WO2022105924A1 - 一种双特异性抗体及其用途 - Google Patents

一种双特异性抗体及其用途 Download PDF

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WO2022105924A1
WO2022105924A1 PCT/CN2021/132195 CN2021132195W WO2022105924A1 WO 2022105924 A1 WO2022105924 A1 WO 2022105924A1 CN 2021132195 W CN2021132195 W CN 2021132195W WO 2022105924 A1 WO2022105924 A1 WO 2022105924A1
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antigen
seq
binding portion
amino acid
heavy chain
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PCT/CN2021/132195
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English (en)
French (fr)
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徐刚
陈博
王常玉
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康诺亚生物医药科技（成都）有限公司
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Priority to JP2023530940A priority Critical patent/JP2023550171A/ja
Priority to CA3199721A priority patent/CA3199721A1/en
Priority to CN202180078861.XA priority patent/CN116490614A/zh
Priority to US18/038,159 priority patent/US20240002540A1/en
Priority to AU2021383901A priority patent/AU2021383901A1/en
Priority to KR1020237021199A priority patent/KR20230117160A/ko
Priority to EP21894071.6A priority patent/EP4249514A1/en
Publication of WO2022105924A1 publication Critical patent/WO2022105924A1/zh

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Definitions

  • the present disclosure relates to a bispecific antibody and uses thereof, particularly a bispecific antibody that binds CD3 and another antigen and uses thereof.
  • T cell bispecific antibody (or T cell adapter) is a special antibody molecule that can recognize the target cell surface antigen (antigen arm) through one end and bind T cell CD3 receptor (CD3 arm) through the other end.
  • target cell surface antigen antigen arm
  • CD3 arm T cell CD3 receptor
  • the aggregation of CD3 on T cells activates T cells and kills tumors.
  • bispecific antibodies were used to kill tumor cells (Staerz UD., Nature. 1985 Apr 18-24; 314(6012): 628-31; Perez P. et al. Nature. 1985 Jul 25-31; 316(6026):354-6).
  • catumaxomab which was marketed in the early days, has been withdrawn from the market due to the binding of the Fc segment to the Fc ⁇ receptor expressed by liver Kupffer cells, causing rapid cytokine release; while blinatumomab, which was launched in 2014, uses Fv antibody fragments, and its biological half-life is only 2 hours, low-dose continuous intravenous infusion, and FDA-approved boxed warnings with cytokine release syndrome and neurotoxicity.
  • TCR binds with low affinity (approximately 1-100 ⁇ M) to exogenous peptide-human leukocyte antigen complex (HLA) on infected or mutated cells, via CD3 signaling complexes (including CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ ) , the activation signal is transduced into the nucleus, and the expression of transcription factors and their downstream proteins (cytokines, granzymes, perforin, etc.) is activated, and the signal strength generated by the TCR complex will determine the fate of T cells.
  • HLA peptide-human leukocyte antigen complex
  • the CD3 bispecific antibodies developed in the early stage are mostly based on a few mouse-derived antibodies such as OKT3, L2K, UCHT1 and TR66, with high affinity, which leads to excessive activation of T cells, the release of a large number of cytokines, and the production of cytokine storm syndrome; at the same time, high affinity also It will lead to the enrichment of bispecific antibodies in secondary lymphoid organs and reduce the exposure in tumor tissue.
  • the binding ability of the Fc part of the antibody to the Fc ⁇ receptor is another important factor affecting drug safety. Since the Fc ⁇ receptor is expressed in a variety of normal tissues, the binding of the bispecific antibody to the Fc ⁇ receptor on the cell membrane through Fc can lead to other factors.
  • the CD3 receptor bound at one end is activated by cross-linking due to Fc ⁇ receptor aggregation, resulting in severe off-target toxicity.
  • Newman et al introduced mutations Ser228Pro and Leu235Glu in IgG4 to stabilize the IgG4 structure while reducing the binding to Fc ⁇ receptors (Newman R, et al, Modification of the Fc region of a primatized IgG antibody to human CD4retains its ability to modulate CD4receptors but does not deplete CD4(+)T cells in chimpanzees.Clin Immunol.2001Feb;98(2):164-74), Idusogie et al.
  • Interchain mismatch is the main technological difficulty in the development of natural IgG-like bispecific antibodies.
  • the common light chain invented by Merchant AM et al. (Merchant AM, et al, An efficient route to human bispecific IgG. Nat Biotechnol. 1998. PMID: 9661204), or the common heavy chain developed by Fischer N et al. (Fischer N, et al, Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG. Nat Commun. 2015 Feb 12;6:6113), often requiring complex protein engineering or production using transgenic animals (McWhirter J, et al, Common light chain mouse. WO2011097603.2011); Carter P et al.
  • the inventors unexpectedly found that by combining a humanized CD3 antibody with a ⁇ light chain and a targeting antibody with a ⁇ light chain, the CD3 antibody ⁇ light chain is more likely to be homologous.
  • CD3 heavy chains are paired, whereas targeting antibody kappa light chains tend to pair with cognate targeting antibody heavy chains.
  • the correct pairing efficiency can be improved.
  • experiments have shown that the novel T cell connector constructed by using multiple target antibodies such as CD20, BCMA and GPC3 and humanized CD3 antibody can achieve 98-100% monomer purity after three-step purification, and the mismatch ratio is extremely high. Low ( ⁇ 1%).
  • the present disclosure provides a novel T cell connector, which is designed using different types of light chain ⁇ bispecific antibodies, and a full-length IgG configuration, wherein: the antibody arms that bind target cells and T cell CD3 use kappa light chain and lambda light chain, respectively. Chain, pairing with its homologous heavy chain, and introducing complementary charge pairs to enhance the correct pairing rate; through affinity optimization, the new T cell connector can recruit activated T cells at low concentrations, effectively killing target cells, without target cells T cells are not activated when present; at the same time, the novel T cell ⁇ bispecific antibody does not bind to Fc ⁇ R receptors, reducing the risk of cytokine storms.
  • the novel CD20 ⁇ CD3 ⁇ bispecific antibody, BCMA ⁇ CD3 ⁇ bispecific antibody and GPC3 ⁇ CD3 ⁇ bispecific antibody constructed by the disclosed method have high purification yields, and can achieve >99% purity through three-step purification. Animals tolerated the novel CD20-CD3 ⁇ bispecific antibody well, and the efficacy and safety of the novel T-cell connector were superior to comparable antibodies.
  • the present disclosure provides a bispecific antibody or antigen-binding portion thereof.
  • the present disclosure provides nucleic acids encoding bispecific antibodies or antigen-binding portions thereof according to the preceding aspects.
  • the present disclosure provides vectors comprising the nucleic acids of the preceding aspects.
  • the present disclosure provides cells comprising the vector of the preceding aspects.
  • the present disclosure provides a pharmaceutical composition or kit comprising an antibody, or antigen-binding portion thereof, or nucleic acid encoding thereof, according to any of the preceding aspects, and a pharmaceutically acceptable carrier.
  • the present disclosure provides antibody-drug conjugates comprising an antibody or antigen-binding portion thereof, bispecific or multispecific molecule of the foregoing of any of the foregoing aspects covalently attached to a therapeutic moiety.
  • the present disclosure provides a method of treating and related disorders comprising the step of administering to the above-described mammal a therapeutically effective amount of an antibody or antigen-binding fragment thereof, nucleic acid, vector, cell and/or of any of the foregoing aspects. or pharmaceutical compositions.
  • the present disclosure provides the antibody or antigen-binding fragment thereof, nucleic acid, vector, cell and/or pharmaceutical composition of any of the preceding aspects in the manufacture of a medicament or kit for the treatment of a disorder associated with a tumor antigen in a mammal use in.
  • the antibodies of the present disclosure can be used in a variety of applications, including the detection of tumor antigens, and the diagnosis, treatment or prevention of diseases associated with tumor antigens.
  • Figure 1 shows the first antigen x CD3 ⁇ bispecific antibody of the present disclosure.
  • Figure 2 shows the binding of CD3 humanized antibodies to human CD3 ⁇ protein.
  • Figure 3 shows the binding of CD3 humanized antibodies to Jurkat cells.
  • Figure 4 shows the binding of CD3 humanized antibodies to human CD3 ⁇ and cynomolgus CD3 ⁇ proteins.
  • Figure 5 shows the structures of ⁇ 001, ⁇ 002, ⁇ 003, ⁇ 004, ⁇ 005 of the present disclosure.
  • FIG. 6 shows the results of protein A purification of the CD20 ⁇ CD3 ⁇ bispecific antibody.
  • FIG. 7 shows the results of SEC-HPLC detection of CD20 ⁇ CD3 ⁇ bispecific antibody.
  • FIG. 8 shows the detection results of CD20 ⁇ CD3 ⁇ bispecific antibody homodimer.
  • Figure 9 shows the binding of CD20xCD3 ⁇ bispecific antibody to CD20 stably transduced cells.
  • Figure 10 shows the binding of CD20xCD3 ⁇ bispecific antibody to tumor cells SU-DHL-4, Raji and NALM-6.
  • Figure 11 shows the binding of the CD20xCD3 ⁇ bispecific antibody to Jurkat cells.
  • Figure 12 shows the binding of CD20xCD3 ⁇ bispecific antibody to peripheral blood T cells.
  • Figure 13 shows the TDCC effect mediated by the CD20xCD3 ⁇ bispecific antibody
  • Figure 13A shows the killing of Nalm-6 cells
  • Figure 13B shows the activation of T cells.
  • Figure 14 shows the TDCC effect mediated by the CD20xCD3 ⁇ bispecific antibody
  • Figure 14A shows the killing of TMD-8 cells
  • Figure 14B shows the activation of T cells.
  • Figure 15 shows the TDCC effect mediated by the CD20xCD3 ⁇ bispecific antibody
  • Figure 15A shows the killing of Toledo cells
  • Figure 15B shows the activation of T cells.
  • Figure 16 shows the effect of CD20xCD3 ⁇ bispecific antibody on T cell NFAT signaling pathway.
  • FIG. 17 shows the inhibitory effect of CD20 ⁇ CD3 ⁇ bispecific antibody in immune reconstitution mouse Raji xenograft model.
  • FIG. 18 shows the inhibitory effect of CD20 ⁇ CD3 ⁇ bispecific antibody in the mixed tumor model of subcutaneous Raji and human PBMC in immunodeficient mice.
  • Figure 19 shows the efficacy of CD20 ⁇ CD3 ⁇ bispecific antibody in cynomolgus monkeys.
  • Figure 20 shows the binding of BCMA x CD3 ⁇ bispecific antibody to BCMA stably transfected cells.
  • Figure 21 shows the binding of BCMAxCD3 ⁇ bispecific antibody to tumor cells NCI-H929 and RPMI-8226.
  • Figure 22 shows the binding of BCMAxCD3 ⁇ bispecific antibody to Jurkat cells.
  • Figure 23 shows the binding of BCMAxCD3 ⁇ bispecific antibody to peripheral blood T cells.
  • Figure 24 shows the TDCC effect mediated by the BCMAxCD3 ⁇ bispecific antibody
  • Figure 24A shows the killing of NCI-H929 cells
  • Figure 24B shows the activation of T cells.
  • Figure 25 shows BCMAxCD3 ⁇ bispecific antibody-mediated TDCC effect
  • Figure 25A shows killing of RPMI-8226 cells
  • Figure 25B shows activation of T cells.
  • Figure 26 shows the effect of BCMAxCD3 ⁇ bispecific antibody on T cell NFAT signaling pathway.
  • Figure 27 shows non-specific activation of PBMCs by BCMAxCD3 ⁇ bispecific antibodies.
  • Figure 28 shows the binding of BCMAxCD3 ⁇ bispecific antibody to Fc receptors.
  • Figure 29 shows the inhibitory effect of BCMAxCD3 ⁇ bispecific antibody in an immunodeficient mouse subcutaneous NCI-H929 xenograft model.
  • Figure 30 shows the binding of GPC3xCD3 ⁇ bispecific antibody to GPC3 stably transfected cells.
  • Figure 31 shows the binding of GPC3xCD3 ⁇ bispecific antibody to tumor cell HepG2.
  • Figure 32 shows binding of GPC3xCD3 ⁇ bispecific antibody to Jurkat cells.
  • Figure 33 shows the binding of GPC3xCD3 ⁇ bispecific antibody to peripheral blood T cells.
  • Figure 34 shows GPC3xCD3 ⁇ bispecific antibody-mediated TDCC effect
  • Figure 34A shows killing of HepG2 cells
  • Figure 34B shows activation of T cells.
  • Figure 35 shows the effect of GPC3xCD3 ⁇ bispecific antibody on T cell NFAT signaling pathway.
  • Figure 36 shows non-specific activation of PBMCs by GPC3xCD3 ⁇ bispecific antibodies.
  • FIG. 37 shows the inhibitory effect of GPC3 ⁇ CD3 ⁇ bispecific antibody in the subcutaneous HepG2 xenograft model of immune reconstituted mice.
  • Figure 38 shows the inhibitory effect of GPC3 ⁇ CD3 ⁇ bispecific antibody in CD3 humanized murine Hepa1-6/human GPC3 xenograft model.
  • tumor antigen preferably refers to any antigen or antigenic determinant that is present on (or binds to) tumor cells, but not normally present on normal cells, or that is more abundant than normal (non-tumor) cells. Antigens or antigenic determinants present on or bound to tumor cells in greater amounts, or present on tumor cells in a form different from that found on normal (non-tumor) cells.
  • TSAs tumor-specific antigens
  • TAAs tumor-associated antigens
  • tumor-associated membrane antigens embryonic antigens on tumors, growth factor receptors, growth factor ligands, and cancer-related antigens of any other type of antigen.
  • Tumor antigens can be, for example, B-cell differentiation antigens (eg, CD19, CD20, and CD37), B-cell maturation antigens (BCMA), Glypican 3 (GPC3), epithelial cancer antigens (eg, breast cancer, gastrointestinal cancer, lung cancer), prostate specific cancer antigen (PSA) or prostate specific membrane antigen (PSMA), bladder cancer antigen, lung (eg, small cell lung) cancer antigen, colon cancer antigen, ovary Cancer antigen, brain cancer antigen, gastric cancer antigen, renal cell cancer antigen, pancreatic cancer antigen, liver cancer antigen, esophagus cancer antigen, head and neck cancer antigen or colorectal cancer antigen.
  • B-cell differentiation antigens eg, CD19, CD20, and CD37
  • B-cell maturation antigens BCMA
  • Glypican 3 Glypican 3
  • epithelial cancer antigens eg, breast cancer, gastrointestinal cancer, lung cancer
  • PSA prostate specific
  • TSA is (or is thought to be) unique to tumor cells and does not occur on other cells in the body (eg, does not occur on other cells to a significant extent).
  • TAAs are not unique to tumor cells, and are instead expressed on normal cells (eg, under conditions that fail to induce a state of immune tolerance to the antigen).
  • TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond, or they may be normally present at very low levels on normal cells but at very low levels on tumor cells Antigens expressed at higher levels.
  • TSA or TAA antigens include differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, HER-2 /neu; unique tumor antigens caused by chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein-Barr virus antigen EBVA and human papilloma Viral (HPV) antigens E6 and E7.
  • differentiation antigens such as MART-1/MelanA (MART-I), gp100 (Pmel 17),
  • 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, ⁇ -catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, ⁇ -HCG, BCA225 , BTAA, CA 125, CA 15-3 ⁇ CA 27.29 ⁇ BCAA, CA 195, CA242, CA-50, CAM43, CD68 ⁇ P1, CO-029, FGF-5, G250, Ga733 ⁇ EpCAM, HTgp-175, M344 , MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 ⁇ Mac
  • tumor antigens include CD19, CD20, CD22, CD30, CD72, CD180, CD171 (L1CAM), CD123, CD133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166, CD71, CLL-1/CLECK12A, ROR1, BCMA, Glypican 3 (GPC3), Mesothelin, CD33/IL3Ra, c-Met, PSCA, PSMA, Glycolipid F77, EGFRvIII, GD-2, MY-ESO-1 or MAGEA3.
  • CD20 refers to any native CD20 from any vertebrate source, including mammals, such as primates (eg, humans) and rodents (eg, mice and rats).
  • anti-CD20 antibody and “antibody that binds CD20” refer to an antibody capable of binding CD20 with sufficient affinity such that the antibody is useful in targeting CD20 as a diagnostic and/or therapeutic agent.
  • the degree of binding of an anti-CD20 antibody to an unrelated non-CD20 protein is less than about 10% of the binding of the antibody to CD20, as measured by, eg, a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • the CD20-binding antibody has ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (eg, 10 ⁇ 8 M or less, eg, 10 ⁇ 8 M to 10" 13M , eg 10" 9M to 10" 13M ) dissociation constant ( Kd ).
  • the anti-CD20 antibody binds to a CD20 epitope that is conserved among CD20s from different species.
  • BCMA may refer to a concept collectively referring to BCMA itself and any variants, isoforms and paralogs present in animals and preferably in humans.
  • human BCMA refers to BCMA of human origin, and may preferably have, but is not limited to, the amino acid sequence of Genbank Accession No. AB052772.1.
  • anti-BCMA antibody and "BCMA-binding antibody” refer to an antibody capable of binding BCMA with sufficient affinity such that the antibody is useful in targeting BCMA as a diagnostic and/or therapeutic agent.
  • the degree of binding of an anti-BCMA antibody to an unrelated non-BCMA protein is less than about 10% of the binding of the antibody to BCMA, as measured by, eg, a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • the antibody that binds BCMA has ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (eg, 10 ⁇ 8 M or less, eg, 10 ⁇ 8 M to 10" 13M , eg 10" 9M to 10" 13M ) dissociation constant ( Kd ).
  • anti-BCMA antibodies bind to BCMA epitopes that are conserved among BCMAs from different species.
  • GPC3 may refer to a concept that collectively refers to GPC3 itself and any of its variants, isoforms and paralogs present in animals and preferably in humans.
  • human GPC3 refers to GPC3 of human origin.
  • anti-GPC3 antibody and "anti-GPC3-binding antibody” refer to an antibody capable of binding GPC3 with sufficient affinity such that the antibody is useful in targeting GPC3 as a diagnostic and/or therapeutic agent.
  • the degree of binding of an anti-GPC3 antibody to an unrelated non-GPC3 protein is less than about 10% of the binding of the antibody to GPC3, as measured by, eg, a radioimmunoassay (RIA).
  • RIA radioimmunoassay
  • the antibody that binds GPC3 has ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (eg, 10 ⁇ 8 M or less, eg, 10 ⁇ 8 M to 10" 13M , eg 10" 9M to 10" 13M ) dissociation constant ( Kd ).
  • anti-GPC3 antibodies bind to GPC3 epitopes that are conserved among GPC3s from different species.
  • CD3 refers to any native CD3 from any vertebrate source, including mammals, such as primates (eg, humans), non-human primates (eg, cynomolgus monkeys), and rodents (eg, mice and rats), unless otherwise specified.
  • the term encompasses "full-length” unprocessed CD3 as well as any form of CD3 derived from processing in a cell.
  • the term also encompasses naturally occurring variants of CD3, such as splice variants or allelic variants.
  • the CD3 is human CD3, particularly the epsilon subunit of human CD3 (CD3 ⁇ ).
  • the amino acid sequence of human CD3 ⁇ is shown in UniProt (www.uniprot.org) accession number P07766 (version 144), or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_000724.1.
  • the amino acid sequence of cynomolgus monkey [Macaca fascicularis] CD3 ⁇ is shown in NCBI GenBank no.BAB71849.1.
  • Cell surface is used according to its normal meaning in the art and thus includes the exterior of the cell accessible by binding to proteins and other molecules.
  • the term “about” or “approximately” means within plus or minus 10% of the given value or range. Where a whole number is required, the term refers to within plus or minus 10% of the given value or range, rounded up or down to the nearest whole number.
  • the phrase "substantially identical" can be understood as exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, Antibody chains of 97%, 98%, 99% or more sequence identity.
  • nucleic acid sequences the term is to be understood as exhibiting at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98 A nucleotide sequence of %, 99% or greater sequence identity.
  • sequence identity has an art-recognized meaning, and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using published techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along regions of the molecule. While many methods exist for measuring the identity between two polynucleotides or polypeptides, the term “identity” is well known to the skilled artisan (Carrillo, H. & Lipman, D., SIAM J Applied Math 48:1073 (1988) ).
  • substitutional variant is one in which at least one amino acid residue in the native sequence has been removed and a different amino acid inserted in its same position.
  • the substitutions can be single, wherein only one amino acid is substituted in the molecule, or multiple, wherein the same molecule has two or more amino acids substituted. Multiple substitutions can be made at consecutive sites.
  • one amino acid can be substituted by multiple residues, wherein such variants include both substitutions and insertions.
  • An “insertional” variant is one in which one or more amino acids are inserted into an amino acid immediately adjacent to a specific position in a native sequence. Immediately adjacent to an amino acid means attachment to the alpha-carboxyl or alpha-amino functional group of the amino acid.
  • a “deletion” variant is one in which one or more amino acids in the native amino acid sequence have been removed. Typically, deletion variants have one or two amino acids deleted in a specific region of their molecule.
  • variable domains of antibodies refers to certain portions of related molecules that differ widely in sequence between antibodies and are used for the specific recognition and binding of a particular antibody against its specific target. However, the variability is not evenly distributed throughout the variable domains of antibodies. Variability is concentrated in three segments called complementarity determining regions (CDRs; ie CDR1, CDR2 and CDR3) or hypervariable regions, all located within the variable domains of light and heavy chains. The more conserved portions of the variable domains are referred to as framework (FR) regions or framework sequences.
  • CDRs complementarity determining regions
  • FR framework regions
  • Each variable domain of native heavy and light chains includes four FR regions, predominantly in a beta-sheet configuration, linked by three CDRs that form loops that connect the beta-sheet structure and Partial ⁇ -sheet structures are formed in some cases.
  • the CDRs of each chain are usually linked in proximity by FR regions and, with the aid of CDRs from other chains, contribute to the formation of antibody target binding sites (epitopes or determinants).
  • the numbering of immunoglobulin amino acid residues is according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated.
  • a CDR can have the ability to specifically bind to the cognate epitope.
  • an "antibody fragment” or “antigen-binding fragment” of an antibody refers to any portion of a full-length antibody that is less than full-length, but which comprises at least a portion of the variable region (eg, one or more of the variable regions of said antibody that binds an antigen) CDRs and/or one or more antibody binding sites), and thus retain binding specificity and at least part of the specific binding capacity of the full-length antibody.
  • an antigen-binding fragment refers to an antibody fragment comprising an antigen-binding portion that binds to the same antigen as the antibody from which the antibody fragment is derived.
  • Antibody fragments include antibody derivatives produced by enzymatic treatment of full-length antibodies, as well as synthetically produced derivatives, eg, recombinantly produced derivatives.
  • Antibodies include antibody fragments. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2 , single-chain Fv (scFv), Fv, dsFv, diabodies, Fd and Fd' fragments, and other fragments, including modified fragments (see, For example, Methods in Molecular Biology, Vol 207: Recombinant Antibodies for Cancer Therapy Methods and Protocols (2003); Chapter 1; p 3-25, Kipriyanov).
  • the fragments may comprise multiple chains linked together, eg, by disulfide bonds and/or by peptide linkers.
  • Antibody fragments generally comprise at least or about 50 amino acids, and typically at least or about 200 amino acids.
  • Antigen-binding fragments include any antibody fragment that, when inserted into the antibody framework (eg, by substituting the corresponding region), results in an antibody that immunospecifically binds (ie, exhibits a Ka of at least or at least about 107-108 M- 1 ) to an antigen .
  • a "functional fragment” or “analog of an antibody” is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction.
  • functional fragments generally have the same meaning as "antibody fragments” and, in the case of antibodies, may refer to fragments that prevent or substantially reduce the ability of the receptor to bind a ligand or initiate signal transduction, eg, Fv, Fab , F(ab') 2 , and so on.
  • "Fv" fragments consist of a dimer ( VH - VL dimer) formed by non-covalent association of the variable domains of a heavy chain and the variable domains of a light chain. In this configuration, the three CDRs of each variable domain interact to define the target binding site on the surface of the VH - VL dimer, as is the case with intact antibodies. The six CDRs collectively confer the target-binding specificity of the intact antibody. However, even a single variable domain (or half of an Fv that includes only 3 target-specific CDRs) can still have the ability to recognize and bind targets.
  • BsAb Bispecific antibody
  • a bispecific antibody and/or antigen-binding molecule Contains two antigen binding sites, each of which is specific for a different antigenic determinant.
  • the bispecific antibody and/or antigen binding molecule is capable of binding two antigenic determinants simultaneously, particularly two antigenic determinants expressed on two different cells.
  • monoclonal antibody refers to a population of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody population is identical to other antibody molecules. This property is in contrast to that of polyclonal populations of antibodies, which comprise antibodies with a variety of different sequences.
  • Monoclonal antibodies can be prepared by a number of well-known methods (Smith et al. (2004) J. Clin. Pathol. 57, 912-917; and Nelson et al., J Clin Pathol (2000), 53, 111-117).
  • monoclonal antibodies can be prepared by immortalizing B cells, eg, by fusion with myeloma cells to generate hybridoma cell lines or by infecting B cells with a virus such as EBV.
  • Recombinant techniques can also be used to prepare antibodies from clonal populations of host cells in vitro by transforming the host cells with a plasmid carrying an artificial sequence of nucleotides encoding the antibody.
  • hybridomas refers to a cell or cell line (usually myeloma or lymphoma cells) produced by fusing antibody-producing lymphocytes and non-antibody-producing cancer cells.
  • hybridomas can proliferate and provide continuous supply to produce specific monoclonal antibodies. Methods for generating hybridomas are known in the art (see, eg, Harlow & Lane, 1988).
  • hybridomas it also includes subclones and progeny cells of hybridomas.
