Arg Asp Asn Ala Lys

fifteen

<210> 53

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 53

Leu Tyr Leu Gln Met Asn Ser Leu Arg

fifteen

<210> 54

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 54

Tyr Leu Gln Met Asn Ser Leu Arg Ala

fifteen

<210> 55

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 55

Leu Gln Met Asn Ser Leu Arg Ala Glu

fifteen

<210> 56

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 56

Ile Gln Met Thr Gln Ser Pro Ser Ser

fifteen

<210> 57

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 57

Met Thr Gln Ser Pro Ser Ser Leu Ser

fifteen

<210> 58

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 58

Ile Thr Cys Thr Ser Ser Gln Ser Leu

fifteen

<210> 59

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 59

Val Arg Ser Gln Lys Asn Tyr Leu Ala

fifteen

<210> 60

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 60

Trp Tyr Gln Gln Lys Pro Gly Lys Ala

fifteen

<210> 61

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 61

Leu Leu Ile Tyr Trp Ala Ser Thr Arg

fifteen

<210> 62

<211> 9

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 62

Phe Thr Leu Thr Ile Ser Ser Leu Gln

fifteen

<210> 63

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 63

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro

1 5 10 15

<210> 64

<211> 5

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 64

Cys Pro Pro Cys Pro

fifteen

<210> 65

<211> 16

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 65

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

<210> 66

<211> 186

<212> Protein

<213> Homo sapiens

<400> 66

Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys

1 5 10 15

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

20 25 30

Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp

35 40 45

Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys

50 55 60

Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg

65 70 75 80

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

85 90 95

Val Cys Glu Asn Cys Met Glu Met Asp Val Met Ser Val Ala Thr Ile

100 105 110

Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu Leu Leu Leu Val Tyr

115 120 125

Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys Pro Val Thr Arg Gly

130 135 140

Ala Gly Ala Gly Gly Arg Gln Arg Gly Glyn Asn Lys Glu Arg Pro Pro

145 150 155 160

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

165 170 175

Leu Tyr Ser Gly Leu Asn Gln Arg Arg Ile

180 185

<210> 67

<211> 1534

<212> DNA

<213> Homo sapiens

<400> 67

tattgtcaga gtcctcttgt ttggccttct aggaaggctg tgggacccag ctttcttcaa 60

ccagtccagg tggaggcctc tgccttgaac gtttccaagt gaggtaaaac ccgcaggccc 120

agaggcctct ctacttcctg tgtggggttc agaaaccctc ctcccctccc agcctcaggt 180

gcctgcttca gaaaatgaag tagtaagtct gctggcctcc gccatcttag taaagtaaca 240

gtcccatgaa acaaagatgc agtcgggcac tcactggaga gttctgggcc tctgcctctt 300

atcagttggc gtttgggggc aagatggtaa tgaagaaatg ggtggtatta cacagacacc 360

atataaagtc tccatctctg gaaccacagt aatattgaca tgccctcagt atcctggatc 420

tgaaatacta tggcaacaca atgataaaaa cataggcggt gatgaggatg ataaaaacat 480

aggcagtgat gaggatcacc tgtcactgaa ggaattttca gaattggagc aaagtggtta 540

ttatgtctgc taccccagag gaagcaaacc agaagatgcg aacttttatc tctacctgag 600

ggcaagagtg tgtgagaact gcatggagat ggatgtgatg tcggtggcca caattgtcat 660

