Bispecific antibodies and uses thereof

Abstract

To provide means for solving mismatches between heavy chains in bispecific antibodies.SOLUTION: The disclosure provides a bispecific antibody or its antigen binding fragment, a coding nucleic acid thereof, a cell comprising the nucleic acid, a composition comprising the bispecific antibody or antigen binding fragment thereof, the nucleic acid and / or cell, and related use of the bispecific antibody or antigen-binding fragment thereof. Also disclosed is a novel T-cell adaptor designed by using the bispecific antibody with light chains of different types κλ, and a full-length IgG conformation, where, as the antibody arms that bind to a target cell and T-cell CD3, a kappa light chain and a lambda light chain are used respectively to pair with their homologous heavy chains, and complementary charge pairs are introduced to enhance the correct pairing rate to optimize affinity.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to bispecific antibodies and uses thereof, in particular bispecific antibodies that bind to CD3 and one other antigen, and uses thereof. [Background technology]

[0002] T cell bispecific antibodies (also called T cell adaptors) are special antibody molecules that recognize the surface antigen (antigen arm) of a target cell through one end and bind to the CD3 receptor (CD3 arm) of a T cell through the other end, activating the T cell and killing the tumor by agglutinating the CD3 of the T cell in a manner similar to TCR / peptide / HLA. In the 1980s, the use of bispecific antibodies to kill tumor cells was reported (Staerz UD., Nature. 1985 Apr 18-24; 314(6012):628-31; Perez P. et al. Nature. 1985 Jul 25-31; 316(6026):354-6). After more than 30 years of research, the problem of antibody mismatch has been basically solved, and three bispecific antibody drugs have been approved one after another. Although they have shown excellent efficacy for the approved indications, the early widespread use of these bispecific antibodies has been hindered by the associated side effects and restrictions on use. For example, catumaxomab, which was approved earlier, is now off the market because its Fc segment binds to Fcγ receptors expressed on liver Kupffer cells, causing rapid cytokine release, and blinatumomab, which was approved in 2014, uses an Fv antibody fragment, so it has a biological half-life of only 2 hours and requires continuous intravenous infusion at low doses, and the FDA has now required a black box warning due to cytokine release syndrome and neurotoxicity.

[0003] In the normal immune response process, TCR binds to exogenous peptide-human leukocyte antigen complex (HLA) of infected or mutated cells with low affinity (about 1-100μM), and transmits activation signals into the nucleus through CD3 signaling complex (CD3εγ, CD3εδ and CD3ζζ), activating the expression of transcription factors and their downstream proteins (cytokines, granzymes, perforin, etc.), among which the signal strength generated by the TCR complex determines the fate of T cells. The CD3 bispecific antibodies developed in the early days were mostly based on a few mouse antibodies such as OKT3, L2K, UCHT1 and TR66, which have high affinity, leading to overactivation of T cells, releasing a large number of cytokines, producing cytokine storm syndrome, and the high affinity allows bispecific antibodies to be recruited in secondary lymphoid organs, reducing their exposure to tumor tissues.

[0004] The binding ability of the antibody Fc portion to Fcγ receptors is another factor affecting drug safety. Fcγ receptors are expressed in multiple normal tissues. Therefore, after bispecific antibodies bind to Fcγ receptors on the cell membrane via Fc, the CD3 receptors bound to the other end are cross-linked and activated by Fcγ receptor aggregation, resulting in severe off-target toxicity. Human IgG2 or IgG4 subtypes, which have a weak ability to bind to Fcγ receptors, can be used, or amino acid substitutions can be made at the corresponding sites of CH2. For example, Armour et al. replaced positions 233-236 (EU sequence number) of IgG1 and IgG4 with the corresponding sequences of IgG2 to reduce binding to Fcγ receptors (Armour KL, et al., Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities, Eur J Immunol. 1999 Aug; 29(8): 2613-24), and Newman et al. introduced the mutations Ser228Pro and Leu235Glu into IgG4 to stabilize the IgG4 structure and reduce binding to Fcγ receptors (Newman R, et al., Modification of the Fc region of a primatized IgG antibody to human CD4 retains its ability to modulate CD4 receptors but does not deplete CD4(+) T cells in chimpanzees. Clin. Immunol. 2001 Feb;98(2):164-74), and Idusogie et al. found that the binding of IgG1 to complement C1q can be reduced by substituting Asp270, Lys322, Pro329, or Pro331 with Ala (Idusogie EE,et al,Mapping of the C1q binding site on rituxan,a chimeric antibody with a human IgG1 Fc. J Immunol. 2000 Apr 15;164(8):4178-84).

[0005] Interchain mismatches are a major process bottleneck during the development of native IgG-like bispecific antibodies. The common light chain invented by Merchant AM et al. (Merchant AM, et al, An efficient route to human bispecific IgG. Nat Biotechnol. 1998. PMID:9661204) or the common heavy chain developed by Fischer N et al. (Fischer N, et al, Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG. Nat Commun. 2015 Feb 12;6:6113) usually requires complex protein engineering or production using genetically modified animals (McWhirter J, et al, Common light chain mouse. WO2011097603.2011), Carter P et al. (Atwell S, et al, Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library, J Mol Biol. 1997 Jul 4;270(1):26-35). To solve the mismatch between heavy chains, a knobs-into-holes complementary mutation was introduced into the antibody Fc segment, and Schaefer G et al. (Schaefer W, et al., Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies, Proc Natl Acad Sci US A. 2011) developed the CrossMab technology to replace the Fab portion or the entire length of the light chain or heavy chain to solve the problem of light chain mismatch. However, in order to achieve correct pairing, CrossMab technology that replaces a part of the heavy chain is not available. VH-VL and CrossMab CH1-CLIt is necessary to introduce an additional peptide segment into the Fab fragment, and to replace the entire Fab fragment. Fab has an efficiency of correct pairing less than 50%. Summary of the Invention

[0006] In the process of expressing bispecific antibodies, the inventors unexpectedly found that when a humanized anti-CD3 antibody having a λ light chain and a target antibody having a κ light chain are combined, the λ light chain of the anti-CD3 antibody tends to pair with the cognate CD3 heavy chain, and the κ light chain of the target antibody tends to pair with the cognate heavy chain of the target antibody. Furthermore, the efficiency of accurate pairing can be further improved by setting up a complementary charge pair between the light chain and the heavy chain. In addition, the novel T cell adapters constructed with multiple target antibodies such as CD20, BCMA, and GPC3 and humanized anti-CD3 antibodies all achieved 98-100% monomer purity through three stages of purification, and were demonstrated to have extremely low mismatch rates (<1%).

[0007] The present disclosure provides a novel T cell adaptor that adopts a different type of light chain κλ bispecific antibody design and full-length IgG conformation, in which the antibody arms that bind to target cells and T cell CD3 adopt κ light chain and λ light chain, respectively, to pair with their cognate heavy chains, and introduce complementary charge pairs to improve the efficiency of precise pairing and optimize affinity, so that the novel T cell adaptor can recruit activated T cells at low concentrations and produce effective killing of target cells, and in the absence of target cells, T cells will not be activated, and the novel T cell κλ bispecific antibody will not bind to FcγR receptors, reducing the risk of cytokine storm. The novel CD20×CD3κλ bispecific antibody, BCMA×CD3κλ bispecific antibody and GPC3×CD3κλ bispecific antibody constructed by the method of the present disclosure have high purification yields and can achieve a purity of over 99% through three-step purification. The novel CD20-CD3κλ bispecific antibody was well tolerated by animals, and the therapeutic efficacy and safety of the novel T cell adaptor are superior to those of the same type of antibody.

[0008] In one aspect, the disclosure provides a bispecific antibody, or antigen-binding portion thereof.

[0009] In another aspect, the present disclosure provides a nucleic acid encoding a bispecific antibody or antigen-binding portion thereof according to the previous aspect.

[0010] In another aspect, the present disclosure provides a vector comprising a nucleic acid according to the previous aspect.

[0011] In another aspect, the present disclosure provides a cell comprising the vector according to the previous aspect.

[0012] According to any of the above-mentioned aspects of the antibody or antigen-binding portion thereof, the antibody or antigen-binding portion thereof is humanized.

[0013] In another aspect, the present disclosure provides a pharmaceutical composition or kit comprising an antibody or antigen-binding portion thereof according to any of the previous aspects, or an encoding nucleic acid thereof, and a pharma- ceutically acceptable carrier.

[0014] In another aspect, the disclosure provides an antibody-drug conjugate comprising the antibody or antigen-binding portion thereof, bispecific or multispecific molecule according to any of the above aspects covalently linked to a therapeutic moiety.

[0015] In another aspect, the disclosure provides a method of treating an associated disease comprising administering to the mammal a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, vector, cell and / or pharmaceutical composition according to any of the preceding aspects.

[0016] In another aspect, the present disclosure provides the use of an antibody or antigen-binding fragment thereof, nucleic acid, vector, cell and / or pharmaceutical composition according to any of the preceding aspects in the manufacture of a medicament or kit for treating a tumor antigen-associated disease in said mammal.

[0017] The antibodies of the present disclosure find use in a number of applications, including detection of tumor antigens, diagnosis, treatment or prevention of tumor antigen-associated diseases. [Brief description of the drawings]

[0018] [Figure 1] FIG. 1 shows a first antigen×CD3κλ bispecific antibody according to the present disclosure. [Diagram 2] FIG. 1 shows binding of CD3 humanized antibodies to human CD3 εγ protein. [Diagram 3] FIG. 1 shows binding of CD3 humanized antibodies to Jurkat cells. [Figure 4] FIG. 1 shows the binding of CD3 humanized antibodies to human CD3-γ and cynomolgus monkey CD3-γ proteins. [Diagram 5] FIG. 1 shows the structures of κλ001, κλ002, κλ003, κλ004, and κλ005 according to the present disclosure. [Figure 6] FIG. 1 shows the results of purification of CD20×CD3κλ bispecific antibody using Protein A. [Figure 7] FIG. 1 shows the results of SEC-HPLC detection of CD20×CD3κλ bispecific antibody. [Figure 8] FIG. 1 shows the results of homodimer detection of CD20×CD3κλ bispecific antibody. [Figure 9] FIG. 1 shows binding of CD20×CD3κλ bispecific antibodies to CD20 stably transfected cells. [Figure 10] FIG. 1 shows binding of CD20×CD3κλ bispecific antibody to tumor cells SU-DHL-4, Raji and NALM-6. [Figure 11] FIG. 1 shows binding of CD20×CD3κλ bispecific antibody to Jurkat cells. [Figure 12] FIG. 1 shows binding of CD20×CD3κλ bispecific antibodies to peripheral blood T cells. [Figure 13]FIG. 13 shows the TDCC effect of CD20×CD3κλ bispecific antibody, with FIG. 13A showing killing of Nalm-6 cells and FIG. 13B showing activation of T cells. [Figure 14] FIG. 14 shows the TDCC effect of CD20×CD3κλ bispecific antibody, with FIG. 14A showing killing of TMD-8 cells and FIG. 14B showing activation of T cells. [Figure 15] FIG. 15 shows the TDCC effect of CD20×CD3κλ bispecific antibody, with FIG. 15A showing killing of Toledo cells and FIG. 15B showing activation of T cells. [Figure 16] FIG. 1 shows the effect of a CD20×CD3κλ bispecific antibody on the T cell NFAT signal pathway. [Figure 17] FIG. 1 shows the inhibitory effect of CD20×CD3κλ bispecific antibody in an immune reconstituted mouse Raji tumor xenograft model. [Figure 18] FIG. 1 shows the inhibitory effect of CD20×CD3κλ bispecific antibody in subcutaneous Raji and human PBMC mixed transplant tumor models in immunodeficient mice. [Figure 19] FIG. 1 shows the efficacy of CD20×CD3κλ bispecific antibodies in cynomolgus monkeys. [Figure 20] FIG. 1 shows binding of BCMAxCD3κλ bispecific antibodies to BCMA stably transfected cells. [Figure 21] FIG. 1 shows binding of BCMA×CD3κλ bispecific antibodies to tumor cells NCI-H929 and RPMI-8226. [Figure 22] FIG. 1 shows binding of BCMA×CD3κλ bispecific antibodies to Jurkat cells. [Figure 23] FIG. 1 shows binding of BCMA×CD3κλ bispecific antibodies to peripheral blood T cells. [Figure 24] FIG. 24 shows the TDCC effect of BCMA×CD3κλ bispecific antibody, with FIG. 24A showing killing of NCI-H929 cells and FIG. 24B showing activation of T cells. [Diagram 25]FIG. 25 shows the TDCC effect of BCMA×CD3κλ bispecific antibodies, with FIG. 25A showing killing of RPMI-8226 cells and FIG. 25B showing activation of T cells. [Figure 26] FIG. 1 shows the effect of BCMA×CD3κλ bispecific antibody on the T cell NFAT signal pathway. [Figure 27] FIG. 1 shows non-specific activation of PBMCs by BCMA×CD3κλ bispecific antibodies. [Figure 28] FIG. 1 shows binding of BCMA×CD3κλ bispecific antibodies to Fc receptors. [Figure 29] FIG. 1 shows the inhibitory effect of BCMA×CD3κλ bispecific antibody in a subcutaneous NCI-H929 tumor transplant model in immunodeficient mice. [Diagram 30] FIG. 1 shows binding of GPC3×CD3κλ bispecific antibody to GPC3 stably transfected cells. [Diagram 31] FIG. 1 shows binding of GPC3×CD3κλ bispecific antibody to tumor cells HepG2. [Diagram 32] FIG. 1 shows binding of GPC3×CD3κλ bispecific antibody to Jurkat cells. [Diagram 33] FIG. 1 shows binding of GPC3×CD3κλ bispecific antibody to peripheral blood T cells. [Diagram 34] FIG. 34 shows the TDCC effect of the GPC3×CD3κλ bispecific antibody, with FIG. 34A showing the killing of HepG2 cells and FIG. 34B showing the activation of T cells. [Diagram 35] FIG. 1 shows the effect of GPC3×CD3κλ bispecific antibody on the T cell NFAT signal pathway. [Diagram 36] FIG. 13 shows non-specific activation of PBMCs by GPC3×CD3κλ bispecific antibody. [Figure 37] FIG. 13 shows the inhibitory effect of the GPC3×CD3κλ bispecific antibody in an immune reconstituted mouse subcutaneous HepG2 tumor xenograft model. [Figure 38]FIG. 13 shows the inhibitory effect of the GPC3×CD3κλ bispecific antibody in a CD3-humanized mouse Hepa1-6 / human GPC3-xenograft tumor model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology used herein are terms and routine procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0020] As used herein, "tumor antigen" preferably refers to any antigen or antigenic determinant present (or associated) on tumor cells that is not generally present on normal cells, or an antigen or antigenic determinant present or associated on tumor cells in greater amounts than on normal (non-tumor) cells, or an antigen or antigenic determinant present on tumor cells in a form that differs from that expressed on normal (non-tumor) cells. The term includes tumor specific antigens (TSA), which include tumor specific antigens, or tumor associated antigens (TAA), which include tumor associated membrane antigens, embryonic antigens on tumors, growth factor receptors, growth factor ligands, and any other type of antigen associated with cancer. The tumor antigen may be, for example, a B cell differentiation antigen (e.g., CD19, CD20 and CD37), B cell maturation antigen (BCMA), phosphatidylinositol proteoglycan 3 (GPC3), an epithelial cancer antigen (e.g., breast cancer, gastrointestinal cancer, lung cancer), prostate specific cancer antigen (PSA) or prostate specific membrane antigen (PSMA), a bladder cancer antigen, a lung (e.g., small cell lung) cancer antigen, a colon cancer antigen, an ovarian cancer antigen, a brain cancer antigen, a gastric cancer antigen, a renal cell cancer antigen, a pancreatic cancer antigen, a liver cancer antigen, an esophageal cancer antigen, a head and neck cancer antigen or a colorectal cancer antigen.

[0021] A TSA is unique to (or considered to be unique to) a tumor cell and does not occur in (e.g., does not occur to a significant extent in) other cells in vivo. A TAA is not unique to tumor cells, but rather is also expressed in normal cells (e.g., is expressed under conditions that cannot induce a state of immune tolerance to the antigen). For example, a TAA may be an antigen that is expressed in normal cells during fetal development when the immune system is immature and unable to respond, or a TAA may be an antigen that is normally present at very low levels in normal cells, but is expressed at higher levels by tumor cells.

[0022] 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, tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, overexpressed fetal 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 pathogenic antigens such as the 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, α-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / CBCAA, 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 / cyclophilin C-related protein, TAAL6, TAG72, TLP, MUC16, IL13Rα2, FRα, VEGFR2, Lewis These include Y, FAP, EphA2, CEACAM5, EGFR, CA6, CA9, GPNMB, EGP1, FOLR1, endothelial receptors, STEAP1, SLC44A4, nectin-4, AGS-16, guanidinocyclase C, MUC-1, CFC1B, integrin α3 chain (a3b1 chain, i.e., laminin receptor chain), and TPS.Other tumor antigens further 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.

[0023] As used herein, the term "CD20" refers to any native CD20 from any vertebrate, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats).

[0024] The terms "anti-CD20 antibody" and "antibody that binds to CD20" refer to an antibody that can bind to CD20 with sufficient affinity such that the antibody is useful in targeting CD20 as a diagnostic and / or therapeutic agent. In one embodiment, the binding of an anti-CD20 antibody to an unrelated, non-CD20 protein is about 10% lower than the binding of the antibody to CD20, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that binds to CD20 has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Dissociation constant (K d In some embodiments, the anti-CD20 antibody binds to a conserved CD20 epitope among CD20 from different species.

[0025] As used herein, the term "BCMA" collectively refers to BCMA itself and any variants, isotypes, and paralogs thereof present in animals, preferably humans.

[0026] The term "human BCMA" refers to BCMA of human origin and may preferably, but is not limited to, have the amino acid sequence of Genbank Accession No. AB052772.1.

[0027] The terms "anti-BCMA antibody" and "antibody that binds BCMA" refer to an antibody that can bind to BCMA with sufficient affinity such that the antibody is useful in targeting BCMA as a diagnostic and / or therapeutic agent. In one embodiment, the binding of the anti-BCMA antibody to an unrelated non-BCMA protein is about 10% lower than the binding of the antibody to BCMA, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that binds BCMA has an affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Dissociation constant (K d In some embodiments, the anti-BCMA antibody binds to a conserved BCMA epitope among BCMA from different species.

[0028] As used herein, the term "GPC3" refers collectively to GPC3 itself and any of its variants, isotypes, and paralogs present in animals and preferably in humans.

[0029] The term "human GPC3" refers to GPC3 derived from humans.

