Bispecific antibodies and their use

Abstract

The present disclosure provides bispecific antibodies or antigen-binding fragments thereof, their encoding nucleic acids, cells containing said nucleic acids, compositions comprising said bispecific antibodies or antigen-binding fragments thereof, nucleic acids and / or cells, and related uses of said bispecific antibodies or antigen-binding fragments thereof in the treatment of cancer.

Description

[Technical Field]

[0001] This disclosure relates to bispecific antibodies and their uses, particularly bispecific antibodies that bind to CD3 and one other antigen, and their uses. [Background technology]

[0002] T cell bispecific antibodies (also called T cell adapters) are special antibody molecules that recognize the surface antigen (antigen arm) of target cells via one end and bind to the CD3 receptor (CD3 arm) of T cells via the other end. They activate T cells and kill tumors by agglutinating CD3 on T cells 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 resolved, and three bispecific antibody drugs have been approved one after another. Although they show excellent efficacy for the approved indications, the associated side effects and limitations on use have hindered the early, broad use of these bispecific antibodies. For example, catumaxomab, an early approved drug, has been withdrawn from the market because its Fc segment binds to Fcγ receptors expressed on hepatic Kupffer cells, causing rapid cytokine release. Blinatumomab, approved in 2014, employs an Fv antibody fragment, resulting in a biological half-life of only two hours and requiring continuous low-dose intravenous infusion. The FDA has also begun requiring black-box warnings regarding cytokine release syndrome and neurotoxicity.

[0003] In a normal immune response, the TCR binds to the exogenous peptide-human leukocyte antigen complex (HLA) of infected or mutated cells with low affinity (approximately 1-100 μM), transmitting an activation signal into the nucleus via the CD3 signaling complex (which includes CD3εγ, CD3εδ, and CD3ζζ), activating the expression of transcription factors and their downstream proteins (cytokines, granzymes, perforins, etc.). The signal intensity produced by the TCR complex determines the fate of T cells. Early CD3 bispecific antibodies were mostly based on a small number of mouse antibodies such as OKT3, L2K, UCHT1, and TR66. Due to their high affinity, they lead to T cell overactivation, releasing numerous cytokines and producing cytokine storm syndrome. However, their high affinity also leads to the recruitment of bispecific antibodies in secondary lymphoid organs, reducing exposure to tumor tissue.

[0004] The ability of the antibody's Fc portion to bind to the Fcγ receptor is another factor affecting drug safety. Because the Fcγ receptor is expressed in multiple normal tissues, bispecific antibodies, after binding to the Fcγ receptor on the cell membrane via Fc, can lead to serious off-target toxicity when the CD3 receptor bound to the other end is cross-linked and activated by the aggregation of the Fcγ receptor. By using human IgG2 or IgG4 subtypes with weak binding ability to the Fcγ receptor, or by further making amino acid substitutions at the corresponding site of CH2, for example, Armour et al. reduced binding to the Fcγ receptor by substituting positions 233-236 (EU sequence number) of IgG1 and IgG4 with the corresponding sequence of IgG2 (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. stabilized the IgG4 structure and reduced binding to the Fcγ receptor by introducing the mutations Ser228Pro and Leu235Glu into IgG4 (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 Idusogie et al. found that the binding of IgG1 to complement C1q could 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] Inter-chain mismatch is a major process challenge in the development of natural IgG-like bispecific antibodies. The co-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 co-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 modifications 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 resolve the mismatch between heavy chains, knobs-into-hole complementary mutations were introduced into the antibody Fc segment. 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 CrossMab technology to replace the Fab portion or the entire length of the light chain and heavy chain to solve the light chain mismatch problem. However, to achieve correct pairing, CrossMab technology that replaces only a portion of the chain is also used. VH-VL and CmarsMab CH1-CLIt is necessary to introduce an additional peptide segment, and also to replace the entire Fab length with CrossMab. Fab The efficiency of accurate pair formation is less than 50%. [Overview of the Initiative]

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

[0007] This disclosure provides a novel T cell adapter employing different types of light chain κλ bispecific antibody designs and full-length IgG conformations. The antibody arms that bind to target cells and T cell CD3 employ κ and λ light chains, respectively, and pair with their corresponding heavy chains. By introducing complementary charge pairs, the efficiency of precise pairing is enhanced, optimizing affinity. As a result, the novel T cell adapter can recruit activated T cells at low concentrations, produce effective killing of target cells, and prevent T cell activation in the absence of target cells. Furthermore, the novel T cell κλ bispecific antibody does not bind to the FcγR receptor, reducing the risk of cytokine storms. The novel CD20×CD3κλ bispecific antibody, BCMA×CD3κλ bispecific antibody, and GPC3×CD3κλ bispecific antibody constructed by the method described herein exhibit high purification yields, achieving purity exceeding 99% in three-step purification. Animals tolerate the novel CD20-CD3κλ bispecific antibody well, and the therapeutic efficacy and safety of the novel T cell adapter are superior to those of the same type of antibody.

[0008] In one embodiment, the present disclosure provides a bispecific antibody or an antigen-binding moiety thereof.

[0009] In another embodiment, the present disclosure provides a bispecific antibody or a nucleic acid encoding an antigen-binding moiety thereof according to the above embodiment.

[0010] In another embodiment, the Disclosure provides a vector comprising nucleic acid according to the above embodiment.

[0011] In another embodiment, the present disclosure provides cells comprising the vector according to the above embodiment.

[0012] According to the antibody or antigen-binding portion relating to any of the above embodiments, the antibody or antigen-binding portion is humanized.

[0013] In another embodiment, the Disclosure provides a drug composition or kit comprising an antibody or its antigen-binding moiety or its coding nucleic acid according to any of the above embodiments, and a pharmaceutically acceptable carrier.

[0014] In another embodiment, the present disclosure provides an antibody-drug conjugate comprising the antibody or its antigen-binding portion, bispecific or multispecific molecule, according to any of the above embodiments, covalently bound to a therapeutic portion.

[0015] In another embodiment, the present disclosure provides a method for treating an associated disease, comprising the step of administering to a mammal a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any of the above embodiments, nucleic acids, vectors, cells and / or drug compositions.

[0016] In another embodiment, the present disclosure provides the use of antibodies or antigen-binding fragments thereof, nucleic acids, vectors, cells and / or drug compositions according to any of the above embodiments in the manufacture of drugs or kits for treating the mammalian tumor antigen-associated diseases.

[0017] The antibodies relating to this disclosure can be used in multiple applications, including the detection of tumor antigens, and the diagnosis, treatment, or prevention of tumor antigen-related diseases. [Brief explanation of the drawing]

[0018] [Figure 1] This figure shows the first antigen × CD3κλ bispecific antibody related to this disclosure. [Figure 2] This figure shows the binding of a CD3 humanized antibody to the human CD3εγ protein. [Figure 3] This figure shows the binding of CD3 humanized antibodies to Jurkat cells. [Figure 4] This figure shows the binding of CD3 humanized antibodies to human CD3εγ and cynomolgus monkey CD3εγ proteins. [Figure 5] This figure shows the structures of κλ001, κλ002, κλ003, κλ004, and κλ005 related to this disclosure. [Figure 6] This figure shows the results of purification of the CD20×CD3κλ bispecific antibody using ProteinA. [Figure 7] This figure shows the SEC-HPLC detection results for the CD20×CD3κλ bispecific antibody. [Figure 8] This figure shows the results of homodimer detection of CD20×CD3κλ bispecific antibodies. [Figure 9] This figure shows the binding of a CD20 × CD3κλ bispecific antibody to CD20-stable transfected cells. [Figure 10] This figure shows the binding of CD20×CD3κλ bispecific antibodies to tumor cells SU-DHL-4, Raji, and NALM-6. [Figure 11] This figure shows the binding of the CD20×CD3κλ bispecific antibody to Jurkat cells. [Figure 12] This figure shows the binding of CD20×CD3κλ bispecific antibodies to peripheral blood T cells. [Figure 13]Figure 13A shows the TDCC activity of the CD20×CD3κλ bispecific antibody, and Figure 13B shows the killing of Nalm-6 cells and the activation of T cells, respectively. [Figure 14] Figure 14A shows the TDCC activity of the CD20×CD3κλ bispecific antibody, and Figure 14B shows the killing of TMD-8 cells and the activation of T cells, respectively. [Figure 15] Figure 15A shows the TDCC activity of the CD20×CD3κλ bispecific antibody, and Figure 15B shows the killing of Toledo cells and the activation of T cells, respectively. [Figure 16] This figure shows the effect of the CD20×CD3κλ bispecific antibody on the T cell NFAT signaling pathway. [Figure 17] This figure shows the inhibitory effect of a CD20×CD3κλ bispecific antibody in an immunoconstitutional mouse Raji transplanted tumor model. [Figure 18] This figure shows the inhibitory effect of a CD20×CD3κλ bispecific antibody in immunodeficient mouse subcutaneous Raji and human PBMC mixed transplant tumor models. [Figure 19] This figure shows the efficacy of the CD20×CD3κλ bispecific antibody in cynomolgus monkeys. [Figure 20] This figure shows the binding of the BCMA × CD3κλ bispecific antibody to BCMA-stable transfected cells. [Figure 21] This figure shows the binding of BCMA × CD3κλ bispecific antibodies to tumor cells NCI-H929 and RPMI-8226. [Figure 22] This figure shows the binding of the BCMA × CD3κλ bispecific antibody to Jurkat cells. [Figure 23] This figure shows the binding of BCMA × CD3κλ bispecific antibody to peripheral blood T cells. [Figure 24] Figure 24A shows the TDCC activity of the BCMA × CD3κλ bispecific antibody, and Figure 24B shows the killing of NCI-H929 cells and the activation of T cells. [Figure 25]Figure 25A shows the TDCC activity of the BCMA × CD3κλ bispecific antibody, and Figure 25B shows the killing of RPMI-8226 cells, while Figure 25B shows the activation of T cells. [Figure 26] This figure shows the effect of BCMA × CD3κλ bispecific antibody on the T cell NFAT signaling pathway. [Figure 27] This figure shows nonspecific activation of PBMCs by a BCMA × CD3κλ bispecific antibody. [Figure 28] This figure shows the binding of the BCMA × CD3κλ bispecific antibody to the Fc receptor. [Figure 29] This figure shows the inhibitory effect of a BCMA × CD3κλ bispecific antibody in a subcutaneous NCI-H929 transplanted tumor model in immunodeficient mice. [Figure 30] This figure shows the binding of the GPC3 × CD3κλ bispecific antibody to GPC3-stable transfected cells. [Figure 31] This figure shows the binding of the GPC3×CD3κλ bispecific antibody to tumor cells HepG2. [Figure 32] This figure shows the binding of the GPC3×CD3κλ bispecific antibody to Jurkat cells. [Figure 33] This figure shows the binding of the GPC3 × CD3κλ bispecific antibody to peripheral blood T cells. [Figure 34] Figure 34A shows the TDCC activity of the GPC3 × CD3κλ bispecific antibody, and Figure 34B shows the killing of HepG2 cells and the activation of T cells. [Figure 35] This figure shows the action of the GPC3×CD3κλ bispecific antibody on the T cell NFAT signaling pathway. [Figure 36] This figure shows nonspecific activation of PBMCs by a GPC3 × CD3κλ bispecific antibody. [Figure 37] This figure shows the inhibitory effect of a GPC3 × CD3κλ bispecific antibody in an immunoconstitutional mouse subcutaneous HepG2 transplanted tumor model. [Figure 38]This figure shows the inhibitory effect of a GPC3 × CD3κλ bispecific antibody in a CD3-humanized mouse Hepa1-6 / human GPC3 transplanted tumor model. [Modes for carrying out the invention]

[0019] In this invention, unless otherwise specified, the scientific and technical terms used herein have meanings that are generally understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are widely used terms and routine procedures in their respective fields. On the other hand, to better understand this invention, definitions and explanations of the relevant terms are provided below.

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

[0021] TSA is unique to tumor cells (or is thought to be specific to tumor cells) and does not occur in other cells in vivo (e.g., does not occur to a significant extent in other cells). TAA is not specific to tumor cells and is rather expressed in normal cells as well (e.g., expressed under conditions that do not induce immune tolerance to the antigen). For example, TAA may be an antigen expressed in normal cells during fetal development when the immune system is immature and unable to respond, or TAA may be an antigen that is normally present in normal cells at very low levels but expressed at higher levels in 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, and TRP-2; tumor-specific multiseries antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed fetal antigens such as CEA; overexpressed oncogenes and mutant tumor suppressor genes such as p53, Ras, and HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein-Barr virus antigen (EBVA) and human papillomavirus (HPV) antigens E6 and E7. Other tumor antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, erbB, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, 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 / cyclophyllin C-related protein, TAAL6, TAG72, TLP, MUC16, IL13Rα2, FRα, VEGFR2, Lewis It contains Y, FAP, EphA2, CEACAM5, EGFR, CA6, CA9, GPNMB, EGP1, FOLR1, endothelial receptor, STEAP1, SLC44A4, nectin-4, AGS-16, guanidinocyclase C, MUC-1, CFC1B, integrin α3 chain (a3b1 chain, i.e., laminin receptor chain), and TPS.As other tumor antigens, it further includes 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 derived 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 and is useful for targeting CD20 as a diagnostic agent and / or therapeutic agent. In one embodiment, for example, the binding degree of the anti-CD20 antibody to an irrelevant non-CD20 protein measured by radioimmunoassay (RIA) is reduced by about 10% compared to the binding degree of the antibody to CD20. In some embodiments, the antibody that binds to CD20 has a dissociation constant (K -8 M) 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 -13 M to 10 -9 M, e.g., 10 -13 M). In some embodiments, the anti-CD20 antibody binds to a conserved CD20 epitope among CD20s derived from different species.

[0025] As used herein, the term "BCMA" collectively refers to BCMA itself and any of its variants, isotypes, and paralogs that exist in animals, preferably in humans.​​

[0026] The term "human BCMA" refers to BCMA derived from humans, and preferably has the amino acid sequence of Genbank registration number AB052772.1, but is not limited thereto.

[0027] The terms "anti-BCMA antibody" and "antibody that binds to BCMA" refer to antibodies that can bind to BCMA with sufficient affinity and are useful for targeting BCMA as a diagnostic and / or therapeutic agent. In one embodiment, for example, the degree of binding of the anti-BCMA antibody to unrelated non-BCMA proteins, as measured by radioimmunoassay (RIA), is about 10% lower than the degree of binding of the antibody to BCMA. In some embodiments, the antibody that binds to BCMA has a molecular weight 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, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 The dissociation constant (K) of M d ) has. In some embodiments, anti-BCMA antibodies bind to conserved BCMA epitopes between BCMAs from different species.

[0028] As used herein, the term "GPC3" refers collectively to GPC3 itself, as well as any 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 and is useful for targeting GPC3 as a diagnostic and / or therapeutic agent. In one embodiment, for example, the degree of binding of the anti-GPC3 antibody to unrelated non-GPC3 proteins, as measured by radioimmunoassay (RIA), is about 10% lower than the degree of binding of the antibody to GPC3. In some embodiments, the antibody that binds to GPC3 has a molecular weight 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, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 The dissociation constant (K) of M d ) has. In some embodiments, anti-GPC3 antibodies bind to conserved GPC3 epitopes among GPC3s originating from different species.

[0031] "CD3" refers to any natural CD3 derived from any vertebrate, including primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and mammals such as rodents (e.g., mice and rats), unless otherwise specified. The term covers CD3 in its "full length" and raw form, as well as any form of CD3 that has been processed intracellularly. The term further covers spontaneously occurring variants of CD3, such as splice variants or alleles. In one embodiment, CD3 is human CD3, and more specifically, the ε subunit of human CD3 (CD3ε). The amino acid sequence of human CD3ε is shown in UniProt (www.uniprot.org) registry 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 usual meaning in this field, where it includes the extracellular space accessible by binding to proteins and other molecules.