agtggacatc tgcatcactg ggggcttgct gctgctggtt tactactgga gcaagaatag 720

aaaggccaag gccaagcctg tgacacgagg agcgggtgct ggcggcaggc aaaggggaca 780

aaacaaggag aggccaccac ctgttcccaa cccagactat gagcccatcc ggaaaggcca 840

gcgggacctg tattctggcc tgaatcagag acgcatctga ccctctggag aacactgcct 900

cccgctggcc caggtctcct ctccagtccc cctgcgactc cctgtttcct gggctagtct 960

tggaccccac gagagagaat cgttcctcag cctcatggtg aactcgcgcc ctccagcctg 1020

atcccccgct ccctcctccc tgccttctct gctggtaccc agtcctaaaa tattgctgct 1080

tcctcttcct ttgaagcatc atcagtagtc acaccctcac agctggcctg ccctcttgcc 1140

aggatattta tttgtgctat tcactccctt ccctttggat gtaacttctc cgttcagttc 1200

cctccttttc ttgcatgtaa gttgtccccc atcccaaagt attccatcta cttttctatc 1260

gccgtcccct tttgcagccc tctctgggga tggactgggt aaatgttgac agaggccctg 1320

ccccgttcac agatcctggc ccctgagccag ccctgtgctc ctccctcccc caacactccc 1380

taccaacccc ctaatcccct actccctcca ccccccctcc actgtaggcc actggatggt 1440

catttgcatc tccgtaaatg tgctctgctc ctcagctgag agagaaaaaa ataaactgta 1500

tttggctgca agaaaaaaaa aaaaaaaaaa aaaa 1534

<210> 68

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotide sequence

<400> 68

gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60

atcacctgca caagctcaca gtcactgttt aatgtccgca gccggaaaaa ctatcttgcg 120

tggtatcagc agaagcctgg caaggctccc aagctgctga tctactgggc cagtacacga 180

gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240

atctcctccc tccagcctga ggatttcgcc acctactact gcaaacagtc ttacgacctt 300

ttcacttttg gcggcggaac aaaggtggag atcaag 336

<210> 69

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotide sequence

<400> 69

gaagtgcagc ttgttgaatc tggcggcggt ttggttcagc ccggtggatc actgcgactc 60

agttgcgcag ctagcggctt caccttttct gattactaca tgacatgggt acgacaggcg 120

ccaggcaagg gtttggaatg ggtagcattc atacgcaata gggcacgcgg gtacacttca 180

gaccacaatc cctcagtaaa aggaagattt accatctcaa gagacaatgc caaaaattca 240

ctctacctgc aaatgaactc acttcgcgcc gaggataccg ccgtgtatta ctgtgccaga 300

gacagaccat cttattacgt gctggactat tggggacagg gcactacagt caccgtcagc 360

tct 363

<210> 70

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotide sequence

<400> 70

gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60

atcacctgca caagctcaca gtcactgttt aatgtccgca gccagaaaaa ctatcttgcg 120

tggtatcagc agaagcctgg caaggctccc aagctgctga tctactgggc cagtacacga 180

gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240

atctcctccc tccagcctga ggatttcgcc acctactact gcaaacagtc ttacgacctt 300

ttcacttttg gcggcggaac aaaggtggag atcaag 336

<210> 71

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotide sequence

<400> 71

gaagtgcagc ttgttgaatc tggcggcggt ttggttcagc ccggtggatc actgcgactc 60

agttgcgcag ctagcggctt caccttttct gattactaca tgacatgggt acgacaggcg 120

ccaggcaagg gtttggaatg ggtagcattc atacgcaatc aggcacgcgg gtacacttca 180

gaccacaatc cctcagtaaa aggaagattt accatctcaa gagacaatgc caaaaattca 240

ctctacctgc aaatgaactc acttcgcgcc gaggataccg ccgtgtatta ctgtgccaga 300

gacagaccat cttattacgt gctggactat tggggacagg gcactacagt caccgtcagc 360

tct 363

<210> 72

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotide sequence

<400> 72

gaagtgcagc ttgttgaatc tggcggcggt ttggttcagc ccggtggatc actgcgactc 60

agttgcgcag ctagcggctt caccttttct gattactaca tgacatgggt acgacaggcg 120

ccaggcaagg gtttggaatg ggtagcattc atacgcaata gggcacaggg gtacacttca 180

gaccacaatc cctcagtaaa aggaagattt accatctcaa gagacaatgc caaaaattca 240

