A bispecific antibody and uses thereof

Abstract

The present disclosure relates to a bispecific antibody and use thereof. The bispecific antibody or antigen-binding fragment thereof comprises: (a) a first antigen-binding moiety or antigen-binding fragment thereof, the first antigen-binding moiety comprising a first light chain and a first heavy chain, the first light chain being a kappa type light chain, the first antigen-binding moiety comprising a first binding domain that binds to a first antigen; and (b) a second antigen-binding moiety or antigen-binding fragment thereof, the second antigen-binding moiety comprising a second light chain and a second heavy chain, the second light chain being a lambda type light chain, the second antigen-binding moiety comprising a second binding domain that binds to a second antigen. The bispecific antibody has a low mispairing rate of heavy and light chains, a high yield in purification, does not bind to Fc gamma R receptors, reduces the risk of cytokine storm, is well tolerated, and has better efficacy and safety than antibodies of the same kind.

Description

Technical Field

[0001] This disclosure relates to a bispecific antibody and its use, particularly a bispecific antibody that binds to CD3 and another antigen and its use. Background Technology

[0002] T-cell bispecific antibodies (or T-cell connectors) are special antibody molecules that recognize target cell surface antigens (antigen arm) at one end and bind to the T-cell CD3 receptor (CD3 arm) at the other end. This causes CD3 aggregation on T cells in a manner similar to TCR / peptide / HLA, thereby activating T cells and killing tumor cells. Reports of using bispecific antibodies to kill tumor cells were published in the 1980s (Nature. 1985 Apr 18-24; 314(6012):628-31; Nature. 1985 Jul 25-31; 316(6026):354-6). After more than 30 years of research, the antibody mismatch problem has been largely solved, and three bispecific antibody drugs have been approved for marketing. Although they have shown very good efficacy in their approved indications, the accompanying side effects and limitations in use hindered the widespread application of these bispecific antibodies in the early stages. For example, catumaxomab, which was launched early on, has been withdrawn from the market because its Fc fragment binds to the Fcγ receptor expressed by Kupffer cells in the liver, triggering a rapid release of cytokines. blinatumomab, launched in 2014, uses an Fv antibody fragment and has a biological half-life of only 2 hours, requiring continuous low-dose intravenous infusion. It also received a black box warning from the FDA regarding cytokine release syndrome and neurotoxicity.

[0003] During a normal immune response, the TCR binds to the exogenous peptide-human leukocyte antigen (HLA) complex on infected or mutated cells with low affinity (approximately 1-100 μM). This HLA complex, via the CD3 signaling complex (including CD3εγ, CD3εδ, and CD3ζζ), transmits the activation signal to the nucleus, activating the expression of transcription factors and their downstream proteins (cytokines, granzymes, perforin, etc.). The signal strength generated by the TCR complex determines the fate of the T cell. Early CD3 bispecific antibodies, mostly based on a few murine antibodies such as OKT3, L2K, UCHT1, and TR66, exhibited high affinity, leading to T cell overactivation, the release of large amounts of cytokines, and cytokine storm syndrome. Simultaneously, high affinity also results in the accumulation of bispecific antibodies in secondary lymphoid organs, reducing their exposure in tumor tissues.

[0004] The binding ability of the antibody Fc portion to the Fcγ receptor is another important factor affecting drug safety. Since the Fcγ receptor is expressed in a variety of normal tissues, after a bispecific antibody binds to the Fcγ receptor on the cell membrane via the Fc portion, the CD3 receptor bound at the other end can be cross-linked and activated due to the aggregation of the Fcγ receptor, thereby producing severe off-target toxicity. By using human IgG2 or IgG4 subtypes with weaker Fcγ receptor binding affinity, or by further substituting amino acids at the corresponding CH2 sites, such as Armour et al. replacing sites 233-236 (EU sequence number) of IgG1 and IgG4 with the corresponding IgG2 sequence, the binding to the Fcγ receptor was reduced (Eur. J. Immunol. 1999); Newman et al. introduced the mutations Ser228Pro and Leu235Glu into IgG4 to stabilize the IgG4 structure while reducing binding to the Fcγ receptor (Clin Immunol. 2001); Idusogie et al. found that replacing Asp270, Lys322, Pro329, or Pro331 with Ala, respectively, could reduce the binding of IgG1 to complement C1q (J Immunol. 2000), etc.

[0005] Interchain mismatch is a major technological challenge in the development of natural IgG-like bispecific antibodies. Common light chains invented by Merchant AM et al. (1998) or common heavy chains developed by Fischer N et al. (2015) typically require complex protein engineering modifications or the use of transgenic animals (McWhirter J, 2012). Carter P et al. (1997) addressed heavy chain mismatch by introducing knobs-into-holes complementary mutations into the antibody Fc segment. Schaefer G et al. (2011) developed CrossMab technology, which involves exchanging the Fab portion or the entire length of the light and heavy chains, to solve the light chain mismatch problem. Partially exchanged CrossMabs... VH-VL and CrossMab CH1-CL Additional peptides need to be introduced to achieve proper pairing, while CrossMab with full-length Fab exchange... Fab The correct matching rate is less than 50%. Summary of the Invention

[0006] During the expression of bispecific antibodies, the inventors unexpectedly discovered that when combining a humanized CD3 antibody with a λ light chain and a targeting antibody with a κ light chain, the CD3 antibody's λ light chain tends to pair with the homologous CD3 heavy chain, while the targeting antibody's kappa light chain tends to pair with the homologous targeting antibody's heavy chain. Further designing complementary charge pairs between the light and heavy chains can improve the correct pairing efficiency. Furthermore, experiments demonstrated that novel T-cell connectors constructed using antibodies targeting multiple targets such as CD20, BCMA, and GPC3, along with humanized CD3 antibodies, achieved 98-100% monomer purity after three-step purification, with an extremely low mismatch rate (<1%).

[0007] This disclosure provides a novel T-cell connector designed with different types of light chain κλ bispecific antibodies and a full-length IgG configuration. Specifically, the antibody arms binding to target cells and T-cell CD3 utilize kappa and lambda light chains, respectively, pairing with their homologous heavy chains. Complementary charge pairs are introduced to enhance the correct pairing rate. Through affinity optimization, the novel T-cell connector can recruit and activate T cells at low concentrations, effectively killing target cells, and does not activate T cells in the absence of target cells. Furthermore, the novel T-cell κλ bispecific antibody does not bind to the FcγR receptor, reducing the risk of cytokine storm. The novel CD20×CD3 κλ bispecific antibody, BCMA×CD3 κλ bispecific antibody, and GPC3xCD3κλ bispecific antibody constructed using the method disclosed in this disclosure have high purification yields, achieving >99% purity through a three-step purification method. Animals tolerate the novel CD20-CD3 κλ bispecific antibody well, and the efficacy and safety of the novel T-cell connector are superior to similar antibodies.

[0008] Therefore, in one respect, this disclosure provides a bispecific antibody or its antigen-binding portion thereof.

[0009] In one respect, this disclosure provides nucleic acids encoding bispecific antibodies or their antigen-binding portions as described above.

[0010] In one respect, this disclosure provides a vector containing nucleic acids as described above.

[0011] In one respect, this disclosure provides cells comprising a vector containing the foregoing aspects.

[0012] The antibody or its antigen-binding portion according to any of the foregoing aspects, wherein the antibody or its antigen-binding portion is humanized.

[0013] In one aspect, this disclosure provides pharmaceutical compositions or kits comprising an antibody or its antigen-binding portion or its encoded nucleic acid as described above and a pharmaceutically acceptable carrier.

[0014] In one aspect, this disclosure provides antibody-drug conjugates comprising an antibody or its antigen-binding portion, a bispecific or multispecific molecule, covalently attached to a therapeutic portion as described in any of the preceding aspects.

[0015] In one aspect, this disclosure provides a method for treating and related conditions, comprising the steps of administering to the mammal a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, vector, cell and / or pharmaceutical composition of any of the foregoing aspects.

[0016] In one aspect, this disclosure provides the use of antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells and / or pharmaceutical compositions of any of the foregoing aspects in the preparation of medicaments or kits for treating tumor antigen-related diseases in mammals.

[0017] The antibodies disclosed herein can be used for a variety of applications, including detecting tumor antigens, diagnosing, treating or preventing diseases associated with tumor antigens. Attached Figure Description

[0018] Figure 1 The first antigen × CD3 κλ bispecific antibody of this disclosure is shown.

[0019] Figure 2 The binding of the humanized CD3 antibody to the human CD3εγ protein was demonstrated.

[0020] Figure 3 The binding of the CD3 humanized antibody to Jurkat cells was demonstrated.

[0021] Figure 4 The binding of the humanized CD3 antibody to human CD3α and cynomolgus monkey CD3α proteins was demonstrated.

[0022] Figure 5 The structures of κλ001, κλ002, κλ003, κλ004, and κλ005 of this disclosure are shown.

[0023] Figure 6 The results of purification of the CD20×CD3κλ bispecific antibody with Protein A are shown.

[0024] Figure 7 The SEC-HPLC detection results of the CD20×CD3κλ bispecific antibody are shown.

[0025] Figure 8 The results of the detection of CD20×CD3κλ bispecific antibody homodimer are shown.

[0026] Figure 9 The binding of the CD20×CD3κλ bispecific antibody to CD20 stable cells was demonstrated.

[0027] Figure 10 The binding of the CD20×CD3κλ bispecific antibody to tumor cells SU-DHL-4, Raji, and NALM-6 was demonstrated.

[0028] Figure 11 The binding of the CD20×CD3κλ bispecific antibody to Jurkat cells is shown.

[0029] Figure 12 The binding of the CD20×CD3κλ bispecific antibody to peripheral blood T cells was demonstrated.

[0030] Figure 13 This demonstrates the TDCC mediated by the CD20×CD3κλ bispecific antibody. Figure 13 A showed cytotoxicity against Nalm-6 cells. Figure 13 B shows activation of T cells.

[0031] Figure 14 This demonstrates the TDCC mediated by the CD20×CD3κλ bispecific antibody. Figure 14 A shows cytotoxicity against TMD-8 cells. Figure 14 B shows activation of T cells.

[0032] Figure 15 This demonstrates the TDCC mediated by the CD20×CD3κλ bispecific antibody. Figure 15 A showed cytotoxicity against Toledo cells. Figure 15 B shows activation of T cells.

[0033] Figure 16 The effect of the CD20×CD3κλ bispecific antibody on the T cell NFAT signaling pathway was demonstrated.

[0034] Figure 17 The inhibitory effect of the CD20×CD3κλ bispecific antibody on the Raji xenograft model in an immune-reconstituted mouse model was demonstrated.

[0035] Figure 18 The inhibitory effect of the CD20×CD3κλ bispecific antibody on a subcutaneous Raji and human PBMC mixed tumor model in immunodeficient mice was demonstrated.

[0036] Figure 19 The efficacy of the CD20×CD3κλ bispecific antibody in cynomolgus monkeys is shown.

[0037] Figure 20 The binding of the BCMA×CD3κλ bispecific antibody to BCMA-stable cells was demonstrated.

[0038] Figure 21The binding of the BCMA×CD3κλ bispecific antibody to tumor cells NCI-H929 and RPMI-8226 was demonstrated.

[0039] Figure 22 The binding of the BCMA×CD3κλ bispecific antibody to Jurkat cells is shown.

[0040] Figure 23 The binding of the BCMA×CD3κλ bispecific antibody to peripheral blood T cells was demonstrated.

[0041] Figure 24 This demonstrates the TDCC mediated by the BCMA×CD3κλ bispecific antibody. Figure 24 A showed cytotoxicity against NCI-H929 cells. Figure 24 B shows activation of T cells.

[0042] Figure 25 This demonstrates the TDCC mediated by the BCMA×CD3κλ bispecific antibody. Figure 25 A showed killing effect on RPMI-8226 cells. Figure 25 B shows activation of T cells.

[0043] Figure 26 The effect of the BCMA×CD3κλ bispecific antibody on the T cell NFAT signaling pathway was demonstrated.

[0044] Figure 27 The non-specific activation of PBMCs by the BCMA×CD3κλ bispecific antibody was demonstrated.

[0045] Figure 28 The binding of the BCMA×CD3κλ bispecific antibody to the Fc receptor is shown.

[0046] Figure 29 The inhibitory effect of BCMA×CD3κλ bispecific antibody in a subcutaneous NCI-H929 xenograft model in immunodeficient mice was demonstrated.

[0047] Figure 30 The binding of the GPC3×CD3κλ bispecific antibody to GPC3 stable cells was demonstrated.

[0048] Figure 31 The binding of the GPC3×CD3κλ bispecific antibody to HepG2 tumor cells was demonstrated.

[0049] Figure 32 The binding of the GPC3×CD3κλ bispecific antibody to Jurkat cells was demonstrated.

[0050] Figure 33The binding of the GPC3×CD3κλ bispecific antibody to peripheral blood T cells was demonstrated.

[0051] Figure 34 This demonstrates the TDCC mediated by the GPC3×CD3κλ bispecific antibody. Figure 34 A showed cytotoxicity against HepG2 cells. Figure 34 B shows activation of T cells.

[0052] Figure 35 This study demonstrates the effect of the GPC3×CD3κλ bispecific antibody on the NFAT signaling pathway in T cells.

[0053] Figure 36 The non-specific activation of PBMCs by the GPC3×CD3κλ bispecific antibody was demonstrated.

[0054] Figure 37 The inhibitory effect of the GPC3×CD3κλ bispecific antibody in the subcutaneous HepG2 xenograft model of immune-reconstituted mice was demonstrated.

[0055] Figure 38 The inhibitory effect of the GPC3×CD3κλ bispecific antibody on the CD3 humanized mouse Hepa1-6 / human GPC3 xenograft model was demonstrated. Detailed Implementation

[0056] I. Definition

[0057] In this disclosure, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this disclosure, definitions and explanations of relevant terms are provided below.

[0058] As used herein, "tumor antigen" preferably refers to any antigen or antigenic determinant present on (or bound to) tumor cells but not generally present on normal cells, or present on or bound to tumor cells in a greater amount than on normal (non-tumor) cells, or present on tumor cells in a form different from that found on normal (non-tumor) cells. The term therefore includes tumor-specific antigens, including tumor-specific antigens (TSA) or tumor-associated antigens (TAA), including tumor-associated membrane antigens, embryonic antigens on tumors, growth factor receptors, growth factor ligands, and any other types of antigens related to cancer. Tumor antigens can be, for example, B-cell differentiation antigens (e.g., CD19, CD20, and CD37), B-cell maturation antigen (BCMA), phosphatidylinositol proteoglycan 3 (GPC3), epithelial cancer antigens (e.g., breast cancer, gastrointestinal cancer, lung cancer), prostate-specific cancer antigen (PSA) or prostate-specific membrane antigen (PSMA), bladder cancer antigen, lung (e.g., small cell lung) cancer antigen, colon cancer antigen, ovarian cancer antigen, brain cancer antigen, gastric cancer antigen, renal cell carcinoma antigen, pancreatic cancer antigen, liver cancer antigen, esophageal cancer antigen, head and neck cancer antigen, or colorectal cancer antigen.

[0059] TSAs are (or are considered) unique to tumor cells and do not occur on other cells in the body (e.g., not to a significant extent on other cells). TAAs are not unique to tumor cells and are instead expressed on normal cells (e.g., under conditions that do not induce an immune tolerance state to the antigen). For example, when the immune system is immature and unable to respond, TAAs can be antigens that are expressed on normal cells during fetal development, or they can be antigens that are normally present at very low levels on normal cells but expressed at higher levels on tumor cells.

[0060] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other tumor antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, erbB, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3, CA 27.29, BCAA, CA 195, CA242, CA-50, CAM43, CD68, P1, CO-029, FGF-5, G250, Ga733, EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, Mac-2 binding protein, cyclophilic C-related protein, TAAL6, TAG72, TLP, MUC16, IL13Rα2, FRα, VEGFR2, Lewis Y, FAP, EphA2, CEACAM5, EGFR, CA6, CA9, GPNMB, EGP1, FOLR1, endothelial receptor, STEAP1, SLC44A4, conjugin-4, AGS-16, guanidinyl cyclase C, MUC-1, CFC1B, integrin α3 chain (a3b1 chain, i.e. laminin receptor chain) and TPS.Other tumor antigens include CD19, CD20, CD22, CD30, CD72, CD180, CD171 (L1CAM), CD123, CD133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166, CD71, CLL-1 / CLECK12A, ROR1, BCMA, phosphatidylinositol proteoglycan 3 (GPC3), mesothelin, CD33 / IL3Ra, c-Met, PSCA, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1, or MAGEA3.

[0061] As used herein, the term "CD20" refers to any natural CD20 from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats).

[0062] The terms "anti-CD20 antibody" and "CD20-binding antibody" refer to antibodies capable of binding to CD20 with sufficient affinity, making the antibody useful as a diagnostic and / or therapeutic agent targeting CD20. In one embodiment, the anti-CD20 antibody binds to less than about 10% of its binding to unrelated non-CD20 proteins, as measured by, for example, radioimmunoassay (RIA). In some embodiments, the CD20-binding antibody has a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 μM). -8 M or lower, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (K) of M) d In some implementations, anti-CD20 antibodies bind to conserved CD20 epitopes from different species.

[0063] As used herein, the term “BCMA” may refer to the concept of BCMA itself, which is present in animals and preferably in humans, as well as any variants, isotypes and paralogs.

[0064] The term "human BCMA" refers to BCMA of human origin, and preferably has an amino acid sequence having, but not limited to, Genbank accession number AB052772.1.

[0065] The terms "anti-BCMA antibody" and "BCMA-binding antibody" refer to antibodies capable of binding to BCMA with sufficient affinity, making the antibody useful as a diagnostic and / or therapeutic agent targeting BCMA. In one embodiment, the anti-BCMA antibody binds to less than about 10% of its binding to unrelated non-BCMA proteins, as measured by, for example, radioimmunoassay (RIA). In some embodiments, the BCMA-binding antibody has a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 μM). -8 M or lower, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (K) of M) d In some implementations, anti-BCMA antibodies bind to conserved BCMA epitopes across different species of BCMA.

[0066] As used herein, the term “GPC3” may refer to the concept of GPC3 itself, which is present in animals and preferably in humans, as well as any variants, isotypes and paralogs.

[0067] The term "human GPC3" refers to GPC3 derived from humans.

[0068] The terms "anti-GPC3 antibody" and "GPC3-binding antibody" refer to antibodies capable of binding to GPC3 with sufficient affinity, making the antibody useful as a diagnostic and / or therapeutic agent targeting GPC3. In one embodiment, the anti-GPC3 antibody binds to less than about 10% of its binding to unrelated non-GPC3 proteins, as measured by, for example, radioimmunoassay (RIA). In some embodiments, the GPC3-binding antibody has a concentration of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 μM). -8 M or lower, such as 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 The dissociation constant (K) of M) d In some implementations, anti-GPC3 antibodies bind to conserved GPC3 epitopes from different species of GPC3.

[0069] “CD3” refers to any native CD3 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats), unless otherwise specified. The term covers “full-length” unprocessed CD3 as well as any form of CD3 derived from cell processing. The term also covers naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, CD3 is human CD3, specifically the epsilon subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in UniProt (www.uniprot.org) accession number P07766 (version 144), or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_000724.1. The amino acid sequence of cynomolgus monkey [Macaca fascicularis] CD3ε is shown in NCBI GenBank no. BAB71849.1.

[0070] The term “cell surface” is used according to its normal meaning in this field, and therefore includes the cell exterior that can be accessed by binding to proteins and other molecules.

[0071] As used herein and unless otherwise stated, the terms “about” or “approximately” mean within 10% of a given value or range. Where an integer is required, the term means within 10% of a given value or range, rounded up or down to the nearest integer.

[0072] Regarding antibody chain polypeptide sequences, the phrase "substantially identical" can be understood as an antibody chain exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a reference polypeptide sequence. Regarding nucleic acid sequences, this term can be understood as a nucleotide sequence exhibiting at least greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher sequence identity with a reference nucleic acid sequence.

[0073] Sequence “identity” or “identity” has a generally accepted meaning in the art, and the percentage of sequence similarity between two nucleic acid or polypeptide molecules or regions can be calculated using publicly available techniques. Sequence similarity can be measured along the full length of a polynucleotide or polypeptide or along a region of that molecule (see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991). Although there are many methods for measuring the similarity between two polynucleotides or polypeptides, the term “similarity” is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0074] A "substitution" variant is a variant in which at least one amino acid residue is removed from the natural sequence and replaced by a different amino acid at the same position. The substitution can be single, where only one amino acid is substituted in the molecule; or it can be multiple, where two or more amino acids are substituted in the same molecule. Multiple substitutions can be located at consecutive sites. Similarly, an amino acid can be substituted by multiple residues, and such variants include both substitution and insertion. An "insertion" variant is a variant in which one or more amino acids are inserted into an amino acid immediately adjacent to a specific position in the natural sequence. An adjacent amino acid is defined as one attached to the α-carboxyl or α-amino functional group of that amino acid. A "deletion" variant is a variant in which one or more amino acids are removed from the natural amino acid sequence. Typically, deletion variants have one or two amino acids missing from a specific region of their molecule.

[0075] Regarding the variable domains of antibodies, the term "variable" refers to certain portions of related molecules that exhibit extensive sequence differences between antibodies and are used for the specific recognition and binding of a particular antibody to its specific target. However, variability is not uniformly distributed throughout the entire variable domain of an antibody. Variability is concentrated in three segments known as complementarity-determining regions (CDRs; namely CDR1, CDR2, and CDR3) or hypervariable regions, all located within the variable domains of the light and heavy chains. More conserved portions within the variable domain are called framework (FR) regions or framework sequences. Each variable domain of the natural heavy and light chains comprises four FR regions, primarily employing a β-sheet configuration, linked by three CDRs forming loops that connect the β-sheet structure and, in some cases, partially form a β-sheet. CDRs on each chain are typically linked together in proximity by FR regions, and the presence of CDRs from other chains contributes to the formation of antibody target binding sites (epitopes or determinants) (see Kabat et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)). As used herein, immunoglobulin amino acid residues are numbered according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise stated. A CDR may have the ability to specifically bind associated epitopes.

[0076] As used herein, an “antibody fragment” or “antigen-binding fragment” refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the antibody’s variable region (e.g., one or more CDRs and / or one or more antibody-binding sites) that binds to an antigen, and thus retains binding specificity as well as at least a portion of the full-length antibody’s specific binding capacity. Therefore, an antigen-binding fragment refers to an antibody fragment containing an antigen-binding portion that binds to the same antigen as an antibody fragment derived from the antibody fragment. Antibody fragments include antibody derivatives produced by enzymatic treatment of a full-length antibody, as well as synthetically produced derivatives, such as recombinant derivatives. Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single-chain Fv (scFv), Fv, dsFv, biantibodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, for example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragment may comprise multiple chains linked together, for example by disulfide bonds and / or by peptide linkers. Antibody fragments generally contain at least or about 50 amino acids, and typically at least or about 200 amino acids. Antigen-binding fragments include any antibody fragment that acquires immune-specific binding (i.e., exhibiting at least or at least about 10) upon insertion into an antibody framework (e.g., by replacing the corresponding region). 7 -10 8 Antibodies against the Ka antigen of M-1. A “functional fragment” or “antibody analog” is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction. As used herein, a functional fragment generally has the same meaning as “antibody fragment,” and in the context of antibodies, it can refer to a fragment that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction, such as Fv, Fab, F(ab')2, etc. An “Fv” fragment is a dimer (V) formed by the non-covalent binding of a variable domain of a heavy chain and a variable domain of a light chain. H -V L (Dimer) composition. In this configuration, the three CDRs of each variable domain interact to determine V. H -V L The target binding sites on the surface of the dimer are the same as in the case of the intact antibody. The six CDRs together confer target binding specificity to the intact antibody. However, even a single variable domain (or half the Fv of only including three target-specific CDRs) can still have the ability to recognize and bind to the target.

[0077] As used herein, the term "bispecific antibody" (BsAb) refers to an antibody and / or antigen-binding molecule that specifically binds to two different antigenic determinants. Typically, a bispecific antibody and / or antigen-binding molecule contains two antigen-binding sites, each specific to a different antigenic determinant. In some embodiments, the bispecific antibody and / or antigen-binding molecule is capable of binding to two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different cells.

[0078] As used herein, “monoclonal antibody” refers to a population of identical antibodies, meaning that each individual antibody molecule in a population of monoclonal antibodies is identical to the others. This characteristic contrasts with that of a polyclonal population of antibodies, which contains antibodies with a variety of different sequences. Monoclonal antibodies can be prepared by a number of well-known methods (Smith et al. (2004) J.Clin.Pahol.57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared from immortalized B cells, for instance, by fusing with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV. Recombinant technologies can also be used to prepare antibodies in vitro from a clonal population of host cells by transforming host cells with plasmids carrying artificial sequences of nucleotides encoding the antibody.

[0079] As used herein, the term "hybridoma" or "hybridoma cell" refers to a cell or cell line (typically myeloma or lymphoma cells) resulting from the fusion of antibody-producing lymphocytes and non-antibody-producing cancer cells. As is known to those skilled in the art, a hybridoma can proliferate and continuously supply cells that produce a specific monoclonal antibody. Methods for generating hybridomas are known in the art (see, for example, Harlow & Lane, 1988). When referring to the term "hybridoma" or "hybridoma cell," it also includes subclones and progeny cells of the hybridoma.

[0080] As used herein, a full-length antibody is an antibody having two full-length heavy chains (e.g., VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region, such as antibodies naturally produced by antibody-secreting B cells and synthetically produced antibodies with the same domains.

[0081] The term "chimeric antibody" refers to an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0082] "Humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) containing minimal sequences derived from non-human immunoglobulins. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues of the complementarity-determining region (CDR) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) with the desired specificity, affinity, and capability, such as mouse, rat, or rabbit.

[0083] Furthermore, in humanization, amino acid residues in the CDR1, CDR2, and / or CDR3 regions of VH and / or VL may be mutated to improve one or more binding properties (e.g., affinity) of the antibody. Mutations can be introduced, for example, through PCR-mediated mutations, and their effects on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conserved mutations are introduced. Such mutations can be amino acid substitutions, additions, or deletions. Additionally, mutations within the CDRs typically do not exceed one or two. Therefore, the humanized antibodies described in this disclosure also cover antibodies containing one or two amino acid mutations within the CDRs.

[0084] As used herein, the term "CDR" refers to the complementarity-determining region. Each heavy and light chain of an antibody molecule is known to have three CDRs. CDRs, also known as hypervariable regions, are located in the variable regions of each heavy and light chain of the antibody and are highly variable sites in the primary structure of the CDR. In this specification, the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 from the N-terminal sequence of the heavy chain, and the CDRs of the light chain are represented by CDR1, CDR2, and CDR3 from the N-terminal sequence of the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen to which the antibody binds.

[0085] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which an antibody binds at its complementary site. Epitope determinants typically comprise chemically active surface subtypes of a molecule, such as amino acid or sugar side chains, and often possess specific three-dimensional structural features as well as specific charge characteristics.

[0086] As used herein, the terms “specific binding” and “immune-specific binding” for antibodies or their antigen-binding fragments are used interchangeably and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with the same antigen through a non-covalent interaction between the antibody and the antigen’s antibody-binding site. The antigen may be an isolated antigen or present in tumor cells. Typically, antibodies that immune-specifically bind (or specifically bind) antigens are present in quantities of approximately 1 × 10⁻⁶. 7 M -1 Or 1×10 8 M -1 Or a larger affinity constant Ka (or 1×10) -7 M or 1×10 -8 The affinity constant (M or lower) binds to the antigen. The affinity constant can be determined by standard kinetic methods of antibody reaction, such as immunoassay, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art (see, for example, Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336 (1989); also see U.S. Patent No. 7,229,619, which describes exemplary SPR and ITC methods for calculating the binding affinity of an antibody). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences; Malmqvist (2000) Biochem.Soc.Trans. 27:335).