[0033] As used herein, the term “approximately” means within ±10% of a given value or range, unless otherwise specified. Where an integer is required, the term means 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" can be understood as an antibody chain exhibiting sequence identity of 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, or 99% or more with respect to a reference polypeptide sequence. With respect to nucleic acid sequences, the aforementioned term can be understood as a nucleotide sequence exhibiting sequence identity of at least 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, with respect to a reference nucleic acid sequence.

[0035] The “homology” or “identity” of sequences has the meanings well known in the art, and the percentage of sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using the disclosed techniques. Sequence identity can be measured along the entire length of a polynucleotide or polypeptide, or along a region of such molecule. There are many methods for measuring 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] A "substitutional" variant is one in which at least one amino acid residue in the native sequence is removed and a different amino acid is inserted in the same position. The substitution may be a single substitution in which only one amino acid is substituted within the molecule, or multiple substitutions in which two or more amino acids are substituted within the same molecule. Multiple substitutions may be located in consecutive sites. Similarly, one amino acid may be substituted by multiple residues, and such variants include both substitutions and insertions. An "insertional" variant is one in which one or more amino acids are inserted directly adjacent to an amino acid at a specific position in the native sequence. Directly adjacent to an amino acid means binding to the α-carboxyl or α-amino functional group of that amino acid. A "deletion" variant is one in which one or more amino acids are removed from the native amino acid sequence. Typically, a deletion variant has one or two amino acids deleted in a specific region of the molecule.

[0037] Regarding the variable domains of antibodies, the term "variable" refers to specific parts of the relevant molecule whose sequences differ significantly among antibodies, used for the specific recognition and binding of a particular antibody to a particular target. However, variability is not evenly distributed throughout the variable domains of antibodies. Variability is concentrated in three segments within the variable domains of all light and heavy chains, called complementarity-determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) or hypervariable regions. The more conserved portions within the variable domains are called framework (FR) regions or framework sequences. Each variable domain of the native heavy and light chains contains four FR regions, each linked by three CDRs and primarily adopting a β-sheet configuration, where the CDRs form loops that link to the β-sheet structure, and these loops, in some cases, form part of the β-sheet structure. The CDRs of each chain are usually linked adjacently by FR regions, and CDRs from other chains contribute to the formation of the antibody's target binding site (epitope or determinant). The numbering of immunoglobulin amino acid residues used herein follows the Kabat et al. immunoglobulin amino acid residue numbering system unless otherwise specified. A single CDR may have the ability to specifically bind to the relevant epitope.

[0038] As used herein, "antibody fragment" or "antigen-binding fragment" refers to any portion of a full-length antibody that is less than the full length but contains at least a portion of the variable region of the antibody that binds to an antigen (e.g., one or more CDRs and / or one or more antibody-binding sites), thereby retaining at least a portion of the binding specificity and the specific binding ability of the full-length antibody. Accordingly, 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 originates. Antibody fragments include antibody derivatives produced by enzymatic catalysis of full-length antibodies, as well as derivatives produced by synthesis, such as derivatives produced by recombinant synthesis. An antibody includes an antibody fragment. 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, for example, 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 typically contain 50 or more amino acids or about 50 amino acids, and typically 200 or more amino acids or about 200 amino acids. Antigen-binding fragments are immunospecifically inserted into the antibody framework (e.g., by substituting the corresponding region) (i.e., at least 10 7 ~10 8 M -1 Ka of at least about 10 7 ~10 8 M -1The material comprises any antibody fragment that yields an antibody that binds to an antigen (showing the Ka of ). 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 to initiate signaling. As used herein, a functional fragment is usually synonymous with 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 to initiate signaling, e.g., Fv, Fab, F(ab')2, etc. An “Fv” fragment is a dimer (V) formed by a non-covalent bond between a variable domain of one heavy chain and a variable domain of one light chain. H -V L It consists of a dimer. In this configuration, as with intact antibodies, the three CDRs of each variable domain interact, V H -V L The target binding site on the surface of the dimer is determined. The six CDRs mentioned above confer target binding specificity to the intact antibody. However, a single variable domain (or half of the Fv containing only the three target-specific CDRs) can also recognize and bind to the target.

[0039] As used herein, the term “bispecific antibody” (BsAb) refers to an antibody and / or antigen-binding molecule that can specifically bind 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, particularly two antigenic determinants expressed on two different cells.

[0040] As used herein, "monoclonal antibody" refers to a group of identical antibodies, meaning that each individual antibody molecule within the monoclonal antibody group is identical to other antibody molecules. This characteristic is the opposite of the characteristic of polyclonal antibodies, which contain antibodies with multiple different sequences. Monoclonal antibodies can be prepared by many well-known methods (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, for example, by fusing with myeloma cells to produce a hybridoma cell line, or by infecting B cells with a virus such as EBV. Recombinant technology can also be used to prepare antibodies in vitro from a clonal population of host cells by transforming host cells with a plasmid containing an artificial sequence of nucleotides encoding the antibody.

[0041] As used herein, the terms “hybridoma” or “hybridoma cell” refer to cells or cell lines produced by the fusion of antibody-producing lymphocytes and non-antibody-producing cancer cells (usually myeloma or lymphoma cells). As is known to those skilled in the art, hybridomas can proliferate and produce and continuously supply specific monoclonal antibodies. Methods for producing hybridomas are known in the art (see, for example, Harlow & Lane, 1988). Where the terms “hybridoma” or “hybridoma cell” are used, subclones and progeny cells of hybridomas are also included.

[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 antibodies naturally produced by antibody-secreting B cells or synthetically produced antibodies having the same domains.

[0043] The term "chimeric antibody" refers to an antibody in which the variable region sequence originates from one species and the constant region sequence originates from another species; for example, an antibody in which the variable region sequence originates from a mouse antibody and the constant region sequence originates from a human antibody.

[0044] A "humanized" antibody refers to a non-human (e.g., mouse) antibody form that is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, or a sub-sequence that binds to other antigens of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. Preferably, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues in the complementarity-determining region (CDR) of the recipient antibody are replaced with CDR residues of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and ability.

[0045] Furthermore, in humanization, it is possible to improve one or more binding properties (e.g., affinity) of the antibody by mutating amino acid residues within the CDR1, CDR2, and / or CDR3 regions of the VH and / or VL. For example, mutations can be introduced by PCR-mediated mutations, and the effect on antibody binding or other functional properties can be evaluated by in vitro or in vivo tests as described herein. Typically, conservative mutations are introduced. Such mutations may be amino acid substitutions, additions, or deletions. Also, the number of mutations in the CDR is usually no more than one or two. Accordingly, the humanized antibodies according to this disclosure further include antibodies containing one or two amino acid mutations in the CDR.

[0046] As used herein, the term "CDR" refers to the complementarity-determining region, and it is known that each heavy chain and light chain of an antibody molecule has three CDRs. CDRs, also called hypervariable regions, are located in the variable regions of each heavy chain and light chain of an antibody and contain highly variable sites in the primary structure of the CDR. In this specification, the heavy chain CDRs are denoted by CDR1, CDR2, and CDR3 at the amino terminus of the amino-terminal sequence derived from the heavy chain, and the light chain CDRs are denoted by 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] As used herein, the term “epitope” refers to any antigenic determinant on an antigen to which an antibody paratope binds. Epitope determinants typically consist of a group of chemically active surfaces of molecules, such as amino acids or sugar side chains, and usually possess specific three-dimensional structural and charge characteristics.

[0048] With respect to antibodies or their antigen-binding fragments as used herein, the terms “specifically bind” or “immunospecifically bind” 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 an alloantigen through non-covalent interactions between the antibody-antibody binding site of the antibody and the antigen. The antigen may be an isolated antigen or may be present in tumor cells. Typically, antibodies that immunospecifically bind to (or specifically bind to) an antigen have a capacity of about 1 × 10⁻⁶. 7 M -1 Or approximately 1 × 10 8 M -1 or a higher affinity constant Ka(1 × 10⁻⁶) -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, e.g., immunoassay, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54, Englebienne (1998) Analyst. 123:1599), isothermal titration calorimetry (ITC), or other kinetic interaction measurements well known in the art (see U.S. Patent No. 7,229,619, which describes exemplary SPR and ITC methods for calculating antibody binding affinity). Apparatus and methods for real-time detection and monitoring of binding rates 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 oligomers or polymers comprising two or more linked nucleotides or nucleotide derivatives, and typically include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) linked by phosphate diester bonds. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules and RNA molecules. Nucleic acid molecules may be single-stranded or double-stranded, and may be cDNA.

[0050] The isolated nucleic acid molecules used herein are nucleic acid molecules isolated from other nucleic acid molecules present in the natural source of nucleic acid molecules. For example, an "isolated" nucleic acid molecule of a cDNA molecule, when prepared by recombinant technology, substantially contains no other cellular material or culture medium, or, when chemically synthesized, substantially contains no chemical precursors or other chemical components. The exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibody or antigen-binding fragments.

[0051] With respect to nucleic acid sequences, regions, elements, or domains as used herein, “operably linked” means that the nucleic acid regions are functionally related to one another. For example, a promoter may be operably linked to a nucleic acid encoding a polypeptide so that it can regulate or mediate the transcription of the nucleic acid.

[0052] Provided herein are “conservative sequence modifications” of the sequences in the sequence listings described herein, i.e., nucleotide and amino acid sequence modifications that do not preclude antibody-antigen binding, either encoded by nucleotide sequences or comprising amino acid sequences. 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 listings described 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, where an amino acid residue is substituted with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, predicted non-essential amino acid residues in anti-GCD20 antibodies, anti-BCMA antibodies, or anti-PC3 antibodies are preferably substituted with other amino acid residues from the same side-chain family. Methods for identifying conserved nucleotide and amino acid substitutions that do not exclude antigen binding are well known in this field (see, for example, 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] As an alternative, in another embodiment, for example, mutations can be randomly introduced along all or part of the sequence encoding the anti-GCD20 antibody, anti-BCMA antibody, or anti-PC3 antibody 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] As used herein, “expression” refers to the process of producing polypeptides through the transcription and translation of polynucleotides. Polypeptide expression levels can be assessed using any method well known in the art, including, for example, a method for determining the amount of polypeptide produced from host cells. Such methods may include, but are not limited to, quantitative analysis of polypeptides in cell lysates by ELISA, Coomassie blue staining after gel electrophoresis, Lowry protein assays, and Bradford protein assays.

[0055] As used herein, “host cell” refers to a cell used to receive, maintain, replicate, and amplify a vector. The host cell may also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acids contained in the vector are replicated and amplified. The host cell may be a eukaryotic cell or a prokaryotic cell. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells, e.g., HEK293 cells.

[0056] As used herein, “vector” is a replicable nucleic acid that, when transformed into a suitable host cell, can express one or more heterologous proteins. Vectors typically include vectors into which nucleic acids encoding polypeptides or fragments thereof can be introduced by restriction digestion and ligation. Vectors further include vectors containing nucleic acids encoding polypeptides. Vectors are used to introduce nucleic acids encoding polypeptides into host cells for nucleic acid amplification or for the expression / presentation of polypeptides encoded by the nucleic acid. Vectors are typically episomes, but may be designed as chromosomes in which genes or parts thereof are integrated into the genome. Vectors that are artificial chromosomes, such as yeast artificial chromosomes and mammalian artificial chromosomes, are also conceivable. The selection and use of such vehicles are well known to those skilled in the art.

[0057] The vectors used herein further include “viral vectors” or “viral vectors.” A viral vector is an engineered virus in which an exogenous gene is operably ligated to an exogenous gene for the purpose of transferring the exogenous gene to a cell (as a vehicle or shuttle).

[0058] As used herein, “expression vector” includes a vector capable of expressing DNA, wherein the DNA is operably ligated to a regulatory sequence that affects the expression of a DNA fragment, for example, a promoter region. 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 generally may be derived from plasmid or viral DNA, or may contain elements of both. Thus, expression carriers refer to recombinant DNA or RNA constructs, e.g., plasmids, phages, recombinant viruses, or other vectors, which, when introduced into a suitable host cell, result in the expression of 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 in the episome or are incorporated into the host cell genome.

[0059] As used herein, “to treat” an individual having a disease or symptoms of a disease means that the symptoms of the individual are partially or completely relieved or remain unchanged after treatment. Accordingly, treatment includes prevention, cure, and / or remission. Prevention refers to the prevention of the potential disease and / or the prevention of the worsening of symptoms or the progression of the disease. Treatment further includes any antibody or its antigen-binding fragment provided, and any pharmaceutical use of the compositions provided herein.

[0060] As used herein, “therapeutic effect” means the effect obtained by treating an individual, and is a change in the symptoms of a disease or disease state, usually an improvement or enhancement of a disease or disease state, or a cure of a disease or disease state.

[0061] As used herein, “therapeutic dose” refers to the amount of a drug, compound, substance, or composition containing a compound that is at least sufficient to achieve a therapeutic effect after administration to a subject. Therefore, it is the amount necessary to prevent, cure, improve, suppress, or partially suppress the symptoms of a disease or condition.

[0062] As used herein, “protective dose” refers to the amount of a substance, compound, material, or composition containing a compound that, at the time of administration to a subject, has a desired preventive effect, such as preventing or delaying the onset or recurrence of a disease or symptom, or reducing the likelihood of the onset or recurrence of a disease or symptom. The complete protective dose does not need to be achieved by administering a single dose, but may be achieved only when a series of doses are administered. Accordingly, the protective dose may be administered in one or more doses.

[0063] As used herein, the term "patient" refers to mammals such as humans. II. Specific Embodiments

[0064] In one embodiment, the Disclosure includes (a) a first antigen-binding moiety or antigen-binding fragment thereof, wherein the first antigen-binding moiety comprises a first light chain and a first heavy chain, the first light chain being a κ-type light chain, and the first antigen-binding moiety includes a first binding domain that binds to a first antigen, and (b) A bispecific antibody or antigen-binding fragment thereof is provided, comprising a second antigen-binding moiety or antigen-binding fragment thereof, wherein the second antigen-binding moiety comprises a second light chain and a second heavy chain, the second light chain being a λ-type light chain, and the second antigen-binding moiety comprising a second binding domain that binds to a second antigen.

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

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

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

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

[0069] In some embodiments, the second light chain CDR of the second binding domain is selected from the following: second light chain CDR1, CDR2, and CDR3 sequences containing amino acid sequence SEQ ID NO: 7, 8, and 9, respectively; second light chain CDR1, CDR2, and CDR3 sequences containing amino acid sequence SEQ ID NO: 14, 15, and 9, respectively; second light chain CDR1, CDR2, and CDR3 sequences containing amino acid sequence SEQ ID NO: 7, 8, and 21, respectively; and / or the heavy chain CDR of the second binding domain is selected from the following: second heavy chain CDR1, CDR2, and CDR3 sequences containing amino acid sequence SEQ ID NO: 26, 27, and 28, respectively; and / or the heavy chain CDR of the second binding domain is selected from the following: second heavy chain CDR1, CDR2, and CDR3 sequences containing amino acid sequence SEQ ID NO: The second heavy chain CDR1, CDR2, and CDR3 sequences are selected from those containing amino acid sequence SEQ ID NO: 31, 27, and 28, respectively; the second heavy chain CDR1, CDR2, and CDR3 sequences containing amino acid sequence SEQ ID NO: 31, 27, and 34, respectively; the second heavy chain CDR1, CDR2, and CDR3 sequences containing amino acid sequence SEQ ID NO: 31, 27, and 37, respectively; the second heavy chain CDR1, CDR2, and CDR3 sequences containing amino acid sequence SEQ ID NO: 31, 27, and 40, respectively; the second heavy chain CDR1, CDR2, and CDR3 sequences containing amino acid sequence SEQ ID NO: 31, 27, and 43, respectively; and the second heavy chain CDR1, CDR2, and CDR3 sequences containing amino acid sequence SEQ ID NO: 46, 47, and 28, respectively.