ctctacctgc aaatgaactc acttcgcgcc gaggataccg ccgtgtatta ctgtgccaga 300

gacagaccat cttattacgt gctggactat tggggacagg gcactacagt caccgtcagc 360

tct 363

<210> 73

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotide sequence

<400> 73

gaagtgcagc ttgttgaatc tggcggcggt ttggttcagc ccggtggatc actgcgactc 60

agttgcgcag ctagcggctt caccttttct gattactaca tgacatgggt acgacaggcg 120

ccaggcaagg gtttggaatg ggtagcattc atacgcaatc aggcacgcgg gtacacttca 180

gaccaccagc cctcagtaaa aggaagattt accatctcaa gagacaatgc caaaaattca 240

ctctacctgc aaatgaactc acttcgcgcc gaggataccg ccgtgtatta ctgtgccaga 300

gacagaccat cttattacgt gctggactat tggggacagg gcactacagt caccgtcagc 360

tct 363

<210> 74

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotide sequence

<400> 74

gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60

atcacctgca caagctcaca gtcactgttt aatgtccgca gccagaaaaa ctatcttgcg 120

tggtatcagc agaagcctgg caaggctccc aagctgctga tctactgggc cagtgatcga 180

gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240

atctcctccc tccagcctga ggatttcgcc acctactact gcaaacagtc ttacgacctt 300

ttcacttttg gcggcggaac aaaggtggag atcaag 336

<210> 75

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotide sequence

<400> 75

gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60

atcacctgca caagcgacca gtcactgttt aatgtccgca gcggcaaaaa ctatcttgcg 120

tggtatcagc agaagcctgg caaggctccc aagctgctga tctactgggc cagtgaccga 180

gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240

atctcctccc tccagcctga ggatttcgcc acctactact gcaaacagtc ttacgacctt 300

ttcacttttg gcggcggaac aaaggtggag atcaag 336

<210> 76

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotide sequence

<400> 76

gaagtgcagc ttgttgaatc tggcggcggt ttggttcagc ccggtggatc actgcgactc 60

agttgcgcag ctagcggctt caccttttct gattactaca tgacatgggt acgacaggcg 120

ccaggcaagg gtttggaatg ggtagcattc atacgcaatc aggaccgcgg gtacacttca 180

gaccaccagc cctcagtaaa aggaagattt accatctcaa gagacaatgc caaaaattca 240

ctctacctgc aaatgaactc acttcgcgcc gaggataccg ccgtgtatta ctgtgccaga 300

gacagaccat cttattacgt gctggactat tggggacagg gcactacagt caccgtcagc 360

tct 363

<210> 77

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotide sequence

<400> 77

gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60

atcacctgca caagctcaca gtcactgttt aatgtccgca gccagaaaaa ctatcttgcg 120

tggtatcagc agaagcctgg caaggctccc aagctgctga tctactgggc cagtacacga 180

gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240

atctcctccc tccagcctga ggatttcgcc acctactact gcaaacagtc ttactacctt 300

ttcacttttg gcggcggaac aaaggtggag atcaag 336

<210> 78

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 78

Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Ser Arg Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 79

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 79

Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 80

<211> 326

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 80

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Arg Val Arg Cys Pro Arg Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 81

<211> 326

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 81

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr

65 70 75 80

Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Thr Val Glu Arg Lys Cys Glu Val Glu Cys Pro Glu Cys Pro Ala Pro