[0030] The terms "anti-GPC3 antibody" and "antibody that binds to GPC3" refer to an antibody that can bind to GPC3 with sufficient affinity such that the antibody is useful for targeting GPC3 as a diagnostic and / or therapeutic agent. In one embodiment, the binding of the anti-GPC3 antibody to an unrelated non-GPC3 protein is about 10% lower than the binding of the antibody to GPC3, as measured, for example, by radioimmunoassay (RIA). In some embodiments, the antibody that binds to GPC3 has a binding affinity of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 Dissociation constant (K d In some embodiments, the anti-GPC3 antibody binds to a conserved GPC3 epitope among GPC3s from different species.

[0031] "CD3" refers to any native CD3 from any vertebrate, 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, and any form of CD3 processed intracellularly. The term further covers naturally occurring variants of CD3, such as splice variants or allelic variants. In one embodiment, the CD3 is human CD3, and in particular the ε 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.

[0032] The term "cell surface" is used according to its ordinary meaning in the art, and includes the exterior of the cell that is accessible for binding to proteins and other molecules.

[0033] As used herein, the term "about" or "approximately" means, unless otherwise specified, within ±10% of a given value or range. When required to be an integer, the term shall be rounded up or down to the nearest integer within ±10% of the given value or range.

[0034] With respect to antibody chain polypeptide sequences, the term "substantially identical" may be understood as antibody chains exhibiting 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more sequence identity to a reference polypeptide sequence. With respect to nucleic acid sequences, the term may be understood as a nucleotide sequence exhibiting at least 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% or more sequence identity to a reference nucleic acid sequence.

[0035] Sequence "homology" or "identity" has its meaning as known in the art, and the disclosed techniques can be used to calculate the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide, or along a region of the molecule. There are many ways to measure homology between two polynucleotides or polypeptides, but the term "homology" is well known to those skilled in the art (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988)).

[0036] "Substitutional" variants are those in which at least one amino acid residue in a native sequence has been removed and a different amino acid has been inserted in the same position. The substitutions may be single, where only one amino acid has been replaced in the molecule, or multiple, where two or more amino acids have been replaced in the same molecule. Multiple substitutions are present at contiguous sites. Similarly, an amino acid may be replaced by multiple residues, and such variants include both substitutions and insertions. "Insertional" variants are those in which one or more amino acids have been inserted immediately adjacent to an amino acid at a particular position in a native sequence. Immediately adjacent to an amino acid means bonded to the α-carboxyl or α-amino functionality of that amino acid. "Deletional" variants are those in which one or more amino acids in a native amino acid sequence have been removed. Typically, deletional variants are those in which one or two amino acids have been deleted in a particular region of the molecule.

[0037] With respect to the variable domain of an antibody, the term "variable" refers to the specific portions of the related molecule that differ greatly in sequence among antibodies and are used for the specific recognition and binding of a particular antibody to a particular target. However, variability is not evenly distributed throughout the variable domain of an antibody. The variability is concentrated in three segments called complementarity determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) or hypervariable regions, which are present in all light and heavy chain variable domains. The more highly conserved parts of the variable domains are called framework (FR) regions or framework sequences. Each naturally occurring heavy and light chain variable domain, respectively, contains four FR regions that are primarily in a β-sheet configuration, connected by three CDRs, which form loops that connect to, and in some cases form part of, the β-sheet structure. The CDRs of each chain are usually adjacently connected by FR regions and contribute to the formation of the target binding site (epitope or determinant) of the antibody with the CDRs from the other chain. As used herein, immunoglobulin amino acid residue numbering is according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise specified. A CDR may have the ability to specifically bind to an associated epitope.

[0038] As used herein, an "antibody fragment" or "antigen-binding fragment" of an antibody refers to any portion of a full-length antibody that is less than full-length but contains at least a portion of the variable region of said antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antibody binding sites), thereby retaining the binding specificity and at least a portion of the specific binding ability of said full-length antibody. Thus, an antigen-binding fragment refers to an antibody fragment that contains an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived. Antibody fragments include antibody derivatives produced by enzyme-catalyzed treatment of a full-length antibody, as well as synthetically produced derivatives, e.g., recombinantly produced derivatives. Antibodies include antibody fragments. Illustrative examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, single chain Fv (scFv), Fv, dsFv, diabody, Fd, and Fd' fragments, as well as other fragments, including modified fragments (see, e.g., Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov). The fragments may include, for example, multiple chains linked by disulfide bonds and / or peptide linkers. Antibody fragments usually contain more than or about 50 amino acids, and typically more than or about 200 amino acids. Antigen-binding fragments immunospecifically (i.e., at least 10 amino acids) when inserted into an antibody framework (e.g., by replacing the corresponding region). 7 ~10 8 M -1 or at least about 10 7 ~10 8 M -1The term "functional fragment" or "antibody analog" includes any antibody fragment that provides an antibody that binds to an antigen (indicating a Ka of 0.1 to 1.0). A "functional fragment" or "antibody analog" is a fragment or analog that inhibits or substantially reduces the ability of the receptor to bind to a ligand or initiate signal transduction. As used herein, a functional fragment generally has the same meaning as an "antibody fragment" and, with respect to an antibody, may refer to a fragment that inhibits or substantially reduces the ability of the receptor to bind to 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 association of one heavy chain variable domain and one light chain variable domain. H -V L In this configuration, the three CDRs of each variable domain interact to form a V H -V L The six CDRs define a target binding site on the surface of the dimer. The six CDRs confer target binding specificity to the intact antibody. However, a single variable domain (or half of an Fv containing only three CDRs specific for a target) can also function to recognize and bind to a target.

[0039] The term "bispecific antibody" (BsAb) as used herein refers to an antibody and / or antigen-binding molecule capable of specifically binding to two different antigenic determinants, and typically a bispecific antibody and / or antigen-binding molecule comprises two antigen-binding sites, each having specificity for a different antigenic determinant. In some embodiments, the bispecific antibody and / or antigen-binding molecule can simultaneously bind to two antigenic determinants, in particular two antigenic determinants expressed on two different cells.

[0040] As used herein, "monoclonal antibody" refers to a population of identical antibodies, meaning that each individual antibody molecule in a monoclonal antibody population is identical to the other antibody molecules. This property is the opposite of that of a polyclonal population of antibodies, which contains antibodies with multiple distinct sequences. Monoclonal antibodies can be prepared in many well-known ways (Smith et al. (2004) J. Clin. Pathol. 57, 912-917, and Nelson et al., J Clin Pathol (2000), 53, 111-117). For example, monoclonal antibodies can be prepared by immortalizing B cells, e.g., by fusing with myeloma cells to produce hybridoma cell lines, or by infection of B cells with a virus such as EBV. Recombinant techniques can also be used to prepare antibodies from a clonal population of host cells in vitro by transforming the host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.

[0041] The term "hybridoma" or "hybridoma cell" as used herein refers to a cell or cell line (usually a myeloma or lymphoma cell) produced by the fusion of an antibody-producing lymphocyte with a non-antibody-producing cancer cell. As known to those skilled in the art, a hybridoma can grow and produce and continuously supply a specific monoclonal antibody. Methods for producing hybridomas are known in the art (see, for example, Harlow & Lane, 1988). When the term "hybridoma" or "hybridoma cell" is referred to, it also includes subclones and progeny cells of the hybridoma.

[0042] 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 an antibody produced naturally by an antibody-secreting B cell, or an antibody having the same domains produced synthetically.

[0043] The term "chimeric antibody" refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, e.g., the variable region sequences are derived from a murine antibody and the constant region sequences are derived from a human antibody.

[0044] "Humanized" antibodies refer to forms of non-human (e.g., murine) antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. Preferably, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the complementarity determining regions (CDRs) of the recipient antibody are replaced by CDR residues from a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity.

[0045] In humanization, it is also possible to improve one or more binding properties (e.g., affinity) of the antibody by mutating amino acid residues in the CDR1, CDR2 and / or CDR3 regions of VH and / or VL. For example, mutations can be introduced by PCR-mediated mutagenesis, and the effect on antibody binding or other functional properties can be evaluated by in vitro or in vivo testing as described herein. Typically, conservative mutations are introduced. Such mutations may be amino acid substitutions, additions, or deletions. Also, mutations in the CDRs usually do not exceed one or two. Thus, the humanized antibody of the present disclosure further includes antibodies containing one or two amino acid mutations in the CDRs.

[0046] The term "CDR" as used herein refers to a complementarity-determining region, and each heavy and light chain of an antibody molecule is known to have three CDRs. CDRs, also called hypervariable regions, are present in the variable regions of each heavy and light chain of an antibody, and are highly variable sites in the primary structure of the CDRs. Herein, the CDRs of the heavy chain are designated CDR1, CDR2, and CDR3 at the amino terminus of the amino terminal sequence derived from the heavy chain, and the CDRs of the light chain are designated CDR1, CDR2, and CDR3 at the amino terminus of the amino terminal sequence derived from the light chain. These sites are adjacent to each other in the tertiary structure and determine the specificity of the antigen that binds to the antibody.

[0047] The term "epitope" as used herein refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.

[0048] As used herein, with respect to an antibody or antigen-binding fragment thereof, the terms "specifically binds" or "immunospecifically binds" are used interchangeably herein and refer to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with a cognate antigen by non-covalent interactions between the antibody and the antibody-binding site of the antigen. The antigen may be an isolated antigen or may be present on tumor cells. Typically, an antibody that immunospecifically binds (or specifically binds) to an antigen is present at approximately 1×10 7 M -1 Or about 1 x 10 8 M -1 or higher affinity constant Ka (1×10 -7 Or 1×10 -8The antibody binds to the antigen with a dissociation constant (Kd) of M or less. The affinity constant can be measured by standard kinetic methods of antibody reactions, such as immunoassays, 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 measurements well known in the art, see U.S. Pat. No. 7,229,619, which describes exemplary SPR and ITC methods for calculating the binding affinity of an antibody. Devices and methods for real-time detection and monitoring of binding kinetics are known and commercially available (see Malmqvist (2000) Biochem. Soc. Trans. 27:335).

[0049] As used herein, the terms "polynucleotide" and "nucleic acid molecule" refer to an oligomer or polymer containing two or more linked nucleotides or nucleotide derivatives, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), typically linked by phosphodiester bonds. As used herein, the term "nucleic acid molecule" is intended to include DNA molecules, RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, and may be cDNA.

[0050] As used herein, an isolated nucleic acid molecule is a nucleic acid molecule that is isolated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. For example, an "isolated" nucleic acid molecule of a cDNA molecule is substantially free of other cellular material or culture medium when prepared by recombinant techniques, or is substantially free of chemical precursors or other chemical components when chemically synthesized. Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules that encode the provided antibodies or antigen-binding fragments.

[0051] As used herein, "operably linked" with respect to a nucleic acid sequence, region, element, or domain means 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 such that it can regulate or mediate transcription of the nucleic acid.

[0052] "Conservative sequence modifications" of the sequences in the sequence listing herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate the binding of the antibody encoded by the nucleotide sequence or containing the amino acid sequence to the antigen, are also provided. These conservative sequence modifications include conservative nucleotide and amino acid substitutions, and nucleotide and amino acid additions and deletions. For example, modifications can be introduced into the sequence listing herein by standard techniques well known in the art (e.g., site-directed mutagenesis and PCR-mediated mutagenesis). Conservative sequence modifications include conservative amino acid substitutions, in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted non-essential amino acid residue in an anti-GCD20 antibody, anti-BCMA antibody, or anti-PC3 antibody is preferably replaced with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not eliminate antigen binding are well known in the art (see, e.g., Blommell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94:412-417 (1997)).

[0053] Alternatively, in another embodiment, mutations can be introduced randomly along all or part of the sequence encoding the anti-GCD20 antibody, anti-BCMA antibody or anti-PC3 antibody, for example by saturation mutagenesis, and the resulting modified anti-CD20 antibody, anti-BCMA antibody or anti-GPC3 antibody can be screened for improved binding activity.

[0054] "Expression" as used herein refers to the process of producing a polypeptide by transcription and translation of a polynucleotide. The expression level of a polypeptide can be assessed using any method known in the art, including, for example, a method for determining the amount of a polypeptide produced from a host cell. Such methods can include, but are not limited to, quantification of a polypeptide in a cell lysate by ELISA, gel electrophoresis followed by Coomassie blue staining, Lowry protein assay, and Bradford protein assay.

[0055] As used herein, a "host cell" is a cell used to receive, maintain, replicate, and amplify a vector. A host cell can also be used to express a polypeptide encoded by a vector. When a host cell divides, the nucleic acid contained in the vector is replicated and the nucleic acid is amplified. A host cell can be a eukaryotic or prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells, such as HEK293 cells.

[0056] A "vector" as used herein is a replicable nucleic acid from which one or more heterologous proteins can be expressed when the vector is transformed into a suitable host cell. Vectors include vectors that can introduce a nucleic acid encoding a polypeptide or a fragment thereof, typically by restriction digestion and ligation. Vectors further include vectors that contain a nucleic acid encoding a polypeptide. Vectors are used to introduce a nucleic acid encoding a polypeptide into a host cell for amplification of the nucleic acid or for expression / display of the polypeptide encoded by the nucleic acid. Vectors usually remain episomal, but may also be engineered into chromosomes where a gene or a part thereof is integrated into the genome. Also contemplated are vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.

[0057] Vector as used herein further includes "viral vectors" or "viral vectors." Viral vectors are engineered viruses that are operably linked to exogenous genes to transfer the exogenous genes into cells (as a vehicle or shuttle).

[0058] As used herein, an "expression vector" includes a vector capable of expressing DNA, said DNA being operably linked to a regulatory sequence, e.g., a promoter region, that affects the expression of the DNA fragment. Such additional fragments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, etc. Expression carriers can generally be derived from plasmid or viral DNA, or contain elements of both. Thus, an expression carrier refers to a recombinant DNA or RNA construct, e.g., a plasmid, phage, recombinant virus, or other vector, that, when introduced into a suitable host cell, results in the expression of the cloned DNA. Suitable expression vectors are well known to those skilled in the art and include expression vectors that are replicable in eukaryotic and / or prokaryotic cells, and expression vectors that remain episomal or that integrate into the host cell genome.

[0059] As used herein, "treating" an individual with a disease or a symptom of a disease means that the individual's symptoms are partially or totally ameliorated or remain unchanged following treatment. Thus, treatment includes prevention, therapy and / or cure. Prevention refers to the prevention of underlying disease and / or the prevention of worsening of symptoms or progression of disease. Treatment further includes any pharmaceutical use of any of the antibodies or antigen-binding fragments thereof provided and compositions provided herein.

[0060] As used herein, "therapeutic benefit" means the benefit obtained by treating an individual, which is an alteration in the symptoms of a disease or disease state, typically an improvement or amelioration of the disease or disease state, or a cure of the disease or disease state.

[0061] As used herein, a "therapeutically effective amount" or a "therapeutically effective dose" refers to an amount of a drug, compound, substance, or composition containing a compound that is at least sufficient to achieve a therapeutic effect following administration to a subject, and thus is the amount necessary to prevent, cure, ameliorate, suppress or partially suppress the symptoms of a disease or condition.

[0062] As used herein, a "prophylactically effective amount" or a "prophylactically effective dose" refers to the amount of a substance, compound, material, or composition containing a compound that, upon administration to a subject, has a desired prophylactic effect, such as, for example, preventing or delaying the onset or recurrence of a disease or condition, or reducing the likelihood of the onset or recurrence of a disease or condition. A complete prophylactically effective dose need not be achieved by administering a single dose, but may be achieved only when a series of doses are administered. Thus, a prophylactically effective amount may be administered in one or more administrations.

[0063] As used herein, the term "patient" refers to a mammal, such as a human. II. Specific Embodiments

[0064] In one aspect, the disclosure provides a method for the preparation of a medicament for a medicament comprising: (a) a first antigen-binding portion or antigen-binding fragment thereof, the first antigen-binding portion comprising a first light chain and a first heavy chain, the first light chain being a kappa-type light chain, the first antigen-binding portion comprising a first binding domain that binds to a first antigen; and (b) a second antigen-binding portion or antigen-binding fragment thereof, the second antigen-binding portion comprising a second light chain and a second heavy chain, the second light chain being a lambda light chain and the second antigen-binding portion comprising a second binding domain that binds to a second antigen.

[0065] In some embodiments, the second antigen is a CD3 antigen.

[0066] In some embodiments, the second light chain variable region of the second antigen binding portion comprises the amino acid sequence of Gln 40 Glu mutation (Vλ CD3 :Gln 40 Glu), and the second heavy chain variable region of the second antigen-binding portion has Gln 39 Lys mutation (VH CD3 :Gln 39 Lys).

[0067] In some embodiments, the second binding domain comprises a second light chain CDR selected from the amino acid sequence of SEQ ID NO:7-9, 14, 15, 20, 21, or any variant thereof, and / or a second heavy chain CDR selected from the amino acid sequence of SEQ ID NO:26-28, 31, 34, 40, 43, 46, 47, or any variant thereof.

[0068] In some embodiments, the second binding domain comprises a second light chain CDR1 selected from the amino acid sequence SEQ ID NO:7, 14, or any variant thereof, a second light chain CDR2 selected from the amino acid sequence SEQ ID NO:8, 15, 20, or any variant thereof, a second light chain CDR3 selected from the amino acid sequence SEQ ID NO:9, 21, or any variant thereof, and / or a second heavy chain CDR1 selected from the amino acid sequence SEQ ID NO:26, 31, 46, or any variant thereof, a second heavy chain CDR2 selected from the amino acid sequence SEQ ID NO:27, 47, or any variant thereof, and a second heavy chain CDR3 selected from the amino acid sequence SEQ ID NO:28, 34, 37, 40, 43, or any variant thereof.

[0069] In some embodiments, the second light chain CDRs of the second binding domain are selected from the second light chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs:7, 8, 9, respectively; the second light chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs:14, 15, 9, respectively; the second light chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs:7, 8, 21, respectively; the second light chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs:7, 20, 21, respectively; and / or the heavy chain CDRs of the second binding domain are selected from the second heavy chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs:26, 27, 28, respectively; the second heavy chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs:31, 27, 28, respectively; the second heavy chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 31, 27 and 34, the second heavy chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 31, 27 and 37, respectively; the second heavy chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 31, 27 and 40, respectively; the second heavy chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 31, 27 and 43, respectively; and the second heavy chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences of SEQ ID NOs: 46, 47 and 28, respectively.

[0070] In some embodiments, the second binding domain comprises a second light chain variable region selected from the amino acid sequence of SEQ ID NO:5, 10, 12, 16, 18, 22, or any variant thereof, and / or a second heavy chain variable region selected from the amino acid sequence of SEQ ID NO:24, 29, 32, 35, 38, 41, 44, 48, 50, 52, or any variant thereof.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] In some embodiments, the second light chain of the second antigen binding moiety is selected from the amino acid sequence SEQ ID NO:58 and 66, and / or the second heavy chain of the second antigen binding moiety is selected from the amino acid sequence SEQ ID NO:60 and 68. In some preferred embodiments, the second light chain of the second antigen binding moiety has the amino acid sequence SEQ ID NO:58 and the second heavy chain of the second antigen binding moiety has the amino acid sequence SEQ ID NO:60. In some preferred embodiments, the second light chain of the second antigen binding moiety has the amino acid sequence SEQ ID NO:66 and the second heavy chain of the second antigen binding moiety has the amino acid sequence SEQ ID NO:68.