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

[0071] In some embodiments, the second binding domain includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 includes 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 amino acid sequences SEQ ID NO: 58 and 66, and / or the second heavy chain of the second antigen-binding moiety is selected from amino acid sequences SEQ ID NO: 60 and 68. In some preferred embodiments, the second light chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 58, and the second heavy chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 60. In some preferred embodiments, the second light chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 66, and the second heavy chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 68.

[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 the CD20 antigen.

[0083] In some preferred embodiments, the first light chain variable region of the first antigen-binding moiety is Gln 38 Lys mutation (Vκ) CD20 :Gln 38 It has Lys). In some preferred embodiments, the first heavy chain variable region of the first antigen-binding moiety is Gln 39 Glu mutation (VH CD20 :Gln 39 It has Glu.

[0084] In some preferred embodiments, the first light chain variable region of the first antigen-binding moiety is Gln 38 Lys mutation (Vκ) CD20 :Gln 38 It has Lys, and the first light chain constant region is Glu 123 Lys and Gln 124 Lys mutation (Vκ-Ck CD20 : Gln 38 Lys / Glu 123 Lys / Gln 124 It has Lys). In some preferred embodiments, the first heavy chain variable region of the first antigen-binding moiety is Gln 39 Glu mutation (VH CD20 :Gln 39 It has Glu, and the first heavy chain constant region is Lys 152 Glu and Lys 218 Glu mutation (V H -C H 1 CD20 :Gln 39 Glu / Lys 152 Glu / Lys 218 It has Glu.

[0085] In some preferred embodiments, the first light chain of the first antigen-binding moiety is selected from amino acid sequences SEQ ID NO: 54, 62, and 70, and / or the first heavy chain of the first antigen-binding moiety is selected from amino acid sequences SEQ ID NO: 56, 64, and 72.

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

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

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

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

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

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

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

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

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

[0095] In some preferred embodiments, the first light chain variable region of the first antigen-binding moiety is Gln 42 Lys mutation (Vκ) BCMA :Gln 42 It has Lys). In some preferred embodiments, the first heavy chain variable region of the first antigen-binding moiety is Gln 39 Glu mutation (VH BCMA :Gln 39 It has Glu.

[0096] In some preferred embodiments, the first light chain of the first antigen-binding moiety is selected from amino acid sequences SEQ ID NO: 80 and 84, and / or the first heavy chain of the first antigen-binding moiety is selected from amino acid sequences SEQ ID NO: 82 and 86.

[0097] In some preferred embodiments, the first light chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO:80, and the first heavy chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO:82.

[0098] In some preferred embodiments, the first light chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO:84, and the first heavy chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO:82.

[0099] In some preferred embodiments, the first light chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO:80, and the first heavy chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO:86.

[0100] In some preferred embodiments, the first light chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO:84, and the first heavy chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO:86.

[0101] In some preferred embodiments, the first light chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO: 80, the first heavy chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO: 82, the second light chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 66, and the second heavy chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 68.

[0102] In some preferred embodiments, the first light chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO: 84, the first heavy chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO: 82, the second light chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 66, and the second heavy chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 68.

[0103] In some preferred embodiments, the first light chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO: 80, the first heavy chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO: 86, the second light chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 66, and the second heavy chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 68.

[0104] In some preferred embodiments, the first light chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO: 84, the first heavy chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO: 86, the second light chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 66, and the second heavy chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 68.

[0105] In some embodiments, the first antigen is the GPC3 antigen.

[0106] In some preferred embodiments, the first light chain variable region of the first antigen-binding moiety is Gln 43 Lys and Gln 39 Glu mutation (Vκ GPC3 :Gln 43 Lys;VH GPC3 :Gln 39 It has Glu.

[0107] In some preferred embodiments, the first light chain of the first antigen-binding moiety is selected from amino acid sequences SEQ ID NO: 88 and 92, and / or the first heavy chain of the first antigen-binding moiety is selected from amino acid sequences SEQ ID NO: 90 and 94.

[0108] In some preferred embodiments, the first light chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO:88, and the first heavy chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO:90.

[0109] In some preferred embodiments, the first light chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO:92, and the first heavy chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO:94.

[0110] In some preferred embodiments, the first light chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO: 88, the first heavy chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO: 90, the second light chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 66, and the second heavy chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO: 68.

[0111] In some preferred embodiments, the first light chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO:92, the first heavy chain of the first antigen-binding moiety is amino acid sequence SEQ ID NO:94, the second light chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO:66, and the second heavy chain of the second antigen-binding moiety is amino acid sequence SEQ ID NO:68.

[0112] In some embodiments, the Fc portion of the first antigen-binding moiety and / or the second antigen-binding moiety of the bispecific antibody employs a knob-into-hole structure. In some preferred embodiments, a human IgG4 knob-into-hole structure is employed.

[0113] In some embodiments, the first antigen-binding moiety and / or the second antigen-binding moiety of the bispecific antibody is Ser 228 Pro, Leu 235 Glu and / or Pro 329 It also has an Ala mutation.

[0114] In one embodiment, the present disclosure provides a nucleic acid encoding the bispecific antibody or its antigen-binding moiety.

[0115] In some preferred embodiments, the second antigen-binding moiety binds to the CD3 antigen, the coding nucleic acid of the second light chain variable region of the second antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 6, 11, 13, 17, 19, and 23, and / or the coding nucleic acid of the second heavy chain variable region of the second antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 25, 30, 33, 36, 39, 42, 45, 49, 51, and 53. In some preferred embodiments, the coding nucleic acid of the second light chain of the second antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 59 and 67, and / or the coding nucleic acid of the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 61 and 69. In some preferred embodiments, the coding nucleic acid for the second light chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 59, and the coding nucleic acid for the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 61. In some preferred embodiments, the coding nucleic acid for the second light chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 67, and the coding nucleic acid for the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 69.

[0116] In some preferred embodiments, the first antigen-binding moiety binds to the CD20 antigen, the coding nucleic acid of the first light chain of the first antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 55, 63, and 71, and / or the coding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 57, 65, and 73. In some preferred embodiments, the coding nucleic acid of the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 55, and the coding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 57. In some preferred embodiments, the coding nucleic acid of the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 63, and the coding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 65. In some preferred embodiments, the coding nucleic acid for the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:71, and the coding nucleic acid for the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:73.

[0117] In some preferred embodiments, the first antigen-binding moiety binds to a BCMA antigen, the coding nucleic acid of the first light chain of the first antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 81 and 85, and / or the coding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 83 and 87. In some preferred embodiments, the coding nucleic acid of the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 81, and the coding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 83. In some preferred embodiments, the coding nucleic acid of the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 85, and the coding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 83. In some preferred embodiments, the coding nucleic acid of the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:81, and the coding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:87. In some preferred embodiments, the coding nucleic acid of the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:85, and the coding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:87.

[0118] In some preferred embodiments, the first antigen-binding moiety binds to the GPC3 antigen, the coding nucleic acid of the first light chain of the first antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 89 and 93, and / or the coding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequences SEQ ID NO: 91 and 95. In some preferred embodiments, the coding nucleic acid of the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 89, and the coding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 91. In some preferred embodiments, the coding nucleic acid of the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 93, and the coding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 95.

[0119] In some preferred embodiments, the first antigen-binding moiety of the bispecific antibody binds to the CD20 antigen, the second antigen-binding moiety binds to the CD3 antigen, the coding nucleic acid of the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 55, the coding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 57, the coding nucleic acid of the second light chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 59, and the coding nucleic acid of the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 61. In some preferred embodiments, the coding nucleic acid for the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:63, the coding nucleic acid for the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:65, the coding nucleic acid for the second light chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:67, the coding nucleic acid for the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:69, the coding nucleic acid for the second light chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:59, and the coding nucleic acid for the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:61. In some preferred embodiments, the coding nucleic acid for the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:71, the coding nucleic acid for the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:73, the coding nucleic acid for the second light chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:67, and the coding nucleic acid for the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:69.In some preferred embodiments, the coding nucleic acid for the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 63, the coding nucleic acid for the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 65, the coding nucleic acid for the second light chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 59, and the coding nucleic acid for the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 61. In some preferred embodiments, the coding nucleic acid for the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 55, the coding nucleic acid for the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 57, the coding nucleic acid for the second light chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 67, and the coding nucleic acid for the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 69.

[0120] In some preferred embodiments, the first antigen-binding moiety of the bispecific antibody binds to the BCMA antigen, the second antigen-binding moiety binds to the CD3 antigen, the coding nucleic acid of the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:81, the coding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:83, the coding nucleic acid of the second light chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:67, and the coding nucleic acid of the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:69. In some preferred embodiments, the coding nucleic acid for the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 85, the coding nucleic acid for the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 83, the coding nucleic acid for the second light chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 67, and the coding nucleic acid for the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 69. In some preferred embodiments, the coding nucleic acid for the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 81, the coding nucleic acid for the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 87, the coding nucleic acid for the second light chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 67, and the coding nucleic acid for the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO: 69. In some preferred embodiments, the coding nucleic acid for the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:85, the coding nucleic acid for the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:87, the coding nucleic acid for the second light chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:67, and the coding nucleic acid for the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:69.

[0121] In some preferred embodiments, the first antigen-binding moiety of the bispecific antibody binds to the GPC3 antigen, the second antigen-binding moiety binds to the CD3 antigen, the coding nucleic acid of the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:89, the coding nucleic acid of the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:91, the coding nucleic acid of the second light chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:67, and the coding nucleic acid of the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:69. In some preferred embodiments, the coding nucleic acid for the first light chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:93, the coding nucleic acid for the first heavy chain of the first antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:95, the coding nucleic acid for the second light chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:67, and the coding nucleic acid for the second heavy chain of the second antigen-binding moiety is selected from nucleotide sequence SEQ ID NO:69.

[0122] In one embodiment, the present disclosure provides a vector comprising the nucleic acid.

[0123] In one embodiment, the present disclosure provides cells comprising the nucleic acid or vector.

[0124] In one embodiment, the present disclosure provides a composition comprising the bispecific antibody or its antigen-binding moiety, nucleic acid, vector, and / or cell.

[0125] In one embodiment, the present disclosure provides an antibody-drug conjugate comprising a bispecific antibody or an antigen-binding moiety therein that is covalently bound to a therapeutic portion.

[0126] In some embodiments, the therapeutic portion is selected from a cytotoxic portion, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a degraded peptide, or a radioisotope.

[0127] The antibodies relating to this disclosure are useful as therapeutic or diagnostic tools for various diseases in which various tumor antigens are unfavorably expressed or represented.

[0128] In one embodiment of a tumor antigen-related disease, the expression of the tumor antigen in cells of a diseased tissue or organ is increased compared to its state in a healthy tissue or organ. This increase means an increase of 10% or more, particularly 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, the expression is found only in the diseased tissue, while the expression in the corresponding healthy tissue is suppressed. According to this disclosure, tumor antigen-related diseases include tumors.

[0129] In some embodiments, the tumor antigen-related disease is a CD20-related disease. In some preferred embodiments, the CD20-related disease includes, for example, B-cell proliferative disorders, particularly CD20-positive B-cell disorders, and preferably the disease is selected from non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), multiple myeloma (MM), and Hodgkin lymphoma (HL).

[0130] In some embodiments, the disease associated with the tumor antigen is a BCMA-related disease, preferably a B-cell disease, preferably a cancer, more preferably a cancer, including multiple myeloma, malignant plasmacytoma, Hodgkin lymphoma, nodular lymphocyte-predominant Hodgkin lymphoma, Karrel disease and myeloid leukemia, plasma cell leukemia, plasmacytoma, B-cell prelymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin 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, Waldenström macroglobulinemia, diffuse large B cell lymphoma, follicular lymphoma, marginal zone lymphoma, mucosa-associated lymphoid tissue lymphoma, small cell lymphocytic lymphoma, mantle cell lymphoma, Burkitt lymphoma, primary mediastinal (thymic) large B cell lymphoma, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, nodal marginal zone B cell lymphoma Lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary exudative 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), senile 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 occurring in HHV8-associated multicentric Castleman disease, diffuse large B-cell lymphoma An unclassified B-cell lymphoma having intermediate characteristics between and Burkitt lymphoma, an unclassified B-cell lymphoma having intermediate characteristics between diffuse large B-cell lymphoma and classical Hodgkin lymphoma, and a B-cell-associated cancer selected from other B-cell-associated lymphomas, more preferably a B-cell disorder, and preferably a plasmacytosis, including multiple myeloma, plasmacytoma, plasmacytoma, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary plasmacytoma of bone, extramyeloma, sclerosing myeloma, heavy chain disease,The disease is selected from monoclonal gammaglobulinemia of unknown significance and smoldering multiple myeloma, and preferably the disease is an autoimmune disease such as systemic lupus erythematosus or rheumatoid arthritis.

[0131] In some embodiments, the therapeutic agent comprises an antibody that specifically binds to an activated T cell antigen.

[0132] In one embodiment, the therapeutic agent includes an antibody that specifically binds to CD3, particularly CD3ε.

[0133] A method for treating diseases and symptoms using a bispecific antibody according to this disclosure comprises the step of administering to a mammal an amount of an antibody or antigen-binding fragment thereof according to any of the above embodiments, or a nucleic acid molecule, vector, cell, or drug composition that is therapeutically effective.

[0134] In some embodiments, the present disclosure provides a method for treating or preventing cancer, comprising administering to a patient an antibody capable of binding to GPC3 to a serum level of 40 μg / ml or higher. In different embodiments, the antibody is administered to a serum level of 50 μg / ml or higher, 150 μg / ml or higher, 300 μg / ml or higher, 400 μg / ml or higher, or 500 μg / ml or higher. In different embodiments, the antibody is administered to a serum level of 800 μg / ml or lower, 700 μg / ml or lower, 600 μg / ml or lower, 550 μg / ml or lower, or 500 μg / ml or lower. 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. As used herein, the term “serum level” means the concentration of the substance under consideration in serum. In one embodiment, a serum level of 7 days or more, or 14 days or more, is provided. In one embodiment, the method is 300 mg / m³ 2 For example, 600 mg / m² 2Above, and preferably 1500 mg / m 2 Below, 1200 mg / m 2 Below, or 1000 mg / m 2 Below, and includes administering an antibody dosage as described above.

[0135] In some embodiments, the present disclosure provides a method for treating or preventing a cancer disease, including administering to a patient an antibody that can bind to GPC3 at a dosage of 300 mg / m 2 Above, for example, 600 mg / m 2 Above, and preferably 1500 mg / m 2 Below, 1200 mg / m 2 Below, or 1000 mg / m 2 Below.

[0136] In some embodiments, the present disclosure provides a method for treating or preventing a cancer disease, including administering to a patient an antibody that can bind to GPC3. In this method, 50% or more, preferably 60% or more, 70% or more, 80% or more, or 90% or more of the cancer cells of the patient are GPC3-positive, and / or 40% or more, preferably 50% or more, or 60% or more of the cancer cells of the patient are positive for surface expression of GPC3. In this regard, the present disclosure further provides a method for treating or preventing a cancer disease, including: a. identifying a patient suffering from cancer cells with 50% or more, preferably 60% or more, 70% or more, 80% or more, or 90% or more GPC3-positive, and / or 40% or more, preferably 50% or more, or 60% or more cancer cells positive for surface expression of GPC3; and b. administering to the patient an antibody that can bind to GPC3. In one embodiment, 95% or more, or 98% or more of the cancer cells of the patient are GPC3-positive. In one embodiment, 70% or more, 80% or more, or 90% or more of the cancer cells of the patient are positive for surface expression of GPC3.

[0137] In one embodiment of the method of any aspect of the present invention, the treatment result of the cancer disease is to achieve stabilization of the disease state. In one embodiment, the stabilization of the disease state reaches 2 months or more, 3 months or more, or 6 months or more.

[0138] In some embodiments, the disclosure provides a method for stabilizing the condition of cancer patients, comprising administering an antibody capable of binding to GPC3 to the patient. In one embodiment, the stabilization of the condition reaches two months or more, three months or more, or six months or more.