100 105 110

Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp

115 120 125

Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Ala

130 135 140

Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly

145 150 155 160

Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn

165 170 175

Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp

180 185 190

Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro

195 200 205

Ser Ser Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu

210 215 220

Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met Thr Lys Asn

225 230 235 240

Gln Val Ser Leu Thr Cys Glu Val Lys Gly Phe Tyr Pro Ser Asp Ile

245 250 255

Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr

260 265 270

Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys

275 280 285

Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys

290 295 300

Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu

305 310 315 320

Ser Leu Ser Pro Gly Lys

325

<210> 82

<211> 216

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 82

Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys

1 5 10 15

Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val

20 25 30

Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr

35 40 45

Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu

50 55 60

Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His

65 70 75 80

Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys

85 90 95

Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln

100 105 110

Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Ser Arg Glu Glu Met

115 120 125

Thr Lys Asn Gln Val Ser Leu Ser Cys Leu Val Lys Gly Phe Tyr Pro

130 135 140

Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn

145 150 155 160

Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu

165 170 175

Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val

180 185 190

Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln

195 200 205

Lys Ser Leu Ser Leu Ser Pro Gly

210 215

<210> 83

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotide sequence

<400> 83

gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60

atcacctgca caagctcaca gtcactgttt aatgtccgca gcggaaaaaa ctatcttgcg 120

tggtatcagc agaagcctgg caaggctccc aagctgctga tctactgggc cagtacacga 180

gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240

atctcctccc tccagcctga ggatttcgcc acctactact gcaaacagtc ttacgacctt 300

ttcacttttg gcggcggaac aaaggtggag atcaag 336

<210> 84

<211> 363

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotide sequence

<400> 84

gaagtgcagc ttgttgaatc tggcggcggt ttggttcagc ccggtggatc actgcgactc 60

agttgcgcag ctagcggctt caccttttct gattactaca tgacatgggt acgacaggcg 120

ccaggcaagg gtttggaatg ggtagcattc atacgcaatc aggcacgcgg gtacacttca 180

gaccacaatc cctcagtaaa aggaagattt accatctcaa gagacaatgc caaaaattca 240

ctctacctgc aaatgaactc acttcgcgcc gaggataccg ccgtgtatta ctgtgccaga 300

gacagacact cttattacgt gctggactat tggggacagg gcactacagt caccgtcagc 360

tct 363

<210> 85

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 85

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Phe Asn Val

20 25 30

Arg Ser Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Ser Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Leu Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 86

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotide sequence

<400> 86

gacatcgtga tgacccagag ccccgatagc ctggccgtgt ctctgggaga gagagccacc 60

atcaactgca agagcagcca gagcctgttc aacgtgagaa gccggaagaa ctacctggcc 120

tggtatcagc agaaacccgg ccagcccccc aagctgctga tcagctgggc cagcaccaga 180

gaaagcggcg tgcccgatag attcagcggc agcggaagcg gcaccgactt caccctgaca 240

atcagctccc tgcaggccga ggacgtggcc gtgtactact gcaagcagag ctacgacctg 300

ttcaccttcg gcagcggcac caagctggaa atcaaa 336

<210> 87

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 87

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Glu Ser Leu Phe Asn Val

20 25 30

Arg Ser Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Asp Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 88

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotide sequence

<400> 88

gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60

atcacctgca caagctcaga gtcactgttt aatgtccgca gcggcaaaaa ctatcttgcg 120

tggtatcagc agaagcctgg caaggctccc aagctgctga tctactgggc cagtgaccga 180

gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240

atctcctccc tccagcctga ggatttcgcc acctactact gcaaacagtc ttacgacctt 300

ttcacttttg gcggcggaac aaaggtggag atcaag 336

<210> 89

<211> 112

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 89

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val

20 25 30

Arg Ser Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Asp Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 90

<211> 336

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic nucleotide sequence

<400> 90

gacatccaga tgacccagtc cccctcttct ctgtctgcct ctgtgggcga cagagtgacc 60

atcacctgca caagctcaca gtcactgttt aatgtccgca gcggcaaaaa ctatcttgcg 120

tggtatcagc agaagcctgg caaggctccc aagctgctga tctactgggc cagtgaccga 180

gaatccggcg tgccttccag attctccggc tctggctctg gcaccgattt caccctgacc 240

atctcctccc tccagcctga ggatttcgcc acctactact gcaaacagtc ttacgacctt 300

ttcacttttg gcggcggaac aaaggtggag atcaag 336

<210> 91

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 91

Thr Ser Ser Glu Ser Leu Phe Asn Val Arg Ser Gly Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 92

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 92

Thr Ser Ser Gln Ser Leu Phe Asn Val Arg Ser Gly Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 93

<211> 461

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 93

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Tyr Tyr

20 25 30

Gly Ser Ser Leu Leu Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Lys Leu Ala Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Arg

85 90 95

Lys Ala Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly

115 120 125

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

130 135 140

Phe Thr Phe Ser Asp Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly

145 150 155 160

Lys Gly Leu Glu Trp Val Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr

165 170 175

Thr Ser Asp His Asn Pro Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

180 185 190

Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala

195 200 205

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr

210 215 220

Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly

225 230 235 240

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe

245 250 255

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

260 265 270

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

275 280 285

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

290 295 300

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

305 310 315 320

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

325 330 335

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

340 345 350

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

355 360 365

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

370 375 380

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

385 390 395 400

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

405 410 415

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

420 425 430

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

435 440 445

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

450 455 460

<210> 94

<211> 465

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 94

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val

20 25 30

Arg Ser Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser

115 120 125

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Ser Ser Tyr Trp Met His Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Lys Pro Ser Asn