[0079] In some embodiments, the first antigen is a tumor antigen.

[0080] 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.

[0081] In one specific embodiment, the tumor-associated antigen is selected from CD20, BCMA and GPC3.

[0082] In some embodiments, the first antigen is a CD20 antigen.

[0083] In some preferred embodiments, the first light chain variable region of the first antigen binding portion comprises the amino acid sequence of Gln 38 Lys mutation (Vκ CD20 :Gln38 In some preferred embodiments, the first heavy chain variable region of the first antigen-binding portion has a Gln 39 Glu mutation (VH CD20 :Gln 39 Glu).

[0084] In some preferred embodiments, the first light chain variable region of the first antigen binding portion comprises the amino acid sequence of Gln 38 Lys mutation (Vκ CD20 :Gln 38 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 In some preferred embodiments, the first heavy chain variable region of the first antigen-binding portion has a 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 H 1 CD20 :Gln 39 Glu / Lys 152 Glu / Lys 218 Glu).

[0085] In some preferred embodiments, the first light chain of the first antigen-binding portion is selected from the amino acid sequences of SEQ ID NOs:54, 62, and 70, and / or the first heavy chain of the first antigen-binding portion is selected from the amino acid sequences of SEQ ID NOs:56, 64, and 72.

[0086] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:54 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:56.

[0087] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:62 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:64.

[0088] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:70 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:72.

[0089] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:54 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:56, the second light chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:58 and the second heavy chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:60.

[0090] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:62, and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:64, the second light chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:66, and the second heavy chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:68, the second light chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:58, and the second heavy chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:60.

[0091] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:70 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:72, the second light chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:66 and the second heavy chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:68.

[0092] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:62 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:64, the second light chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:58 and the second heavy chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:60.

[0093] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:54 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:56, the second light chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:66 and the second heavy chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:68.

[0094] In some embodiments, the first antigen is a BCMA antigen.

[0095] In some embodiments, the first binding domain comprises a first light chain CDR selected from the amino acid sequence of SEQ ID NO:99-101, 134-135, or any variant thereof; and / or a first heavy chain CDR selected from the amino acid sequence of SEQ ID NO:96-98, or any variant thereof.

[0096] In some preferred embodiments, the first binding domain comprises a first light chain CDR1 selected from the amino acid sequence SEQ ID NO:99, 134, or any variant thereof, a first light chain CDR2 selected from the amino acid sequence SEQ ID NO:100, 135, or any variant thereof, a first light chain CDR3 selected from the amino acid sequence SEQ ID NO:101, or any variant thereof; and / or a first heavy chain CDR1 selected from the amino acid sequence SEQ ID NO:96, or any variant thereof, a first heavy chain CDR2 selected from the amino acid sequence SEQ ID NO:97, or any variant thereof, and a first heavy chain CDR3 selected from the amino acid sequence SEQ ID NO:98, or any variant thereof.

[0097] In some preferred embodiments, the first light chain CDR of the first binding domain comprises the first light chain CDR1, CDR2, and CDR3 sequences of the amino acid sequences SEQ ID NO:99, 100, 101, respectively; and / or the heavy chain CDR of the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of the amino acid sequences SEQ ID NO:96, 97, 98, respectively.

[0098] In some preferred embodiments, the first light chain CDR of the first binding domain comprises the first light chain CDR1, CDR2, and CDR3 sequences of the amino acid sequences SEQ ID NO:134, 135, 101, respectively; and / or the heavy chain CDR of the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of the amino acid sequences SEQ ID NO:96, 97, 98, respectively.

[0099] In some preferred embodiments, the first light chain variable region of the first antigen binding portion comprises the amino acid sequence of Gln 42 Lys mutation (Vκ BCMA :Gln 42 In some preferred embodiments, the first heavy chain variable region of the first antigen-binding portion has a Gln 39 Glu mutation (VH BCMA :Gln 39 Glu).

[0100] In some preferred embodiments, the first binding domain comprises a first light chain variable region selected from the amino acid sequence of SEQ ID NO:122, 124, or any variant thereof; and / or a first heavy chain variable region selected from the amino acid sequence of SEQ ID NO:108, 120, or any variant thereof.

[0101] In some preferred embodiments, the first binding domain comprises a first light chain variable region of amino acid sequence SEQ ID NO:122, or any variant thereof; and a first heavy chain variable region of amino acid sequence SEQ ID NO:108, or any variant thereof.

[0102] In some preferred embodiments, the first binding domain comprises a first light chain variable region of amino acid sequence SEQ ID NO:124, or any variant thereof; and a first heavy chain variable region of amino acid sequence SEQ ID NO:108, or any variant thereof.

[0103] In some preferred embodiments, the first binding domain comprises a first light chain variable region of amino acid sequence SEQ ID NO:122, or any variant thereof; and a first heavy chain variable region of amino acid sequence SEQ ID NO:120, or any variant thereof.

[0104] In some preferred embodiments, the first binding domain comprises a first light chain variable region of amino acid sequence SEQ ID NO:124, or any variant thereof; and a first heavy chain variable region of amino acid sequence SEQ ID NO:120, or any variant thereof.

[0105] In some preferred embodiments, the first binding domain comprises a first light chain variable region of amino acid sequence SEQ ID NO:122, or any variant thereof; and a first heavy chain variable region of amino acid sequence SEQ ID NO:108, or any variant thereof; 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.

[0106] In some preferred embodiments, the first binding domain comprises a first light chain variable region of amino acid sequence SEQ ID NO:124, or any variant thereof; and a first heavy chain variable region of amino acid sequence SEQ ID NO:108, or any variant thereof; 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.

[0107] In some preferred embodiments, the first binding domain comprises a first light chain variable region of amino acid sequence SEQ ID NO:122, or any variant thereof; and a first heavy chain variable region of amino acid sequence SEQ ID NO:120, or any variant thereof; 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.

[0108] In some preferred embodiments, the first binding domain comprises a first light chain variable region of amino acid sequence SEQ ID NO:124, or any variant thereof; and a first heavy chain variable region of amino acid sequence SEQ ID NO:120, or any variant thereof; 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.

[0109] In some preferred embodiments, the first light chain of the first antigen-binding portion is selected from the amino acid sequences of SEQ ID NOs:80 and 84, and / or the first heavy chain of the first antigen-binding portion is selected from the amino acid sequences of SEQ ID NOs:82 and 86.

[0110] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:80 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:82.

[0111] In some preferred embodiments, the first light chain of the first antigen-binding portion has the amino acid sequence SEQ ID NO:84 and the first heavy chain of the first antigen-binding portion has the amino acid sequence SEQ ID NO:82.

[0112] In some preferred embodiments, the first light chain of the first antigen-binding portion has the amino acid sequence SEQ ID NO:80 and the first heavy chain of the first antigen-binding portion has the amino acid sequence SEQ ID NO:86.

[0113] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:84 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:86.

[0114] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:80 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:82, the second light chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:66 and the second heavy chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:68.

[0115] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:84 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:82, the second light chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:66 and the second heavy chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:68.

[0116] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:80 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:86, the second light chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:66 and the second heavy chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:68.

[0117] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:84 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:86, the second light chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:66 and the second heavy chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:68.

[0118] In some embodiments, the first antigen is a GPC3 antigen.

[0119] In some preferred embodiments, the first binding domain comprises a first light chain CDR selected from the amino acid sequence of SEQ ID NO:102-104, or any variant thereof; and / or a first heavy chain CDR selected from the amino acid sequence of SEQ ID NO:105-107, or any variant thereof.

[0120] In some preferred embodiments, the first binding domain comprises a first light chain CDR1 selected from the amino acid sequence of SEQ ID NO:102 or any variant thereof, a first light chain CDR2 selected from the amino acid sequence of SEQ ID NO:103 or any variant thereof, a first light chain CDR3 selected from the amino acid sequence of SEQ ID NO:104 or any variant thereof; and / or a first heavy chain CDR1 selected from the amino acid sequence of SEQ ID NO:105 or any variant thereof, a first heavy chain CDR2 selected from the amino acid sequence of SEQ ID NO:106 or any variant thereof, and a first heavy chain CDR3 selected from the amino acid sequence of SEQ ID NO:107 or any variant thereof.

[0121] In some preferred embodiments, the first light chain CDR of the first binding domain comprises the first light chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NOs:102, 103, and 104, respectively; and / or the heavy chain CDR of the first binding domain comprises the first heavy chain CDR1, CDR2, and CDR3 sequences of amino acid sequences SEQ ID NOs:105, 106, and 107, respectively.

[0122] In some preferred embodiments, the first light chain variable region of the first antigen binding portion comprises the amino acid sequence of Gln 43 Lys and Gln 39 Glu mutation (Vκ GPC3 :Gln 43 Lys;VH GPC3 :Gln 39 Glu).

[0123] In some preferred embodiments, the first binding domain comprises a first light chain variable region selected from the amino acid sequence of SEQ ID NO:126, or any variant thereof; and / or a first heavy chain variable region selected from the amino acid sequence of SEQ ID NO:128, or any variant thereof.

[0124] In some preferred embodiments, the first light chain of the first antigen-binding portion is selected from the amino acid sequences of SEQ ID NOs:88 and 92, and / or the first heavy chain of the first antigen-binding portion is selected from the amino acid sequences of SEQ ID NOs:90 and 94.

[0125] In some preferred embodiments, the first light chain of the first antigen-binding portion has the amino acid sequence SEQ ID NO:88 and the first heavy chain of the first antigen-binding portion has the amino acid sequence SEQ ID NO:90.

[0126] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:92 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:94.

[0127] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:88 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:90, the second light chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:66 and the second heavy chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:68.

[0128] In some preferred embodiments, the first light chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:92 and the first heavy chain of the first antigen-binding moiety has the amino acid sequence SEQ ID NO:94, the second light chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:66 and the second heavy chain of the second antigen-binding moiety has the amino acid sequence SEQ ID NO:68.

[0129] In some embodiments, the Fc portion of the first antigen-binding portion and / or the second antigen-binding portion of the bispecific antibody adopts a knob-into-hole conformation, hi some preferred embodiments, a human IgG4 knob-into-hole conformation.

[0130] In some embodiments, the first antigen-binding portion and / or the second antigen-binding portion of the bispecific antibody comprises Ser 228 Pro, Leu 235 Glu and / or Pro 329 It also has an Ala mutation.

[0131] In one aspect, the disclosure provides a nucleic acid encoding the bispecific antibody or an antigen-binding portion thereof.

[0132] In some preferred embodiments, the second antigen binding portion binds to the CD3 antigen, and the encoding nucleic acid for the second light chain variable region of the second antigen binding portion is selected from the nucleotide sequence SEQ ID NO:6, 11, 13, 17, 19, and 23, and / or the encoding nucleic acid for the second heavy chain variable region of the second antigen binding portion is selected from the nucleotide sequence SEQ ID NO:25, 30, 33, 36, 39, 42, 45, 49, 51, and 53.

[0133] In some preferred embodiments, the encoding nucleic acid for the second light chain of the second antigen binding moiety is selected from the nucleotide sequences SEQ ID NOs:59 and 67, and / or the encoding nucleic acid for the second heavy chain of the second antigen binding moiety is selected from the nucleotide sequences SEQ ID NOs:61 and 69.

[0134] In some preferred embodiments, the encoding nucleic acid for the second light chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:59, and the encoding nucleic acid for the second heavy chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:61.

[0135] In some preferred embodiments, the encoding nucleic acid for the second light chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:67, and the encoding nucleic acid for the second heavy chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:69.

[0136] In some preferred embodiments, the first antigen-binding portion binds to a CD20 antigen, and the encoding nucleic acid of the first light chain of the first antigen-binding portion is selected from the nucleotide sequences SEQ ID NOs:55, 63, and 71, and / or the encoding nucleic acid of the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequences SEQ ID NOs:57, 65, and 73.

[0137] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:55, and the encoding nucleic acid of the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:57.

[0138] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:63, and the encoding nucleic acid of the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:65.

[0139] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:71, and the encoding nucleic acid of the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:73.

[0140] In some preferred embodiments, the first antigen binding portion binds to a BCMA antigen, and the nucleic acid encoding the first light chain variable region of the first antigen binding portion is selected from the nucleotide sequences SEQ ID NOs:123 and 125; and / or the nucleic acid encoding the first heavy chain variable region of the first antigen binding portion is selected from the nucleotide sequences SEQ ID NOs:109 and 121.

[0141] In some preferred embodiments, the nucleic acid encoding the first light chain variable region of the first antigen binding portion has the nucleotide sequence SEQ ID NO:123, and the nucleic acid encoding the first heavy chain variable region of the first antigen binding portion has the nucleotide sequence SEQ ID NO:109.

[0142] In some preferred embodiments, the nucleic acid encoding the first light chain variable region of the first antigen binding portion has the nucleotide sequence SEQ ID NO:125, and the nucleic acid encoding the first heavy chain variable region of the first antigen binding portion has the nucleotide sequence SEQ ID NO:109.

[0143] In some preferred embodiments, the nucleic acid encoding the first light chain variable region of the first antigen binding portion has the nucleotide sequence SEQ ID NO:123, and the nucleic acid encoding the first heavy chain variable region of the first antigen binding portion has the nucleotide sequence SEQ ID NO:121.

[0144] In some preferred embodiments, the nucleic acid encoding the first light chain variable region of the first antigen binding portion has the nucleotide sequence SEQ ID NO:125, and the nucleic acid encoding the first heavy chain variable region of the first antigen binding portion has the nucleotide sequence SEQ ID NO:121.

[0145] In some preferred embodiments, the encoding nucleic acid for the first light chain of the first antigen-binding moiety is selected from the nucleotide sequences SEQ ID NOs:81 and 85, and / or the encoding nucleic acid for the first heavy chain of the first antigen-binding moiety is selected from the nucleotide sequences SEQ ID NOs:83 and 87.

[0146] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:81, and the encoding nucleic acid of the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:83.

[0147] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:85, and the encoding nucleic acid of the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:83.

[0148] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:81, and the encoding nucleic acid of the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:87.

[0149] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:85, and the encoding nucleic acid of the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:87.

[0150] In some preferred embodiments, the first antigen-binding portion binds to a GPC3 antigen, and the nucleic acid encoding the first light chain variable region of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:127; and / or the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:129.

[0151] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen-binding moiety is selected from the nucleotide sequences SEQ ID NOs:89 and 93, and / or the encoding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from the nucleotide sequences SEQ ID NOs:91 and 95.

[0152] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:89, and the encoding nucleic acid of the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:91.

[0153] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:93, and the encoding nucleic acid of the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:95.

[0154] In some preferred embodiments, a first antigen-binding portion of the bispecific antibody binds to a CD20 antigen and a second antigen-binding portion binds to a CD3 antigen, and the encoding nucleic acid of a first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:55, the encoding nucleic acid of a first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:57, the encoding nucleic acid of a second light chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:59, and the encoding nucleic acid of a second heavy chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:61.

[0155] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:63, and the encoding nucleic acid of the first heavy chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:65, the encoding nucleic acid of the second light chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:67, and the encoding nucleic acid of the second heavy chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:69, the encoding nucleic acid of the second light chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:59, and the encoding nucleic acid of the second heavy chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:61.

[0156] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen-binding moiety is selected from the nucleotide sequence SEQ ID NO:71 and the encoding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from the nucleotide sequence SEQ ID NO:73, the encoding nucleic acid of the second light chain of the second antigen-binding moiety is selected from the nucleotide sequence SEQ ID NO:67 and the encoding nucleic acid of the second heavy chain of the second antigen-binding moiety is selected from the nucleotide sequence SEQ ID NO:69.

[0157] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen-binding moiety is selected from the nucleotide sequence SEQ ID NO:63 and the encoding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from the nucleotide sequence SEQ ID NO:65, the encoding nucleic acid of the second light chain of the second antigen-binding moiety is selected from the nucleotide sequence SEQ ID NO:59 and the encoding nucleic acid of the second heavy chain of the second antigen-binding moiety is selected from the nucleotide sequence SEQ ID NO:61.

[0158] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:55, the encoding nucleic acid of the first heavy chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:57, the encoding nucleic acid of the second light chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:67, and the encoding nucleic acid of the second heavy chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:69.

[0159] In some preferred embodiments, a first antigen-binding portion of the bispecific antibody binds a BCMA antigen and a second antigen-binding portion binds a CD3 antigen, wherein the nucleic acid encoding the first light chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:123, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:109; the nucleic acid encoding the second light chain variable region of the second antigen-binding portion is the nucleotide sequence SEQ ID NO:19, and the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion is the nucleotide sequence SEQ ID NO:51.

[0160] In some preferred embodiments, the nucleic acid encoding the first light chain variable region of the first antigen binding portion has the nucleotide sequence SEQ ID NO:125, the nucleic acid encoding the first heavy chain variable region of the first antigen binding portion has the nucleotide sequence SEQ ID NO:109; the nucleic acid encoding the second light chain variable region of the second antigen binding portion has the nucleotide sequence SEQ ID NO:19, and the nucleic acid encoding the second heavy chain variable region of the second antigen binding portion has the nucleotide sequence SEQ ID NO:51.

[0161] In some preferred embodiments, the nucleic acid encoding the first light chain variable region of the first antigen binding portion has the nucleotide sequence SEQ ID NO:123, the nucleic acid encoding the first heavy chain variable region of the first antigen binding portion has the nucleotide sequence SEQ ID NO:121; the nucleic acid encoding the second light chain variable region of the second antigen binding portion has the nucleotide sequence SEQ ID NO:19, and the nucleic acid encoding the second heavy chain variable region of the second antigen binding portion has the nucleotide sequence SEQ ID NO:51.

[0162] In some preferred embodiments, the nucleic acid encoding the first light chain variable region of the first antigen binding portion has the nucleotide sequence SEQ ID NO:125, the nucleic acid encoding the first heavy chain variable region of the first antigen binding portion has the nucleotide sequence SEQ ID NO:121; the nucleic acid encoding the second light chain variable region of the second antigen binding portion has the nucleotide sequence SEQ ID NO:19, and the nucleic acid encoding the second heavy chain variable region of the second antigen binding portion has the nucleotide sequence SEQ ID NO:51.

[0163] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:81, the encoding nucleic acid of the first heavy chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:83, the encoding nucleic acid of the second light chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:67, and the encoding nucleic acid of the second heavy chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:69.