  • a full-length antibody is one that has two full-length heavy chains (eg, VH-CH1-CH2-CH3 or VH-CH1-CH2-CH3-CH4) and two full-length light chains (VL-CL) and a hinge region antibodies, such as those naturally produced by antibody-secreting B cells and those produced synthetically with the same domains.
  • chimeric antibody refers to an antibody in which the variable region sequences are derived from one species and the constant region sequences are derived from another species, such as in which the variable region sequences are derived from a mouse antibody and the constant region sequences are derived from a human antibody of antibodies.
  • Humanized antibodies refer to non-human (eg, mouse) forms of antibodies that are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (eg, Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) containing minimal sequence derived from non-human immunoglobulins.
  • the humanized antibody is a human immunoglobulin (recipient antibody) in which the complementarity determining region (CDR) residues of the recipient antibody are derived from a non-human species with the desired specificity, affinity and capacity ( donor antibody) such as mouse, rat or rabbit CDR residue substitutions.
  • CDR complementarity determining region
  • telomeres can be mutated amino acid residues within the CDR1, CDR2 and/or CDR3 regions of VH and/or VL, thereby improving one or more binding properties (eg, affinity) of the antibody .
  • PCR-mediated mutagenesis can be performed to introduce mutations whose effect on antibody binding or other functional properties can be assessed using the in vitro or in vivo assays described herein. Typically, conservative mutations are introduced. Such mutations can be amino acid substitutions, additions or deletions.
  • CDR refers to a complementarity-determining region
  • each of the heavy and light chains of antibody molecules is known to have 3 CDRs.
  • the CDRs are also referred to as hypervariable regions, and are present in the variable regions of each heavy and light chain of antibodies, with sites of very high variability in the primary structure of the CDRs.
  • the CDRs of the heavy chain are represented by CDR1, CDR2, and CDR3 derived from the amino terminal of the amino terminal sequence of the heavy chain
  • CDRs of the light chain are represented by CDR1, CDR2, and CDR3 derived from the amino terminal of the amino terminal sequence of the light chain.
  • epitopic determinants refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants typically comprise chemically active surface profiles of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics.
  • an antibody that immunospecifically binds (or specifically binds) an antigen has an affinity constant Ka of about or 1 ⁇ 10 7 M -1 or 1 ⁇ 10 8 M -1 or greater (or 1 ⁇ 10-7 M or 1 ⁇ 10 ⁇ 8 M or lower dissociation constant (Kd)) binds to the antigen.
  • Affinity constants can be determined by standard kinetic methods of antibody responses, eg, immunoassays, surface plasmon resonance (SPR) (Rich and Myszka (2000) Curr. Opin. Biotechnol 11:54; Englebienne (1998) Analyst. 123: 1599), isothermal titration calorimetry (ITC), or other kinetic interaction assays known in the art; see also U.S. Pat. No. 7,229,619 describing exemplary SPR and ITC methods for calculating binding affinity of antibodies No). Instruments and methods for real-time detection and monitoring of binding rates are known and commercially available (see, Malmqvist (2000) Biochem. Soc. Trans. 27:335).
  • nucleic acid molecules refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives, including usually linked together by phosphodiester bonds Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleic acid molecule is intended to include DNA molecules and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.
  • an isolated nucleic acid molecule is one that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule.
  • An "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material or culture medium when prepared by recombinant techniques, or substantially free of chemical precursors or other chemical components when chemically synthesized.
  • Exemplary isolated nucleic acid molecules provided herein include isolated nucleic acid molecules encoding the provided antibodies or antigen-binding fragments.
  • operably linked in reference to nucleic acid sequences, regions, elements or domains means that the nucleic acid regions are functionally related to each other.
  • a promoter can be operably linked to a nucleic acid encoding a polypeptide such that the promoter regulates or mediates transcription of the nucleic acid.
  • conservative sequence modifications of the sequences described in the Sequence Listing described herein, i.e., nucleotide and amino acid sequence modifications that do not eliminate binding of the antibody to the antigen encoded by the nucleotide sequence or containing the amino acid sequence.
  • conservative sequence modifications include conservative nucleotide and amino acid substitutions and nucleotide and amino acid additions and deletions.
  • modifications can be introduced into the Sequence Listing described herein by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative sequence modifications include conservative amino acid substitutions in which amino acid residues are replaced with amino acid residues having similar side chains. Families of amino acid residues with similar side chains are defined in the art.
  • amino acids with basic side chains eg, lysine, arginine, histidine
  • amino acids with acidic side chains eg, aspartic acid, glutamic acid
  • amino acids with uncharged polar side chains amino acids e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • amino acids with non-polar side chains e.g. alanine, valine
  • leucine, isoleucine, proline, phenylalanine, methionine amino acids with beta branched side chains
  • a predicted non-essential amino acid residue in an anti-CD20, BCMA or GPC3 antibody is preferably replaced by another amino acid residue from the same side chain family.
  • Methods for identifying conservative substitutions of nucleotides and amino acids that do not abolish antigen binding are well known in the art (for example, see Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12 ( 10): 879-884 (1999); Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997)).
  • mutations can be introduced randomly along all or a portion of the anti-GCD20, BCMA or PC3 antibody coding sequence, eg, by saturation mutagenesis, and the resulting modified anti-CD20, BCMA or PC3 antibodies can be screened for improved binding activity.
  • BCMA or GPC3 antibody can be introduced randomly along all or a portion of the anti-GCD20, BCMA or PC3 antibody coding sequence, eg, by saturation mutagenesis, and the resulting modified anti-CD20, BCMA or PC3 antibodies can be screened for improved binding activity.
  • expression refers to the process by which a polypeptide is produced by transcription and translation of a polynucleotide. Expression levels of a polypeptide can be assessed using any method known in the art, including, for example, methods that determine the amount of polypeptide produced from a host cell. Such methods may include, but are not limited to, quantification of polypeptides in cell lysates by ELISA, Coomassie blue staining followed by gel electrophoresis, Lowry protein assay, and Bradford protein assay.
  • a "host cell” is a cell used to receive, maintain, replicate and amplify a vector. Host cells can also be used to express the polypeptide encoded by the vector. When the host cell divides, the nucleic acid contained in the vector replicates, thereby amplifying the nucleic acid.
  • Host cells can be eukaryotic cells or prokaryotic cells. Suitable host cells include, but are not limited to, CHO cells, various COS cells, HeLa cells, HEK cells such as HEK 293 cells.
  • a "vector" is a replicable nucleic acid from which one or more heterologous proteins can be expressed when transformed into an appropriate host cell.
  • References to vectors include those into which nucleic acids encoding polypeptides or fragments thereof can be introduced, usually by restriction digestion and ligation. Reference to vectors also includes those vectors comprising nucleic acid encoding a polypeptide. Vectors are used to introduce nucleic acid encoding a polypeptide into a host cell, to amplify the nucleic acid, or to express/display the polypeptide encoded by the nucleic acid. Vectors generally remain episomal, but can be designed to integrate the gene or portion thereof into the chromosome of the genome. Also contemplated are artificial chromosome vectors, such as yeast artificial vectors and mammalian artificial chromosomes. The selection and use of such vehicles is well known to those skilled in the art.
  • a vector also includes a "viral vector” or “viral vector.”
  • a viral vector is an engineered virus that is operably linked to a foreign gene to transfer (either as a vehicle or shuttle) the foreign gene into a cell.
  • an "expression vector” includes a vector capable of expressing DNA operably linked to regulatory sequences, such as promoter regions, capable of affecting the expression of such DNA fragments. Such additional fragments may include promoter and terminator sequences, and optionally, one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are typically derived from plasmid or viral DNA, or may contain elements of both. Thus, an expression vector refers to a recombinant DNA or RNA construct, such as a plasmid, phage, recombinant virus, or other vector, which, when introduced into an appropriate host cell, results in the expression of cloned DNA. Appropriate expression vectors are well known to those skilled in the art and include those that are replicable in eukaryotic and/or prokaryotic cells as well as those that remain episomal or that integrate into the host cell genome.
  • treating an individual with a disease or condition means that the individual's symptoms are partially or completely alleviated, or remain unchanged following treatment.
  • treatment includes prevention, treatment and/or cure.
  • Prevention refers to preventing an underlying disease and/or preventing the worsening of symptoms or the development of a disease.
  • Treatment also includes any provided antibodies or antigen-binding fragments thereof and any pharmaceutical uses of the compositions provided herein.
  • therapeutic effect refers to an effect resulting from treatment of an individual that alters, generally ameliorates or ameliorates the symptoms of a disease or condition, or cures the disease or condition.
  • a “therapeutically effective amount” or “therapeutically effective dose” refers to an amount of a substance, compound, material, or composition comprising a compound that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, retard or partially retard the symptoms of a disease or disorder.
  • a prophylactically effective amount or “prophylactically effective dose” refers to an amount of a substance, compound, material, or composition comprising a compound that, when administered to a subject, will have the desired prophylactic effect, eg, prevent or delay a disease or symptom occurrence or recurrence, and reduce the likelihood of occurrence or recurrence of disease or symptoms.
  • a fully prophylactically effective dose need not occur by administering one dose, and may occur only after administering a series of doses. Thus, a prophylactically effective amount can be administered in one or more administrations.
  • the term "patient” refers to a mammal, such as a human.
  • the present disclosure provides a bispecific antibody or antigen-binding fragment thereof comprising:
  • a first antigen-binding portion or an antigen-binding fragment thereof comprises a first light chain and a first heavy chain, the first light chain is a kappa-type light chain, and the first antigen-binding portion comprises a first antigen-binding portion the first binding domain that binds;
  • a second antigen-binding portion or an antigen-binding fragment thereof comprising a second light chain and a second heavy chain, the second light chain being a ⁇ -type light chain, and the second antigen-binding portion comprising a second antigen-binding portion Binding of the second binding domain.
  • the second antigen is the CD3 antigen.
  • the second light chain variable region of the second antigen-binding portion has a Gln 40 Glu mutation ( V ⁇ CD3 :Gln 40 Glu); the second heavy chain variable region of the second antigen-binding portion has Gln 39 Lys Mutation (VH CD3 :Gln 39 Lys).
  • the second binding domain comprises a second light chain CDR selected from the amino acid sequence of SEQ ID NOs: 7-9, 14, 15, 20, 21 or any variant thereof; and/or selected from the amino acid sequence The second heavy chain CDRs of SEQ ID NOs: 26-28, 31, 34, 40, 43, 46, 47 or any variant thereof.
  • the second binding domain comprises a second light chain CDRl selected from the amino acid sequence of SEQ ID NO: 7, 14, or any variant thereof, selected from the amino acid sequence of SEQ ID NO: 8, 15, 20, or any variant thereof the second light chain CDR2 of any variant selected from the second light chain CDR3 of the amino acid sequence SEQ ID NO: 9, 21 or any variant thereof; and/or selected from the amino acid sequence SEQ ID NO: 26, 31, 46 or The second heavy chain CDR1 of any variant thereof, selected from the second heavy chain CDR2 of the amino acid sequence SEQ ID NO: 27, 47 or any variant thereof, selected from the amino acid sequence SEQ ID NO: 28, 34, 37, 40, 43 or the second heavy chain CDR3 of any variant thereof.
  • the second light chain CDRs of the second binding domain are selected from the group consisting of: the second light chain CDR1, CDR2 and CDR3 sequences comprising the amino acid sequences SEQ ID NOs: 7, 8, 9, respectively; comprising the amino acid sequences SEQ ID NOs: 7, 8, and 9, respectively;
  • the second light chain CDR1, CDR2 and CDR3 sequences of ID NOs: 7, 20, 21; and/or the heavy chain CDRs of the second binding domain are selected from the group consisting of: the sequences comprising the amino acid sequences of SEQ ID NOs: 26, 27, 28, respectively Double chain CDR1, CDR2 and CDR3 sequences; comprising the second heavy chain CDR1, CDR2 and
  • the second binding domain comprises a second light chain variable region selected from the amino acid sequence of SEQ ID NO: 5, 10, 12, 16, 18, 22, or any variant thereof; and/or selected from The second heavy chain variable region of the amino acid sequence of SEQ ID NO: 24, 29, 32, 35, 38, 41, 44, 48, 50, 52 or any variant thereof.
  • the second binding domain comprises the second light chain variable region of amino acid sequence SEQ ID NO: 18 or any variant thereof; and the second heavy chain of amino acid sequence SEQ ID NO: 24 or any variant thereof chain variable region.
  • the second binding domain comprises the second light chain variable region of the amino acid sequence of SEQ ID NO:5, or any variant thereof; and the second heavy chain of the amino acid sequence of SEQ ID NO:48, or any variant thereof chain variable region.
  • the second binding domain comprises the second light chain variable region of amino acid sequence SEQ ID NO: 18 or any variant thereof; and the second heavy chain of amino acid sequence SEQ ID NO: 48 or any variant thereof chain variable region.
  • the second binding domain comprises the second light chain variable region of amino acid sequence SEQ ID NO:5 or any variant thereof; and the second heavy chain of amino acid sequence SEQ ID NO:50 or any variant thereof chain variable region.
  • the second binding domain comprises the second light chain variable region of amino acid sequence SEQ ID NO: 10 or any variant thereof; and the second heavy chain of amino acid sequence SEQ ID NO: 50 or any variant thereof chain variable region.
  • the second binding domain comprises the second light chain variable region of amino acid sequence SEQ ID NO: 12 or any variant thereof; and the second heavy chain of amino acid sequence SEQ ID NO: 50 or any variant thereof chain variable region.
  • the second binding domain comprises the second light chain variable region of amino acid sequence SEQ ID NO: 18 or any variant thereof; and the second heavy chain of amino acid sequence SEQ ID NO: 50 or any variant thereof chain variable region.
  • the second light chain of the second antigen-binding portion is selected from the amino acid sequence of SEQ ID NOs: 58 and 66; and/or the second heavy chain of the second antigen-binding portion is selected from the amino acid sequence of SEQ ID NO: 60 and 68.
  • the second light chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:58; and the second heavy chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:60.
  • the second light chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:66; and the second heavy chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:68.
  • the first antigen is a tumor antigen.
  • the tumor antigen is selected from the group consisting of: 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.
  • the tumor associated antigen is selected from CD20, BCMA and GPC3.
  • the first antigen is the CD20 antigen.
  • the first light chain variable region of the first antigen binding moiety has a Gln38Lys mutation ( V ⁇ CD20 : Gln38Lys ). In some preferred embodiments, the first heavy chain variable region of the first antigen binding moiety has a Gln 39 Glu mutation (VH CD20 :Gln 39 Glu).
  • the first light chain variable region of the first antigen binding moiety has a Gln 38 Lys mutation (V ⁇ CD20 :Gln 38 Lys), and the first light chain constant region has Glu 123 Lys and Gln 124 Lys mutations (V ⁇ -Ck CD20 : Gln38Lys ⁇ Glu123Lys ⁇ Gln124Lys ).
  • the first heavy chain variable region of the first antigen binding moiety has a Gln39Glu mutation (VH CD20 : Gln39Glu ) and the first heavy chain constant region has Lys152Glu and Lys218Glu Mutation ( VH - CH1CD20 : Gln39Glu ⁇ Lys152Glu ⁇ Lys218Glu ).
  • the first light chain of the first antigen-binding portion is selected from the amino acid sequence of SEQ ID NOs: 54, 62 and 70; and/or the first heavy chain of the first antigen-binding portion is selected from the amino acid sequence of SEQ ID NO: 56, 64 and 72.
  • the first light chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:54
  • the first heavy chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:56.
  • the first light chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:62
  • the first heavy chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:64.
  • the first light chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:70
  • the first heavy chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:72.
  • the first light chain of the first antigen-binding moiety is of amino acid sequence SEQ ID NO:54
  • the first heavy chain of the first antigen-binding moiety is of amino acid sequence SEQ ID NO:56
  • the second antigen-binding moiety has the amino acid sequence of SEQ ID NO:56.
  • the second light chain of the portion is the amino acid sequence of SEQ ID NO:58
  • the second heavy chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:60.
  • the first light chain of the first antigen-binding moiety is of amino acid sequence SEQ ID NO:62
  • the first heavy chain of the first antigen-binding moiety is of amino acid sequence SEQ ID NO:64
  • the second antigen-binding moiety has the amino acid sequence of SEQ ID NO:64.
  • the second light chain of the portion is the amino acid sequence of SEQ ID NO:66
  • the second heavy chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:68
  • the second light chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:68 NO:58
  • the second heavy chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:60.
  • the first light chain of the first antigen-binding portion is of amino acid sequence SEQ ID NO:70
  • the first heavy chain of the first antigen-binding portion is of amino acid sequence SEQ ID NO:72
  • the second antigen-binding portion is of amino acid sequence SEQ ID NO:72.
  • the second light chain of the portion is the amino acid sequence of SEQ ID NO:66
  • the second heavy chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:68.
  • the first light chain of the first antigen-binding moiety is of amino acid sequence SEQ ID NO:62
  • the first heavy chain of the first antigen-binding moiety is of amino acid sequence SEQ ID NO:64
  • the second antigen-binding moiety has the amino acid sequence of SEQ ID NO:64.
  • the second light chain of the portion is the amino acid sequence of SEQ ID NO:58
  • the second heavy chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:60.
  • the first light chain of the first antigen-binding moiety is of amino acid sequence SEQ ID NO:54
  • the first heavy chain of the first antigen-binding moiety is of amino acid sequence SEQ ID NO:56
  • the second antigen-binding moiety has the amino acid sequence of SEQ ID NO:56.
  • the second light chain of the portion is the amino acid sequence of SEQ ID NO:66
  • the second heavy chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:68.
  • the first antigen is a BCMA antigen.
  • the first light chain variable region of the first antigen binding moiety has a Gln42Lys mutation ( V ⁇ BCMA : Gln42Lys ).
  • the first heavy chain variable region of the first antigen binding moiety has a Gln 39 Glu mutation (VH BCMA : Gln 39 Glu).
  • the first light chain of the first antigen-binding portion is selected from the amino acid sequences of SEQ ID NOs: 80 and 84; and/or the first heavy chain of the first antigen-binding portion is selected from the amino acid sequences of SEQ ID NO: 82 and 86.
  • the first light chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:80; and the first heavy chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:82.
  • the first light chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:84; and the first heavy chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:82.
  • the first light chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:80; and the first heavy chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:86.
  • the first light chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:84; and the first heavy chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:86.
  • the first light chain of the first antigen-binding portion is of amino acid sequence SEQ ID NO:80
  • the first heavy chain of the first antigen-binding portion is of amino acid sequence SEQ ID NO:82
  • the second antigen-binding portion is of amino acid sequence SEQ ID NO:82.
  • the second light chain of the portion is the amino acid sequence of SEQ ID NO:66
  • the second heavy chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:68.
  • the first light chain of the first antigen-binding portion is of amino acid sequence SEQ ID NO:84
  • the first heavy chain of the first antigen-binding portion is of amino acid sequence SEQ ID NO:82
  • the second antigen-binding portion is of amino acid sequence SEQ ID NO:82.
  • the second light chain of the portion is the amino acid sequence of SEQ ID NO:66
  • the second heavy chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:68.
  • the first light chain of the first antigen-binding moiety is of amino acid sequence SEQ ID NO:80
  • the first heavy chain of the first antigen-binding moiety is of amino acid sequence SEQ ID NO:86
  • the second antigen-binding moiety has the amino acid sequence of SEQ ID NO:86.
  • the second light chain of the portion is the amino acid sequence of SEQ ID NO:66
  • the second heavy chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:68.
  • the first light chain of the first antigen-binding moiety is of amino acid sequence SEQ ID NO:84
  • the first heavy chain of the first antigen-binding moiety is of amino acid sequence SEQ ID NO:86
  • the second antigen-binding moiety has the amino acid sequence of SEQ ID NO:86.
  • the second light chain of the portion is the amino acid sequence of SEQ ID NO:66
  • the second heavy chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:68.
  • the first antigen is a GPC3 antigen.
  • the first light chain variable region of the first antigen binding moiety has Gln43Lys and Gln39Glu mutations ( V ⁇ GPC3 : Gln43Lys ; VH GPC3 : Gln39Glu ).
  • the first light chain of the first antigen-binding moiety is selected from the amino acid sequence of SEQ ID NOs: 88 and 92; and/or the first heavy chain of the first antigen-binding moiety is selected from the amino acid sequence of SEQ ID NO: 90 and 94.
  • the first light chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:88, and the first heavy chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:90.
  • the first light chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:92
  • the first heavy chain of the first antigen-binding portion is the amino acid sequence of SEQ ID NO:94.
  • the first light chain of the first antigen-binding portion is of amino acid sequence SEQ ID NO:88
  • the first heavy chain of the first antigen-binding portion is of amino acid sequence SEQ ID NO:90
  • the second antigen-binding portion is of amino acid sequence SEQ ID NO:90.
  • the second light chain of the portion is the amino acid sequence of SEQ ID NO:66
  • the second heavy chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:68.
  • the first light chain of the first antigen-binding moiety is of amino acid sequence SEQ ID NO:92
  • the first heavy chain of the first antigen-binding moiety is of amino acid sequence SEQ ID NO:94
  • the second antigen-binding moiety has the amino acid sequence of SEQ ID NO:94.
  • the second light chain of the portion is the amino acid sequence of SEQ ID NO:66
  • the second heavy chain of the second antigen-binding portion is the amino acid sequence of SEQ ID NO:68.
  • the Fc portion of the first antigen-binding portion and/or the second antigen-binding portion of the bispecific antibody employs a knob-into-hole structure.
  • the human IgG4 knob-into-hole structure is employed.
  • the first antigen-binding portion and/or the second antigen-binding portion of the bispecific antibody further has Ser 228 Pro, Leu 235 Glu, and/or Pro 329 Ala mutations.
  • the present disclosure provides nucleic acids encoding the aforementioned bispecific antibodies or antigen-binding portions thereof.
  • the second antigen-binding portion binds the CD3 antigen
  • the nucleic acid encoding the second light chain variable region of the second antigen-binding portion is selected from the group consisting of nucleotide sequences SEQ ID NOs: 6, 11, 13, 17, 19 and 23; and/or the nucleic acid encoding the second heavy chain variable region of the second antigen binding portion is selected from the group consisting of nucleotide sequences SEQ ID NOs: 25, 30, 33, 36, 39, 42, 45, 49, 51 and 53.
  • the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the group consisting of the nucleotide sequences of SEQ ID NOs: 59 and 67; and/or the nucleic acid encoding the second heavy chain of the second antigen-binding portion Selected from the nucleotide sequences of SEQ ID NOs: 61 and 69.
  • the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:59, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the group consisting of nucleosides Acid sequence SEQ ID NO:61.
  • the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the group consisting of nucleosides Acid sequence SEQ ID NO:69.
  • the first antigen-binding portion binds the CD20 antigen
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the group consisting of nucleotide sequences SEQ ID NOs: 55, 63, and 71; and/or the first The nucleic acid encoding the first heavy chain of the antigen binding portion is selected from the group consisting of nucleotide sequences SEQ ID NOs: 57, 65 and 73.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO: 55, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleosides Acid sequence SEQ ID NO:57.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:63, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleosides Acid sequence SEQ ID NO:65.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:71, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleosides Acid sequence SEQ ID NO:73.
  • the first antigen binding moiety binds the BCMA antigen
  • the nucleic acid encoding the first light chain of the first antigen binding moiety is selected from the group consisting of nucleotide sequences SEQ ID NOs: 81 and 85; and/or the first antigen binding
  • the nucleic acid encoding part of the first heavy chain is selected from the nucleotide sequences of SEQ ID NOs: 83 and 87.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO: 81, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleosides Acid sequence SEQ ID NO:83.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO: 85, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleosides Acid sequence SEQ ID NO:83.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO: 81, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleosides Acid sequence SEQ ID NO:87.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO: 85, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleosides Acid sequence SEQ ID NO:87.
  • the first antigen binding moiety binds the GPC3 antigen
  • the nucleic acid encoding the first light chain of the first antigen binding moiety is selected from the group consisting of nucleotide sequences SEQ ID NOs: 89 and 93; and/or the first antigen binds
  • the nucleic acid encoding part of the first heavy chain is selected from the nucleotide sequences of SEQ ID NOs: 91 and 95.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO: 89, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleosides Acid sequence SEQ ID NO:91.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:93, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleosides Acid sequence SEQ ID NO:95.
  • the first antigen-binding portion of the bispecific antibody binds the CD20 antigen
  • the second antigen binds the CD3 antigen
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO.
  • the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 57
  • the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 57 : 59
  • the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO: 61.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:63, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleosides
  • the acid sequence of SEQ ID NO:65, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nuclear
  • the nucleotide sequence of SEQ ID NO:69, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:59, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from Nucleotide sequence SEQ ID NO:61.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:71, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleosides
  • the acid sequence of SEQ ID NO:73, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nuclear nucleotide sequence SEQ ID NO:69.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:63, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleosides
  • the acid sequence of SEQ ID NO:65, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:59, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nuclear nucleotide sequence SEQ ID NO:61.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:55, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleotides The sequence SEQ ID NO:57, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleoside Acid sequence SEQ ID NO:69.
  • the first antigen-binding portion of the bispecific antibody binds the BCMA antigen
  • the second antigen binds the CD3 antigen
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO.
  • the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 83
  • the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 83 : 67
  • the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO: 69.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO: 85, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleotides The sequence SEQ ID NO:83, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleoside Acid sequence SEQ ID NO:69.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO: 81, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleotides
  • the sequence SEQ ID NO:87, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleoside Acid sequence SEQ ID NO:69.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO: 85, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleotides
  • the sequence SEQ ID NO:87, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleoside Acid sequence SEQ ID NO:69.
  • the first antigen-binding portion of the bispecific antibody binds the GPC3 antigen
  • the second antigen binds the CD3 antigen
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO.
  • the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the nucleotide sequence SEQ ID NO:91
  • the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence SEQ ID NO: 91 : 67
  • the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO: 69.