[0087] As used herein, the terms “polynucleotide” and “nucleic acid molecule” refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) typically linked together by phosphodiester bonds. As used herein, the term “nucleic acid molecule” is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0088] As used herein, isolated nucleic acid molecules are nucleic acid molecules isolated from other nucleic acid molecules present in natural sources of nucleic acid molecules. “Isolated” nucleic acid molecules, such as cDNA molecules, may be substantially free of other cellular material or culture medium when prepared by recombinant technology, or substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding provided antibody or antigen-binding fragments.

[0089] As used herein, “operably linked” in relation to a nucleic acid sequence, region, element, or domain indicates that the nucleic acid regions are functionally related to each other. For example, a promoter can be operably linked to a nucleic acid encoding a polypeptide, thereby regulating or mediating the transcription of that nucleic acid.

[0090] Also provided are “conserved sequence modifications” of the sequences listed herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate the binding of antibodies to antigens encoded by nucleotide sequences or containing amino acid sequences. These conserved sequence modifications include conserved nucleotide and amino acid substitutions, as well as nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listings herein using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved sequence modifications include conserved amino acid substitutions, wherein amino acid residues are replaced with amino acid residues having similar side chains. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with non-polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, the predicted non-essential amino acid residues in anti-CD20, BCMA, or GPC3 antibodies are preferably replaced by another amino acid residue from the same side chain family. Methods for identifying nucleotides and conserved amino acid substitutions that do not eliminate antigen binding are well known in the field (e.g., see Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); Burks et al., Proc.Natl.Acad.Sci.USA 94: 412-417 (1997)).

[0091] As an alternative, in another embodiment, mutations can be randomly introduced along all or part of the coding sequence of the anti-GCD20, BCMA, or PC3 antibody, for example, through saturation mutagenesis, and the modified anti-CD20, BCMA, or GPC3 antibody can be screened for improved binding activity.

[0092] As used herein, “expression” refers to the process by which a polypeptide is produced through the transcription and translation of polynucleotides. The expression level of a polypeptide can be evaluated using any method known in the art, including, for example, methods for determining the amount of polypeptide produced from host cells. Such methods may include, but are not limited to, quantifying polypeptides in cell lysates by ELISA, Coomassie blue staining following gel electrophoresis, Lowry protein assays, and Bradford protein assays.

[0093] As used herein, a “host cell” is a cell used to receive, maintain, replicate, and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When a host cell divides, the nucleic acids contained in the vector replicate, thereby amplifying the nucleic acids. Host cells can be eukaryotic or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, and HEK cells such as HEK 293 cells.

[0094] As used herein, a "vector" is a reproducible nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. Vectors include those into which nucleic acids encoding polypeptides or fragments thereof can typically be introduced via restriction enzyme digestion and ligation. Vectors also include those containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for amplification of nucleic acids or for expression / display of the polypeptide encoded by the nucleic acid. Vectors are typically kept free but can be designed to integrate genes or portions thereof into the chromosome of the genome. Vectors for artificial chromosomes, such as yeast artificial vectors and mammalian artificial chromosomes, are also considered. The selection and use of such vectors are well known to those skilled in the art.

[0095] As used in this article, vectors also include “viral vectors” or “vectors of viruses.” Viral vectors are engineered viruses that are operatively linked to a foreign gene to transfer (as a medium or shuttle) the foreign gene into cells.

[0096] As used herein, "expression vector" includes a vector capable of expressing DNA operatively linked to regulatory sequences, such as promoter regions, that influence the expression of such DNA fragments. These additional fragments may include promoter and terminator sequences and optionally include one or more origins of replication, one or more selection markers, enhancers, polyadenylation signals, etc. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. Therefore, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, bacteriophage, recombinant virus, or other vector, which, when introduced into a suitable host cell, results in the expression of clonal DNA. Suitable expression vectors are well known to those skilled in the art and include reproducible expression vectors in eukaryotic and / or prokaryotic cells, as well as expression vectors that remain free or are integrated into the host cell genome.

[0097] As used herein, “treatment” for an individual suffering from a disease or disease condition means that the individual’s symptoms are partially or completely relieved, or remain unchanged after treatment. Therefore, treatment includes prevention, treatment, and / or cure. Prevention refers to preventing underlying disease and / or preventing the worsening of symptoms or the development of disease. Treatment also includes any antibodies or antigen-binding fragments thereof provided, and any pharmaceutical use of the compositions provided herein.

[0098] As used in this article, “therapeutic effect” refers to the effect resulting from treatment of an individual, which alters, usually improves or enhances the symptoms of a disease or condition, or cures a disease or condition.

[0099] As used herein, "therapeutic effective amount" or "therapeutic effective dose" refers to an amount of substance, compound, material, or composition containing a compound that, when applied to a subject, is at least sufficient to produce a therapeutic effect. Therefore, it is the amount necessary to prevent, cure, improve, block, or partially block the symptoms of a disease or condition.

[0100] As used herein, "preventive effective dose" or "preventive effective amount" refers to the amount of a substance, compound, material, or composition containing a compound that, when applied to a subject, would have the intended preventive effect, such as preventing or delaying the onset or recurrence of a disease or symptom, or reducing the likelihood of the onset or recurrence of a disease or symptom. A fully preventive effective dose does not necessarily occur through the administration of a single dose and can occur only after a series of doses have been administered. Therefore, a preventive effective dose can be administered in one or more applications.

[0101] As used in this article, the term "patient" refers to mammals, such as humans.

[0102] II. Detailed Implementation Plan

[0103] In one aspect, this disclosure provides a bispecific antibody or an antigen-binding fragment thereof, comprising:

[0104] (a) A first antigen-binding moiety or an antigen-binding fragment thereof, the first antigen-binding moiety comprising a first light chain and a first heavy chain, the first light chain being a κ-type light chain, the first antigen-binding moiety comprising a first binding domain for binding to a first antigen; and

[0105] (b) A second antigen-binding moiety or an antigen-binding fragment thereof, wherein the second antigen-binding moiety comprises a second light chain and a second heavy chain, the second light chain being a λ-type light chain, and the second antigen-binding moiety comprises a second binding domain that binds to the second antigen.

[0106] In some implementations, the second antigen is the CD3 antigen.

[0107] In some embodiments, the second light chain variable region of the second antigen-binding moiety has Gln 40 Glu mutation (Vλ) CD3 Gln 40 Glu); the second heavy chain variable region of the second antigen-binding moiety has Gln 39 Lys mutation (VH) CD3 Gln 39 Lys).

[0108] In some embodiments, the second binding domain comprises a second light chain CDR selected from amino acid sequences SEQ ID NO: 7-9, 14, 15, 20, 21 or any variant thereof; and / or a second heavy chain CDR selected from amino acid sequences SEQ ID NO: 26-28, 31, 34, 40, 43, 46, 47 or any variant thereof.

[0109] In some embodiments, the second binding domain comprises a second light chain CDR1 selected from amino acid sequences SEQ ID NO: 7, 14 or any variant thereof, a second light chain CDR2 selected from amino acid sequences SEQ ID NO: 8, 15, 20 or any variant thereof, a second light chain CDR3 selected from amino acid sequences SEQ ID NO: 9, 21 or any variant thereof; and / or a second heavy chain CDR1 selected from amino acid sequences SEQ ID NO: 26, 31, 46 or any variant thereof, a second heavy chain CDR2 selected from amino acid sequences SEQ ID NO: 27, 47 or any variant thereof, and a second heavy chain CDR3 selected from amino acid sequences SEQ ID NO: 28, 34, 37, 40, 43 or any variant thereof.

[0110] In some embodiments, the second light chain CDR of the second binding domain is selected from: second light chain CDR1, CDR2, and CDR3 sequences comprising amino acid sequences SEQ ID NO: 7, 8, and 9, respectively; second light chain CDR1, CDR2, and CDR3 sequences comprising amino acid sequences SEQ ID NO: 14, 15, and 9, respectively; second light chain CDR1, CDR2, and CDR3 sequences comprising amino acid sequences SEQ ID NO: 7, 8, and 21, respectively; second light chain CDR1, CDR2, and CDR3 sequences comprising amino acid sequences SEQ ID NO: 7, 20, and 21, respectively; and / or the heavy chain CDR of the second binding domain is selected from: second heavy chain CDR1, CDR2, and CDR3 sequences comprising amino acid sequences SEQ ID NO: 26, 27, and 28, respectively; second heavy chain CDR1, CDR2, and CDR3 sequences comprising amino acid sequences SEQ ID NO: 31 ... The second heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 31, 27, and 37, respectively; the second heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 31, 27, and 40, respectively; the second heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 31, 27, and 43, respectively; and the second heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NO: 46, 47, and 28, respectively.

[0111] In some embodiments, the second binding domain comprises a second light chain variable region selected from amino acid sequences SEQ ID NO: 5, 10, 12, 16, 18, 22 or any variant thereof; and / or a second heavy chain variable region selected from amino acid sequences SEQ ID NO: 24, 29, 32, 35, 38, 41, 44, 48, 50, 52 or any variant thereof.

[0112] In some embodiments, the second binding domain comprises a second light chain variable region of amino acid sequence SEQ ID NO: 18 or any variant thereof; and a second heavy chain variable region of amino acid sequence SEQ ID NO: 24 or any variant thereof.

[0113] In some embodiments, the second binding domain comprises a second light chain variable region of amino acid sequence SEQ ID NO: 5 or any variant thereof; and a second heavy chain variable region of amino acid sequence SEQ ID NO: 48 or any variant thereof.

[0114] In some embodiments, the second binding domain comprises a second light chain variable region of amino acid sequence SEQ ID NO: 18 or any variant thereof; and a second heavy chain variable region of amino acid sequence SEQ ID NO: 48 or any variant thereof.

[0115] In some embodiments, the second binding domain comprises a second light chain variable region of amino acid sequence SEQ ID NO: 5 or any variant thereof; and a second heavy chain variable region of amino acid sequence SEQ ID NO: 50 or any variant thereof.

[0116] In some embodiments, the second binding domain comprises a second light chain variable region of amino acid sequence SEQ ID NO: 10 or any variant thereof; and a second heavy chain variable region of amino acid sequence SEQ ID NO: 50 or any variant thereof.

[0117] In some embodiments, the second binding domain comprises a second light chain variable region of amino acid sequence SEQ ID NO: 12 or any variant thereof; and a second heavy chain variable region of amino acid sequence SEQ ID NO: 50 or any variant thereof.

[0118] In some embodiments, the second binding domain comprises a second light chain variable region of amino acid sequence SEQ ID NO: 18 or any variant thereof; and a second heavy chain variable region of amino acid sequence SEQ ID NO: 50 or any variant thereof.

[0119] In some embodiments, the second light chain of the second antigen-binding moiety is selected from amino acid sequences SEQ ID NO: 58 and 66; and / or the second heavy chain of the second antigen-binding moiety is selected from amino acid sequences SEQ ID NO: 60 and 68. In some preferred embodiments, the second light chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 58; and the second heavy chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 60. In some preferred embodiments, the second light chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 66; and the second heavy chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 68.

[0120] In some implementations, the first antigen is a tumor antigen.

[0121] In some preferred embodiments, the tumor antigen is selected from: CD19, CD20, CD22, CD30, CD38, CD72, CD180, CD171 (L1CAM), CD123, CD133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166, CD71, CLL-1 / CLECK12A, ROR1, BCMA, GPC3, mesothelin, CD33 / IL3Ra, c-Met, PSCA, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1, Her2, Her3, MUC1, MUC17, Claudin18, or MAGEA3.

[0122] In one specific implementation, the tumor-associated antigen is selected from CD20, BCMA, and GPC3.

[0123] In some implementations, the first antigen is the CD20 antigen.

[0124] In some preferred embodiments, the first light chain variable region of the first antigen-binding moiety has Gln 38 Lys mutation (Vκ) CD20 Gln 38 Lys). In some preferred embodiments, the first heavy chain variable region of the first antigen-binding moiety has Gln. 39 Glu mutation (VH) CD20 Gln 39 Glu).

[0125] In some preferred embodiments, the first light chain variable region of the first antigen-binding moiety has Gln 38 Lys mutation (Vκ) CD20 Gln 38 Lys), and the first light chain constant region has Glu 123 Lys and Gln 124 Lys mutation (Vκ-Ck) CD20 Gln 38 Lys\Glu 123 Lys\Gln 124 Lys). In some preferred embodiments, the first heavy chain variable region of the first antigen-binding moiety has Gln. 39 Glu mutation (VH) CD20 Gln 39 Glu), and the first heavy chain constant region has Lys 152 Glu and Lys 218 Glu mutation (V H -C H1 CD20 Gln 39 Glu\Lys 152 Glu\Lys 218 Glu).

[0126] In some preferred embodiments, the first light chain of the first antigen-binding portion is selected from amino acid sequences SEQ ID NO: 54, 62 and 70; and / or the first heavy chain of the first antigen-binding portion is selected from amino acid sequences SEQ ID NO: 56, 64 and 72.

[0127] In some preferred embodiments, the first light chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 54, and the first heavy chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 56.

[0128] In some preferred embodiments, the first light chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 62, and the first heavy chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 64.

[0129] In some preferred embodiments, the first light chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 70, and the first heavy chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 72.

[0130] In some preferred embodiments, the first light chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 54, and the first heavy chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 56; the second light chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 58, and the second heavy chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 60.

[0131] In some preferred embodiments, the first light chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 62, and the first heavy chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 64; the second light chain of the second antigen-binding portion is an amino acid sequence SEQ ID NO: 66, and the second heavy chain of the second antigen-binding portion is an amino acid sequence SEQ ID NO: 68; the second light chain of the second antigen-binding portion is an amino acid sequence SEQ ID NO: 58, and the second heavy chain of the second antigen-binding portion is an amino acid sequence SEQ ID NO: 60.

[0132] In some preferred embodiments, the first light chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 70, and the first heavy chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 72; the second light chain of the second antigen-binding portion is an amino acid sequence SEQ ID NO: 66, and the second heavy chain of the second antigen-binding portion is an amino acid sequence SEQ ID NO: 68.

[0133] In some preferred embodiments, the first light chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 62, and the first heavy chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 64; the second light chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 58, and the second heavy chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 60.

[0134] In some preferred embodiments, the first light chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 54, and the first heavy chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 56; the second light chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 66, and the second heavy chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 68.

[0135] In some implementations, the first antigen is the BCMA antigen.

[0136] In some preferred embodiments, the first light chain variable region of the first antigen-binding moiety has Gln 42 Lys mutation (Vκ) BCMA Gln 42 Lys). In some preferred embodiments, the first heavy chain variable region of the first antigen-binding moiety has Gln. 39 Glu mutation (VH) BCMA Gln 39 Glu).

[0137] In some preferred embodiments, the first light chain of the first antigen-binding portion is selected from amino acid sequences SEQ ID NO: 80 and 84; and / or the first heavy chain of the first antigen-binding portion is selected from amino acid sequences SEQ ID NO: 82 and 86.

[0138] In some preferred embodiments, the first light chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 80; and the first heavy chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 82.

[0139] In some preferred embodiments, the first light chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 84; and the first heavy chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 82.

[0140] In some preferred embodiments, the first light chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 80; and the first heavy chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 86.

[0141] In some preferred embodiments, the first light chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 84; and the first heavy chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 86.

[0142] In some preferred embodiments, the first light chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 80, and the first heavy chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 82; the second light chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 66, and the second heavy chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 68.

[0143] In some preferred embodiments, the first light chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 84, and the first heavy chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 82; the second light chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 66, and the second heavy chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 68.

[0144] In some preferred embodiments, the first light chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 80, and the first heavy chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 86; the second light chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 66, and the second heavy chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 68.

[0145] In some preferred embodiments, the first light chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 84, and the first heavy chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 86; the second light chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 66, and the second heavy chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 68.

[0146] In some implementations, the first antigen is the GPC3 antigen.

[0147] In some preferred embodiments, the first light chain variable region of the first antigen-binding moiety has Gln 43 Lys and Gln 39 Glu mutation (Vκ) GPC3 Gln 43 Lys;VH GPC3 Gln 39 Glu).

[0148] In some preferred embodiments, the first light chain of the first antigen-binding portion is selected from amino acid sequences SEQ ID NO: 88 and 92; and / or the first heavy chain of the first antigen-binding portion is selected from amino acid sequences SEQ ID NO: 90 and 94.

[0149] In some preferred embodiments, the first light chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 88, and the first heavy chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 90.

[0150] In some preferred embodiments, the first light chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 92, and the first heavy chain of the first antigen-binding portion is an amino acid sequence SEQ ID NO: 94.

[0151] In some preferred embodiments, the first light chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 88, and the first heavy chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 90; the second light chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 66, and the second heavy chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 68.

[0152] In some preferred embodiments, the first light chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 92, and the first heavy chain of the first antigen-binding portion has an amino acid sequence SEQ ID NO: 94; the second light chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 66, and the second heavy chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 68.

[0153] In some embodiments, the Fc portion of the first antigen-binding portion and / or the second antigen-binding portion of the bispecific antibody employs a knob-into-hole structure. In some preferred embodiments, a human IgG4 knob-into-hole structure is employed.

[0154] In some embodiments, the first antigen-binding portion and / or the second antigen-binding portion of the bispecific antibody further comprises Ser 228 Pro, Leu 235 Glu and / or Pro 329 Ala mutation.

[0155] In one respect, this disclosure provides nucleic acids encoding the aforementioned bispecific antibodies or their antigen-binding portions.

[0156] In some preferred embodiments, the second antigen-binding moiety binds to the CD3 antigen, and the nucleic acid encoding the second light chain variable region of the second antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 6, 11, 13, 17, 19, and 23; and / or the nucleic acid encoding the second heavy chain variable region of the second antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 25, 30, 33, 36, 39, 42, 45, 49, 51, and 53. In some preferred embodiments, the nucleic acid encoding the second light chain of the second antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 59 and 67; and / or the nucleic acid encoding the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 61 and 69. In some preferred embodiments, the nucleic acid encoding the second light chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 59, and the nucleic acid encoding the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 61. In some preferred embodiments, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 69.

[0157] In some preferred embodiments, the first antigen-binding moiety binds to the CD20 antigen, and the nucleic acid encoding the first light chain of the first antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 55, 63, and 71; and / or the nucleic acid encoding the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 57, 65, and 73. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 55, and the nucleic acid encoding the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 57. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 63, and the nucleic acid encoding the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 65. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 71, and the nucleic acid encoding the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 73.

[0158] In some preferred embodiments, the first antigen-binding moiety binds to the BCMA antigen, and the nucleic acid encoding the first light chain of the first antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 81 and 85; and / or the nucleic acid encoding the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 83 and 87. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 81, and the nucleic acid encoding the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 83. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 85, and the nucleic acid encoding the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 83. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 81, and the nucleic acid encoding the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 87. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 85, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 87.

[0159] In some preferred embodiments, the first antigen-binding moiety binds to the GPC3 antigen, and the nucleic acid encoding the first light chain of the first antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 89 and 93; and / or the nucleic acid encoding the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 91 and 95. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 89, and the nucleic acid encoding the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 91. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 93, and the nucleic acid encoding the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 95.

[0160] In some preferred embodiments, the first antigen-binding portion of the bispecific antibody binds to the CD20 antigen, and the second antigen binds to the CD3 antigen. The nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 55, the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 57, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 59, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 61. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 63, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 65; the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 69; the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 59, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 61. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from nucleotide sequence SEQ ID NO: 71, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from nucleotide sequence SEQ ID NO: 73; the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from nucleotide sequence SEQ ID NO: 67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from nucleotide sequence SEQ ID NO: 69. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from nucleotide sequence SEQ ID NO: 63, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from nucleotide sequence SEQ ID NO: 65; the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from nucleotide sequence SEQ ID NO: 59, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from nucleotide sequence SEQ ID NO: 61.In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 55, the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 57, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 69.

[0161] In some preferred embodiments, the first antigen-binding portion of the bispecific antibody binds to the BCMA antigen, the second antigen binds to the CD3 antigen, and the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 81, the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 83, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 69. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 85, the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 83, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 69. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 81, the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 87, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 69. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 85, the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 87, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 69.

[0162] In some preferred embodiments, the first antigen-binding portion of the bispecific antibody binds to the GPC3 antigen, and the second antigen binds to the CD3 antigen. The nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 89, the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 91, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 69. In some preferred embodiments, the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 93, the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 95, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 69.

[0163] On the one hand, this disclosure provides a vector containing the aforementioned nucleic acid.

[0164] In one respect, this disclosure provides cells containing the aforementioned nucleic acids or vectors.

[0165] In one aspect, this disclosure provides compositions comprising the aforementioned bispecific antibody or its antigen-binding portion, nucleic acid, vector, and / or cells.

[0166] In one respect, this disclosure provides antibody-drug conjugates comprising the aforementioned bispecific antibody or its antigen-binding portion covalently attached to a therapeutic portion.

[0167] Preferably, the therapeutic component is selected from cytotoxic components, chemotherapeutic agents, cytokines, immunosuppressants, immunostimulants, cleavage peptides, or radioisotopes.

[0168] The antibodies disclosed herein can be used as therapeutic or diagnostic tools in diseases in which various tumor antigens are adversely expressed or discovered.

[0169] In one embodiment of a tumor antigen-related disease, the expression of the tumor antigen in cells of a diseased tissue or organ is increased compared to its state in a healthy tissue or organ. An increase is defined as an increase of at least 10%, particularly at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000%, or even more. In one embodiment, expression is found only in the diseased tissue, while expression is suppressed in the corresponding healthy tissue. According to this disclosure, tumor antigen-related diseases include tumors.

[0170] In some embodiments, the tumor antigen-associated disease is a CD20-associated disease. In some preferred embodiments, the CD20-associated disease includes B-cell diseases, such as B-cell proliferative disorders, particularly CD20-positive B-cell disorders; preferably, the disease is selected from non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and multiple myeloma (MM) and Hodgkin lymphoma (HL).

[0171] In some embodiments, the tumor antigen-related disease is a BCMA-related disease; preferably, the BCMA-related disease includes B-cell diseases; preferably, the disease is cancer; more preferably, the cancer is a B-cell-related cancer selected from multiple myeloma, malignant plasmacytoma, Hodgkin lymphoma, nodular lymphocytic Hodgkin lymphoma, Kahler's disease, myeloid leukemia, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), and chronic lymphocytic leukemia (CLL). Acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-cell lymphoma, myeloid leukemia, Waldenström macroglobulinemia, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B-cell lymphoma, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, marginal zone B-cell lymphoma of lymph nodes, marginal zone lymphoma of spleen, blood Intravascular large B-cell lymphoma, primary exudative lymphoma, lymphomatoid granulomatosis, T-cell / histocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma in the elderly, inflammation-associated diffuse large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising from HHV8-associated multicentric Castleman's disease, unclassified B-cell lymphoma with intermediate features between diffuse large B-cell lymphoma and Burkitt lymphoma. B-cell lymphoma, unclassified B-cell lymphoma with intermediate features between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and other B-cell-associated lymphomas; more preferably, the B-cell disease is a B-cell disorder; preferably, the plasma cell disorder is selected from: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary osteoplasmacytoma, extramedullary plasmacytoma, osteosclerosing myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and condensational multiple myeloma; preferably, the disease is an autoimmune disease, such as systemic lupus erythematosus or rheumatoid arthritis.

[0172] In some implementations, the therapeutic agent contains an antibody that specifically binds to an activated T-cell antigen.

[0173] In one embodiment, the therapeutic agent comprises an antibody that specifically binds to CD3, particularly CD3ε.

[0174] Methods for treating diseases and symptoms using the bispecific antibodies of this disclosure include the following steps: administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof, or nucleic acid molecule, or carrier, or cell, or pharmaceutical composition to the mammal.

[0175] In some embodiments, this disclosure provides a method of treating or preventing cancer, comprising administering an antibody capable of binding to GPC3 to a patient, wherein the antibody is administered to provide a serum level of at least 40 μg / ml. In various embodiments, the antibody is administered to provide a serum level of at least 50 μg / ml, at least 150 μg / ml, at least 300 μg / ml, at least 400 μg / ml, or at least 500 μg / ml. In various embodiments, the antibody is administered to provide a serum level not exceeding 800 μg / ml, 700 μg / ml, 600 μg / ml, 550 μg / ml, or 500 μg / ml. In one embodiment, the provided serum level is from 40 μg / ml to 700 μg / ml, preferably from 40 μg / ml to 600 μg / ml, preferably from 50 μg / ml to 500 μg / ml, such as from 150 μg / ml to 500 μg / ml or from 300 μg / ml to 500 μg / ml. As used herein, the term "serum level" refers to the concentration of the substance in serum. In one embodiment, said serum level is provided for at least 7 days or at least 14 days. In one embodiment, the method includes administering at least 300 mg / m². 2 Antibody dosage, such as at least 600 mg / m² 2 And preferably up to 1500 mg / m² 2 At most 1200 mg / m 2 Or up to 1000 mg / m 2 .

[0176] In some embodiments, this disclosure provides a method of treating or preventing cancer, comprising administering to a patient an antibody capable of binding to GPC3, wherein the antibody is at a concentration of at least 300 mg / m². 2 such as at least 600mg / m 2 And preferably up to 1500 mg / m² 2 At most 1200 mg / m 2 Or up to 1000 mg / m 2 The antibody was administered at the prescribed dose.

[0177] In some embodiments, this disclosure provides a method for treating or preventing cancer, comprising administering an antibody capable of binding to GPC3 to a patient, wherein at least 50%, preferably 60%, 70%, 80%, or 90% of the patient's cancer cells are GPC3 positive and / or at least 40%, preferably 50%, or 60% of the patient's cancer cells are GPC3 surface-expressing positive. In this respect, this disclosure also provides a method for treating or preventing cancer, the method comprising: a. identifying a patient showing at least 50%, preferably 60%, 70%, 80%, or 90% GPC3-positive cancer cells and / or at least 40%, preferably 50%, or 60% of cancer cells that are GPC3 surface-expressing positive; and b. administering an antibody capable of binding to GPC3 to the patient. In one embodiment, at least 95% or at least 98% of the patient's cancer cells are GPC3 positive. In one embodiment, at least 70%, at least 80%, or at least 90% of the patient's cancer cells are GPC3 surface-expressing positive.

[0178] In one embodiment of the methods described herein, the treatment outcome for cancer is the achievement of disease stabilization. In one embodiment, disease stabilization is achieved for at least 2 months, at least 3 months, or at least 6 months.

[0179] In some embodiments, this disclosure provides a method for achieving disease stabilization in a cancer patient, comprising administering an antibody capable of binding to GPC3 to the patient. In one embodiment, disease stabilization is achieved for at least 2 months, at least 3 months, or at least 6 months.

[0180] In one embodiment of the method described herein, the antibody is administered in a single dose or multiple doses.

[0181] In some embodiments, this disclosure provides a method for treating or preventing cancer, which includes administering an antibody capable of binding to GPC3 to a patient, wherein the antibody is administered in multiple doses.

[0182] If the antibody is administered in multiple doses according to this disclosure, it is preferred to administer the antibody in at least 3, 4, 5, 6, 7, 8, 9, or 10 doses, and more preferably in up to 30, 25, 20, 15, or 10 doses. It is preferred to administer the antibody at intervals of at least 7, 10, 14, or 20 days. It is also preferred to administer the antibody at intervals of 7 to 30 days, 10 to 20 days, and most preferably about 14 days.

[0183] In one embodiment, the antibody is administered to provide a serum level of at least 40 μg / ml. In various embodiments, the antibody is administered to provide a serum level of at least 50 μg / ml, at least 150 μg / ml, at least 300 μg / ml, at least 400 μg / ml, or at least 500 μg / ml. In various embodiments, the antibody is administered to provide a serum level not exceeding 800 μg / ml, 700 μg / ml, 600 μg / ml, 550 μg / ml, or 500 μg / ml. In one embodiment, the provided serum level is from 40 μg / ml to 700 μg / ml, preferably from 40 μg / ml to 600 μg / ml, preferably from 50 μg / ml to 500 μg / ml, such as from 150 μg / ml to 500 μg / ml or from 300 μg / ml to 500 μg / ml. In one embodiment, the serum level is provided for at least 7 days or at least 14 days. In one embodiment, the method includes administering at least 300 mg / ml. 2 such as at least 600mg / m 2 And preferably up to 1500 mg / m² 2 Up to 1200mg / m 2 Or up to 1000 mg / m 2 The dosage of antibodies.