[0164] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:85, the encoding nucleic acid of the first heavy chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:83, the encoding nucleic acid of the second light chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:67, and the encoding nucleic acid of the second heavy chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:69.

[0165] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:81, the encoding nucleic acid of the first heavy chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:87, the encoding nucleic acid of the second light chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:67, and the encoding nucleic acid of the second heavy chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:69.

[0166] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:85, the encoding nucleic acid of the first heavy chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:87, the encoding nucleic acid of the second light chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:67, and the encoding nucleic acid of the second heavy chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:69.

[0167] In some preferred embodiments, a first antigen-binding portion of the bispecific antibody binds to the GPC3 antigen and a second antigen-binding portion binds to the CD3 antigen, wherein the nucleic acid encoding the first light chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:127, the nucleic acid encoding the first heavy chain variable region of the first antigen-binding portion is the nucleotide sequence SEQ ID NO:129; the nucleic acid encoding the second light chain variable region of the second antigen-binding portion is the nucleotide sequence SEQ ID NO:19, and the nucleic acid encoding the second heavy chain variable region of the second antigen-binding portion is the nucleotide sequence SEQ ID NO:51.

[0168] In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:89, the encoding nucleic acid of the first heavy chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:91, the encoding nucleic acid of the second light chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:67, and the encoding nucleic acid of the second heavy chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:69. In some preferred embodiments, the encoding nucleic acid of the first light chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:93, the encoding nucleic acid of the first heavy chain of the first antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:95, the encoding nucleic acid of the second light chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:67, and the encoding nucleic acid of the second heavy chain of the second antigen binding moiety is selected from the nucleotide sequence SEQ ID NO:69.

[0169] In one aspect, the disclosure provides a vector comprising the nucleic acid.

[0170] In one aspect, the disclosure provides a cell comprising the nucleic acid or vector.

[0171] In one aspect, the disclosure provides a composition comprising said bispecific antibody or antigen-binding portion thereof, nucleic acid, vector, and / or cell.

[0172] In one aspect, the disclosure provides an antibody-drug conjugate comprising said bispecific antibody or an antigen-binding portion thereof covalently linked to a therapeutic moiety.

[0173] In some embodiments, the therapeutic moiety is selected from a cytotoxic moiety, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a degradative peptide, or a radioisotope.

[0174] The antibodies of the present disclosure are useful as therapeutic or diagnostic tools for a variety of diseases in which various tumor antigens are adversely expressed or expressed.

[0175] In one embodiment of the disease associated with a tumor antigen, expression of the tumor antigen in cells of a diseased tissue or organ is increased compared to the state in a healthy tissue or organ. By increased, it is meant increased by 10% or more, in particular 20% or more, 50% or more, 100% or more, 200% or more, 500% or more, 1000% or more, 10000% or more or more. In one embodiment, expression is found only in the diseased tissue and expression in the corresponding healthy tissue is suppressed. According to the present disclosure, the disease associated with a tumor antigen includes tumors.

[0176] In some embodiments, the disease associated with a tumor antigen is a CD20-associated disease. In some preferred embodiments, the CD20-associated disease comprises a B cell disease, such as a B cell proliferative disease, in particular a CD20 positive B cell disease, preferably the disease is selected from non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), and Hodgkin's lymphoma (HL).

[0177] In some embodiments, the disease associated with a tumor antigen is a BCMA associated disease, preferably the BCMA associated disease comprises a B cell disease, preferably the disease is cancer, more preferably the cancer is multiple myeloma, malignant plasmacytoma, Hodgkin's lymphoma, nodular lymphocyte predominant Hodgkin's lymphoma, Kahler's disease and myeloid leukemia, plasma cell leukemia, plasmacytoma, B cell prolymphocytic leukemia, hairy cell leukemia, B cell non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), ), chronic myeloid leukemia (CML), follicular lymphoma, Burkitt lymphoma, marginal zone lymphoma, mantle cell lymphoma, large cell lymphoma, precursor B-lymphocytic lymphoma, myeloid leukemia, Waldenstrom 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, Waldenstrom macroglobulinemia, nodal marginal zone B-cell lymphoma lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymphoma, lymphomatoid granulomatosis, T-cell / histiocyte-rich large B-cell lymphoma, primary central nervous system lymphoma, primary cutaneous diffuse large B-cell lymphoma (leg type), elderly EBV-positive diffuse large B-cell lymphoma, 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 in HHV8-associated multicentric Castleman disease, diffuse large B-cell lymphoma and Burkitt's lymphoma, unclassified B-cell lymphoma with characteristics intermediate between diffuse large B-cell lymphoma and classical Hodgkin's lymphoma, and other B-cell associated lymphomas; more preferably, the B-cell disease is a B-cell disorder, and preferably, the plasma cell disorder is multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenström's macroglobulinemia, solitary plasmacytoma of bone, extramedullary plasmacytoma, osteosclerosing myeloma, heavy chain disease,selected from monoclonal gammopathy of undetermined significance and smoldering multiple myeloma, preferably the disease is an autoimmune disease such as systemic lupus erythematosus or rheumatoid arthritis;

[0178] In some embodiments, the therapeutic agent comprises an antibody that specifically binds to an activating T cell antigen.

[0179] In one embodiment, the therapeutic agent comprises an antibody that specifically binds to CD3, in particular CD3ε.

[0180] Methods for treating diseases and conditions with the bispecific antibodies of the present disclosure include administering to a mammal a therapeutically effective amount of an antibody or antigen-binding fragment thereof, or a nucleic acid molecule, vector, cell, or pharmaceutical composition according to any of the preceding aspects.

[0181] In some embodiments, the disclosure provides a method of treating or preventing a cancer disease, comprising administering to a patient an antibody capable of binding to GPC3 to a serum level of 40 μg / ml or more. In different embodiments, the antibody is administered to a serum level of 50 μg / ml or more, 150 μg / ml or more, 300 μg / ml or more, 400 μg / ml or more, or 500 μg / ml or more. In different embodiments, the antibody is administered to a serum level of 800 μg / ml or less, 700 μg / ml or less, 600 μg / ml or less, 550 μg / ml or less, or 500 μg / ml or less. In one embodiment, the serum level provided is between 40μg / ml and 700μg / ml, preferably between 40μg / ml and 600μg / ml, preferably between 50μg / ml and 500μg / ml, for example between 150μg / ml and 500μg / ml, or between 300μg / ml and 500μg / ml. As used herein, the term "serum level" refers to the concentration in the serum of the substance under consideration. In one embodiment, serum levels are provided for 7 days or more, or 14 days or more. In one embodiment, the method comprises providing a serum level of 300mg / ml or more. 2 More than 600 mg / m 2or more, and preferably 1500 mg / m 2 Below 1200mg / m 2 Less than or equal to 1000 mg / m 2 This involves administering the following doses of antibody:

[0182] In some embodiments, the present disclosure provides a method for administering a dose of 300 mg / m 2 More than 600 mg / m 2 or more, and preferably 1500 mg / m 2 Below 1200mg / m 2 Less than or equal to 1000 mg / m 2 The present invention provides a method for treating or preventing a cancer disease, comprising administering an antibody capable of binding to GPC3 at the following doses:

[0183] In some embodiments, the present disclosure provides a method for treating or preventing a cancer disease, comprising administering to a patient an antibody capable of binding to GPC3, wherein 50% or more, preferably 60% or more, 70% or more, 80% or more or 90% or more of the patient's cancer cells are GPC3 positive, and / or 40% or more, preferably 50% or more or 60% or more of the patient's cancer cells are positive for surface expression of GPC3. In this regard, the present disclosure further provides a method for treating or preventing a cancer disease, comprising: a. identifying a patient suffering from 50% or more, preferably 60% or more, 70% or more, 80% or more or 90% or more of GPC3 positive cancer cells, and / or 40% or more, preferably 50% or more or 60% or more of GPC3 positive cancer cells, and b. administering to the patient an antibody capable of binding to GPC3. In one embodiment, 95% or more or 98% or more of the patient's cancer cells are positive for GPC3. In one embodiment, 70% or more, 80% or more, or 90% or more of the cancer cells in the patient are positive for surface expression of GPC3.

[0184] In one embodiment of the method of any aspect of the present invention, the outcome of treating the cancer disease is achieving stabilization of the disease, hi one embodiment, the stabilization of the disease lasts for 2 months or more, 3 months or more, or 6 months or more.

[0185] In some embodiments, the present disclosure provides a method of stabilizing a disease state in a cancer patient, comprising administering to the patient an antibody capable of binding to GPC3. In one embodiment, the stabilization of the disease state lasts for 2 months or more, 3 months or more, or 6 months or more.

[0186] In one embodiment of the method of any aspect described herein, the antibody is administered in a single dose or in multiple doses.

[0187] In some embodiments, the present disclosure provides a method of treating or preventing a cancer disease comprising administering to a patient multiple doses of an antibody capable of binding to GPC3.

[0188] According to the present disclosure, when the antibody is administered in multiple doses, the antibody is preferably administered in 3 or more doses, 4 or more doses, 5 or more doses, 6 or more doses, 7 or more doses, 8 or more doses, 9 or more doses, or 10 or more doses, and preferably 30 or less doses, 25 or less doses, 20 or less doses, 15 or less doses, or 10 or less doses. Preferably, the antibody doses are administered at intervals of 7 or more days, 10 or more days, 14 or more days, or 20 or more days. Preferably, the antibody doses are administered at intervals of 7 to 30 days, 10 to 20 days, or preferably about 14 days.

[0189] In one embodiment, the antibody is administered to provide a serum level of 40 μg / ml or more. In a different embodiment, the antibody is administered to provide a serum level of 50 μg / ml or more, 150 μg / ml or more, 300 μg / ml or more, 400 μg / ml or more, or 500 μg / ml or more. In a different embodiment, the antibody is administered to provide a serum level of 800 μg / ml or less, 700 μg / ml or less, 600 μg / ml or less, 550 μg / ml or less, or 500 μg / ml or less. In one embodiment, the serum level provided is 40 μg / ml to 700 μg / ml, preferably 40 μg / ml to 600 μg / ml, preferably 50 μg / ml to 500 μg / ml, for example 150 μg / ml to 500 μg / ml or 300 μg / ml to 500 μg / ml. In one embodiment, the serum level is provided for 7 days or more, or 14 days or more. In one embodiment, the method comprises administering a concentration of 300 mg / m 2 More than 600 mg / m 2 or more, and preferably 1500 mg / m 2 Below 1200mg / m 2 Less than or equal to 1000 mg / m 2 This includes administering the following doses of antibody:

[0190] There is provided use of the antibody or antigen-binding fragment thereof, or the nucleic acid molecule, or the vector, or the cell, or the pharmaceutical composition according to any of the preceding aspects in the manufacture of a medicament for treating a GPC3-related disease in a mammal.

[0191] According to any of the above aspects, optionally the antibody is conjugated to another drug, for example a labeled or cytotoxic conjugate.

[0192] In one aspect, the present disclosure further includes kits including, for example, the antibodies, fragments, homologs, derivatives, nucleic acids, vectors, cells, compositions, etc., of the present disclosure, such as labeled or cytotoxic conjugates, and antibody instructions, conjugates that kill specific types of cells, etc. The instructions may include a guide to use the antibodies, conjugates, etc., in vitro, in vivo, or ex vivo. The antibodies may be in liquid or solid form, and are typically lyophilized. The kits may further include other suitable reagents, such as buffers, reconstitution solutions, and other components required depending on the intended use. Combinations of packaged reagents in predetermined amounts with instructions for their use, such as therapeutic or diagnostic assay use, are also contemplated. If the antibody is labeled, for example with an enzyme, the kit may include a substrate and a cofactor required for the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), etc., may also be included. The relative amounts of the various reagents can be varied to provide a concentrate of the reagent solution, thereby providing user flexibility, space savings, reagent savings, etc. These reagents can also be provided in the form of a dry powder, typically provided lyophilized and containing excipients that, when dissolved, provide a solution of the appropriate concentration of the reagent.

[0193] The present invention provides use of the antibody or functional fragment thereof, or the nucleic acid molecule, or the vector, or the cell, or the pharmaceutical composition, or the kit according to any one of the above aspects in the manufacture of a reagent for inhibiting the binding of GPC3.

[0194] The antibodies of the present disclosure may also be used in immunoassays, purification methods, and other methods that employ immunoglobulins or fragments thereof, such uses being well known to those of skill in the art.

[0195] Correspondingly, the present disclosure further provides a composition comprising an anti-GPC3 antibody or a fragment thereof according to the present disclosure, which can be conveniently combined with a pharma- ceutically acceptable carrier, diluent or excipient, which is a means known in the art.

[0196] The term "pharmaceutical composition" as used in this disclosure refers to a formulation of various preparations. The formulations containing a therapeutically effective amount of a multivalent antibody may be in a sterile liquid solution, liquid suspension, or lyophilized form, and may optionally contain stabilizers or excipients.

[0197] The antibodies of the present disclosure may be used as compositions administered alone or in combination with other active agents.

[0198] In some embodiments, the humanized antibodies of the present disclosure are conjugated to a therapeutic moiety (i.e., a drug). The therapeutic moiety may be, for example, a cytotoxin, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a degradative peptide, or a radioisotope. Such conjugates are also referred to herein as "antibody-drug conjugates" or "ADCs."

[0199] In some embodiments, the antibody is conjugated to a cytotoxic moiety, which may be selected from the group consisting of taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine (cephaeline), mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracenedione, tubulin inhibitors such as maytansine or an analogue or derivative thereof, mitotic inhibitors such as monomethyl auristatin E or F or an analogue or derivative thereof, dolastatin 10 or 15 or an analogue thereof, irinotecan or an analogue thereof, mitoxantrone, mithramycin, actinomycin D, D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, calicheamicin or its analogs or derivatives, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, antimetabolites such as gemcitabine or cladribine, e.g., mechlorethamine, thiopurine, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclosporine, ... alkylating agents such as rofosfamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, and mitomycin C; platinum derivatives such as cisplatin or carboplatin; duocarmycin A, duocarmycin SA, rachelmycin (CC-1065) or analogs or derivatives thereof; antibiotics such as actinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, primycin, and anthramycin (AMC);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 toxins, cholera toxin, shiga-like toxins such as SLT I, SLT II, ​​SLT IIV, LT toxin, C3 toxin, shiga toxin, pertussis toxin, tetanus toxin, Bowman-Birk soybean protease inhibitor, Pseudomonas exotoxins, allorin, saporin, modeccin, geranin, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins such as PAPI, PAPII and PAP-S, momordica charantia inhibitors, curcin, crotin, saponaria officinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin and enomycin toxins, ribonuclease (RNase), DNase I, Staphylococcus aureus endotoxin A, pokeweed antiviral protein, diphtheria toxin, and Pseudomonas endotoxin.

[0200] In some embodiments, the antibody is conjugated to an auristatin or its peptide analog, derivative, or prodrug. Auristatins affect microtubule dynamics, GTP hydrolysis, and nuclear and cell division, and have been shown to have anticancer and antifungal activity. For example, auristatin E can react with p-acetylbenzoic acid or benzoylvaleric acid to generate AEB and AEVB, respectively. Other exemplary auristatin derivatives include AFP, MMAF (monomethyl auristatin F), and MMAE (monomethyl auristatin E). Suitable auristatins, analogs, derivatives and prodrugs of auristatins, as well as linkers suitable for conjugating auristatins with Abs, are described, for example, in U.S. Pat. Nos. 5,635,483, 5,780,588, 6,214,345 and International Patent Publications WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968 and WO205082023.

[0201] In some embodiments, the antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine (PDB), its peptide analogs, derivatives, or prodrugs. Suitable PDBs, 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 PWet al., Bioorg Med Chem Lett 2009;19:6463-6466, and Sagnou et al., Bioorg Med Chem Lett 2000;10(18):2083-2086.

[0202] In some embodiments, the antibody is conjugated to a cytotoxic moiety selected from an anthracycline antibiotic, mertansine, a calicheamicin, a duocarmycin, rachelmycin (CC-1065), dolastatin 10, dolastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, PDB, or an analog, derivative, or prodrug of any of these.

[0203] In some embodiments, the antibody is conjugated to an anthracycline antibiotic or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to mertansine or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to calicheamicin or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to duocarmycin or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to rachelmycin (CC-1065) or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 10 or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 15 or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to monomethyl auristatin E or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to monomethylauristatin F or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to irinotecan or an analog, derivative, or prodrug thereof.

[0204] In some embodiments, the antibody is conjugated to a cytokine (e.g., 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, ancestim, and TNFa).

[0205] In some embodiments, the antibody is conjugated to a radioisotope or a chelate containing a radioisotope. For example, the antibody can be conjugated to a chelating linker (e.g., DOTA, DTPA, or tiuxetan). The antibody can also, or alternatively, include 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, radioisotopes that emit beta or alpha particle radiation, e.g. 131 I, 90 Y, 211 At, 212 Bi, 67 Cu, 186 Re, 188 Re and 212 Pb may also be used.

[0206] Techniques for conjugating molecules to antibodies are well known in the art. Usually, nucleic acid molecules are covalently linked to lysines or cysteines in antibodies via N-hydroxysuccinimide esters or maleimide functional groups, respectively. It has been reported that conjugation methods using engineered cysteines or incorporating unnatural amino acids can improve the identity of conjugates. Those skilled in the art will particularly consider Fc-containing polypeptides engineered with acyl donor glutamine-containing tags (e.g., Gin-containing peptide tags or Q-tags) or endogenous glutamines made reactive by polypeptide engineering (e.g., by amino acid deletion, insertion, substitution, or polypeptide mutation). Transglutaminase can then covalently cross-link amine donating agents (e.g., small molecules that contain or are coupled to reactive amines) to form a stable and homogenous population of engineered Fc-containing polypeptide conjugates in which the amine donating agents are site-specifically conjugated to Fc-containing polypeptides via acyl donor glutamine-containing tags or accessible / exposed / reactive endogenous glutamines (WO2012059882).

[0207] It will be understood that the therapeutic agents of the embodiments are administered with suitable pharma- ceutically acceptable carriers, excipients, and other agents that are incorporated into the formulation to provide improved transfer, delivery, tolerance, etc. These formulations may include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic)-containing carriers (e.g., Lipofectin, TM ), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycols (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. Any of the above mixtures may be used in the treatment or method of treatment of the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation, is physiologically compatible, and is tolerable to the route of administration.