  • the nucleic acid encoding the first light chain of the first antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:93, and the nucleic acid encoding the first heavy chain of the first antigen-binding portion is selected from the group consisting of nucleotides The sequence of SEQ ID NO:95, the nucleic acid encoding the second light chain of the second antigen-binding portion is selected from the nucleotide sequence of SEQ ID NO:67, and the nucleic acid encoding the second heavy chain of the second antigen-binding portion is selected from the nucleoside Acid sequence SEQ ID NO:69.
  • the present disclosure provides vectors comprising the aforementioned nucleic acids.
  • the present disclosure provides cells comprising the aforementioned nucleic acids or vectors.
  • compositions comprising the aforementioned bispecific antibodies or antigen-binding portions thereof, nucleic acids, vectors, and/or cells.
  • the present disclosure provides antibody-drug conjugates comprising the aforementioned bispecific antibodies, or antigen-binding portions thereof, covalently attached to a therapeutic moiety.
  • the therapeutic moiety is selected from a cytotoxic moiety, a chemotherapeutic agent, a cytokine, an immunosuppressant, an immunostimulatory agent, a lytic peptide, or a radioisotope.
  • the antibodies of the present disclosure are useful as therapeutic or diagnostic tools in diseases in which various tumor antigens are unfavorably expressed or found.
  • a disease associated with a tumor antigen the expression of the tumor antigen in cells of a diseased tissue or organ is increased compared to the state in a healthy tissue or organ.
  • To increase means to increase by at least 10%, especially at least 20%, at least 50%, at least 100%, at least 200%, at least 500%, at least 1000%, at least 10000% or even more.
  • expression is found only in diseased tissue, whereas expression in corresponding healthy tissue is suppressed.
  • diseases associated with tumor antigens include tumors.
  • the tumor antigen-related disease is a CD20-related disease.
  • the disease associated with CD20 comprises a B cell disease, such as a B cell proliferative disorder, particularly a CD20 positive B cell disorder; preferably, the disease is selected from non-Hodgkin's lymphoma (NHL), acute Lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma ( MZL), as well as multiple myeloma (MM) and Hodgkin lymphoma (HL).
  • NHL non-Hodgkin's lymphoma
  • ALL acute Lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • FL mantle cell lymphoma
  • MZL marginal zone lymphoma
  • the tumor antigen-associated disease is a BCMA-associated disease; preferably, the BCMA-associated disease includes a B-cell disease; preferably, the disease is cancer; more preferably, the cancer is a B-cell-associated cancer, It is selected from the group consisting of multiple myeloma, malignant plasmacytoma, Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's lymphoma, Kahler's disease and myeloid leukemia, plasma cell leukemia, plasmacytoma, B- prolymphocytic leukemia, hairy cell leukemia, B-cell non-Hodgkin lymphoma (NHL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia Leukemia (CML), Follicular Lymphoma, Burkitt Lymphoma, Marginal Zone Lymphoma, Man
  • the therapeutic agent comprises an antibody that specifically binds an activating T cell antigen.
  • the therapeutic agent comprises an antibody that specifically binds CD3, particularly CD3 ⁇ .
  • Methods of treating diseases and symptoms with the bispecific antibodies of the present disclosure include the steps of administering to a mammal a therapeutically effective amount of an antibody or antigen-binding fragment or nucleic acid molecule or vector or cell or pharmaceutical composition of any of the foregoing aspects.
  • the present disclosure provides a method of treating or preventing a cancer disease comprising administering to a patient an antibody capable of binding GPC3, wherein the antibody is administered to provide a serum level of at least 40 ⁇ g/ml.
  • the antibody is administered to provide serum levels of at least 50 ⁇ g/ml, at least 150 ⁇ g/ml, at least 300 ⁇ g/ml, at least 400 ⁇ g/ml, or at least 500 ⁇ g/ml.
  • the antibody is administered to provide a serum level of no more than 800 ⁇ g/ml, 700 ⁇ g/ml, 600 ⁇ g/ml, 550 ⁇ g/ml or 500 ⁇ g/ml.
  • the provided serum level is 40 ⁇ g/ml to 700 ⁇ g/ml, preferably 40 ⁇ g/ml to 600 ⁇ g/ml, preferably 50 ⁇ g/ml to 500 ⁇ g/ml, such as 150 ⁇ g/ml to 500 ⁇ g/ml or 300 ⁇ g /ml to 500 ⁇ g/ml.
  • serum level as used in this specification means the concentration of the substance in question in serum.
  • serum levels are provided for at least 7 days or for at least 14 days.
  • the method comprises administering an antibody dose of at least 300 mg/m 2 , such as at least 600 mg/m 2 , and preferably at most 1500 mg/m 2 , at most 1200 mg/m 2 or at most 1000 mg/m 2 .
  • the present disclosure provides a method of treating or preventing a cancer disease comprising administering to a patient an antibody capable of binding GPC3 at a concentration of at least 300 mg/m 2 , such as at least 600 mg/m 2 , and preferably at most 1500 mg/m 2 , the antibody is administered at a dose of up to 1200 mg/m 2 or up to 1000 mg/m 2 .
  • the present disclosure provides a method of treating or preventing a cancer disease comprising administering to a patient an antibody capable of binding GPC3, wherein at least 50%, preferably 60%, 70%, 80% or 90% of the patient has cancer
  • the cells are GPC3 positive and/or at least 40%, preferably 50% or 60% of the cancer cells of the patient are positive for surface expression of GPC3.
  • the present disclosure also provides a method of treating or preventing a cancer disease, the method comprising: a. identifying at least 50%, preferably 60%, 70%, 80% or 90% of GPC3 positive cancer cells and/or at least 40% %, preferably 50% or 60% of the patient with cancer cells positive for surface expression of GPC3; and b. administering to the patient an antibody capable of binding GPC3.
  • at least 95% or at least 98% of the cancer cells of the patient are GPC3 positive.
  • at least 70%, at least 80% or at least 90% of the cancer cells of the patient are positive for surface expression of GPC3.
  • the outcome of the treatment of the cancer disease is the achievement of stable disease.
  • disease stabilization is achieved for at least 2 months, at least 3 months, or at least 6 months.
  • the present disclosure provides methods of achieving disease stabilization in a cancer patient comprising administering to the patient an antibody capable of binding GPC3.
  • disease stabilization is achieved for at least 2 months, at least 3 months, or at least 6 months.
  • the antibody is administered in a single dose or multiple doses.
  • the present disclosure provides a method of treating or preventing a cancer disease comprising administering to a patient an antibody capable of binding GPC3, wherein the antibody is administered in multiple doses.
  • the antibody is administered in multiple doses according to the present disclosure, preferably at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, at least 8 doses, at least 9 doses, or at least 9 doses
  • the antibody is administered in 10 doses and preferably up to 30 doses, 25 doses, 20 doses, 15 doses or 10 doses.
  • Doses of the antibody are preferably administered at intervals of at least 7 days, at least 10 days, at least 14 days, or at least 20 days.
  • Doses of antibody are preferably administered at intervals of 7 to 30 days, 10 to 20 days, and preferably about 14 days.
  • the antibody is administered so as to provide a serum level of at least 40 ⁇ g/ml. In various embodiments, the antibody is administered so as to provide serum levels of at least 50 ⁇ g/ml, at least 150 ⁇ g/ml, at least 300 ⁇ g/ml, at least 400 ⁇ g/ml, or at least 500 ⁇ g/ml. In various embodiments, the antibody is administered so as to provide serum levels of no more than 800 ⁇ g/ml, 700 ⁇ g/ml, 600 ⁇ g/ml, 550 ⁇ g/ml or 500 ⁇ g/ml.
  • the provided serum level is 40 ⁇ g/ml to 700 ⁇ g/ml, preferably 40 ⁇ g/ml to 600 ⁇ g/ml, preferably 50 ⁇ g/ml to 500 ⁇ g/ml, such as 150 ⁇ g/ml to 500 ⁇ g/ml or 300 ⁇ g /ml to 500 ⁇ g/ml.
  • serum levels are provided for at least 7 days or for at least 14 days.
  • the method comprises administering a dose of the antibody of at least 300 mg/m 2 , such as at least 600 mg/m 2 and preferably at most 1500 mg/m 2 , at most 1200 mg/m 2 or at most 1000 mg/m 2 .
  • the antibody is conjugated to other drugs, such as labeled or cytotoxic conjugates.
  • kits eg, kits comprising antibodies, fragments, homologues, derivatives thereof, nucleic acids, vectors, cells, compositions, etc., of the disclosure, eg, labeled or cytotoxic conjugates conjugates, as well as antibody instructions, conjugates that kill specific types of cells, and more.
  • the instructions may include instructions for using the antibodies, conjugates, etc. in vitro, in vivo or ex vivo.
  • Antibodies can be in liquid form or solid, usually lyophilized.
  • the kit may contain other suitable reagents, such as buffers, reconstitution solutions, and other necessary components for the intended use.
  • kits may include a substrate and cofactors required by the enzyme (eg, a substrate precursor that provides a detectable chromophore or fluorophore).
  • a substrate precursor that provides a detectable chromophore or fluorophore
  • other additives such as stabilizers, buffers (eg, blocking buffers or lysis buffers), etc., may also be included.
  • the relative amounts of the various reagents can be varied to provide concentrates of reagent solutions, which provides user flexibility, space savings, reagent savings, and the like.
  • These reagents can also be provided in dry powder form, usually lyophilized, including excipients which, when dissolved, provide a solution of the reagents of appropriate concentrations.
  • antibodies of the present disclosure can also be used in immunoassays, purification methods, and other methods using immunoglobulins or fragments thereof. Such uses are well known in the art.
  • compositions comprising the anti-GPC3 antibodies or fragments thereof of the present disclosure, the antibodies being conveniently combined with a pharmaceutically acceptable carrier, diluent or excipient, which is a routine practice in the art.
  • the term "pharmaceutical composition” refers to a formulation of various preparations. Formulations containing a therapeutically effective amount of the multivalent antibody are in sterile liquid solutions, liquid suspensions, or lyophilized forms, optionally containing stabilizers or excipients.
  • the antibodies of the present disclosure can be used as compositions for administration alone, or can be used in combination with other active agents.
  • the humanized antibodies of the present disclosure are conjugated to a therapeutic moiety (ie, a drug).
  • Therapeutic moieties can be, for example, cytotoxins, chemotherapeutic agents, cytokines, immunosuppressive agents, immunostimulatory agents, lytic peptides, or radioisotopes.
  • conjugates are referred to herein as "antibody-drug conjugates" or "ADCs".
  • the antibody is conjugated to a cytotoxic moiety.
  • Cytotoxic moieties may, for example, be selected from the following: paclitaxel; cytochalasin B; gramicidin D; ethidium bromide; ipecine; mitomycin; etoposide; teniposide; vincristine; vinblastine ; Colchicine; Doxorubicin; Daunorubicin; F or its analogues or derivatives; Dolastatin 10 or 15 or its analogues; Irinotecan or its analogues; Mitoxantrone; corticosteroids; procaine; tetracaine; lidocaine; propranolol; puromycin; calicheamicin or its analogs or derivatives; antimetabolites such as methotrexate, 6-mercaptopurine , 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, sebadiazine, hydroxy
  • the antibody is conjugated to auristatin or a peptide analog, derivative or prodrug thereof.
  • Auristatin has been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division and has anticancer and antifungal activity.
  • auristatin E can be reacted 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 auristatin and auristatin analogs, derivatives and prodrugs, as well as suitable linkers for conjugating auristatin to the Ab are described, for example, in US Pat. Nos. 5,635,483, 5,780,588 and 6,214,345 and International Patent Application Publication WO02088172 , WO2004010957, WO2005081711, WO2005084390, WO2006132670, WO03026577, WO200700860, WO207011968 and WO205082023.
  • the antibody is conjugated to pyrrolo[2,1-c][1,4]-benzodiazepine (PDB) or a peptide analog, derivative or prodrug thereof.
  • PDB pyrrolo[2,1-c][1,4]-benzodiazepine
  • Suitable PDBs and PDB derivatives and related techniques are described, for example, in Hartley J.A. et al., Cancer Res 2010; 70(17):6849-6858; Antonow D. et al., Cancer J 2008; 14(3):154-169; Howard P.W. et al, Bioorg Med Chem Lett 2009; 19:6463-6466 and Sagnou et al, Bioorg Med Chem Lett 2000; 10(18):2083-2086.
  • the antibody is conjugated to a cytotoxic moiety selected from the group consisting of anthracyclines, maytansines, calicheamicin, dukamycin, racheomycin (CC-1065), dolastatin 10.
  • a cytotoxic moiety selected from the group consisting of anthracyclines, maytansines, calicheamicin, dukamycin, racheomycin (CC-1065), dolastatin 10.
  • Dolastatin 15 irinotecan, monomethylauristatin E, monomethylauristatin F, PDB, or any analog, derivative or prodrug thereof.
  • the antibody is conjugated to an anthracycline or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to maytansine or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to calicheamicin or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to Dokamycin or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to Rachelmycin (CC-1065) or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 10 or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to dolastatin 15 or an analog, derivative or prodrug thereof.
  • the antibody is conjugated to monomethyl auristatin E or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to monomethylauristatin F or an analog, derivative or prodrug thereof. In some embodiments, the antibody is conjugated to a pyrrolo[2,1-c][1,4]-benzodiazepine or an analog, derivative, or prodrug thereof. In some embodiments, the antibody is conjugated to irinotecan or an analog, derivative or prodrug thereof.
  • the antibody is conjugated with a cytokine (eg, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL- 23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa, IFN3, IFNy, GM-CSF, CD40L, Flt3 ligand, stem cell factor, axigrastim and TNFa) couple link.
  • a cytokine eg, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL- 23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNa, IFN3, IFNy, GM-CSF, CD40L, Flt3 ligand, stem cell factor, axigrastim and TNFa
  • the antibody is conjugated to a radioisotope or radioisotope-containing chelate.
  • the antibody can be conjugated to a chelator linker (eg, DOTA, DTPA, or tiracetam) that allows complexation of the antibody with the radioisotope.
  • Antibodies may also or alternatively contain or be conjugated to one or more radiolabeled amino acids or other radiolabeled molecules.
  • radioisotopes include3H , 14C , 15N , 35S , 90Y , 99Tc , 125I , 131I , 186Re , 213Bi , 225Ac , and227Th .
  • radioisotopes that emit beta or alpha particle radiation such as131I , 90Y , 211At , 212Bi , 67Cu , 186Re , 188Re and212Pb , can be used.
  • nucleic acid molecules are covalently linked to lysine or cysteine on the antibody through an N-hydroxysuccinimide ester or maleimide functional group, respectively.
  • Conjugation methods using engineered cysteines or incorporating unnatural amino acids have been reported to improve the homogeneity of the conjugates.
  • acyl-donor glutamine-containing tags eg Gin peptide-containing tags or Q-tags
  • polypeptide engineering eg by amino acid deletions, insertions, substitutions or mutations on polypeptides
  • Transglutaminase can then be covalently cross-linked with an amine-donating agent (eg, a small molecule comprising or linked to a reactive amine) to form a stable and homogeneous population of engineered Fc-containing polypeptide conjugates, wherein Amine donor agents are site-specifically coupled to Fc-containing polypeptides via an acyl-donor glutamine-containing tag or an accessible/exposed/reactive endogenous glutamine (WO2012059882).
  • an amine-donating agent eg, a small molecule comprising or linked to a reactive amine
  • the therapeutic agents according to the above-described embodiments will be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents that are incorporated into the formulation to provide improved transfer, delivery, tolerability, and the like.
  • suitable pharmaceutically acceptable carriers include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing (cationic or anionic) carriers (eg, Lipofectin TM ), DNA conjugates, anhydrous slurries, oil-in-water and water-in-oil emulsions, emulsion polyethylene glycols (polyethylene glycols of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycols. Any of the foregoing mixtures may be suitable for use in treatment or therapy according to the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and that the formulation is physiologically compatible and tolerated by the route of administration.
  • the above-described antibodies can be used as therapeutic agents. Such agents will typically be used to treat, alleviate and/or prevent a disease or pathology associated with aberrant tumor antigen expression, activity and/or signaling in a subject.
  • Therapeutic regimens can be implemented using standard methods by identifying a subject, such as a human patient having (or at risk or developing) a disease or disorder associated with aberrant tumor antigen expression, activity and/or signaling, such as a tumor antigen-related disorder .
  • An antibody preparation preferably one with high specificity and high affinity for its target antigen, is administered to a subject and will generally have an effect due to its binding to the target.
  • Administered antibodies can eliminate or inhibit or interfere with the expression, activity and/or signaling function of the target (eg, tumor antigen).
  • Administered antibodies can eliminate or inhibit or prevent the target (eg, tumor antigen) from binding to the endogenous ligand to which it naturally binds.
  • an antibody binds to a target and modulates, blocks, inhibits, reduces, antagonizes, neutralizes and/or otherwise interferes with tumor antigen expression, activity and/or signaling.
  • an antibody having heavy and light chain CDRs can be administered to a subject.
  • antibodies directed against tumor antigens can be used in methods known in the art related to tumor antigen localization and/or quantification (eg, for the determination of tumor antigens and/or tumor antigens in appropriate physiological samples). levels, for diagnostic methods, for protein imaging, etc.).
  • an antibody comprising an antigen-binding domain derived from an antibody, specific for a tumor antigen or a derivative, fragment, analog or homolog thereof, is used as a pharmaceutically active compound (hereinafter referred to as as "therapeutic agent").
  • tumor antigen polypeptides can be isolated by standard techniques such as immunoaffinity, chromatography or immunoprecipitation using antibodies specific for the tumor antigen.
  • Antibodies (or fragments thereof) directed against tumor antigen proteins can be used to detect proteins in biological samples.
  • tumor antigens can be detected in biological samples as part of a clinical testing procedure, eg, to determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (ie, physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase;
  • suitable prosthetic complexes include streptavidin/biotin and avidin/ Biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine aminofluorescein, dansyl chloride, or phycoerythrin;
  • luminescent materials include Mino;
  • bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include125I, 131I , 35S , or3H .
  • antibodies according to the present disclosure can be used as reagents for detecting the presence of tumor antigens or protein fragments thereof in a sample.
  • the antibody comprises a detectable label.
  • the antibody is a polyclonal antibody, or more preferably a monoclonal antibody. Whole antibodies or fragments thereof (eg Fab, scFv or F(ab') 2 ) are used.
  • labeling in reference to an antibody is intended to include direct labeling of the antibody by conjugating (ie, physically linking) a detectable substance to the antibody, as well as indirect labeling of the antibody by reaction with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody, and end-labeling the antibody with biotin to enable detection with fluorescently-labeled streptavidin.
  • bio sample is intended to include tissues, cells, and biological fluids isolated from a subject, as well as tissues, cells, and fluids present in a subject.
  • biological sample is used to include blood and fractions or components in blood, including serum, plasma, or lymph.
  • the detection methods of the above-described embodiments can be used to detect analyte mRNA, protein or genomic DNA in biological samples in vitro and in vivo.
  • in vitro detection techniques for analyte mRNA include Norhtern hybridization and in situ hybridization.
  • Analyte protein in vitro detection techniques include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence.
  • In vitro detection techniques for analyte genomic DNA include Southern hybridization. Procedures for performing immunoassays are described, for example, in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, JRCrowther (ed.) Human Press, Totowa, NJ, 1995.
  • in vivo detection techniques for analyte proteins include introducing into a subject a labeled anti-analyte protein antibody.
  • an antibody can be labeled with a radiolabel, and the presence and location of the radiolabel in a subject can then be detected by standard imaging techniques.
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • the principles and considerations involved in preparing such compositions and guidelines for selecting components are well known in the art.
  • compositions typically comprise the antibody and a pharmaceutically acceptable carrier.
  • antibody fragments When antibody fragments are used, the smallest inhibitory fragment that specifically binds to the target protein binding domain may be preferred.
  • peptide molecules can be designed that retain the ability to bind target protein sequences. Such peptides can be chemically synthesized and/or produced by recombinant DNA techniques (see, eg, Marasco et al., Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)).
  • the term "pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration .
  • Suitable pharmaceutically acceptable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard bibliography in the art, which is incorporated herein by reference.
  • Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
  • Liposomes and non-aqueous vehicles, such as fixed oils, can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. In addition to any conventional media or reagents that are incompatible with the antibody, its use in compositions is contemplated.
  • compositions of the above-described embodiments are formulated to be compatible with their intended route of administration.
  • routes of administration include parenteral, eg, intravenous, intradermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal.
  • Solutions or suspensions for parenteral, intradermal or subcutaneous administration may include the following components: sterile injectable diluents such as water, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; Antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate Or phosphate, and agents to adjust osmotic pressure, such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be packaged in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (herein water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • suitable pharmaceutically acceptable carriers include physiological saline, bacteriostatic water, Cremophor EL TM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that it is easy to inject. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by the use of coatings such as lecithin to maintain the desired particle size in the case of dispersions, and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents such as sugars, polyols (such as mannitol, sorbitol), sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the antibody in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization.
  • dispersions are prepared by incorporating the antibody into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the methods of preparation are vacuum drying and freeze-drying to obtain a powder containing the active ingredient and any additional desired ingredient from a sterile-filtered solution of those previously enumerated. .
  • the compounds are delivered as an aerosol spray from a pressurized container or dispenser containing a gas of a suitable propellant, such as carbon dioxide, or a nebulizer.
  • a gas of a suitable propellant such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the permeation barrier are used in the formulation.
  • penetrants are generally known in the art and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • one or more antibodies can be formulated into an ointment, ointment, gel, or cream as generally known in the art.
  • the compounds can also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter or other glycerides) or retention enemas.
  • suppositories eg, with conventional suppository bases such as cocoa butter or other glycerides
  • retention enemas e.g., retention enemas.
  • the antibody may be prepared with a carrier that will prevent its rapid elimination from the body, such as a sustained/controlled release formulation, including implants and microencapsulated delivery systems.
  • a sustained/controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparing such formulations will be apparent to those skilled in the art.
  • Dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier one or more antibodies.
  • the specifications for the dosage unit forms of the above-described embodiments are indicated by and are directly dependent on the unique characteristics of the antibody and the particular therapeutic effect to be achieved, and the limitations inherent in the art of formulation of such antibodies for the treatment of individuals.
  • compositions can be placed in a container, pack, or dispenser with instructions for administration.
  • compositions described herein may also contain more than one antibody, preferably those that have complementary activities but do not negatively affect each other, depending on the particular condition to be treated.
  • the composition may, for example, comprise an agent that enhances its function, such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent.
  • an agent that enhances its function such as a cytotoxic agent, a cytokine, a chemotherapeutic agent, or a growth inhibitory agent.
  • Such molecules are suitably combined in amounts effective for the intended purpose. For example, it can be combined in a kit, and can also be combined in use.
  • one or more antibodies may be administered in combination therapy, ie, with other agents such as therapeutic agents, which are useful in the treatment of pathological conditions or disorders, such as various forms of cancer, autoimmune disorders and inflammation sexually transmitted diseases) combined.
  • agents such as therapeutic agents, which are useful in the treatment of pathological conditions or disorders, such as various forms of cancer, autoimmune disorders and inflammation sexually transmitted diseases
  • the term "combination” as used herein refers to the administration of the agents substantially simultaneously, simultaneously or sequentially. If administered sequentially, at the start of administration of the second compound, the first of the two compounds is still preferably detected at an effective concentration at the treatment site. In one instance, “combination” can also be the simultaneous inclusion of an antibody of the present disclosure and other therapeutic agents in a kit.
  • a combination therapy can comprise one or more antibodies described herein with one or more additional therapeutic agents (eg, one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors) , enzyme inhibitors, and/or cytotoxins or cytostatics, as described in more detail below) are co-formulated and/or co-administered.
  • additional therapeutic agents eg, one or more cytokine and growth factor inhibitors, immunosuppressants, anti-inflammatory agents, metabolic inhibitors
  • enzyme inhibitors e.g., enzyme inhibitors, and/or cytotoxins or cytostatics
  • the treatment regimen is effective to reduce cytokine release associated with administration of the T cell activating therapeutic agent in the subject compared to a corresponding treatment regimen without administration of an anti-tumor antigen antibody.
  • Figure 1 shows the structure of a novel first antigen x CD3 ⁇ bispecific antibody.
  • the experiments of the present disclosure demonstrate that in the case of free combination, the lambda light chain of the humanized CD3 arm tends to pair with the homologous heavy chain, and the pairing ratio with the heterologous heavy chain is lower; The kappa light chain also tends to pair with the homologous heavy chain, with a very low pairing ratio with the humanized CD3 heavy chain; meanwhile, the introduction of complementary charge variants in the Fv further reduces possible light chain mismatches.
  • the Fc part of the CD20 ⁇ CD3 ⁇ bispecific antibody adopts the structure of human IgG4knob-into-hole. By mutating Ser 228 Pro, Leu 235 Glu and Pro 329 Ala, the hinge region is kept stable and the interaction with Fc ⁇ receptors and C1q is weakened.
  • the amino acid sequence of (hole) is shown in SEQ ID NO. 3, and the amino acid sequence of cynomolgus monkey CD3 ⁇ IgG Fc (knob) is shown in SEQ ID NO. 4).
  • the recombinant plasmids expressing CD3 ⁇ -Fc and CD3 ⁇ -Fc were mixed with 3 mg/mL PEI (Polysciences, #24765-2) and co-transfected into HEK293E cells (medium OPM-293CD03DPM), after culturing for 7 days at 37°C 120rpm and 5% CO2 , collect the supernatant of the medium, and purify it by Protein A affinity chromatography to obtain human or cynomolgus CD3 ⁇ -Fc recombinant protein.