[0139] In one embodiment of the method described herein, the antibody is administered in a single dose or multiple doses.

[0140] In some embodiments, the present disclosure provides a method for treating or preventing cancerous disease, which involves administering to a patient an antibody capable of binding to GPC3 in multiple doses.

[0141] According to this disclosure, when administering antibodies in multiple doses, preferably the antibodies are 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 fewer doses, 25 or fewer doses, 20 or fewer doses, 15 or fewer doses, or 10 or fewer doses. Preferably the antibody doses are administered at time 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 time intervals of 7 to 30 days, 10 to 20 days, and most preferably about 14 days.

[0142] In one embodiment, the antibody is administered to achieve a serum level of 40 μg / ml or higher. In different embodiments, the antibody is administered to achieve a serum level of 50 μg / ml or higher, 150 μg / ml or higher, 300 μg / ml or higher, 400 μg / ml or higher, or 500 μg / ml or higher. In different embodiments, the antibody is administered to achieve a serum level of 800 μg / ml or lower, 700 μg / ml or lower, 600 μg / ml or lower, 550 μg / ml or lower, or 500 μg / ml or lower. In one embodiment, the provided serum level 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, it is provided at a serum level for 7 days or more, or 14 days or more. In one embodiment, the method is 300 mg / m 2 or more, for example, 600 mg / m 2 or more, and preferably, 1500 mg / m 2 or less, 1200 mg / m 2 or less, or 1000 mg / m 2 or less of the antibody dosage.

[0143] Provided is the use of the antibody or its antigen-binding fragment, or nucleic acid molecule, or vector, or cell, or pharmaceutical composition according to any of the above aspects in the manufacture of a drug for treating mammalian GPC3-related diseases.

[0144] According to any of the above aspects, optionally, the antibody is conjugated to another drug, for example, a conjugate with a label or having cytotoxicity.

[0145] In one embodiment, the Disclosure further includes, for example, an antibody relating to the Disclosure, its fragments, homologs, its derivatives, nucleic acids, vectors, cells, compositions, etc., such as labeled or cytotoxic conjugates, and a kit containing an antibody instruction manual, a conjugate that kills a specific type of cell, etc. The instruction manual may include a guide for the in vitro, in vivo, or ex vivo use of the antibody, conjugate, etc. The antibody may be in liquid or solid form and is usually lyophilized. The kit may further include other appropriate reagents such as buffers, reconstitution solutions, and other components required depending on the intended use. A combination of reagents packaged in predetermined amounts and an instruction manual for use such as therapeutic use or diagnostic assay use is also conceivable. If the antibody is labeled, for example, enzyme-labeled, the kit may include a substrate and cofactors required for the enzyme (e.g., a substrate precursor that provides a detectable chromophore or fluorophore). Furthermore, other additives such as stabilizers and buffers (e.g., blocking buffer or lysis buffer) may also be included. By varying the relative amounts of various reagents, concentrated reagent solutions can be provided, offering user flexibility, space savings, and reagent conservation. These reagents can also be supplied in the form of dry powders, typically lyophilized, and containing excipients, allowing for the provision of reagent solutions of appropriate concentrations upon dissolution.

[0146] The present invention provides the use of an antibody or functional fragment thereof, or a nucleic acid molecule, or a vector, or a cell, or a drug composition, or a kit according to any of the above embodiments, in the manufacture of a reagent for inhibiting GPC3 binding.

[0147] Furthermore, the antibodies relating to this disclosure may be used in immunoassays, purification methods, and other methods using immunoglobulins or fragments thereof. Such uses are well known to those skilled in the art.

[0148] Correspondingly, the present disclosure further provides compositions comprising an anti-GPC3 antibody or a fragment thereof relating to the present disclosure, the antibody being conveniently combined with a pharmaceutically acceptable carrier, diluent or excipient, which is a means known in the art.

[0149] As used in this disclosure, the term “drug composition” refers to a formulation of various preparations. A formulation containing a therapeutically effective amount of polyvalent antibody may be in the form of a sterile liquid solution, liquid suspension, or lyophilized material, and may optionally contain stabilizers or excipients.

[0150] The antibody relating to this disclosure may be used as a composition administered alone or in combination with other activators.

[0151] In some embodiments, the humanized antibody relating to this disclosure is 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 degraded peptide, or a radioisotope. Such a conjugate is also referred to herein as an “antibody-drug conjugate” or “ADC”.

[0152] In some embodiments, the antibody is conjugated to the cytotoxic moiety. The cytotoxic moiety includes tubulin inhibitors such as taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine (cephaeline), mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracendione, mytansine or its analogs or derivatives; mitotic inhibitors such as monomethyl auristatin E or F or its analogs or derivatives; drastatin 10 or 15 or its analogs; irinotecan or its analogs; mitoxantrone, mitramycin, actinomycin D. D) Antimetabolites such as 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, calichemycin or its analogs or derivatives, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine or cladribine, for example, mechloretamine, thiopurine, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophorexate Alkylating agents such as lophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, platinum derivatives such as cisplatin or carboplatin, duocalmycin A, duocalmycin SA, rakelmycin (CC-1065) or its analogs or derivatives, antibiotics such as actinomycin, bleomycin, daunorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, primycin, anthramycin (AMC), pyrrolo[2,1-c][1,4]-Benzodiazepines (PDB), diphtheria toxin and related molecules such as diphtheria A chain and its active fragments and hybrid molecules, lysine such as lysine A or deglycosylated lysine A chain toxin, cholera toxin, Shiga-like toxins such as SLT I, SLT II, ​​SLT IIV, LT toxin, C3 toxin, Shiga toxin, pertussis toxin, tetanus toxin, Bowman-Bark soybean protease inhibitors, Pseudomonas exotoxin, allolin, saporin, modeccin, geranine, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthin protein, Phytolacca americana protein such as PAPI, PAPII and PAP-S, Momordica charantia inhibitors, curcin, crotin, saponaria cone The following may be selected from officinalis inhibitors, geronin, mitogellin, restrictosin, phenomycin and enomycin toxins, ribonuclease (RNase), DNase I, Staphylococcus endotoxin A, pokeweed antiviral protein, diphtheria toxin, and Pseudomonas endotoxin.

[0153] In some embodiments, antibodies are conjugated to auristatin or its peptide analogs, derivatives, or prodrugs. Auristatin has been shown to affect microtubule dynamics, GTP hydrolysis, and nuclear and cell division, and to possess anticancer and antifungal activity. For example, auristatin E can react with p-acetylbenzoic acid or benzoylvaleric acid to produce AEB and AEVB, respectively. Other typical auristatin derivatives include AFP, MMAF (monomethyl auristatin F), and MMAE (monomethyl auristatin E). Suitable auristatins, auristatin analogs, derivatives and prodrugs, and linkers suitable for conjugating auristatin with Ab are described, for example, in U.S. Patents 5,635,483, 5,780,588, 6,214,345 and International Patent Application Publications WO02088172, WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968 and WO205082023.

[0154] In some embodiments, the antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine (PDB), its peptide analogue, derivative, or prodrug. Suitable PDBs, PDB derivatives, and related technologies are described, for example, by 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.

[0155] In some embodiments, the antibody is conjugated to a cytotoxic moiety selected from anthracycline antibiotics, meltansine, calichemycin, duocalmycin, rakelmycin (CC-1065), drastatin 10, drastatin 15, irinotecan, monomethyl auristatin E, monomethyl auristatin F, PDB, or any analog, derivative, or prodrug thereof.

[0156] In some embodiments, the antibody is conjugated to an anthracycline antibiotic or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated to meltansine or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated to calichemycin or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated to duocalmycin or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated to rakelmycin (CC-1065) or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated to drastatin 10 or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated to drastatin 15 or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated to monomethyl auristatin E or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated to monomethyl auristatin F or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine or its analogues, derivatives, or prodrugs. In some embodiments, the antibody is conjugated to irinotecan or its analogues, derivatives, or prodrugs.

[0157] In some embodiments, the antibody is conjugated to cytokines (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 factors, ancestim, and TNFa).

[0158] 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 chelate linker (e.g., DOTA, DTPA, or tiuxetan). The antibody may further, or alternatively, contain or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecules. Non-limiting examples of radioisotopes include: 3 H, 14 C, 15 N, 35 S, 90 Y, 99 Tc, 125 I, 131 I, 186 Re, 213 Bi, 225 American and 227 Contains Th. For therapeutic purposes, radioactive isotopes that emit β or α particle radiation, for example, 131 I, 90 Y, 211 At, 212 Bi, 67 Cu, 186 Re, 188 Re and 212 You may use Pb.

[0159] The technique of conjugating molecules into antibodies is well known in this field. Typically, nucleic acid molecules are covalently linked to lysine or cysteine ​​in antibodies via N-hydroxysuccinimide ester or maleimide functional groups, respectively. It has been reported that conjugation methods using modified cysteine ​​or incorporating non-natural amino acids can improve the identity of the conjugate. Those skilled in the art may particularly consider Fc-containing polypeptides modified with endogenous glutamine activated by acyl donor glutamine-containing tags (e.g., Gin-containing peptide tags or Q tags) or polypeptide manipulation (e.g., by amino acid deletion, insertion, substitution, or polypeptide mutation). Subsequently, transglutaminase can covalently crosslink with an amine donor (e.g., a small molecule containing or bound to a reactive amine), and the amine donor can site-specifically conjugate with the Fc-containing polypeptide via an acyl donor glutamine-containing tag or a contactable / exposed / reactive endogenous glutamine, forming a stable and homogeneous population of manipulated Fc-containing polypeptide conjugates (WO2012059882).

[0160] It is understood that the therapeutic agents according to the above embodiment are administered together with appropriate pharmaceutically acceptable carriers, excipients, and other reagents incorporated into the formulation to provide improved transfer, delivery, tolerance, etc. These formulations may include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, and lipid (cationic or anionic)-containing carriers (e.g., Lipofectin). TM Examples include DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. Any of these mixtures is applicable to the treatment or method of treatment according to the present invention, provided that the active ingredient in the formulation is not inactivated by the formulation, that it is physiologically compatible, and that it can withstand the route of administration.

[0161] In one embodiment, the antibody may be used as a therapeutic agent. Such agents are typically used to treat, induce remission of, and / or prevent diseases or conditions associated with the abnormal expression, activity, and / or signaling of tumor antigens in a subject. The treatment regimen can be implemented by identifying a subject, for example, a human patient suffering from a disease or disorder associated with abnormal tumor antigen expression, activity, and / or signaling, such as tumor antigen-related disorder (or a patient at risk of or already suffering from it), using standard methods. An antibody preparation, preferably one having high specificity and high affinity for the target antigen, is administered to the subject and generally exerts its effect by binding to the target. The administered antibody may resolve, inhibit, or interfere with the expression, activity, and / or signaling function of the target (e.g., tumor antigen). The administered antibody may resolve, inhibit, or interfere with the binding of the target (e.g., tumor antigen) to an endogenous ligand that naturally binds to it. For example, antibodies bind to a target and modulate, inhibit, suppress, reduce, antagonize, neutralize, and / or interfere with the expression, activity, and / or signaling of tumor antigens. In some embodiments, antibodies having heavy-chain and light-chain CDRs can be administered to subjects to treat diseases or disorders associated with abnormal tumor antigen expression.

[0162] In another embodiment, antibodies against tumor antigens are used in methods known in the art related to the localization and / or quantification of tumor antigens (e.g., for determining the level of tumor antigens and / or tumor antigens in a suitable biological sample, diagnostic methods, protein imaging, etc.). In a particular embodiment, an antibody containing an antigen-binding domain derived from the antibody that is specific to a tumor antigen or its derivatives, fragments, analogs, or homologs is used as a pharmaceutically active compound (hereinafter referred to as "therapeutic agent").

[0163] In another embodiment, tumor antigen polypeptides can be isolated using antibodies specific to the tumor antigen by standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies (or fragments thereof) against tumor antigen proteins are used to detect proteins in biological samples. In some embodiments, for example, tumor antigens in biological samples are detected as part of a clinical trial procedure to determine the effectiveness of a particular treatment regimen. Conjugating (i.e., physically binding) antibodies to detectable substances 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 substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminofluorescein, dansylamide chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, fluorescein, and aequorin; and examples of suitable radioactive materials include, 125 I, 131 I, 35 S, or 3 Includes H.

[0164] In another embodiment, the antibodies relating to this disclosure can be used as reagents for detecting the presence of tumor antigens or their protein fragments in a sample. In some embodiments, the antibodies include detectable labeling. The antibodies are polyclonal antibodies, or more preferably monoclonal antibodies. Intact antibodies or their fragments (e.g., Fab, scFv, or F(ab')2) are used. The term “labeling” with respect to an antibody includes directly labeling the antibody by conjugating (i.e., physically binding) a detectable substance to the antibody, and indirectly labeling the antibody by reaction with another directly labeled reagent. Examples of indirect labeling include detection of a primary antibody by a fluorescently labeled secondary antibody, and terminal labeling of an antibody with biotin to enable detection by 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 in the body of a subject. Accordingly, the term “biological sample” includes blood and fractions or components in blood, including serum, plasma, or lymph. In other words, the detection methods according to the above embodiments are used in vitro and in vivo to detect analytes such as mRNA, protein, or genomic DNA in biological samples. For example, in vitro detection techniques for the analyte mRNA include Northern hybridization and in situ hybridization. In vitro detection techniques for the analyte protein include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. In vitro detection techniques for the analyte genomic DNA include Southern hybridization. Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology," Vol. 42, JRCrowther (ed.), Human Press, Totowa, NJ, 1995. In vivo detection techniques for the analyte protein include introducing labeled anti-analyte protein antibodies into the subject.For example, an antibody is labeled with a radioactive label, and the presence and location of the radioactive label in the subject's body are detected using standard imaging techniques.

[0165] The antibodies and their derivatives, fragments, analogs, and homologs relating to this specification can be incorporated into pharmaceutical compositions suitable for administration. The principles, considerations, and guidelines for selecting constituent components of such compositions are well known in the art.

[0166] Such compositions typically include an antibody and a pharmaceutically acceptable carrier. When antibody fragments are used, preferably minimal repressive fragments that specifically bind to the binding domain of the target protein. For example, based on the variable region sequence of an antibody, peptide molecules that retain the ability to bind to the target protein sequence can be designed. Such peptides can be produced by chemical synthesis and / or recombinant DNA technology (see, e.g., Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).

[0167] As used herein, the term “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic agents and absorption retarders, etc., that are acceptable for drug administration. Suitable pharmaceutically acceptable carriers are listed in the latest edition of Remington's Pharmaceutical Sciences, a standard reference bibliography in the art, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vectors such as liposomes and immobilized oils may also be used. The use of such media and reagents with pharmaceutically active substances is well known in the art. Their use in compositions is envisioned, other than in conventional culture media or reagents that are incompatible with antibodies.

[0168] The drug compositions of the embodiments described above are prepared to suit their intended routes of administration. Examples of routes of administration include, for example, parenteral administration, such as 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 sterile diluents for injection, such as water, saline solutions, fixatives, polyethylene glycol-based solvents, glycerol, propylene glycol, or other synthetic solvents, antimicrobial agents, such as benzyl alcohol or methyl p-hydroxybenzoate, antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid (EDTA), buffers, such as acetates, citrates, or phosphates, and osmotic regulators, such as sodium chloride or dextrose. The pH can be adjusted with an acid or base, such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be packaged in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0169] The drug composition suitable for injection includes a sterile aqueous solution (wherein it is water-soluble) or dispersion, and a sterile powder for immediate preparation of a sterile injection solution or dispersion. The pharmaceutically acceptable carrier suitable for intravenous administration includes physiological saline, sterile water, and Cremophor EL. TMThe composition may contain (BASF, Parsippany, NJ) or phosphate-buffered saline (PBS). In all cases, the composition must be sterile and have a fluidity that is easy to inject. It must also be stable under manufacturing and storage conditions and be able to prevent contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or a suitable mixture thereof. Coatings, such as lecithin, can be used to maintain the desired particle size in the case of dispersions, and surfactants can be used to maintain appropriate fluidity. Prevention of microbial activity can be achieved by various antimicrobial and antifungal agents such as parahydroxybenzoic acid esters, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents such as sugars, polyols (e.g., mannitol, sorbitol), and sodium chloride in the composition. The extension of absorption of an injectable composition can be achieved, for example, by including an absorption-delaying reagent in the composition, such as aluminum monostearate or gelatin.