[0208] In one embodiment, the antibody can be used as a therapeutic agent. Such agents are typically used to treat, ameliorate, and / or prevent a disease or condition associated with aberrant tumor antigen expression, activity, and / or signaling in a subject. A treatment regimen can be performed by standard methods by identifying a subject, e.g., a human patient suffering from (or at risk of or suffering from) a disease or disorder associated with aberrant tumor antigen expression, activity, and / or signaling, e.g., a tumor antigen-associated disorder. An antibody preparation, preferably an antibody preparation with high specificity and high affinity for a target antigen, is administered to the subject and generally has an effect by binding to the target. The administered antibody can eliminate, inhibit, or interfere with the expression, activity, and / or signaling function of the target (e.g., tumor antigen). The administered antibody can eliminate, inhibit, or interfere with the binding of the target (e.g., tumor antigen) to an endogenous ligand that naturally binds to it. For example, the antibody binds to a target and modulates, inhibits, suppresses, reduces, antagonizes, neutralizes, and / or otherwise interferes with tumor antigen expression, activity, and / or signaling. In some embodiments, an antibody having heavy and light chain CDRs can be administered to a subject to treat a disease or disorder associated with aberrant tumor antigen expression.

[0209] In another embodiment, antibodies against tumor antigens are used in methods related to tumor antigen localization and / or quantification known in the art (e.g., for determining the level of tumor antigen and / or tumor antigen in an appropriate biological sample, diagnostic methods, protein imaging, etc.). In a particular embodiment, antibodies comprising an antigen-binding domain derived from an antibody specific for a tumor antigen or a derivative, fragment, analog or homolog thereof are used as pharma- ceutical active compounds (hereinafter referred to as "therapeutic agents").

[0210] In another embodiment, tumor antigen polypeptides can be isolated using antibodies specific for tumor antigens by standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies (or fragments thereof) against tumor antigen proteins are used to detect the proteins in biological samples. In some embodiments, tumor antigens are detected in biological samples as part of a clinical trial procedure, for example to determine the efficacy of a particular treatment regimen. Conjugating (i.e., physically linking) the antibody to a detectable substance is advantageous for detection. 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, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminofluorescein, dansylamide chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, fluorescein and aequorin; examples of suitable radioactive materials include 125 I, 131 I, 35 S, or 3 Contains H.

[0211] In another embodiment, the antibody of the present disclosure can be used as a reagent to detect the presence of a tumor antigen or a protein fragment thereof in a sample. In some embodiments, the antibody comprises a detectable label. The antibody is a polyclonal antibody or, more preferably, a monoclonal antibody. An intact antibody or a fragment thereof (e.g., Fab, scFv, or F(ab')2) is used. The term "labeling" with respect to an antibody includes direct labeling of the antibody by conjugating (i.e., physically linking) a detectable substance to the antibody, and indirect labeling of the antibody by reaction with another directly labeled reagent. Examples of indirect labeling include detection of a primary antibody with a fluorescently labeled secondary antibody and end-labeling of an antibody with biotin to allow 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's body. Thus, the term "biological sample" includes blood and fractions or components of blood, including serum, plasma, or lymph. In other words, the detection method according to the embodiment is used for detecting analyte mRNA, protein, or genomic DNA in a biological sample in vitro and in vivo. For example, in vitro detection techniques for analyte mRNA include Northern hybridization and in situ hybridization. In vitro detection techniques for analyte protein 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, J.R.Crowther (ed.), Human Press, Totowa, NJ, 1995. In addition, in vivo detection techniques for analyte protein include introducing a labeled anti-analyte protein antibody into a subject.For example, an antibody can be labeled with a radioactive label, and the presence and location of the radioactive label within a subject can be detected by standard imaging techniques.

[0212] The antibodies and derivatives, fragments, analogs and homologs thereof described herein can be incorporated into pharmaceutical compositions suitable for administration. The principles, considerations and guidelines for the preparation of such compositions and for the selection of the components thereof are well known in the art.

[0213] Such compositions usually include an antibody and a pharma- ceutically acceptable carrier. When an antibody fragment is used, it is preferably a minimal inhibitory fragment that specifically binds to the binding domain of the target protein. For example, based on the variable region sequence of an antibody, a peptide molecule can be designed that retains the ability to bind to the target protein sequence. Such peptides can be produced by chemical synthesis and / or recombinant DNA technology (see, for example, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).

[0214] The term "pharmaceutical acceptable carrier" as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, acceptable for pharmaceutical administration. Suitable pharmaceutical acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference text in the field and is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vectors such as liposomes and fixed oils can also be used. The use of such media and agents for pharmaceutical active substances is well known in the art. Except for conventional media or agents that are incompatible with the antibody, their use in the composition is contemplated.

[0215] The pharmaceutical compositions of the above embodiments are prepared to suit their intended route of administration. Examples of routes of administration include, for example, parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration include a sterile injectable diluent, e.g., water, saline, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents, antibacterial agents, e.g., benzyl alcohol or methyl p-hydroxybenzoate, antioxidants, e.g., ascorbic acid or sodium bisulfite, chelating agents, e.g., ethylenediaminetetraacetic acid (EDTA), buffers, e.g., acetates, citrates, or phosphates, and osmolality regulators, e.g., sodium chloride or dextrose. The pH can be adjusted with acids or bases, e.g., hydrochloric acid or sodium hydroxide. Parenteral formulations can be packaged in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0216] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Pharmaceutically acceptable carriers suitable for intravenous administration include saline, sterile water, Cremophor EL, and the like. TM(BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid for easy injection. It must also be stable under the conditions of manufacture and storage and must be capable of preventing the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. A coating, for example, lecithin, can be used to maintain a desired particle size in the case of dispersions, and surfactants can be used to maintain proper fluidity. The prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, such as sugars, polyols (for example, mannitol, sorbitol), sodium chloride, and the like, in the composition. Prolonged absorption of the injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0217] Sterile injectable solutions are prepared by incorporating the antibody in the required amount in a suitable solvent with one or a combination of the above listed ingredients (as needed), as required, and then filtered and sterilized. Usually, dispersions are prepared by incorporating the antibody in a sterile carrier containing a basic dispersion medium and other required ingredients from those listed above. For sterile powders for preparing sterile injectable solutions, the preparation method includes vacuum drying and freeze-drying of the powder containing the active ingredient and any additional desired ingredients from a sterile-filtered solution of the above ingredients.

[0218] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser or nebulizer that is suitably supplied with a propellant, e.g., a gas such as carbon dioxide.

[0219] Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for permeating the barrier is used in the formulation. Such penetrants are well known in the art and include, for example, detergents for transmucosal administration, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal drops or suppositories. For transdermal administration, one or more antibodies can be formulated into pastes, ointments, gels, or creams well known in the art.

[0220] The compounds can also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas.

[0221] In one embodiment, the antibody can be prepared with a carrier that will protect it from rapid elimination from the body, such as a sustained / controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations are readily apparent to those skilled in the art.

[0222] It is particularly advantageous to prepare parenteral compositions in unit dosage form for ease of administration and uniformity of dosage.As used herein, unit dosage form refers to a physically discrete unit suitable for single administration to a subject to be treated, each unit containing a specific amount of one or more of the above antibodies calculated to produce a desired therapeutic effect in combination with the required pharmaceutical carrier.The specifications of the unit dosage form of the above embodiment are determined and directly depend on the inherent characteristics of the antibody and the specific therapeutic effect to be achieved, as well as the limitations inherent in the technology of preparing such antibodies to treat individuals.

[0223] The pharmaceutical compositions may be included in a container, pack, or dispenser together with instructions for administration.

[0224] The formulations herein may further comprise more than one of the above antibodies depending on the specific condition to be treated, preferably antibodies with complementary activities but not adversely affecting each other. Additionally or alternatively, the composition may comprise an agent that enhances the function of the composition, such as a cytotoxin, cytokine, chemotherapeutic agent, or growth inhibitor. Such molecules may be present in an appropriate combination in an effective amount for the intended purpose. For example, they may be present in a kit or in a combination at the time of use.

[0225] In one embodiment, one or more of the antibodies are used in combination therapy, i.e., in combination with other agents, e.g., therapeutic agents (for treating pathological conditions or disorders, e.g., various forms of cancer, autoimmune disorders, and inflammatory diseases). As used herein, the term "combination" refers to the administration of agents substantially simultaneously, simultaneously, or sequentially. When administered sequentially, preferably, the first compound of the two compounds can still be detected at effective concentrations at the treatment site when administration of the second compound begins. In some cases, "combination" may include the antibody of the present disclosure and another therapeutic agent simultaneously in a kit.

[0226] For example, combination therapy includes the co-preparation and / or co-administration of one or more antibodies according to the present disclosure 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 proliferation inhibitors, as described in more detail below.) Such combination therapy may utilize lower dosages of the administered therapeutic agents, thus avoiding possible toxicities or complications associated with various monotherapies.

[0227] In one embodiment, the therapeutic regimen can effectively reduce cytokine release associated with administration of the T cell activating therapeutic agent to the subject, as compared to a therapeutic regimen in which the corresponding anti-tumor antigen antibody is not administered.

[0228] Although technical features are described herein as part of the same or separate embodiments for clarity and conciseness of description, it will be understood that the scope of the invention may include embodiments consisting of combinations of all or some of the described features.

[0229] FIG. 1 shows the structure of a novel first antigen×CD3κλ bispecific antibody.

[0230] In the present disclosure, as demonstrated by experiments, when freely combined, the λ light chain of the humanized CD3 arm has a tendency to pair with the homologous heavy chain, and the corresponding ratio of the heterologous heavy chain is relatively low; similarly, the κ light chain of the humanized antigen arm also has a tendency to pair with the homologous heavy chain, and the pairing ratio with the humanized CD3 heavy chain is extremely low; the introduction of complementary charge mutants into the Fv further reduces the possibility of mismatch. The Fc portion of the CD20×CD3κλ bispecific antibody adopts a human IgG4 knob-into-hole structure and the mutant Ser 228 Pro, Leu 235 Glu and Pro 329 The Ala maintained stability of the hinge region and reduced interaction with the Fcγ receptor, C1q.

[0231] Working Example

[0232] Example 1: Optimization of anti-CD3 antibody and its activation effect on T cells

[0233] 1. Recombinant Protein Synthesis Human CD3γ (UniProt P09693, Gln23-Asn116) and CD3ε (UniProt P07766, Gln23-Asp126) extracellular domain nucleotide sequences were synthesized, and the C-terminus was fused to human IgG Fc hole or Fc knob, respectively, to form human CD3εγ-Fc heterodimer (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 the C-terminus was fused to cynomolgus monkey IgG Fc hole or Fc knob, respectively. The CD3γ-Fc and CD3ε-Fc heterodimers were expressed by fusing the IgG Fc (hole) to knob (knob) (the amino acid sequence of the IgG Fc (hole) is shown in SEQ ID NO. 3, and the amino acid sequence of the IgG Fc (knob) is shown in SEQ ID NO. 4). The recombinant plasmids expressing CD3γ-Fc and CD3ε-Fc were mixed with 3 mg / mL PEI (Polysciences, #24765-2) and co-transfected into HEK293E cells (medium OPM-293 CD03 DPM), and cultured at 37°C, 120 rpm, 5% CO2 for 7 days. The culture supernatants were collected and purified by Protein A affinity chromatography to obtain human or cynomolgus CD3εγ-Fc recombinant proteins.

[0234] 2. Humanization of anti-CD3 antibodies A mouse-derived hybridoma anti-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) recognized human and cynomolgus monkey CD3 receptors, and the sequences were as follows:

[0235] Anti-CD3 Mouse Monoclonal Antibody Light Chain Amino Acid Sequence (SEQ ID NO.96): [ka]

[0236] Anti-CD3 Mouse Monoclonal Antibody Heavy Chain Amino Acid Sequence (SEQ ID NO.97): [ka]

[0237] Anti-CD3 mouse monoclonal antibody was humanized, the most identical human germline gene IMGT_hVL7-43 was selected, light chain CDR grafting was performed, human IGLJ3*02 was used as FM4, and human IMGT_hVH3-73 was selected, heavy chain CDR grafting was performed, human IGHJ4*01 was used as FM4. Different heavy and light chain variants were designed to obtain (Table 1). [Table 1]

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

[0239] Nucleic acid sequence [ka]

[0240] The amino acid sequence of hVL2 is shown in SEQ ID NO.10, its encoding nucleic acid is shown in SEQ ID NO.11, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.7, 8 and 9, respectively. [ka]

[0241] Nucleic acid sequence [ka]

[0242] The amino acid sequence of hVL3 is shown in SEQ ID NO.12, its encoding nucleic acid is shown in SEQ ID NO.13, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.14, 15 and 9, respectively. [ka]

[0243] Nucleic acid sequence [ka]

[0244] The amino acid sequence of hVL4 is shown in SEQ ID NO.16, its encoding nucleic acid is shown in SEQ ID NO.17, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.14, 15 and 9, respectively. [ka]

[0245] Nucleic acid sequence [ka]

[0246] The amino acid sequence of hVL5 is shown in SEQ ID NO.18, its encoding nucleic acid is shown in SEQ ID NO.19, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.7, 8 and 21, respectively. [ka]

[0247] Nucleic acid sequence [ka]

[0248] The amino acid sequence of hVL6 is shown in SEQ ID NO.22, its encoding nucleic acid is shown in SEQ ID NO.23, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.7, 20 and 21, respectively. [ka]

[0249] Nucleic acid sequence [ka]

[0250] The amino acid sequence of hVH1 is shown in SEQ ID NO.24, its encoding nucleic acid is shown in SEQ ID NO.25, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.26, 27 and 28, respectively. [ka]

[0251] Nucleic acid sequence [ka]

[0252] 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 NOs.31, 27 and 28, respectively. [ka]

[0253] Nucleic acid sequence [ka]

[0254] 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 NOs.31, 27 and 34, respectively. [ka]

[0255] Nucleic acid sequence [ka]

[0256] The amino acid sequence of hVH4 is shown in SEQ ID NO.35, its encoding nucleic acid is shown in SEQ ID NO.36, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.31, 27 and 37, respectively. [ka]

[0257] Nucleic acid sequence [ka]

[0258] The amino acid sequence of hVH5 is shown in SEQ ID NO.38, its encoding nucleic acid is shown in SEQ ID NO.39, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.31, 27 and 40, respectively. [ka]

[0259] Nucleic acid sequence [ka]

[0260] The amino acid sequence of hVH6 is shown in SEQ ID NO.41, its encoding nucleic acid is shown in SEQ ID NO.42, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.31, 27 and 43, respectively. [ka]

[0261] Nucleic acid sequence [ka]

[0262] 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 NOs.46, 47 and 28, respectively. [ka]

[0263] Nucleic acid sequence [ka]

[0264] 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 NOs. 26, 27 and 28, respectively. [ka]

[0265] Nucleic acid sequence [ka]

[0266] 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 NOs.26, 27 and 28, respectively. [ka]

[0267] Nucleic acid sequence [ka]

[0268] The amino acid sequence of hVH10 is shown in SEQ ID NO.52, its encoding nucleic acid is shown in SEQ ID NO.53, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.26, 27 and 28, respectively. [ka]

[0269] Nucleic acid sequence [ka]

[0270] The entire sequences of the humanized light and heavy chain mutants were synthesized, and then cloned into eukaryotic expression vectors containing the antibody λ light chain constant region or the human IgG4 heavy chain constant region CH1-CH3, and co-transfected into HEK293E cells. After culturing at 37°C, 120 rpm, and 5% CO2 for 5 to 6 days, the culture supernatant was collected and purified using a Protein A chromatography column.

[0271] 3. Affinity of CD3 humanized antibody Human CD3εγ protein was coated overnight at 4℃. After blocking with 2% skim milk, anti-CD3 antibodies were added to each well at different dilutions and incubated for 1 hour. HPR-labeled goat anti-human IgG Fc was added as the secondary antibody, and the color was developed with TMB solution. The reaction was then terminated with concentrated sulfuric acid, and the absorbance was read at 450 nm. Figure 2 shows that CD3 humanized antibodies (including aCD3-hVH1 / VL5, aCD3-hVH8 / VL1, aCD3-hVH8 / VL5, aCD3-hVH9 / VL1, aCD3-hVH9 / VL2, aCD3-hVH9 / VL3, aCD3-hVH9 / VL5) bind to human CD3εγ protein, and CD3 humanized antibodies bind to CD3εγ recombinant protein with high affinity.

[0272] Jurkat cells in the logarithmic growth phase were blocked with 3% BSA for 30 min and plated in a U-shaped 96-well plate at 5 × 10 4Cells / well were added, centrifuged, and the supernatant was discarded. 50 μL of gradient-diluted antibody (antibody concentration was diluted 3-fold in a 5-fold gradient from 30 μg / ml) was added to each well and incubated for 1 hour at 4° C. After washing to remove the primary antibody, Alexa Fluro647-labeled goat anti-human IgG Fc (Jackson ImmunoResearch, 109-606-170) diluted 1:300 was added as the secondary antibody and incubated for 45 minutes at 4° C. After washing, each well was resuspended in 50 μl of PBS and subjected to FACS (iQue, Intellicyt) detection. The results are shown in Figure 3. The CD3 humanized antibodies bound to Jurkat cells, and among them, the CD3 humanized antibodies hVH9 / VL5 (aCD3-hVH9 / VL5) and hVH9 / VL2 (aCD3-hVH9 / VL2) bound to Jurkat cells with medium affinity, both significantly weaker than the reference antibody OKT3.

[0273] Table 2 shows the affinity of the CD3 humanized antibodies to the CD3 recombinant protein and Jurkat cells. [Table 2]

[0274] 4. Cross-recognition of human and cynomolgus monkey CD3 antigens by CD3 humanized antibodies Human CD3εγ protein and cynomolgus CD3εγ protein were coated overnight at 4℃. After blocking with 2% skim milk, anti-CD3 antibodies were added to each well at different dilutions and incubated for 1 hour. HPR-labeled goat anti-human IgG Fc was added as the secondary antibody, and the color was developed with TMB solution. The reaction was then terminated with concentrated sulfuric acid, and the absorbance was read at 450nm. Figure 4 shows that the CD3 humanized antibodies hVH9 / VL5 (aCD3-hVH9 / VL5) and hVH9 / VL2 (aCD3-hVH9 / VL2) both simultaneously bound to human CD3γ and cynomolgus CD3γ proteins.

[0275] Example 2 Construction of a CD20×CD3κλ bispecific antibody formed from different types of light chains

[0276] 1. Construction of CD20×CD3κλ bispecific antibody A novel T cell κλ bispecific antibody with native IgG conformation was constructed using CD3 humanized antibody hVH9 / VL5 (heavy chain paired with λ light chain) and humanized CD20 antibody (heavy chain paired with κ light chain).