[0184] The use of any of the foregoing antibodies or their antigen-binding fragments or nucleic acid molecules or carriers or cells or pharmaceutical compositions in the preparation of a medicament for the treatment of GPC3-related diseases in mammals.

[0185] Optionally, according to any of the foregoing aspects, the antibody may be conjugated to other drugs, such as labeled or cytotoxic conjugates.

[0186] In one aspect, this disclosure also includes kits, such as those comprising antibodies, fragments thereof, homologs thereof, derivatives thereof, nucleic acids, vectors, cells, compositions thereof, etc., as disclosed herein, such as labeled or cytotoxic conjugates, and instructions for use of antibodies, conjugates that kill specific cell types, etc. These instructions may include guidance on the use of antibodies, conjugates, etc., in vitro, in vivo, or ex vivo. Antibodies may be in liquid or solid form, typically lyophilized. The kit may contain other suitable reagents, such as buffers, reconstitution solutions, and other necessary components for the intended use. Consideration is given to reagent combinations packaged in predetermined quantities with instructions for their use, such as for therapeutic purposes or for diagnostic assays. When the antibody is labeled, for example, enzyme-labeled, the kit may include substrates and cofactors required for the enzyme (e.g., providing substrate precursors for detecting chromophores or fluorophores). Furthermore, other additives, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), may also be included. The relative amounts of various reagents can be varied to provide concentrated reagent solutions, which provides user flexibility, space savings, reagent savings, etc. These reagents may also be provided in dry powder form, typically lyophilized, and include excipients that, when dissolved, provide a reagent solution of appropriate concentration.

[0187] The use of any of the foregoing antibodies or their functional fragments or nucleic acid molecules or vectors or cells or pharmaceutical compositions or kits in the preparation of reagents for inhibiting GPC3 binding.

[0188] Furthermore, the antibodies disclosed herein can also be used in immunoassays, purification methods, and other methods that utilize immunoglobulins or fragments thereof. Such uses are well known in the art.

[0189] Accordingly, this disclosure also provides compositions comprising an antibody or fragment thereof against GPC3 of this disclosure, said antibody being conveniently combined with a pharmaceutically acceptable carrier, diluent or excipient, as is common practice in the art.

[0190] As used in this disclosure, the term "pharmaceutical composition" refers to a formulation of a variety of preparations. Formulations containing a therapeutically effective amount of a multivalent antibody are in the form of a sterile liquid solution, liquid suspension, or lyophilized form, optionally containing a stabilizer or excipient.

[0191] The antibodies disclosed herein can be used as a single-use composition or in combination with other active agents.

[0192] In some embodiments, the humanized antibody of this disclosure is conjugated to a therapeutic portion (i.e., a drug). The therapeutic portion may be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a cleaved peptide, or a radioisotope. Such conjugates are referred to herein as “antibody-drug conjugates” or “ADCs”.

[0193] In some implementations, the antibody is conjugated to the cytotoxic portion. The cytotoxic portion may be selected, for example, from the following: paclitaxel; cytochalasin B; bacitracin D; ethidium bromide; emetine; mitomycin; etoposide; teniposide; vincristine; vinblastine; colchicine; doxorubicin; daunorubicin; dihydroxyanthradinone; microtubule inhibitors such as maytansin or their analogues or derivatives; antimitotic agents such as monomethylolpropionate E or F or their analogues or derivatives; salivarius toxin 10 or 15 or... Its analogues; irinotecan or its analogues; mitoxantrone; sclerosomycin; actinomycin D; 1-dehydrotestosterone; glucocorticoids; procaine; tetracaine; lidocaine; propranolol; puromycin; chachomycin or its analogues or derivatives; antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, sebazine, hydroxyurea, asparaginase, gemcitabine or cladribine; alkyl Antibiotics such as dichloromethyldiethylamine, thiopurine, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C; platinum derivatives such as cisplatin or carboplatin; docamycin A, docamycin SA, resveratrol (CC-1065) or their analogues or derivatives; antibiotics such as actinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, chloramphenicol, mitomycin, mitoxantrone, praziamic acid, benzomycin (AMC); pyrrolo[2,1-c][1,4]-benzodiazepines (PDB); diphtheria toxin and related molecules such as diphtheria A chain and its active fragments and hybrid molecules, ricin such as ricin A or deglycosylated ricin A chain toxin, cholera toxin, shiga-like toxins such as SLT. I, SLT II, ​​SLT III, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, alarin, saponins, saccharin, gellingin, absinthecin A chain, saccharin A chain, α-sarcin, Aleurites fordii protein, caryophyllin protein, American pokeweed proteins such as PAPI, PAPII and PAP-S, momordica charantia inhibitor, lacrimalin, croton toxin, sapaonaria officinalis inhibitor, white tree toxin, mitomycin, localized aspergillin, phenolmycin and enoxamycin toxin; ribonuclease (RNase); DNase I, staphylococcal endotoxin A; pokeweed antiviral protein; diphtheria toxin and Pseudomonas endotoxin.

[0194] In some embodiments, the antibody is conjugated to oligristatin or its peptide analogs, derivatives, or prodrugs. Oligstatin has been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division, and to possess anticancer and antifungal activities. For example, oligristatin E can react with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEB, respectively. Other typical oligristatin derivatives include AFP, MMAF (monomethyl oligristatin F), and MMAE (monomethyl oligristatin E). Suitable olistatin and its analogues, derivatives and prodrugs, as well as suitable adapters for conjugating olistatin to Ab, are described, for example, in U.S. Patent Nos. 5,635,483, 5,780,588 and 6,214,345 and International Patent Application Publications WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968 and WO205082023.

[0195] In some implementations, the antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine (PDB) or its peptide analogs, derivatives, or prodrugs. Suitable PDBs and PDB derivatives and related techniques are described, for example, in Hartley JA et al., Cancer Res 2010; 70(17):6849-6858; Antonow D. et al., Cancer J 2008; 14(3):154-169; Howard PW et al., Bioorg Med Chem Lett 2009; 19:6463-6466; and Sagnou et al., Bioorg Med Chem Lett 2000; 10(18):2083-2086.

[0196] In some implementations, the antibody is conjugated to a cytotoxic moiety selected from the following: anthracycline antibiotics, maytansine, chachomycosis, docamycosis, resveratrol (CC-1065), salipodoxomil 10, salipodoxomil 15, irinotecan, monomethylolpropionate E, monomethylolpropionate F, PDB, or any analogues, derivatives or prodrugs thereof.

[0197] In some embodiments, the antibody is conjugated with anthracycline antibiotics or their analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with maytansine or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with chachiomycin or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with docalomycin or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with resveratrol (CC-1065) or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with salinomycin 10 or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with salinomycin 15 or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with monomethylolpropionate E or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with monomethylolpropionate F or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated with pyrrolo[2,1-c][1,4]-benzodiazepine or its analogues, derivatives, or prodrugs. In some implementations, the antibody is conjugated with irinotecan or its analogues, derivatives, or prodrugs.

[0198] In some implementations, the antibody is conjugated to cytokines such as IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa, IFN3, IFNy, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ansistatin, and TNFα.

[0199] In some embodiments, the antibody is conjugated to a radioisotope or a chelate containing a radioisotope. For example, the antibody may be conjugated to a chelating agent linker (e.g., DOTA, DTPA, or thiacetam) that allows the antibody to complex with the radioisotope. The antibody may also, or optionally, contain or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecules. Non-limiting examples of radioisotopes include... 3 H, 14 C 15 N、 35 S, 90 Y、 99 Tc, 125 I, 131 I, 186 Re、 213 Bi、 225 Ac and 227 For therapeutic purposes, radioactive isotopes that emit beta or alpha particle radiation, such as... 131 I, 90 Y、 211 At、212 Bi、 67 Cu、 186 Re、 188 Re and 212 Pb.

[0200] The technique of conjugating molecules to antibodies is well known in the art. Typically, nucleic acid molecules are covalently linked to lysine or cysteine ​​residues on antibodies via N-hydroxysuccinimide or maleimide functional groups, respectively. It has been reported that conjugation methods using engineered cysteine ​​residues or integrating non-natural amino acids can improve the homogeneity of conjugates. In particular, those skilled in the art can also anticipate generating reactive, endogenous glutamine-engineered Fc-containing peptides using tags containing acyl donor glutamine (e.g., tags containing Gin peptides or Q-tags) or through peptide engineering (e.g., through amino acid deletion, insertion, substitution, or mutation on the peptide). Transglutaminase can then be covalently crosslinked with an amine donor agent (e.g., a small molecule containing or linked to a reactive amine) to form a stable and homogeneous pool of engineered Fc-containing peptide conjugates, wherein the amine donor agent specifically conjugates the Fc-containing peptide via an acyl donor glutamine tag or an accessible / exposed / reactive endogenous glutamine site (WO2012059882).

[0201] It should be understood that the therapeutic agent according to the described embodiment will be administered together with a suitable pharmaceutically acceptable carrier, excipient, and other agents incorporated into the formulation to provide improved transfer, delivery, tolerability, etc. A large number of suitable formulations can be found in all pharmacopoeias known to medicinal chemists: Remington's Pharmaceutical Sciences (15th edition, Mack Publishing Company, Easton, Pa. (1975)), particularly Chapter 87 of Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, and lipid-containing (cationic or anionic) carriers (e.g., Lipofectin). TM ( ), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycol (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. Any of the foregoing mixtures may be used for treatments or therapies according to this disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerable for the route of administration.

[0202] In one embodiment, the antibody may be used as a therapeutic agent. Such agents are typically used to treat, alleviate, and / or prevent diseases or pathologies in subjects associated with abnormal tumor antigen expression, activity, and / or signaling. Treatment regimens can be administered using standard methods by identifying subjects, such as those with (or at risk of developing) diseases or disorders associated with abnormal tumor antigen expression, activity, and / or signaling, such as tumor antigen-related disorders. An antibody preparation, preferably one with high specificity and high affinity for its target antigen, is administered to the subject and will typically produce an effect due to its binding to the target. The administered antibody may eliminate, inhibit, or impede the expression, activity, and / or signaling function of the target (e.g., tumor antigen). The administered antibody may eliminate, inhibit, or impede the binding of the target (e.g., tumor antigen) to its naturally bound endogenous ligand. For example, the antibody binds to the target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes, and / or otherwise impedes tumor antigen expression, activity, and / or signaling. In some implementations, antibodies with heavy and light chain CDRs may be administered to subjects to treat diseases or disorders associated with abnormal tumor antigen expression.

[0203] In another embodiment, antibodies against tumor antigens can be used in methods known in the art related to the localization and / or quantification of tumor antigens (e.g., for determining the level of tumor antigens and / or tumor antigens in appropriate physiological samples, for diagnostic methods, for protein imaging, etc.). In a given embodiment, an antibody that is specific to a tumor antigen or its derivatives, fragments, analogs, or homologues and comprises an antigen-binding domain derived from the antibody is used as a pharmaceutically active compound (hereinafter referred to as a "therapeutic agent").

[0204] In another embodiment, tumor antigen peptides can be isolated using antibodies specific to tumor antigens via standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies (or fragments thereof) targeting tumor antigen proteins can be used to detect proteins in biological samples. In some embodiments, the detection of tumor antigens in biological samples is part of a clinical testing process, for example, to determine the efficacy of a given treatment regimen. Conjugating (i.e., physically linking) antibodies to detectable substances can facilitate detection. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriazine fluorescein, dansyl chloride, or phycoerythrin; one example of a luminescent material is luminol; examples of bioluminescent materials include luciferase, luciferin, and jellyfish protein; and examples of suitable radioactive materials include...125 I, 131 I, 35 S or 3 H.

[0205] In another embodiment, the antibody according to this disclosure can be used as a reagent to detect the presence of tumor antigens or protein fragments thereof in a sample. In some embodiments, the antibody contains a detectable label. The antibody is a polyclonal antibody, or more preferably a monoclonal antibody. A complete antibody or fragment thereof (e.g., Fab, scFv, or F(ab')2) is used. The term "label" with respect to the antibody is intended to include both direct labeling of the antibody by conjugation (i.e., physical linking) to the antibody by a detectable substance and indirect labeling of the antibody by reaction with another directly labeled reagent. Examples of indirect labeling include detecting a first antibody using a fluorescently labeled second antibody, and end-labeling an antibody with biotin to enable detection with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present within the subject. Thus, the term "biological sample" as used includes blood and fractions or components of blood, including serum, plasma, or lymph. In other words, the detection method of the embodiments described can be used to detect analytes mRNA, protein, or genomic DNA in biological samples in vitro and in vivo. For example, in vitro detection techniques for analyte mRNA include Norhtern hybridization and in situ hybridization. In vitro detection techniques for analyte proteins include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vitro detection techniques for analyte genomic DNA include Southern hybridization. Procedures for performing immunoassays are described, for example, in “ELISA: Theory and Practice: Methods in Molecular Biology,” Vol. 42, JRCrowther (ed.), Human Press, Totowa, NJ, 1995; “Immunoassay,” E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif., 1996; and “Practice and Theory of Enzyme Immunoassays,” P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo detection techniques for analyte proteins involve introducing labeled anti-analyte protein antibodies into the subject. For example, antibodies can be labeled with radioactive markers, and then the presence and location of the radiolabel in the subject's body can be detected using standard imaging techniques.

[0206] The antibodies and their derivatives, fragments, analogs, and homologues described herein can be incorporated into pharmaceutical compositions suitable for administration. The principles and considerations involved in preparing such compositions, as well as guidelines for selecting components, are well known in the art, for example, see Remington's Pharmaceutical Sciences: The Science and Practice of Pharmacy, 19th edition (edited by Alfonso R. Gennaro et al.), Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, and Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances in Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

[0207] Such compositions typically comprise an antibody and a pharmaceutically acceptable carrier. When using antibody fragments, the minimally inhibitory fragment that specifically binds to the target protein binding domain is preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be chemically synthesized and / or produced via recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).

[0208] As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delayers compatible with drug administration. Suitable pharmaceutically acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, the standard bibliography in the art, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, glucose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers, such as immobilized oils, may also be used. The use of such media and reagents for pharmaceutically active substances is well known in the art. The use of any conventional media or reagent in the composition is contemplated, except that it may be incompatible with the antibody.

[0209] The pharmaceutical composition of the embodiments described herein is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., local), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: sterile diluents for injection, such as water, saline solutions, fixative oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates; and osmotic pressure adjusting agents, such as sodium chloride or dextran. pH may be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral formulations may be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0210] Suitable pharmaceutical compositions for injection include sterile aqueous solutions (in this case, water-soluble) or dispersions, as well as sterile powders for immediate preparation of sterile injections or dispersions. For intravenous administration, suitable pharmaceutically acceptable carriers include physiological saline, antibacterial water, Cremophor EL... TM (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and should be fluid enough for easy injection. It must be stable under manufacturing and storage conditions and must be resistant to contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by using a coating such as lecithin to maintain the desired particle size in the dispersion case, and by using surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols (such as mannitol, sorbitol), and sodium chloride. Prolonged absorption of the injectable composition can be achieved by including absorbance-retarding agents such as aluminum monostearate and gelatin in the composition.

[0211] As needed, a sterile injectable solution can be prepared by incorporating the antibody in the required amount into a suitable solvent having one or a combination of the components listed above (as required), followed by filtration sterilization. Generally, a dispersion is prepared by incorporating the antibody into a sterile carrier containing an alkaline dispersion medium and any other desired components listed above. For sterile powders used to prepare sterile injectable solutions, the preparation method involves obtaining a powder containing the active ingredient and any other desired components derived from a sterile filtration solution of the aforementioned components through vacuum drying and freeze-drying.

[0212] For inhalation administration, the compound is delivered in the form of an aerosol spray from a pressurized container or dispenser or nebulizer containing a suitable propellant such as carbon dioxide.

[0213] Systemic administration can also be achieved via mucosal or transdermal routes. For mucosal or transdermal administration, a permeabilizing agent suitable for the permeability barrier is used in the formulation. Such permeabilizing agents are generally known in the art and include detergents, bile salts, and fusidic acid derivatives, such as those used for mucosal administration. Mucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, one or more of the antibodies can be formulated into ointments, ointments, gels, or creams as commonly known in the art.

[0214] The compound can also be prepared in the form of suppositories (e.g., having a conventional suppository base, such as cocoa butter or other glycerides) or retention enemas for rectal delivery.

[0215] In one embodiment, the antibody can be prepared using a carrier that prevents it from being rapidly eliminated by the body, such as a sustained-release / controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene-vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art.

[0216] Particularly advantageous is the formulation of parenteral compositions in unit dosage form for ease of administration and dosage consistency. As used herein, unit dosage form refers to physically separable units suitable as unit doses for use in the subject to be treated; each unit contains a predetermined amount of one or more of the antibodies calculated to bind with the desired drug carrier to produce the desired therapeutic effect. The specifications of the unit dosage form of the embodiments are indicated by and directly depend on the unique characteristics of the antibody and the specific therapeutic effect to be achieved, and the inherent limitations in the field of formulation of such antibodies for the individual being treated.

[0217] The pharmaceutical composition may be placed in a container, package, or dispenser together with the instructions for use.

[0218] The formulations described herein may also contain more than one of the antibodies described, depending on the specific condition to be treated, preferably those with complementary activities that do not negatively affect each other. Alternatively or in addition, the composition may, for example, contain agents that enhance its function, such as cytotoxic agents, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules are appropriately combined in amounts effective for the intended purpose. For example, they may be combined in a kit or in use.

[0219] In one embodiment, one or more of the antibodies may be administered in combination therapy, i.e., in combination with other agents, such as therapeutic agents (which can be used to treat pathological conditions or disorders, such as various forms of cancer, autoimmune disorders, and inflammatory diseases). The term "combination" herein refers to the administration of the agents substantially synchronously, simultaneously, or sequentially. If administered sequentially, the first of the two compounds is still preferably detected at an effective concentration at the therapeutic site when the second compound is initiated. In one case, "combination" may also mean that the antibody of this disclosure and other therapeutic agents are simultaneously contained in a kit.

[0220] For example, combination therapy may comprise one or more antibodies described herein co-formulated and / or co-administered with one or more additional therapeutic agents (e.g., one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors, enzyme inhibitors, and / or cytotoxins or cell growth inhibitors, as detailed below). Such combination therapies can advantageously utilize lower doses of the administered therapeutic agent, thus avoiding the potential toxicities or complications associated with various monotherapy approaches.

[0221] In one implementation, the treatment regimen effectively reduces the release of cytokines associated with the administration of the T-cell activating therapeutic agent in the subject compared to a corresponding treatment regimen that does not administer antitumor antigen antibodies.

[0222] For the purpose of clarity and concise description, the features are described herein as part of some identical or separate embodiments; however, it will be understood that the scope of this disclosure may include some embodiments having a combination of all or some of the features described.

[0223] Figure 1 The structure of a novel first antigen × CD3 κλ bispecific antibody is shown.

[0224] This experimental disclosure demonstrates that, under free assortment, the λ light chain of the humanized CD3 arm tends to pair with homologous heavy chains, with a low pairing rate with heterologous heavy chains; similarly, the κ light chain of the humanized antigen arm also tends to pair with homologous heavy chains, with an extremely low pairing rate with the humanized CD3 heavy chain; furthermore, the introduction of complementary charge variants in the Fv region further reduces the potential for light chain mismatches. The Fc region of the CD20×CD3 κλ bispecific antibody adopts a human IgG4 knob-into-hole structure, achieved by mutating Ser... 228 Pro, Leu 235 Glu and Pro 329 Ala maintains the stability of the hinge region and weakens its interaction with Fcγ receptors and C1q.

[0225] Example

[0226] Example 1: Optimization of CD3 antibody and its activation effect on T cells

[0227] 1. Synthesis of recombinant proteins

[0228] The extracellular nucleotide sequences of human CD3γ (UniProt P09693, Gln23-Asn116) and CD3ε (UniProt P07766, Gln23-Asp126) were synthesized, and their C-termini were fused with human IgG Fc holes or Fc knobs, respectively, to form human CD3εγ-Fc heterodimers (the amino acid sequence of human CD3γ IgG Fc (hole) is shown in SEQ ID NO. 1, and the amino acid sequence of human CD3ε IgG Fc (knob) is shown in SEQ ID NO. 2). Similarly, cynomolgus monkey CD3γ (UniProt Q95LI7, Gln23-Asn110) and CD3ε (UniProt Q95LI5, Gln22-Asp117) were synthesized, and their C-termini were fused with cynomolgus monkey IgG Fc holes or Fc knobs, respectively, to form cynomolgus monkey CD3εγ-Fc heterodimers (cynomolgus monkey CD3γ IgG). The amino acid sequence of Fc (hole) is shown in SEQ ID NO. 3, and the amino acid sequence of cynomolgus monkey CD3γ IgG Fc (knob) is shown in SEQ ID NO. 4. Recombinant plasmids expressing CD3γ-Fc and CD3ε-Fc were mixed with 3 mg / mL PEI (Polysciences, #24765-2) and co-transfected into HEK293E cells. culture medium After culturing at 37℃, 120 rpm, and 5% CO2 for 7 days (OPM-293 CD03 DPM), the supernatant was collected and purified by Protein A affinity chromatography to obtain recombinant human or cynomolgus monkey CD3εγ-Fc protein.

[0229] 2. Humanization of CD3 antibodies

[0230] A murine hybridoma CD3 antibody (EMBO J.1985.4(2): 337-344; J.Immunol.1986,137(4): 1097-100; J.Exp.Med.1991,174: 319-326; J.Immunol.1991,147(9): 3047-52) recognizes the human and cynomolgus monkey CD3 receptors. Its sequence is as follows:

[0231] The light chain amino acid sequence of the anti-CD3 mouse monoclonal antibody:

[0232] QAVVTQESALTTSPGETVTLTCR SSTGAVTTSNYAN WVQQKPDHLFTGLIG GTNKRAP GVPARFSGSLIGDKAALTITGAQTEDEAIYFCA LWYSNLWV FGGGTKLTVL

[0233] Anti-CD3 mouse monoclonal antibody heavy chain amino acid sequence:

[0234] EVQLVESGGGLVQPKGSLKLSCAASGFTFN TYAMN WVRQAPGKGLEWVAR IRSKYNNYATYYADS VKDRFTISRDDSQSILYLQMNNLKTEDTAMYYCVR HGNFGNSYVSWFAY WGQGTLVTVSS

[0235] The anti-CD3 mouse monoclonal antibody was humanized. The human germline gene IMGT_hVL7-43, with the highest homology, was selected for light chain CDR transplantation, and human IGLJ3*02 was used for FM4. Human IMGT_hVH3-73 was selected for heavy chain CDR transplantation, and human IGHJ4*01 was used for FM4. Different heavy chain and light chain variants were obtained (Table 1).

[0236] Table 1. Variable region sequence of CD3 humanized antibody

[0237]

[0238] The amino acid sequence of hVL1 is shown in SEQ ID NO. 5, its encoded nucleic acid is shown in SEQ ID NO. 6, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO. 7, 8 and 9, respectively.

[0239] QAVVTQEPSLTVSPGGTVTLTC RSSTGAVTTSNYAN WVQQKPGQAPRGLI G GTNKRAP WTPARFSGSLLGGKAA

[0240] LTLSGAQPEDEAEYYC ALWYSNLWV FGGGTKLTVL

[0241] Nucleic acid sequence

[0242] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTAGATCTTCTACAG

[0243] GCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGCAGAAGCCCGGCCAGGTCCCTAGAGGACTGATCGGCGGAAC

[0244] AAACAAGAGAGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACACTTTCTGGT

[0245] GCTCAGCCTGAGGACGAGGCCGAGTACTATTGTGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAAC

[0246] TGACAGTTCTG

[0247] The amino acid sequence of hVL2 is shown in SEQ ID NO. 10, and its encoded nucleic acid is shown in SEQ ID NO. 11. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO. 7, 8 and 9, respectively.

[0248] QAVVTQEPSLTVSPGGTVTLTC RSSTGAVTTSNYAN WVQEKPGQAPRGLI G GTNKRAP WTPARFSGSLLGGKAA

[0249] LTLSGAQPEDEAEYYC ALWYSNLWV FGGGTKLTVL

[0250] Nucleic acid sequence

[0251] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTAGATCTTCTACAG

[0252] GCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGgAGAAGCCCGGCCAGGTCCCTAGAGGACTGATCGGCGGAAC

[0253] AAACAAGAGAGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACACTTTCTGGT

[0254] GCTCAGCCTGAGGACGAGGCCGAGTACTATTGTGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAAC

[0255] TGACAGTTCTG

[0256] The amino acid sequence of hVL3 is shown in SEQ ID NO. 12, its encoded nucleic acid is shown in SEQ ID NO. 13, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO. 14, 15 and 9, respectively.

[0257] EAVVTQEPSLTVSPGGTVTLTC ESSDGAVTTSNYAN WVQEKPGQAPRGLI G GTNKEAP WTPARFSGSLLGGKAA

[0258] LTLSGAQPEDEAEYYC ALWYSNLWV FGGGTKLTVL

[0259] Nucleic acid sequence

[0260] GAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTGAGTCTTCTGACG

[0261] GCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGGAGAAGCCCGGCCAGGTCCCTAGAGGACTGATCGGCGGAAC

[0262] AAACAAGGAGGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACACTTTCTGGT

[0263] GCTCAGCCTGAGGACGAGGCCGAGTACTATTGTGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAAC

[0264] TGACAGTTCTG

[0265] The amino acid sequence of hVL4 is shown in SEQ ID NO. 16, and its encoded nucleic acid is shown in SEQ ID NO. 17. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO. 14, 15 and 9, respectively.

[0266] QAVVTQEPSLTVSPGGTVTLTC ESSDGAVTTSNYAN WVQEKPGQAPRGLI G GTNKEAP WTPARFSGSLLGGKAA

[0267] LTLSGAQPEDEAEYYC ALWYSNLWV FGGGTKLTVL

[0268] Nucleic acid sequence

[0269] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTGAGTCTTCTGACG

[0270] GCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGGAGAAGCCCGGCCAGGTCCCTAGAGGACTGATCGGCGGAAC

[0271] AAACAAGGAGGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACACTTTCTGGT

[0272] GCTCAGCCTGAGGACGAGGCCGAGTACTATTGTGCCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAAC

[0273] TGACAGTTCTG

[0274] The amino acid sequence of hVL5 is shown in SEQ ID NO. 18, its encoded nucleic acid is shown in SEQ ID NO. 19, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO. 7, 8 and 21, respectively.

[0275] QAVVTQEPSLTVSPGGTVTLTC RSSTGAVTTSNYAN WVQEKPGQAPRGLI G GTNKRAP WTPARFSGSLLGGKAA

[0276] LTITGAQAEDEAEYYC VLWYSNLWV FGGGTKLTVL

[0277] Nucleic acid sequence

[0278] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTAGATCTTCTACAG

[0279] GCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGgAGAAGCCCGGCCAGGTCCCTAGAGGACTGATCGGCGGAAC

[0280] AAACAAGAGAGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACAATCACTGGT

[0281] GCTCAGGCTGAGGACGAGGCCGAGTACTATTGTGTGCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAAC

[0282] TGACAGTTCTG

[0283] The amino acid sequence of hVL6 is shown in SEQ ID NO. 22, and its encoded nucleic acid is shown in SEQ ID NO. 23. Its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO. 7, 20 and 21, respectively.