  • Murine hybridoma CD3 antibody (EMBO J.1985.4(2):337-344; J.Immunol.1986,137(4):1097-100; J.Exp.Med.1991,174:319-326; J. Immunol. 1991, 147(9):3047-52) recognizes human and cynomolgus monkey CD3 receptors, the sequence of which is as follows:
  • Anti-CD3 mouse monoclonal antibody light chain amino acid sequence (SEQ ID NO.96):
  • Anti-CD3 mouse monoclonal antibody heavy chain amino acid sequence (SEQ ID NO.97):
  • Anti-CD3 mouse monoclonal antibody was humanized, the human germline gene IMGT_hVL7-43 with the highest homology was selected for light chain CDR transplantation, human IGLJ3*02 was selected for FM4; human IMGT_hVH3-73 was selected for heavy chain CDR transplantation, and human FM4 was selected for transplantation IGHJ4*01.
  • the designs resulted in different heavy and light chain variants (Table 1).
  • hVL1 The amino acid sequence of hVL1 is shown in SEQ ID NO.5, its encoding nucleic acid is shown in SEQ ID NO.6, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.7, 8, and 9 respectively.
  • the amino acid sequence of hVL2 is shown as SEQ ID NO.10, its encoding nucleic acid is shown as SEQ ID NO.11, and its LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO.7, 8, 9 respectively.
  • the amino acid sequence of hVL3 is shown in SEQ ID NO.12
  • its encoding nucleic acid is shown in SEQ ID NO.13
  • its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.14, 15, 9 respectively.
  • the amino acid sequence of hVL4 is shown in SEQ ID NO.16, its encoding nucleic acid is shown in SEQ ID NO.17, and its LCDR1, LCDR2 and LCDR3 are shown in SEQ ID NO.14, 15, 9 respectively.
  • the amino acid sequence of hVL5 is shown as SEQ ID NO.18, its encoding nucleic acid is shown as SEQ ID NO.19, and its LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO.7, 8, 21 respectively.
  • the amino acid sequence of hVL6 is shown as SEQ ID NO.22, its encoding nucleic acid is shown as SEQ ID NO.23, and its LCDR1, LCDR2 and LCDR3 are shown as SEQ ID NO.7, 20, 21 respectively.
  • hVH1 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, and 28, respectively.
  • hVH2 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.
  • hVH3 The amino acid sequence of hVH3 is shown in SEQ ID NO.32, its encoding nucleic acid is shown in SEQ ID NO.33, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.31, 27, 34 respectively.
  • hVH4 The amino acid sequence of hVH4 is shown in SEQ ID NO.35, its encoding nucleic acid is shown in SEQ ID NO.36, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.31, 27, 37 respectively.
  • hVH5 The amino acid sequence of hVH5 is shown in SEQ ID NO.38, its encoding nucleic acid is shown in SEQ ID NO.39, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.31, 27, 40 respectively.
  • hVH6 The amino acid sequence of hVH6 is shown in SEQ ID NO.41, its encoding nucleic acid is shown in SEQ ID NO.42, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.31, 27, 43 respectively.
  • the amino acid sequence of hVH7 is shown in SEQ ID NO.44, its encoding nucleic acid is shown in SEQ ID NO.45, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.46, 47, 28 respectively.
  • hVH8 The amino acid sequence of hVH8 is shown in SEQ ID NO.48, its encoding nucleic acid is shown in SEQ ID NO.49, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.26, 27, 28 respectively.
  • the amino acid sequence of hVH9 is shown in SEQ ID NO.50, its encoding nucleic acid is shown in SEQ ID NO.51, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.26, 27, 28, respectively.
  • the amino acid sequence of hVH10 is shown in SEQ ID NO.52, its encoding nucleic acid is shown in SEQ ID NO.53, and its HCDR1, HCDR2 and HCDR3 are shown in SEQ ID NO.26, 27, 28, respectively.
  • the light and heavy chain humanized variants were synthesized in full sequence, cloned into a eukaryotic expression vector containing antibody lambda light chain constant region or human IgG4 heavy chain constant region CH1-CH3, and co-transfected into HEK293E cells at 37°C After 5-6 days of culture at 120 rpm 5% CO 2 , the medium supernatant was collected and purified by a Protein A column.
  • CD3 antibodies Coated with human CD3 ⁇ recombinant protein, overnight at 4°C. After blocking with 2% skim milk, CD3 antibodies of different dilutions were added to each well and incubated for 1 hour; the secondary antibody was added with HPR-labeled goat anti-human IgG Fc, after TMB solution developed color, the reaction was terminated with concentrated sulfuric acid and read at 450nm absorbance.
  • Figure 2 shows CD3 humanized antibodies (including aCD3-hVH1/VL5, aCD3-hVH8/VL1, aCD3-hVH8/VL5, aCD3-hVH9/VL1, aCD3-hVH9/VL2, aCD3-hVH9/VL3, aCD3-hVH9/ VL5) binding to human CD3 ⁇ protein, CD3 humanized antibody binds CD3 ⁇ recombinant protein with high affinity.
  • CD3 humanized antibodies including aCD3-hVH1/VL5, aCD3-hVH8/VL1, aCD3-hVH8/VL5, aCD3-hVH9/VL1, aCD3-hVH9/VL2, aCD3-hVH9/VL3, aCD3-hVH9/ VL5
  • the results are shown in Figure 3.
  • the CD3 humanized antibodies bind to Jurkat cells, and the CD3 humanized antibodies hVH9/VL5 (aCD3-hVH9/VL5) and hVH9/VL2 (aCD3-hVH9/VL2) are significantly weaker than the control antibodies.
  • OKT3, binds Jurkat cells with moderate affinity.
  • Table 2 shows the affinity of CD3 humanized antibodies to CD3 recombinant protein and Jurkat cells.
  • CD20 ⁇ CD3 ⁇ 001 retain the natural sequences of CD3 arm and CD20 antigen arm;
  • CD20 ⁇ CD3 ⁇ 002 charge variants ( V ⁇ CD20 : Gln 38 Lys; VH CD20 : Gln 39 Glu; V ⁇ CD3 : Gln 40 Glu; VH CD3 : Gln 39 Lys) were introduced into the CD20 antigen arm and CD3 arm at the same time;
  • CD20 ⁇ CD3 ⁇ 003 Based on CD20 ⁇ CD3 ⁇ 002 , a complementary charge pair is added between CH1/C ⁇ (V ⁇ - CkCD20 : Gln38Lys ⁇ Glu123Lys ⁇ Gln124Lys ; VH - CH1CD20 : Gln39Glu ⁇ Lys 152 Glu ⁇ Lys 218 Glu; V ⁇ CD3 : Gln 40 Glu; VH CD3 : Gln 39 Lys);
  • CD20 ⁇ CD3 ⁇ 004 only charge variants (V ⁇ CD20 : Gln 38 Lys; VH CD20 : Gln 39 Glu) are introduced in the CD20 antigen arm;
  • CD20 ⁇ CD3 ⁇ 005 charge variants (V ⁇ CD3: Gln 40 Glu; VH CD3 : Gln 39 Lys) were introduced only on the CD3 arm.
  • control antibody CD20 ⁇ CD3-crossFab was constructed with reference to the CrossFab method (Schaefer W et al., PNAS 2011).
  • CD20 ⁇ CD3 ⁇ 001 CD20 ⁇ CD3 ⁇ 001:
  • CD20 arm heavy chain (heavy chain 1) SEQ ID NO.56
  • CD3 arm heavy chain (heavy chain 2) SEQ ID NO.60
  • CD20 ⁇ CD3 ⁇ 002 CD20 ⁇ CD3 ⁇ 002:
  • CD20 arm heavy chain (heavy chain 1) SEQ ID NO.64
  • CD3 arm heavy chain (heavy chain 2) SEQ ID NO.68
  • CD20 ⁇ CD3 ⁇ 003 CD20 ⁇ CD3 ⁇ 003:
  • CD20 arm heavy chain (heavy chain 1) SEQ ID NO.72
  • CD3 arm heavy chain (heavy chain 2) SEQ ID NO.68
  • CD20 ⁇ CD3 ⁇ 004 CD20 ⁇ CD3 ⁇ 004:
  • CD20 arm heavy chain (heavy chain 1) SEQ ID NO.64
  • CD3 arm heavy chain (heavy chain 2) SEQ ID NO.60
  • CD20 ⁇ CD3 ⁇ 005 CD20 ⁇ CD3 ⁇ 005:
  • CD20 arm heavy chain (heavy chain 1) SEQ ID NO.56
  • CD3 arm heavy chain (heavy chain 2) SEQ ID NO.68
  • CD20 arm heavy chain (heavy chain 1) SEQ ID NO.74
  • CD3 arm heavy chain (heavy chain 2) SEQ ID NO.78
  • CHO-S cells were co-transfected and cultured in 500 mL CD CHO AGT medium (Gibco #12490-001) at 37°C, 5% CO 2 150 rpm, on days 2, 4 and 6 of transient transfection, respectively.
  • 4% CHO Feed C + feed (Gibco #A25031-05) was added.
  • the CD20 ⁇ CD3 ⁇ bispecific antibody was further purified and eluted by gradient elution of 50-300mM NaCl, 50mM phosphate, pH 6.4, and the elution peaks were combined, SEC-HPLC showed high monomer content at 99% (Figure 7).
  • the light chain mismatch ratio was extremely low ( ⁇ 1%), and no CD3 homodimers or CD20 homodimers were detected ( Figure 8).
  • Jurkat cells in logarithmic growth phase were taken, 200 ⁇ g/mL mouse IgG (Jackson ImmunoResearch, 115-005-03) was added, and the cells were ice-bathed for 30 minutes.
  • the cells were adjusted to 5 ⁇ 10 5 cells/mL with 4% calf serum, 100 ⁇ L per well was added to a 96-well U-plate, centrifuged at 300 g to remove the supernatant, and 100 ⁇ L of serially diluted antibodies were added to each well (initial concentration 1800nM, 3-fold dilution). , 10 gradients), incubated at 4°C for 60 minutes.
  • the test results are shown in Figure 11 and Table 6.
  • the CD20 ⁇ CD3 ⁇ bispecific antibody binds to the human leukemia T cell line Jurkat cells with moderate affinity, and the EC 50 is about 71-120nM, which is about 10 times lower than the binding force of the CD20 antigen arm to the CD20 receptor.
  • PBMCs were obtained by separation by Ficoll.Paque Plu (GE, 17-1440-03). PBMC were adjusted to 5 ⁇ 10 5 cells/mL with 4% calf serum (Hyclone, SH30626.06), 100 ⁇ L/well was added to a 96-well U-shaped plate, the supernatant was discarded by centrifugation, and 100 ⁇ L of serially diluted antibody was added to each well. (initial concentration 1800nM, 3-fold dilution, 10 gradients), incubated at 4°C for 60 minutes.
  • the test results are shown in Figure 12 and Table 6.
  • the CD20 ⁇ CD3 ⁇ bispecific antibody recognizes human peripheral blood CD4 + T and CD8 + T cells, and the affinity for human T cells is about 65-98nM, which is weaker than the binding force of the CD20 antigen arm to the CD20 receptor. About 10 times, which is favorable for the preferential enrichment of bispecific antibodies to tumor cells.
  • FIGS. 13A and 13B show the killing of human B lymphoid leukemia cell Nalm-6 and the activation of T cells by CD20 ⁇ CD3 ⁇ bispecific antibody, respectively.
  • Figures 14A and 14B show the killing of TMD-8 cells and the activation of T cells by CD20 ⁇ CD3 ⁇ bispecific antibody, respectively.
  • Figures 15A and 15B show the killing of Toledo cells and the activation of T cells by CD20 ⁇ CD3 ⁇ bispecific antibody, respectively.
  • tumor cells Nalm-6, TMD-8 and Toledo with different expression levels of CD20 both CD20 ⁇ CD3 ⁇ 002 and CD20 ⁇ CD3 ⁇ 003 can mediate effective killing of T cells, and the killing activity is comparable to or slightly stronger than that of the control antibody bsAB1. Activation of cells is milder than the latter.
  • Jurkat-NFAT-luc reporter cells and CD20-positive target cells (SU-DHL-4, Raji and NALM-6 cells) in logarithmic growth phase were taken, the supernatant was discarded by centrifugation, and resuspended to 2 ⁇ 10 6 cells/ml.
  • 50 ⁇ l/well of target cells were seeded into 96-well plates, centrifuged at 300g for 5 minutes to discard the supernatant, 50 ⁇ l/well of Jurkat-NFAT-luc reporter cells were seeded into 96-well plates, and 50 ⁇ L of serially diluted CD20 ⁇ CD3 ⁇ bispecific antibody or control was added to each well.
  • Antibody KLH ⁇ CD3 (initial concentration 20 ⁇ g/ml, 10-fold dilution, 10 gradients), 5% CO 2 , and incubated at 37° C. for 6 hours. After the incubation, according to the instructions of ONE-Glo Luciferase Assay System, 100 ⁇ L of detection reagent was added to each well, placed at room temperature for 3 minutes, and detected on a microplate reader (Biotek Synergy HT). The test results are shown in Figure 16 and Table 7. When the CD20 ⁇ CD3 ⁇ bispecific antibody targets tumor cells with different CD20 expression levels, it can activate the NFAT signaling pathway of T cells.
  • His-Tag antibody 50 ⁇ g/ml His-Tag antibody was coupled to the CM5 chip through amino groups to capture the His-tagged Fc ⁇ RI, Fc ⁇ RIIA H131 and Fc ⁇ RIIIA V158 recombinant proteins (Sino Biological, #10256-H08H/10374-H08H1/10389-H08H1), respectively, The capture time was 40 seconds, the flow rate was 10 ⁇ L/min. After the baseline was stable, the serially diluted antibody (initial concentration 37.5 ⁇ g/mL, 2-fold dilution) was flowed through the chip at a flow rate of 30 ⁇ L/min. The binding time was 120 seconds, and the dissociation time was 120 seconds.
  • the affinity constants were obtained by fitting with Biacore evaluation software. It can be seen from Table 8 that the CD20 ⁇ CD3 ⁇ bispecific antibody does not bind to Fc ⁇ RI, Fc ⁇ RIIA H131 and Fc ⁇ RIIIA V158 ; the wild-type IgG4 control antibody binds to Fc ⁇ RI with strong affinity and weakly binds to Fc ⁇ RIIA H131 .
  • mice 6-8 weeks old female B-NGD mice (Biositu Biotechnology Co., Ltd.) were selected, and 3 ⁇ 10 6 Raji cells were subcutaneously inoculated.
  • the tumor grew to 60 mm 3 , they were randomly divided into groups and set to the administration group of 3.0 mg respectively. /kg, administration group 0.6 mg/kg, administration group 0.12 mg/kg and negative control group KLH ⁇ CD3 3 mg/kg.
  • 1 ⁇ 10 7 PBMC cells were injected into the tail vein of each mouse, and the first mouse administration was started 3 days later, and the administration interval was once every 5 days, and the administration was administered 3 times in total. The tumor volume and body weight of the mice were monitored.
  • mice were sacrificed by cervical dislocation, and the tumors were weighed and recorded. The results are shown in Figure 17.
  • the in vivo efficacy of the CD20 ⁇ CD3 ⁇ bispecific antibody is dose-dependent, and the tumor inhibition rates at the middle and high doses are 82% and 89%, respectively.
  • the tumor-bearing mice tolerated the above doses well without adverse reactions such as weight loss.
  • cynomolgus monkeys were assigned to 4 dose groups, each of which consisted of 2 monkeys, half male and half female.
  • the doses received by each dose group were 0.3, 1, 3 mg/kg (once a week for 3 weeks, a total of 4 doses) and 1 mg/kg (single dose) CD20 ⁇ CD3 ⁇ 002 bispecific antibody , the dosing schedule is shown in Table 9.
  • all monkeys in each group were in good general condition, no toxic reaction, no death or dying.
  • Example 2 Using the BCMA humanized antibody containing ⁇ light chain, and the humanized CD3 antibody containing ⁇ light chain, refer to Example 2 to construct a new BCMA-CD3 ⁇ humanized bispecific antibody with a natural IgG configuration, and at the same time in the BCMA antigen.
  • V ⁇ BCMA Gln 42 Lys
  • VH BCMA Gln 39 Glu
  • V ⁇ CD3 Gln 40 Glu
  • VH CD3 Gln 39 Lys
  • Fc portion of bispecific antibody A human IgG4 knob-into-hole structure was employed to achieve heterodimeric pairing, and by mutating Ser 228 Pro, Leu 235 Glu, and Pro 329 Ala, the hinge region was stabilized and interactions with Fc ⁇ R receptors and C1q were attenuated.
  • BCMA arm kappa light chain SEQ ID NO.80
  • BCMA arm heavy chain (heavy chain 1): SEQ ID NO.82
  • CD3 arm lambda light chain SEQ ID NO.66
  • CD3 arm heavy chain (heavy chain 2): SEQ ID NO.68
  • BCMA arm kappa light chain SEQ ID NO.84
  • BCMA arm heavy chain (heavy chain 1): SEQ ID NO.86
  • CD3 arm lambda light chain SEQ ID NO.66
  • CD3 arm heavy chain (heavy chain 2): SEQ ID NO.68
  • BCMA arm kappa light chain SEQ ID NO.80
  • BCMA arm heavy chain (heavy chain 1): SEQ ID NO.86
  • CD3 arm lambda light chain SEQ ID NO.66
  • CD3 arm heavy chain (heavy chain 2): SEQ ID NO.68
  • BCMA arm kappa light chain SEQ ID NO.84
  • BCMA arm heavy chain (heavy chain 1): SEQ ID NO.86
  • CD3 arm lambda light chain SEQ ID NO.66
  • CD3 arm heavy chain (heavy chain 2): SEQ ID NO.68
  • CHO-human BCMA stably transfected cells CHO-hBCMA
  • CHO-cynomolgus monkey BCMA stably transfected cells CHO-cynoBCMA
  • tumor cells NCI-H929 and RPMI-8226 in logarithmic growth phase were taken, and each well was blocked.
  • Add 100 ⁇ L of serially diluted antibodies initial concentration 1800 nM, 3-fold dilution, 10 gradients), and incubate at 4° C. for 60 minutes.
  • FIG. 20 shows that BCMA ⁇ CD3 ⁇ bispecific antibody binds to human and cynomolgus monkey BCMA stably transfected cells with high affinity;
  • Figure 21 shows that BCMA ⁇ CD3 ⁇ bispecific antibody binds BCMA+ tumor cells NCI-H929 and RPMI-8226 with high affinity.
  • the binding constant EC50 of BCMA ⁇ CD3 ⁇ bispecific antibody to cells is shown in Table 13.
  • Jurkat cells in logarithmic growth phase were taken, 200 ⁇ g/mL mouse IgG (Jackson ImmunoResearch, 115-005-03) was added, and the cells were ice-bathed for 30 minutes.
  • the cells were adjusted to 5 ⁇ 10 5 cells/mL with 4% calf serum, 100 ⁇ L per well was added to a 96-well U-plate, centrifuged at 300 g to remove the supernatant, and 100 ⁇ L of serially diluted antibodies were added to each well (initial concentration 1800nM, 3-fold dilution). , 10 gradients), incubated at 4°C for 60 minutes.
  • PBMCs were separated by Ficoll.Paque Plu (GE, 17-1440-03). PBMC were adjusted to 5 ⁇ 10 5 cells/mL with 4% calf serum (Hyclone, SH30626.06), 100 ⁇ L/well was added to a 96-well U-shaped plate, the supernatant was discarded by centrifugation, and 100 ⁇ L of serially diluted antibody was added to each well. (initial concentration 1800nM, 3-fold dilution, 10 gradients), incubated at 4°C for 60 minutes.
  • FIGS. 24A and 24B show the killing of NCI-H929 cells and the activation of T cells by BCMA ⁇ CD3 ⁇ bispecific antibodies, respectively.
  • Figures 25A and 25B show the killing of RPMI-8226 cells and the activation of T cells by BCMA ⁇ CD3 ⁇ bispecific antibodies, respectively.
  • BCMA ⁇ CD3 ⁇ bispecific antibody can mediate effective killing of T cells, and the killing activity is comparable to that of control antibody REGN5458.
  • Jurkat-NFAT-luc reporter cells and BCMA-positive target cells RPMI-8226 in logarithmic growth phase were taken, centrifuged to discard the supernatant, and resuspended to 2 ⁇ 10 6 cells/ml.
  • 50 ⁇ l/well of target cells were seeded into 96-well plates, centrifuged at 300g for 5 minutes to discard the supernatant, 50 ⁇ l/well of Jurkat-NFAT-luc reporter cells were seeded into 96-well plates, and 50 ⁇ L of serially diluted BCMA ⁇ CD3 ⁇ bispecific antibody or control was added to each well.
  • Antibody KLH ⁇ CD3 (initial concentration 20 ⁇ g/ml, 10-fold dilution, 10 gradients), 5% CO 2 , and incubated at 37° C. for 6 hours. After the incubation, according to the instructions of ONE-Glo Luciferase Assay System, 100 ⁇ L of detection reagent was added to each well, placed at room temperature for 3 minutes, and detected on a microplate reader (Biotek Synergy HT). The test results are shown in Figure 26.
  • the BCMA ⁇ CD3 ⁇ bispecific antibody targets RPMI-8226 tumor cells, it can activate the NFAT signaling pathway of T cells; when there are no target cells, it does not activate the NFAT signaling pathway.
  • His-Tag antibody 50 ⁇ g/ml His-Tag antibody was coupled to the CM5 chip through amino groups, and the Fc ⁇ RI, Fc ⁇ RIIA H131 and Fc ⁇ RIIIA V158 recombinant proteins with His6 tags were captured respectively.
  • the capture time was 40 seconds, and the flow rate was 10 ⁇ L/min.
  • the antibody (initial concentration 37.5 ⁇ g/mL, 2-fold dilution) flowed through the chip at a flow rate of 30 ⁇ L/min, the binding time was 120 seconds, the dissociation time was 200 seconds, and the affinity constant was obtained by fitting with Biacore evaluation software.
  • the BCMA ⁇ CD3 ⁇ bispecific antibody did not bind to Fc ⁇ RI, Fc ⁇ RIIA H131 and Fc ⁇ RIIIA V158 ; the wild-type IgG4 control antibody bound Fc ⁇ RI with strong affinity and weakly bound to Fc ⁇ RIIA H131 .
  • mice 6-8 week old female B-NGD mice (Biositu Biotechnology Co., Ltd.) were selected and inoculated subcutaneously with 2 ⁇ 10 6 NCI-H929 cells (mixed with Matrigel 1:1), and the tumors grew to 60 mm 3
  • the patients were randomly divided into groups, they were 3.0 mg/kg in the administration group, 0.6 mg/kg in the administration group and KLH ⁇ CD33 mg/kg in the negative control group.
  • Each mouse was injected with 1 ⁇ 10 7 PBMC cells through the tail vein, and the first mouse administration was started 3 days later, and the administration interval was once every 5 days, with a total of 2 administrations. The tumor volume and body weight of the mice were monitored every 2 days.
  • mice were sacrificed by neck dislocation, and the tumors were weighed and recorded. The results are shown in Figure 29.
  • the in vivo efficacy of the BCMA ⁇ CD3 ⁇ bispecific antibody is dose-dependent.
  • the tumor inhibition rates in the 3.0 mg/kg and 0.6 mg/kg groups were 95% and 108%, respectively (BCMA ⁇ CD3 ⁇ 005) and 94% and 108% (BCMA ⁇ CD3 ⁇ 006).
  • the tumor-bearing mice tolerated the above doses well without adverse reactions such as weight loss.
  • the humanized GPC3 antibody containing ⁇ light chain and the humanized CD3 antibody containing ⁇ light chain were constructed with reference to Example 2 to construct a new GPC3-CD3 ⁇ humanized bispecific antibody with natural IgG configuration.
  • Arm and CD3 arm introduced charge variants (V ⁇ GPC3 : Gln 43 Lys; VH GPC3 : Gln 39 Glu; V ⁇ CD3 : Gln 40 Glu; VH CD3 : Gln 39 Lys).
  • the Fc part of the bispecific antibody adopts the human IgG4 knob-into-hole structure to achieve heterodimer pairing, and by mutating Ser 228 Pro, Leu 235 Glu and Pro 329 Ala, the hinge region is stabilized and the interaction with Fc ⁇ receptors and Fc ⁇ receptors is weakened. Interaction of C1q.
  • GPC3 arm heavy chain (heavy chain 1) SEQ ID NO.90
  • CD3 arm heavy chain (heavy chain 2) SEQ ID NO.68
  • GPC3 arm heavy chain (heavy chain 1) SEQ ID NO.94
  • CD3 arm heavy chain (heavy chain 2) SEQ ID NO.68
  • CHO-human GPC3, CHO-cynomolgus monkey GPC3 stable transfected cells or human hepatocellular carcinoma HepG2 tumor cells in logarithmic growth phase, adjust the cells to 5 ⁇ 10 5 cells/ml after blocking, and add 100 ⁇ l/well of cell suspension Transfer to a 96-well U-shaped plate, centrifuge at 300g for 5 minutes, discard the supernatant, add 100 ⁇ L of serially diluted antibody (initial concentration 1800nM, 3-fold dilution, 10 gradients) to each well, and incubate at 4°C for 60 minutes.
  • Jurkat cells in logarithmic growth phase were taken, 200 ⁇ g/mL mouse IgG (Jackson ImmunoResearch, 115-005-03) was added, and the cells were ice-bathed for 30 minutes.
  • the cells were adjusted to 5 ⁇ 10 5 cells/mL with 4% calf serum, 100 ⁇ L per well was added to a 96-well U-plate, centrifuged at 300 g to remove the supernatant, and 100 ⁇ L of serially diluted antibodies were added to each well (initial concentration 1800nM, 3-fold dilution). , 10 gradients), incubated at 4°C for 60 minutes.
  • the GPC3 ⁇ CD3 ⁇ bispecific antibody binds to the human leukemia T cell line Jurkat cells with moderate affinity, with an EC 50 of about 20-40 nM.
  • PBMCs were isolated by Ficoll.Paque Plus (GE, 17-1440-03). PBMC were adjusted to 5 ⁇ 10 5 cells/mL with 4% calf serum (Hyclone, SH30626.06), 100 ⁇ L/well was added to a 96-well U-shaped plate, the supernatant was discarded by centrifugation, and 100 ⁇ L of serially diluted antibody was added to each well. (initial concentration 1800nM, 3-fold dilution, 10 gradients), incubated at 4°C for 60 minutes.