[0277] As shown in FIG. 5, five CD20×CD3κλ bispecific antibodies were designed and constructed as follows: 1) CD20×CD3κλ001: native sequence with CD3 arm and CD20 antigen arm retained; 2) CD20×CD3κλ002: CD20 antigen arm and CD3 arm are charged mutant (Vκ CD20 :Gln 38 Lys;VH CD20 :Gln 39 Glu; Vλ CD3 :Gln 40 Glu;VH CD3 :Gln 39 Bispecific antibodies co-injected with Lys; 3) CD20×CD3κλ003: Based on CD20×CD3κλ002, a complementary charge pair between CH1 / Cκ (Vκ-Ck CD20 :Gln 38 Lys / Glu 123 Lys / Gln 124 Lys;V H -C H 1 CD20 :Gln 39 Glu / Lys 152 Glu / Lys 218 Glu;Vλ CD3 :Gln 40 Glu;VH CD3 :Gln 39 bispecific antibodies with additional Lys; 4) CD20×CD3κλ004: CD20 antigen arm only contains a charge variant (Vκ CD20 :Gln 38 Lys;VH CD20 :Gln39 Bispecific antibodies incorporating Glu; 5) CD20×CD3κλ005: A charge variant in the CD3 arm (Vλ CD3 :Gln 40 Glu;VH CD3 :Gln 39 A bispecific antibody incorporating Lys.

[0278] The corresponding sequences are shown in Table 3, and the reference antibody CD20xCD3-crossFab was constructed according to the CrossFab method (Schaefer W et al., PNAS 2011). [Table 3]

[0279] CD20×CD3κλ001: CD20 arm kappa light chain SEQ ID NO.54 [ka]

[0280] Nucleotide sequence SEQ ID NO.55 [ka]

[0281] CD20 arm heavy chain (heavy chain 1) SEQ ID NO.56 [ka]

[0282] Nucleotide sequence SEQ ID NO. 57 [ka]

[0283] CD3 arm lambda light chain SEQ ID NO.58 [ka]

[0284] Nucleotide sequence SEQ ID NO.59 [ka]

[0285] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.60 [ka]

[0286] Nucleotide sequence SEQ ID NO.61 [ka]

[0287] CD20×CD3κλ002: CD20 arm kappa light chain SEQ ID NO.62 [ka]

[0288] Nucleotide sequence SEQ ID NO.63 [ka]

[0289] CD20 arm heavy chain (heavy chain 1) SEQ ID NO.64 [ka]

[0290] Nucleotide sequence SEQ ID NO.65 [ka]

[0291] CD3 arm lambda light chain SEQ ID NO.66 [ka]

[0292] Nucleotide sequence SEQ ID NO.67 [ka]

[0293] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.68 [ka]

[0294] Nucleotide sequence SEQ ID NO.69 [ka]

[0295] CD20×CD3κλ003: CD20 arm kappa light chain SEQ ID NO.70 [ka]

[0296] Nucleotide sequence SEQ ID NO.71 [ka]

[0297] CD20 arm heavy chain (heavy chain 1) SEQ ID NO.72 [ka]

[0298] Nucleotide sequence SEQ ID NO. 73 [ka]

[0299] CD3 arm lambda light chain SEQ ID NO.66 [ka]

[0300] Nucleotide sequence SEQ ID NO.67 [ka]

[0301] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.68 [ka]

[0302] Nucleotide sequence SEQ ID NO.69 [ka]

[0303] CD20×CD3κλ004: CD20 arm kappa light chain SEQ ID NO.62 [ka]

[0304] Nucleotide sequence SEQ ID NO.63 [ka]

[0305] CD20 arm heavy chain (heavy chain 1) SEQ ID NO.64 [ka]

[0306] Nucleotide sequence SEQ ID NO.65 [ka]

[0307] CD3 arm lambda light chain SEQ ID NO.58 [ka]

[0308] Nucleotide sequence SEQ ID NO.59 [ka]

[0309] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.60 [ka]

[0310] Nucleotide sequence SEQ ID NO.61 [ka]

[0311] CD20×CD3κλ005: CD20 arm kappa light chain SEQ ID NO.54 [ka]

[0312] Nucleotide sequence SEQ ID NO.55 [ka]

[0313] CD20 arm heavy chain (heavy chain 1) SEQ ID NO.56 [ka]

[0314] Nucleotide sequence SEQ ID NO.57 [ka]

[0315] CD3 arm lambda light chain SEQ ID NO.66 [ka]

[0316] Nucleotide sequence SEQ ID NO.67 [ka]

[0317] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.68 [ka]

[0318] Nucleotide sequence SEQ ID NO.69 [ka]

[0319] CD20×CD3 crossFab CD20 arm kappa light chain SEQ ID NO.70 [ka]

[0320] Nucleotide sequence SEQ ID NO.71 [ka]

[0321] CD20 arm heavy chain (heavy chain 1) SEQ ID NO.74 [ka]

[0322] Nucleotide sequence SEQ ID NO.75 [ka]

[0323] CD3 arm lambda light chain SEQ ID NO.76 [ka]

[0324] Nucleotide sequence SEQ ID NO.77 [ka]

[0325] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.78 [ka]

[0326] Nucleotide sequence SEQ ID NO. 79 [ka]

[0327] 2. Expression and Purification of CD20×CD3κλ Bispecific Antibody Plasmids encoding the corresponding antibody fragments were mixed in 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, mixed with 3 mg / mL PEI, and then co-transfected into CHO-S cells. The cells were cultured in 500 mL of CD CHO AGT medium (Gibco # 12490-001) at 37 ° C, 5% CO2, and 150 rpm, and after each transient transfection, 4% CHO Feed C + feed (Gibco # A25031-05) was added on the 2nd, 4th, and 6th days. When the cell activity decreased to about 85%, the fermentation liquid was harvested, filtered, and purified by Protein A affinity chromatography. The CD20xCD3κλ bispecific antibodies constructed based on different light chain types had monomeric purity approaching or exceeding 90% after one-step purification with Protein A, whereas the reference antibody CD20xCD3-crossFab had a monomeric purity below 80% (Table 4) and a κλ light chain ratio approaching 1:1 (Figure 6). [Table 4]

[0328] The CD20xCD3κλ bispecific antibodies were further purified by Capto S ImpAct ion exchange chromatography, gradient elution from 50 to 300 mM NaCl, 50 mM phosphate, pH 6.4, and the elution peaks were pooled and shown to have >99% monomer content by SEC-HPLC (Figure 7). The light chain mismatch ratios were extremely low (<1%) in purified samples of CD20xCD3κλ002 and CD20xCD3κλ003, and no CD3 or CD20 homodimers were detected (Figure 8).

[0329] 3. Binding activity of CD20×CD3κλ bispecific antibodies The affinity of the bispecific antibody CD20 antigen arm was determined by detecting the binding to CD20-overexpressed stably transfected cells or CD20+ tumor cells, respectively, and the affinity of the bispecific antibody CD3 arm was determined by detecting the binding to CD3 recombinant antigen, Jurkat cells or freshly isolated peripheral blood T cells, respectively. The detection results showed that the affinity of the novel CD20×CD3κλ bispecific antibody to tumor cells was about 3-5 times higher than that to T cells. The positive control antibody bsAB1 was synthesized, expressed and prepared according to the literature US20170174781.

[0330] (1) Binding of CD20×CD3κλ bispecific antibodies to human and cynomolgus monkey CD20 stably transfected cells The CHO-human CD20 and CHO-cynomolgus monkey CD20 stably transfected cells produced in Example 1 in the logarithmic growth phase were harvested and cultured at 5 × 10 5The cell suspension was adjusted to 100 cells / ml, and 100 μl / well of the cell suspension was added to a U-shaped 96-well plate, centrifuged at 300 g for 5 minutes, the supernatant was discarded, and 100 μL of gradient-diluted antibody (initial concentration 1800 nM, 3-fold dilution, 10 gradient) was added to each well and incubated at 4 ° C for 60 minutes. Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added as a secondary antibody at 50 μL / well, incubated on ice for 20 minutes, washed once, and then propidium iodide (PI) solution (1:300) was added at 50 μL / well, incubated for 5 minutes, and detected by flow cytometry. As shown in Figure 9 and Table 5, the CD20 × CD3 κ λ bispecific antibody bound to the cellular CD20 receptor with high affinity, and the affinity to human CD20 stably transfected cells was equivalent to the affinity to cynomolgus monkey CD20 stably transfected cells. [Table 5]

[0331] (2) Binding of CD20×CD3κλ bispecific antibodies to human CD20+ tumor cells SU-DHL-4, Raji, and NALM-6 cells in the logarithmic growth phase were taken, and 200 μg / mL of mouse IgG (Jackson ImmunoResearch, 115-005-03) was added, blocked in an ice bath for 30 minutes, and the cells were diluted with 4% fetal bovine serum at 5 × 10 5The cells / mL were adjusted, 100 μL / well was added to a U-shaped 96-well plate, centrifuged at 300 g for 5 minutes, the supernatant was discarded, and 100 μL of gradient diluted antibody (initial concentration 1800 nM, 3-fold dilution, 10 gradient) was added to each well and incubated at 4 ° C for 60 minutes. The primary antibody was removed by washing, and Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) 50 μL / well was added, incubated on ice for 20 minutes, washed once, and then PI 50 μL / well was added, incubated for 5 minutes, and detected by flow cytometry. The detection results are shown in Figure 10 and Table 6, and the CD20 × CD3 κ λ bispecific antibody bound to CD20 + tumor cells SU-DHL-4, Raji and NALM-6 with high affinity.

[0332] (3) Binding of CD20×CD3κλ bispecific antibodies to Jurkat cells Jurkat cells in the logarithmic growth phase were taken, and 200 μg / mL of mouse IgG (Jackson ImmunoResearch, 115-005-03) was added and blocked in an ice bath for 30 minutes. The cells were diluted to 5 × 10 5 The cells were adjusted to 100 μL / mL, added to a U-shaped 96-well plate at 100 μL / well, centrifuged at 300 g to discard the supernatant, and 100 μL of gradient diluted antibody (initial concentration 1800 nM, 3-fold dilution, 10 gradient) was added to each well and incubated at 4 ° C for 60 minutes. Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) 50 μL / well was added as a secondary antibody, incubated on ice for 20 minutes, washed once, added PI 50 μL / well, incubated for 5 minutes, and detected by flow cytometry (BD C6). As a detection result, as shown in Figure 11 and Table 6, the CD20 × CD3 κλ bispecific antibody bound to human leukemia T cell line Jurkat cells with medium affinity and inhibited EC 50 was approximately 71 to 120 nM, which was approximately 10 times lower than the binding affinity of the CD20 antigen arm to the CD20 receptor.

[0333] (4) Binding of CD20×CD3κλ bispecific antibodies to human peripheral blood T cells Fresh human peripheral blood was collected and PBMCs were isolated using Ficoll.Paque Plus (GE, 17-1440-03). PBMCs were diluted with 4% fetal bovine serum (Hyclone, SH30626.06) at 5 × 10 5 The concentration was adjusted to 100 cells / mL, and 100 μL / well was added to a U-shaped 96-well plate. The plate was centrifuged to discard the supernatant, and 100 μL of gradient diluted antibody (initial concentration 1800 nM, 3-fold dilution, 10 gradient) was added to each well and incubated at 4°C for 60 minutes. Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added as a secondary antibody at 50 μL / well, incubated in an ice bath for 20 minutes, washed once, and then PI at 50 μL / well was added, incubated for 5 minutes, and detected by flow cytometry (BD C6). The detection results are shown in Figure 12 and Table 6. The CD20 × CD3 κ λ bispecific antibody was found to inhibit human peripheral blood CD4 + T and CD8 + It recognized T cells and had an affinity for human T cells of approximately 65-98 nM, which was approximately 10-fold weaker than the binding affinity of the CD20 antigen arm to the CD20 receptor, favoring preferential recruitment of the bispecific antibody to tumor cells. [Table 6]

[0334] 4. TDCC effect of CD20×CD3κλ bispecific antibody Freshly isolated PBMCs were taken and mixed with target cells NALM-6, TMD-8 and Toledo cells in the logarithmic growth phase, respectively, with effector cells / target cells = 8:1, gradient diluted antibodies (antibody concentration was diluted 10-fold with 7 gradients from 66.7 nM) 50 μL / well were added, and the mixture was cultured for 24 hours at 5% CO2 and 37 °C. After the culture was completed, 50 μL of the supernatant was transferred to a new black immunoplate, and 50 μL / well of LDH detection substrate was added, and after 10 minutes, the reaction was terminated and LDH release was detected. The remaining cells in the wells were washed twice with 4% fetal bovine serum, and then human IgG 100 μg / mL was added, incubated for 10 minutes, and then antibodies for detecting T cell activation (CD25-PE, CD4-APC, CD69-FITC and CD8-APC) were added and incubated on ice for 20 minutes. After washing, the supernatant was discarded, PI (60 μL / well) was added, incubated on ice for 5 minutes, and detected by flow cytometry. Figures 13A and 13B show the killing of human B lymphoma leukemia cells Nalm-6 and the activation of T cells by CD20×CD3κλ bispecific antibody, respectively. Figures 14A and 14B show the killing of TMD-8 cells and the activation of T cells by CD20×CD3κλ bispecific antibody, respectively. Figures 15A and 15B show the killing of Toledo cells and the activation of T cells by CD20×CD3κλ bispecific antibody, respectively. For tumor cells Nalm-6, TMD-8, and Toledo with different CD20 expression levels, CD20×CD3κλ002 and CD20×CD3κλ003 could contribute to effective killing by T cells, and their killing activity was equivalent to or slightly stronger than that of the control antibody bsAB1, and their activation of T cells was milder than that of the latter.

[0335] 5. Activation of T cell activation pathways by CD20×CD3 κλ bispecific antibodies Jurkat-NFAT-luc reporter cells and CD20-positive target cells (SU-DHL-4, Raji, and NALM-6 cells) in the logarithmic growth phase were harvested and centrifuged, the supernatant was discarded, and 2 × 10 6The cells were resuspended at 100 μL / ml. 50 μL / well of target cells were inoculated into a 96-well plate, centrifuged at 300 g for 5 minutes, the supernatant was discarded, and 50 μL / well of Jurkat-NFAT-luc reporter cells were inoculated into a 96-well plate. 50 μL / well of gradient-diluted CD20×CD3 κλ bispecific antibody or reference antibody KLH×CD3 (initial concentration 20 μg / ml, 10-fold dilution, 10 gradient) was added, and the cells were cultured at 5% CO2 and 37°C for 6 hours. After the culture was completed, 100 μL / well of detection reagent was added according to the ONE-Glo Luciferase Assay System instruction manual, left at room temperature for 3 minutes, and detected with a microplate reader (Biotek Synergy HT). The detection results are shown in Figure 16 and Table 7. When tumor cells with different CD20 expression levels were used as target cells, both CD20×CD3 κλ bispecific antibodies were able to activate the NFAT signal pathway of T cells. [Table 7]

[0336] 6. Binding of CD20×CD3κλ Bispecific Antibody to Fcγ Receptors The His-Tag antibody (50 μg / ml) was coupled to the CM5 chip via the amino group, and the His-tagged FcγRI and FcγRIIA antibodies were H131 and FcγRIIIA V158 Recombinant protein (Sino Biological, #10256-H08H / 10374-H08H1 / 10389-H08H1) was captured, capture time was 40 seconds, flow rate was 10 μL / min, after baseline stabilization, gradient diluted antibody (initial concentration 37.5 μg / mL, 2-fold dilution) was run 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 by fitting with Biacore evaluation software. As can be seen from Table 8, CD20×CD3κλ bispecific antibody has the following affinity constants: FcγRI, FcγRIIA H131 and FcγRIIIA V158The wild-type IgG4 control antibody bound FcγRI with relatively high affinity and FcγRIIA H131 It bound slightly weakly to [Table 8]

[0337] 7. Immune reconstituted mouse subcutaneous Raji tumor model Six to eight-week-old B-NGD female mice (Biocytogen Pharmaceuticals Co., Ltd.) were selected and subcutaneously injected with 3 × 10 Raji cells. 6 The tumor was 60 mm 3 When the mice reached the target concentration, they were randomly divided into groups: 3.0 mg / kg, 0.6 mg / kg, 0.12 mg / kg, and the negative control group, KLH×CD3 3 mg / kg. Each mouse was administered 1×10 PBMC cells. 7 The mice were injected with 100 mg of CD20×CD3κλ bispecific antibody into the tail vein, and the first administration was started 3 days later. The administration interval was once every 5 days, and the mice were administered 3 times in total. The tumor volume and the weight of the mice were monitored, and after the experiment, the mice were killed by decapitation, and the tumors were weighed and recorded. The results are shown in FIG. 17, and the CD20×CD3κλ bispecific antibody showed a dose-dependent in vivo drug efficacy, with tumor inhibition rates of 82% and 89% at medium and high doses, respectively. The tumor-bearing mice tolerated the above doses well and did not show any adverse effects such as weight loss.

[0338] 8. Subcutaneous tumor inoculation model in immunodeficient mice with a mixture of Raji and human PBMCs Select 6- to 8-week-old B-NGD female mice (Biocytogen Pharmaceuticals Co., Ltd.) and inoculate them with Raji (3 × 10 6 ) and human PBMCs (5 × 10 6 The mixture was subcutaneously inoculated into mice to produce tumors with a volume of 60 to 100 mm. 3When the tumor volume reached 100 mg / mL, the mice were randomly divided into groups. The doses were 3.0 mg / mL, 0.6 mg / mL, and 0.12 mg / mL, respectively, and the negative control group was KLH×CD3 3 mg / kg. The administration interval was once every 5 days, and the mice were administered twice in total. The tumor volume and body weight of the mice were monitored, and after the experiment, the mice were killed by decapitation, and the tumors were weighed and recorded. The results are shown in FIG. 18, and the CD20×CD3κλ bispecific antibody showed dose-dependent efficacy in vivo, with tumor inhibition rates of 65%, 98%, and 162% at low, medium, and high doses, respectively, and tumors were completely inhibited or regressed in the high and medium dose groups.