[0170] If necessary, a sterile injection solution is prepared by incorporating the required amount of antibody into a suitable solvent containing one or a combination of the above-mentioned components (if necessary), and then filtration and disinfection is performed. Typically, a dispersion is prepared by incorporating the antibody into a sterile carrier containing a basic dispersion medium and other necessary components from those listed above. For sterile powders for preparing sterile injection solutions, the preparation method involves vacuum drying and lyophilization of a powder containing the active component and any additional desired components from a sterile filtration solution of the above-mentioned components.

[0171] For inhalation administration, the compound is delivered in aerosol spray form from a pressurized container, distributor, or sprayer that appropriately supplies a propellant, such as carbon dioxide or other gas.

[0172] Furthermore, the drug may be administered systemically by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrating agent suitable for barrier penetration into the formulation is used. Such penetrating agents are well known in the art and include, for example, transmucosal administration detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved using nasal sprays or suppositories. For transdermal administration, one or more antibodies can be prepared in the form of pastes, ointments, gels, or creams, which are well known in the art.

[0173] The compound can further be prepared in the form of suppositories or retained enemas (for example, having a conventional suppository base such as cocoa butter or other glycerides) and delivered rectally.

[0174] In one embodiment, the antibody may be prepared using a carrier that prevents rapid excretion from the body, such as a sustained-release / controlled-release formulation including an implant and a microencapsulation delivery system. Examples of biodegradable, biocompatible polymers that can be used include ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations are obvious to those skilled in the art.

[0175] For ease of administration and dose uniformity, preparing parenteral compositions in unit dosage forms is particularly advantageous. As used herein, a unit dosage form refers to a physically separated unit suitable for a single dose for a subject being treated, each unit containing a specific amount of one or more of the antibodies calculated to produce the desired therapeutic effect in combination with the required pharmaceutical carrier. The specifications of the unit dosage forms in the embodiments are determined and directly depend on the inherent properties of the antibodies, the specific therapeutic effect to be achieved, and the limitations inherent in the techniques for preparing such antibodies for treating an individual.

[0176] The drug composition may be placed in a container, pack, or dispenser along with instructions for administration.

[0177] The formulations described herein may further contain more than one of the aforementioned antibodies, preferably antibodies having complementary activity but not adversely affecting each other, depending on the specific condition to be treated. Alternatively, the composition may also contain reagents that enhance the function of the composition, such as cytotoxins, cytokines, chemotherapeutic agents, or growth inhibitors. Such molecules may be present in appropriate combinations in amounts effective for the intended purpose. For example, they may be present in combination in a kit, or they may be present in combination at the time of use.

[0178] In one embodiment, one or more of the antibodies are used in combination therapy, i.e., in combination with other reagents, such as 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” means administering the reagents substantially simultaneously, concurrently, or sequentially. When administered sequentially, preferably, when administration of the second compound is initiated, the first compound in both compounds can still be detected at an effective concentration at the treatment site. In some cases, “combination” may mean that the kit contains the antibodies and other therapeutic agents according to this disclosure simultaneously.

[0179] For example, combination therapy includes the simultaneous preparation and / or administration of one or more antibodies relating to this disclosure and 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 can avoid possible toxicity or complications associated with various monotherapies because the administered therapeutic agents can be used at lower doses.

[0180] In one embodiment, the treatment regimen can effectively reduce cytokine release in a subject in response to the administration of the T-cell activating agent compared to a treatment regimen that does not administer the corresponding antitumor antigen antibody.

[0181] For the sake of clarity and conciseness, technical features are described herein as part of the same or distinct embodiments; however, the scope of the present invention may be understood to include embodiments comprising all or some of the described features in combination.

[0182] Figure 1 shows the structure of a novel first antigen × CD3κλ bispecific antibody.

[0183] This disclosure demonstrates, as experimentally demonstrated, that when freely combined, the λ light chain of the humanized CD3 arm tends to pair with the same type of heavy chain, with a relatively low pairing ratio with heterologous heavy chains. Similarly, the κ light chain of the humanized antigen arm also tends to pair with the same type of heavy chain, with an extremely low pairing ratio with the humanized CD3 heavy chain. Introducing complementary charge mutants to 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 mutant Ser 228 Pro, Leu 235 Glu and Pro 329 Ala maintained the stability of the hinge region and reduced interaction with the Fcγ receptor and C1q.

[0184] Examples

[0185] Example 1: Optimization of anti-CD3 antibody and its activating effect on T cells

[0186] 1. Synthesis of recombinant proteins Human CD3γ (UniProt P09693, Gln23-Asn116) and CD3ε (UniProt P07766, Gln23-Asp126) extracellular domain nucleotide sequences were synthesized, and their C-terminuses were fused to human IgG Fc hole or Fc knob, respectively. Human CD3εγ-Fc heterodimers (the amino acid sequence of human CD3γ IgG Fc(hole) is shown in SEQ ID NO.1, and the amino acid sequence of human CD3ε IgG Fc(knob) is shown in SEQ ID NO.2) were expressed to form these heterodimers. Similarly, cynomolgus monkey CD3γ (UniProt Q95LI7, Gln23-Asn110) and CD3ε (UniProt Q95LI5, Gln22-Asp117) were synthesized, and their C-terminuses were fused to cynomolgus monkey IgG Fc hole or Fc knob, respectively. The CD3εγ-Fc heterodimer was formed by fusing it to a knob and expressing it (the amino acid sequence of cynomolgus monkey CD3γ IgG Fc (hole) is shown in SEQ ID NO.3, and the amino acid sequence of cynomolgus monkey CD3γ IgG Fc (knob) is shown in SEQ ID NO.4). Recombinant plasmids expressing CD3γ-Fc and CD3ε-Fc were mixed with 3 mg / mL of PEI (Polysciences, #24765-2), cotransfected into HEK293E cells (OPM-293 CD03 DPM medium), cultured at 37°C at 120 rpm in 5% CO2 for 7 days, then the supernatant was collected and purified by Protein A affinity chromatography to obtain human or cynomolgus monkey CD3εγ-Fc recombinant protein.

[0187] 2. Humanization of anti-CD3 antibodies Mouse-derived hybridoma anti-CD3 antibodies (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 their sequences were as follows.

[0188] Amino acid sequence of the light chain of an anti-CD3 mouse monoclonal antibody (SEQ ID NO.96):

Chemical formula

[0189] Amino acid sequence of the heavy chain of an anti-CD3 mouse monoclonal antibody (SEQ ID NO.97):

Chemical formula

[0190] The anti-CD3 mouse monoclonal antibody was humanized, the human germline gene IMGT_hVL7-43 with the highest identity was selected, light chain CDR transplantation was performed, human IGLJ3*02 was used as FM4, human IMGT_hVH3-73 was selected, heavy chain CDR transplantation was performed, and human IGHJ4*01 was used as FM4. Different heavy chain mutants and light chain mutants were obtained by design (Table 1).

Table 1

[0191] The amino acid sequence of hVL1 is shown in SEQ ID NO.5, its coding nucleic acid is shown in SEQ ID NO.6, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.7, 8, 9 respectively.

Chemical formula

[0192] Nucleic acid sequence

Chemical formula

[0193] The amino acid sequence of hVL2 is shown in SEQ ID NO.10, its coding nucleic acid is shown in SEQ ID NO.11, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.7, 8, 9 respectively. [ka]

[0194] nucleic acid sequence [ka]

[0195] The amino acid sequence of hVL3 is shown in SEQ ID NO. 12, its coding nucleic acid is shown in SEQ ID NO. 13, and its LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 14, 15, and 9, respectively. [ka]

[0196] nucleic acid sequence [ka]

[0197] The amino acid sequence of hVL4 is shown in SEQ ID NO. 16, its coding nucleic acid is shown in SEQ ID NO. 17, and its LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 14, 15, and 9, respectively. [ka]

[0198] nucleic acid sequence [ka]

[0199] The amino acid sequence of hVL5 is shown in SEQ ID NO. 18, its coding nucleic acid is shown in SEQ ID NO. 19, and its LCDR1, LCDR2, and LCDR3 are shown in SEQ ID NO. 7, 8, and 21, respectively. [ka]

[0200] Nucleic acid sequence

Chem.

[0201] 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 NO.7, 20, 21 respectively.

Chem.

[0202] Nucleic acid sequence

Chem.

[0203] 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 NO.26, 27, 28 respectively.

Chem.

[0204] Nucleic acid sequence

Chem.

[0205] The amino acid sequence of hVH2 is shown in SEQ ID NO.29, its encoding nucleic acid is shown in SEQ ID NO.30, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.31, 27, 28 respectively.

Chem.

[0206] nucleic acid sequence [ka]

[0207] The amino acid sequence of hVH3 is shown in SEQ ID NO. 32, its coding nucleic acid is shown in SEQ ID NO. 33, and its HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO. 31, 27, and 34, respectively. [ka]

[0208] nucleic acid sequence [ka]

[0209] The amino acid sequence of hVH4 is shown in SEQ ID NO. 35, its coding nucleic acid is shown in SEQ ID NO. 36, and its HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO. 31, 27, and 37, respectively. [ka]

[0210] nucleic acid sequence [ka]

[0211] The amino acid sequence of hVH5 is shown in SEQ ID NO. 38, its coding nucleic acid is shown in SEQ ID NO. 39, and its HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO. 31, 27, and 40, respectively. [ka]

[0212] nucleic acid sequence [ka]

[0213] The amino acid sequence of hVH6 is shown in SEQ ID NO. 41, its coding nucleic acid is shown in SEQ ID NO. 42, and its HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO. 31, 27, and 43, respectively. [ka]

[0214] nucleic acid sequence [ka]

[0215] The amino acid sequence of hVH7 is shown in SEQ ID NO. 44, its coding nucleic acid is shown in SEQ ID NO. 45, and its HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO. 46, 47, and 28, respectively. [ka]

[0216] nucleic acid sequence [ka]

[0217] The amino acid sequence of hVH8 is shown in SEQ ID NO. 48, its coding nucleic acid is shown in SEQ ID NO. 49, and its HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO. 26, 27, and 28, respectively. [ka]

[0218] nucleic acid sequence [ka]

[0219] The amino acid sequence of hVH9 is shown in SEQ ID NO. 50, its coding nucleic acid is shown in SEQ ID NO. 51, and its HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO. 26, 27, and 28, respectively. [ka]

[0220] nucleic acid sequence [ka]

[0221] The amino acid sequence of hVH10 is shown in SEQ ID NO. 52, its coding nucleic acid is shown in SEQ ID NO. 53, and its HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO. 26, 27, and 28, respectively. [ka]

[0222] nucleic acid sequence [ka]

[0223] After synthesizing the complete sequences of humanized light chain and heavy chain mutants, they were cloned into eukaryotic expression vectors containing the antibody λ light chain constant region or the human IgG4 heavy chain constant region CH1-CH3. These vectors were cotransfected into HEK293E cells and cultured at 37°C at 120 rpm in 5% CO2 for 5-6 days. The supernatant was collected and purified by Protein A chromatography.

[0224] 3. Affinity of CD3 humanized antibodies Human CD3εγ proteins were coated overnight at 4°C. After blocking with 2% skim milk, anti-CD3 antibodies of different dilutions were added to each well and incubated for 1 hour. HPR-labeled goat anti-human IgG Fc was added as a secondary antibody, and the reaction was chromogenically developed with TMB solution. The reaction was then terminated with concentrated sulfuric acid, and the absorbance at 450 nm was read. 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, and aCD3-hVH9 / VL5) bound to human CD3εγ proteins, demonstrating that CD3 humanized antibodies bind to the CD3εγ recombinant protein with high affinity.

[0225] Jurkat cells in the logarithmic growth phase were isolated, blocked with 3% BSA for 30 minutes, and then plated in 5 × 10⁶ U-shaped 96-well plates. 4 Cells were added to each well, centrifuged, and the supernatant was discarded. 50 μL of gradient-diluted antibody (antibody concentration 3-fold diluted from 30 μg / ml via 5 gradients) was added to each well and incubated at 4°C for 1 hour. 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 at 4°C for 45 minutes. After washing, each well was resuspended in 50 μL of PBS and detected by FACS (iQue, Intellicyt). The results are shown in Figure 3. 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 significantly weaker affinity compared to the reference antibody OKT3.

[0226] Table 2 shows the affinity of CD3 humanized antibodies to CD3 recombinant protein and Jurkat cells. [Table 2]

[0227] 4. Cross-recognition of human and cynomolgus monkey CD3 antigens by humanized CD3 antibodies Human CD3εγ protein and cynomolgus monkey CD3εγ protein were coated overnight at 4°C. After blocking with 2% skim milk, anti-CD3 antibody at different dilutions was added to each well and incubated for 1 hour. HPR-labeled goat anti-human IgG Fc was added as a secondary antibody, and the reaction was chromogenically developed with TMB solution. The reaction was then terminated with concentrated sulfuric acid, and the absorbance at 450 nm was read. Figure 4 shows that both the CD3 humanized antibodies hVH9 / VL5 (aCD3-hVH9 / VL5) and hVH9 / VL2 (aCD3-hVH9 / VL2) simultaneously bind to human CD3εγ and cynomolgus monkey CD3εγ proteins.

[0228] Example 2: Construction of CD20 × CD3κλ bispecific antibodies formed from different types of light chains

[0229] 1. Construction of a CD20 × CD3κλ bispecific antibody A novel T cell κλ bispecific antibody possessing the natural IgG conformation was constructed using the CD3 humanized antibody hVH9 / VL5 (heavy chain pairing with the λ light chain) and the humanized CD20 antibody (heavy chain pairing with the κ light chain).

[0230] As shown in Figure 5, the following five types of CD20×CD3κλ bispecific antibodies were designed and constructed. 1) CD20×CD3κλ001: Natural sequence retaining the CD3 arm and CD20 antigen arm; 2) CD20×CD3κλ002: Charge variant (Vκ) in the CD20 antigen arm and CD3 arm. CD20 :Gln 38 Lys;VH CD20 :Gln 39 Glu; Vλ CD3 :Gln 40 Glu;VH CD3 :Gln 39 A bispecific antibody containing Lys) introduced simultaneously; 3) Based on CD20×CD3κλ003:CD20×CD3κλ002, a complementary charge pair (Vκ-Ck) is formed between CH1 / Cκ. CD20 :Gln 38 Lys / Glu 123 Lys / Gln 124 Lys;V H -C H 1 CD20 :Gln 39 Glu / Lys 152 Glu / Lys 218 Glu;Vλ CD3 :Gln 40 Glu;VH CD3 :Gln 39 A bispecific antibody with added Lys; 4) CD20×CD3κλ004: Charge variant (Vκ) only in the CD20 antigen arm CD20 :Gln 38 Lys;VH CD20 :Gln 39 Bispecific antibodies incorporating Glu; 5) CD20×CD3κλ005: Charge mutant (Vλ) only in the CD3 arm CD3 :Gln 40 Glu;VH CD3 :Gln 39 A bispecific antibody incorporating Lys.