  • FIGS. 34A and 34B show the killing of HepG2 cells and the activation of T cells by GPC3 ⁇ CD3 ⁇ bispecific antibody, respectively.
  • the target cell CHO-human GPC3 in logarithmic growth phase was taken, the supernatant was discarded by centrifugation, and resuspended to 2 ⁇ 10 5 cells/ml. 50 ⁇ L/well of target cells were seeded into a 96-well plate, 5% CO 2 , and cultured at 37°C overnight.
  • Jurkat-NFAT-luc reporter cells Take the Jurkat-NFAT-luc reporter cells in logarithmic growth phase, centrifuge at 300g for 5 minutes, discard the supernatant, resuspend to 4 ⁇ 10 6 cells/ml, remove the 96-well plate, discard the supernatant, and inoculate Jurkat-NFAT- luc reporter cells were transferred to a 96-well plate, and 25 ⁇ L of serially diluted GPC3 ⁇ CD3 ⁇ bispecific antibody or control antibody KLH ⁇ CD3 was added to each well (initial concentration 20 ⁇ g/ml, 3-fold dilution, 10 gradients), 5% CO 2 , 37 Incubate for 6 hours.
  • Freshly isolated PBMCs were taken, added with 100 ⁇ L of antibody (10 ⁇ g/mL), and incubated at 37° C. with 5% CO 2 for 24 hours. After the cells in the well were washed twice with 4% calf blood, 100 ⁇ g/mL human IgG was added to incubate for 10 minutes, and then T cell activation detection antibodies (CD25-BV421, CD4-FITC, CD69-BV605 and CD8-APC) were added, and the cells were kept on ice. Incubate for 20 minutes. Wash and discard the supernatant, add 60 ⁇ L/well of PI, incubate on ice for 5 minutes, and use a flow cytometer for detection.
  • T cell activation detection antibodies CD25-BV421, CD4-FITC, CD69-BV605 and CD8-APC
  • the positive control antibody ERY974 was prepared with reference to US20170267783. The test results are shown in Figure 36. In the absence of target cells, GPC3 ⁇ CD3 ⁇ bispecific antibody has no activating effect on peripheral blood T cells, which is equivalent to the negative control KLH ⁇ CD3.
  • mice 6-8 weeks old female B-NGD mice (Biositu Biotechnology Co., Ltd.) were selected and subcutaneously inoculated with HepG2 cells (7 ⁇ 10 6 /mice). When the tumor grew to 60-100 mm 3 , they were randomly divided into groups and set separately.
  • the high-dose administration group was 3.0 mg/kg
  • the middle-dose administration group was 1.0 mg/kg
  • the low-dose administration group was 0.3 mg/kg
  • Each mouse was injected with 1 ⁇ 10 7 PBMC cells through the tail vein, and the first mouse administration was started 3 days later, and the administration interval was once every 5 days, and the administration was administered twice in total.
  • mice The tumor volume and body weight of the mice were monitored. After the experiment, the mice were sacrificed by cervical dislocation, and the tumors were weighed and recorded. The results are shown in Figure 37.
  • the in vivo efficacy of GPC3 ⁇ CD3 ⁇ bispecific antibody is dose-dependent, and the tumor inhibition rates (low dose to high dose) are: 76.7%, 81.3% and 95.9%, respectively.
  • the tumor-bearing mice tolerated the above doses well without adverse reactions such as weight loss.
  • mice Biositu Biotechnology Co., Ltd.
  • Hepa1-6/human GPC3 (6 ⁇ 10 6 / mouse) subcutaneously in the mice, until the tumor volume reaches 60- 100mm 3 , random grouping. They were set as 10 mg/mL in the high-dose group, 3 mg/mL in the middle-dose group, 1 mg/mL in the low-dose group, ERY974 in the positive control group and KLH ⁇ CD3 10 mg/kg in the negative control group.
  • the dosing interval was once every 3 days, and a total of 3 doses were administered.
  • the tumor volume and body weight of the mice were monitored.
  • the GPC3 ⁇ CD3 ⁇ bispecific antibody can significantly mediate immune cells to kill tumor cells and reduce the tumor-bearing volume; its 10 mg/kg dose is equivalent to ERY974.

Abstract

提供了双特异性抗体或其抗原结合片段、其编码核酸、包含所述核酸的细胞、包含所述双特异性抗体或其抗原结合片段、核酸和/或细胞的组合物，以及所述双特异性抗体或其抗原片段在治疗癌症的相关应用。

Description

一种双特异性抗体及其用途 技术领域
本公开涉及一种双特异性抗体及其用途，特别是结合CD3及另一种抗原的双特异性抗体及其用途。
背景技术
T细胞双特异抗体(或称T细胞衔接器)，是一种特殊的抗体分子，可通过其一端识别靶细胞表面抗原(抗原臂)，通过另一端结合T细胞CD3受体(CD3臂)，以一种类似于TCR/肽/HLA的方式，使T细胞上的CD3发生聚集，从而活化T细胞并杀伤肿瘤。上世纪80年代，即有报道利用双特异抗体杀伤肿瘤细胞(Staerz UD.,Nature.1985 Apr 18-24；314(6012):628-31；Perez P.et al.Nature.1985Jul 25-31；316(6026):354-6)。经过30多年的研究，基本解决了抗体的错配问题，有3个双特异抗体药物陆续获得批准上市。尽管在批准的适应症中显示了非常好的疗效，但随之带来的副作用和使用上的局限性，阻碍了早期这些双特异抗体的广泛应用。例如：早期上市的catumaxomab由于Fc段与肝脏Kupffer细胞表达的Fcγ受体结合，引发了快速的细胞因子释放，目前已退市；而2014年上市的blinatumomab，由于采用Fv抗体片段，生物半衰期仅为2小时，需低剂量持续静脉输注，同时FDA批准伴有细胞因子释放综合症和神经毒性的黑框警告。
正常免疫应答过程中，TCR以低亲和力(约1-100μM)结合感染或突变细胞上的外源肽-人白细胞抗原复合物(HLA)，通过CD3信号传导复合物(包括CD3εγ、CD3εδ和CD3ζζ)，将活化信号传导到核内，激活转录因子及其下游蛋白(细胞因子、颗粒酶、穿孔素等)的表达，其中TCR复合物产生的信号强度将决定T细胞的命运。早期开发的CD3双特异抗体，多基于OKT3、L2K、UCHT1和TR66等少数鼠源抗体，亲和力较高，导致T细胞过度活化，释放大量细胞因子，产生细胞因子风暴综合征；同时，高亲和力也会导致双特异抗体在二级淋巴器官的富集，减低在肿瘤组织的暴露。
抗体Fc部分与Fcγ受体的结合能力是另一个对影响药物安全的重要因素，由于Fcγ受体在多种正常组织表达，双特异抗体通过Fc与细胞膜上的Fcγ受体结合后，可导致另一端结合的CD3受体因Fcγ受体聚集而交联活化，从而产生严重的脱靶毒性。通过使用Fcγ受体结合能力较弱的人IgG2亚型或IgG4亚型，或者进一步在CH2相应位点进行氨基酸替换，如：Armour等将IgG1和IgG4的第233-236位点(EU序列编号)替换成IgG2相应序列，减少了和Fcγ受体的结合(Armour KL,et al,Recombinant human IgG molecules lacking Fcgamma receptor I binding and monocyte triggering activities,Eur J Immunol.1999Aug；29(8):2613-24)，Newman等在IgG4引入突变Ser228Pro和Leu235Glu，稳定IgG4结构同时减少与Fcγ受体的结合(Newman R,et al,Modification of the Fc region of a primatized IgG antibody to human CD4retains its ability to modulate CD4receptors but does not deplete CD4(+)T cells in chimpanzees.Clin Immunol.2001Feb；98(2):164-74)，Idusogie等发现将Asp270、Lys322、Pro329或Pro331分别替换成Ala，可减少IgG1与补体C1q的结合(Idusogie EE,et al,Mapping of the C1q binding site on rituxan,a chimeric antibody with a human IgG1Fc.J Immunol.2000Apr 15；164(8):4178-84)等。
链间错配是天然IgG样双特异抗体开发的主要工艺难点。Merchant AM等发明的共同轻链(Merchant AM,et al,An efficient route to human bispecific IgG.Nat Biotechnol.1998.PMID:9661204)，或Fischer N等开发的共同重链(Fischer N,et al,Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG.Nat Commun.2015Feb 12；6:6113)，通常都需要进行复杂的蛋白质工程改造，或者利用转基因动物产生(McWhirter J,et al,Common light chain mouse.WO2011097603.2011)；Carter P等(Atwell S,et al,Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library,J Mol Biol.1997Jul 4；270(1):26-35)通过在抗体Fc段引入knobs-into-holes互补突变，以解决重链间的错配；Schaefer G等(Schaefer W,et al,Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies,Proc Natl Acad Sci U S A.2011)开发了将轻、重链的Fab部分或全长进行交换的CrossMab技术，解决轻链错配问题，其中部分交换的CrossMab VH-VL和CrossMab CH1-CL需要引入额外肽段以实现正确配对，而Fab全长交换的CrossMab Fab正确配对率不足50％。
发明内容
在表达双特异抗体的过程中，本发明人意外发现，将具有λ轻链的人源化CD3抗体和具有κ轻链的靶向抗体进行组合，CD3抗体λ轻链更倾向于和同源的CD3重链进行配对，而靶向抗体kappa轻链倾向于和同源的靶向抗体重链配对。通过进一步在轻、重链间设计互补电荷对，可以提高正确配对效率。并且，实验证明，利用CD20、BCMA和GPC3等多个靶点抗体和人源化CD3抗体构建的新型T细胞连接器，经三步纯化均实现98-100％的单体纯度，错配比例极低(<1％)。
本公开提供了一种新型T细胞连接器，采用不同类型轻链κλ双特异抗体设计，和全长IgG构型，其中：结合靶细胞和T细胞CD3的抗体臂分别采用kappa轻链和lambda轻链，与其同源的重链进行配对，同时引入互补电荷对增强正确配对率；通过亲和力优化，新型T细胞连接器低浓度下即可募集活化T细胞，对靶细胞产生有效杀伤，无靶细胞存在时不激活T细胞；同时，新型T细胞κλ双特异抗体不与FcγR受体结合，减低了产生细胞因子风暴的风险。采用本公开方法构建的新型CD20×CD3κλ双特异抗体、BCMA×CD3κλ双特异抗体和GPC3xCD3κλ双特异 抗体纯化收率高，通过三步法纯化可实现>99％纯度。动物对新型CD20-CD3κλ双特异抗体耐受良好，新型T细胞连接器的疗效和安全性均优于同类抗体。
在一方面，本公开提供了一种双特异性抗体或其抗原结合部分。
在另一方面，本公开提供了编码如前述方面的双特异性抗体或其抗原结合部分的核酸。
在另一方面，本公开提供了包含前述方面的核酸的载体。
在另一方面，本公开提供了包含前述方面的载体的细胞。
根据前述任一方面的抗体或其抗原结合部分，其中上述抗体或其抗原结合部分是人源化的。
在另一方面，本公开提供了包含如前述任一方面的抗体或其抗原结合部分或其编码核酸和药学上可接受的载体的药物组合物或试剂盒。
在另一方面，本公开提供了包含共价附着至治疗部分的前述任一方面上述抗体或其抗原结合部分、双特异性或多特异性分子的抗体-药物偶联物。
在另一方面，本公开提供了治疗与相关病症的方法，其包括下述步骤：向上述哺乳动物施用治疗有效量的前述任一方面的抗体或其抗原结合片段、核酸、载体、细胞和/或药物组合物。
在另一方面，本公开提供了前述任一方面的抗体或其抗原结合片段、核酸、载体、细胞和/或药物组合物在制备用于治疗哺乳动物中与肿瘤抗原相关病症的药物或试剂盒中的用途。
本公开的抗体可以用于多种应用，包括检测肿瘤抗原，诊断、治疗或预防与肿瘤抗原相关的疾病。
附图说明
图1示出了本公开的第一抗原×CD3κλ双特异抗体。
图2示出了CD3人源化抗体与人CD3εγ蛋白的结合。
图3示出了CD3人源化抗体与Jurkat细胞的结合。
图4示出了CD3人源化抗体与人CD3εγ和食蟹猴CD3εγ蛋白的结合。
图5示出了本公开的κλ001、κλ002、κλ003、κλ004、κλ005的结构。
图6示出了CD20×CD3κλ双特异抗体经Protein A纯化的结果。
图7示出了CD20×CD3κλ双特异抗体的SEC-HPLC检测结果。
图8示出了CD20×CD3κλ双特异抗体同源二聚体检测结果。
图9示出了CD20×CD3κλ双特异抗体与CD20稳转细胞的结合。
图10示出了CD20×CD3κλ双特异抗体与肿瘤细胞SU-DHL-4、Raji和NALM-6的结合。
图11示出了CD20×CD3κλ双特异抗体与Jurkat细胞的结合。
图12示出了CD20×CD3κλ双特异抗体与外周血T细胞的结合。
图13示出了CD20×CD3κλ双特异抗体介导的TDCC作用，图13A显示对Nalm-6细胞的杀伤，图13B显示对T细胞的活化。
图14示出了CD20×CD3κλ双特异抗体介导的TDCC作用，图14A显示对TMD-8细胞的杀伤，图14B显示对T细胞的活化。
图15示出了CD20×CD3κλ双特异抗体介导的TDCC作用，图15A显示对Toledo细胞的杀伤，图15B显示对T细胞的活化。
图16示出了CD20×CD3κλ双特异抗体对T细胞NFAT信号通路的作用。
图17示出了CD20×CD3κλ双特异抗体在免疫重建小鼠Raji移植瘤模型中的抑制作用。
图18示出了CD20×CD3κλ双特异抗体在免疫缺陷型小鼠皮下Raji与人PBMC混合接瘤模型中的抑制作用。
图19示出了CD20×CD3κλ双特异抗体在食蟹猴中的药效.
图20示出了BCMA×CD3κλ双特异抗体与BCMA稳转细胞的结合。
图21示出了BCMA×CD3κλ双特异抗体与肿瘤细胞NCI-H929和RPMI-8226的结合。
图22示出了BCMA×CD3κλ双特异抗体与Jurkat细胞的结合。
图23示出了BCMA×CD3κλ双特异抗体与外周血T细胞的结合。
图24示出了BCMA×CD3κλ双特异抗体介导的TDCC作用，图24A显示对NCI-H929细胞的杀伤，图24B显示对T细胞的活化。
图25示出了BCMA×CD3κλ双特异抗体介导的TDCC作用，图25A显示对RPMI-8226细胞的杀伤，图25B显示对T细胞的活化。
图26示出了BCMA×CD3κλ双特异抗体对T细胞NFAT信号通路的作用。
图27示出了BCMA×CD3κλ双特异抗体对PBMC的非特异激活。
图28示出了BCMA×CD3κλ双特异抗体与Fc受体的结合。
图29示出了BCMA×CD3κλ双特异抗体在免疫缺陷型小鼠皮下NCI-H929移植瘤模型中的抑制作用。
图30示出了GPC3×CD3κλ双特异抗体与GPC3稳转细胞的结合。
图31示出了GPC3×CD3κλ双特异抗体与肿瘤细胞HepG2的结合。
图32示出了GPC3×CD3κλ双特异抗体与Jurkat细胞的结合。
图33示出了GPC3×CD3κλ双特异抗体与外周血T细胞的结合。
图34示出了GPC3×CD3κλ双特异抗体介导的TDCC作用，图34A显示对HepG2细胞的杀伤，图34B显示对T细胞的活化。
图35示出了GPC3×CD3κλ双特异抗体对T细胞NFAT信号通路的作用。
图36示出了GPC3×CD3κλ双特异抗体对PBMC的非特异激活。
图37示出了GPC3×CD3κλ双特异抗体在免疫重建小鼠皮下HepG2移植瘤模型中的抑制作用。
图38示出了GPC3×CD3κλ双特异抗体在CD3人源化鼠Hepa1-6/人GPC3移植瘤模型中的抑制作用。
具体实施方式
在本公开中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本公开，下面提供相关术语的定义和解释。
如本文中使用的，″肿瘤抗原″优选地是指存在于(或结合于)肿瘤细胞上而一般不存在于正常细胞上的任何抗原或抗原决定簇，或以比正常(非肿瘤)细胞上更大的量存在于或结合于肿瘤细胞上的抗原或抗原决定簇，或以不同于在正常(非肿瘤)细胞上发现的形式存在于肿瘤细胞上的抗原或抗原决定簇。该术语因此包括肿瘤特异性抗原，包括肿瘤特异性抗原(TSA)或肿瘤相关抗原(TAA)，包括肿瘤相关膜抗原、肿瘤上的胚胎抗原、生长因子受体、生长因子配体和与癌症有关的任何其它类型的抗原。肿瘤抗原可以是，例如，B细胞分化抗原(例如，CD19、CD20和CD37)、B细胞成熟抗原(B-cell maturation antigen，BCMA)、磷脂酰肌醇蛋白聚糖3(GPC3)、上皮癌抗原(例如，乳癌、胃肠癌、肺癌)、前列腺特异性癌抗原(PSA)或前列腺特异性膜抗原(PSMA)、膀胱癌抗原、肺(例如，小细胞肺)癌抗原、结肠癌抗原、卵巢癌抗原、脑癌抗原、胃癌抗原、肾细胞癌抗原、胰腺癌抗原、肝癌抗原、食道癌抗原、头颈癌抗原或结肠直肠癌抗原。
TSA对于肿瘤细胞是(或者被认为是)独特的，并且不发生在体内的其他细胞上(例如，在其他细胞上不在显著的程度上发生)。TAA对于肿瘤细胞不是独特的，并且相反也在正常细胞上表达(例如，在不能诱导对抗原的免疫耐受状态的条件下表达)。例如，当免疫系统不成熟并且不能应答时，TAA可以是在胎儿发育过程中在正常细胞上表达的抗原，或者它们可以是正常地在正常细胞上在极低水平下存在但是在肿瘤细胞上在更高水平下表达的抗原。
TSA或TAA抗原的非限制性实例包括分化抗原，诸如MART-1/MelanA(MART-I)、gp100(Pmel 17)、酪氨酸酶、TRP-1、TRP-2和肿瘤特异性多谱系抗原诸如MAGE-1、MAGE-3、BAGE、GAGE-1、GAGE-2、p15；过表达的胚胎抗原，诸如CEA；过表达的致癌基因和突变的肿瘤抑制基因，诸如p53、Ras、HER-2/neu；由染色体易位导致的独特肿瘤抗原，诸如BCR-ABL、E2A-PRL、H4-RET、IGH-IGK、MYL-RAR；以及病毒抗原，诸如爱泼斯坦巴尔病毒抗原EBVA以及人类乳头瘤病毒(HPV)抗原E6和E7。其他肿瘤抗原包括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-连环蛋白、CDK4、Mum-1、p 15、p 16、43-9F、5T4、791Tgp72、甲胎蛋白、β-HCG、BCA225、BTAA、CA 125、CA 15-3\CA 27.29\BCAA、CA 195、CA242、CA-50、CAM43、CD68\P1、CO-029、FGF-5、G250、Ga733\EpCAM、HTgp-175、M344、MA-50、MG7-Ag、MOV18、NB/70K、NY-CO-1、RCAS1、SDCCAG16、TA-90\Mac-2结合蛋白\亲环蛋白C-相关蛋白、TAAL6、TAG72、TLP、MUC16、IL13Rα2、FRα、VEGFR2、Lewis Y、FAP、EphA2、CEACAM5、EGFR、CA6、CA9、GPNMB、EGP1、FOLR1、内皮受体、STEAP1、SLC44A4、结合素-4、AGS-16、胍基环化酶C、MUC-1、CFC1B、整联蛋白α3链(a3b1的链，即层粘连蛋白受体链)和TPS。其他肿瘤抗原还包括CD19、CD20、CD22、CD30、CD72、CD180、CD171(L1CAM)、CD123、CD133、CD138、CD37、CD70、CD79a、CD79b、CD56、CD74、CD166、CD71、CLL-1/CLECK12A、ROR1、BCMA、磷脂酰肌醇蛋白聚糖3(GPC3)、间皮素、CD33/IL3Ra、c-Met、PSCA、PSMA、糖脂F77、EGFRvIII、GD-2、MY-ESO-1或MAGEA3。
如本文中使用的，术语“CD20”指来自任何脊椎动物来源，包括哺乳动物，诸如灵长动物(例如人)和啮齿动物(例如小鼠和大鼠)的任何天然CD20。
术语“抗CD20抗体”和“结合CD20的抗体”指能够以足够亲和力结合CD20，使得该抗体作为诊断剂和/或治疗剂在靶向CD20中是有用的抗体。在一个实施方案中，抗CD20抗体对无关非CD20蛋白的结合程度小于该抗体对CD20的结合的约10％，如通过例如放射免疫测定法(RIA)测量的。在某些实施方案中，结合CD20的抗体具有≤1μM，≤100nM，≤10nM，≤1nM，≤0.1nM，≤0.01nM，或≤0.001nM(例如10 -8M或更低，例如10 -8M至10 -13M，例如10 -9M至10 -13M)的解离常数(K d)。在某些实施方案中，抗CD20抗体结合在来自不同物种的CD20间保守的CD20表位。
如本文中使用的，术语“BCMA”可以指共同指代存在于动物中并且优选地存在于人体中的BCMA本身及其任何变体、同种型和旁系同源物的概念。
术语“人BCMA”是指人来源的BCMA，并且可以优选具有但不限于Genbank登录号AB052772.1的氨基酸序列。
术语“抗BCMA抗体”和“结合BCMA的抗体”指能够以足够亲和力结合BCMA，使得该抗体作为诊断剂和/或治疗剂在靶向BCMA中是有用的抗体。在一个实施方案中，抗BCMA抗体对无关非BCMA蛋白的结合程度小于该抗 体对BCMA的结合的约10％，如通过例如放射免疫测定法(RIA)测量的。在某些实施方案中，结合BCMA的抗体具有≤1μM，≤100nM，≤10nM，≤1nM，≤0.1nM，≤0.01nM，或≤0.001nM(例如10 -8M或更低，例如10 -8M至10 -13M，例如10 -9M至10 -13M)的解离常数(K d)。在某些实施方案中，抗BCMA抗体结合在来自不同物种的BCMA间保守的BCMA表位。
如本文中使用的，术语“GPC3”可以指共同指代存在于动物中并且优选地存在于人体中的GPC3本身及其任何变体、同种型和旁系同源物的概念。
术语“人GPC3”是指人来源的GPC3。
术语“抗GPC3抗体”和“结合GPC3的抗体”指能够以足够亲和力结合GPC3，使得该抗体作为诊断剂和/或治疗剂在靶向GPC3中是有用的抗体。在一个实施方案中，抗GPC3抗体对无关非GPC3蛋白的结合程度小于该抗体对GPC3的结合的约10％，如通过例如放射免疫测定法(RIA)测量的。在某些实施方案中，结合GPC3的抗体具有≤1μM，≤100nM，≤10nM，≤1nM，≤0.1nM，≤0.01nM，或≤0.001nM(例如10 -8M或更低，例如10 -8M至10 -13M，例如10 -9M至10 -13M)的解离常数(K d)。在某些实施方案中，抗GPC3抗体结合在来自不同物种的GPC3间保守的GPC3表位。
“CD3”指来自任何脊椎动物来源，包括哺乳动物，诸如灵长动物(例如人)，非人灵长动物(例如食蟹猴)和啮齿动物(例如小鼠和大鼠)的任何天然CD3，除非另外指明。该术语涵盖“全长”未加工的CD3以及源自细胞中加工的任何形式的CD3。该术语还涵盖CD3的天然发生变体，例如剪接变体或等位变体。在一个实施方案中，CD3是人CD3，特别是人CD3的厄普西隆亚基(CD3ε)。人CD3ε的氨基酸序列显示于UniProt(www.uniprot.org)登录号P07766(版本144)，或NCBI(www.ncbi.nlm.nih.gov/)RefSeq NP_000724.1。食蟹猴[Macaca fascicularis]CD3ε的氨基酸序列显示于NCBI GenBank no.BAB71849.1。
“细胞表面”的使用是根据其在本领域的正常含义，因此包括可通过与蛋白和其它分子结合而接近的细胞外部。
如本文使用的和除非另作说明，术语“约”或“大约”是指在给定值或范围的加或减10％之内。在需要整数的情况下，该术语是指在给定值或范围的加或减10％之内、向上或向下舍入到最接近的整数。
就抗体链多肽序列而言，短语“基本相同”可理解为表现出与参照多肽序列至少60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更多的序列同一性的抗体链。就核酸序列而言，该术语可理解为表现出与参照核酸序列至少大于60％、65％、70％、75％、80％、85％、90％、95％、96％、97％、98％、99％或更高的序列同一性的核苷酸序列。
序列“相同性”或“同一性”具有本领域公认的含义，并且可以利用公开的技术计算两个核酸或多肽分子或区域之间序列相同性的百分比。可以沿着多核苷酸或多肽的全长或者沿着该分子的区域测量序列相同性。虽然存在许多测量两个多核苷酸或多肽之间的相同性的方法，但是术语“相同性”是技术人员公知的(Carrillo,H.&Lipman,D.,SIAM J Applied Math 48:1073(1988))。
“取代型”变体是天然序列中至少一个氨基酸残基被除去并被不同的氨基酸插入其相同位置的变体。所述取代可为单个的，其中该分子中仅有一个氨基酸被取代；或可为多个的，其中该相同分子有两个或更多的氨基酸被取代。多个取代可位于连续的位点。同样，一个氨基酸可被多个残基取代，其中这样的变体包括取代和插入二者。“插入型”变体是一个或多个氨基酸被插入到紧邻一段天然序列某个特定位置处的氨基酸的变体。紧邻氨基酸意指与该氨基酸的α-羧基或α-氨基官能团连接。“缺失型”变体是天然氨基酸序列中一个或多个氨基酸被除去的变体。通常情况下，缺失型变体在其分子的特定区域内有一个或两个氨基酸被缺失。