165 170 175

Glu Leu Thr Asn Val His Glu Lys Phe Lys Asp Arg Phe Thr Ile Ser

180 185 190

Val Asp Lys Ala Lys Asn Ser Ala Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Thr Ile Thr Thr Thr

210 215 220

Glu Gly Tyr Trp Phe Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr

225 230 235 240

Val Ser Ser Gly Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

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

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

gly

465

<210> 95

<211> 461

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 95

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Tyr Tyr

20 25 30

Gly Ser Ser Leu Leu Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Lys Leu Ala Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Arg

85 90 95

Lys Ala Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly

115 120 125

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

130 135 140

Phe Thr Phe Ser Asp Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly

145 150 155 160

Lys Gly Leu Glu Trp Val Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr

165 170 175

Thr Ser Asp His Asn Pro Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

180 185 190

Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala

195 200 205

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr

210 215 220

Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly

225 230 235 240

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe

245 250 255

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

260 265 270

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

275 280 285

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

290 295 300

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

305 310 315 320

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

325 330 335

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

340 345 350

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

355 360 365

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

370 375 380

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

385 390 395 400

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

405 410 415

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

420 425 430

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

435 440 445

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

450 455 460

<210> 96

<211> 465

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 96

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Thr Ser Asp Gln Ser Leu Phe Asn Val

20 25 30

Arg Ser Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Asp Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser

115 120 125

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Ser Ser Tyr Trp Met His Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Lys Pro Ser Asn

165 170 175

Glu Leu Thr Asn Val His Glu Lys Phe Lys Asp Arg Phe Thr Ile Ser

180 185 190

Val Asp Lys Ala Lys Asn Ser Ala Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Thr Ile Thr Thr Thr

210 215 220

Glu Gly Tyr Trp Phe Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr

225 230 235 240

Val Ser Ser Gly Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

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

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

gly

465

<210> 97

<211> 461

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 97

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Tyr Tyr

20 25 30

Gly Ser Ser Leu Leu Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Lys Leu Ala Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Arg

85 90 95

Lys Ala Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly

115 120 125

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

130 135 140

Phe Thr Phe Ser Asp Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly

145 150 155 160

Lys Gly Leu Glu Trp Val Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr

165 170 175

Thr Ser Asp His Asn Pro Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

180 185 190

Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala

195 200 205

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr

210 215 220

Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly

225 230 235 240

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe

245 250 255

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

260 265 270

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

275 280 285

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

290 295 300

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

305 310 315 320

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

325 330 335

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

340 345 350

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

355 360 365

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

370 375 380

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

385 390 395 400

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

405 410 415

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

420 425 430

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

435 440 445

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

450 455 460

<210> 98

<211> 465

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 98

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Gln Ser Leu Phe Asn Val

20 25 30

Arg Ser Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Asp Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser

115 120 125

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Ser Ser Tyr Trp Met His Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Lys Pro Ser Asn

165 170 175

Glu Leu Thr Asn Val His Glu Lys Phe Lys Asp Arg Phe Thr Ile Ser

180 185 190

Val Asp Lys Ala Lys Asn Ser Ala Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Thr Ile Thr Thr Thr

210 215 220

Glu Gly Tyr Trp Phe Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr

225 230 235 240

Val Ser Ser Gly Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

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

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

gly

465

<210> 99

<211> 461

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 99

Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Tyr Tyr

20 25 30

Gly Ser Ser Leu Leu Gln Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Lys Leu Ala Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Arg

85 90 95

Lys Ala Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly

100 105 110

Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser Gly Gly Gly

115 120 125

Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

130 135 140

Phe Thr Phe Ser Asp Tyr Tyr Met Thr Trp Val Arg Gln Ala Pro Gly

145 150 155 160

Lys Gly Leu Glu Trp Val Ala Phe Ile Arg Asn Gln Ala Arg Gly Tyr

165 170 175

Thr Ser Asp His Asn Pro Ser Val Lys Gly Arg Phe Thr Ile Ser Arg

180 185 190

Asp Asn Ala Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala

195 200 205

Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Arg Pro Ser Tyr Tyr

210 215 220

Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly

225 230 235 240

Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe

245 250 255

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

260 265 270

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

275 280 285

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

290 295 300

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

305 310 315 320

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

325 330 335

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

340 345 350

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro

355 360 365

Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val

370 375 380

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

385 390 395 400

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

405 410 415

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

420 425 430

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

435 440 445

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

450 455 460

<210> 100

<211> 465

<212> Protein

<213> Artificial sequence

<220>

<223> Synthetic peptide sequence

<400> 100

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Thr Ser Ser Glu Ser Leu Phe Asn Val