[0284] QAVVTQEPSLTVSPGGTVTLTC RSSTGAVTTSNYAN WFQEKPGQAPRGLI Y GTNKRAP WTPARFSGSLLGGKAA

[0285] LTLSGAQAEDEAEYYC VLWYSNLWV FGGGTKLTVL

[0286] Nucleic acid sequence

[0287] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTAGATCTTCTACAG

[0288] GCGCCGTGACCACCAGCAACTACGCTAATTGGTTCCAGGAGAAGCCCGGCCAGGTCCCTAGAGGACTGATCTACGGAAC

[0289] AAACAAGAGAGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACACTTTCTGGT

[0290] GCTCAGGCTGAGGACGAGGCCGAGTACTATTGTGTCCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAAC

[0291] TGACAGTTCTG

[0292] The amino acid sequence of hVH1 is shown in SEQ ID NO. 24, the encoded nucleic acid is shown in SEQ ID NO. 25, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO. 26, 27 and 28, respectively.

[0293] EVQLVESGGGLVQPGGSLRLSCAASGFTF NTYAMN WVRQAPGKGLEWVSR IRSKYNNYATYYADSVKD RFTISRDDSKNTLYLQMNSLRAEDTAVYYCVR HGNFGNSYVSWFAY WGQGTLVTVSS

[0294] Nucleic acid sequence

[0295] GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCT

[0296] TCACCTTCAACACCTACGCTATGAACTGGGTCCGACAGGCCCCTGGCAAAGGACTGGAATGGGTGTCCAGAATCAGGTC

[0297] CAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACAGATTCACCATCAGCAGGGACGACAGCAAGAAC

[0298] ACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGTCAGACACGGCAACTTCGGCA

[0299] ACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACACTGGTCACAGTTAGCTCT

[0300] The amino acid sequence of hVH2 is shown in SEQ ID NO. 29, its encoding nucleic acid is shown in SEQ ID NO. 30, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO. 31, 27, 28 respectively.

[0301] EVQLVESGGGLVQPGGSLRLSCAASGFTF STYAMN WVRKAPGKGLEWVSR IRSKYNNYATYYADSVKD RFTISRDDSKNTLYLQMNSLRAEDTAVYYCVR HGNFGNSYVSWFAY WGQGTLVTVSS

[0302] Nucleic acid sequence

[0303] GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCT

[0304] TCACCTTCtcCACCTACGCTATGAACTGGGTCCGAaagGCCCCTGGCAAAGGACTGGAATGGGTGTCCAGAATCAGGTC

[0305] CAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACAGATTCACCATCAGCAGGGACGACAGCAAGAAC

[0306] ACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGTCAGACACGGCAACTTCGGCA

[0307] ACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACACTGGTCACAGTTAGCTCT

[0308] The amino acid sequence of hVH3 is shown in SEQ ID NO. 32, its encoding nucleic acid is shown in SEQ ID NO. 33, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO. 31, 27, 34 respectively.

[0309] EVQLVESGGGLVQPGGSLRLSCAASGFTF STYAMN WVRKAPGKGLEWVSR IRSKYNNYATYYADSVKD RFTISRDDSKNTLYLQMNSLRAEDTAVYYCVR HGNFGESYVSWFAY WGQGTLVTVSS

[0310] Nucleic acid sequence

[0311] GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCT

[0312] TCACCTTCTCCACCTACGCTATGAACTGGGTCCGAAAGGCCCCTGGCAAAGGACTGGAATGGGTGTCCAGAATCAGGTC

[0313] CAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACAGATTCACCATCAGCAGGGACGACAGCAAGAAC

[0314] ACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGTCAGACACGGCAACTTCGGCG

[0315] AGAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACACTGGTCACAGTTAGCTCT

[0316] The amino acid sequence of hVH4 is shown in SEQ ID NO. 35, and its encoded nucleic acid is shown in SEQ ID NO. 36. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO. 31, 27 and 37, respectively.

[0317] EVQLVESGGGLVQPGGSLRLSCAASGFTF STYAMN WVRKAPGKGLEWVSR IRSKYNNYATYYADSVKD RFTISRDDSKNTLYLQMNSLRAEDTAVYYCVR HGNFGQSYVSWFAY WGQGTLVTVSS

[0318] Nucleic acid sequence

[0319] GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCT

[0320] TCACCTTCTCCACCTACGCTATGAACTGGGTCCGAAAGGCCCCTGGCAAAGGACTGGAATGGGTGTCCAGAATCAGGTC

[0321] CAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACAGATTCACCATCAGCAGGGACGACAGCAAGAAC

[0322] ACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGTCAGACACGGCAACTTCGGCC

[0323] AGAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACACTGGTCACAGTTAGCTCT

[0324] The amino acid sequence of hVH5 is shown in SEQ ID NO. 38, the encoded nucleic acid is shown in SEQ ID NO. 39, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO. 31, 27 and 40, respectively.

[0325] EVQLVESGGGLVQPGGSLRLSCAASGFTF STYAMN WVRKAPGKGLEWVSR IRSKYNNYATYYADSVKD RFTISRDDSKNTLYLQMNSLRAEDTAVYYCVR HGNFGDSYVSWFAY WGQGTLVTVSS

[0326] Nucleic acid sequence

[0327] GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCT

[0328] TCACCTTCTCCACCTACGCTATGAACTGGGTCCGAAAGGCCCCTGGCAAAGGACTGGAATGGGTGTCCAGAATCAGGTC

[0329] CAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACAGATTCACCATCAGCAGGGACGACAGCAAGAAC

[0330] ACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGTCAGACACGGCAACTTCGGCG

[0331] ACAGCTAGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACACTGGTCACAGTTAGCTCT

[0332] The amino acid sequence of hVH6 is shown in SEQ ID NO. 41, and its encoded nucleic acid is shown in SEQ ID NO. 42. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO. 31, 27 and 43, respectively.

[0333] EVQLVESGGGLVQPGGSLRLSCAASGFTF STYAMN WVRKAPGKGLEWVSR IRSKYNNYATYYADSVKD RFTISRDDSKNTLYLQMNSLRAEDTAVYYCVR HGNFGTSYVSWFAY WGQGTLVTVSS

[0334] Nucleic acid sequence

[0335] GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCT

[0336] TCACCTTCTCCACCTACGCTATGAACTGGGTCCGAAAGGCCCCTGGCAAAGGACTGGAATGGGTGTCCAGAATCAGGTC

[0337] CAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACAGATTCACCATCAGCAGGGACGACAGCAAGAAC

[0338] ACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGTCAGACACGGCAACTTCGGCA

[0339] CCAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACACTGGTCACAGTTAGCTCT

[0340] The amino acid sequence of hVH7 is shown in SEQ ID NO. 44, its encoding nucleic acid is shown in SEQ ID NO. 45, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO. 46, 47, 28 respectively.

[0341] EVQLVESGGGLVQPGGSLRLSCAASGFTF SDYAMN WVRKAPGKGLEWVSR IRSKYNNYATYYADSVED RFTISRDDSKNTLYLQMNSLRAEDTAVYYCVR HGNFGNSYVSWFAY WGQGTLVTVSS

[0342] Nucleic acid sequence

[0343] GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCT

[0344] TCACCTTCTCCGACTACGCTATGAACTGGGTCCGAAAGGCCCCTGGCAAAGGACTGGAATGGGTGTCCAGAATCAGGTC

[0345] CAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGGAGGACAGATTCACCATCAGCAGGGACGACAGCAAGAAC

[0346] ACCCTGTACCTGCAGATGAACAGCCTGAGAGCCGAGGACACCGCCGTGTACTACTGTGTCAGACACGGCAACTTCGGCA

[0347] ACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACACTGGTCACAGTTAGCTCT

[0348] The amino acid sequence of hVH8 is shown in SEQ ID NO. 48, its encoding nucleic acid is shown in SEQ ID NO. 49, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO. 26, 27, and 28 respectively.

[0349] EVQLVESGGGLVQPGGSLRLSCAASGFTF NTYAMN WVRKAPGKGLEWVGR IRSKYNNYATYYADSVKD RFTISRDDSKNSLYLQMNSLKTEDTAVYYCAR HGNFGNSYVSWFAY WGQGTLVTVSS

[0350] Nucleic acid sequence

[0351] GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCT

[0352] TCACCTTCAACACCTACGCTATGAACTGGGTCCGAAAGGCCCCTGGCAAAGGACTGGAATGGGTGGGAAGAATCAGGTC

[0353] CAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACAGATTCACCATCAGCAGGGACGACAGCAAGAAC

[0354] AGCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGTGCCAGACACGGCAACTTCGGCA

[0355] ACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACACTGGTCACAGTTAGCTCT

[0356] The amino acid sequence of hVH9 is shown in SEQ ID NO. 50, its encoding nucleic acid is shown in SEQ ID NO. 51, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO. 26, 27, 28 respectively.

[0357] EVQLVESGGGLVQPGGSLRLSCAASGFTF NTYAMN WVRKAPGKGLEWVGR IRSKYNNYATYYADSVKD RFTISRDDSKNSLYLQMNSLKTEDTAVYYCVR HGNFGNSYVSWFAY WGQGTLVTVSS

[0358] Nucleic acid sequence

[0359] GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCT

[0360] TCACCTTCAACACCTACGCTATGAACTGGGTCCGAAAGGCCCCTGGCAAAGGACTGGAATGGGTGGGAAGAATCAGGTC

[0361] CAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACAGATTCACCATCAGCAGGGACGACAGCAAGAAC

[0362] AGCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGTGTCAGACACGGCAACTTCGGCA

[0363] ACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACACTGGTCACAGTTAGCTCT

[0364] The amino acid sequence of hVH10 is shown in SEQ ID NO. 52, and its encoded nucleic acid is shown in SEQ ID NO. 53. Its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO. 26, 27 and 28, respectively.

[0365] EVQLVESGGGLVQPGGSLRLSCAASGFTF NTYAMN WVRKAPGKGLEWVAR IRSKYNNYATYYADSVKD RFTISRDDSKNSLYLQMNSLKTEDTAVYYCVR HGNFGNSYVSWFAY WGQGTLVTVSS

[0366] Nucleic acid sequence

[0367] GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCT

[0368] TCACCTTCAACACCTACGCTATGAACTGGGTCCGAAAGGCCCCTGGCAAAGGACTGGAATGGGTGGCCAGAATCAGGTC

[0369] CAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACAGATTCACCATCAGCAGGGACGACAGCAAGAAC

[0370] AGCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGTGTCAGACACGGCAACTTCGGCA

[0371] ACAGCTAGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACACTGGTCACAGTTAGCTCT

[0372] After synthesizing the full sequences of the humanized light and heavy chain variants, they were cloned into eukaryotic expression vectors containing the constant region of the antibody lambda light chain or the constant region CH1-CH3 of the human IgG4 heavy chain. These vectors were then co-transfected into HEK293E cells and cultured at 37°C, 120 rpm, and 5% CO2 for 5-6 days. The supernatant was then collected and purified using a Protein A chromatography column.

[0373] 3. Affinity of CD3 humanized antibodies

[0374] The recombinant human CD3εγ protein was coated and incubated overnight at 4°C. After blocking with 2% skim milk, CD3 antibody of different dilutions was added to each well and incubated for 1 hour. HPR-labeled goat anti-human IgG Fc was added to the secondary antibody, and after color development with TMB solution, the reaction was terminated with concentrated sulfuric acid and the absorbance was read at 450 nm. Figure 2 The binding of humanized CD3 antibodies (including aCD3-hVH1 / VL5, aCD3-hVH8 / VL1, aCD3-hVH8 / VL5, aCD3-hVH9 / VL1, aCD3-hVH9 / VL2, aCD3-hVH9 / VL3, and aCD3-hVH9 / VL5) to human CD3εγ protein was demonstrated, and the humanized CD3 antibodies bound the recombinant CD3εγ protein with high affinity.

[0375] Jurkat cells in the logarithmic growth phase were blocked with 3% BSA for 30 minutes, and then seeded at a density of 5 × 10⁶ cells per well. 4 Cells were added to 96-well U-plates, centrifuged, and the supernatant was discarded. 50 μL of serially diluted antibody (antibody concentration starting at 30 μg / mL, 5 three-fold dilutions) was added to each well, and the plates were incubated at 4°C for 1 hour. After washing away the primary antibody, secondary antibody was added to 1:300 diluted Alexa Fluro647-labeled goat anti-human IgG Fc (Jackson ImmunoResearch, 109-606-170), and the plates were incubated at 4°C for 45 minutes. After washing, each well was resuspended in 50 μL PBS for FACS (iQue, Intellicyt) detection. Results are as follows: Figure 3 As shown, the humanized CD3 antibodies bound to Jurkat cells. The humanized CD3 antibodies hVH9 / VL5 (aCD3-hVH9 / VL5) and hVH9 / VL2 (aCD3-hVH9 / VL2) were significantly weaker than the control antibody OKT3, binding to Jurkat cells with moderate affinity.

[0376] Table 2 shows the affinity of the humanized CD3 antibody for recombinant CD3 protein and Jurkat cells.

[0377] Table 2. Affinity of CD3 humanized antibodies

[0378]

[0379] ND: Not detected.

[0380] -: Not combined

[0381] 4. Cross-recognition between humanized CD3 antibodies and human and cynomolgus monkey CD3εγ antigens

[0382] Human CD3εγ protein and cynomolgus monkey CD3εγ protein were coated separately and incubated overnight at 4°C. After blocking with 2% skim milk, CD3 antibodies of different dilutions were added to each well and incubated for 1 hour. HPR-labeled goat anti-human IgG Fc was added as secondary antibody, and after color development with TMB solution, the reaction was terminated with concentrated sulfuric acid and the absorbance was read at 450 nm. Figure 4 The humanized CD3 antibodies hVH9 / VL5 (aCD3-hVH9 / VL5) and hVH9 / VL2 (aCD3-hVH9 / VL2) can both bind to human CD3α and cynomolgus monkey CD3α proteins simultaneously.

[0383] Example 2: Construction of CD20×CD3κλ bispecific antibodies formed by different types of light chains

[0384] 1. Construction of CD20×CD3κλ bispecific antibody

[0385] A novel T-cell κλ bispecific antibody with a natural IgG conformation was constructed using the humanized CD3 antibody hVH9 / VL5 (λ light chain and paired heavy chain) and the humanized CD20 antibody (κ light chain and paired heavy chain).

[0386] like Figure 5 As shown, the following five CD20×CD3κλ bispecific antibodies were designed and constructed:

[0387] 1) CD20×CD3κλ001: Retains the native sequences of the CD3 arm and the CD20 antigen arm;

[0388] 2) CD20×CD3κλ002: Simultaneous introduction of a charge variant (Vκ) into both the CD20 antigen arm and the CD3 arm. CD20 Gln 38 Lys;VH CD20 Gln 39 Glu; Vλ CD3 Gln 40 Glu; VH CD3 Gln 39 Lys);

[0389] 3) CD20×CD3κλ003: Based on CD20×CD3κλ002, a complementary charge pair (Vκ-Ck) is added between CH1 / Cκ. CD20 Gln 38 Lys\Glu 123 Lys\Gln 124 Lys;V H -C H 1 CD20 Gln 39 Glu\Lys 152 Glu\Lys 218Glu; Vλ CD3 Gln 40 Glu; VH CD3 Gln 39 Lys);

[0390] 4) CD20×CD3κλ004: Introducing a charge variant (Vκ) only in the CD20 antigen arm. CD20 Gln 38 Lys;VH CD20 Gln 39 Glu);

[0391] 5) CD20×CD3κλ005: A charge variant (Vλ) is introduced only in the CD3 arm. CD3 Gln 40 Glu; VH CD3 Gln 39 Lys).

[0392] The corresponding sequences are shown in Table 3. The control antibody CD20×CD3-crossFab was constructed using the CrossFab method (Schaefer W et al., PNAS 2011).

[0393] Table 3. Sequences of CD20×CD3κλ bispecific antibodies

[0394]

[0395] CD20×CD3 κλ001:

[0396] CD20 arm κ light chain SEQ ID NO. 54

[0397] EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSNWPITFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0398] Nucleotide sequence SEQ ID NO. 55

[0399] GAGATCGTGCTGACACAGAGCCCTGGCACACTGTCACTGTCTCCAGGCGAGAGAGCCACACTGAGCTGTAGAGCCAGCCAGAGCGTGTCCTCTTACCTGGCCTGGTATCAGCAGAAGCCTGGACAGGCTCCCAGACTGCTGATCTACGACGCCAGCAACAGAGCCACAGGCATCCCCGATAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGAAGCAACTGGCCCATCACATTCGGCCAGGGCACCAAGCTGGAAATCAAGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0400] CD20 Arm Heavy Chain (Heavy Chain 1) SEQ ID NO. 56

[0401] EVQLLESGGGVVQPGGSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIQYGNYYYGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0402] Nucleotide sequence SEQ ID NO. 57

[0403]

[0404] CD3 arm lambda light chain SEQ ID NO. 58

[0405] QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEAEYYCVLWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0406] Nucleotide sequence SEQ ID NO. 59

[0407] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTAGATCTTCTACAGGCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGCAGAAGCCCGGCCAGGCTCCTAGAGGACTGATCGGCGGAACAAACAAGAGAGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACAATCACTGGTGCTCAGGCTGAGGACGAGGCCGAGTACTATTGTGTGCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAACTGACAGTTCTGGGTCAGCCCAAGGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTATCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0408] CD3 Arm Heavy Chain (Heavy Chain 2) SEQ ID NO. 60

[0409] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0410] Nucleotide sequence SEQ ID NO.61

[0411]

[0412] CD20×CD3 κλ002:

[0413] CD20 Arm κ Light Chain SEQ ID NO. 62

[0414] EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQKKPGQAPRLLIYDASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSNWPITFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0415] Nucleotide Sequence SEQ ID NO. 63

[0416] GAGATCGTGCTGACACAGAGCCCTGGCACACTGTCACTGTCTCCAGGCGAGAGAGCCACACTGAGCTGTAGAGCCAGCCAGAGCGTGTCCTCTTACCTGGCCTGGTATCAGAAGAAGCCTGGACAGGCTCCCAGACTGCTGATCTACGACGCCAGCAACAGAGCCACAGGCATCCCCGATAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGAAGCAACTGGCCCATCACATTCGGCCAGGGCACCAAGCTGGAAATCAAGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0417] CD20 Arm Heavy Chain (Heavy Chain 1) SEQ ID NO. 64

[0418] EVQLLESGGGVVQPGGSLRLSCAASGFTFNDYAMHWVREAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIQYGNYYYGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0419] Nucleotide sequence SEQ ID NO. 65

[0420]

[0421] CD3 arm λ light chain SEQ ID NO. 66

[0422] QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEAEYYCVLWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0423] Nucleotide sequence SEQ ID NO. 67

[0424] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTAGATCTTCTACAGGCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGGAGAAGCCCGGCCAGGCTCCTAGAGGACTGATCGGCGGAACAAACAAGAGAGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACAATCACTGGTGCTCAGGCTGAGGACGAGGCCGAGTACTATTGTGTGCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAACTGACAGTTCTGGGTCAGCCCAAGGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTATCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0425] CD3 Arm Heavy Chain (Heavy Chain 2) SEQ ID NO. 68

[0426] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRKAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0427] Nucleotide sequence SEQ ID NO. 69

[0428]

[0429] CD20×CD3 κλ003:

[0430] CD20 Arm κ Light Chain SEQ ID NO. 70

[0431] EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQKKPGQAPRLLIYDASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSNWPITFGQGTKLEIKRTVAAPSVFIFPPSDKKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0432] Nucleotide Sequence SEQ ID NO. 71

[0433] GAGATCGTGCTGACACAGAGCCCTGGCACACTGTCACTGTCTCCAGGCGAGAGAGCCACACTGAGCTGTAGAGCCAGCCAGAGCGTGTCCTCTTACCTGGCCTGGTATCAGAAGAAGCCTGGACAGGCTCCCAGACTGCTGATCTACGACGCCAGCAACAGAGCCACAGGCATCCCCGATAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGAAGCAACTGGCCCATCACATTCGGCCAGGGCACCAAGCTGGAAATCAAGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATAAGAAATTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0434] CD20 arm heavy chain (heavy chain 1) SEQ ID NO. 72

[0435] EVQLLESGGGVVQPGGSLRLSCAASGFTFNDYAMHWVREAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIQYGNYYYGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDERVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0436] Nucleotide sequence SEQ ID NO. 73

[0437]

[0438] CD3 arm lambda light chain SEQ ID NO. 66

[0439] QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEAEYYCVLWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0440] Nucleotide sequence SEQ ID NO. 67

[0441] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTAGATCTTCTACAGGCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGGAGAAGCCCGGCCAGGCTCCTAGAGGACTGATCGGCGGAACAAACAAGAGAGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACAATCACTGGTGCTCAGGCTGAGGACGAGGCCGAGTACTATTGTGTGCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAACTGACAGTTCTGGGTCAGCCCAAGGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTATCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0442] CD3 Arm Heavy Chain (Heavy Chain 2) SEQ ID NO. 68

[0443] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRKAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0444] Nucleotide sequence SEQ ID NO. 69

[0445]

[0446] CD20×CD3 κλ004:

[0447] CD20 Arm κ Light Chain SEQ ID NO. 62

[0448] EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQKKPGQAPRLLIYDASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSNWPITFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0449] Nucleotide Sequence SEQ ID NO. 63

[0450] GAGATCGTGCTGACACAGAGCCCTGGCACACTGTCACTGTCTCCAGGCGAGAGAGCCACACTGAGCTGTAGAGCCAGCCAGAGCGTGTCCTCTTACCTGGCCTGGTATCAGAAGAAGCCTGGACAGGCTCCCAGACTGCTGATCTACGACGCCAGCAACAGAGCCACAGGCATCCCCGATAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGAAGCAACTGGCCCATCACATTCGGCCAGGGCACCAAGCTGGAAATCAAGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0451] CD20 Arm Heavy Chain (Heavy Chain 1) SEQ ID NO. 64

[0452] EVQLLESGGGVVQPGGSLRLSCAASGFTFNDYAMHWVREAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIQYGNYYYGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0453] Nucleotide sequence SEQ ID NO. 65

[0454]

[0455] CD3 arm lambda light chain SEQ ID NO. 58

[0456] QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEAEYYCVLWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0457] Nucleotide sequence SEQ ID NO. 59

[0458] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTAGATCTTCTACAGGCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGCAGAAGCCCGGCCAGGCTCCTAGAGGACTGATCGGCGGAACAAACAAGAGAGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACAATCACTGGTGCTCAGGCTGAGGACGAGGCCGAGTACTATTGTGTGCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAACTGACAGTTCTGGGTCAGCCCAAGGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTATCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0459] CD3 Arm Heavy Chain (Heavy Chain 2) SEQ ID NO. 60

[0460] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0461] Nucleotide sequence SEQ ID NO. 61

[0462]

[0463] CD20×CD3 κλ005:

[0464] CD20 arm κ light chain SEQ ID NO. 54

[0465] EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSNWPITFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0466] Nucleotide sequence SEQ ID NO. 55

[0467] GAGATCGTGCTGACACAGAGCCCTGGCACACTGTCACTGTCTCCAGGCGAGAGAGCCACACTGAGCTGTAGAGCCAGCCAGAGCGTGTCCTCTTACCTGGCCTGGTATCAGCAGAAGCCTGGACAGGCTCCCAGACTGCTGATCTACGACGCCAGCAACAGAGCCACAGGCATCCCCGATAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGAAGCAACTGGCCCATCACATTCGGCCAGGGCACCAAGCTGGAAATCAAGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0468] CD20 Arm Heavy Chain (Heavy Chain 1) SEQ ID NO. 56

[0469] EVQLLESGGGVVQPGGSLRLSCAASGFTFNDYAMHWVRQAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIQYGNYYYGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0470] Nucleotide sequence SEQ ID NO. 57

[0471]

[0472] CD3 arm λ light chain SEQ ID NO. 66

[0473] QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEAEYYCVLWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0474] Nucleotide sequence SEQ ID NO. 67

[0475] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTAGATCTTCTACAGGCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGGAGAAGCCCGGCCAGGCTCCTAGAGGACTGATCGGCGGAACAAACAAGAGAGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACAATCACTGGTGCTCAGGCTGAGGACGAGGCCGAGTACTATTGTGTGCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAACTGACAGTTCTGGGTCAGCCCAAGGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTATCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0476] CD3 Arm Heavy Chain (Heavy Chain 2) SEQ ID NO. 68

[0477] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRKAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0478] Nucleotide sequence SEQ ID NO. 69

[0479]

[0480] CD20×CD3-crossFab

[0481] CD20 Arm κ Light Chain SEQ ID NO. 70

[0482] EIVLTQSPGTLSLSPGERATLSCRASQSVSSYLAWYQKKPGQAPRLLIYDASNRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQRSNWPITFGQGTKLEIKRTVAAPSVFIFPPSDKKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0483] Nucleotide Sequence SEQ ID NO. 71

[0484] GAGATCGTGCTGACACAGAGCCCTGGCACACTGTCACTGTCTCCAGGCGAGAGAGCCACACTGAGCTGTAGAGCCAGCCAGAGCGTGTCCTCTTACCTGGCCTGGTATCAGAAGAAGCCTGGACAGGCTCCCAGACTGCTGATCTACGACGCCAGCAACAGAGCCACAGGCATCCCCGATAGATTCAGCGGCTCTGGCTCTGGCACCGACTTCACCCTGACAATCAGCAGACTGGAACCCGAGGACTTCGCCGTGTACTACTGCCAGCAGAGAAGCAACTGGCCCATCACATTCGGCCAGGGCACCAAGCTGGAAATCAAGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATAAGAAATTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0485] CD20 Arm Heavy Chain (Heavy Chain 1) SEQ ID NO. 74

[0486] EVQLLESGGGVVQPGGSLRLSCAASGFTFNDYAMHWVREAPGKGLEWVSTISWNSGSIGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDIQYGNYYYGMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDERVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0487] Nucleotide sequence SEQ ID NO. 75

[0488]

[0489] CD3 arm lambda light chain SEQ ID NO. 76

[0490] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRKAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV

[0491] Nucleotide sequence SEQ ID NO. 77

[0492] GAGGTGCAGCTGGTTGAATCTGGCGGAGGACTGGTTCAGCCTGGCGGATCTCTGAGACTGTCTTGTGCCGCCAGCGGCTTCACCTTCAACACCTACGCTATGAACTGGGTCCGAAAGGCCCCTGGCAAAGGACTGGAATGGGTGGGAAGAATCAGGTCCAAGTACAACAACTACGCCACCTACTACGCCGACAGCGTGAAGGACAGATTCACCATCAGCAGGGACGACAGCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAAAACCGAGGACACCGCCGTGTACTACTGTGTCAGACACGGCAACTTCGGCAACAGCTATGTGTCTTGGTTTGCCTACTGGGGCCAGGGCACACTGGTCACAGTTAGCTCTGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCTTGCAGCAGAAGCACCAGCGAGAGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGCGCTCTGACCAGCGGCGTGCATACCTTCCCCGCCGTGCTCCAGAGCAGCGGACTGTACTCCCTGAGCAGCGTGGTGACCGTGCCTTCCAGCAGCCTGGGCACCAAGACCTACACCTGCAACGTGGACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTG

[0493] CD3 Arm Heavy Chain (Heavy Chain 2) SEQ ID NO. 78

[0494] QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEAEYYCVLWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECSESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFASTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0495] Nucleotide sequence SEQ ID NO. 79

[0496]

[0497] 2. Expression and purification of CD20×CD3κλ bispecific antibody

[0498] The plasmid encoding the corresponding antibody fragment was mixed with 3 mg / mL PEI at a ratio of CD20 arm light chain: CD3 arm light chain: CD20 arm heavy chain (heavy chain 1): CD3 arm heavy chain (heavy chain 2) = 2:2:1:1, and then co-transfected with CHO-S cells. The cells were cultured in 500 mL CD CHO AGT medium (Gibco #12490-001) at 37°C with 5% CO2 at 150 rpm. On days 2, 4, and 6 after transient transfection, 4% CHO Feed C+ (Gibco #A25031-05) was added. When cell viability dropped to approximately 85%, the fermentation broth was harvested, filtered, and purified by Protein A affinity chromatography. Based on different light chain types, the CD20×CD3κλ bispecific antibodies constructed showed monomer purity close to or higher than 90% after one-step purification of Protein A, while the monomer purity of the control antibody CD20×CD3-crossFab was lower than 80% (Table 4), and the ratio of κλ light chains was close to 1:1. Figure 6 ).