[0339] 9. Efficacy of CD20 × CD3 κλ bispecific antibodies in cynomolgus monkeys Eight cynomolgus monkeys were distributed into four dose groups, with each dose group consisting of two monkeys (half female, half male). The doses received by each dose group were 0.3 mg / kg, 1 mg / kg, 3 mg / kg (once a week for three weeks, a total of four doses) and 1 mg / kg (single dose), respectively, and the dosing regimen is shown in Table 9. During the administration and recovery periods, all monkeys in each group were in good condition, with no toxic reactions, and no deaths or terminal conditions. In each dose group, no obvious abnormal changes were observed in body temperature, the II lead electrocardiogram waveform was normal, and no obvious abnormalities were observed in the indices of heart rate, RR interphase, PR interphase, QT interphase, QRS interval, systolic pressure, diastolic pressure, etc. At different time points after administration, the changes in the numbers of B and T cell populations in peripheral blood were analyzed by flow cytometry, B cells were identified by the cell surface marker CD20 (CD20+ cells) and T cells were identified by CD3 (CD3+ cells). Eight hours after administration, B cells in peripheral blood were rapidly depleted and after 24 hours were below the lower limit of detection (Figure 19). [Table 9]

[0340] Example 3: Construction of a BCMAxCD3κλ bispecific antibody formed from different types of light chains 1. Construction of BCMA×CD3κλ bispecific antibody Using a BCMA humanized antibody containing a κ light chain and a humanized anti-CD3 antibody containing a λ light chain, see Example 2, a novel BCMA-CD3 κ λ humanized bispecific antibody was constructed with a native IgG conformation and a charge variant (Vκ) was added to the BCMA antigen arm and the CD3 arm. BCMA :Gln 42 Lys;VH BCMA :Gln 39 Glu;Vλ CD3 :Gln 40 Glu;VH CD3 :Gln 39 Lys) (sequence shown in Table 10) was introduced into the Fc portion of the bispecific antibody to achieve heterodimeric pairing, and a human IgG4 knob-into-hole structure was employed as the Fc portion of the bispecific antibody to achieve heterodimeric pairing. 228 Pro, Leu 235 Glu and Pro 329 Ala maintains the stability of the hinge region and reduces interaction with FcγR receptors and C1q. [Table 10]

[0341] BCMA arm kappa light chain variable region: SEQ ID NO.122, where LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.99, 100, 101, respectively. [ka]

[0342] BCMA arm kappa light chain variable region nucleotide sequence: SEQ ID NO.123 [ka]

[0343] BCMA×CD3κλ003 BCMA arm kappa light chain: SEQ ID NO.80 [ka]

[0344] Nucleotide sequence: SEQ ID NO.81 [ka]

[0345] BCMA arm heavy chain (heavy chain 1) variable region: SEQ ID NO.108, where HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.96, 97, 98, respectively. [ka]

[0346] BCMA arm heavy chain (heavy chain 1) variable region nucleotide sequence: SEQ ID NO.109 [ka]

[0347] BCMA arm heavy chain (heavy chain 1): SEQ ID NO.82 [ka]

[0348] Nucleotide sequence: SEQ ID NO.83 [ka]

[0349] CD3 arm λ light chain: SEQ ID NO.66 [ka]

[0350] Nucleotide sequence: SEQ ID NO.67 [ka]

[0351] CD3 arm heavy chain (heavy chain 2): SEQ ID NO.68 [ka]

[0352] Nucleotide sequence: SEQ ID NO. 69 [ka]

[0353] BCMA arm kappa light chain variable region: SEQ ID NO.124, where LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.134, 135, 101, respectively. [ka]

[0354] BCMA arm kappa light chain variable region nucleotide sequence: SEQ ID NO.125 [ka]

[0355] BCMA×CD3κλ004 BCMA arm kappa light chain: SEQ ID NO.84 [ka]

[0356] Nucleotide sequence: SEQ ID NO.85 [ka]

[0357] BCMA arm heavy chain (heavy chain 1) variable region: SEQ ID NO.108, where HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.96, 97, 98, respectively. [ka]

[0358] BCMA arm heavy chain (heavy chain 1) variable region nucleotide sequence: SEQ ID NO.109 [ka]

[0359] BCMA arm heavy chain (heavy chain 1): SEQ ID NO.82 [ka]

[0360] Nucleotide sequence: SEQ ID NO.83 [ka]

[0361] CD3 arm λ light chain: SEQ ID NO.66 [ka]

[0362] Nucleotide sequence: SEQ ID NO.67 [ka]

[0363] CD3 arm heavy chain (heavy chain 2): SEQ ID NO.68 [ka]

[0364] Nucleotide sequence: SEQ ID NO.69 [ka]

[0365] BCMA×CD3κλ005 BCMA arm kappa light chain variable region: SEQ ID NO.122, where LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.99, 100, 101, respectively. [ka]

[0366] BCMA arm kappa light chain variable region nucleotide sequence: SEQ ID NO.123 [ka]

[0367] BCMA arm kappa light chain: SEQ ID NO.80 [ka]

[0368] Nucleotide sequence: SEQ ID NO.81 [ka]

[0369] BCMA arm heavy chain (heavy chain 1) variable region: SEQ ID NO.120, where HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.96, 97, 98, respectively. [ka]

[0370] BCMA arm heavy chain (heavy chain 1) variable region nucleotide sequence: SEQ ID NO.121 [ka]

[0371] BCMA arm heavy chain (heavy chain 1): SEQ ID NO.86 [ka]

[0372] Nucleotide sequence: SEQ ID NO.87 [ka]

[0373] CD3 arm λ light chain: SEQ ID NO.66 [ka]

[0374] Nucleotide sequence: SEQ ID NO.67 [ka]

[0375] CD3 arm heavy chain (heavy chain 2): SEQ ID NO.68 [ka]

[0376] Nucleotide sequence: SEQ ID NO.69 [ka]

[0377] BCMA×CD3κλ006 BCMA arm kappa light chain variable region: SEQ ID NO.124, where LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs.134, 135, 101, respectively. [ka]

[0378] BCMA arm kappa light chain variable region nucleotide sequence: SEQ ID NO.125 [ka]

[0379] BCMA arm kappa light chain: SEQ ID NO.84 [ka]

[0380] Nucleotide sequence: SEQ ID NO.85 [ka]

[0381] BCMA arm heavy chain (heavy chain 1) variable region: SEQ ID NO.120, where HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs.96, 97, 98, respectively. [ka]

[0382] BCMA arm heavy chain (heavy chain 1) variable region nucleotide sequence: SEQ ID NO.121 [ka]

[0383] BCMA arm heavy chain (heavy chain 1): SEQ ID NO.86 [ka]

[0384] Nucleotide sequence: SEQ ID NO.87 [ka]

[0385] CD3 arm λ light chain: SEQ ID NO.66 [ka]

[0386] Nucleotide sequence: SEQ ID NO.67 [ka]

[0387] CD3 arm heavy chain (heavy chain 2): SEQ ID NO.68 [ka]

[0388] Nucleotide sequence: SEQ ID NO.69 [ka]

[0389] 2. Expression and Purification of BCMA x CD3κλ Bispecific Antibody Plasmids encoding the corresponding antibody fragments were mixed in 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, mixed with 3 mg / mL PEI, and co-transfected into CHO-S cells. Cultured in 500 mL of CD CHO AGT medium (Gibco #12490-001) at 37°C, 5% CO2, 150 rpm, and supplemented with 4% CHO Feed C+ feed (Gibco #A25031-05) on days 2, 4, and 6 after each transient transfection. When the cell activity dropped to about 85%, the fermentation liquid was harvested, filtered, and pre-purified by Protein A affinity chromatography. SEC-HPLC showed that the monomer content was close to or exceeded 92%, and after passing through Capto S ImpAct ion exchange chromatography, the monomer content was further improved to more than 98-99% (Table 11). [Table 11]

[0390] 3. Binding activity of BCMA×CD3κλ bispecific antibodies (1) Measurement of the affinity of BCMA×CD3κλ bispecific antibodies to antigens Human or cynomolgus monkey BCMA or CD3εγ recombinant antigens were coupled to a CM5 chip (GE healthcare) at 10μg / mL via the amino group, and the antigen binding amount was controlled to about 200RU. After the baseline was stabilized, gradient-diluted antibodies (diluted 2-fold with seven gradients from 10μg / mL) were run through the chip at a flow rate of 30μL / min, and the binding time was 350 seconds and the dissociation time was 600 seconds. The affinity constant was obtained by fitting with a 1:1 binding model using Biacore T200 evaluation software. The affinity measurement results are shown in Table 12. [Table 12]

[0391] (2) Binding of BCMA×CD3κλ bispecific antibodies to BCMA+ cells CHO-human BCMA stable transfected cells (CHO-hBCMA), CHO-cynomolgus monkey BCMA stable transfected cells (CHO-cynoBCMA), tumor cells NCI-H929 and RPMI-8226 in the logarithmic growth phase were taken and blocked, and then 100 μL of gradient diluted antibody (initial concentration 1800 nM, 3-fold dilution, 10 gradient) was added to each well and incubated for 60 minutes at 4 ° C. Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) 50 μL / well was added as a secondary antibody, incubated on ice for 20 minutes, washed once, and then PI solution (1:300) 50 μL / well was added, incubated for 5 minutes, and detected by flow cytometry. Figure 20 shows that BCMAxCD3κλ bispecific antibodies bind with high affinity to human and cynomolgus BCMA stably transfected cells, and Figure 21 shows that BCMAxCD3κλ bispecific antibodies bind with high affinity to BCMA+ tumor cells NCI-H929 and RPMI-8226. Binding constants EC 50 is shown in Table 13. [Table 13]

[0392] (3) Binding of BCMA×CD3κλ bispecific antibodies to Jurkat cells Jurkat cells in the logarithmic growth phase were taken, and 200 μg / mL of mouse IgG (Jackson ImmunoResearch, 115-005-03) was added and blocked in an ice bath for 30 minutes. The cells were diluted to 5 × 10 5 The cells / mL were adjusted, 100 μL / well was added to a U-shaped 96-well plate, centrifuged at 300 g, the supernatant was discarded, and 100 μL of gradient diluted antibody (initial concentration 1800 nM, 3-fold dilution, 10 gradient) was added to each well and incubated at 4 ° C for 60 minutes. Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) 50 μL / well was added as a secondary antibody, incubated on ice for 20 minutes, washed once, and then PI 50 μL / well was added, incubated for 5 minutes, and detected by flow cytometry (BD C6). The detection results are shown in Figure 22 and Table 14, and the BCMA × CD3 κ λ bispecific antibody bound to the human leukemia T cell line Jurkat cells with medium affinity.

[0393] (4) Binding of BCMA×CD3κλ bispecific antibodies to peripheral blood T cells Fresh human peripheral blood was collected and PBMCs were isolated using Ficoll.Paque Plus (GE, 17-1440-03). PBMCs were cultured at 5 × 10 cells / mL in 4% fetal bovine serum (Hyclone, SH30626.06). 5The cells were adjusted to 100 μL / mL, added to a U-shaped 96-well plate at 100 μL / well, centrifuged, the supernatant was discarded, and gradient diluted antibody (initial concentration 1800 nM, 3-fold dilution, 10 gradient) was added at 100 μL / well and incubated at 4 ° C for 60 minutes. Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added as a secondary antibody at 50 μL / well, incubated in an ice bath for 20 minutes, washed once, and then PI was added at 50 μL / well, incubated for 5 minutes, and detected by flow cytometry (BD C6). The reference antibody REGN5458 was synthesized and prepared with reference to US20200024356. The detection results are shown in Figure 23 and Table 14. The BCMA x CD3 κλ bispecific antibody recognized human peripheral blood CD4 + T and CD8 + T cells, and its affinity to human T cells was approximately 60-97 nM, both of which were weaker than the binding strength of the BCMA antigen arm to the BCMA receptor, which was favorable for the preferential recruitment of the bispecific antibody to tumor cells. [Table 14]

[0394] 4. TDCC effect of BCMA×CD3κλ bispecific antibody Freshly isolated PBMCs were taken and mixed with logarithmic growth phase target cells NCI-H929 and RPMI-8226 cells, respectively, with effector cells / target cells = 8:1, gradient diluted antibodies (antibody concentration was diluted 10-fold with 7 gradients from 66.7 nM) were added 50 μL / well, and cultured at 5% CO2 and 37 °C for 24 hours. After the culture was completed, 50 μL of the supernatant was transferred to a new black immunoplate, and 50 μL / well of LDH detection substrate was added. After 10 minutes, the reaction was terminated and LDH release was detected. The remaining cells in the wells were washed twice with 4% fetal bovine serum, and then human IgG 100 μg / mL was added, incubated for 10 minutes, and T cell activation detection antibodies (CD25-PE, CD4-APC, CD69-FITC and CD8-APC) were added and incubated on ice for 20 minutes. After washing, the supernatant was discarded, PI (60 μL / well) was added, incubated on ice for 5 minutes, and detected by flow cytometry. Figures 24A and 24B show the killing of NCI-H929 cells and the activation of T cells by BCMA×CD3κλ bispecific antibodies, respectively. Figures 25A and 25B show the killing of RPMI-8226 cells and the activation of T cells by BCMA×CD3κλ bispecific antibodies, respectively. For tumor cells NCI-H929 and RPMI-8226 with different BCMA expression levels, both BCMA×CD3κλ bispecific antibodies can contribute to the effective killing of T cells, and the killing activity is comparable to that of the reference antibody REGN5458.

[0395] 5. Activation of T cell activation pathways by BCMA × CD3κλ bispecific antibodies Jurkat-NFAT-luc reporter cells and BCMA-positive target cells RPMI-8226 in the logarithmic growth phase were taken, centrifuged, the supernatant was discarded, and 2 × 10 6The cells were resuspended at 100 μl / ml. 50 μl / well of target cells were inoculated into a 96-well plate, centrifuged at 300 g for 5 minutes, the supernatant was discarded, 50 μl / well of Jurkat-NFAT-luc reporter cells were inoculated into a 96-well plate, 50 μl / well of gradient-diluted BCMA×CD3κλ bispecific antibody or reference antibody KLH×CD3 (initial concentration 20 μg / ml, 10-fold dilution, 10 gradients) were added to each well, and the wells were cultured at 5% CO2 and 37°C for 6 hours. After the culture was completed, 100 μL of detection reagent was added to each well according to the ONE-Glo Luciferase Assay System instruction manual, the wells were left at room temperature for 3 minutes, and the wells were detected with a microplate reader (Biotek Synergy HT). The detection results are shown in Figure 26. The BCMAxCD3κλ bispecific antibody could activate the NFAT signal pathway of T cells when RPMI-8226 tumor cells were used as target cells, but did not activate the NFAT signal pathway in the absence of target cells.

[0396] 6. Non-specific activation of PBMCs by BCMA × CD3κλ bispecific antibodies Freshly isolated PBMCs were taken, and 50 μL of gradient-diluted antibody (antibody concentration was diluted 10-fold in 7 gradients from 66.7 nM) was added to each well, and the wells were incubated at 37°C with 5% CO2 for 24 hours. After incubation, 50 μL of the supernatant was transferred to a new black immunoplate, and 50 μL / well of LDH detection substrate was added. After 10 minutes, the reaction was terminated and LDH release was detected. The remaining cells in the wells were washed twice with 4% fetal bovine serum, and then 100 μg / mL of human IgG was added and incubated for 10 minutes. Then, antibodies for detecting T cell activation (CD25-PE, CD4-APC, CD69-FITC and CD8-APC) were added and incubated on ice for 20 minutes. After washing, the supernatant was discarded, and 60 μL / well of PI was added, incubated on ice for 5 minutes, and detected by flow cytometry. The detection results are shown in FIG. 27, which shows that in the absence of target cells, the BCMA×CD3 κλ bispecific antibody had no activating effect on peripheral blood T cells and was comparable to the negative control KLH×CD3.

[0397] 7. Binding of BCMA×CD3κλ humanized bispecific antibodies to Fc receptors The His-Tag antibody was coupled to the CM5 chip at 50 μg / ml via the amino group, and His-tagged FcγRI, FcγRIIA H131 and FcγRIIIA V158 The recombinant proteins were captured, respectively, with a capture time of 40 seconds and a flow rate of 10 μL / min. After the baseline was stabilized, gradient diluted antibodies (initial concentration 37.5 μg / mL, 2-fold diluted) were run through the chip at a flow rate of 30 μL / min, with an association time of 120 seconds and a dissociation time of 200 seconds. The affinity constants were obtained by fitting with the Biacore evaluation software. As can be seen from Figure 28, the BCMA×CD3κλ bispecific antibody binds FcγRI, FcγRIIA H131 and FcγRIIIA V158 The wild-type IgG4 control antibody bound FcγRI with relatively high affinity and FcγRIIA H131 It bound weakly to

[0398] 8. Subcutaneous NCI-H929 tumor transplantation model in immunodeficient mice Six to eight-week-old B-NGD female mice (Biocytogen Pharmaceuticals Co., Ltd.) were selected and subcutaneously inoculated with 2 × 10 NCI-H929 cells (mixed with Matrigel at a ratio of 1:1). 6 The tumor was 60 mm 3 When the mice reached the target concentration, they were randomly divided into groups: 3.0 mg / kg, 0.6 mg / kg, 0.12 mg / kg, and the negative control group, KLH×CD3 3 mg / kg. Each mouse was administered 1×10 PBMC cells. 7The mice were injected into the tail vein with 100 mg of the antibody, and the first administration was started 3 days later, with an administration interval of once every 5 days, for a total of two administrations. The tumor volume and body weight of the mice were monitored once every 2 days, and after the experiment was completed, the mice were killed by decapitation, and the tumors were weighed and recorded. The results are shown in Figure 29, and the BCMA x CD3 κλ bispecific antibody showed a dose-dependent in vivo efficacy, with tumor inhibition rates of 95% and 108% (BCMA x CD3 κλ005) and 94% and 108% (BCMA x CD3 κλ006) in the 3.0 mg / kg and 0.6 mg / kg groups, respectively. The tumor-bearing mice tolerated the above doses well and did not show any adverse effects such as weight loss.