20 25 30

Arg Ser Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys

35 40 45

Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Asp Arg Glu Ser Gly Val

50 55 60

Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Lys Gln

85 90 95

Ser Tyr Asp Leu Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Gly Gly Gly Gly Ser Gly Gly Gly Gly Glu Val Gln Leu Val Glu Ser

115 120 125

Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala

130 135 140

Ala Ser Gly Phe Thr Phe Ser Ser Tyr Trp Met His Trp Val Arg Gln

145 150 155 160

Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly Glu Ile Lys Pro Ser Asn

165 170 175

Glu Leu Thr Asn Val His Glu Lys Phe Lys Asp Arg Phe Thr Ile Ser

180 185 190

Val Asp Lys Ala Lys Asn Ser Ala Tyr Leu Gln Met Asn Ser Leu Arg

195 200 205

Ala Glu Asp Thr Ala Val Tyr Tyr Cys Thr Arg Thr Ile Thr Thr Thr

210 215 220

Glu Gly Tyr Trp Phe Phe Asp Val Trp Gly Gln Gly Thr Leu Val Thr

225 230 235 240

Val Ser Ser Gly Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

245 250 255

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

260 265 270

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

275 280 285

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

290 295 300

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

305 310 315 320

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

325 330 335

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

340 345 350

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

355 360 365

Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

370 375 380

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

385 390 395 400

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

405 410 415

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

420 425 430

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

435 440 445

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

450 455 460

gly

465

<---

Claims (108)