[0499] Table 4. Purity of CD20×CD3κλ bispecific antibody (SEC-HPLC)

[0500]

[0501] The CD20×CD3κλ bispecific antibody was further purified using Capto S ImpAct ion exchange chromatography. Elution was performed using a gradient of 50-300 mM NaCl, 50 mM phosphate, and pH 6.4. The elution peaks were combined, and SEC-HPLC showed a monomer content higher than 99%. Figure 7 In the purified samples of CD20×CD3κλ002 and CD20×CD3κλ003, the light chain mismatch rate was extremely low (<1%), and no CD3 homodimers or CD20 homodimers were detected. Figure 8 ).

[0502] 3. Binding activity of CD20×CD3κλ bispecific antibody

[0503] The affinity of the CD20 antigen arm of the bispecific antibody was determined by detecting its binding to CD20-overexpressing stable cells or CD20+ tumor cells, and the affinity of the CD3 arm was determined by detecting its binding to CD3 recombinant antigen, Jurkat cells, or freshly isolated peripheral blood T cells. The results showed that the novel CD20×CD3κλ bispecific antibody had approximately 3-5 times higher affinity for tumor cells than for T cells. The positive control antibody bsAB1 was synthesized and expressed according to the literature US20170174781.

[0504] (1) Binding of CD20×CD3κλ bispecific antibody to human and cynomolgus monkey CD20 stable cells

[0505] CHO-human CD20 and CHO-cynomolgus monkey CD20 stable transgenic cells prepared in Example 1 during the logarithmic growth phase were taken and adjusted to 5 × 10⁶ cells / year with 4% fetal bovine serum (Hyclone, SH30626.06). 5 Cells / ml were collected, and 100 μl / well of cell suspension was added to a 96-well U-shaped plate. The plate was centrifuged at 300g for 5 minutes, and the supernatant was discarded. 100 μL of serially diluted antibody (starting concentration 1800 nM, 3-fold dilution, 10 gradients) was added to each well, and the plate was incubated at 4°C for 60 minutes. Secondary antibody was added to 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution), and the plate was incubated on ice for 20 minutes. After washing once, 50 μL / well of propidium iodide (PI) solution (1:300) was added, and the plate was incubated for 5 minutes. Detection was performed using flow cytometry. Figure 9 As shown in Table 5, the CD20×CD3κλ bispecific antibody binds to the CD20 receptor in cells with high affinity, and the affinity is comparable in humans and cynomolgus monkeys with stable CD20 transfection.

[0506] Table 5. Binding of CD20×CD3κλ bispecific antibody to CD20 stable cells

[0507]

[0508] "-": Do not combine

[0509] (2) CD20×CD3κλ bispecific antibody and human CD20 + binding of tumor cells

[0510] SU-DHL-4, Raji, and NALM-6 cells in logarithmic growth phase were collected, and 200 μg / mL mouse IgG (Jackson ImmunoResearch, 115-005-03) was added and the cells were blocked on ice for 30 minutes. The cell volume was then adjusted to 5 × 10⁶ cells / mL with 4% fetal bovine serum. 5Cells / mL, 100 μL per well of a 96-well U-shaped plate, centrifuged at 300g for 5 minutes, supernatant discarded, 100 μL of serially diluted antibody (starting concentration 1800 nM, 3-fold dilution, 10 gradients) added to each well, incubated at 4°C for 60 minutes. After washing away the primary antibody, 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added, incubated on ice for 20 minutes, washed once, and then 50 μL / well of PI was added, incubated for 5 minutes, and analyzed by flow cytometry. Results are shown below. Figure 10 According to Table 6, the CD20×CD3κλ bispecific antibody binds with high affinity to CD20+ tumor cells SU-DHL-4, Raji, and NALM-6.

[0511] (3) Binding of CD20×CD3κλ bispecific antibody to Jurkat cells

[0512] Jurkat cells in logarithmic growth phase were taken and 200 μg / mL mouse IgG (Jackson ImmunoResearch, 115-005-03) was added. The cells were then incubated on ice for 30 minutes. The cell volume was adjusted to 5 × 10⁶ cells / mL with 4% fetal bovine serum. 5 Cells / mL, 100 μL per well of a 96-well U-shaped plate, centrifuged at 300g to remove supernatant, 100 μL of serially diluted antibody (starting concentration 1800 nM, 3-fold dilution, 10 gradients) per well, incubated at 4°C for 60 min. Secondary antibody was added to 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution), incubated on ice for 20 min, washed once, and then 50 μL / well of PI was added, incubated for 5 min, and analyzed by flow cytometry (BD C6). Detection results are as follows: Figure 11 According to Table 6, the CD20×CD3κλ bispecific antibody binds with intermediate affinity to the human leukemia T cell line Jurkat cells, EC... 50 It is approximately 71-120 nM, which is about 10 times lower than the binding affinity of the CD20 antigen arm to the CD20 receptor.

[0513] (4) Binding of CD20×CD3κλ bispecific antibody to human peripheral blood T cells

[0514] Fresh peripheral blood was collected, and PBMCs were isolated using Ficoll-Paque Plus (GE, 17-1440-03). The PBMCs were adjusted to 5 × 10⁶ cells / mL with 4% fetal bovine serum (Hyclone, SH30626.06). 5Cells / mL, 100 μL / well added to a 96-well U-shaped plate, centrifuged and supernatant discarded, 100 μL of serially diluted antibody added to each well (starting concentration 1800 nM, 3-fold dilution, 10 gradients), incubated at 4°C for 60 min. Secondary antibody added 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution), incubated on ice for 20 min, washed once, then 50 μL / well of PI added, incubated for 5 min, and analyzed by flow cytometry (BD C6). Results are shown in [link to results]. Figure 12 According to Table 6, the CD20×CD3κλ bispecific antibody recognizes human peripheral blood CD4. + T and CD8 + T cells have an affinity of approximately 65-98 nM for human T cells, which is about 10 times weaker than the binding affinity of the CD20 antigen arm to the CD20 receptor. This makes it easier for bispecific antibodies to preferentially accumulate in tumor cells.

[0515] Table 6. Binding affinity (nM) of CD20×CD3κλ bispecific antibody to human cells.

[0516]

[0517] ND: Not saturated under high concentration conditions

[0518] 4. CD20 × CD3κλ bispecific antibody-mediated TDCC

[0519] Freshly isolated PBMCs were mixed with target cells in logarithmic growth phase (NALM-6, TMD-8, and Toledo cells) at an effector / target cell ratio of 8:1. 50 μL of serially diluted antibody (starting at 66.7 nM, 10-fold dilution, 7 gradients) was added to each well, and the cells were incubated at 37°C with 5% CO2 for 24 hours. After incubation, 50 μL of supernatant was transferred to a new black ELISA plate, and 50 μL / well of LDH detection substrate was added. The reaction was stopped after 10 minutes, and LDH release was detected. The remaining cells in the wells were washed twice with 4% fetal bovine blood, incubated with 100 μg / mL human IgG for 10 minutes, and then T cell activation detection antibodies (CD25-PE, CD4-APC, CD69-FITC, and CD8-APC) were added. The cells were incubated on ice for 20 minutes. After washing and discarding the supernatant, 60 μL / well of PI was added, and the cells were incubated on ice for 5 minutes. Flow cytometry was then used for detection. Figure 13 A and 13B show the killing effect of CD20×CD3κλ bispecific antibody on human B lymphocytic leukemia cells Nalm-6 and the activation of T cells, respectively. Figure 14 A and 14B show the killing effect of CD20×CD3κλ bispecific antibody on TMD-8 cells and the activation of T cells, respectively. Figure 15A and 15B show the killing effect of the CD20×CD3κλ bispecific antibody on Toledo cells and the activation effect on T cells, respectively. For tumor cells Nalm-6, TMD-8, and Toledo with different CD20 expression levels, both CD20×CD3κλ002 and CD20×CD3κλ003 can mediate effective killing of T cells, with killing activity comparable to or slightly stronger than the control antibody bsAB1, and the activation of T cells is milder than that of the latter.

[0520] 5. Activation of T cell activation pathway by CD20×CD3κλ bispecific antibody

[0521] Jurkat-NFAT-luc reporter cells and CD20-positive target cells (SU-DHL-4, Raji, and NALM-6 cells) in logarithmic growth phase were collected, centrifuged, and the supernatant was discarded. The cells were then resuspended to a concentration of 2 × 10⁻⁶. 6 Cells / ml. Seed 50 μl / well of target cells into a 96-well plate, centrifuge at 300g for 5 minutes and discard the supernatant. Seed 50 μl / well of Jurkat-NFAT-luc reporter cells into a 96-well plate. Add 50 μl of serially diluted CD20×CD3κλ bispecific antibody or control antibody KLH×CD3 (starting concentration 20 μg / ml, 10-fold dilution, 10 gradients) to each well. Incubate at 37℃ for 6 hours with 5% CO2. After incubation, add 100 μl of detection reagent to each well according to the ONE-Glo Luciferase Assay System instructions, incubate at room temperature for 3 minutes, and detect using a microplate reader (Biotek Synergy HT). Results are shown below. Figure 16 As shown in Table 7, the CD20×CD3κλ bispecific antibody can activate the NFAT signaling pathway of T cells when targeting tumor cells with different CD20 expression levels.

[0522] Table 7. Activation of the NFAT pathway in T cells by CD20×CD3κλ bispecific antibody.

[0523]

[0524] 6. CD20 × Binding of CD3κλ bispecific antibody to Fcγ receptor

[0525] A 50 μg / ml His-Tag antibody was amino-conjugated to a CM5 chip to capture His-tagged FcγRI and FcγRIIA, respectively. H131 and FcγRIIIA V158Recombinant protein (Sino Biological, #10256-H08H / 10374-H08H1 / 10389-H08H1), with a capture time of 40 seconds and a flow rate of 10 μL / min, after baseline stabilization, serially diluted antibody (initial concentration 37.5 μg / mL, 2-fold dilution) was flowed through the chip at a flow rate of 30 μL / min. Binding time was 120 seconds, dissociation time was 200 seconds, and affinity constants were obtained using Biacore evaluation software. As shown in Table 8, the CD20×CD3κλ bispecific antibody showed affinity for FcγRI and FcγRIIA. H131 and FcγRIIIA V158 No binding was observed; the wild-type IgG4 control antibody bound FcγRI with strong affinity, and FcγRIIA was also observed. H131 There is a weak binding.

[0526] Table 8. CD20×CD3κλ bispecific antibody and Affinity of Fcγ receptor

[0527]

[0528] 7. Immunoreconstituted mouse subcutaneous Raji xenograft model

[0529] Select 6-8 week old female B-NGD mice (Biocytok Biotechnology Co., Ltd.), and subcutaneously inoculate them with 3×10 6 Raji cells, until the tumor grows to 60mm 3 Mice were randomly assigned to four groups: a treatment group (3.0 mg / kg), a treatment group (0.6 mg / kg), a treatment group (0.12 mg / kg), and a negative control group (KLH×CD3 3 mg / kg). Each mouse was injected via tail vein with 1×10⁻⁶ KLH×CD3. 7 Three days after the first administration of PBMC cells to mice, the mice were given the first dose, followed by administration every five days for a total of three doses. Tumor volume and body weight were monitored. Mice were euthanized by cervical dislocation at the end of the experiment, and tumors were collected, weighed, and recorded. Results are shown below. Figure 17 The in vivo efficacy of the CD20×CD3κλ bispecific antibody showed a dose-related relationship, with tumor inhibition rates of 82% and 89% at medium and high doses, respectively. Tumor-bearing mice tolerated the above doses well, with no adverse reactions such as weight loss.

[0530] 8. Subcutaneous Raji and human PBMC co-inoculation model in immunodeficient mice

[0531] Select 6-8 week old female B-NGD mice (Biocytok Biotechnology Co., Ltd.), and feed Raji mice (3×10⁻⁶) to the mice. 6 Individuals) and people's PBMCs (5×10) 6 (The mixture of individual samples) was subcutaneously injected into mice, and the tumor volume was increased to 60-100 mm². 3Mice were randomly assigned to groups: a treatment group (3.0 mg / mL), a treatment group (0.6 mg / mL), a treatment group (0.12 mg / mL), and a negative control group (KLH×CD3 3 mg / kg). Dosing was administered twice, every 5 days. Tumor volume and body weight were monitored. Mice were euthanized by cervical dislocation at the end of the experiment, and tumors were collected, weighed, and recorded. Results are shown below. Figure 18 The in vivo efficacy of the CD20×CD3κλ bispecific antibody showed a dose-related relationship, with low, medium and high doses showing tumor inhibition rates of 65%, 98% and 162%, respectively. In the high and medium dose groups, tumors were completely inhibited or regressed.

[0532] 9. Efficacy of CD20×CD3κλ bispecific antibody in cynomolgus monkeys

[0533] Eight cynomolgus monkeys were assigned to four dosage groups, with each group consisting of two monkeys (half male and half female). The dosage groups received 0.3, 1, and 3 mg / kg (once weekly for 3 weeks, for a total of 4 doses) and 1 mg / kg (single dose) of CD20×CD3κλ002 bispecific antibody, respectively. The dosing regimens are shown in Table 9. During the dosing and recovery periods, all monkeys in each group were in good general condition, with no toxic reactions, deaths, or near-death experiences observed. No significant abnormal changes in body temperature were observed in any dosage group, and the ECG waveforms in lead II were normal. Heart rate, RR interval, PR interval, QT interval, QRS duration, systolic blood pressure, and diastolic blood pressure were all within normal ranges. At different time points after administration, changes in the number of B and T cells in peripheral blood were analyzed using flow cytometry. B cells were identified using the cell surface marker CD20 (CD20+ cells), and T cells were identified using CD3 (CD3+ cells). Eight hours after administration, B was rapidly cleared from peripheral blood, and within 24 hours it was below the detection limit. Figure 19 ).

[0534] Table 9. Dosing regimen for CD20×CD3κλ bispecific antibody

[0535]

[0536] Example 3: Construction of BCMA×CD3κλ bispecific antibodies formed by different types of light chains

[0537] 1. Construction of BCMA×CD3κλ bispecific antibody

[0538] A novel BCMA-CD3 κλ humanized bispecific antibody with a native IgG conformation was constructed using a BCMA humanized antibody containing the κ light chain and a humanized CD3 antibody containing the λ light chain, referring to Example 2. Simultaneously, a charge variant (Vκ) was introduced into both the BCMA antigen arm and the CD3 arm. BCMA Gln 42 Lys;VH BCMA Gln 39 Glu; VλCD3 Gln 40 Glu; VH CD3 Gln 39 Lys (sequence shown in Table 10); the Fc portion of the bispecific antibody adopts a human IgG4 knob-into-hole structure to achieve heterodimer pairing, and through mutation of Ser 228 Pro、Leu 235 Glu and Pro 329 Ala maintains the stability of the hinge region and weakens its interaction with FcγR receptors and C1q.

[0539] Table 10. BCMA×CD3κλ Bispecific Antibody

[0540]

[0541] BCMA×CD3 κλ003

[0542] BCMA arm κ light chain: SEQ ID NO. 80

[0543] DIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQKKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQHSRELPWTFGQGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0544] Nucleotide sequence: SEQ ID NO. 81

[0545] GACATCGTGCTGACACAGAGCCCTGCTTCTCTGGCTGTGTCTCCTGGCCAGAGAGCCACCATCACCTGTAGAGCCAGCAAGAGCGTGTCCACCAGCGGCTACTCTTACATGCACTGGTATCAGAAGAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTACCTGGCTAGCAACCTCGAAAGCGGAGTGCCTGCTAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAACCCCGTGGAAGCCGAAGACACCGCCAACTACTACTGCCAGCACAGCAGAGAGCTGCCCTGGACATTTGGCCAGGGCACCAAGGTGGAAATCAAGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0546] BCMA arm heavy chain (heavy chain 1): SEQ ID NO. 82

[0547] QVQLVQSGSELKKPGASVKVSCKASGYIFTNFGMNWVREAPGQGLEWMGWINTYTGEQIYADGFTGRFVFSLDTSASTAYLQISSLKAEDTAVYFCARGEIYYGYDVGFVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0548] Nucleotide sequence: SEQ ID NO. 83

[0549]

[0550] CD3 arm lambda light chain: SEQ ID NO. 66

[0551] QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEAEYYCVLWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0552] Nucleotide sequence: SEQ ID NO. 67

[0553] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTAGATCTTCTACAGGCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGGAGAAGCCCGGCCAGGCTCCTAGAGGACTGATCGGCGGAACAAACAAGAGAGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACAATCACTGGTGCTCAGGCTGAGGACGAGGCCGAGTACTATTGTGTGCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAACTGACAGTTCTGGGTCAGCCCAAGGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTATCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0554] CD3 Arm Heavy Chain (Heavy Chain 2): SEQ ID NO. 68

[0555] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRKAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0556] Nucleotide sequence: SEQ ID NO. 69

[0557]

[0558] BCMA×CD3 κλ004

[0559] BCMA arm κ light chain: SEQ ID NO. 84

[0560] DIVLTQSPASLAVSPGQRATITCRASKSVTTSGYSYIHWYQKKPGQPPKLLIYLASDLEAGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQHSRELPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0561] Nucleotide sequence: SEQ ID NO. 85

[0562] GACATCGTGCTGACACAGAGCCCTGCTTCTCTGGCTGTGTCTCCTGGCCAGAGAGCCACCATCACCTGTAGAGCCAGCAAGAGCGTGACCACCAGCGGCTACTCTTACATCCACTGGTATCAGAAGAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTACCTGGCCAGCGATCTGGAAGCTGGCGTGCCAGCTAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAACCCCGTGGAAGCCGAAGACACCGCCAACTACTACTGCCAGCACAGCAGAGAGCTGCCCTGGACATTTGGCCAGGGCACCAAGGTGGAAATCAAGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0563] BCMA arm heavy chain (heavy chain 1): SEQ ID NO. 82

[0564] QVQLVQSGSELKKPGASVKVSCKASGYIFTNFGMNWVREAPGQGLEWMGWINTYTGEQIYADGFTGRFVFSLDTSASTAYLQISSLKAEDTAVYFCARGEIYYGYDVGFVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0565] Nucleotide sequence: SEQ ID NO. 83

[0566]

[0567] CD3 arm lambda light chain: SEQ ID NO. 66

[0568] QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEAEYYCVLWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0569] Nucleotide sequence: SEQ ID NO. 67

[0570] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTAGATCTTCTACAGGCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGGAGAAGCCCGGCCAGGCTCCTAGAGGACTGATCGGCGGAACAAACAAGAGAGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACAATCACTGGTGCTCAGGCTGAGGACGAGGCCGAGTACTATTGTGTGCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAACTGACAGTTCTGGGTCAGCCCAAGGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTATCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0571] CD3 Arm Heavy Chain (Heavy Chain 2): SEQ ID NO. 68

[0572] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRKAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0573] Nucleotide sequence: SEQ ID NO. 69

[0574]

[0575] BCMA×CD3 κλ005

[0576] BCMA arm κ light chain: SEQ ID NO. 80

[0577] DIVLTQSPASLAVSPGQRATITCRASKSVSTSGYSYMHWYQKKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQHSRELPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0578] Nucleotide sequence: SEQ ID NO. 81

[0579] GACATCGTGCTGACACAGAGCCCTGCTTCTCTGGCTGTGTCTCCTGGCCAGAGAGCCACCATCACCTGTAGAGCCAGCAAGAGCGTGTCCACCAGCGGCTACTCTTACATGCACTGGTATCAGAAGAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTACCTGGCTAGCAACCTCGAAAGCGGAGTGCCTGCTAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAACCCCGTGGAAGCCGAAGACACCGCCAACTACTACTGCCAGCACAGCAGAGAGCTGCCCTGGACATTTGGCCAGGGCACCAAGGTGGAAATCAAGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0580] BCMA arm heavy chain (heavy chain 1): SEQ ID NO. 86

[0581] QVQLVQSGSELKKPGASVKVSCKASGYIFTNFGMNWVREAPGQGLEWMGWINTYTGEQIYADGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARGEIYYGYDVGFVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0582] Nucleotide sequence: SEQ ID NO. 87

[0583]

[0584] CD3 Arm λ Light Chain: SEQ ID NO. 66

[0585] QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEAEYYCVLWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0586] Nucleotide Sequence: SEQ ID NO. 67

[0587] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTAGATCTTCTACAGGCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGGAGAAGCCCGGCCAGGCTCCTAGAGGACTGATCGGCGGAACAAACAAGAGAGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACAATCACTGGTGCTCAGGCTGAGGACGAGGCCGAGTACTATTGTGTGCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAACTGACAGTTCTGGGTCAGCCCAAGGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTATCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0588] CD3 Arm Heavy Chain (Heavy Chain 2): SEQ ID NO. 68

[0589] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRKAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0590] Nucleotide sequence: SEQ ID NO. 69

[0591]

[0592] BCMA×CD3 κλ006

[0593] BCMA arm κ light chain: SEQ ID NO. 84

[0594] DIVLTQSPASLAVSPGQRATITCRASKSVTTSGYSYIHWYQKKPGQPPKLLIYLASDLEAGVPARFSGSGSGTDFTLTINPVEAEDTANYYCQHSRELPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0595] Nucleotide sequence: SEQ ID NO. 85

[0596] GACATCGTGCTGACACAGAGCCCTGCTTCTCTGGCTGTGTCTCCTGGCCAGAGAGCCACCATCACCTGTAGAGCCAGCAAGAGCGTGACCACCAGCGGCTACTCTTACATCCACTGGTATCAGAAGAAGCCCGGCCAGCCTCCTAAGCTGCTGATCTACCTGGCCAGCGATCTGGAAGCTGGCGTGCCAGCTAGATTTTCTGGCAGCGGCTCTGGCACCGACTTCACCCTGACAATCAACCCCGTGGAAGCCGAAGACACCGCCAACTACTACTGCCAGCACAGCAGAGAGCTGCCCTGGACATTTGGCCAGGGCACCAAGGTGGAAATCAAGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0597] BCMA arm heavy chain (heavy chain 1): SEQ ID NO. 86

[0598] QVQLVQSGSELKKPGASVKVSCKASGYIFTNFGMNWVREAPGQGLEWMGWINTYTGEQIYADGFTGRFVFSLDTSVSTAYLQISSLKAEDTAVYFCARGEIYYGYDVGFVYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0599] Nucleotide sequence: SEQ ID NO. 87

[0600]

[0601] CD3 arm lambda light chain: SEQ ID NO. 66

[0602] QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEAEYYCVLWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0603] Nucleotide sequence: SEQ ID NO. 67

[0604] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTAGATCTTCTACAGGCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGGAGAAGCCCGGCCAGGCTCCTAGAGGACTGATCGGCGGAACAAACAAGAGAGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACAATCACTGGTGCTCAGGCTGAGGACGAGGCCGAGTACTATTGTGTGCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAACTGACAGTTCTGGGTCAGCCCAAGGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTATCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0605] CD3 Arm Heavy Chain (Heavy Chain 2): SEQ ID NO. 68

[0606] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRKAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0607] Nucleotide sequence: SEQ ID NO. 69

[0608]

[0609] 2. Expression and purification of BCMA×CD3κλ bispecific antibody

[0610] The plasmids encoding the corresponding antibody fragments were mixed with 3 mg / mL PEI at a ratio of BCMA arm light chain (κ light chain): CD3 arm light chain (λ light chain): BCMA arm heavy chain (heavy chain 1): CD3 arm heavy chain (heavy chain 2) = 2:2:1:1, and then co-transfected with CHO-S cells in 500 mL CD CHO AGT medium (Gibco #12490-001) at 37°C and 5% CO2 at 150 rpm. On days 2, 4, and 6 after transient transfection, 4% CHO Feed C+ (Gibco #A25031-05) was added. When the cell viability dropped to approximately 85%, the fermentation broth was harvested, filtered, and preliminarily purified by Protein A affinity chromatography. SEC-HPLC showed a monomer content close to or higher than 92%. Capto S ImpAct ion exchange chromatography further increased the monomer content to over 98-99% (Table 11).

[0611] Table 11. Purity of BCMA×CD3κλ bispecific antibody

[0612]

[0613] 3. Binding activity of BCMA×CD3κλ bispecific antibody

[0614] (1) Determination of the affinity between BCMA×CD3κλ bispecific antibody and antigen

[0615] 10 μg / mL of recombinant human or cynomolgus monkey BCMA or CD3εγ antigens were conjugated to a CM5 chip (GE Healthcare) via amino-coupling, controlling the antigen binding amount to approximately 200 RU. After baseline stabilization, serially diluted antibodies (starting from 10 μg / mL, with 7 two-fold dilutions) were flowed through the chip at a flow rate of 30 μL / min, with a binding time of 350 seconds and a dissociation time of 600 seconds. Kinetic constants were obtained by fitting a 1:1 binding model using Biacore T200 evaluation software. Affinity assay results are shown in Table 12.

[0616] Table 12. Affinity of BCMA×CD3κλ bispecific antibody to antigen

[0617]

[0618] (2) Binding of BCMA×CD3κλ bispecific antibody to BCMA+ cells

[0619] Log-phase CHO-human BCMA stable cells (CHO-hBCMA) and CHO-cynomonas BCMA stable cells (CHO-cynomonas BCMA), along with tumor cells NCI-H929 and RPMI-8226, were collected. After blocking, 100 μL of serially diluted antibody (starting concentration 1800 nM, 3-fold dilution, 10 gradients) was added to each well, and incubated at 4°C for 60 minutes. Secondary antibody was added to 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution), and incubated on ice for 20 minutes. After washing once, 50 μL / well of PI solution (1:300) was added, and incubated for 5 minutes. Analyze the results using flow cytometry. Figure 20 The results showed that the BCMA×CD3κλ bispecific antibody bound human and cynomolgus monkey BCMA-stable transgenic cells with high affinity; Figure 21 The results showed that the BCMA×CD3κλ bispecific antibody bound to BCMA+ tumor cells NCI-H929 and RPMI-8226 with high affinity. The binding constant EC50 of the BCMA×CD3κλ bispecific antibody to cells was [not specified]. 50 See Table 13.

[0620] Table 13. Binding of BCMA×CD3κλ bispecific antibody to BCMA stable cells

[0621]

[0622] (3) Binding of BCMA×CD3κλ bispecific antibody to Jurkat cells

[0623] Jurkat cells in logarithmic growth phase were collected and added to 200 μg / mL mouse IgG (Jackson ImmunoResearch, 115-005-03), then incubated on ice for 30 minutes. The cell count was adjusted to 5 × 10⁶ cells / mL with 4% fetal bovine serum. 5 Cells / mL, 100 μL per well of a 96-well U-shaped plate, centrifuged at 300g to remove supernatant, 100 μL of serially diluted antibody (starting concentration 1800 nM, 3-fold dilution, 10 gradients) per well, incubated at 4°C for 60 min. Secondary antibody was added to 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution), incubated on ice for 20 min, washed once, and then 50 μL / well of PI was added, incubated for 5 min, and analyzed by flow cytometry (BD C6). Detection results are as follows: Figure 22 According to Table 14, the BCMA×CD3κλ bispecific antibody binds to the human leukemia T cell line Jurkat cells with medium affinity.