就抗体的可变结构域而言，术语“可变”系指抗体之间有广泛序列差异的相关分子的某些部分，且被用于针对其特异靶的特定抗体的特异识别和结合。但是，可变性在抗体的整个可变结构域内不是均匀分布的。可变性集中在被称为互补决定区域(CDRs；即CDR1、CDR2和CDR3)或超变区的三个区段，它们均位于轻链和重链的可变结构域内。可变结构域内保守程度更高的部分被称为构架(FR)区或构架序列。天然重链和轻链的每个可变结构域均包括四个FR区，其主要采用β-折叠构型，它们籍三个CDRs连接起来，CDRs形成环，所述环连接β-折叠结构并在某些情形下形成部分的β-折叠结构。每条链的CDRs通常被FR区在邻近连接起来，并且借助于来自其它链的CDR，有助于抗体靶结合位点(表位或决定簇)的形成。正如本文所使用，免疫球蛋白氨基酸残基的编号是依据Kabat等人的免疫球蛋白氨基酸残基编号系统而进行的，除非另有说明。一个CDR可具有特异结合关联表位的能力。
如本文所用，抗体的“抗体片段”或“抗原结合片段”指全长抗体的任何部分，其少于全长，但是至少包含结合抗原的所述抗体的部分可变区(例如一个或多个CDR和/或一个或多个抗体结合位点)，并且因此保留结合特异性以及所述全长抗体的至少部分特异性结合能力。因此，抗原结合片段指包含与衍生抗体片段的抗体结合相同抗原的抗原结合部分的抗体片段。抗体片段包括通过酶促处理全长抗体所产生的抗体衍生物，以及合成产生的衍生物，例如重组产生的衍生物。抗体包括抗体片段。抗体片段的实例包括但不限于Fab、Fab'、F(ab') 2、单链Fv(scFv)、Fv、dsFv、双抗体、Fd和Fd'片段以及其他片段，包括修饰的片段(参见，例如，Methods in Molecular Biology，Vol 207:Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003)；Chapter 1；p 3-25,Kipriyanov)。所述片段可以包括连接在一起的多条链，例如通过二硫键和/或通过肽接头。抗体片段一般包含至少或约50个氨基酸，并且典型至少或约200个氨基酸。抗原结合片段包括任何抗体片段，其在被插入抗体框架(例如通过置换相应区域)时获得免疫特异性地结合(即表现出至少或至少约10 7-10 8M-1的Ka)抗原的抗体。“功能片段”或“抗体的类似物”是可防止或实质降低所述受体结合配体或启动信号转导的能力的片段或类似物。正如本文所使用，功能片段一般与“抗体片段″含义相同，且就抗体而论，可指能防止或实质降低所述受体结合配体或启动信号转导的能力的片段，例如Fv、Fab、F(ab') 2等等。“Fv”片段由一条重链的可变结构域和一条轻链的可变结构域籍非共价结合方式而形成的二 聚体(V H-V L二聚体)组成。在该构型中，每个可变结构域的三个CDRs相互作用，以确定V H-V L二聚体表面上的靶结合位点，与完整抗体的情况一样。所述六个CDRs共同赋予完整抗体的靶结合特异性。但是，即使是单个可变结构域(或仅包括3个靶特异的CDRs的Fv的一半)，仍可具有识别和结合靶的能力。
如本文中所用，术语“双特异性”(Bispecific antibody，BsAb)指抗体和/或抗原结合分子能够特异性结合两种不同的抗原性决定簇，通常，双特异性抗体和/或抗原结合分子包含两种抗原结合位点，其中每种特异于不同的抗原性决定簇。在某些实施方案中，所述双特异性抗体和/或抗原结合分子能够同时结合两种抗原决定簇，特别是在两种不同的细胞上表达的两种抗原性决定簇。
如本文所用，“单克隆抗体”指相同抗体的群体，表示单克隆抗体群体中的每个单独的抗体分子与其他抗体分子相同。这种特性与抗体的多克隆群体的特性相反，所述抗体的多克隆群体包含具有多种不同序列的抗体。单克隆抗体可以通过许多公知的方法来制备(Smith et al.(2004)J.Clin.Pathol.57,912-917；和Nelson et al.,J Clin Pathol(2000),53,111-117)。例如，单克隆抗体可以通过永生化B细胞来制备，例如通过与骨髓瘤细胞融合以产生杂交瘤细胞系或者通过用诸如EBV的病毒感染B细胞。重组技术还可以用来在体外通过用携带编码抗体的核苷酸的人工序列的质粒转化宿主细胞来从宿主细胞的克隆群体制备抗体。
如本文中所用，术语“杂交瘤”或“杂交瘤细胞”指由融合产抗体的淋巴细胞和不产抗体的癌细胞而产生的细胞或细胞系(通常为骨髓瘤或淋巴瘤细胞)。如本领域普通技术人员所知的，杂交瘤可增殖并持续供应产生特定单克隆抗体。用于产生杂交瘤的方法为本领域已知的(见例如，Harlow&Lane，1988)。当提及术语“杂交瘤”或“杂交瘤细胞”时，其还包括杂交瘤的亚克隆和后代细胞。
如本文所用，全长抗体是具有两条全长重链(例如VH-CH1-CH2-CH3或VH-CH1-CH2-CH3-CH4)和两条全长轻链(VL-CL)和铰链区的抗体，例如通过抗体分泌B细胞天然产生的抗体以及合成产生的具有相同结构域的抗体。
术语“嵌合抗体”是指这样的抗体，其中可变区序列源自一个物种，恒定区序列源自另一物种，如其中可变区序列源自小鼠抗体及恒定区序列源自人抗体的抗体。
“人源化”抗体是指非人(例如小鼠)抗体形式，其是嵌合的免疫球蛋白、免疫球蛋白链或者其片段(如Fv、Fab、Fab'、F(ab') 2或者抗体的其它抗原结合亚序列)，含有源自非人免疫球蛋白的最小序列。优选地，人源化抗体是人免疫球蛋白(接受者抗体)，其中接受者抗体的互补决定区(CDR)的残基由来自具有希望的特异性、亲和性和能力的非人物种(供体抗体)如小鼠、大鼠或者兔的CDR残基置换。
此外，在人源化中，还可能对VH和/或VL的CDR1、CDR2和/或CDR3区内的氨基酸残基进行突变，由此改善抗体的一或多种结合特性(例如亲和性)。可进行例如PCR介导的突变引入突变，其对抗体结合或其它功能特性的影响可利用本文所述的体外或体内测试评估。通常，引入保守性突变。此类突变可为氨基酸取代、添加或缺失。另外，CDR内的突变通常不超过一个或两个。因此，本公开所述人源化抗体还涵盖CDR内包含1或2两个氨基酸突变的抗体。
如本文所用，术语“CDR”指互补决定区(complementarity-determining region)，已知抗体分子的每个重链和轻链具有3个CDR。CDR也称作高变区，且存在于抗体的每个重链和轻链的可变区中，在CDR的一级结构中具有非常高的变异性位点。本说明书中，重链的CDR由来自重链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示，轻链的CDR由来自轻链的氨基端序列的氨基端的CDR1、CDR2、CDR3表示。这些位点在三级结构中彼此临近，并决定抗体所结合的抗原的特异性。
如本文所用，术语“表位”指抗体的互补位结合的抗原上的任何抗原决定簇。表位决定簇通常包含分子的化学活性表面分型，例如氨基酸或糖侧链，并且通常具有特定的三维结构特征以及特定的电荷特征。
如本文所用，关于抗体或其抗原结合片段的“特异性结合”或“免疫特异性地结合”在本文中可交换使用，并且指抗体或抗原结合片段通过抗体和抗原的抗体结合位点之间的非共价相互作用与同种抗原形成一个或多个非共价键的能力。所述抗原可以是分离的抗原或存在于肿瘤细胞。通常，免疫特异性地结合(或特异性结合)抗原的抗体是以约或1×10 7M -1或1×10 8M -1或更大的亲和常数Ka(或者1×10 -7M或1×10 -8M或更低的解离常数(Kd))结合所述抗原。亲和常数可以通过抗体反应的标准动力学方法来测定，例如，免疫测定、表面等离子共振(SPR)(Rich and Myszka(2000)Curr.Opin.Biotechnol 11:54；Englebienne(1998)Analyst.123:1599)、等温滴定量热法(ITC)或本领域已知的其他动力学相互作用测定；还参见描述用于计算抗体的结合亲和力的示例性SPR和ITC方法的美国专利第7，229，619号)。用于实时检测和监测结合速率的仪器和方法是已知的，并且可商购(参见，Malmqvist(2000)Biochem.Soc.Trans.27:335)。
如本文所用，术语“多核苷酸”和“核酸分子”指包含至少两个连接的核苷酸或核苷酸衍生物的寡聚体或聚合物，包括通常通过磷酸二酯键连接在一起的脱氧核糖核酸(DNA)和核糖核酸(RNA)。如本文所使用，术语“核酸分子”意欲包括DNA分子及RNA分子。核酸分子可为单链或双链，且可为cDNA。
如本文所用，分离的核酸分子是从存在于核酸分子的天然来源中的其他核酸分子分离的核酸分子。诸如cDNA分子的“分离的”核酸分子可以在通过重组技术制备时基本上不含其他细胞物质或培养基，或者在化学合成时基本上不含化学前体或其他化学成分。本文所提供的示例性分离的核酸分子包括编码所提供的抗体或抗原结合片段的分离的核酸分子。
如本文所用，关于核酸序列、区域、元件或结构域的“可操作地连接”表示核酸区域互相功能相关。例如，启动子可以可操作地连接至编码多肽的核酸，从而所述启动子调控或介导所述核酸的转录。
亦提供本文所述序列表中所述序列的“保守序列修饰”，即不消除由核苷酸序列编码或含有氨基酸序列的抗体与 抗原的结合的核苷酸及氨基酸序列修饰。这些保守序列修饰包括保守核苷酸及氨基酸取代以及核苷酸及氨基酸添加及缺失。例如，可通过本领域已知的标准技术(例如定点诱变及PCR介导的诱变)将修饰引入本文所述的序列表中。保守序列修饰包括保守氨基酸取代，其中氨基酸残基被替换为具有类似侧链的氨基酸残基。具有类似侧链的氨基酸残基的家族是本领域中已有定义的。这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电极性侧链的氨基酸(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、具有非极性侧链的氨基酸(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、具有β分枝侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)及具有芳香族侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，抗CD20、BCMA或GPC3抗体中的预测的非必需氨基酸残基优选被来自同一侧链家族的另一氨基酸残基替代。鉴定不消除抗原结合的核苷酸及氨基酸保守取代的方法为本领域所熟知(例如，参见Brummell et al.,Biochem.32:1180-1187(1993)；Kobayashi et al.,Protein Eng.12(10):879-884(1999)；Burks et al.,Proc.Natl.Acad.Sci.USA 94:412-417(1997))。
作为另一选择，在另一实施方案中，可通过例如饱和诱变沿抗GCD20、BCMA或PC3抗体编码序列的全部或一部分随机引入突变，且可针对改良的结合活性筛选所得经修饰抗CD20、BCMA或GPC3抗体。
如本文所用，“表达”指通过多核苷酸的转录和翻译产生多肽的过程。多肽的表达水平可以利用本领域已知的任何方法来评价，包括例如测定从宿主细胞产生的多肽的量的方法。这类方法可以包括但不限于通过ELISA定量细胞裂解物中的多肽，凝胶电泳之后考马斯蓝染色，Lowry蛋白测定以及Bradford蛋白测定。
如本文所用，“宿主细胞”是用于接受、保持、复制和扩增载体的细胞。宿主细胞还可以用来表达载体所编码的多肽。当宿主细胞分裂时，载体中所含的核酸复制，从而扩增核酸。宿主细胞可以是真核细胞或原核细胞。合适的宿主细胞包括但不限于CHO细胞、各种COS细胞、HeLa细胞、HEK细胞例如HEK 293细胞。
如本文所用，“载体”是可复制的核酸，当载体转化入适当的宿主细胞时，可以从该载体表达一种或多种异源蛋白。关于载体包括那些通常通过限制酶切消化和连接可以将编码多肽或其片段的核酸引入其中的载体。关于载体还包括那些包含编码多肽的核酸的载体。载体用来将编码多肽的核酸引入宿主细胞，用于扩增核酸或者用于表达/展示核酸所编码的多肽。载体通常保持游离，但是可以设计为使基因或其部分整合入基因组的染色体。还考虑人工染色体的载体，例如酵母人工载体和哺乳动物人工染色体。这类媒介物的选择和用途是本领域技术人员公知的。
如本文所用，载体还包括“病毒载体”或“病毒的载体”。病毒的载体是工程化的病毒，其可操作地连接至外源基因以将外源基因转移(作为媒介物或穿梭(shuttle))入细胞。
如本文所用，“表达载体”包括能够表达DNA的载体，所述DNA与诸如启动子区的能够影响这类DNA片段表达的调控序列可操作地连接。这类额外的片段可以包括启动子和终止子序列，并且任选地可以包括一个或多个复制起点、一个或多个选择标记、增强子、多腺苷酸化信号等。表达载体一般来源于质粒或病毒DNA，或者可以包含这两者的元件。因此，表达载体指重组DNA或RNA构建体，例如质粒、噬菌体、重组病毒或其他载体，当引入适当的宿主细胞时，导致克隆DNA的表达。适当的表达载体是本领域技术人员公知的，并且包括在真核细胞和/或原核细胞中可复制的表达载体以及保持游离的表达载体或者整合入宿主细胞基因组的表达载体。
如本文所用，“治疗”患有疾病或疾病状况的个体表示所述个体的症状部分或全部缓解，或者在治疗后保持不变。因此，治疗包括预防、治疗和/或治愈。预防指防止潜在疾病和/或防止症状恶化或疾病发展。治疗还包括所提供的任何抗体或其抗原结合片段以及本文所提供的组合物的任何药学用途。
如本文所用，“疗效”表示由个体的治疗所导致的效果，其改变、通常改良或改善疾病或疾病状况的症状，或者治愈疾病或疾病状况。
如本文所用，“治疗有效量”或“治疗有效剂量”指施用于对象之后至少足以产生疗效的物质、化合物、材料或包含化合物的组合物的量。因此，其为防止、治愈、改善、阻滞或部分阻滞疾病或病症的症状所必需的量。
如本文所用，“预防有效量”或“预防有效剂量”指在施用于对象时会具有预期的预防效果的物质、化合物、材料或包含化合物的组合物的量，例如，防止或延迟疾病或症状的发生或复发，减少疾病或症状发生或复发的可能性。完全预防有效剂量不必通过施用一个剂量发生，并且可以仅在施用一系列剂量之后发生。因此，预防有效量可以在一次或多次施用中施用。
如本文中所使用的，术语“患者”是指哺乳动物，例如人。
II.具体实施方案详述
在一方面，本公开提供了一种双特异性抗体或其抗原结合片段，其包含：
(a)第一抗原结合部分或其抗原结合片段，第一抗原结合部分包含第一轻链和第一重链，第一轻链为κ型轻链，第一抗原结合部分包含与第一抗原结合的第一结合结构域；和
(b)第二抗原结合部分或其抗原结合片段，第二抗原结合部分包含第二轻链和第二重链，第二轻链为λ型轻链，第二抗原结合部分包含与第二抗原结合的第二结合结构域。
在一些实施方案中，第二抗原为CD3抗原。
在一些实施方案中，第二抗原结合部分的第二轻链可变区具有Gln 40Glu突变(Vλ CD3:Gln 40Glu)；第二抗原结合部分的第二重链可变区具有Gln 39Lys突变(VH CD3:Gln 39Lys)。
在一些实施方案中，第二结合结构域包含选自氨基酸序列SEQ ID NO:7-9、14、15、20、21或其任何变体的第二轻链CDR；和/或选自氨基酸序列SEQ ID NO:26-28、31、34、40、43、46、47或其任何变体的第二重链CDR。
在一些实施方案中，第二结合结构域包含选自氨基酸序列SEQ ID NO:7、14或其任何变体的第二轻链CDR1，选自氨基酸序列SEQ ID NO:8、15、20或其任何变体的第二轻链CDR2，选自氨基酸序列SEQ ID NO:9、21或其任何变体的第二轻链CDR3；和/或选自氨基酸序列SEQ ID NO:26、31、46或其任何变体的第二重链CDR1，选自氨基酸序列SEQ ID NO:27、47或其任何变体的第二重链CDR2，选自氨基酸序列SEQ ID NO:28、34、37、40、43或其任何变体的第二重链CDR3。
在一些实施方案中，第二结合结构域的第二轻链CDR选自：分别包含氨基酸序列SEQ ID NO:7、8、9的第二轻链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:14、15、9的第二轻链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:7、8、21的第二轻链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:7、20、21的第二轻链CDR1、CDR2及CDR3序列；和/或第二结合结构域的重链CDR选自：分别包含氨基酸序列SEQ ID NO:26、27、28的第二重链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:31、27、28的第二重链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:31、27、34的第二重链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:31、27、37的第二重链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:31、27、40的第二重链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:31、27、43的第二重链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:46、47、28的第二重链CDR1、CDR2及CDR3序列。
在一些实施方案中，第二结合结构域包含选自氨基酸序列SEQ ID NO:5、10、12、16、18、22或其任何变体的第二轻链可变区；和/或选自氨基酸序列SEQ ID NO:24、29、32、35、38、41、44、48、50、52或其任何变体的第二重链可变区。
在一些实施方案中，第二结合结构域包含氨基酸序列SEQ ID NO:18或其任何变体的第二轻链可变区；和氨基酸序列SEQ ID NO:24或其任何变体的第二重链可变区。
在一些实施方案中，第二结合结构域包含氨基酸序列SEQ ID NO:5或其任何变体的第二轻链可变区；和氨基酸序列SEQ ID NO:48或其任何变体的第二重链可变区。
在一些实施方案中，第二结合结构域包含氨基酸序列SEQ ID NO:18或其任何变体的第二轻链可变区；和氨基酸序列SEQ ID NO:48或其任何变体的第二重链可变区。
在一些实施方案中，第二结合结构域包含氨基酸序列SEQ ID NO:5或其任何变体的第二轻链可变区；和氨基酸序列SEQ ID NO:50或其任何变体的第二重链可变区。
在一些实施方案中，第二结合结构域包含氨基酸序列SEQ ID NO:10或其任何变体的第二轻链可变区；和氨基酸序列SEQ ID NO:50或其任何变体的第二重链可变区。
在一些实施方案中，第二结合结构域包含氨基酸序列SEQ ID NO:12或其任何变体的第二轻链可变区；和氨基酸序列SEQ ID NO:50或其任何变体的第二重链可变区。
在一些实施方案中，第二结合结构域包含氨基酸序列SEQ ID NO:18或其任何变体的第二轻链可变区；和氨基酸序列SEQ ID NO:50或其任何变体的第二重链可变区。
在一些实施方案中，第二抗原结合部分的第二轻链选自氨基酸序列SEQ ID NO:58和66；和/或第二抗原结合部分的第二重链选自氨基酸序列SEQ ID NO:60和68。在一些优选的实施方案中，第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:58；和第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:60。在一些优选的实施方案中，第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66；和第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68。
在一些实施方案中，第一抗原为肿瘤抗原。
在一些优选实施方案中，肿瘤抗原选自：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、间皮素、CD33/IL3Ra、c-Met、PSCA、PSMA、糖脂F77、EGFRvIII、GD-2、MY-ESO-1、Her2、Her3、MUC1、MUC17、Claudin18或MAGEA3。
在一个具体实施方案中，肿瘤相关抗原选自CD20、BCMA和GPC3。
在一些实施方案中，第一抗原为CD20抗原。
在一些优选实施方案中，第一抗原结合部分的第一轻链可变区具有Gln 38Lys突变(Vκ CD20:Gln 38Lys)。在一些优选的实施方案中，第一抗原结合部分的第一重链可变区具有Gln 39Glu突变(VH CD20:Gln 39Glu)。
在一些优选实施方案中，第一抗原结合部分的第一轻链可变区具有Gln 38Lys突变(Vκ CD20:Gln 38Lys)，且第一轻链恒定区具有Glu 123Lys和Gln 124Lys突变(Vκ-Ck CD20:Gln 38Lys\Glu 123Lys\Gln 124Lys)。在一些优选的实施方案中，第一抗原结合部分的第一重链可变区具有Gln 39Glu突变(VH CD20:Gln 39Glu)，且第一重链恒定区具有Lys 152Glu和Lys 218Glu突变(V H-C H1 CD20:Gln 39Glu\Lys 152Glu\Lys 218Glu)。
在一些优选实施方案中，第一抗原结合部分的第一轻链选自氨基酸序列SEQ ID NO:54、62和70；和/或第一抗原结合部分的第一重链选自氨基酸序列SEQ ID NO:56、64和72。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:54，和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:56。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:62，和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:64。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:70，和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:72。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:54，和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:56，第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:58，和第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:60。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:62，和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:64，第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68，第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:58，和第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:60。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:70，和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:72，第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:62，和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:64，第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:58，和第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:60。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:54，和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:56，第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68。
在一些实施方案中，第一抗原为BCMA抗原。
在一些优选实施方案中，第一抗原结合部分的第一轻链可变区具有Gln 42Lys突变(Vκ BCMA:Gln 42Lys)。在一些优选的实施方案中，第一抗原结合部分的第一重链可变区具有Gln 39Glu突变(VH BCMA:Gln 39Glu)。
在一些优选实施方案中，第一抗原结合部分的第一轻链选自氨基酸序列SEQ ID NO:80和84；和/或第一抗原结合部分的第一重链选自氨基酸序列SEQ ID NO:82和86。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:80；和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:82。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:84；和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:82。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:80；和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:86。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:84；和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:86。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:80，和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:82，第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:84，和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:82，第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:80，和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:86，第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:84，和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:86，第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68。
在一些实施方案中，第一抗原为GPC3抗原。
在一些优选实施方案中，第一抗原结合部分的第一轻链可变区具有Gln 43Lys和Gln 39Glu突变(Vκ GPC3:Gln 43Lys；VH GPC3:Gln 39Glu)。
在一些优选实施方案中，第一抗原结合部分的第一轻链选自氨基酸序列SEQ ID NO:88和92；和/或第一抗原结合部分的第一重链选自氨基酸序列SEQ ID NO:90和94。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:88，和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:90。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:92，和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:94。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:88，和第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:90，第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68。
在一些优选实施方案中，第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:92，和第一抗原结合部分的 第一重链为氨基酸序列SEQ ID NO:94，第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68。
在一些实施方案中，双特异抗体的第一抗原结合部分和/或第二抗原结合部分的Fc部分采用knob-into-hole结构。在一些优选的实施方案中，采用人IgG4knob-into-hole结构。
在一些实施方案中，双特异抗体的第一抗原结合部分和/或第二抗原结合部分还具有Ser 228Pro、Leu 235Glu和/或Pro 329Ala突变。
在一方面，本公开提供了编码前述双特异性抗体或其抗原结合部分的核酸。
在一些优选实施方案中，第二抗原结合部分结合CD3抗原，第二抗原结合部分的第二轻链可变区的编码核酸选自核苷酸序列SEQ ID NO:6、11、13、17、19和23；和/或第二抗原结合部分的第二重链可变区的编码核酸选自核苷酸序列SEQ ID NO:25、30、33、36、39、42、45、49、51和53。在一些优选实施方案中，第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:59和67；和/或第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:61和69。在一些优选实施方案中，第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:59，和第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:61。在一些优选实施方案中，第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69。