[0399] Example 4: Construction of GPC3×CD3κλ bispecific antibodies formed from different types of light chains 1. Construction of GPC3×CD3κλ bispecific antibody Using a humanized GPC3 antibody containing a κ light chain and a humanized anti-CD3 antibody containing a λ light chain, a novel GPC3-CD3 κλ humanized bispecific antibody having a natural IgG conformation was constructed with reference to Example 2, and a charge mutant (Vκ) was added to 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 To achieve heterodimeric pairing, the Fc portion of the bispecific antibody adopts a human IgG4 knob-into-hole structure and also contains the mutation Ser 228 Pro, Leu 235 Glu and Pro 329 Ala maintains stability of the hinge region and reduces interaction with Fcγ receptors and C1q. [Table 15]

[0400] GPC3×CD3κλ002: GPC3-arm kappa light chain variable region SEQ ID NO.126, where LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 102, 103 and 104, respectively. [ka]

[0401] GPC3 arm kappa light chain variable region nucleotide sequence SEQ ID NO.127 [ka]

[0402] GPC3 arm kappa light chain SEQ ID NO.88 [ka]

[0403] Nucleotide sequence SEQ ID NO.89 [ka]

[0404] GPC3 arm heavy chain (heavy chain 1) variable region SEQ ID NO.128, where HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs. 105, 106 and 107, respectively. [ka]

[0405] GPC3 arm heavy chain (heavy chain 1) variable region nucleotide sequence SEQ ID NO.129 [ka]

[0406] GPC3 arm heavy chain (heavy chain 1) SEQ ID NO.90 [ka]

[0407] Nucleotide sequence SEQ ID NO.91 [ka]

[0408] CD3 arm lambda light chain SEQ ID NO.66 [ka]

[0409] Nucleotide sequence SEQ ID NO.67 [ka]

[0410] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.68 [ka]

[0411] Nucleotide sequence SEQ ID NO.69 [ka]

[0412] GPC3×CD3κλ003: GPC3-arm kappa light chain variable region SEQ ID NO.126, where LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NOs. 102, 103 and 104, respectively. [ka]

[0413] GPC3 arm kappa light chain variable region nucleotide sequence SEQ ID NO.127 [ka]

[0414] GPC3 arm kappa light chain SEQ ID NO.92 [ka]

[0415] Nucleotide sequence SEQ ID NO.93 [ka]

[0416] GPC3 arm heavy chain (heavy chain 1) variable region SEQ ID NO.128, where HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NOs. 105, 106 and 107, respectively. [ka]

[0417] GPC3 arm heavy chain (heavy chain 1) variable region nucleotide sequence SEQ ID NO.129 [ka]

[0418] GPC3 arm heavy chain (heavy chain 1) SEQ ID NO.94 [ka]

[0419] Nucleotide sequence SEQ ID NO.95 [ka]

[0420] CD3 arm lambda light chain SEQ ID NO.66 [ka]

[0421] Nucleotide sequence SEQ ID NO.67 [ka]

[0422] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.68 [ka]

[0423] Nucleotide sequence SEQ ID NO.69 [ka]

[0424] Plasmids encoding the corresponding antibody fragments were mixed in 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, mixed with 3 mg / mL PEI, and then co-transfected into CHO-S cells. The cells were cultured in 500 mL of CD CHO AGT medium (Gibco #12490-001) at 37°C, 5% CO2, and 150 rpm, and 4% CHO Feed C+ feed (Gibco #A25031-05) was added on the 2nd, 4th, and 6th days after transient transfection, respectively. When the cell activity decreased to about 85%, the fermentation liquid was harvested, filtered, and pre-purified by Protein A affinity chromatography. The monomer content was shown to be over 92% by SEC-HPLC, and the monomer content was further improved to over 99.5% by Butyl HP hydrophobic chromatography and Capto Q anion chromatography (Table 16). [Table 16]

[0425] 2. Binding of GPC3 × CD3 κλ bispecific antibodies to GPC3 stably transfected cells CHO-human GPC3, CHO-cynomolgus GPC3 stable transfected cells, or human hepatocellular carcinoma HepG2 tumor cells in the logarithmic growth phase were taken and blocked, and then the cells were diluted to 5 × 10 5The concentration was adjusted to 100 cells / ml, and 100 μl / well of the cell suspension was added to a U-shaped 96-well plate, centrifuged at 300 g for 5 minutes, the supernatant was discarded, and 100 μL of gradient-diluted antibody (diluted 3-fold with a gradient of 10 from an initial concentration of 1800 nM) was added to each well and incubated at 4 ° C for 60 minutes. Alexa Fluro647-labeled goat anti-human IgG Fc (diluted 1:300) 50 μL / well was added as a secondary antibody, incubated on ice for 20 minutes, washed once, and then PI solution (1:300) 50 μL / well was added, incubated for 5 minutes, and detected by flow cytometry. The results are shown in Figures 30-31 and Table 17, and the GPC3 × CD3 κ λ bispecific antibody bound to GPC3 + ​​cells with high affinity. [Table 17]

[0426] 3. Binding of GPC3×CD3κλ bispecific antibody to Jurkat cells Jurkat cells in the logarithmic growth phase were taken, and 200 μg / mL of mouse IgG (Jackson ImmunoResearch, 115-005-03) was added and blocked in an ice bath for 30 minutes. The cells were diluted to 5 × 10 5 The antibody was adjusted to cells / mL, 100 μL / well was added to a U-shaped 96-well plate, centrifuged at 300 g to discard the supernatant, and 100 μL of gradient-diluted antibody (diluted 3-fold with a gradient of 10 from an initial concentration of 1800 nM) was added to each well and incubated at 4 ° C for 60 minutes. Alexa Fluro647-labeled goat anti-human IgG Fc (diluted 1:300) 50 μL / well was added as a secondary antibody, incubated on ice for 20 minutes, washed once, PI 50 μL / well was added, incubated for 5 minutes, and detected by flow cytometry (BD C6). The detection results are shown in Figure 32 and Table 18. The GPC3 × CD3 κ λ bispecific antibody bound to human leukemia T cell line Jurkat cells with medium affinity and EC 50 was 20-40nM.

[0427] 4. Binding of GPC3×CD3κλ bispecific antibody to peripheral blood T cells Fresh human or cynomolgus monkey peripheral blood was collected, and PBMCs were isolated using Ficoll.Paque Plus (GE, 17-1440-03). PBMCs were diluted with 4% fetal bovine serum (Hyclone, SH30626.06) at 5 × 10 5 The concentration was adjusted to 100 cells / mL, 100 μL / well was added to a U-shaped 96-well plate, centrifuged to discard the supernatant, and 100 μL of gradient diluted antibody (diluted 3-fold with a gradient of 10 from an initial concentration of 1800 nM) was added to each well and incubated at 4 ° C for 60 minutes. Alexa Fluro647-labeled goat anti-human IgG Fc (diluted 1:300) 50 μL / well was added as a secondary antibody, incubated in an ice bath for 20 minutes, washed once, PI 50 μL / well was added, incubated for 5 minutes, and detected by flow cytometry (BD Celesta). The detection results are shown in Figure 33 and Table 18, and the GPC3 × CD3 κ λ bispecific antibody bound to human peripheral blood T cells with low affinity. [Table 18]

[0428] 5. TDCC effect of GPC3×CD3κλ bispecific antibody Freshly isolated PBMCs were taken and mixed with target cells HepG2 cells in the logarithmic growth phase, with effector cells / target cells = 10:1, and 50 μL of gradient diluted antibodies (antibody concentration was diluted 10-fold with a 7-fold gradient from 66.7 nM) were added to each well and incubated for 24 hours at 5% CO2 and 37°C. After the incubation, 50 μL of the supernatant was transferred to a new black immunoplate, and 50 μL / well of LDH detection substrate was added. After 10 minutes, the reaction was terminated and LDH release was detected. The remaining cells in the wells were washed twice with 4% fetal bovine serum, and then 100 μg / mL of human IgG was added and incubated for 10 minutes. Then, antibodies for detecting T cell activation (CD25-BV421, CD4-FITC, CD69-BV605 and CD8-APC) were added and incubated on ice for 20 minutes. After washing, the supernatant was discarded, PI (60 μL / well) was added, incubated on ice for 5 minutes, and detected by flow cytometry. Figures 34A and 34B show the killing of HepG2 cells and the activation of T cells by the GPC3×CD3κλ bispecific antibody, respectively.

[0429] 6. Activation of T cell activation pathway by GPC3×CD3κλ bispecific antibody Target cells CHO-human GPC3 in the logarithmic growth phase were taken and centrifuged, the supernatant was discarded, and 2 × 10 5 The target cells were inoculated into a 96-well plate at 50 μl / well and cultured overnight at 5% CO2 and 37°C. Jurkat-NFAT-luc reporter cells in logarithmic growth phase were centrifuged at 300 g for 5 min, the supernatant was discarded, and 4 × 10 6The cells were resuspended at 100 μL / ml, the 96-well plate was removed, the supernatant was discarded, and 25 μL / well of Jurkat-NFAT-luc reporter cells were inoculated into the 96-well plate. 25 μL of gradient-diluted GPC3×CD3 κλ bispecific antibody or control antibody KLH×CD3 (initial concentration 20 μg / ml, 3-fold dilution, 10 gradient) was added to each well, and the cells were cultured at 5% CO2 and 37°C for 6 hours. After the culture was completed, 100 μL of detection reagent was added to each well according to the ONE-Glo Luciferase Assay System instruction manual, and detection was performed using ELISA (MD SpectraMax i3x). The detection results are shown in FIG. 35, and the GPC3×CD3 κλ bispecific antibody was able to activate the NFAT signal pathway of T cells when CHO-human GPC3 was used as the target cell.

[0430] 7. Non-specific activation of PBMCs by GPC3 × CD3κλ bispecific antibodies Freshly isolated PBMCs were taken, 100 μL (10 μg / mL) of antibody was added, and the cells were cultured at 5% CO2 and 37°C for 24 hours. After washing the cells in the wells twice with 4% fetal bovine serum, 100 μg / mL of human IgG was added, incubated for 10 minutes, and T cell activation detection antibodies (CD25-BV421, CD4-FITC, CD69-BV605, and CD8-APC) were added and incubated on ice for 20 minutes. After washing, the supernatant was discarded, 60 μL / well of PI was added, incubated on ice for 5 minutes, and detected by flow cytometry. Referring to US20170267783, a positive control antibody ERY974 was prepared. The detection results are shown in Figure 36. In the absence of target cells, the GPC3×CD3κλ bispecific antibody had no activating effect on peripheral blood T cells, and was equivalent to the negative control KLH×CD3.

[0431] 8. Immune Reconstitution Mouse Subcutaneous HepG2 Tumor Model Six to eight-week-old B-NGD female mice (Biocytogen Pharmaceuticals Co., Ltd.) were selected and subcutaneously inoculated with HepG2 cells (7 × 10 6 / animal) and tumors were 60-100 mm3 When the mice reached the target dose, they were randomly assigned to the following groups: high dose group (3.0 mg / kg), medium dose group (1.0 mg / kg), low dose group (0.3 mg / kg), positive control group (ERY974), and negative control group (KLH×CD3) (3 mg / kg). Each mouse was administered 1×10 PBMC cells. 7 The mice were injected with 100 mg of the drug into the tail vein, and 3 days later, the first administration of the drug was started. The administration interval was once every 5 days, and the mice were administered twice in total. The tumor volume and body weight of the mice were monitored, and after the experiment, the mice were killed by decapitation, and the tumors were weighed and recorded. The results are shown in Figure 37, and the GPC3 x CD3 κλ bispecific antibody showed a dose-dependent in vivo efficacy, with tumor inhibition rates (from medium to high doses) of 76.7%, 81.3%, and 95.9%, respectively. The tumor-bearing mice tolerated the above doses well and did not show any adverse effects such as weight loss.

[0432] 9. CD3 humanized mouse Hepa1-6 / human GPC3 tumor transplant model Six-week-old C57 / BL6-hCD3 female mice (Biocytogen Pharmaceuticals Co., Ltd.) were selected and transfected with Hepa1-6 / human GPC3 (6 × 10 6 / animal) was subcutaneously inoculated into mice, and the tumor volume was 60-100 mm 3 When the total number of mice reached 10 mg / kg, they were randomly divided into groups. The high dose group was 10 mg / kg, the medium dose group was 3 mg / kg, the low dose group was 1 mg / kg, the positive control group was ERY974, and the negative control group was 10 mg / kg. The administration interval was once every 3 days, and the mice were administered three times in total. The tumor volume and the weight of the mice were monitored, and after the experiment, the mice were killed by decapitation, and the tumors were weighed and recorded. The results are shown in Figure 38, and the GPC3 x CD3 κλ bispecific antibody significantly contributed to the killing of tumor cells by immune cells and reduced tumor volume, and the dose of 10 mg / kg was equivalent to the efficacy of ERY974.

Claims

1. An antibody or antigen-binding fragment thereof that binds to BCMA, A first light chain variable domain comprising a light chain complementarity determining region (LCDR) 1, LCDR2, and LCDR3 of the sequence set forth in SEQ ID NO: 80; and and a first heavy chain variable domain comprising heavy chain complementarity determining region (HCDR) 1, HCDR2, and HCDR3 of the sequence set forth in SEQ ID NO:

86.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof comprises a first light chain and a first heavy chain, the first light chain comprising a first light chain variable domain and the first heavy chain comprising a first heavy chain variable domain.

3. i) the first light chain comprises a lysine (K) at amino acid position 42; and ii) the first heavy chain comprises a glutamate (E) at amino acid position 39; The antibody or antigen-binding fragment thereof of claim 2, wherein the numbers refer to the sequences shown in SEQ ID NOs: 80 and 86, respectively.

4. 4. The antibody or antigen-binding fragment thereof of claim 2 or 3, wherein the first light chain comprises a first light chain variable domain of the sequence shown in SEQ ID NO: 80, and the first heavy chain comprises a first heavy chain variable domain of the sequence shown in SEQ ID NO:

86.

5. The antibody or antigen-binding fragment thereof according to any one of claims 2 to 4, wherein the first heavy chain comprises the mutations Y349C, T366S, L368A, and Y407V, wherein the numbering is according to the EU numbering system.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is of the IgG4 isotype.

7. The first light chain comprises the amino acid sequence of SEQ ID NO: 80, and the first heavy chain The antibody or antigen-binding fragment thereof according to any one of claims 2 to 6, comprising the amino acid sequence of SEQ ID NO:

86.

8. The antibody or antigen-binding fragment thereof according to any one of claims 2 to 6, wherein the first light chain consists of the amino acid sequence of SEQ ID NO: 80 and the first heavy chain consists of the amino acid sequence of SEQ ID NO:

86.

9. The antibody or antigen-binding fragment thereof according to any one of claims 2 to 7, wherein the antibody or antigen-binding fragment thereof comprises a second light chain and a second heavy chain.

10. i) the second light chain comprises a glutamate (E) at amino acid position 40; and / or 10. The antibody or antigen-binding fragment thereof of claim 9, wherein ii) the second heavy chain comprises a lysine (K) at amino acid position 39.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the first light chain is a kappa-type light chain.

12. The antibody or antigen-binding fragment thereof according to any one of claims 9 to 11, wherein the second light chain is a lambda-type light chain.

13. The antibody or antigen-binding fragment thereof according to any one of claims 9 to 12, wherein the second heavy chain comprises a S354C mutation, a T366W mutation, and wherein the numbering is according to the EU numbering system.

14. 14. The antibody or antigen-binding fragment thereof of any one of claims 9 to 13, wherein the first heavy chain and the second heavy chain each independently comprise one or more or all of the mutations corresponding to S228P, L235E, and P329A, wherein the numbering is according to the EU numbering system.

15. The antibody or antigen-binding fragment thereof according to any one of claims 9 to 14, wherein the antibody or antigen-binding fragment thereof is multispecific.

16. The antibody or antigen-binding fragment thereof of claim 15, wherein the antibody or antigen-binding fragment thereof further binds to a T cell antigen.

17. The second light chain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 7, SEQ a second light chain variable domain comprising an LCDR2 comprising the amino acid sequence of SEQ ID NO:8, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:21; and The antibody or antigen-binding fragment thereof according to any one of claims 9 to 16, wherein the second heavy chain comprises a second heavy chain variable domain comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO: 26, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:

28.

18. 18. The antibody or antigen-binding fragment thereof of claim 17, wherein the second light chain variable domain comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 18, and the second heavy chain variable domain comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

50.

19. The second light chain variable domain comprises the amino acid sequence of SEQ ID NO: 18, and wherein the second heavy chain variable domain comprises the amino acid sequence of SEQ ID NO:

50.

20. The antibody or antigen-binding fragment thereof according to any one of claims 17 to 19, wherein the second light chain comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 66, and the second heavy chain comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

68.

21. The antibody or antigen-binding fragment thereof according to any one of claims 17 to 20, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 66 and the second heavy chain comprises the amino acid sequence of SEQ ID NO:

68.

22. The antibody or antigen-binding fragment thereof according to any one of claims 17 to 20, wherein the second light chain consists of the amino acid sequence of SEQ ID NO: 66 and the second heavy chain consists of the amino acid sequence of SEQ ID NO:

68.

23. An antibody or antigen-binding fragment thereof that binds to BCMA and CD3, comprising a first heavy chain, a first light chain, a second heavy chain, and a second light chain; wherein said first heavy chain and said first light chain form a first antigen-binding domain, and said second heavy chain and said second light chain form a second antigen-binding domain; wherein the first heavy chain comprises a first heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of the sequence shown in SEQ ID NO: 86; and the first light chain comprises a first heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of the sequence shown in SEQ ID NO:

87. the second heavy chain comprises a second heavy chain variable region comprising an HCDR1 comprising the amino acid sequence of SEQ ID NO:26, an HCDR2 comprising the amino acid sequence of SEQ ID NO:27, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:28; and the second light chain comprises a second light chain variable region comprising an LCDR1 comprising the amino acid sequence of SEQ ID NO:7, an LCDR2 comprising the amino acid sequence of SEQ ID NO:8, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:

21.

24. 24. The antibody or antigen-binding fragment thereof of claim 23, wherein the first heavy chain variable region comprises a heavy chain variable region sequence as set forth in SEQ ID NO: 86; the first light chain variable region comprises a light chain variable region sequence as set forth in SEQ ID NO: 80; the second heavy chain variable domain comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO: 50; and the second light chain variable domain comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

18.

25. 25. The antibody or antigen-binding fragment thereof of claim 23 or 24, wherein the first heavy chain variable region comprises a heavy chain variable region sequence as shown in SEQ ID NO: 86; the first light chain variable region comprises a light chain variable region sequence as shown in SEQ ID NO: 80; the second heavy chain variable domain comprises an amino acid sequence of SEQ ID NO: 50; and the second light chain variable domain comprises an amino acid sequence of SEQ ID NO:

18.

26. The first heavy chain comprises the amino acid sequence of SEQ ID NO: 86; the first light chain comprises the amino acid sequence of SEQ ID NO: 80; and the second heavy chain comprises the amino acid sequence of SEQ ID NO:

26. The antibody or antigen-binding fragment thereof of any one of claims 23 to 25, wherein the second light chain comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO: 68; and the second light chain comprises an amino acid sequence having at least 95% identity with the amino acid sequence of SEQ ID NO:

66.

27. The first heavy chain comprises the amino acid sequence of SEQ ID NO: 86; the first light chain comprises the amino acid sequence of SEQ ID NO: 80; and the second heavy chain comprises the amino acid sequence of SEQ ID NO: The antibody or antigen-binding fragment thereof according to any one of claims 23 to 26, wherein the second light chain comprises the amino acid sequence of SEQ ID NO: 68; and the second light chain comprises the amino acid sequence of SEQ ID NO:

66.

28. 27. The antibody or antigen-binding fragment thereof of any one of claims 23 to 26, wherein the first heavy chain consists of the amino acid sequence of SEQ ID NO: 86; the first light chain consists of the amino acid sequence of SEQ ID NO: 80; the second heavy chain consists of the amino acid sequence of SEQ ID NO: 68; and the second light chain consists of the amino acid sequence of SEQ ID NO:

66.

29. One or more nucleic acid molecules encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 28.

30. 30. A vector comprising one or more nucleic acid molecules according to claim 29.

31. 31. A cell comprising the vector of claim 30 or one or more nucleic acid molecules of claim 29.

32. 32. A method for producing an antibody or antigen-binding fragment thereof, the method comprising: obtaining a cell according to claim 31; and producing the antibody or antigen-binding fragment thereof.

33. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 28 and a pharma- ceutically acceptable carrier.

34. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 28, or a pharmaceutical composition according to claim 33, for use in treating a BCMA-associated disease or disorder in a subject.

35. Use of an antibody or antigen conjugate thereof according to any one of claims 1 to 28, or a pharmaceutical composition according to claim 33, in the manufacture of a medicament for the treatment of a disease or disorder.

36. 36. The use of claim 35, wherein the disease or disorder is an autoimmune disease or disorder.

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