[0624] (4) Binding of BCMAxCD3κλ bispecific antibody to peripheral blood T cells

[0625] Fresh human peripheral blood was collected, and PBMCs were isolated using Ficoll-Paque Plus (GE, 17-1440-03). The PBMCs were adjusted to 5 × 10⁶ cells / mL with 4% fetal bovine serum (Hyclone, SH30626.06). 5 Cells / mL, 100 μL / well added to a 96-well U-shaped plate, centrifuged and supernatant discarded, 100 μL of serially diluted antibody added to each well (starting concentration 1800 nM, 3-fold dilution, 10 gradients), incubated at 4°C for 60 min. Secondary antibody added 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution), incubated on ice for 20 min, washed once, then 50 μL / well of PI added, incubated for 5 min, and detected by flow cytometry (BD C6). Control antibody REGN5458 was synthesized and prepared according to US20200024356. Detection results are shown in […]. Figure 23 As shown in Table 14, the BCMA×CD3κλ bispecific antibody recognizes human peripheral blood CD4+ T and CD8+ T cells, and has an affinity of about 60-97 nM for human T cells, which is weaker than the binding affinity of the BCMA antigen arm to the BCMA receptor, thus favoring the preferential enrichment of the bispecific antibody into tumor cells.

[0626] Table 14. Binding of BCMA×CD3κλ bispecific antibody to Jurkat cells

[0627]

[0628] 4. BCMA×CD3κλ bispecific antibody-mediated TDCC action

[0629] Freshly isolated PBMCs were mixed with target cells NCI-H929 and RPMI-8226 cells in logarithmic growth phase, with an effector / target cell ratio of 8:1. 50 μL of serially diluted antibody (antibody concentration starting at 66.7 nM, 10-fold dilution, 7 gradients) was added to each well, and the cells were incubated at 37°C with 5% CO2 for 24 hours. After incubation, 50 μL of supernatant was transferred to a new black ELISA plate, and 50 μL / well of LDH detection substrate was added. The reaction was stopped after 10 minutes, and LDH release was detected. The remaining cells in the wells were washed twice with 4% fetal bovine blood, incubated with 100 μg / mL human IgG for 10 minutes, and then T cell activation detection antibodies (CD25-PE, CD4-APC, CD69-FITC, and CD8-APC) were added. The cells were incubated on ice for 20 minutes. After washing and discarding the supernatant, 60 μL / well of PI was added, and the cells were incubated on ice for 5 minutes. Flow cytometry was then used for detection. Figure 24 A and 24B show the killing effect of BCMA×CD3κλ bispecific antibody on NCI-H929 cells and the activation effect on T cells, respectively. Figure 25A and 25B show the killing effect of the BCMA×CD3κλ bispecific antibody on RPMI-8226 cells and the activation effect on T cells, respectively. For tumor cells NCI-H929 and RPMI-8226 with different BCMA expression levels, the BCMA×CD3κλ bispecific antibody mediated effective killing of T cells, with killing activity comparable to the control antibody REGN5458.

[0630] 5. Activation of T cell activation pathway by BCMA×CD3κλ bispecific antibody

[0631] Jurkat-NFAT-luc reporter cells and BCMA-positive target cells RPMI-8226 in logarithmic growth phase were collected, centrifuged, and the supernatant was discarded. The cells were then resuspended to a concentration of 2 × 10⁻⁶. 6 Cells / ml. Seed 50 μl / well of target cells into a 96-well plate, centrifuge at 300g for 5 minutes and discard the supernatant. Seed 50 μl / well of Jurkat-NFAT-luc reporter cells into a 96-well plate. Add 50 μl of serially diluted BCMA×CD3 κλ bispecific antibody or control antibody KLH×CD3 (starting concentration 20 μg / ml, 10-fold dilution, 10 gradients) to each well. Incubate at 37℃ for 6 hours with 5% CO2. After incubation, add 100 μl of detection reagent to each well according to the ONE-Glo Luciferase Assay System instructions, incubate at room temperature for 3 minutes, and detect using a microplate reader (Biotek Synergy HT). Results are shown below. Figure 26 When the BCMA×CD3κλ bispecific antibody is used as the target cell of RPMI-8226 tumor cells, it can activate the NFAT signaling pathway of T cells; when there are no target cells, it does not activate the NFAT signaling pathway.

[0632] 6. Non-specific activation of PBMCs by BCMA×CD3κλ bispecific antibody

[0633] Freshly isolated PBMCs were added to each well with 50 μL of serially diluted antibody (antibody concentration starting at 66.7 nM, 10-fold dilution, 7 gradients), and incubated at 37°C with 5% CO2 for 24 hours. After incubation, 50 μL of supernatant was transferred to a new black ELISA plate, and 50 μL / well of LDH detection substrate was added. The reaction was stopped after 10 minutes, and LDH release was detected. The remaining cells in the wells were washed twice with 4% fetal bovine blood, incubated with 100 μg / mL human IgG for 10 minutes, and then T cell activation detection antibodies (CD25-PE, CD4-APC, CD69-FITC, and CD8-APC) were added. The cells were incubated on ice for 20 minutes. The supernatant was discarded after washing, and 60 μL / well of PI was added. The cells were incubated on ice for 5 minutes and detected by flow cytometry. The results are shown in [Figure number missing]. Figure 27In the absence of target cells, the BCMA×CD3 κλ bispecific antibody had no activating effect on peripheral blood T cells, which was comparable to the negative control KLH×CD3.

[0634] 7. BCMA × Binding of CD3κλ humanized bispecific antibody to Fc receptor

[0635] A 50 μg / ml His-Tag antibody was amino-conjugated to a CM5 chip to capture FcγRI and FcγRIIA tagged with His6, respectively. H131 and FcγRIIIA V158 Recombinant protein was captured for 40 seconds at a flow rate of 10 μL / min. After baseline stabilization, serially diluted antibody (initial concentration 37.5 μg / mL, 2-fold dilution) was flowed through the chip at a flow rate of 30 μL / min. Binding time was 120 seconds, and dissociation time was 200 seconds. Affinity constants were obtained by fitting the data using Biacore evaluation software. Figure 28 It can be seen that the BCMA×CD3κλ bispecific antibody is similar to FcγRI and FcγRIIA. H131 and FcγRIIIA V158 No binding was observed; the wild-type IgG4 control antibody bound FcγRI with strong affinity, and FcγRIIA was also observed. H131 There is a weak binding.

[0636] 8. Subcutaneous NCI-H929 xenograft model in immunodeficient mice

[0637] Select 6-8 week old female B-NGD mice (Biocytok Biotechnology Co., Ltd.), and subcutaneously inoculate them with 2×10 6 NCI-H929 cells (mixed with Matrigel 1:1), until the tumor grows to 60mm 3 Mice were randomly assigned to three groups: a treatment group (3.0 mg / kg), a treatment group (0.6 mg / kg), and a negative control group (3 mg / kg) of KLH×CD3. Each mouse received 1×10 mg / kg via tail vein injection. 7 Three days after the first administration of PBMC cells to mice, the mice were given the first dose, followed by two doses every five days. Tumor volume and body weight were monitored every two days. Mice were euthanized by cervical dislocation at the end of the experiment, and tumors were collected, weighed, and recorded. Results are shown below. Figure 29 The in vivo efficacy of the BCMA×CD3κλ bispecific antibody showed a dose-related relationship. The tumor inhibition rates at 3.0 mg / kg and 0.6 mg / kg were 95% and 108% (BCMA×CD3κλ005), and 94% and 108% (BCMA×CD3κλ006), respectively. Tumor-bearing mice tolerated the above doses well, with no adverse reactions such as weight loss.

[0638] Example 4: Construction of GPC3×CD3κλ bispecific antibodies formed by different types of light chains

[0639] 1. Construction of GPC3×CD3κλ bispecific antibody

[0640] A novel GPC3-CD3 κλ humanized bispecific antibody with a native IgG conformation was constructed by combining a humanized GPC3 antibody containing a κ light chain with a humanized CD3 antibody containing a λ light chain, referring to Example 2. Simultaneously, a charge variant (Vκ) was introduced into both the GPC3 antigen arm and the CD3 arm. GPC3 Gln 43 Lys;VH GPC3 Gln 39 Glu; Vλ CD3 Gln 40 Glu; VH CD3 Gln 39 Lys (sequence shown in Table 15). The Fc region of the bispecific antibody adopts a human IgG4 knob-into-hole structure to achieve heterodimer pairing, and is further enhanced by mutating Ser... 228 Pro, Leu 235 Glu and Pro 329 Ala maintains the stability of the hinge region and weakens its interaction with Fcγ receptors and C1q.

[0641] Table 15. Humanized Bispecific Antibodies for GPC3×CD3κλ

[0642]

[0643] GPC3×CD3 κλ002:

[0644] GPC3 arm κ light chain SEQ ID NO.88

[0645] DVVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLKKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCLQVTHVPLTFGQGTKL EIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0646] Nucleotide sequence SEQ ID NO. 89

[0647] GACGTGGTCATGACACAGAGCCCTCTGAGCCTGCCTGTGACACCTGGCGAACCTGCCAGCATCAGCTGTAGAAGCAGCCAGAGCATCGTGCACAGCAACGGCAACACATACCTGGAGTGGTATCTGAAGAAGCCCGGCCAGTCTCCTCAGCTGCTGATCTACAAGGTGTCCAACAGATTCAGCGGCGTGCCCGACAGATTCTCTGGCTCTGGATCTGGCACCGACTTCACCCTGAAGATCTCCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTTCTGTCTCCAGGTCACACACGTGCCCCTGACATTTGGCCAGGGCACCAAGCTGGAAATCAAGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0648] GPC3 arm heavy chain (heavy chain 1) SEQ ID NO. 90

[0649] QVQLVQSGAEVKKPGSSVKVSCKASGYTFADYEIHWVREAPGQGLEWMGAIHPGSGGTAYAQKFQGRVTLTADESSTTAYMELSSLRSEDTAVYYCTRYYSFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0650] Nucleotide sequence SEQ ID NO.91

[0651]

[0652] CD3 arm lambda light chain SEQ ID NO.66

[0653] QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEAEYYCVLWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0654] Nucleotide sequence SEQ ID NO.67

[0655] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTAGATCTTCTACAGGCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGGAGAAGCCCGGCCAGGCTCCTAGAGGACTGATCGGCGGAACAAACAAGAGAGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACAATCACTGGTGCTCAGGCTGAGGACGAGGCCGAGTACTATTGTGTGCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAACTGACAGTTCTGGGTCAGCCCAAGGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTATCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0656] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.68

[0657] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRKAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0658] Nucleotide sequence SEQ ID NO. 69

[0659]

[0660] GPC3×CD3 κλ003:

[0661] GPC3 arm κ light chain SEQ ID NO. 92

[0662] DVVMTQSPLSLPVTPGEPASISCRSSQSIVHSNGNTYLEWYLKKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYFCLQVTHVPLTFGQGTKLEIKRTVAAPSVFIFPPSDKKLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0663] Nucleotide sequence SEQ ID NO.93

[0664] GACGTGGTCATGACACAGAGCCCTCTGAGCCTGCCTGTGACACCTGGCGAACCTGCCAGCATCAGCTGTAGAAGCAGCCAGAGCATCGTGCACAGCAACGGCAACACATACCTGGAGTGGTATCTGAAGAAGCCCGGCCAGTCTCCTCAGCTGCTGATCTACAAGGTGTCCAACAGATTCAGCGGCGTGCCCGACAGATTCTCTGGCTCTGGATCTGGCACCGACTTCACCCTGAAGATCTCCAGAGTGGAAGCCGAGGACGTGGGCGTGTACTTCTGTCTCCAGGTCACACACGTGCCCCTGACATTTGGCCAGGGCACCAAGCTGGAAATCAAGCGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATAAGAAATTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT

[0665] GPC3 arm heavy chain (heavy chain 1) SEQ ID NO. 94

[0666] QVQLVQSGAEVKKPGSSVKVSCKASGYTFADYEIHWVREAPGQGLEWMGAIHPGSGGTAYAQKFQGRVTLTADESSTTAYMELSSLRSEDTAVYYCTRYYSFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVEDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDERVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVCTLPPSQEEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0667] Nucleotide sequence SEQ ID NO.95

[0668]

[0669] CD3 arm lambda light chain SEQ ID NO. 66

[0670] QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTITGAQAEDEAEYYCVLWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS

[0671] Nucleotide sequence SEQ ID NO.67

[0672] CAGGCTGTGGTCACACAAGAGCCTAGCCTGACAGTGTCTCCTGGCGGCACAGTGACCCTGACCTGTAGATCTTCTACAGGCGCCGTGACCACCAGCAACTACGCTAATTGGGTGCAGGAGAAGCCCGGCCAGGCTCCTAGAGGACTGATCGGCGGAACAAACAAGAGAGCCCCTTGGACACCCGCCAGATTCTCTGGATCTCTGCTCGGCGGAAAGGCCGCTCTGACAATCACTGGTGCTCAGGCTGAGGACGAGGCCGAGTACTATTGTGTGCTGTGGTACAGCAACCTGTGGGTGTTCGGCGGAGGCACCAAACTGACAGTTCTGGGTCAGCCCAAGGCGGCGCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTATCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA

[0673] CD3 Arm Heavy Chain (Heavy Chain 2) SEQ ID NO. 68

[0674] EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRKAPGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNSLYLQMNSLKTEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLASSIEKTISKAKGQPREPQVYTLPPCQEEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0675] Nucleotide sequence SEQ ID NO. 69

[0676]

[0677] The plasmids encoding the corresponding antibody fragments were mixed with 3 mg / mL PEI at a ratio of GPC3 arm light chain (κ light chain): CD3 arm light chain (λ light chain): GPC3 arm heavy chain (heavy chain 1): CD3 arm heavy chain (heavy chain 2) = 2:2:1:1, and then co-transfected with CHO-S cells in 500 mL CD CHO AGT medium (Gibco #12490-001) at 37°C and 5% CO2 at 150 rpm. On days 2, 4, and 6 after transient transfection, 4% CHO Feed C+ (Gibco #A25031-05) was added. When the cell viability dropped to approximately 85%, the fermentation broth was harvested, filtered, and preliminarily purified by Protein A affinity chromatography. SEC-HPLC showed a monomer content higher than 92%. Further purification by Butyl HP hydrophobic chromatography and Capto Q anion exchange chromatography further increased the monomer content to over 99.5% (Table 16).

[0678] Table 16. Purification of GPC3-CD3 humanized bispecific antibody

[0679]

[0680] 2. Binding of GPC3×CD3κλ bispecific antibody to GPC3 stable transfected cells

[0681] Log-phase CHO-human GPC3, CHO-cynomolgus monkey GPC3 stable cells, or human hepatocellular carcinoma HepG2 tumor cells were collected, blocked, and adjusted to 5 × 10⁶ cells / year. 5 Cells / ml were collected, and 100 μl / well of cell suspension was added to a 96-well U-shaped plate. The plate was centrifuged at 300g for 5 minutes, the supernatant was discarded, and 100 μL of serially diluted antibody (starting concentration 1800 nM, 3-fold dilution, 10 gradients) was added to each well. The plate was incubated at 4°C for 60 minutes. Secondary antibody was added to 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution). The plate was incubated on ice for 20 minutes, washed once, and then 50 μL / well of PI solution (1:300 dilution) was added. The plate was incubated for 5 minutes, and the results were analyzed by flow cytometry. Results are shown below. Figures 30-31 According to Table 17, the GPC3×CD3κλ bispecific antibody binds to GPC3+ cells with high affinity.

[0682] Table 17. Binding of GPC3×CD3κλ bispecific antibody to GPC3+ cells

[0683]

[0684] 3. GPC3 × Binding of CD3κλ bispecific antibody to Jurkat cells

[0685] Jurkat cells in logarithmic growth phase were collected and added to 200 μg / mL mouse IgG (Jackson ImmunoResearch, 115-005-03), then incubated on ice for 30 minutes. The cell count was adjusted to 5 × 10⁶ cells / mL with 4% fetal bovine serum. 5 Cells / mL, 100 μL per well of a 96-well U-shaped plate, centrifuged at 300g to remove supernatant, 100 μL of serially diluted antibody (starting concentration 1800 nM, 3-fold dilution, 10 gradients) per well, incubated at 4°C for 60 min. Secondary antibody was added to 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution), incubated on ice for 20 min, washed once, and then 50 μL / well of PI was added, incubated for 5 min, and analyzed by flow cytometry (BD C6). Detection results are as follows: Figure 32 According to Table 18, the GPC3×CD3κλ bispecific antibody binds with intermediate affinity to the human leukemia T cell line Jurkat cells, EC... 50 Approximately 20-40 nM.

[0686] 4. GPC3 × Binding of CD3κλ bispecific antibody to peripheral blood T cells

[0687] Peripheral blood from fresh humans or cynomolgus monkeys was collected and PBMCs were isolated using Ficoll.Paque Plus (GE, 17-1440-03). The PBMCs were adjusted to 5 × 10⁶ cells / mL with 4% fetal bovine serum (Hyclone, SH30626.06). 5 Cells / mL, 100 μL / well added to a 96-well U-shaped plate, centrifuged and supernatant discarded, 100 μL of serially diluted antibody (starting concentration 1800 nM, 3-fold dilution, 10 gradients) added to each well, incubated at 4°C for 60 min. Secondary antibody added 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution), incubated on ice for 20 min, washed once, then 50 μL / well of PI added, incubated for 5 min, and analyzed by flow cytometry (BD Celesta). Results are shown below. Figure 33 According to Table 18, the GPC3×CD3κλ bispecific antibody binds to human peripheral blood T cells with low affinity.

[0688] Table 18. Binding of GPC3×CD3κλ bispecific antibody to T cells

[0689]

[0690] 5. GPC3 × CD3κλ bispecific antibody-mediated TDCC

[0691] Freshly isolated PBMCs were mixed with HepG2 target cells in logarithmic growth phase at an effector / target cell ratio of 10:1. 50 μL of serially diluted antibody (antibody concentration starting at 66.7 nM, 10-fold dilution, 7 gradients) was added to each well, and the cells were incubated at 37°C with 5% CO2 for 24 hours. After incubation, 50 μL of supernatant was transferred to a new black ELISA plate, and 50 μL / well of LDH detection substrate was added. The reaction was stopped after 10 minutes, and LDH release was detected. The remaining cells in the wells were washed twice with 4% fetal bovine blood, incubated with 100 μg / mL human IgG for 10 minutes, and then T cell activation detection antibodies (CD25-BV421, CD4-FITC, CD69-BV605, and CD8-APC) were added. The cells were incubated on ice for 20 minutes. After washing and discarding the supernatant, 60 μL / well of PI was added, and the cells were incubated on ice for 5 minutes. Flow cytometry was then used for detection. Figure 34 A and 34B show the killing effect of GPC3×CD3κλ bispecific antibody on HepG2 cells and the activation effect on T cells, respectively.

[0692] 6. GPC3 × Activation of T cell activation pathway by CD3κλ bispecific antibody

[0693] Take target cells (CHO-human GPC3) in the logarithmic growth phase, centrifuge, discard the supernatant, and resuspend to 2×10⁻⁶. 5 Cells / ml. Seed 50 μL / well of target cells into 96-well plates and incubate overnight at 37°C with 5% CO2. Centrifuge Jurkat-NFAT-luc reporter cells in logarithmic growth phase at 300g for 5 minutes, discard the supernatant, and resuspend to 4×10⁶ cells / ml. 6 Cells / ml were collected from a 96-well plate, the supernatant was discarded, and 25 μL / well was seeded with Jurkat-NFAT-luc reporter cells. 25 μL of serially diluted GPC3×CD3 κλ bispecific antibody or control antibody KLH×CD3 (starting concentration 20 μg / ml, 3-fold dilution, 10 gradients) was added to each well. The plate was incubated at 37°C for 6 hours with 5% CO2. After incubation, 100 μL of assay reagent was added to each well according to the ONE-Glo Luciferase Assay System instructions, and the results were analyzed using a microplate reader (MD SpectraMax i3x). Results are shown below. Figure 35 When the GPC3×CD3κλ bispecific antibody targets CHO-human GPC3 cells, it can activate the NFAT signaling pathway in T cells.

[0694] 7. GPC3 × Non-specific activation of CD3κλ bispecific antibody PBMC

[0695] Freshly isolated PBMCs were incubated at 37°C for 24 hours with 100 μL of antibody (10 μg / mL) in 5% CO2. Cells were washed twice with 4% fetal bovine blood, then incubated with 100 μg / mL human IgG for 10 minutes. T-cell activation detection antibodies (CD25-BV421, CD4-FITC, CD69-BV605, and CD8-APC) were added, and the cells were incubated on ice for 20 minutes. The supernatant was discarded after washing, and 60 μL / well of PI was added. The cells were incubated on ice for 5 minutes and then analyzed by flow cytometry. The positive control antibody ERY974 was prepared according to US20170267783. Detection results are shown below. Figure 36 In the absence of target cells, the GPC3×CD3 κλ bispecific antibody had no activating effect on peripheral blood T cells, which was comparable to the negative control KLH×CD3.

[0696] 8. Immunoreconstituted mouse subcutaneous HepG2 xenograft model

[0697] Female B-NGD mice (6-8 weeks old, Biocytogen Biosciences Co., Ltd.) were selected and subcutaneously inoculated with HepG2 cells (7×10⁶ cells per 10 ... 6 / each), until the tumor grows to 60-100mm 3 Mice were randomly assigned to four groups: a high-dose group (3.0 mg / kg), a medium-dose group (1.0 mg / kg), a low-dose group (0.3 mg / kg), a positive control group (ERY974), and a negative control group (KLH×CD3 3 mg / kg). Each mouse was injected intravenously with 1×10 mg / kg of the drug. 7 PBMC cells were administered to mice 3 days later, with dosing intervals of 5 days, for a total of 2 doses. Tumor volume and mouse weight were monitored. Mice were euthanized by cervical dislocation at the end of the experiment, and tumors were collected, weighed, and recorded. Results are shown below. Figure 37 The in vivo efficacy of the GPC3×CD3κλ bispecific antibody showed a dose-related relationship, with tumor inhibition rates (from low to high doses) of 76.7%, 81.3%, and 95.9%, respectively. Tumor-bearing mice tolerated the above doses well, with no adverse reactions such as weight loss.