在一些优选实施方案中，第一抗原结合部分结合CD20抗原，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:55、63和71；和/或第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:57、65和73。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:55，和第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:57。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:63，和第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:65。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:71，和第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:73。
在一些优选实施方案中，第一抗原结合部分结合BCMA抗原，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:81和85；和/或第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:83和87。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:81，和第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:83。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:85，和第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:83。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:81，和第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:87。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:85，和第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:87。
在一些优选实施方案中，第一抗原结合部分结合GPC3抗原，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:89和93；和/或第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:91和95。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:89，和第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:91。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:93，和第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:95。
在一些优选实施方案中，双特异性抗体的第一抗原结合部分结合CD20抗原，第二抗原结合CD3抗原，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:55，第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:57，第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:59，和第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:61。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:63，和第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:65，第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69，第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:59，和第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:61。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:71，和第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:73，第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:63，和第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:65，第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:59，和第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:61。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:55，第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:57，第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69。
在一些优选实施方案中，双特异性抗体的第一抗原结合部分结合BCMA抗原，第二抗原结合CD3抗原，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:81，第一抗原结合部分的第一重链的编码核酸选 自核苷酸序列SEQ ID NO:83，第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:85，第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:83，第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:81，第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:87，第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:85，第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:87，第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69。
在一些优选实施方案中，双特异性抗体的第一抗原结合部分结合GPC3抗原，第二抗原结合CD3抗原，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:89，第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:91，第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69。在一些优选实施方案中，第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:93，第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:95，第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69。
在一方面，本公开提供了包含前述核酸的载体。
在一方面，本公开提供了包含前述核酸或载体的细胞。
在一方面，本公开提供了包含前述双特异性抗体或其抗原结合部分、核酸、载体和/或细胞的组合物。
在一方面，本公开提供了包含共价附着至治疗部分的前述双特异性抗体或其抗原结合部分的抗体-药物偶联物。
在一些实施方案中，治疗部分选自细胞毒性部分、化疗剂、细胞因子、免疫抑制剂、免疫刺激剂、裂解肽或放射性同位素。
本公开的抗体在各种肿瘤抗原被不利地表达或发现的疾病中可用作治疗或诊断工具。
在一个与肿瘤抗原相关的疾病实施方案中，肿瘤抗原在患病组织或器官的细胞中的表达与在健康组织或器官中的状态相比有所增加。增加是指增加至少10％、特别是至少20％、至少50％、至少100％、至少200％、至少500％、至少1000％、至少10000％或甚至更多。在一个实施方案中，表达仅在患病组织中发现，而在相应健康组织中的表达受阻抑。根据本公开，与肿瘤抗原相关的疾病包括肿瘤。
在一些实施方案中，与肿瘤抗原相关的疾病为与CD20相关疾病。在一些优选的实施方案中，与CD20相关的疾病包括B细胞疾病，例如B细胞增殖性病症，特别是CD20阳性B细胞病症；优选地，疾病选自非霍奇金淋巴瘤(NHL)，急性淋巴细胞性白血病(ALL)，慢性淋巴细胞性白血病(CLL)，弥漫性大B细胞淋巴瘤(DLBCL)，滤泡性淋巴瘤(FL)，套细胞淋巴瘤(MCL)，边缘区淋巴瘤(MZL)，以及多发性骨髓瘤(MM)和霍奇金淋巴瘤(HL)。
在一些实施方案中，与肿瘤抗原相关的疾病为与BCMA相关疾病；优选地，与BCMA相关的疾病包括B细胞疾病；优选地，疾病是癌症；更优选地，癌症是B-细胞相关癌症，其选自多发性骨髓瘤、恶性浆细胞瘤、霍奇金淋巴瘤、结节性淋巴细胞为主的霍奇金淋巴瘤、Kahler’s病和骨髓性白血病、浆细胞白血病、浆细胞瘤、B-细胞幼淋巴细胞白血病、毛细胞白血病、B-细胞非霍奇金淋巴瘤(NHL)、急性髓性白血病(AML)、慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL)、慢性髓性白血病(CML)、滤泡性淋巴瘤、伯基特淋巴瘤、边缘区淋巴瘤、套细胞淋巴瘤、大细胞淋巴瘤、前体B-淋巴细胞淋巴瘤、髓性白血病、瓦尔登斯特伦巨球蛋白血症、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、边缘区淋巴瘤、粘膜相关淋巴组织淋巴瘤、小细胞淋巴细胞性淋巴瘤、套细胞淋巴瘤、伯基特淋巴瘤、原发性纵隔(胸腺)大B细胞淋巴瘤、淋巴浆细胞淋巴瘤、瓦尔登斯特伦巨球蛋白血症、淋巴结边缘区B细胞淋巴瘤、脾边缘区淋巴瘤、血管内大B-细胞淋巴瘤、原发性渗出性淋巴瘤、淋巴瘤样肉芽肿病、富含T细胞/组织细胞的大B-细胞淋巴瘤、原发性中枢神经系统淋巴瘤、原发性皮肤弥漫性大B-细胞淋巴瘤(腿型)、老年人EBV阳性弥漫性大B-细胞淋巴瘤、炎症相关的弥漫性大B-细胞淋巴瘤、血管内大B-细胞淋巴瘤、ALK阳性大B-细胞淋巴瘤、浆母细胞淋巴瘤、HHV8相关的多中心Castleman病中产生的大B-细胞淋巴瘤、未分类的具有弥漫性大B-细胞淋巴瘤和伯基特淋巴瘤中间特征的B-细胞淋巴瘤、未分类的具有弥漫性大B-细胞淋巴瘤和经典霍奇金淋巴瘤中间特征的B-细胞淋巴瘤以及其他B-细胞相关淋巴瘤；更优选地，B细胞疾病是B细胞障碍；优选地，浆细胞障碍选自：多发性骨髓瘤、浆细胞瘤、浆细胞白血病、巨球蛋白血症、淀粉样变性、华氏巨球蛋白血症、孤立性骨浆细胞瘤、髓外浆细胞瘤、骨硬化性骨髓瘤、重链病、意义不明确的单克隆丙种球蛋白病以及郁积型多发性骨髓瘤；优选地，疾病是自身免疫性病症，如系统性红斑性狼疮或类风湿性关节炎。
在一些实施方案中，该治疗剂包含特异性结合活化性T细胞抗原的抗体。
在一个实施方案中，该治疗剂包含特异性结合CD3，特别是CD3ε的抗体。
用本公开的双特异性抗体治疗的疾病和症状方法包括下述步骤：向哺乳动物施用治疗有效量的前述任一方面的抗体或其抗原结合片段或核酸分子或载体或细胞或药物组合物。
在一些实施方案中，本公开提供治疗或预防癌症疾病的方法，其包括向患者施用能够结合GPC3的抗体，其中施用抗体以提供至少40μg/ml的血清水平。在不同实施方案中，施用抗体以提供至少50μg/ml、至少150μg/ml、至 少300μg/ml、至少400μg/ml或至少500μg/ml的血清水平。在不同实施方案中，施用抗体以提供不超过800μg/ml、700μg/ml、600μg/ml、550μg/ml或500μg/ml的血清水平。在一个实施方案中，所提供的血清水平为40μg/ml至700μg/ml，优选为40μg/ml至600μg/ml，优选为50μg/ml至500μg/ml，如150μg/ml至500μg/ml或者300μg/ml至500μg/ml。如本说明书中使用的术语“血清水平”，其意指所讨论的物质在血清中的浓度。在一个实施方案中，提供血清水平至少7天或至少14天。在一个实施方案中，方法包括施用至少300mg/m 2的抗体剂量，如至少600mg/m 2，且优选至多1500mg/m 2，至多1200mg/m 2或至多1000mg/m 2。
在一些实施方案中，本公开提供治疗或预防癌症疾病的方法，其包括向患者施用能够结合GPC3的抗体，其中以至少300mg/m 2，如至少600mg/m 2，且优选至多1500mg/m 2，至多1200mg/m 2或至多1000mg/m 2的剂量施用抗体。
在一些实施方案中，本公开提供治疗或预防癌症疾病的方法，其包括向患者施用能够结合GPC3的抗体，其中患者的至少50％，优选为60％、70％、80％或90％的癌细胞为GPC3阳性和/或患者的至少40％，优选为50％或60％的癌细胞为GPC3的表面表达阳性。在该方面，本公开还提供治疗或预防癌症疾病的方法，方法包括：a.鉴定显示至少50％，优选为60％、70％、80％或90％的GPC3阳性癌细胞和/或至少40％，优选为50％或60％的癌细胞的患者，癌细胞为GPC3的表面表达阳性；以及b.向患者施用能够结合GPC3的抗体。在一个实施方案中，患者的至少95％或至少98％的癌细胞为GPC3阳性的。在一个实施方案中，患者的至少70％、至少80％或至少90％的癌细胞为GPC3的表面表达阳性。
在本文的任何方面的方法的一个实施方案中，癌症疾病的治疗结果是实现病情稳定。在一个实施方案中，病情稳定实现至少2个月、至少3个月或至少6个月。
在一些实施方案中，本公开提供实现癌症患者的病情稳定的方法，其包括向患者施用能够结合GPC3的抗体。在一个实施方案中，病情稳定实现至少2个月、至少3个月或至少6个月。
在本文的任何方面的方法的一个实施方案中，以单剂量或多剂量施用抗体。
在一些实施方案中，本公开提供治疗或预防癌症疾病的方法，其包括向患者施用能够结合GPC3的抗体，其中以多剂量施用抗体。
如果根据本公开以多次剂量施用抗体，则优选以至少3次剂量、至少4次剂量、至少5次剂量、至少6次剂量、至少7次剂量、至少8次剂量、至少9次剂量或至少10次剂量且优选至多30次剂量、25次剂量、20次剂量、15次剂量或10次剂量施用抗体。优选以至少7天、至少10天、至少14天或至少20天的时间间隔施用抗体的剂量。优选以7至30天、10至20天且优选为约14天的时间间隔施用抗体的剂量。
在一个实施方案中，施用抗体以便提供至少40μg/ml的血清水平。在不同实施方案中，施用抗体以便提供至少50μg/ml、至少150μg/ml、至少300μg/ml、至少400μg/ml或至少500μg/ml的血清水平。在不同实施方案中，施用抗体以便提供不超过800μg/ml、700μg/ml、600μg/ml、550μg/ml或500μg/ml的血清水平。在一个实施方案中，所提供的血清水平为40μg/ml至700μg/ml，优选为40μg/ml至600μg/ml，优选为50μg/ml至500μg/ml，如150μg/ml至500μg/ml或300μg/ml至500μg/ml。在一个实施方案中，提供血清水平至少7天或至少14天。在一个实施方案中，方法包括施用至少300mg/m 2，如至少600mg/m 2且优选至多1500mg/m 2、至多1200mg/m 2或至多1000mg/m 2的抗体的剂量。
前述任一方面的抗体或其抗原结合片段或核酸分子或载体或细胞或药物组合物在制备用于治疗哺乳动物中GPC3相关病症的药物中的用途。
根据前述任一方面，任选地，抗体偶联其他药物，例如带标记或具有细胞毒性的偶联物。
在一方面，本公开还包括试剂盒，例如试剂盒包括本公开的抗体、其片段、同源物、其衍生物、核酸、载体、细胞、组合物等，例如带标记或具有细胞毒性的缀合物，以及抗体使用说明书、杀死特定类型细胞的偶联物等等。该说明书可包括在体外、体内或离体使用抗体、偶联物等的指导。抗体可以是液体形式或固体，通常是冻干的。该试剂盒可包含其它适宜的试剂，如缓冲液、重构溶液以及为了预定用途的其它必要成分。考虑了以预定量包装好的试剂组合与用于其用途的说明书，用途例如用于治疗用途或用于进行诊断测定。当抗体是带标记的时，例如用酶标记的，那么该试剂盒可包括底物和酶所需的辅因子(例如提供可检测生色团或荧光团的底物前体)。此外，其它添加剂，如稳定剂、缓冲液(例如封闭缓冲液或裂解缓冲液)等也可包括在内。多种试剂的相对量可以改变而提供试剂溶液的浓缩物，这就提供了用户灵活性、节省空间、节省试剂等。这些试剂也可以干粉形式提供，通常是冻干形式，包括赋形剂，它在溶解时可提供具有适当浓度的试剂溶液。
前述任一方面的抗体或其功能片段或核酸分子或载体或细胞或药物组合物或试剂盒在制备用于抑制GPC3结合的试剂中的用途。
此外，本公开的抗体还可用于免疫测定、纯化方法以及其它用到免疫球蛋白或其片段的方法。此类用途在本领域为人所熟知。
相应地，本公开还提供包含本公开的抗GPC3的抗体或其片段的组合物，抗体方便地和药学上可接受的载体、稀释剂或赋形剂组合，这是本领域的常规做法。
本公开所使用的术语“药物组合物”系指多种制备物的制剂。含有治疗有效量的多价抗体的制剂为无菌液体溶液、液体悬浮剂或冻干形式，任选地包含稳定剂或赋形剂。
本公开的抗体可以作为单独施用的组合物使用，或可与其它活性剂联合使用。
在一些实施方案中，本公开的人源化抗体与治疗部分(即药物)缀合。治疗部分可以是例如细胞毒素、化疗剂、细胞因子、免疫抑制剂、免疫刺激剂、裂解肽或放射性同位素。此类偶联物在本文中称为“抗体-药物偶联物”或“ADC”。
在一些实施方案中，抗体与细胞毒性部分偶联。细胞毒性部分可以例如选自以下：紫杉醇；细胞松弛素B；短杆菌肽D；溴化乙锭；吐根碱；丝裂霉素；依托泊苷；替尼泊苷；长春新碱；长春碱；秋水仙碱；多柔比星；柔红霉素；二羟基蒽二酮；微管蛋白抑制剂如美登素或其类似物或衍生物；抗有丝分裂剂如单甲基奥瑞他汀E或F或其类似物或衍生物；海兔毒素10或15或其类似物；伊立替康或其类似物；米托蒽醌；光辉霉素；放线菌素D；1-脱氢睾酮；糖皮质激素；普鲁卡因；丁卡因；利多卡因；普萘洛尔；嘌呤霉素；卡奇霉素或其类似物或衍生物；抗代谢药，如甲氨喋呤、6巯基嘌呤、6硫鸟嘌呤、阿糖胞苷、氟达拉滨、5氟尿嘧啶、癸二嗪、羟基脲、天冬酰胺酶、吉西他滨或克拉屈滨；烷化剂，如二氯甲基二乙胺、硫代嘌呤、苯丁酸氮芥、美法仑、卡莫司汀(BSNU)、洛莫司汀(CCNU)、环磷酰胺、白消安、二溴甘露醇、链脲佐菌素、达卡巴嗪(DTIC)、丙卡巴嗪、丝裂霉素C；铂类衍生物，如顺铂或卡铂；多卡霉素A、多卡霉素SA、雷切霉素(CC-1065)或其类似物或衍生物；抗生素，如放线菌素、博来霉素、柔红霉素、多柔比星、伊达比星、光霉素、丝裂霉素、米托蒽醌、普力霉素、安定霉素(AMC)；吡咯并[2,1-c][1,4]-苯并二氮杂卓(PDB)；白喉毒素及相关分子如白喉A链及其活性片段和杂合分子、蓖麻毒素如蓖麻毒素A或去糖基化蓖麻毒素A链毒素、霍乱毒素、志贺样毒素如SLT I、SLT II、SLT IIV、LT毒素、C3毒素、志贺毒素、百日咳毒素、破伤风毒素、大豆Bowman-Birk蛋白酶抑制剂、假单胞菌外毒素、阿罗林、皂草素、蒴莲根毒素、胶凝蛋白、相思豆毒素A链、蒴莲根毒素A链、α-sarcin、油桐(Aleurites fordii)蛋白、石竹素蛋白、美洲商陆蛋白如PAPI、PAPII和PAP-S、苦瓜(momordica charantia)抑制剂、泻果素、巴豆毒素、肥阜草(sapaonaria officinalis)抑制剂、白树毒素、丝裂霉素、局限曲菌素、酚霉素和依诺霉素毒素；核糖核酸酶(RNase)；DNase I、葡萄球菌内毒素A；商陆抗病毒蛋白；白喉毒素和假单胞菌内毒素。
在一些实施方案中，抗体与奥瑞他汀或其肽类似物、衍生物或前药偶联。已经表明，奥瑞他汀干扰微管动力学、GTP水解以及核和细胞分裂并具有抗癌和抗真菌活性。例如，奥瑞他汀E可以与对乙酰基苯甲酸或苯甲酰基戊酸反应，分别产生AEB和AEVB。其他典型的奥瑞他汀衍生物包括AFP、MMAF(单甲基奥瑞他汀F)和MMAE(单甲基奥瑞他汀E)。合适的奥瑞他汀和奥瑞他汀的类似物、衍生物和前药，以及用于将奥瑞他汀与Ab偶联的合适接头描述于例如美国专利号5,635,483、5,780,588和6,214,345以及国际专利申请公开WO02088172、WO2004010957、WO2005081711、WO2005084390、WO2006132670、WO03026577、WO200700860、WO207011968和WO205082023。
在一些实施方案中，抗体与吡咯并[2,1-c][1,4]-苯并二氮杂卓(PDB)或其肽类似物、衍生物或前药偶联。合适的PDB以及PDB衍生物和相关技术描述于例如Hartley J.A.等,Cancer Res 2010；70(17):6849-6858；Antonow D.等,Cancer J 2008；14(3):154-169；Howard P.W.等,Bioorg Med Chem Lett 2009；19:6463-6466和Sagnou等,Bioorg MedChem Lett2000；10(18):2083-2086。
在一些实施方案中，抗体与选自以下的细胞毒性部分偶联：蒽环类抗生素、美登素、卡奇霉素、多卡霉素、雷切霉素(CC-1065)、海兔毒素10、海兔毒素15、伊立替康、单甲基奥瑞他汀E、单甲基奥瑞他汀F、PDB，或它们任何的类似物、衍生物或前药。
在一些实施方案中，抗体与蒽环类抗生素或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与美登素或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与卡奇霉素或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与多卡霉素或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与雷切霉素(CC-1065)或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与海兔霉素10或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与海兔霉素15或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与单甲基奥瑞他汀E或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与单甲基奥瑞他汀F或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与吡咯并[2,1-c][1,4]-苯并二氮杂卓或其类似物、衍生物或前药偶联。在一些实施方案中，抗体与伊立替康或其类似物、衍生物或前药偶联。
在一些实施方案中，抗体与细胞因子(例如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配体、干细胞因子、安西司亭和TNFa)偶联。
在一些实施方案中，抗体与放射性同位素或含放射性同位素的螯合物偶联。例如，抗体可以与允许抗体与放射性同位素络合的螯合剂接头(例如DOTA、DTPA或噻西坦)偶联。抗体还可以或可选地包含一个或多个放射性标记的氨基酸或其他放射性标记的分子或与之偶联。放射性同位素的非限制性实例包括 3H、 14C、 15N、 35S、 90Y、 99Tc、 125I、 131I、 186Re、 213Bi、 225Ac和 227Th。为了治疗目的，可以使用发射β或α颗粒辐射的放射性同位素，例如 131I、 90Y、 211At、 212Bi、 67Cu、 186Re、 188Re和 212Pb。
将分子与抗体偶联的技术是本领域熟知的。通常，核酸分子分别通过N-羟基琥珀酰亚胺酯或马来酰亚胺官能团与抗体上的赖氨酸或半胱氨酸共价连接。据报道，使用工程化半胱氨酸或整合非天然氨基酸的偶联方法可改进偶联物的同质性。特别地，本领域技术人员还可以预期用含酰基供体谷氨酰胺的标签(例如含有Gin肽的标签或Q-标签)或通过多肽工程(例如通过多肽上的氨基酸缺失、插入、取代或突变)产生反应性的内源性谷氨酰胺工程化的含Fc多肽。然后，转谷氨酰胺酶可与胺供体剂(例如，包含或连接于反应性胺的小分子)共价交联，以形成稳定且均质的工程化含Fc多肽偶联物群，其中胺供体剂通过含酰基供体谷氨酰胺标签或可接近/暴露/反应性的内源性谷氨酰胺位点特异性地与含Fc多肽偶联(WO2012059882)。
应当理解，根据上述实施方案的治疗剂将与合适的药学上可接受的载体、赋形剂、以及其它被掺入制剂中以提供改善的转移、递送、耐受性等的试剂一同施用。这些制剂包括例如粉末、糊剂、膏剂、凝胶剂、蜡、油、脂质、含脂质(阳离子或阴离子)载体(例如Lipofectin TM)、DNA缀合物、无水吸浆、水包油和油包水乳液、乳液聚乙二 醇(各种分子量的聚乙二醇)、半固态凝胶以及含有聚乙二醇的半固态混合物。任何前述混合物均可适用于根据本公开的治疗或疗法，条件是制剂中的活性成分不被制剂灭活并且制剂在生理学上是相容的并耐受给药途径。
在一个实施方案中，可将上述抗体用作治疗剂。此类试剂将通常用于治疗、缓解和/或预防受试者的与异常肿瘤抗原表达、活性和/或信号传导相关的疾病或病理。可使用标准方法通过鉴定受试者，例如患有(或处于风险或发展)与异常肿瘤抗原表达、活性和/或信号传导相关的疾病或障碍，例如肿瘤抗原相关病症的人患者来实施治疗方案。将抗体制剂，优选对其靶抗原有高特异性和高亲和性的抗体制剂施用给受试者并且将通常因其与靶标结合而产生效应。施用的抗体可消除或抑制或妨碍靶标(例如肿瘤抗原)的表达、活性和/或信号传导功能。施用的抗体可消除或抑制或妨碍靶标(例如肿瘤抗原)与其所天然结合的内源性的配体结合。例如，抗体与靶标结合并调节、阻断、抑制、减少、拮抗、中和/或以其它方式妨碍肿瘤抗原表达、活性和/或信号传导。在一些实施方案中，为治疗与异常肿瘤抗原表达相关的疾病或障碍，可将具有重链和轻链CDR的抗体施用给受试者。
在另一个实施方案中，针对肿瘤抗原的抗体可用于本领域中已知的与肿瘤抗原定位和/或定量相关的方法(例如，用于测定适当生理样品中的肿瘤抗原和/或肿瘤抗原的水平，用于诊断方法，用于蛋白成像等等)。在一个给定实施方案中，对肿瘤抗原或其衍生物、片段、类似物或同系物具有特异性的、包含源于抗体的抗原结合结构域的抗体，被用作药物学活性化合物(下文称为“治疗剂”)。
在另一个实施方案中，可通过标准技术例如免疫亲和、色谱或免疫沉淀，使用对肿瘤抗原具有特异性的抗体来分离肿瘤抗原多肽。针对肿瘤抗原蛋白质的抗体(或其片段)可用于检测生物样品中的蛋白质。在一些实施方案中，在生物样品中可检测肿瘤抗原作为临床测试过程的一部分，例如，用于确定给定治疗方案的功效。将抗体偶联(即物理连接)到可检测物质可有利于检测。可检测物质的示例包括各种酶、辅基、荧光材料、发光材料、生物发光材料和放射性材料。合适的酶的示例包括辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶或乙酰胆碱酯酶；合适的辅基复合物的示例包括链霉亲和素/生物素和亲和素/生物素；合适的荧光材料的示例包括伞形酮、荧光素、异硫氰酸荧光素、罗丹明、二氯三嗪氨荧光素、丹磺酰氯或藻红蛋白；发光材料的一个示例包括鲁米诺；生物发光材料的示例包括荧光素酶、荧光素和水母蛋白，并且合适放射性材料的示例包括 125I、 131I、 35S或 3H。
在另一个实施方案中，根据本公开的抗体可用作检测样品中肿瘤抗原或其蛋白质片段存在的试剂。在一些实施方案中，抗体包含可检测标记。抗体为多克隆抗体，或更优选单克隆抗体。使用完整的抗体或其片段(例如Fab、scFv或F(ab') 2)。关于抗体的术语“标记”旨在包括通过将可检测物质偶联(即物理连接)到该抗体来直接标记该抗体，以及通过与直接标记的另一种试剂反应来间接标记该抗体。间接标记的示例包括使用荧光标记的第二抗体检测第一抗体，以及用生物素进行末端标记抗体，以便能够用荧光标记的链霉亲和素进行检测。术语“生物样品”旨在包括从受试者分离的组织、细胞和生物学流体，以及受试者体内存在的组织、细胞和流体。因此，使用的术语“生物样品”包括血液和血液中的级分或组分，包括血清、血浆、或淋巴液。换言之，上述实施方案的检测方法可用于在体外及体内检测生物样品中的分析物mRNA、蛋白质或基因组DNA。例如，分析物mRNA体外检测技术包括Norhtern杂交和原位杂交。分析物蛋白质体外检测技术包括酶联免疫吸附测定(ELISA)、Western印迹、免疫沉淀、以及免疫荧光。分析物基因组DNA体外检测技术包括Southern杂交。用于进行免疫测定的过程描述于例如“ELISA:Theory and Practice:Methods in Molecular Biology”，第42卷，J.R.Crowther(编辑)Human Press，Totowa，N.J.，1995。此外，分析物蛋白质的体内检测技术包括向受试者体内导入标记的抗分析物蛋白抗体。例如，可以用放射性标记标记抗体，然后可以通过标准成像技术检测受试者体内该放射性标记物的存在和位置。