[0231] The corresponding sequences are shown in Table 3, and the reference antibody CD20×CD3-crossFab was constructed according to the CrossFab method (Schaefer W et al., PNAS 2011). [Table 3]

[0232] CD20×CD3κλ001: CD20 arm κ light chain SEQ ID NO.54 [ka]

[0233] Nucleotide sequence SEQ ID NO.55 [ka]

[0234] CD20 arm heavy chain (heavy chain 1) SEQ ID NO. 56 [ka]

[0235] Nucleotide sequence SEQ ID NO. 57 [ka]

[0236] CD3 arm λ light chain SEQ ID NO.58 [ka]

[0237] Nucleotide sequence SEQ ID NO.59 [ka]

[0238] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.60 [ka]

[0239] Nucleotide sequence SEQ ID NO.61 [ka]

[0240] CD20×CD3κλ002: CD20 arm κ light chain SEQ ID NO.62 [ka]

[0241] CiNii sequence SEQ ID NO.63 [ka]

[0242] CD20 arm heavy chain (heavy chain 1) SEQ ID NO.64 [ka]

[0243] Nucleotide sequence SEQ ID NO.65 [ka]

[0244] CD3 arm λ light chain SEQ ID NO.66 [ka]

[0245] CiNii sequence SEQ ID NO.67 [ka]

[0246] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.68 [ka]

[0247] Nucleotide sequence SEQ ID NO.69 [ka]

[0248] CD20×CD3κλ003: CD20 arm κ light chain SEQ ID NO.70 [ka]

[0249] Nucleotide sequence SEQ ID NO.71 [ka]

[0250] CD20 arm heavy chain (heavy chain 1) SEQ ID NO.72 [ka]

[0251] nucleotide sequence SEQ ID NO. 73 [ka]

[0252] CD3 arm λ light chain SEQ ID NO.66 [ka]

[0253] CiNii sequence SEQ ID NO.67 [ka]

[0254] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.68 [ka]

[0255] Nucleotide sequence SEQ ID NO.69 [ka]

[0256] CD20×CD3κλ004: CD20 arm κ light chain SEQ ID NO.62 [ka]

[0257] CiNii sequence SEQ ID NO.63 [ka]

[0258] CD20 arm heavy chain (heavy chain 1) SEQ ID NO.64 [ka]

[0259] nucleotide sequence SEQ ID NO.65 [ka]

[0260] CD3 arm λ light chain SEQ ID NO.58 [ka]

[0261] Nucleotide sequence SEQ ID NO.59 [ka]

[0262] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.60 [ka]

[0263] Nucleotide sequence SEQ ID NO.61 [ka]

[0264] CD20×CD3κλ005: CD20 arm κ light chain SEQ ID NO.54 [ka]

[0265] Nucleotide sequence SEQ ID NO.55 [ka]

[0266] CD20 arm heavy chain (heavy chain 1) SEQ ID NO. 56 [ka]

[0267] Nucleotide sequence SEQ ID NO.57 [ka]

[0268] CD3 arm λ light chain SEQ ID NO.66 [ka]

[0269] CiNii sequence SEQ ID NO.67 [ka]

[0270] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.68 [ka]

[0271] Nucleotide sequence SEQ ID NO.69 [ka]

[0272] CD20×CD3-cross Fa CD20 arm κ light chain SEQ ID NO.70 [ka]

[0273] Nucleotide sequence SEQ ID NO.71 [ka]

[0274] CD20 arm heavy chain (heavy chain 1) SEQ ID NO.74 [ka]

[0275] Nucleotide sequence SEQ ID NO.75 [ka]

[0276] CD3 arm λ light chain SEQ ID NO.76 [ka]

[0277] nucleotide sequence SEQ ID NO.77 [ka]

[0278] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.78 [ka]

[0279] Nucleotide sequence SEQ ID NO. 79 [ka]

[0280] 2. Expression and purification of CD20×CD3κλ bispecific antibodies Plasmids encoding the corresponding antibody fragments were mixed in the ratio 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 of 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 at 150 rpm with 5% CO2, and transiently transfected. On days 2, 4, and 6, 4% CHO Feed C+ feed (Gibco #A25031-05) was added. When the cell activity decreased to approximately 85%, the fermentation broth was harvested, filtered, and purified by Protein A affinity chromatography. CD20×CD3κλ bispecific antibodies constructed based on different light chain types had monomer purity close to or exceeding 90% after one-step purification with Protein A, while the reference antibody CD20×CD3-crossFab had monomer purity less than 80% (Table 4) and a κλ light chain ratio close to 1:1 (Figure 6). [Table 4]

[0281] The CD20×CD3κλ bispecific antibody was further purified by Capto S ImpAct ion-exchange chromatography, and gradient elution was performed with 50-300 mM NaCl and 50 mM phosphate at pH 6.4. The elution peaks were matched, indicating that the SEC-HPLC monomer content exceeded 99% (Figure 7). In the purified samples of CD20×CD3κλ002 and CD20×CD3κλ003, the light chain mismatch ratio was extremely low (<1%), and neither CD3 homodimer nor CD20 homodimer was detected (Figure 8).

[0282] 3. Binding activity of CD20×CD3κλ bispecific antibody The affinity of the bispecific antibody CD20 antigen arm was determined by detecting its binding to stable transfected cells overexpressing CD20 or CD20+ tumor cells, respectively. The affinity of the bispecific antibody CD3 arm was determined by detecting its binding to CD3 recombinant antigen, Jurkat cells, or isolated fresh peripheral blood T cells, respectively. The detection results showed that the novel CD20 × CD3κλ bispecific antibody exhibited approximately 3 to 5 times higher affinity to tumor cells compared to T cells. The positive control antibody bsAB1 was synthesized, expressed, and prepared according to reference US20170174781.

[0283] (1) Binding of CD20 × CD3κλ bispecific antibodies to human and cynomolgus monkey CD20 stable transfected cells Stable transfected cells of CHO-human CD20 and CHO-cynomolgus monkey CD20 produced in Example 1 during the logarithmic growth phase were taken and inoculated with 4% fetal bovine serum (Hyclone, SH30626.06) in a 5 × 10⁻¹⁶ ratio. 5 The cell suspension was adjusted to individual cells / ml, and 100 μl / well was added to a U-type 96-well plate. The plate was 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 gradients) was added to each well. The plates were incubated at 4°C for 60 minutes. As a secondary antibody, 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added, incubated on ice for 20 minutes, washed once, and then 50 μL / well of propidium iodide (PI) solution (1:300) was added. The plates were incubated for 5 minutes and detected by flow cytometry. As shown in Figure 9 and Table 5, the CD20 × CD3κλ bispecific antibody bound to the cellular CD20 receptor with high affinity, and its affinity to human CD20 stable transfected cells corresponded to its affinity to cynomolgus monkey CD20 stable transfected cells. [Table 5]

[0284] (2) Binding of CD20 × CD3κλ bispecific antibody to human CD20+ tumor cells SU-DHL-4, Raji, and NALM-6 cells in the logarithmic growth phase were isolated, 200 μg / mL of mouse IgG (Jackson ImmunoResearch, 115-005-03) was added, the cells were blocked in an ice bath for 30 minutes, and the cells were inoculated with 4% fetal bovine serum in a 5 × 10⁶ ratio. 5 The solution was adjusted to cells / mL, added 100 μL / well to a U-type 96-well plate, centrifuged at 300 g for 5 minutes, discarded the supernatant, and 100 μL of gradient-diluted antibody (initial concentration 1800 nM, 3-fold dilution, 10 gradients) was added to each well and incubated at 4°C for 60 minutes. The primary antibody was removed by washing, 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added, incubated on ice for 20 minutes, washed once, and 50 μL / well of PI was added, incubated for 5 minutes, and detected by flow cytometry. The detection results are shown in Figure 10 and Table 6. The CD20 × CD3κλ bispecific antibody bound to CD20+ tumor cells SU-DHL-4, Raji, and NALM-6 with high affinity.

[0285] (3) Binding of CD20×CD3κλ bispecific antibody to Jurkat cells Jurkat cells in the logarithmic growth phase were isolated, 200 μg / mL of mouse IgG (Jackson ImmunoResearch, 115-005-03) was added, and the cells were blocked in an ice bath for 30 minutes. Cells were then divided into 5 × 10⁻¹⁶ cells in 4% fetal bovine serum. 5The solution was adjusted to cells / mL, 100 μL / well was added to a U-type 96-well plate, centrifuged at 300 g, and the supernatant was discarded. 100 μL of gradient-diluted antibody (initial concentration 1800 nM, 3-fold dilution, 10 gradients) was added to each well and incubated at 4°C for 60 minutes. 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added as a secondary antibody, incubated on ice for 20 minutes, washed once, and then 50 μL / well of PI was added and incubated for 5 minutes. Detection was performed by flow cytometry (BD C6). 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 EC 50 The binding strength was approximately 71-120 nM, which was about 10 times lower than the binding strength of the CD20 antigen arm to the CD20 receptor.

[0286] (4) Binding of CD20×CD3κλ bispecific antibody to human peripheral blood T cells Fresh human peripheral blood was taken, and PBMCs were isolated using Ficoll. Paque Plu (GE, 17-1440-03). The PBMCs were then inoculated with 4% fetal bovine serum (Hyclone, SH30626.06) to a cell count of 5 × 10⁻¹⁶ cells. 5 The antibody was adjusted to individual cells / mL, added 100 μL / well to a U-type 96-well plate, centrifuged, and the supernatant was discarded. 100 μL of gradient-diluted antibody (initial concentration 1800 nM, 3-fold dilution, 10 gradients) was added to each well and incubated at 4°C for 60 minutes. 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added as a secondary antibody, incubated in an ice bath for 20 minutes, washed once, and then 50 μL / well of PI was added and incubated for 5 minutes. Detection was performed by flow cytometry (BD C6). The detection results are shown in Figure 12 and Table 6. The CD20 × CD3κλ bispecific antibody was found to be human peripheral blood CD4 + T and CD8 + The antibody recognized T cells and exhibited an affinity for human T cells of approximately 65-98 nM, which was about 10 times weaker than the binding affinity of the CD20 antigen arm to the CD20 receptor, thus favoring the preferential recruitment of the bispecific antibody to tumor cells. [Table 6]

[0287] 4. TDCC activity by CD20×CD3κλ bispecific antibodies Fresh isolated PBMCs were taken and mixed with target cells NALM-6, TMD-8, and Toledo cells, each in the logarithmic growth phase, in an effector cell / target cell ratio of 8:1. 50 μL / well of gradient-diluted antibody (antibody concentration 66.7 nM, diluted 10-fold with 7 gradients) was added, and the cells were incubated at 5% CO2, 37°C for 24 hours. After incubation, 50 μL of the supernatant was transferred to a new black immunosorbent plate, 50 μL / well of LDH detection substrate was added, and the reaction was terminated after 10 minutes. LDH release was detected, residual cells in the wells were washed twice with 4% fetal bovine serum, 100 μg / mL of human IgG was added, and the cells were incubated for 10 minutes. Then, antibodies for T cell activation detection (CD25-PE, CD4-APC, CD69-FITC, and CD8-APC) were added, and the cells were incubated on ice for 20 minutes. The cells were washed, the supernatant was discarded, 60 μL / well of PI was added, and the cells were incubated on ice for 5 minutes before detection by flow cytometry. Figures 13A and 13B show the killing of human B lymphocyte leukemia cells Nalm-6 and their activation into T cells by the CD20×CD3κλ bispecific antibody, respectively. Figures 14A and 14B show the killing of TMD-8 cells and their activation into T cells by the CD20×CD3κλ bispecific antibody, respectively. Figures 15A and 15B show the killing of Toledo cells and their activation into T cells by the CD20×CD3κλ bispecific antibody, respectively. Against tumor cells Nalm-6, TMD-8, and Toledo with different CD20 expression levels, CD20×CD3κλ002 and CD20×CD3κλ003 were able to contribute to effective killing by T cells. Their killing activity was equivalent to or slightly stronger than that of the control antibody bsAB1, and their T cell activation was milder compared to the latter.

[0288] 5. Activation of the T cell activation pathway by CD20×CD3κλ 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 are isolated, centrifuged, and the supernatant is discarded. 6 The cells were resuspended at individual cells / ml. 50 μl / well of target cells was inoculated into a 96-well plate, centrifuged at 300 g for 5 minutes, and the supernatant was discarded. 50 μl / well of Jurkat-NFAT-luc reporter cells was then inoculated into the 96-well plate, and 50 μl / well of gradient-diluted CD20×CD3κλ bispecific antibody or reference antibody KLH×CD3 (initial concentration 20 μg / ml, 10-fold dilution, 10 gradients) was added. The cells were cultured at 5% CO2, 37°C for 6 hours. After culturing, 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 using a microplate reader (Biotek Synergy HT). The detection results are shown in Figure 16 and Table 7. The CD20×CD3κλ bispecific antibody was able to activate the NFAT signaling pathway of T cells in both cases, even when targeting tumor cells with different CD20 expression levels. [Table 7]

[0289] 6. Binding of CD20×CD3κλ bispecific antibody to Fcγ receptor A 50 μg / ml His-Tag antibody was coupled to a CM5 chip using an amino group, resulting in His-tagged FcγRI and FcγRIIA, respectively. H131 and FcγRIIIA V158Recombinant proteins (Sino Biological, #10256-H08H / 10374-H08H1 / 10389-H08H1) were captured for 40 seconds at a flow rate of 10 μL / min. After baseline stabilization, gradient-diluted antibodies (initial concentration 37.5 μg / mL, 2-fold dilution) were flowed through the tip at a flow rate of 30 μL / min. The binding time was 120 seconds, and the dissociation time was 200 seconds. Affinity constants were obtained by fitting with Biacore evaluation software. As can be seen from Table 8, the CD20×CD3κλ bispecific antibodies were FcγRI and FcγRIIA. H131 or FcγRIIIA V158 The wild-type IgG4 control antibody does not bind to FcγRI, but binds to FcγRIIA with relatively strong affinity. H131 It was weakly bonded to it. [Table 8]

[0290] 7. Immunologically reconstituted mouse subcutaneous Raji transplant tumor model Select 6-8 week old B-NGD female mice (Biocytogen Pharmaceuticals Co., Ltd.) and subcutaneously inoculate them with 3 x 10⁶ Raji cells. 6 After inoculation, the tumor reached 60mm 3 Upon reaching this point, the mice were randomly divided into groups: a treatment group of 3.0 mg / kg, a treatment group of 0.6 mg / kg, a treatment group of 0.12 mg / kg, and a negative control group of 3 mg / kg of KLH×CD3. Each mouse received 1 × 10¹⁶ PBMC cells. 7 The drug was injected into the tail vein, and the first administration to mice began 3 days later. The administration interval was once every 5 days, for a total of 3 administrations. The tumor volume and body weight of the mice were monitored. After the experiment, the mice were executed by decapitation, and the tumors were weighed and recorded. The results are shown in Figure 17. The CD20×CD3κλ bispecific antibody showed dose-dependent efficacy in vivo, with tumor suppression rates of 82% and 89% at medium and high doses, respectively. Tumor-bearing mice showed good tolerability to the above doses and did not exhibit adverse effects such as weight loss.

[0291] 8. A subcutaneous tumor inoculation model of immunodeficient mice mixed with Raji and human PBMCs. Select 6-8 week old B-NGD female mice (Biocytogen Pharmaceuticals Co., Ltd.) and perform Raji (3 × 10⁻¹⁰) 6 (pieces) and human PBMC (5 x 10) 6 The mixture (individual units) was injected subcutaneously into mice, and the tumor volume was 60-100 mm. 3 Upon reaching a certain level, the mice were randomly divided into groups. The treatment groups were 3.0 mg / mL, 0.6 mg / mL, 0.12 mg / mL, and a negative control group of KLH×CD3 3 mg / kg. The administration interval was once every 5 days, for a total of two doses. The tumor volume and body weight of the mice were monitored. After the experiment, the mice were executed by decapitation, and the tumors were weighed and recorded. The results are shown in Figure 18. The CD20×CD3κλ bispecific antibody showed dose-dependent efficacy in vivo, with tumor suppression rates of 65%, 98%, and 162% at low, medium, and high doses, respectively. In the high and medium dose groups, the tumors were completely suppressed or regressed.