[0698] 9. CD3 humanized mouse Hepa1-6 / human GPC3 xenograft model

[0699] Six-week-old female C57 / BL6-hCD3 mice (Biocytok Biotechnology Co., Ltd.) were selected, and Hepa1-6 / human GPC3 (6×10⁻⁶) was introduced. 6 (Each mouse) was subcutaneously inoculated until the tumor volume reached 60-100 mm. 3Mice were randomly assigned to groups: a high-dose group (10 mg / mL), a medium-dose group (3 mg / mL), a low-dose group (1 mg / mL), a positive control group (ERY974), and a negative control group (KLH×CD3 10 mg / kg). Dosing was administered every 3 days for a total of 3 doses. Tumor volume and body weight were monitored. Mice were euthanized by cervical dislocation at the end of the experiment, and tumors were collected, weighed, and recorded. Results are shown below. Figure 38 The GPC3×CD3κλ bispecific antibody can significantly mediate the killing of tumor cells by immune cells and reduce tumor volume; its 10 mg / kg dose is comparable to the efficacy of ERY974. SEQUENCE LISTING <110> Connoya Biomedical Technology (Chengdu) Co., Ltd. <120> A bispecific antibody and its uses <130> MTI20179 <140> 202011325843.0 <141> 2020-11-23 <160> 95 <170> PatentIn version 3.5 <210> 1 <211> 306 <212> PRT <213> Homo sapiens <400> 1 Gln Ser Ile Lys Gly Asn His Leu Val Lys Val Tyr Asp Tyr Gln Glu 1 5 10 15 Asp Gly Ser Val Leu Leu Thr Cys Asp Ala Glu Ala Lys Asn Ile Thr 20 25 30 Trp Phe Lys Asp Gly Lys Met Ile Gly Phe Leu Thr Glu Asp Lys Lys 35 40 45 Lys Trp Asn Leu Gly Ser Asn Ala Lys Asp Pro Arg Gly Met Tyr Gln 50 55 60 Cys Lys Gly Ser Gln Asn Lys Ser Lys Pro Leu Gln Val Tyr Tyr Arg 65 70 75 80 Met Cys Gln Asn Cys Ile Glu Leu Asn Ala Pro Glu Ala Ala Gly Gly 85 90 95 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 100 105 110 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 115 120 125 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 130 135 140 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 145 150 155 160 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 165 170 175 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu 180 185 190 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys 195 200 205 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 210 215 220 Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 225 230 235 240 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 245 250 255 Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp 260 265 270 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 275 280 285 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 290 295 300 Gly Lys 305 <210> 2 <211> 339 <212> PRT <213> Homo sapiens <400> 2 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 Ala Pro Glu Ala Ala Gly Gly 100 105 110 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 115 120 125 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 130 135 140 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 145 150 155 160 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 165 170 175 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 180 185 190 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu 195 200 205 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 210 215 220 Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 225 230 235 240 Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 245 250 255 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 260 265 270 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 275 280 285 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 290 295 300 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 305 310 315 320 Gly Lys Gly Ser Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu 325 330 335 Trp His Glu <210> 3 <211> 305 <212> PRT <213> Macaca fascicularis <400> 3 Gln Ser Phe Glu Glu Asn Arg Lys Leu Asn Val Tyr Asn Gln Glu Asp 1 5 10 15 Gly Ser Val Leu Leu Thr Cys His Val Lys Asn Thr Asn Ile Thr Trp 20 25 30 Phe Lys Glu Gly Lys Met Ile Asp Ile Leu Thr Ala His Lys Asn Lys 35 40 45 Trp Asn Leu Gly Ser Asn Thr Lys Asp Pro Arg Gly Val Tyr Gln Cys 50 55 60 Lys Gly Ser Lys Asp Lys Ser Lys Thr Leu Gln Val Tyr Tyr Arg Met 65 70 75 80 Cys Gln Asn Cys Ile Glu Leu Asn Ala Pro Glu Ala Ala Gly Gly Pro 85 90 95 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 100 105 110 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 115 120 125 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 130 135 140 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 145 150 155 160 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 165 170 175 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ser Ile Glu Lys 180 185 190 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr 195 200 205 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser 210 215 220 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 225 230 235 240 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 245 250 255 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys 260 265 270 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 275 280 285 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 Lys 305 <210> 4 <211> 330 <212> PRT <213> Macaca fascicularis <400> 4 Gln Asp Gly Asn Glu Glu Met Gly Ser Ile Thr Gln Thr Pro Tyr Gln 1 5 10 15 Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Ser Gln His Leu 20 25 30 Gly Ser Glu Ala Gln Trp Gln His Asn Gly Lys Asn Lys Glu Asp Ser 35 40 45 Gly Asp Arg Leu Phe Leu Pro Glu Phe Ser Glu Met Glu Gln Ser Gly 50 55 60 Tyr Tyr Val Cys Tyr Pro Arg Gly Ser Asn Pro Glu Asp Ala Ser His 65 70 75 80 His Leu Tyr Leu Lys Ala Arg Val Cys Glu Asn Cys Met Glu Met Asp 85 90 95 Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 100 105 110 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 115 120 125 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 130 135 140 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 145 150 155 160 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 165 170 175 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 180 185 190 Ala Leu Pro Ala Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 195 200 205 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu Leu 210 215 220 Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro 225 230 235 240 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 245 250 255 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 260 265 270 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 275 280 285 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 290 295 300 Lys Ser Leu Ser Leu Ser Pro Gly Lys Gly Ser Gly Leu Asn Asp Ile 305 310 315 320 Phe Glu Ala Gln Lys Ile Glu Trp His Glu 325 330 <210> 5 <211> 109 <212> PRT <213> Homo sapiens <400> 5 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 6 <211> 327 <212> DNA <213> Homo sapiens <400> 6 caggctgtgg tcacacaaga gcctagcctg acagtgtctc ctggcggcac agtgaccctg 60 acctgtagat cttctacagg cgccgtgacc accagcaact acgctaattg ggtgcagcag 120 aagcccggcc aggctcctag aggactgatc ggcggaacaa acaagagagc cccttggaca 180 cccgccagat tctctggatc tctgctcggc ggaaaggccg ctctgacact ttctggtgct 240 cagcctgagg acgaggccga gtactattgt gccctgtggt acagcaacct gtgggtgttc 300 ggcggaggca ccaaactgac agttctg 327 <210> 7 <211> 14 <212> PRT <213> Homo sapiens <400> 7 Arg Ser Ser Thr Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 8 <211> 8 <212> PRT <213> Homo sapiens <400> 8 Gly Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 9 <211> 9 <212> PRT <213> Homo sapiens <400> 9 Ala Leu Trp Tyr Ser Asn Leu Trp Val 1 5 <210> 10 <211> 109 <212> PRT <213> Homo sapiens <400> 10 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 11 <211> 327 <212> DNA <213> Homo sapiens <400> 11 caggctgtgg tcacacaaga gcctagcctg acagtgtctc ctggcggcac agtgaccctg 60 acctgtagat cttctacagg cgccgtgacc accagcaact acgctaattg ggtgcaggag 120 aagcccggcc aggctcctag aggactgatc ggcggaacaa acaagagagc cccttggaca 180 cccgccagat tctctggatc tctgctcggc ggaaaggccg ctctgacact ttctggtgct 240 cagcctgagg acgaggccga gtactattgt gccctgtggt acagcaacct gtgggtgttc 300 ggcggaggca ccaaactgac agttctg 327 <210> 12 <211> 109 <212> PRT <213> Homo sapiens <400> 12 Glu Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Glu Ser Ser Asp Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Glu Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 13 <211> 327 <212> DNA <213> Homo sapiens <400> 13 gaggctgtgg tcacacaaga gcctagcctg acagtgtctc ctggcggcac agtgaccctg 60 acctgtgagt cttctgacgg cgccgtgacc accagcaact acgctaattg ggtgcaggag 120 aagcccggcc aggctcctag aggactgatc ggcggaacaa acaaggaggc cccttggaca 180 cccgccagat tctctggatc tctgctcggc ggaaaggccg ctctgacact ttctggtgct 240 cagcctgagg acgaggccga gtactattgt gccctgtggt acagcaacct gtgggtgttc 300 ggcggaggca ccaaactgac agttctg 327 <210> 14 <211> 14 <212> PRT <213> Homo sapiens <400> 14 Glu Ser Ser Asp Gly Ala Val Thr Thr Ser Asn Tyr Ala Asn 1 5 10 <210> 15 <211> 8 <212> PRT <213> Homo sapiens <400> 15 Gly Gly Thr Asn Lys Glu Ala Pro 1 5 <210> 16 <211> 109 <212> PRT <213> Homo sapiens <400> 16 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Glu Ser Ser Asp Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Glu Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Pro Glu Asp Glu Ala Glu Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 17 <211> 327 <212> DNA <213> Homo sapiens <400> 17 caggctgtgg tcacacaaga gcctagctg acagtgctc ctggcggcac agtgaccctg 60 acctgtgagt cttctgacgg cgccgtgacc accaccact acctattg ggtgcaggag 120 aagcccggcc aggctcctag aggactgatc ggcggacaaaaggaggc cccttggaca 180 cccgccagat tctctgatc tctgctcggc ggaaggccg ctctgacact tctgtgct 240 cagcctgagg acgaggccga gtactattgt gccctgtggt acagcaacct gtgggtgttc 300 ggcggaggca ccaaactgac agttctg 327 <210> 18 <211> 109 <212> PRT <213> Homo sapiens <400> 18 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 19 <211> 327 <212> DNA <213> Homo sapiens <400> 19 caggctgtgg tcacacaaga gcctagcctg acagtgtctc ctggcggcac agtgaccctg 60 acctgtagat cttctacagg cgccgtgacc accagcaact acgctaattg ggtgcaggag 120 aagcccggcc aggctcctag aggactgatc ggcggaacaa acaagagagc cccttggaca 180 cccgccagat tctctggatc tctgctcggc ggaaaggccg ctctgacaat cactggtgct 240 caggctgagg acgaggccga gtactattgt gtgctgtggt acagcaacct gtgggtgttc 300 ggcggaggca ccaaactgac agttctg 327 <210> 20 <211> 8 <212> PRT <213> Homo sapiens <400> 20 Tyr Gly Thr Asn Lys Arg Ala Pro 1 5 <210> 21 <211> 9 <212> PRT <213> Homo sapiens <400> 21 Val Leu Trp Tyr Ser Donkey Leu Trp Val 1 5 <210> 22 <211> 109 <212> PRT <213> Homo sapiens <400> 22 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Phe Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Tyr Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 23 <211> 327 <212> DNA <213> Homo sapiens <400> 23 caggctgtgg tcacacaaga gcctagcctg acagtgtctc ctggcggcac agtgaccctg 60 acctgtagat cttctacagg cgccgtgacc accaccact accctattg gttccaggag 120 aagcccggcc aggctcctag aggactgatc tacggacaaaagagagc cccttggaca 180 cccgccagat tctctgatc tctgctcggc ggaaggccg ctctgacact tctgtgct 240 caggctgagg acgaggccga gtactattgt gtcctgtggt acaccaacct gtgggtgttc 300 ggcggaggca ccaactgac agttctg 327 <210> 24 <211> 125 <212> PRT <213> Homo sapiens <400> 24 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 Asn Thr Tyr 20 25 30 Only Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Free Mp3 Download 85 90 95 Tyrant Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Free Mp3 Download 115 120 125 <210> 25 <211> 375 <212> DNA <213> Homo sapiens <400> 25 60. gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg tcttgtgccg ccagcggctt caccttcaac acctacgcta tgaactgggt ccgacaggcc 120 cctggcaaag gactggaatg ggtgtccaga atcaggtcca agtacaacaa ctacgccacc tactacgccg acagcgtga ggacagattc accatcagca gggacgacag caagacacc ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ctgtgtcaga cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 26 <211> 6 <212> PRT <213> Homo sapiens <400> 26 Asn Thr Tyr Ala Met Asn 1 5 <210> 27 <211> 18 <212> PRT <213> Homo sapiens <400> 27 Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Lys Asp <210> 28 <211> 14 <212> PRT <213> Homo sapiens <400> 28 His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 29 <211> 125 <212> PRT <213> Homo sapiens <400> 29 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 Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Tyr and Gln Met Asn Served by Arg and Glu Asp Thr and Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Only Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 30 <211> 375 <212> DNA <213> Homo sapiens <400> 30 gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60 tcttgtgccg ccagcggctt caccttctcc acctacgcta tgaactgggt ccgaaggcc 120 cctggcaaag gactggaatg gtgtccaga atcaggtcca agtacaacaa ctacgccacc 180 tactacgccg acagcgtga ggacagattc accatcagca gggacgacag caagacacc ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ctgtgtcaga cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 31 <211> 6 <212> PRT <213> Homo sapiens <400> 31 Ser Thr Tyr Ala With Asn 1 5 <210> 32 <211> 125 <212> PRT <213> Homo sapiens <400> 32 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 Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Light Tire Asn Asn Tire White Thr Tyre Tyre White Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 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 Val Arg His Gly Asn Phe Gly Glu Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 33 <211> 375 <212> DNA <213> Homo sapiens <400> 33 gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60 tcttgtgccg ccagcggctt caccttctcc acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgtccaga atcaggtcca agtacaacaa ctacgccacc 180 tactacgccg acagcgtgaa ggacagattc accatcagca gggacgacag caagaacacc 240 ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ctgtgtcaga 300 cacggcaact tcggcgagag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 34 <211> 14 <212> PRT <213> Homo sapiens <400> 34 His Gly Asn Phe Gly Glu Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 35 <211> 125 <212> PRT <213> Homo sapiens <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 Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Free Mp3 Download 85 90 95 Tyrant Cys Val Arg His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Free Mp3 Download 115 120 125 <210> 36 <211> 375 <212> DNA <213> Homo sapiens <400> 36 60. gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg tcttgtgccg ccagcggctt caccttctcc acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgtccaga atcaggtcca agtacaacaa ctacgccacc tactacgccg acagcgtga ggacagattc accatcagca gggacgacag caagacacc ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ctgtgtcaga cacggcaact tcggccagag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 37 <211> 14 <212> PRT <213> Homo sapiens <400> 37 His Gly Asn Phe Gly Gln Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 38 <211> 125 <212> PRT <213> Homo sapiens <400> 38 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 Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 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 Val Arg His Gly Asn Phe Gly Asp Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Free Mp3 Download 115 120 125 <210> 39 <211> 375 <212> DNA <213> Homo sapiens <400> 39 60. gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg tcttgtgccg ccagcggctt caccttctcc acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgtccaga atcaggtcca agtacaacaa ctacgccacc tactacgccg acagcgtga ggacagattc accatcagca gggacgacag caagacacc ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ctgtgtcaga 360. cacggcaact tcggcgacag ctatgtgtct tggtttgcct actggggcca gggcacactg gtcacagtta gctct 375 <210> 40 <211> 14 <212> PRT <213> Homo sapiens <400> 40 His Gly Asn Phe Gly Asp Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 41 <211> 125 <212> PRT <213> Homo sapiens <400> 41 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 Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 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 Val Arg His Gly Asn Phe Gly Thr Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 42 <211> 375 <212> DNA <213> Homo sapiens <400> 42 60. gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg tcttgtgccg ccagcggctt caccttctcc acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgtccaga atcaggtcca agtacaacaa ctacgccacc tactacgccg acagcgtga ggacagattc accatcagca gggacgacag caagacacc ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ctgtgtcaga 360. cacggcaact tcggcaccag ctatgtgtct tggtttgcct actggggcca gggcacactg gtcacagtta gctct 375 <210> 43 <211> 14 <212> PRT <213> Homo sapiens <400> 43 His Gly Asn Phe Gly Thr Ser Tyr Val Ser Trp Phe Ala Tyr 1 5 10 <210> 44 <211> 125 <212> PRT <213> Homo sapiens <400> 44 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 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Glu Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 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 Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 45 <211> 375 <212> DNA <213> Homo sapiens <400> 45 gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60 tcttgtgccg ccagcggctt caccttctcc gactacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgtccaga atcaggtcca agtacaacaa ctacgccacc tactacgccg acagcgtgga ggacagattc accatcagca gggacgacag caagaacacc ctgtacctgc agatgaacag cctgagagcc gaggacaccg ccgtgtacta ctgtgtcaga cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 46 <211> 6 <212> PRT <213> Homo sapiens <400> 46 Ser Asp Tyr Wing With Ass 1 5 <210> 47 <211> 18 <212> PRT <213> Homo sapiens <400> 47 Ile Arg Ser Light Tire Asn Asn Tire Ala Thr Tyr Tyr Ala Asp Ser Val 1 5 10 15 Glu Asp <210> 48 <211> 125 <212> PRT <213> Homo sapiens <400> 48 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 Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 49 <211> 375 <212> DNA <213> Homo sapiens <400> 49 60. gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg tcttgtgccg ccagcggctt caccttcaac acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgggaaga atcaggtcca agtacaacaa ctacgccacc tactacgccg acagcgtgaa ggacagattc accatcagca gggacgacag caagaacagc ctgtacctgc agatgaacag cctgaaaacc gaggacaccg ccgtgtacta ctgtgccaga cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 50 <211> 125 <212> PRT <213> Homo sapiens <400> 50 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 Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 51 <211> 375 <212> DNA <213> Homo sapiens <400> 51 gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60 tcttgtgccg ccagcggctt caccttcaac acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgggaaga atcaggtcca agtacaacaa ctacgccacc 180 tactacgccg acagcgtgaa ggacagattc accatcagca gggacgacag caagaacagc 240 ctgtacctgc agatgaacag cctgaaaacc gaggacaccg ccgtgtacta ctgtgtcaga 300 cacggcaact tcggcacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 52 <211> 125 <212> PRT <213> Homo sapiens <400> 52 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 Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Only Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Only Thr Tyr Tyr Only Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Tyr Leu Gln Met Asn Served Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Free Mp3 Download 115 120 125 <210> 53 <211> 375 <212> DNA <213> Homo sapiens <400> 53 60. gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg tcttgtgccg ccagcggctt caccttcaac acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtggccaga atcaggtcca agtacaacaa ctacgccacc tactacgccg acagcgtgaa ggacagattc accatcagca gggacgacag caagaacagc ctgtacctgc agatgaacag cctgaaaacc gaggacaccg ccgtgtacta ctgtgtcaga cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctct 375 <210> 54 <211> 214 <212> PRT <213> Homo sapiens <400> 54 Glu Ile Val Leu Thr Gln Ser Pro Gly 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 Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 55 <211> 642 <212> DNA <213> Homo sapiens <400> 55 gagatcgtgc tgacacagag ccctggcaca ctgtcactgt ctccaggcga gagagccaca 60 ctgagctgta gagccagcca gagcgtgtcc tcttacctgg cctggtatca gcagaagcct 120 ggacaggctc ccagactgct gatctacgac gccagcaaca gagccacagg catccccgat 180 agattcagcg gctctggctc tggcaccgac ttcaccctga caatcagcag actggaaccc 240 gaggacttcg ccgtgtacta ctgccagcag agaagcaact ggcccatcac attcggccag 300 ggcaccaagc tggaaatcaa gcgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaagtaca gtggaagttg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaag cagactacga gaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaa gagctcaac agggaggt gt 642 <210> 56 <211> 449 <212> PRT <213> Homo sapiens <400> 56 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Arg Asp Asn Serves 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 Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Ala Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr 340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser 355 360 365 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 Lys <210> 57 <211> 1347 <212> DNA <213> Homo sapiens <400> 57 gaagtgcagc tgctggaatc tggtggcgga gttgttcagc ctggcggctc tctgagactg 60 tcttgtgctg ccagcggctt caccttcaac gactacgcta tgcactgggt ccgacaggcc 120 cctggcaaag gacttgaatg ggtgtccacc atcagctgga acagcggctc tatcggctac 180 gccgattccg tgaagggcag attcaccatc tccagagaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag agccgaggac accgccgtgt actactgtgc caaggacatc 300 cagtacggca actactacta cggcatggac tactggggcc agggaacact ggttaccgtt 360 agctctgcta gcaccaaggg ccccagcgtg ttccccctgg ccccttgcag cagaagcacc 420 agcgagagca cagccgccct gggctgcctg gtgaaggact acttccccga gcccgtgacc 480 gtgtcctgga acagcggcgc tctgaccagc ggcgtgcata ccttccccgc cgtgctccag 540 agcagcggac tgtactccct gagcagcgtg gtgaccgtgc cttccagcag cctgggcacc 600 aagacctaca cctgcaacgt ggaccacaag cccagcaaca ccaaggtgga caagagagtg 660 gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcga gggcggacct 720 agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag aacccccgag 780 gtgacctgcg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt taattggtac 840 gtggacggcg tggaagtgca taacgccaag accaagccca gagaggagca gttcaacagc 900 acctacagag tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa 960 tacaagtgca aggtctccaa caagggcctg gccagcagca tcgagaagac catcagcaag 1020 gccaagggcc agccacggga gccccaggtc tgcaccctgc cacctagcca agaggagatg 1080 accaagaacc aggtgtccct gagctgtgcc gtgaaaggct tctatcccag cgatatcgcc 1140 gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 1200 gacagcgacg gcagcttctt cctggtttcc aagctgaccg tggacaagtc cagatggcag 1260 gagggcaacg tcttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1320 aagtccctga gcctgagcct gggcaag 1347 <210> 58 <211> 215 <212> PRT <213> Homo sapiens <400> 58 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Gln Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115 120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr Glu Cys Ser 210 215 <210> 59 <211> 645 <212> DNA <213> Homo sapiens <400> 59 caggctgtgg tcacacaaga gcctagcctg acagtgtctc ctggcggcac agtgaccctg 60 acctgtagat cttctacagg cgccgtgacc accagcaact acgctaattg ggtgcagcag 120 aagcccggcc aggctcctag aggactgatc ggcggaacaa acaagagagc cccttggaca 180 cccgccagat tctctggatc tctgctcggc ggaaaggccg ctctgacaat cactggtgct 240 caggctgagg acgaggccga gtactattgt gtgctgtggt acagcaacct gtgggtgttc 300 ggcggaggca ccaaactgac agttctgggt cagcccaagg cggcgccctc ggtcactctg 360 ttcccgccct cctctgagga gcttcaagcc aacaaggcca cactggtgtg tctcataagt 420 gacttctatc cgggagccgt gacagtggcc tggaaggcag atagcagccc cgtcaaggcg 480 ggagtggaga ccaccacacc ctccaaacaa agcaacaaca agtacgcggc cagcagctac 540 ctgagcctga cgcctgagca gtggaagtcc cacagaagct acagctgcca ggtcacgcat 600 gaagggagca ccgtggagaa gacagtggcc cctacagaat gttca 645 <210> 60 <211> 452 <212> PRT <213> Homo sapiens <400> 60 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 Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Ala Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Leu Gly Lys 450 <210> 61 <211> 1356 <212> DNA <213> Homo sapiens <400> 61 60. gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg tcttgtgccg ccagcggctt caccttcaac acctacgcta tgaactgggt ccgacaggcc 120 cctggcaaag gactggaatg ggtgggaaga atcaggtcca agtacaacaa ctacgccacc tactacgccg acagcgtgaa ggacagattc accatcagca gggacgacag caagaacagc ctgtacctgc agatgaacag cctgaaaacc gaggacaccg ccgtgtacta ctgtgtcaga cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctctgctag caccaagggc cccagcgtgt tccccctggc cccttgcagc 420 agaagcacca gcgagagcac agccgccctg ggctgcctgg tgaaggacta cttccccgag 480 cccgtgaccg tgtcctggaa cagcggcgct ctgaccagcg gcgtgcatac cttccccgcc 540 gtgctccaga gcagcggact gtactccctg agcagcgtgg tgaccgtgcc ttccagcagc 600 ctgggcacca agacctacac ctgcaacgtg gaccacaagc ccagcaacc caaggtggac aagagagtgg agagcaagta cggccctccc tgcccccctt gccctgcccc cgagttcgag 720 ggcggaccta gcgtgttcct gttccccccc aagcccaagg acaccctgat gatcagcaga 780 acccccgagg tgacctgcgt ggtggtggac gtgtcccagg aggaccccga ggtccagttt 840 aattggtacg tggacggcgt ggaagtgcat aacgccaaga ccaagcccag agaggagcag 900 ttcaacagca cctacagagt ggtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 960 ggcaaggaat acaagtgcaa ggtctccaac aagggcctgg ccagcagcat cgagaagacc 1020 atcagcaagg ccaagggcca gccacgggag ccccaggtct acaccctgcc accttgtcaa 1080 gaggagatga ccaagaacca ggtgtccctg tggtgtctgg tgaaaggctt ctatcccagc 1140 gatatcgccg tggagtggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 1200 cctgtgctgg acagcgacgg cagcttcttc ctgtactcca agctgaccgt ggacaagtcc 1260 agatggcagg agggcaacgt cttcagctgc tccgtgatgc acgaggccct gcacaaccac 1320 tacacccaga agtccctgag cctgagcctg ggcaag 1356 <210> 62 <211> 214 <212> PRT <213> Homo sapiens <400> 62 Glu Ile Val Leu Thr Gln Ser Pro Gly 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 Tyr 20 25 30 Leu Ala Trp Tyr Gln Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 63 <211> 642 <212> DNA <213> Homo sapiens <400> 63 gagatcgtgc tgacacagag ccctggcaca ctgtcactgt ctccaggcga gagagccaca 60 ctgagctgta gagccagcca gagcgtgtcc tcttacctgg cctggtatca gaagaagcct 120 ggacaggctc ccagactgct gattacgac gccagcaca gagccacagg catccccgat 180 agattcagcg gctctggctc tggcaccgac ttcaccctga caatcagcag actggaaccc 240 gaggacttcg ccgtgtacta ctgccagcag agaagcaact ggcccatcac attcggccag 300 ggcaccaagc tggaaatcaa gcgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 ccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540 ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642 <210> 64 <211> 449 <212> PRT <213> Homo sapiens <400> 64 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr 20 25 30 Ala Met His Trp Val Arg Glu Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly 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 Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Ala Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr 340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser 355 360 365 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Serving Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Only Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 Lys <210> 65 <211> 1347 <212> DNA <213> Homo sapiens <400> 65 gaagtgcagc tgctggaatc tggtggcgga gttgttcagc ctggcggctc tctgagactg 60 tcttgtgctg ccagcggctt caccttcac gactacgcta tgcactggt ccgagaggcc 120 cctggcaaag gacttgaatg gtgtccacc atcagctgga acagcggctc tatcggctac 180 gccgattccg tgaagggcag attcaccatc tccagagaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag agccgaggac accgccgtgt actactgtgc caaggacatc 300 footcggca actactacta cggcatggac tactggggcc agggaacact ggttaccgtt 360 agctctgcta gcaccaaggg ccccagcgtg ttccccctgg ccccttgcag cagaagcacc 420 agcgagagca cagccgccct gggctgcctg gtgaaggact acttccccga gcccgtgacc 480 gtgtcctgga acagcggcgc tctgaccagc ggcgtgcata ccttccccgc cgtgctccag 540 agcagcggac tgtactccct gagcagcgtg gtgaccgtgc cttccagcag cctgggcacc 600 aagacctaca cctgcaacgt ggaccacaag cccagcaaca ccaaggtgga caagagagtg 660 gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcga gggcggacct 720 agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag aacccccgag 780 gtgacctgcg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt taattggtac 840 gtggacggcg tggaagtgca taacgccaag accaagccca gagaggagca gttcaacagc 900 acctacagag tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa 960 tacaagtgca aggtctccaa caagggcctg gccagcagca tcgagaagac catcagcaag 1020 gccaagggcc agccacggga gcccccaggtc tgcaccctgc cacctagcca agaggagatg 1080 accaagaacc aggtgtccct gagctgtgcc gtgaaaggct tctatcccag cgatatcgcc 1140 gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 1200 gacagcgacg gcagcttctt cctggtttcc aagctgaccg tggacaagtc cagatggcag 1260 gagggcaacg tcttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1320 aagtccctga gcctgagcct gggcaag 1347 <210> 66 <211> 215 <212> PRT <213> Homo sapiens <400> 66 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115 120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr Glu Cys Ser 210 215 <210> 67 <211> 645 <212> DNA <213> Homo sapiens <400> 67 caggctgtgg tcacacaaga gcctagcctg acagtgtctc ctggcggcac agtgaccctg 60 acctgtagat cttctacagg cgccgtgacc accagcaact acgctaattg ggtgcaggag 120 aagcccggcc aggctcctag aggactgatc ggcggaacaa acaagagagc cccttggaca 180 cccgccagat tctctggatc tctgctcggc ggaaaggccg ctctgacaat cactggtgct 240 caggctgagg acgaggccga gtactattgt gtgctgtggt acagcaacct gtgggtgttc 300 ggcggaggca ccaaactgac agttctgggt cagcccaagg cggcgccctc ggtcactctg 360 ttccgccct cctctgagga gcttcaagcc aacaaggcca cactggtgtg tctcataagt 420 gacttctatc cgggagccgt gacagtggcc tggaaggcag atagcagccc cgtcaaggcg 480 ggagtggaga caccacacc ctccaaaaa agcacaaca agtacgcggc cagcagctac 540 ctgagcctga cgcctgagca gtggaagtcc cacagaagct acagctgcca ggtcacgcat 600 gaagggagca ccgtggagaa gaagtggcc cctacagaat gttca 645 <210> 68 <211> 452 <212> PRT <213> Homo sapiens <400> 68 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 Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg With Arg Ser Tyr Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Tyr Leu Gln Met Asn Served Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu 210 215 220 Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser 260 265 270 Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr 290 295 300 Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310 315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Ala Ser Ser 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys Asn Gln Val 355 360 365 Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val 420 425 430 Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435 440 445 Ser Leu Gly Lys 450 <210> 69 <211> 1356 <212> DNA <213> Homo sapiens <400> 69 gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60 tcttgtgccg ccagcggctt caccttcaac acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgggaaga atcaggtcca agtacaacaa ctacgccacc 180 tactacgccg acagcgtgaa ggacagattc accatcagca gggacgacag caagaacagc 240 ctgtacctgc agatgaacag cctgaaaacc gaggacaccg ccgtgtacta ctgtgtcaga 300 cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctctgctag caccaagggc cccagcgtgt tccccctggc cccttgcagc 420 agaagcacca gcgagagcac agccgccctg ggctgcctgg tgaaggacta cttccccgag 480 cccgtgaccg tgtcctggaa cagcggcgct ctgaccagcg gcgtgcatac cttccccgcc 540 gtgctccaga gcagcggact gtactccctg agcagcgtgg tgaccgtgcc ttccagcagc 600 ctgggcacca agacctacac ctgcaacgtg gaccacaagc ccaggcaccac caggtggac 660 aagagagtgg agcaagta cggccctccc tgccccctt gccctgcccc cgagttcgag 720 ggcggaccta gcgtgttcct gttcccccc aagcccagg acacctgat gatcagcaga 780 acccccgagg tgacctgcgt ggtgtggac gtgtcccagg aggaccccga ggtccagttt 840 aattggtacg tggacgggt ggaagtgcat aacgccaga ccaagcccag agaggagcag 900 ttcacagca cctacagagt gtgtccgtg ctgaccgtgc tgcaccagga ctggctgaac 960 ggcaggaat acagtgcaa ggtctccaac aagggcctgg ccagcagcat cgagaagacc 1020 atcagcagg ccaagggcca gccacgggag ccccaggtct acaccctgcc accttgtcaa 1080 gaggagatga ccaagaacca gtgtccctg tggtgtctgg tgaaaggctt ctacccagc 1140 gatatcgccg tggagtggga gagcaacggc cagcccgaga acaactacaa gaccaccccc 1200 cctgtgctgg acagcgacgg cagcttcttc ctgtactcca agctgaccgt ggacaagtcc 1260 agatggcagg agggcaacgt cttcagctgc tccgtgatgc acgaggccct gcacaaccac 1320 taacacccaga agtccctgag cctgagcctg ggcaag 1356 <210> 70 <211> 214 <212> PRT <213> Homo sapiens <400> 70 Glu Ile Val Leu Thr Gln Ser Pro Gly 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 Tyr 20 25 30 Leu Ala Trp Tyr Gln Lys Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Lys Lys Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 71 <211> 642 <212> DNA <213> Homo sapiens <400> 71 gagatcgtgc tgacacagag ccctggcaca ctgtcactgt ctccaggcga gagagccaca 60 ctgagctgta gagccagcca gagcgtgtcc tcttacctgg cctggtatca gaagaagcct 120 ggacaggctc ccagactgct gatctacgac gccagcaaca gagccacagg catccccgat 180 agattcagcg gctctggctc tggcaccgac ttcaccctga caatcagcag actggaaccc 240 gaggacttcg ccgtgtacta ctgccagcag agaagcaact ggcccatcac attcggccag 300 ggcaccaagc tggaaatcaa gcgaactgtg gctgcaccat ctgtcttcat cttcccgcca 360 tctgataaga aattgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420 cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480 gagagtgtca cagagcag cagcaggac agcacctaca gcctcagcag caccctgacg 540 600 ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642 <210> 72 <211> 449 <212> PRT <213> Homo sapiens <400> 72 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr 20 25 30 Ala Met His Trp Val Arg Glu Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly 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 Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Glu Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Ala Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr 340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser 355 360 365 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 Lys <210> 73 <211> 1347 <212> DNA <213> Homo sapiens <400> 73 gaagtgcagc tgctgggaatc tggtggcgga gttgttcagc ctggcggctc tctgagactg 60 tcttgtgctg ccagcggctt caccttcaac gactacgcta tgcactgggt ccgagaggcc 120 cctggcaaag gacttgaatg ggtgtccacc atcagctgga acagcggctc tatcggctac 180 gccgattccg tgaagggcag attcaccatc tccagagaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag agccgaggac accgccgtgt actactgtgc caaggacatc 300 footcggca actactacta cggcatggac tactggggcc agggaacact ggttaccgtt 360 agctctgcta gcaccaaggg ccccagcgtg ttccccctgg ccccttgcag cagaagcacc 420 agcgagagca cagccgccct gggctgcctg gtggaggact acttccccga gcccgtgacc 480 gtgtcctgga acagcggcgc tctgaccagc ggcgtgcata ccttccccgc cgtgctccag 540 agcagcggac tgtactccct gagcagcgtg gtgaccgtgc cttccagcag cctgggcacc 600 aagacctaca cctgcaacgt ggaccacaag cccagcaaca ccaaggtgga cgagagagtg 660 gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcga aggcggacct 720 agcgtgttcc tgttcccccc caagcccaag gaccctga tgatcagcag aacccccgag 780 gtgacctgcg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt taattggtac 840 gtggacggcg tggaagtgca taacgccaag accaagcccca gagaggagca gttcaacagc 900 acctacagag tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaggaa 960 tacaagtgca aggtctccaa caagggcctg gccagcagca tcgagaagac catcagcaag 1020 gccaagggcc agccacggga gccccaggtc tgcaccctgc cacctagcca agaggagatg 1080 accagaacc aggtgtccct gagctgtgcc gtgaaaggct tctatcccag cgatatcgcc 1140 gtggagtggg agacaacgg ccagccgag aacaactaca agaccacccc ccctgtgctg 1200 gagagcgacg gcagctctt cctggtttcc aagctgaccg tggacaagtc cagatggcag 1260 gagggcaacg tcttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1320 aagtccctga gcctgagcct gggcaag 1347 <210> 74 <211> 449 <212> PRT <213> Homo sapiens <400> 74 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asp Tyr 20 25 30 Ala Met His Trp Val Arg Glu Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Thr Ile Ser Trp Asn Ser Gly Ser Ile Gly 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 Lys Asp Ile Gln Tyr Gly Asn Tyr Tyr Tyr Gly Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Glu Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Glu Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr 340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser 355 360 365 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 Lys <210> 75 <211> 1347 <212> DNA <213> Homo sapiens <400> 75 gaagtgcagc tgctgggaatc tggtggcgga gttgttcagc ctggcggctc tctgagactg 60 tcttgtgctg ccagcggctt caccttcaac gactacgcta tgcactgggt ccgagaggcc 120 cctggcaaag gacttgaatg ggtgtccacc atcagctgga acagcggctc tatcggctac 180 gccgattccg tgaagggcag attcaccatc tccagagaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag agccgaggac accgccgtgt actactgtgc caaggacatc 300 footcggca actactacta cggcatggac tactggggcc agggaacact ggttaccgtt 360 agctctgcta gcaccaaggg ccccagcgtg ttccccctgg ccccttgcag cagaagcacc 420 agcgagagca cagccgccct gggctgcctg gtggaggact acttccccga gcccgtgacc 480 gtgtcctgga acagcggcgc tctgaccagc ggcgtgcata ccttccccgc cgtgctccag 540 agcagcggac tgtactccct gagcagcgtg gtgaccgtgc cttccagcag cctgggcacc 600 aagacctaca cctgcaacgt ggaccacaag cccagcaaca ccaaggtgga cgagagagtg 660 gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcga aggcggacct 720 agcgtgttcc tgttcccccc caagcccaag gaccctga tgatcagcag aacccccgag 780 gtgacctgcg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt taattggtac 840 gtggacggcg tggaagtgca taacgccaag accaagcccca gagaggagca gttcgccagc 900 acctacagag tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaggaa 960 tacaagtgca aggtctccaa caagggcctg cctagcagca tcgagaagac catcagcaag 1020 gccaagggcc agccacggga gccccaggtc tgcaccctgc cacctagcca agaggagatg 1080 accagaacc aggtgtccct gagctgtgcc gtgaaaggct tctatcccag cgatatcgcc 1140 gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 1200 gagagcgacg gcagctctt cctggtttcc aagctgaccg tggacaagtc cagatggcag 1260 gagggcaacg tcttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1320 aagtccctga gcctgagcct gggcaag 1347 <210> 76 <211> 223 <212> PRT <213> Homo sapiens <400> 76 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 Asn Thr Tyr 20 25 30 Ala Met Asn Trp Val Arg Lys Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110 Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys 195 200 205 Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val 210 215 220 <210> 77 <211> 669 <212> DNA <213> Homo sapiens <400> 77 gaggtgcagc tggttgaatc tggcggagga ctggttcagc ctggcggatc tctgagactg 60 tcttgtgccg ccagcggctt caccttcaac acctacgcta tgaactgggt ccgaaaggcc 120 cctggcaaag gactggaatg ggtgggaaga atcaggtcca agtacaacaa ctacgccacc 180 tactacgccg acagcgtgaa ggacagattc accatcagca gggacgacag caagaacagc 240 ctgtacctgc agatgaacag cctgaaaacc gaggacaccg ccgtgtacta ctgtgtcaga 300 cacggcaact tcggcaacag ctatgtgtct tggtttgcct actggggcca gggcacactg 360 gtcacagtta gctctgctag caccaagggc cccagcgtgt tccccctggc cccttgcagc 420 agaagcacca gcgagagcac agccgccctg ggctgcctgg tgaaggacta cttccccgag 480 cccgtgaccg tgtcctggaa cagcggcgct ctgaccagcg gcgtgcatac cttccccgcc 540 gtgctccaga gcagcggact gtactccctg agcagcgtgg tgaccgtgcc ttccagcagc 600 ctgggcacca agacctacac ctgcaacgtg gaccacaagc ccagcaacac caaggtggac 660 aagagagtg 669 <210> 78 <211> 444 <212> PRT <213> Homo sapiens <400> 78 Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly 35 40 45 Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe 50 55 60 Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln Ala Glu Asp Glu Ala Glu Tyr Tyr Cys Val Leu Trp Tyr Ser Asn 85 90 95 Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro 100 105 110 Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu 115 120 125 Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro 130 135 140 Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala 145 150 155 160 Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala 165 170 175 Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg 180 185 190 Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr 195 200 205 Val Ala Pro Thr Glu Cys Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro 210 215 220 Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 245 250 255 Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe 260 265 270 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285 Arg Glu Glu Gln Phe Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320 Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala 325 330 335 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln 340 345 350 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly 355 360 365 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 370 375 380 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400 Phe Phe Leu Tyr Ser Ser Leu Thr Val Asp Lys Ser Arg Trp Gln Glu 405 410 415 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435,440 <210> 79 <211> 1332 <212> DNA <213> Homo sapiens <400> 79 caggctgtgg tcacacaaga gcctagctg acagtgctc ctggcggcac agtgaccctg 60 acctgtagat cttctacagg cgccgtgacc accaccact acgctattg ggtgcaggag 120 aagcccggcc aggctcctag aggactgatc ggcggacaaaagagagc cccttggaca 180 cccgccagat tctctggatc tctgctcggc ggaaggccg ctctgacaat cactggtgct 240 caggctgagg acgaggccga gtactattgt gtgctgtggt acagcaacct gtgggtgttc 300 ggcggaggca ccaaactgac agttctgggt cagcccaagg cggcgccctc ggtcactctg 360 ttcccgccct cctctgagga gcttcaagcc aacaaggcca cactggtgtg tctcataagt 420 gacttctatc cgggagccgt gacagtggcc tggaaggcag atagcagccc cgtcaaggcg 480 ggagtggaga ccaccacacc ctccaaacaa agcaacaaca agtacgcggc cagcagctac 540 ctgagcctga cgcctgagca gtggaagtcc cacagaagct acagctgcca ggtcacgcat 600 gaagggagca ccgtggagaa gacagtggcc cctacagaat gttcagagag caagtacggc 660 cctccctgcc ccccttgccc tgcccccgag ttcgagggcg gacctagcgt gttcctgttc 720 ccccccaagc ccaaggacac cctgatgatc agcagaaccc ccgaggtgac ctgcgtggtg 780 gtggacgtgt cccaggagga ccccgaggtc cagtttaatt ggtacgtgga cggcgtggaa 840 gtgcataacg ccaagaccaa gcccagagag gagcagttcg ccagcaccta cagagtggtg 900 tccgtgctga ccgtgctgca ccaggactgg ctgaacggca aggaatacaa gtgcaaggtc 960 tccaacaagg gcctgcctag cagcatcgag aagaccatca gcaaggccaa gggccagcca 1020 cgggagcccc aggtctacac cctgccacct tgtcaagagg agatgaccaa gaaccaggtg 1080 tccctgtggt gtctggtgaa aggcttctat cccagcgata tcgccgtgga gtgggagagc 1140 aacggccagc ccgagaacaa ctacaagacc accccccctg tgctggacag cgacggcagc 1200 ttcttcctgt actccaagct gaccgtggac aagtccagat ggcaggaggg caacgtcttc 1260 agctgctccg tgatgcacga ggccctgcac aaccactaca cccagaagtc cctgagcctg 1320 agcctgggca ag 1332 <210> 80 <211> 218 <212> PRT <213> Homo sapiens <400> 80 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Met His Trp Tyr Gln Lys Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 81 <211> 654 <212> DNA <213> Homo sapiens <400> 81 60. gacatcgtgc tgacacagag ccctgcttct ctggctgtgt ctcctggcca gagagccacc atcacctgta gagccagcaa gagcgtgtcc accagcggct actcttacat gcactggtat 120 cagaagagc ccggccagcc tcctaagctg ctgatctacc tggctagcaa cctcgaaagc 240. ctagtgcctg ctagtttc tggcagcggc tctggcaccg acttcaccct gacaatcaac 300. cccgtggaag ccgaagacac cgccaactac tactgccagc acagcagaga gctgccctgg acatttggcc agggcaccaa ggtggaaatc aagcgaactg tggctgcacc atctgtcttc atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420 aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg ggtaactccc aggagagtgt cacagagcag cacagcaagg acagcaccta cagcctcagc 540 agcaccctga cgctgagcaa agcagactac gagaaacaca aagtctacgc ctgcgaagtc 600 acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt 654 <210> 82 <211> 449 <212> PRT <213> Homo sapiens <400> 82 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asn Phe 20 25 30 Gly Met Asn Trp Val Arg Glu Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Gln Ile Tyr Ala Asp Gly Phe 50 55 60 Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Glu Ile Tyr Tyr Gly Tyr Asp Val Gly Phe Val Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Ala Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr 340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser 355 360 365 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 Lys <210> 83 <211> 1347 <212> DNA <213> Homo sapiens <400> 83 caggttcagc tggtgcagtc tggcagcgag ctgaagaaac ctggcgcctc tgtgaaggtg 60 tcctgcaagg ctagcggcta catcttcacc aacttcggca tgaactgggt ccgagaggct 120 cctggacagg gactcgaatg gatgggctgg atcaacacct acaccggcga gcagatctac 180 gccgatggct tcacaggcag attcgtgttc agcctggaca ccagcgccag cacagcttac 240 ctgcagatca gctctctgaa ggccgaggat accgccgtgt acttctgtgc cagaggcgag 300 atctactacg gctacgacgt gggctttgtg tactggggcc agggaacact ggtcaccgtt 360 agctctgcta gcaccaaggg ccccagcgtg ttccccctgg ccccttgcag cagaagcacc 420 agcgagagca cagccgccct gggctgcctg gtgaaggact acttccccga gcccgtgacc 480 gtgtcctgga acagcggcgc tctgaccagc ggcgtgcata ccttccccgc cgtgctccag 540 agcagcggac tgtactccct gagcagcgtg gtgaccgtgc cttccagcag cctgggcacc 600 aagacctaca cctgcaacgt ggaccacaag cccagcaaca ccaaggtgga caagagagtg 660 gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcga gggcggacct 720 agcgtgttcc tgttcccccc caagcccaag gacaccctga tgatcagcag aacccccgag 780 gtgacctgcg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt taattggtac 840 gtggacggcg tggaagtgca taacgccaag accaagccca gagaggagca gttcaacagc 900 acctacagag tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggaa 960 tacaagtgca aggtctccaa caagggcctg gccagcagca tcgagaagac catcagcaag 1020 gccaagggcc agccacggga gccccaggtc tgcaccctgc cacctagcca agaggagatg 1080 accaagaacc aggtgtccct gagctgtgcc gtgaaaggct tctatcccag cgatatcgcc 1140 gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 1200 gagagcgacg gcagctctt cctggtttcc aagctgaccg tggacaagtc cagatggcag 1260 gagggcaacg tcttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1320 aagtccctga gcctgagcct gggcaag 1347 <210> job <211> 218 <212> PRT <213> Homo sapiens <400> job Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg With Thr Ile Thr Cys Arg With Lys Ser Val Val Thr Thr Ser 20 25 30 Gly Tyr Ser Tyr Ile His Trp Tyr Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Wing Ser Asp Leu Glu Wing Gly Val Pro Wing 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Glu Asp Thr Ala Asn Tyr Tyr Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 85 <211> 654 <212> DNA <213> Homo sapiens <400> 85 gacatcgtgc tgacacagag ccctgcttct ctggctgtgt ctcctggcca gagagccacc 60 atcacctgta gagccagcaa gagcgtgacc accagcggct actcttacat ccactggtat 180. cagaagagc ccggccagcc tcctaagctg ctgatctacc tggccagcga tctggaagct 240. ggcgtgccag ctagatttc tggcagcggc tctggcaccg acttcaccct gacaatcaac 300. cccgtggaag ccgaagacac cgccaactac tactgccagc acagcagaga gctgccctgg acatttggcc agggcaccaa ggtggaaatc aagcgaactg tggctgcacc atctgtcttc atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420 aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg ggtaactccc aggagagtgt cacagagcag cacagcaagg acagcaccta cagcctcagc 540 agcaccctga cgctgagcaa agcacctc gagaaacaca aagtctacgc ctgcgaagtc acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt 654 <210> 86 <211> 449 <212> PRT <213> Homo sapiens <400> 86 Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asn Phe 20 25 30 Gly Met Asn Trp Val Arg Glu Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Gln Ile Tyr Ala Asp Gly Phe 50 55 60 Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Gly Glu Ile Tyr Tyr Gly Tyr Asp Val Gly Phe Val Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Glu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Ala Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr 340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser 355 360 365 Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 Lys <210> 87 <211> 1347 <212> DNA <213> Homo sapiens <400> 87 caggttcagc tggtgcagtc tggcagcgag ctgaagaaac ctggcgcctc tgtgaaggtg 60 tcctgcaagg ctagcggcta catcttcacc aacttcggca tgaactgggt ccgagaggct 120 cctggacagg gactcgaatg gatgggctgg atcaacacct acaccggcga gcagatctac 180 gccgatggct tcacaggcag attcgtgttc agcctggaca ccagcgtcag cacagcttac 240 ctgcagatca gctctctgaa ggccgaggat accgccgtgt acttctgtgc cagaggcgag 300 atctactacg gctacgacgt gggctttgtg tactggggcc agggaacact ggtcaccgtt 360 agctctgcta gcaccaaggg ccccagcgtg ttccccctgg ccccttgcag cagaagcacc 420 agcgagagca cagccgccct gggctgcctg gtgaaggact acttccccga gcccgtgacc 480 gtgtcctgga acagcggcgc tctgaccagc ggcgtgcata ccttccccgc cgtgctccag 540 agcagcggac tgtactccct gagcagcgtg gtgaccgtgc cttccagcag cctgggcacc 600 aagacctaca cctgcaacgt ggaccacaag cccagcaaca ccaaggtgga caagagagtg 660 gagagcaagt acggccctcc ctgcccccct tgccctgccc ccgagttcga gggcggacct 720 agcgtgttcc tgttcccccc caagcccaag gaccctga tgatcagcag aacccccgag 780 gtgacctgcg tggtggtgga cgtgtcccag gaggaccccg aggtccagtt taattggtac 840 gtggacggcg tggaagtgca taacgccaag accaagcccca gagaggagca gttcaacagc 900 acctacagag tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaggaa 960 tacaagtgca aggtctccaa caagggcctg gccagcagca tcgagaagac catcagcaag 1020 gccaagggcc agccacggga gccccaggtc tgcaccctgc cacctagcca agaggagatg 1080 accagaacc aggtgtccct gagctgtgcc gtgaaaggct tctatcccag cgatatcgcc 1140 gtggagtggg agagcaacgg ccagcccgag aacaactaca agaccacccc ccctgtgctg 1200 gagagcgacg gcagctctt cctggtttcc aagctgaccg tggacaagtc cagatggcag 1260 gagggcaacg tcttcagctg ctccgtgatg cacgaggccc tgcacaacca ctacacccag 1320 aagtccctga gcctgagcct gggcaag 1347 <210> 88 <211> 219 <212> PRT <213> Homo sapiens <400> 88 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Lys Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Leu Gln Val 85 90 95 Thr His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 89 <211> 657 <212> DNA <213> Homo sapiens <400> 89 gacgtggtca tgacacagag ccctctgagc ctgcctgtga cacctggcga acctgccagc 60 atcagctgta gaagcagcca gagcatcgtg cacagcaacg gcaacacata cctggagtgg 120 tatctgaaga agcccggcca gtctcctcag ctgctgatct acaaggtgtc caacagattc 180 agcggcgtgc ccgacagatt ctctggctct ggatctggca ccgacttcac cctgaagatc 240 tccagagtgg aagccgagga cgtgggcgtg tacttctgtc tccaggtcac acacgtgccc 300 ctgacatttg gccagggcac caagctggaa atcaagcgaa ctgtggctgc accatctgtc 360 ttcatcttcc cgccatctga tgagcagttg aaatctggaa ctgcctctgt tgtgtgcctg 420 ctgaataact tctatcccag agaggccaaa gtacagtgga aggtggataa cgccctccaa 480 tcgggtaact cccaggagag tgtcacagag caggacagca aggacagcac ctacagcctc 540 agcagcaccc tgacgctgag caaagcagac tacgagaaac acaaagtcta cgcctgcgaa 600 gtcacccatc agggcctgag ctcgcccgtc acaaagagct tcaacagggg agagtgt 657 <210> 90 <211> 442 <212> PRT <213> Homo sapiens <400> 90 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ala Asp Tyr 20 25 30 Glu Ile His Trp Val Arg Glu Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Ile His Pro Gly Ser Gly Gly Thr Ala Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Tyr Tyr Ser Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys 210 215 220 Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 225 230 235 240 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 245 250 255 Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp 260 265 270 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 275 280 285 Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 290 295 300 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 305 310 315 320 Lys Gly Leu Ala Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 325 330 335 Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu 340 345 350 Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gl...