1. An antibody that specifically binds to CD3, where the antibody contains: a) a heavy chain variable region (VH) comprising a VH complementarity determining region one (VH CDR1), a VH complementarity determining region two (VH CDR2), and a VH complementarity determining region three (VH CDR3) of the VH sequence of SEQ ID NO: 4; and a light chain variable region (VL) comprising a VL complementarity determining region one (VL CDR1), a VL complementarity determining region two (VL CDR2), and a VL complementarity determining region three (VL CDR3) of the VL sequence of SEQ ID NO: 3; or b) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, where VH and VL have the sequences (i) SEQ ID NO: 2 and 1, respectively; (ii) SEQ ID NO: 2 and 3, respectively; (iii) SEQ ID NOs: 5 and 3, respectively; (iv) SEQ ID NOs: 7 and 6, respectively; (v) SEQ ID NO: 7 and 8, respectively; (vi) SEQ ID NO: 4 and 6, respectively; (vii) SEQ ID NOs: 4 and 8, respectively; (viii) SEQ ID NOs: 10 and 9, respectively; (ix) SEQ ID NO: 7 and 9, respectively; (x) SEQ ID NOs: 12 and 11, respectively; (xi) SEQ ID NOs: 35 and 34, respectively; (xii) SEQ ID NOs: 4 and 9, respectively; (xiii) SEQ ID NOs: 4 and 87, respectively; or (xiiii) SEQ ID NOs: 4 and 89, respectively. 2. The antibody according to claim 1, where the antibody contains: a) VH CDR1 containing the sequence of SEQ ID NO: 13, 14 or 15; VH CDR2 containing the sequence of SEQ ID NO: 19, 20; and VH CDR3 containing the sequence of SEQ ID NO: 18; and VL CDR1 containing the sequence of SEQ ID NO: 29; VL CDR2 containing the sequence of SEQ ID NO: 27; and VL CDR3 containing the sequence of SEQ ID NO: 28; b) VH CDR1 containing the sequence of SEQ ID NO: 13, 14 or 15, VH CDR2 containing the sequence of SEQ ID NO: 16 or 17, VH CDR3 containing the sequence of SEQ ID NO: 18, and VL CDR1, VL CDR2 and VL CDR3 containing the sequences of SEQ ID NOs: 26, 27 and 28, respectively; c) VH CDR1 containing the sequence of SEQ ID NO: 13, 14 or 15, VH CDR2 containing the sequence of SEQ ID NO: 16 or 17, VH CDR3 containing the sequence of SEQ ID NO: 18, and VL CDR1, VL CDR2 and VL CDR3 containing the sequences of SEQ ID NOs: 29, 27 and 28, respectively; d) VH CDR1 containing the sequence of SEQ ID NO: 13, 14 or 15, VH CDR2 containing the sequence of SEQ ID NO: 21 or 22, VH CDR3 containing the sequence of SEQ ID NO: 18, and VL CDR1, VL CDR2 and VL CDR3 containing the sequences of SEQ ID NOs: 29, 27 and 28, respectively; e) VH CDR1 containing the sequence of SEQ ID NO: 13, 14 or 15, VH CDR2 containing the sequence of SEQ ID NO: 23 or 20, VH CDR3 containing the sequence of SEQ ID NO: 18, and VL CDR1, VL CDR2 and VL CDR3 containing the sequences of SEQ ID NOs: 30, 27 and 28, respectively; f) VH CDR1 containing the sequence of SEQ ID NO: 13, 14 or 15, VH CDR2 containing the sequence of SEQ ID NO: 23 or 20, VH CDR3 containing the sequence of SEQ ID NO: 18, and VL CDR1, VL CDR2 and VL CDR3 containing the sequences of SEQ ID NOs: 29, 31 and 28, respectively; g) VH CDR1 containing the sequence of SEQ ID NO: 13, 14 or 15, VH CDR2 containing the sequence of SEQ ID NO: 19 or 20, VH CDR3 containing the sequence of SEQ ID NO: 18, and VL CDR1, VL CDR2 and VL CDR3 containing the sequences of SEQ ID NOs: 30, 27 and 28, respectively; h) VH CDR1 containing the sequence of SEQ ID NO: 13, 14 or 15, VH CDR2 containing the sequence of SEQ ID NO: 19 or 20, VH CDR3 containing the sequence of SEQ ID NO: 18, and VL CDR1, VL CDR2 and VL CDR3 containing the sequences of SEQ ID NOs: 29, 31 and 28, respectively; i) VH CDR1 containing the sequence of SEQ ID NO: 13, 14 or 15, VH CDR2 containing the sequence of SEQ ID NO: 24 or 20, VH CDR3 containing the sequence of SEQ ID NO: 18, and VL CDR1, VL CDR2 and VL CDR3 containing the sequences of SEQ ID NOs: 32, 31 and 28, respectively; j) VH CDR1 containing the sequence of SEQ ID NO: 13, 14 or 15, VH CDR2 containing the sequence of SEQ ID NO: 19 or 20, VH CDR3 containing the sequence of SEQ ID NO: 25, and VL CDR1, VL CDR2 and VL CDR3 containing the sequences of SEQ ID NOs: 29, 27 and 33, respectively; k) VH CDR1 containing the sequence of SEQ ID NO: 13, 14 or 15, VH CDR2 containing the sequence of SEQ ID NO: 19 or 20, VH CDR3 containing the sequence of SEQ ID NO: 18, and VL CDR1, VL CDR2 and VL CDR3 containing the sequences of SEQ ID NOs: 26, 27 and 28, respectively; l) VH CDR1 containing the sequence of SEQ ID NO: 13, 14 or 15, VH CDR2 containing the sequence of SEQ ID NO: 19 or 20, VH CDR3 containing the sequence of SEQ ID NO: 18, and VL CDR1, VL CDR2 and VL CDR3 containing the sequences of SEQ ID NOs: 32, 31 and 28, respectively; m) VH CDR1 containing the sequence of SEQ ID NO: 13, 14 or 15, VH CDR2 containing the sequence of SEQ ID NO: 19 or 20, VH CDR3 containing the sequence of SEQ ID NO: 18, and VL CDR1, VL CDR2 and VL CDR3 containing the sequences of SEQ ID NOs: 91, 31 and 28, respectively; or n) VH CDR1 containing the sequence of SEQ ID NO: 13, 14 or 15, VH CDR2 containing the sequence of SEQ ID NO: 19 or 20, VH CDR3 containing the sequence of SEQ ID NO: 18, and VL CDR1, VL CDR2 and VL CDR3 containing the sequences of SEQ ID NOs: 92, 31 and 28, respectively. 