[0292] 9. The efficacy of CD20 × CD3κλ bispecific antibodies in cynomolgus monkeys. Eight cynomolgus monkeys were divided into four dose groups, with each group consisting of two monkeys (half female, half male). The doses administered to each group were 0.3 mg / kg, 1 mg / kg, 3 mg / kg (administered once a week for a total of four times over three weeks) and 1 mg / kg (single dose) of CD20×CD3κλ002 bispecific antibody, respectively. The administration regimens are shown in Table 9. During the administration and recovery phases, all monkeys in each group were in good condition, showing no toxic reactions, deaths, or terminal conditions. None of the dose groups showed any significant abnormal changes in body temperature, and their lead II electrocardiogram waveforms were normal. No significant abnormalities were observed in indicators such as heart rate, RR interphase, PR interphase, QT interphase, QRS duration, systolic pressure, or diastolic pressure. Changes in the number of B cells and T cells in peripheral blood were analyzed by flow cytometry at different time points after administration. B cells were identified using the cell surface label CD20 (CD20+ cells), and T cells were identified using CD3 (CD3+ cells). Eight hours after administration, B cells were rapidly eliminated from peripheral blood, and after 24 hours, they fell below the lower limit of detection (Figure 19). [Table 9]

[0293] Example 3: Construction of a BCMA × CD3κλ bispecific antibody formed from different types of light chains 1. Construction of a BCMA × CD3κλ bispecific antibody Referring to Example 2, a novel BCMA-CD3κλ humanized bispecific antibody with a natural IgG conformation was constructed using a BCMA humanized antibody containing a κ light chain and a humanized anti-CD3 antibody containing a λ light chain, and a charge variant (Vκ) was added to the BCMA antigen arm and CD3 arm. BCMA :Gln 42 Lys;VH BCMA :Gln 39 Glu;Vλ CD3 :Gln 40 Glu;VH CD3 :Gln 39To achieve heterodimer pairing, the human IgG4 knob-into-hole structure was adopted as the Fc portion of the bispecific antibody, and mutant Ser 228 Pro, Leu 235 Glu and Pro 329 Ala maintains the stability of the hinge region and reduces interaction with the FcγR receptor and C1q. [Table 10]

[0294] BCMA×CD3κλ003 BCMA arm κ light chain: SEQ ID NO.80 JPEG0007846110000093.jpg12141

[0295] Nucleotide sequence: SEQ ID NO.81 [ka]

[0296] BCMA Arm Heavy Chain (Heavy Chain 1): SEQ ID NO.82 [ka]

[0297] Nucleotide sequence: SEQ ID NO.83 [ka]

[0298] CD3 arm λ light chain: SEQ ID NO.66 [ka]

[0299] Nucleotide sequence: SEQ ID NO.67 [ka]

[0300] CD3 arm heavy chain (heavy chain 2): SEQ ID NO. 68 [ka]

[0301] Nucleotide sequence: SEQ ID NO. 69 [ka]

[0302] BCMA×CD3κλ004 BCMA arm κ light chain: SEQ ID NO.84 [ka]

[0303] Nucleotide sequence: SEQ ID NO.85 [ka]

[0304] BCMA Arm Heavy Chain (Heavy Chain 1): SEQ ID NO.86 [ka]

[0305] Nucleotide sequence: SEQ ID NO.87 [ka]

[0306] CD3 arm λ light chain: SEQ ID NO.66 [ka]

[0307] Nucleotide sequence: SEQ ID NO.67 [ka]

[0308] CD3 arm heavy chain (heavy chain 2): SEQ ID NO. 68 [ka]

[0309] Nucleotide sequence: SEQ ID NO.69 [ka]

[0310] BCMA×CD3κλ005 BCMA arm κ light chain: SEQ ID NO.80 [ka]

[0311] Nucleotide sequence: SEQ ID NO.81 [ka]

[0312] BCMA Arm Heavy Chain (Heavy Chain 1): SEQ ID NO.86 [ka]

[0313] Nucleotide sequence: SEQ ID NO.87 [ka]

[0314] CD3 arm λ light chain: SEQ ID NO.66 [ka]

[0315] Nucleotide sequence: SEQ ID NO.67 [ka]

[0316] CD3 arm heavy chain (heavy chain 2): SEQ ID NO. 68 [ka]

[0317] Nucleotide sequence: SEQ ID NO.69 [ka]

[0318] BCMA×CD3κλ006 BCMA arm κ light chain: SEQ ID NO.84 [ka]

[0319] Nucleotide sequence: SEQ ID NO.85 [ka]

[0320] BCMA Arm Heavy Chain (Heavy Chain 1): SEQ ID NO.86 [ka]

[0321] Nucleotide sequence: SEQ ID NO.87 [ka]

[0322] CD3 arm λ light chain: SEQ ID NO.66 [ka]

[0323] Nucleotide sequence: SEQ ID NO.67 [ka]

[0324] CD3 arm heavy chain (heavy chain 2): SEQ ID NO. 68 [ka]

[0325] Nucleotide sequence: SEQ ID NO.69 [ka]

[0326] 2. Expression and purification of BCMA × CD3κλ bispecific antibody Plasmids encoding the corresponding antibody fragments were mixed in the 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 of 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 at 150 rpm with 5% CO2, and transiently transfected. On days 2, 4, and 6, 4% CHO Feed C+ feed (Gibco #A25031-05) was added. When the cell activity rate decreases to about 85%, the fermentation liquid is harvested, filtered, and then pre-purified by Protein A affinity chromatography. SEC-HPLC is then used to show a monomer content of nearly 92% or more, and the monomer content is further increased to 98-99% or higher through Capto S ImpAct ion exchange chromatography (Table 11). [Table 11]

[0327] 3. Binding activity of BCMA × CD3κλ bispecific antibody (1) Measurement of affinity of BCMA × CD3κλ bispecific antibody to antigen Human or cynomolgus monkey BCMA or CD3εγ recombinant antigen at 10 μg / mL was coupled to a CM5 chip (GE Healthcare) using an amino group, and the antigen binding amount was controlled to approximately 200 RU. After baseline stabilization, gradient-diluted antibody (diluted 2-fold from 10 μg / mL with 7 gradients) was flowed through the chip at a flow rate of 30 μL / min. The binding time was 350 seconds, and the dissociation time was 600 seconds. Affinity constants were obtained by fitting a 1:1 binding model using Biacore T200 evaluation software. The affinity measurement results are shown in Table 12. [Table 12]

[0328] (2) Binding of BCMA × CD3κλ bispecific antibody 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 were isolated in the logarithmic growth phase, blocked, and then 100 μL of gradient-diluted antibody (initial concentration 1800 nM, 3-fold dilution, 10 gradients) was added to each well and incubated at 4°C for 60 minutes. As a secondary antibody, 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added and incubated on ice for 20 minutes. After one wash, 50 μL / well of PI solution (1:300) was added and incubated for 5 minutes, and detection was performed by flow cytometry. Figure 20 shows that the BCMA×CD3κλ bispecific antibody binds to human and cynomolgus monkey BCMA-stable transfected cells with high affinity, and Figure 21 shows that the BCMA×CD3κλ bispecific antibody binds to BCMA+ tumor cells NCI-H929 and RPMI-8226 with high affinity. The binding constant EC of the BCMA×CD3κλ bispecific antibody to cells. 50 This is shown in Table 13. [Table 13]

[0329] (3) Binding of BCMA × CD3κλ bispecific antibody to Jurkat cells Jurkat cells in the logarithmic growth phase were isolated, 200 μg / mL of mouse IgG (Jackson ImmunoResearch, 115-005-03) was added, and the cells were blocked in an ice bath for 30 minutes. Cells were then divided into 5 × 10⁻¹⁶ cells in 4% fetal bovine serum. 5The antibody was adjusted to cells / mL, added 100 μL / well to a U-type 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 gradients) was added to each well and incubated at 4°C for 60 minutes. As a secondary antibody, 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added, incubated on ice for 20 minutes, washed once, and then 50 μL / well of PI was added and incubated for 5 minutes. Detection was performed by flow cytometry (BD C6). The detection results are shown in Figure 22 and Table 14. The BCMA × CD3κλ bispecific antibody bound to the human leukemia T cell line Jurkat cells with medium affinity.

[0330] (4) Binding of BCMA × CD3κλ bispecific antibody to peripheral blood T cells Fresh human peripheral blood was collected, and PBMCs were isolated using Ficoll. Paque Plu (GE, 17-1440-03). The PBMCs were then treated with 4% fetal bovine serum (Hyclone, SH30626.06) to divide the cells into 5 × 10⁻¹⁶ cells. 5 The antibody was adjusted to individual cells / mL, added 100 μL / well to a U-type 96-well plate, centrifuged, the supernatant discarded, and 100 μL / well of gradient-diluted antibody (initial concentration 1800 nM, 3-fold dilution, 10 gradients) was added, and incubated at 4°C for 60 minutes. As a secondary antibody, 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added, incubated in an ice bath for 20 minutes, washed once, and then 50 μL / well of PI was added, 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 × CD3κλ bispecific antibody recognized human peripheral blood CD4+ T and CD8+ T cells, and its affinity for human T cells was approximately 60-97 nM. This was weaker than the binding affinity of the BCMA antigen arm to the BCMA receptor, which was advantageous for the preferential recruitment of tumor cells by the bispecific antibody. [Table 14]

[0331] 4. TDCC activity by BCMA × CD3κλ bispecific antibody Fresh isolated PBMCs were taken and mixed with target cells NCI-H929 and RPMI-8226, respectively, in the logarithmic growth phase, resulting in an effector cell / target cell ratio of 8:1. 50 μL / well of gradient-diluted antibody (antibody concentration 10-fold diluted from 66.7 nM with 7 gradients) was added, and the cells were incubated at 5% CO2, 37°C for 24 hours. After incubation, 50 μL of the supernatant was transferred to a new black immunosorbent plate, 50 μL / well of LDH detection substrate was added, and the reaction was terminated after 10 minutes to detect LDH release. Residual cells in the wells were washed twice with 4% fetal bovine serum, then 100 μg / mL of human IgG was added and incubated for 10 minutes. T cell activation detection antibodies (CD25-PE, CD4-APC, CD69-FITC, and CD8-APC) were added and incubated on ice for 20 minutes. The cells were washed, the supernatant was discarded, 60 μL / well of PI was added, and the cells were incubated on ice for 5 minutes before detection by flow cytometry. Figures 24A and 24B show the killing of NCI-H929 cells and their activation into T cells by the BCMA×CD3κλ bispecific antibody, respectively. Figures 25A and 25B show the killing of RPMI-8226 cells and their activation into T cells by the BCMA×CD3κλ bispecific antibody, respectively. Against tumor cells NCI-H929 and RPMI-8226 with different BCMA expression levels, the BCMA×CD3κλ bispecific antibody was able to contribute to effective killing by T cells, and its killing activity was equivalent to that of the reference antibody REGN5458.

[0332] 5. Activation of the T cell activation pathway by BCMA × CD3κλ bispecific antibody Jurkat-NFAT-luc reporter cells and BCMA-positive target cells RPMI-8226 in the logarithmic growth phase were taken, centrifuged, and the supernatant was discarded. 6The cells were resuspended at individual cells / ml. 50 μl / well of target cells was inoculated into a 96-well plate, centrifuged at 300 g for 5 minutes, and the supernatant was discarded. 50 μl / well of Jurkat-NFAT-luc reporter cells was inoculated into the 96-well plate, and 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) was added to each well. The cells were cultured at 5% CO2, 37°C for 6 hours. After culturing, 100 μL of detection reagent was added to each well according to the ONE-Glo Luciferase Assay System instruction manual, left at room temperature for 3 minutes, and detected using a microplate reader (Biotek Synergy HT). The detection results are shown in Figure 26. The BCMA × CD3κλ bispecific antibody was able to activate the NFAT signaling pathway of T cells when RPMI-8226 tumor cells were targeted, but did not activate the NFAT signaling pathway when no target cells were present.

[0333] 6. Nonspecific activation of PBMCs by BCMA × CD3κλ bispecific antibody Fresh isolated PBMCs were taken, and 50 μL of gradient-diluted antibody (antibody concentration 10-fold diluted with 7 gradients from 66.7 nM) was added to each well. The cells were incubated at 37°C in 5% CO2 for 24 hours. After incubation, 50 μL of the supernatant was transferred to a new black immunosorbent plate, 50 μL / well of LDH detection substrate was added, and the reaction was terminated after 10 minutes. LDH release was detected, and residual cells in the wells were washed twice with 4% fetal bovine serum. Human IgG 100 μg / mL was added and incubated for 10 minutes. Then, T cell activation detection antibodies (CD25-PE, CD4-APC, CD69-FITC, and CD8-APC) were added and incubated on ice for 20 minutes. The cells were washed, the supernatant was discarded, PI 60 μL / well was added, and the cells were incubated on ice for 5 minutes and detected by flow cytometry. The detection results are shown in Figure 27. Under conditions where target cells were absent, the BCMA×CD3κλ bispecific antibody did not have an activating effect on peripheral blood T cells and was equivalent to the negative control KLH×CD3.

[0334] 7. Binding of BCMA × CD3κλ humanized bispecific antibody to the Fc receptor The amino group is used to couple 50 μg / ml of His-Tag antibody to the CM5 chip, resulting in His-tagged FcγRI and FcγRIIA. H131 and FcγRIIIA V158 Recombinant proteins were captured individually for 40 seconds at a flow rate of 10 μL / min. After baseline stabilization, gradient-diluted antibodies (initial concentration 37.5 μg / mL, 2-fold dilution) were flowed through the tip at a flow rate of 30 μL / min. The binding time was 120 seconds, and the dissociation time was 200 seconds. Affinity constants were obtained by fitting with Biacore evaluation software. As can be seen in Figure 28, the BCMA × CD3κλ bispecific antibodies were FcγRI and FcγRIIA. H131 and FcγRIIIA V158 The wild-type IgG4 control antibody does not bind to FcγRI, but binds to FcγRIIA with relatively strong affinity. H131 It bonded to it only slightly and weakly.

[0335] 8. Immunodeficient mouse subcutaneous NCI-H929 transplanted tumor model Select 6-8 week old B-NGD female mice (Biocytogen Pharmaceuticals Co., Ltd.) and subcutaneously inoculate 2 × 10⁶ NCI-H929 cells (mixed with Matrigel in a 1:1 ratio). 6 Individual doses were administered, and the tumor reached 60mm. 3 Upon reaching this point, the mice were randomly divided into groups: a treatment group of 3.0 mg / kg, a treatment group of 0.6 mg / kg, a treatment group of 0.12 mg / kg, and a negative control group of 3 mg / kg of KLH×CD3. Each mouse received 1 × 10¹⁶ PBMC cells. 7The antibody was injected into the tail vein, and the first administration to mice began 3 days later. The administration interval was 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. After the experiment, the mice were executed by decapitation, and the tumors were weighed and recorded. The results are shown in Figure 29. The BCMA×CD3κλ bispecific antibody showed dose-dependent efficacy in vivo, with tumor suppression rates of 95% and 108% (BCMA×CD3κλ005) and 94% and 108% (BCMA×CD3κλ006) in the 3.0 mg / kg dose group and the 0.6 mg / kg dose group, respectively. Tumor-bearing mice tolerated the above doses well and did not show adverse effects such as weight loss.