Claims

1. A bispecific antibody or its antigen-binding fragment, comprising: (a) A first antigen-binding moiety or an antigen-binding fragment thereof, the first antigen-binding moiety comprising a first light chain and a first heavy chain, the first light chain being a κ-type light chain, the first antigen-binding moiety comprising a first binding domain for binding to a first antigen; and (b) A second antigen-binding moiety or an antigen-binding fragment thereof, wherein the second antigen-binding moiety comprises a second light chain and a second heavy chain, the second light chain being a λ-type light chain, and the second antigen-binding moiety comprises a second binding domain that binds to the second antigen. The second antigen is the CD3 antigen; The second light chain CDR of the second binding domain is: The second light chain CDR1, consisting of the amino acid sequence SEQ ID NO: 7; The second light chain CDR2, consisting of the amino acid sequence SEQ ID NO: 8; The second light chain CDR3, consisting of the amino acid sequence SEQ ID NO: 21; The second heavy chain CDR of the second binding domain is: The second heavy chain CDR1, consisting of the amino acid sequence SEQ ID NO: 26; The second heavy chain CDR2, consisting of the amino acid sequence SEQ ID NO: 27; The second heavy chain CDR3 is composed of the amino acid sequence SEQ ID NO:

28.

2. The bispecific antibody or its antigen-binding fragment according to claim 1, wherein, The second light chain variable region of the second antigen-binding moiety has a Gln40Glu mutation (VλCD3: Gln40Glu); the second heavy chain variable region of the second antigen-binding moiety has a Gln39Lys mutation (VHCD3: Gln39Lys).

3. The bispecific antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The second binding domain comprises a second light chain variable region of amino acid sequence SEQ ID NO: 18 or any variant thereof; and a second heavy chain variable region of amino acid sequence SEQ ID NO: 50 or any variant thereof.

4. The bispecific antibody or its antigen-binding fragment according to claim 1, wherein, The second light chain of the second antigen-binding portion has the amino acid sequence SEQ ID NO: 58; and the second heavy chain of the second antigen-binding portion has the amino acid sequence SEQ ID NO: 60; or The second light chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO: 66; and the second heavy chain of the second antigen-binding portion has an amino acid sequence SEQ ID NO:

68.

5. The bispecific antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The first antigen is a tumor antigen.

6. The bispecific antibody or its antigen-binding fragment according to claim 5, wherein, The tumor antigens are selected from: CD19, CD20, CD22, CD30, CD38, CD72, CD180, CD171 (L1CAM), CD123, CD133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166, CD71, CLL-1 / CLECK12A, ROR1, BCMA, GPC3, mesothelin, CD33 / IL3Ra, c-Met, PSCA, PSMA, glycolipid F77, EGFRvIII, GD-2, MY-ESO-1, Her2, Her3, MUC1, MUC17, Claudin18, or MAGEA3.

7. The bispecific antibody or its antigen-binding fragment according to claim 5, wherein, The tumor antigen is CD20, BCMA, or GPC3.

8. The bispecific antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The Fc portion of the bispecific antibody has the Ser228Pro, Leu235Glu, and Pro329Ala mutations.

9. A nucleic acid encoding the bispecific antibody or its antigen-binding fragment according to any one of claims 1-8.

10. The nucleic acid according to claim 9, wherein, The nucleic acid encoding the second light chain of the second antigen-binding portion is the nucleotide sequence SEQ ID NO: 59, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is the nucleotide sequence SEQ ID NO: 61; or The nucleic acid encoding the second light chain of the second antigen-binding portion is a nucleotide sequence SEQ ID NO: 67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is a nucleotide sequence SEQ ID NO:

69.

11. A vector containing the nucleic acid of claim 9 or 10.

12. Cells containing the nucleic acid of claim 9 or 10 or the vector of claim 11.

13. A composition comprising the bispecific antibody or antigen-binding fragment thereof as described in any one of claims 1-8, the nucleic acid as described in claim 9 or 10, the vector as described in claim 11, and / or the cell as described in claim 12.

14. An antibody-drug conjugate comprising a bispecific antibody or an antigen-binding fragment thereof as described in any one of claims 1-8, covalently attached to a therapeutic portion.

15. The antibody-drug conjugate according to claim 14, wherein, The therapeutic component may be a cytotoxic component, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a cleaved peptide, or a radioactive isotope.

16. The antibody-drug conjugate according to claim 15, wherein, The cytotoxic component is selected from the following: Paclitaxel; Cytochalasin B; Bacitracin D; Ethidium bromide; Emetidine; Mitomycin; Etoposide; Teniposide; Vincristine; Colchicine; Doxorubicin; Daunorubicin; Dihydroxyanthradinone; Maytansin; Antimitotic agents; Saccharoside 10 or 15; Irinotecan; Mitoxanthraquinone; Glucocorticoids; Actinomycin D; 1-Dehydrotestosterone; Glucocorticoids; Procaine; Tetracaine; Lidocaine; Propranolol; Puromycin; Carbazin; Antimetabolites; Dichloromethyldiethylamine, thiopurine, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine; Platinum derivatives; Docamycin A, Docamycin SA, Rechemycin (CC-1065); Actinomycin, bleomycin, idarubicin, chloromycin, mitoxantrone, proprammycin, benzomycin (AMC); pyrrolo[2,1-c][1,4]-benzodiazepine (PDB); Diphtheria toxin and related molecules, ricin, cholera toxin, shiga-like toxin, pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas exotoxin, aloin, saponins, saccharin, gellingin, absinthecin A chain, saccharin A chain, α-sarcin, PAPI, PAPII, PAP-S laxative, croton toxin, sapaonaria officinalis inhibitor, white tree toxin, localized aspergillin, phenolmycin, enoxacin toxin; ribonuclease (RNase); DNase I, staphylococcal endotoxin A; pokeweed antiviral protein; Pseudomonas endotoxin; The cytokines are selected from IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa, IFN3, IFNy, GM-CSF, CD40L, Flt3 ligand, ansistatin, and TNFα; The radioactive isotopes are selected from 3H, 14C, 15N, 35S, 67Cu, 90Y, 99Tc, 125I, 131I, 186Re, 188Re, 211At, 212Bi, 212Pb, 213Bi, 225Ac and 227Th.

17. The antibody-drug conjugate according to claim 16, wherein, The antimitotic agent is monomethylolpropionate E or F; The antimetabolites are methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, sebazine, hydroxyurea, asparaginase, gemcitabine, or cladribine. The platinum derivative is cisplatin or carboplatin; The diphtheria toxin and related molecules are the diphtheria A chain and its active fragments and hybrid molecules; The ricin is ricin A or deglycosylated ricin A streptotoxin; The Shiga-like toxins are SLT I, SLT II, ​​SLT IIV, LT toxin, C3 toxin, or Shiga toxin.

18. A kit comprising a bispecific antibody or antigen-binding fragment thereof as claimed in any one of claims 1-8, a nucleic acid as claimed in claim 9 or 10, a vector as claimed in claim 11, or a cell as claimed in claim 12, a composition as claimed in claim 13, and / or an antibody-drug conjugate as claimed in any one of claims 14-17.

19. Use of a bispecific antibody or antigen-binding fragment thereof according to any one of claims 1-8, a nucleic acid according to claim 9 or 10, a vector according to claim 11, or a cell according to claim 12, a composition according to claim 13, and / or an antibody-drug conjugate according to any one of claims 14-17 in the preparation of a medicament or kit for the diagnosis, treatment, or prevention of diseases related to tumor antigens.

20. The use according to claim 19, wherein, The tumor antigen is CD20, and the tumor antigen-related disease is a CD20-related disease; CD20-related diseases include B-cell diseases, selected from non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), and multiple myeloma (MM) and Hodgkin lymphoma (HL); or The tumor antigen is BCMA, and the tumor antigen-related diseases are BCMA-related diseases. BCMA-related diseases are B-cell-related cancers, selected from multiple myeloma, malignant plasmacytoma, Hodgkin lymphoma, Hodgkin lymphoma (predominantly nodular lymphocytes), Kahler's disease, myeloid leukemia, plasma cell leukemia, plasmacytoma, B-cell prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), and chronic myeloid leukemia. Leukemia (CML), Follicular Lymphoma, Burkitt Lymphoma, Marginal Zone Lymphoma, Mantle Cell Lymphoma, Large Cell Lymphoma, Precursor B-cell Lymphoma, Myeloid Leukemia, Waldenström Macroglobulinemia, Diffuse Large B-cell Lymphoma, Follicular Lymphoma, Marginal Zone Lymphoma, Mucosa-associated Lymphoid Tissue Lymphoma, Small Cell Lymphocytic Lymphoma, Mantle Cell Lymphoma, Burkitt Lymphoma, Primary Mediastinal (Thymic) Large B-cell Lymphoma, Lymphoplasmacytic Lymphoma, Waldenström Macroglobulinemia, Marginal Zone B-cell Lymphoma of Lymph Nodes, Marginal Zone Lymphoma of the Spleen, Intravascular Large B-cell Lymphoma Large B-cell lymphoma, primary exudative lymphoma, lymphomatoid granulomatosis, large B-cell lymphoma rich in T cells / histocytes, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), EBV-positive diffuse large B-cell lymphoma in the elderly, inflammation-associated diffuse large B-cell lymphoma, intravascular large B-cell lymphoma, ALK-positive large B-cell lymphoma, plasmablastic lymphoma, large B-cell lymphoma arising from HHV8-associated multicentric Castleman's disease, unclassified intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma. B-cell lymphoma of the type, unclassified B-cell lymphoma with intermediate features between diffuse large B-cell lymphoma and classical Hodgkin's lymphoma, and other B-cell-associated lymphomas; or, the B-cell disease is a B-cell disorder; plasma cell disorder is selected from: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary osteoplasmacytoma, extramedullary plasmacytoma, osteosclerosing myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and condensational multiple myeloma; the disease is systemic lupus erythematosus or rheumatoid arthritis; or The tumor antigen is GPC3, and the tumor antigen-related diseases are GPC3-related diseases; GPC3-related diseases include tumors such as GPC3-positive liver cancer, GPC3-positive hepatocellular carcinoma, GPC3-positive pancreatic cancer, GPC3-positive lung cancer, GPC3-positive colon cancer, GPC3-positive breast cancer, GPC3-positive prostate cancer, GPC3-positive leukemia, or GPC3-positive lymphoma.

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