3. The antibody of claim 1, wherein the antibody comprises a VL/VH amino acid sequence pair selected from the group consisting of SEQ ID NOs: 1 and 2; SEQ ID NO: 3 and 2; SEQ ID NOs: 3 and 4; SEQ ID NOs: 3 and 5; SEQ ID NOs: 6 and 7; SEQ ID NOs: 8 and 7; SEQ ID NOs: 6 and 4; SEQ ID NOs: 8 and 4; SEQ ID NOs: 9 and 10; SEQ ID NOs: 9 and 7; SEQ ID NOs: 11 and 12; SEQ ID NOs: 34 and 35; SEQ ID NOs: 9 and 4; SEQ ID NO: 87 and 4; and SEQ ID NOs: 89 and 4. 4. The antibody according to claim 1, where the antibody contains VH containing the sequence of SEQ ID NO: 4, and VL containing the sequence SEQ ID NO: 3. 5. An antibody according to any one of claims 1 to 4, which is selected from the group consisting of a monoclonal antibody, a chimeric antibody, a polyclonal antibody, a labeled antibody, a humanized antibody, a human antibody, and fragments thereof. 6. Bispecific antibody that specifically binds to CD3 and contains VH CDR1 containing SEQ ID NO: 13, 14 or 15; VH CDR2 containing SEQ ID NO: 19, 20; and VH CDR3 containing SEQ ID NO: 18; and VL CDR1 containing SEQ ID NO: 29; VL CDR2 containing SEQ ID NO: 27; and VL CDR3 containing SEQ ID NO: 28, wherein the bispecific antibody also specifically binds to the tumor antigen. 7. A bispecific antibody according to claim 6, containing a VH containing SEQ ID NO: 4 and a VL containing SEQ ID NO: 3. 8. An antibody or bispecific antibody according to any one of claims 1 to 7, further comprising a human or humanized VH framework and a human or humanized VL framework. 9. An antibody or bispecific antibody according to any one of claims 1-8, wherein the antibody is a humanized antibody. 10. A pharmaceutical composition for the treatment of cancer, comprising an effective amount of an antibody or a bispecific antibody according to any one of claims 1 to 9 and a pharmaceutically acceptable carrier. 11. A method for modulating a T cell-mediated immune response in a patient, comprising administering to the patient an antibody or a bispecific antibody according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 10, thereby modulating the immune response in the patient. 12. A method for inhibiting the growth of tumor cells in an individual, comprising administering to the individual an antibody or a bispecific antibody according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 10 in an amount effective to inhibit the growth of tumor cells. 13. The method of claim 12, wherein the tumor cells are cancer cells. 14. The method of claim 13, wherein the cancer is selected from the group consisting of breast, ovarian, thyroid, prostate, cervix, lung, bladder, endometrial, head and neck, testicular, glioblastoma, and digestive system cancers. 15. The method of claim 14, wherein the cancer of the digestive system is selected from the group consisting of cancer of the esophagus, stomach, small intestine, colon, rectum, anus, liver, gallbladder, appendix, bile duct, and pancreas. 16. A polynucleotide for producing an antibody or a bispecific antibody according to any one of claims 1 to 9, wherein the polynucleotide contains a nucleotide sequence encoding an antibody according to any one of claims 1 to 9. 17. The use of an antibody or bispecific antibody according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 10 in a method for modulating a T-cell mediated immune response in a patient. 18. The use of an antibody or bispecific antibody according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 10 in a method for inhibiting the growth of tumor cells in an individual. 19. The use of an antibody or bispecific antibody according to any one of claims 1 to 9 or a pharmaceutical composition according to claim 10 in a method of treating cancer, optionally, where the cancer is selected from the group consisting of breast, ovarian, thyroid, prostate, cervical cancer , lung, bladder, endometrium, head and neck, testis, glioblastoma, and cancer of the digestive system. 20. Use according to claim 19, wherein the T-cell mediated immune response is modulated or where tumor cell growth is inhibited.

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