[0336] Example 4: Construction of a GPC3 × CD3κλ bispecific antibody formed from different types of light chains 1. Construction of a GPC3 × CD3κλ bispecific antibody Referring to Example 2, a novel GPC3-CD3κλ humanized bispecific antibody with a natural IgG conformation was constructed using a humanized GPC3 antibody containing a κ light chain and a humanized anti-CD3 antibody containing a λ light chain, and a charge variant (Vκ) was added to the GPC3 antigen arm and CD3 arm. GPC3 :Gln 43 Lys;VH GPC3 :Gln 39 Glu;Vλ CD3 :Gln 40 Glu;VH CD3 :Gln 39 Lys) (sequence shown in Table 15) was introduced. To achieve heterodimer pairing, the Fc portion of the bispecific antibody adopted a human IgG4 knob-into-hole structure, and mutant Ser 228 Pro, Leu 235 Glu and Pro 329 Ala maintains the stability of the hinge region and reduces interaction with the Fcγ receptor and C1q. [Table 15]

[0337] GPC3×CD3κλ002: GPC3 arm κ light chain SEQ ID NO.88 [ka]

[0338] Nucleotide sequence SEQ ID NO.89 [ka]

[0339] GPC3 Arm Heavy Chain (Heavy Chain 1) SEQ ID NO.90 [ka]

[0340] Nucleotide sequence SEQ ID NO.91 [ka]

[0341] CD3 arm λ light chain SEQ ID NO.66 [ka]

[0342] CiNii sequence SEQ ID NO.67 [ka]

[0343] CD3 arm heavy chain (heavy chain 2) SEQ ID NO.68 [ka]

[0344] Nucleotide sequence SEQ ID NO.69 [ka]

[0345] GPC3×CD3κλ003: GPC3 arm κ light chain SEQ ID NO.92 [ka]

[0346] Nucleotide sequence SEQ ID NO.93 [ka]

[0347] GPC3 Arm Heavy Chain (Heavy Chain 1) SEQ ID NO. 94 [ka]

[0348] Nucleotide sequence SEQ ID NO.95 [ka]

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

[0350] CiNii 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] Plasmids encoding the corresponding antibody fragments were mixed in the 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 of 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 at 150 rpm with 5% CO2, and transiently transfected. On days 2, 4, and 6, 4% CHO Feed C+ feed (Gibco #A25031-05) was added. When the cell activity rate decreases to approximately 85%, the fermentation liquid is harvested, filtered, and then pre-purified by Protein A affinity chromatography. SEC-HPLC shows that the monomer content exceeds 92%, and Butyl HP hydrophobic chromatography and Capto Q anion chromatography further increase the monomer content to over 99.5% (Table 16). [Table 16]

[0354] 2. Binding of GPC3 × CD3κλ bispecific antibody to GPC3 stable transfected cells CHO-human GPC3, CHO-cynomolgus monkey GPC3 stable transfected cells, or human hepatocellular carcinoma (HepG2) tumor cells in the logarithmic growth phase were taken, blocked, and then the cells were divided into 5 × 10⁻¹⁴ cells. 5The cell suspension was adjusted to individual cells / ml, and 100 μl / well was added to a U-type 96-well plate. The plate was centrifuged at 300 g for 5 minutes, the supernatant was discarded, and 100 μL of gradient-diluted antibody (diluted 3-fold from an initial concentration of 1800 nM with 10 gradients) was added to each well. The plates were incubated at 4°C for 60 minutes. As a secondary antibody, 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added, incubated on ice for 20 minutes, washed once, and then 50 μL / well of PI solution (1:300) was added. The plates were incubated for 5 minutes and detected by flow cytometry. The results are shown in Figures 30-31 and Table 17. The GPC3 × CD3κλ bispecific antibody bound to GPC3+ cells with high affinity. [Table 17]

[0355] 3. Binding of GPC3 × CD3κλ bispecific antibody to Jurkat cells Jurkat cells in the logarithmic growth phase were isolated, 200 μg / mL of mouse IgG (Jackson ImmunoResearch, 115-005-03) was added, and the cells were blocked in an ice bath for 30 minutes. Cells were then divided into 5 × 10⁻¹⁶ cells in 4% fetal bovine serum. 5 The antibody was adjusted to cells / mL, added 100 μL / well to a U-type 96-well plate, centrifuged at 300 g, and the supernatant was discarded. 100 μL of gradient-diluted antibody (diluted 3-fold from an initial concentration of 1800 nM with 10 gradients) was added to each well and incubated at 4°C for 60 minutes. 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added as a secondary antibody, incubated on ice for 20 minutes, washed once, and then 50 μL / well of PI was added and incubated for 5 minutes. Detection was performed by flow cytometry (BD C6). The detection results are shown in Figure 32 and Table 18. The GPC3 × CD3κλ bispecific antibody bound to human leukemia T cell line Jurkat cells with medium affinity and EC 50 The intensity was 20-40 nM.

[0356] 4. Binding of GPC3 × CD3κλ bispecific antibody to peripheral blood T cells Fresh human or cynomolgus monkey peripheral blood was taken, and PBMCs were isolated using Ficoll.Paque Plus (GE, 17-1440-03). PBMCs were then inoculated with 4% fetal bovine serum (Hyclone, SH30626.06) for a total cell count of 5 × 10⁶ cells. 5 The antibody was adjusted to individual cells / mL, 100 μL / well was added to a U-type 96-well plate, centrifuged, and the supernatant was discarded. 100 μL of gradient-diluted antibody (diluted 3-fold from an initial concentration of 1800 nM with 10 gradients) was added to each well and incubated at 4°C for 60 minutes. 50 μL / well of Alexa Fluro647-labeled goat anti-human IgG Fc (1:300 dilution) was added as the secondary antibody, incubated in an ice bath for 20 minutes, washed once, and then 50 μL / well of PI was added and incubated for 5 minutes. Detection was performed by flow cytometry (BD Celesta). The detection results are shown in Figure 33 and Table 18. The GPC3 × CD3κλ bispecific antibody bound to human peripheral blood T cells with low affinity. [Table 18]

[0357] 5. TDCC activity by GPC3 × CD3κλ bispecific antibody Fresh isolated PBMCs were taken and mixed with target cells (HepG2 cells) in the logarithmic growth phase, resulting in an effector cell / target cell ratio of 10:1. 50 μL of gradient-diluted antibody (antibody concentration 10-fold dilution from 66.7 nM via a 7-gradient) was added to each well, and the cells were incubated at 5% CO2, 37°C for 24 hours. After incubation, 50 μL of the supernatant was transferred to a new black immunosorbent plate, 50 μL / well of LDH detection substrate was added, and the reaction was terminated after 10 minutes. LDH release was detected, and residual cells in the wells were washed twice with 4% fetal bovine serum. Then, 100 μg / mL of human IgG was added and incubated for 10 minutes. Finally, antibodies for T cell activation detection (CD25-BV421, CD4-FITC, CD69-BV605, and CD8-APC) were added and incubated on ice for 20 minutes. The cells were washed, the supernatant was discarded, 60 μL / well of PI was added, and the cells were incubated on ice for 5 minutes. Detection was then performed by flow cytometry. Figures 34A and 34B show the killing of HepG2 cells and their activation into T cells by the GPC3 × CD3κλ bispecific antibody, respectively.

[0358] 6. Activation of the T cell activation pathway by GPC3 × CD3κλ bispecific antibody Target cells CHO-human GPC3 in the logarithmic growth phase are taken, centrifuged, the supernatant is discarded, and 2 × 10⁻⁶ cells are collected. 5 The cells were resuspended at individual cells / ml. 50 μl / well of target cells were inoculated into a 96-well plate and cultured overnight at 5% CO2 and 37°C. Jurkat-NFAT-luc reporter cells in the logarithmic growth phase were centrifuged at 300 g for 5 minutes, the supernatant was discarded, and 4 × 10⁶ cells were collected. 6The cells were resuspended at individual cells / 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 gradients) was added to each well, and the cells were cultured at 5% CO2, 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 Figure 35, and the GPC3×CD3 κλ bispecific antibody was able to activate the NFAT signaling pathway of T cells when targeting CHO-human GPC3.

[0359] 7. Nonspecific activation of GPC3×CD3κλ bispecific antibody PBMCs Fresh isolated PBMCs were taken, 100 μL (10 μg / mL) of antibody was added, and they were cultured at 5% CO2, 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 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, 60 μL / well of PI was added, and the cells were incubated on ice for 5 minutes and detected by flow cytometry. A positive control antibody ERY974 was prepared referring to US20170267783. The detection results are shown in Figure 36. Under conditions where target cells were absent, the GPC3×CD3κλ bispecific antibody did not have an activating effect on peripheral blood T cells and corresponded to the negative control KLH×CD3.

[0360] 8. Immune-reconstituted mouse subcutaneous HepG2 transplant tumor model Select 6-8 week old B-NGD female mice (Biocytogen Pharmaceuticals Co., Ltd.) and subcutaneously inoculate HepG2 cells (7 × 10⁴). 6 ( / animal) is inoculated, and the tumor is 60-100 mm3 Upon reaching this point, mice were randomly divided into groups: a high-dose group of 3.0 mg / kg, a medium-dose group of 1.0 mg / kg, a low-dose group of 0.3 mg / kg, a positive control group of ERY974, and a negative control group of KLH×CD3 at 3 mg / kg. Each mouse received 1 × 10¹⁶ PBMC cells. 7 The antibody was injected into the tail vein, and the first administration to mice began 3 days later. The administration interval was once every 5 days, for a total of two administrations. The tumor volume and body weight of the mice were monitored. After the experiment, the mice were executed by decapitation, and the tumors were weighed and recorded. The results are shown in Figure 37. The GPC3×CD3κλ bispecific antibody showed dose-dependent efficacy in vivo, with tumor suppression rates of 76.7%, 81.3%, and 95.9% (from medium to high doses), respectively. Tumor-bearing mice showed good tolerability to the above doses and did not exhibit adverse effects such as weight loss.

[0361] 9. CD3-humanized mouse Hepa1-6 / human GPC3 transplant tumor model Six-week-old C57 / BL6-hCD3 female mice (Biocytogen Pharmaceuticals Co., Ltd.) were selected, and Hepa1-6 / human GPC3 (6×10) was administered. 6 ( / mouse) was inoculated subcutaneously, and the tumor volume was 60-100 mm 3 Upon reaching a certain level, the mice were randomly divided into groups. These groups consisted of a high-dose group (10 mg / kg), a medium-dose group (3 mg / kg), a low-dose group (1 mg / kg), a positive control group (ERY974), and a negative control group (KLH×CD3) (10 mg / kg). Administration was performed once every three days, for a total of three doses. Tumor volume and body weight were monitored. After the experiment, the mice were executed by decapitation, and the tumors were weighed and recorded. The results are shown in Figure 38. The GPC3×CD3κλ bispecific antibody significantly contributed to the killing of tumor cells by immune cells, reducing tumor volume, and its 10 mg / kg dose was equivalent to the efficacy of ERY974.

Claims

1. (a) A first antigen-binding moiety or antigen-binding fragment thereof, wherein the first antigen-binding moiety comprises a first light chain and a first heavy chain, the first light chain being a κ-type light chain, and the first antigen-binding moiety comprises a first binding domain that binds to a first antigen, and (b) A second antigen-binding moiety or antigen-binding fragment thereof, wherein the second antigen-binding moiety comprises a second light chain and a second heavy chain, the second light chain being a λ-type light chain, and the second antigen-binding moiety comprises a second binding domain that binds to a second antigen, The second antigen mentioned above is the CD3 antigen, The second light chain CDR of the second binding domain is CDR1 consists of the amino acid sequence indicated by SEQ ID NO:

7. CDR2 consisting of the amino acid sequence indicated by SEQ ID NO: 8, This is CDR3 consisting of the amino acid sequence indicated by SEQ ID NO: 21, The second heavy chain CDR of the second binding domain is CDR1, consisting of the amino acid sequence indicated by SEQ ID NO: 26, CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 27, This is CDR3 consisting of the amino acid sequence indicated by SEQ ID NO: 28, The second light chain of the second antigen-binding portion is the amino acid sequence indicated by SEQ ID NO: 66, and the second heavy chain of the second antigen-binding portion is the amino acid sequence indicated by SEQ ID NO: 68, and the first antigen is not the CD20 antigen. A bispecific antibody or its antigen-binding fragment.

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

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

4. The bispecific antibody or antigen-binding fragment thereof according to claim 2, wherein the tumor antigen is GPC3.

5. A nucleic acid encoding a bispecific antibody or an antigen-binding fragment thereof, as described in any one of claims 1 to 4.

6. The nucleic acid according to claim 5, wherein the coding nucleic acid of the second light chain of the second antigen-binding portion is the nucleotide sequence shown in SEQ ID NO: 67, and the coding nucleic acid of the second heavy chain of the second antigen-binding portion is the nucleotide sequence shown in SEQ ID NO:

69.

7. A vector comprising the nucleic acid described in claim 5 or 6.

8. A cell comprising the nucleic acid according to claim 5 or 6 or the vector according to claim 7.

9. A composition comprising a bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, a nucleic acid according to claim 5 or 6, a vector according to claim 7, and / or a cell according to claim 8.

10. An antibody-drug conjugate comprising a bispecific antibody or an antigen-binding fragment thereof according to any one of claims 1 to 4, which is covalently bound to the treatment site.

11. The antibody-drug conjugate according to claim 10, wherein the therapeutic portion is a cytotoxic portion, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulant, a degraded peptide, or a radioisotope.

12. The cytotoxic portion is Taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine (cephaeline), mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracendione, mytansine, mitotic inhibitors, drastatin 10 or 15, irinotecan, mitoxantrone, mitramycin, actinomycin D D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, calichemycin, antimetabolites, mechloretamine, thiopurine, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine or mitomycin C, platinum derivatives, duocalmycin A, duocalmycin SA, rakelmycin (CC-1065), actinomycin, bleomycin, da Unorubicin, doxorubicin, idarubicin, mithramycin, mitomycin, mitoxantrone, primycin, anthramycin (AMC), pyrrolo[2,1-c][1,4]-benzodiazepine (PDB), diphtheria toxin and related molecules, lysine toxin, cholera toxin, Shiga-like toxin, pertussis toxin, tetanus toxin, Bowman-Burke soybean protease inhibitors, Pseudomonas exotoxin, allorin, saporin, modeccin, geranine, abrin A chain, modeccin A chain, α-sarcin, PAPI, PAPI II and PAP-S, laxatives, croton toxin, saponaria cone Selected from officinalis inhibitors, leukotoxins, restrictocin, phenomycin and enomycin toxins, ribonuclease (RNase), DNase I, Staphylococcus endotoxin A, pokeweed antiviral protein, and Pseudomonas endotoxin, The cytokines are selected from IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa, IFN3, IFNy, GM-CSF, CD40L, Flt3 ligand, ancestim, and TNFA. The radioactive isotope is ２２５ , ２２７ , ２１２ , ２１３ , ２１２ H, １４ C, １５ N, ３５ S, ６７ Cu, ９０ Y, ９９ Tc, １２５ I, １３１ I, １８６ Re, １８８ Re, ２１１ At, ２１２ Bi, ２１２ Pb, ２１３ Bi, ２２５ Ac and ２２７ The antibody-drug conjugate according to claim 11, selected from Th.

13. The mitotic inhibitor is monomethyl auristatin E or F. The aforementioned antimetabolites are methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine. The platinum-based derivative is cisplatin or carboplatin. The aforementioned diphtheria toxin and related molecules are diphtheria A chain and its active fragment and hybrid molecules, The lysine toxin is lysine toxin A or deglycosylated lysine toxin A chain toxin. The antibody-drug conjugate according to claim 12, wherein the Ciga-like toxin is SLT I, SLT II, ​​SLT IIV, LT toxin, C3 toxin, or Ciga toxin.

14. A kit comprising a bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, a nucleic acid according to claim 5 or 6, a vector according to claim 7, or a cell according to claim 8, a composition according to claim 9, and / or an antibody-drug conjugate according to any one of claims 10 to 13.

15. A bispecific antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, a nucleic acid according to claim 5 or 6, a vector according to claim 7, or a cell according to claim 8, a composition according to claim 9, and / or an antibody-drug conjugate according to any one of claims 10 to 13, for use in the diagnosis, treatment, or prevention of tumor antigen-related diseases.

16. The bispecific antibody or antigen-binding fragment thereof, nucleic acid, vector, cell, composition, and / or antibody-drug conjugate according to claim 15, wherein the tumor antigen is GPC3, the tumor antigen-related disease is a GPC3-related disease, preferably the GPC3-related disease includes a tumor, the tumor is cancer, and is GPC3-positive liver cancer, GPC3-positive hepatocellular carcinoma, GPC3-positive pancreatic cancer, GPC3-positive lung cancer, GPC3-positive colon cancer, GPC3-positive breast cancer, GPC3-positive prostate cancer, GPC3-positive leukemia, or GPC3-positive lymphoma.

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