可将本文所述抗体和其衍生物、片段、类似物和同系物掺入适于施用的药物组合物中。制备此类组合物所涉及的原理和考虑事项以及选择组分的指南在本领域中是熟知的。
此类组合物通常包含抗体和药学上可接受的载体。当使用抗体片段时，与靶蛋白结合结构域特异性结合的最小抑制片段可为优选的。例如，基于抗体的可变区序列，可以设计保留结合靶蛋白质序列能力的肽分子。此类肽可化学合成和/或通过重组DNA技术产生(参见例如Marasco等人，Proc.Natl.Acad.Sci.USA，90:7889-7893(1993))。
如本文所用，术语“药学上可接受的载体”旨在包括与药物给药相容的任何和所有溶剂、分散介质、包衣、抗细菌剂和抗真菌剂、等渗剂和吸收延缓剂等。合适的药学上可接受的载体描述于最新版的Remington's Pharmaceutical Sciences中，这是本领域的标准参考书目，其以引用方式并入本文。此类载体或稀释剂的优选示例包括但不限于水、盐水、林格氏溶液、葡萄糖溶液和5％的人血清白蛋白。也可以使用脂质体和非水性载体，例如固定化油。将此类介质和试剂用于药物活性物质是本领域熟知的。除去任何常规的介质或试剂与抗体不相容之外，设想其在组合物中的用途。
将上述实施方案的药物组合物配制成与其预期施用途径相容。给药途径的示例包括肠胃外，例如静脉内、皮内、皮下、经口(例如吸入)、经皮(即局部的)、经粘膜和直肠给药。用于肠胃外、皮内或皮下施用的溶液或悬浮液可包括以下组分：注射用无菌稀释剂例如水、盐溶液、固定油、聚乙二醇类、甘油、丙二醇或其它合成溶剂；抗细菌剂，例如苄醇或对羟基苯甲酸甲酯；抗氧化剂，例如抗坏血酸或亚硫酸氢钠；螯合剂，例如乙二胺四乙酸(EDTA)；缓冲剂，例如乙酸盐、柠檬酸盐或磷酸盐、以及调节渗透压的试剂，例如氯化钠或右旋糖。pH可用酸或碱进行调节，例如盐酸或氢氧化钠。可将肠胃外制剂包装在安瓿、一次性注射器或玻璃或塑料制多剂量小瓶内。
适于注射用途的药物组合物包括无菌水性溶液(在此是水溶性的)或分散体以及用于即时制备无菌注射液或分散体的无菌粉末。对于静脉内施用，合适的药学上可接受的载体包括生理盐水、抑菌水、Cremophor EL TM(BASF，Parsippany，N.J.)或磷酸盐缓冲盐水(PBS)。在所有情况下，组合物必须是无菌的并且应当为流动性达到易于注射的程度。其在制造和储存条件下必须是稳定的并且必须能防止微生物例如细菌和真菌的污染作用。载体可以是含有 例如水、乙醇、多元醇(例如，甘油、丙二醇和液体聚乙二醇等)的溶剂或分散介质，及其适宜的混合物。例如通过利用涂层例如卵磷脂，在分散体情况下维持所需颗粒尺寸，以及利用表面活性剂，可以保持适宜的流动性。对微生物作用的防止可以通过各种抗细菌剂和抗真菌剂例如对羟基苯甲酸酯、氯代丁醇、苯酚、抗坏血酸、硫柳汞等来实现。在许多情况下，将优选在组合物中包含等渗剂，例如糖、多元醇(诸如甘露糖醇、山梨醇)、氯化钠。注射用组合物的延长吸收可通过在组合物中包含延缓吸收的试剂例如单硬脂酸铝和明胶来达到。
根据需要，可以通过将抗体以所需量掺入具有上文所列成分中的一种或组合(按需要)的合适溶剂中来制备无菌注射溶液，然后过滤消毒。一般来讲，通过将抗体掺入含有碱性分散介质和上文所列那些中的所需其它成分的无菌载体中来制备分散体。就用于制备无菌注射溶液的无菌粉末而言，制备方法是获得粉末的真空干燥和冷冻干燥，该粉末包含活性成分和任何另外的期望成分，它们来自前述的这些成分的无菌过滤溶液。
对于吸入给药，从包含合适推进剂如二氧化碳等气体的加压容器或分配器或者喷雾器以气溶胶喷雾形式递送化合物。
还可以通过经粘膜或透皮方式全身给药。对于经粘膜或透皮给药，在制剂中使用适于渗透屏障的渗透剂。此类渗透剂通常在本领域是通常所知的，并且包括如用于经粘膜给药的去污剂、胆盐和夫西地酸衍生物。经粘膜给药可以通过使用喷鼻剂或栓剂来实现。对于透皮给药，可将一种或多种抗体配制成如本领域通常所知的膏剂、软膏、凝胶、或霜膏。
还可将化合物以栓剂(例如，具有常规栓剂基质，如可可脂或其它甘油酯)或滞留性灌肠剂形式进行制备以用于经直肠递送。
在一个实施方案中，抗体可用防止其不被身体迅速消除的载体制备，例如缓释/控释制剂，包括植入体和微胶囊化递送体系。可使用可生物降解、可生物相容的聚合物，例如乙烯-乙酸乙烯酯、聚酐、聚乙醇酸、胶原、聚原酸酯和聚乳酸。用于制备此类制剂的方法对于本领域技术人员而言是显而易见的。
尤其有利的是以剂量单位形式配制肠胃外组合物以易于施用和剂量的一致性。如本文所用，剂量单位形式是指用于待治疗的受试者，适合作为单位剂量的物理上可分离的单位；每个单位含有经计算与所需药物载体结合产生期望治疗效果的预定量的一种或多种抗体。上述实施方案的剂量单位形式的规格由以下指示并直接取决于：抗体的独特特征和待实现的具体治疗效果，和用于治疗个体的此类抗体的调配领域中固有的局限性。
药物组合物可与给药说明书一起放于容器、包装、或分配器中。
本文所述制剂还可根据要治疗的具体情况而包含多于一种抗体，优选具有互补活性但对彼此无负面影响的那些。另选地或除此之外，组合物可例如包含增强其功能的试剂，诸如细胞毒素试剂、细胞因子、化学治疗剂、或生长抑制剂。此类分子以对预期目的有效的量适当地联合存在。例如，可以在试剂盒中联合存在，也可以在使用中联合存在。
在一个实施方案中，一种或多种抗体可在联合治疗中施用，即与其它试剂例如治疗剂(其可用于治疗病理学病症或障碍，例如各种形式的癌症、自身免疫性障碍和炎性疾病)联合。术语“联合”在本文中是指将试剂基本上同步地，同时地或顺次地给予。如果顺次给予，则在开始施用第二种化合物时，两种化合物中的第一种仍优选在治疗位点处以有效浓度被检测到。在一种情况下，“联合”也可以是在试剂盒中同时包含本公开的抗体和其他治疗剂。
例如，联合治疗可包含本文所述一种或多种抗体与一种或多种附加治疗剂(例如一种或多种细胞因子和生长因子抑制剂、免疫抑制剂、抗炎剂、代谢抑制剂、酶抑制剂、和/或细胞毒素或细胞生长抑制剂，如下更详述的)共同配制和/或共同施用。此类联合治疗可有利地利用较低剂量的施用的治疗剂，因而避免了与各种单一疗法相关的可能毒性或并发症。
在一个实施方案中，与相应的不施用抗肿瘤抗原抗体的治疗方案相比该治疗方案有效降低该受试者中与该T细胞活化性治疗剂的施用相关的细胞因子释放。
为了达到清楚和简洁描述的目的，本文中作为相同的或分开的一些实施方案的一部分来描述特征，然而，将要理解的是，本公开的范围可包括具有所描述的所有或一些特征的组合的一些实施方案。
图1示出了新型第一抗原×CD3κλ双特异抗体的结构。
本公开实验证明，在自由组合情况下，人源化CD3臂的λ轻链倾向于与同源的重链进行配对，与异源重链的配对比率较低；同样，人源化抗原臂的κ轻链也倾向于与同源的重链进行配对，与人源化CD3重链的配对比率极低；同时，在Fv引入互补电荷变异体，进一步减少了可能产生的轻链错配。CD20×CD3κλ双特异抗体的Fc部分采用人IgG4knob-into-hole结构，通过突变Ser 228Pro、Leu 235Glu和Pro 329Ala，保持铰链区稳定并减弱与Fcγ受体以及C1q的相互作用。
实施例
实施例1：CD3抗体的优化和对T细胞的激活作用
1.重组蛋白的合成
合成人CD3γ(UniProt P09693，Gln23-Asn116)和CD3ε(UniProt P07766，Gln23-Asp126)胞外区核苷酸序列，C端分别与人IgG Fc hole或Fc knob融合，表达形成人CD3εγ-Fc异源二聚体(人CD3γIgG Fc(hole)的氨基酸序列如SEQ ID NO.1所示，人CD3εIgG Fc(knob)的氨基酸序列如SEQ ID NO.2所示)；同样合成食蟹猴CD3γ(UniProt Q95LI7，Gln23-Asn110)和CD3ε(UniProt Q95LI5，Gln22-Asp117)，C端分别与食蟹猴IgG Fc hole或Fc knob融合，表达形成食蟹猴CD3εγ-Fc异源二聚体(食蟹猴CD3γIgG Fc(hole)的氨基酸序列如SEQ ID NO.3所示，食蟹 猴CD3γIgG Fc(knob)的氨基酸序列如SEQ ID NO.4)。将表达CD3γ-Fc和CD3ε-Fc的重组质粒与3mg/mL PEI(Polysciences，#24765-2)混合，共转染HEK293E细胞(培养基OPM-293CD03DPM)，37℃120rpm 5％CO 2培养7天后，收集培养基上清液，经Protein A亲和层析纯化，得到人或食蟹猴CD3εγ-Fc重组蛋白。
2.CD3抗体人源化
鼠源杂交瘤CD3抗体(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)识别人和食蟹猴CD3受体，其序列如下：
抗CD3鼠单抗轻链氨基酸序列(SEQ ID NO.96)：
Figure PCTCN2021132195-appb-000001
抗CD3鼠单抗重链氨基酸序列(SEQ ID NO.97)：
Figure PCTCN2021132195-appb-000002
将抗CD3鼠单抗人源化，选择同源性最高的人胚系基因IMGT_hVL7-43进行轻链CDR移植，FM4选用人IGLJ3*02；选择人IMGT_hVH3-73进行重链CDR移植，FM4选用人IGHJ4*01。设计共得到不同的重链变体和轻链变体(表1)。
表1、CD3人源化抗体的可变区序列
Figure PCTCN2021132195-appb-000003
hVL1的氨基酸序列如SEQ ID NO.5所示，其编码核酸如SEQ ID NO.6所示，其LCDR1、LCDR2和LCDR3分别如SEQ ID NO.7、8、9所示。
Figure PCTCN2021132195-appb-000004
核酸序列
Figure PCTCN2021132195-appb-000005
hVL2的氨基酸序列如SEQ ID NO.10所示，其编码核酸如SEQ ID NO.11所示，其LCDR1、LCDR2和LCDR3分别如SEQ ID NO.7、8、9所示。
Figure PCTCN2021132195-appb-000006
核酸序列
Figure PCTCN2021132195-appb-000007
Figure PCTCN2021132195-appb-000008
hVL3的氨基酸序列如SEQ ID NO.12所示，其编码核酸如SEQ ID NO.13所示，其LCDR1、LCDR2和LCDR3分别如SEQ ID NO.14、15、9所示。
Figure PCTCN2021132195-appb-000009
核酸序列
Figure PCTCN2021132195-appb-000010
hVL4的氨基酸序列如SEQ ID NO.16所示，其编码核酸如SEQ ID NO.17所示，其LCDR1、LCDR2和LCDR3分别如SEQ ID NO.14、15、9所示。
Figure PCTCN2021132195-appb-000011
核酸序列
Figure PCTCN2021132195-appb-000012
hVL5的氨基酸序列如SEQ ID NO.18所示，其编码核酸如SEQ ID NO.19所示，其LCDR1、LCDR2和LCDR3分别如SEQ ID NO.7、8、21所示。
Figure PCTCN2021132195-appb-000013
核酸序列
Figure PCTCN2021132195-appb-000014
hVL6的氨基酸序列如SEQ ID NO.22所示，其编码核酸如SEQ ID NO.23所示，其LCDR1、LCDR2和LCDR3分别如SEQ ID NO.7、20、21所示。
Figure PCTCN2021132195-appb-000015
核酸序列
Figure PCTCN2021132195-appb-000016
hVH1的氨基酸序列如SEQ ID NO.24所示，其编码核酸如SEQ ID NO.25所示，其HCDR1、HCDR2和HCDR3分别如SEQ ID NO.26、27、28所示。
Figure PCTCN2021132195-appb-000017
核酸序列
Figure PCTCN2021132195-appb-000018
hVH2的氨基酸序列如SEQ ID NO.29所示，其编码核酸如SEQ ID NO.30所示，其HCDR1、HCDR2和HCDR3分别如SEQ ID NO.31、27、28所示。
Figure PCTCN2021132195-appb-000019
核酸序列
Figure PCTCN2021132195-appb-000020
hVH3的氨基酸序列如SEQ ID NO.32所示，其编码核酸如SEQ ID NO.33所示，其HCDR1、HCDR2和HCDR3分别如SEQ ID NO.31、27、34所示。
Figure PCTCN2021132195-appb-000021
核酸序列
Figure PCTCN2021132195-appb-000022
hVH4的氨基酸序列如SEQ ID NO.35所示，其编码核酸如SEQ ID NO.36所示，其HCDR1、HCDR2和HCDR3分别如SEQ ID NO.31、27、37所示。
Figure PCTCN2021132195-appb-000023
核酸序列
Figure PCTCN2021132195-appb-000024
hVH5的氨基酸序列如SEQ ID NO.38所示，其编码核酸如SEQ ID NO.39所示，其HCDR1、HCDR2和HCDR3分别如SEQ ID NO.31、27、40所示。
Figure PCTCN2021132195-appb-000025
核酸序列
Figure PCTCN2021132195-appb-000026
hVH6的氨基酸序列如SEQ ID NO.41所示，其编码核酸如SEQ ID NO.42所示，其HCDR1、HCDR2和HCDR3分别如SEQ ID NO.31、27、43所示。
Figure PCTCN2021132195-appb-000027
核酸序列
Figure PCTCN2021132195-appb-000028
hVH7的氨基酸序列如SEQ ID NO.44所示，其编码核酸如SEQ ID NO.45所示，其HCDR1、HCDR2和HCDR3分别如SEQ ID NO.46、47、28所示。
Figure PCTCN2021132195-appb-000029
核酸序列
Figure PCTCN2021132195-appb-000030
hVH8的氨基酸序列如SEQ ID NO.48所示，其编码核酸如SEQ ID NO.49所示，其HCDR1、HCDR2和HCDR3分别如SEQ ID NO.26、27、28所示。
Figure PCTCN2021132195-appb-000031
核酸序列
Figure PCTCN2021132195-appb-000032
hVH9的氨基酸序列如SEQ ID NO.50所示，其编码核酸如SEQ ID NO.51所示，其HCDR1、HCDR2和HCDR3 分别如SEQ ID NO.26、27、28所示。
Figure PCTCN2021132195-appb-000033
核酸序列
Figure PCTCN2021132195-appb-000034
hVH10的氨基酸序列如SEQ ID NO.52所示，其编码核酸如SEQ ID NO.53所示，其HCDR1、HCDR2和HCDR3分别如SEQ ID NO.26、27、28所示。
Figure PCTCN2021132195-appb-000035
核酸序列
Figure PCTCN2021132195-appb-000036
将轻、重链人源化变体分别进行全序列合成后，克隆到含有抗体lambda轻链恒定区或人IgG4重链恒定区CH1-CH3的真核表达载体，共转染HEK293E细胞，37℃120rpm 5％CO 2培养5-6天后，收集培养基上清液，通过Protein A层析柱进行纯化。
3.CD3人源化抗体的亲和力
包被人CD3εγ重组蛋白，4℃过夜。2％脱脂牛奶封闭后，每孔加入不同稀释倍数的CD3抗体，孵育1小时；二抗加入HPR标记的山羊抗人IgG Fc，TMB溶液显色后，以浓硫酸终止反应并在450nm处读出吸光度。图2显示CD3人源化抗体(包括aCD3-hVH1/VL5、aCD3-hVH8/VL1、aCD3-hVH8/VL5、aCD3-hVH9/VL1、aCD3-hVH9/VL2、aCD3-hVH9/VL3、aCD3-hVH9/VL5)与人CD3εγ蛋白的结合，CD3人源化抗体高亲和力结合CD3εγ重组蛋白。
取对数生长期的Jurkat细胞，3％BSA封闭30分钟，以每孔5×10 4细胞加入96孔U型板，离心弃上清，每孔加入50μL梯度稀释的抗体(抗体浓度从30μg/mL起，3倍稀释5个梯度)，4℃孵育1小时。洗去一抗后，二抗加入1:300稀释的Alexa Fluro647标记的山羊抗人IgG Fc(Jackson ImmunoResearch，109-606-170)，4℃孵育45分钟，洗涤后每孔重悬于50μL PBS进行FACS(iQue，Intellicyt)检测。结果如图3所示，CD3人源化抗体与Jurkat细胞结合，其中CD3人源化抗体hVH9/VL5(aCD3-hVH9/VL5)和hVH9/VL2(aCD3-hVH9/VL2)均显著弱于对照抗体OKT3，以中度亲和力结合Jurkat细胞。
表2显示CD3人源化抗体与CD3重组蛋白和Jurkat细胞的亲和力。
表2、CD3人源化抗体的亲和力
  ELISA(CD3εγ) FACS(Jurkat)
aCD3-hVH1/VL5 0.60nM 10nM
aCD3-hVH8/VL1 0.65nM 15nM
aCD3-hVH8/VL5 0.74nM 较弱
aCD3-hVH9/VL1 0.49nM 7nM
aCD3-hVH9/VL2 0.44nM 25nM
aCD3-hVH9/VL3 0.70nM 弱
aCD3-hVH9/VL5 0.42nM 20nM
OKT3-hIgG1 ND 0.27nM
KLH-hIgG4 - -
ND：未检测。
-：不结合
4.CD3人源化抗体与人、食蟹猴CD3抗原的交叉识别
分别包被人CD3εγ蛋白和食蟹猴CD3εγ蛋白，4℃过夜。2％脱脂牛奶封闭后，每孔加入不同稀释倍数的CD3抗体，孵育1小时；二抗加入HPR标记的山羊抗人IgG Fc，TMB溶液显色后，以浓硫酸终止反应并在450nm处读出吸光度。图4显示CD3人源化抗体hVH9/VL5(aCD3-hVH9/VL5)和hVH9/VL2(aCD3-hVH9/VL2)均可同时结合人CD3εγ和食蟹猴CD3εγ蛋白。
实施例2构建不同类型轻链形成的CD20×CD3κλ双特异抗体
1.CD20×CD3κλ双特异抗体的构建
以CD3人源化抗体hVH9/VL5(λ轻链和配对重链)和人源化CD20抗体(κ轻链和配对重链)，构建具有天然IgG构型的新型T细胞κλ双特异抗体。
如图5所示，设计并构建了下述5种CD20×CD3κλ双特异抗体：
1)CD20×CD3κλ001：保留CD3臂和CD20抗原臂的天然序列；
2)CD20×CD3κλ002：同时在CD20抗原臂和CD3臂引入电荷变异体(Vκ CD20:Gln 38Lys；VH CD20:Gln 39Glu；Vλ CD3:Gln 40Glu；VH CD3:Gln 39Lys)；
3)CD20×CD3κλ003：基于CD20×CD3κλ002，在CH1/Cκ之间增加互补电荷对(Vκ-Ck CD20:Gln 38Lys\Glu 123Lys\Gln 124Lys；V H-C H1 CD20:Gln 39Glu\Lys 152Glu\Lys 218Glu；Vλ CD3:Gln 40Glu；VH CD3:Gln 39Lys)；
4)CD20×CD3κλ004：仅在CD20抗原臂引入电荷变异体(Vκ CD20:Gln 38Lys；VH CD20:Gln 39Glu)；
5)CD20×CD3κλ005：仅在CD3臂引入电荷变异体(Vλ CD3:Gln 40Glu；VH CD3:Gln 39Lys)。
相应序列见表3，对照抗体CD20×CD3-crossFab参考CrossFab方法构建(Schaefer W等，PNAS 2011)。
表3、CD20×CD3κλ双特异抗体的序列
  CD20臂轻链 CD20臂重链 CD3臂轻链 CD3臂重链
CD20×CD3κλ001 SEQ ID NO.54 SEQ ID NO.56 SEQ ID NO.58 SEQ ID NO.60
CD20×CD3κλ002 SEQ ID NO.62 SEQ ID NO.64 SEQ ID NO.66 SEQ ID NO.68
CD20×CD3κλ003 SEQ ID NO.70 SEQ ID NO.72 SEQ ID NO.66 SEQ ID NO.68
CD20×CD3κλ004 SEQ ID NO.62 SEQ ID NO.64 SEQ ID NO.58 SEQ ID NO.60
CD20×CD3κλ005 SEQ ID NO.54 SEQ ID NO.56 SEQ ID NO.66 SEQ ID NO.68
CD20×CD3-crossFab SEQ ID NO.70 SEQ ID NO.74 SEQ ID NO.76 SEQ ID NO.78
CD20×CD3κλ001：
CD20臂κ轻链SEQ ID NO.54
Figure PCTCN2021132195-appb-000037
核苷酸序列SEQ ID NO.55
Figure PCTCN2021132195-appb-000038
CD20臂重链(重链1)SEQ ID NO.56
Figure PCTCN2021132195-appb-000039
核苷酸序列SEQ ID NO.57
Figure PCTCN2021132195-appb-000040
CD3臂λ轻链SEQ ID NO.58
Figure PCTCN2021132195-appb-000041
核苷酸序列SEQ ID NO.59
Figure PCTCN2021132195-appb-000042
CD3臂重链(重链2)SEQ ID NO.60
Figure PCTCN2021132195-appb-000043
核苷酸序列SEQ ID NO.61
Figure PCTCN2021132195-appb-000044
CD20×CD3κλ002：
CD20臂κ轻链SEQ ID NO.62
Figure PCTCN2021132195-appb-000045
核苷酸序列SEQ ID NO.63
Figure PCTCN2021132195-appb-000046
CD20臂重链(重链1)SEQ ID NO.64
Figure PCTCN2021132195-appb-000047
Figure PCTCN2021132195-appb-000048
核苷酸序列SEQ ID NO.65
Figure PCTCN2021132195-appb-000049
CD3臂λ轻链SEQ ID NO.66
Figure PCTCN2021132195-appb-000050
核苷酸序列SEQ ID NO.67
Figure PCTCN2021132195-appb-000051
CD3臂重链(重链2)SEQ ID NO.68
Figure PCTCN2021132195-appb-000052
核苷酸序列SEQ ID NO.69
Figure PCTCN2021132195-appb-000053
Figure PCTCN2021132195-appb-000054
CD20×CD3κλ003：
CD20臂κ轻链SEQ ID NO.70
Figure PCTCN2021132195-appb-000055
核苷酸序列SEQ ID NO.71
Figure PCTCN2021132195-appb-000056
CD20臂重链(重链1)SEQ ID NO.72
Figure PCTCN2021132195-appb-000057
核苷酸序列SEQ ID NO.73
Figure PCTCN2021132195-appb-000058
CD3臂λ轻链SEQ ID NO.66
Figure PCTCN2021132195-appb-000059
核苷酸序列SEQ ID NO.67
Figure PCTCN2021132195-appb-000060
CD3臂重链(重链2)SEQ ID NO.68
Figure PCTCN2021132195-appb-000061
Figure PCTCN2021132195-appb-000062
核苷酸序列SEQ ID NO.69
Figure PCTCN2021132195-appb-000063
CD20×CD3κλ004：
CD20臂κ轻链SEQ ID NO.62
Figure PCTCN2021132195-appb-000064
核苷酸序列SEQ ID NO.63
Figure PCTCN2021132195-appb-000065
CD20臂重链(重链1)SEQ ID NO.64
Figure PCTCN2021132195-appb-000066
核苷酸序列SEQ ID NO.65
Figure PCTCN2021132195-appb-000067
Figure PCTCN2021132195-appb-000068
CD3臂λ轻链SEQ ID NO.58
Figure PCTCN2021132195-appb-000069
核苷酸序列SEQ ID NO.59
Figure PCTCN2021132195-appb-000070
CD3臂重链(重链2)SEQ ID NO.60
Figure PCTCN2021132195-appb-000071
核苷酸序列SEQ ID NO.61
Figure PCTCN2021132195-appb-000072
CD20×CD3κλ005：
CD20臂κ轻链SEQ ID NO.54
Figure PCTCN2021132195-appb-000073
核苷酸序列SEQ ID NO.55
Figure PCTCN2021132195-appb-000074
Figure PCTCN2021132195-appb-000075
CD20臂重链(重链1)SEQ ID NO.56
Figure PCTCN2021132195-appb-000076
核苷酸序列SEQ ID NO.57
Figure PCTCN2021132195-appb-000077
CD3臂λ轻链SEQ ID NO.66
Figure PCTCN2021132195-appb-000078
核苷酸序列SEQ ID NO.67
Figure PCTCN2021132195-appb-000079
CD3臂重链(重链2)SEQ ID NO.68
Figure PCTCN2021132195-appb-000080
核苷酸序列SEQ ID NO.69
Figure PCTCN2021132195-appb-000081
Figure PCTCN2021132195-appb-000082
CD20×CD3-crossFab
CD20臂κ轻链SEQ ID NO.70
Figure PCTCN2021132195-appb-000083
核苷酸序列SEQ ID NO.71
Figure PCTCN2021132195-appb-000084
CD20臂重链(重链1)SEQ ID NO.74
Figure PCTCN2021132195-appb-000085
核苷酸序列SEQ ID NO.75
Figure PCTCN2021132195-appb-000086
CD3臂λ轻链SEQ ID NO.76
Figure PCTCN2021132195-appb-000087
核苷酸序列SEQ ID NO.77
Figure PCTCN2021132195-appb-000088
Figure PCTCN2021132195-appb-000089
CD3臂重链(重链2)SEQ ID NO.78
Figure PCTCN2021132195-appb-000090
核苷酸序列SEQ ID NO.79
Figure PCTCN2021132195-appb-000091
2.CD20×CD3κλ双特异抗体的表达和纯化
将编码相应抗体片段的质粒，按CD20臂轻链：CD3臂轻链：CD20臂重链(重链1)：CD3臂重链(重链2)＝2:2:1:1的比率混合，与3mg/mL PEI混合后，共同转染CHO-S细胞，在500mL CD CHO AGT培养基(Gibco#12490-001)37℃5％CO 2 150rpm培养，分别在瞬转第2、4和6天，加入4％CHO Feed C+补料(Gibco#A25031-05)。当细胞活率降至85％左右，收获发酵液，过滤后经Protein A亲和层析纯化。基于不同轻链类型构建的CD20×CD3κλ双特异抗体，Protein A一步纯化后单体纯度接近或高于90％，而对照抗体CD20×CD3-crossFab的单体纯度低于80％(表4)，κλ轻链的比率接近1:1(图6)。
表4、CD20×CD3κλ双特异抗体的纯度(SEC-HPLC)
Figure PCTCN2021132195-appb-000092
使用Capto S ImpAct离子交换层析，对CD20×CD3κλ双特异抗体进一步精制纯化，通过50-300mM NaCl，50mM磷酸盐，pH6.4梯度洗脱，合并洗脱峰，SEC-HPLC单体含量显示高于99％(图7)。CD20×CD3κλ002和CD20×CD3κλ003纯化样品中，轻链错配比率极低(<1％)，未检测到CD3同源二聚体或CD20同源二聚体(图8)。
3.CD20×CD3κλ双特异抗体的结合活性
分别通过检测与CD20过表达稳转细胞或CD20+肿瘤细胞的结合，确定双特异抗体CD20抗原臂的亲和力，通过检测与CD3重组抗原、Jurkat细胞或新鲜分离的外周血T细胞的结合，确定双特异抗体CD3臂的亲和力。检测结果显示，新型CD20×CD3κλ双特异抗体与肿瘤细胞的亲和力高于与T细胞的亲和力约3～5倍。阳性对照抗体bsAB1按文献US20170174781合成并表达制备。
(1)CD20×CD3κλ双特异抗体与人和食蟹猴CD20稳转细胞的结合
取对数生长期的实施例1制备的CHO-人CD20和CHO-食蟹猴CD20稳转细胞，用4％小牛血清(Hyclone，SH30626.06)调整细胞至5×10 5个细胞/ml，加入100μl/孔细胞悬液至96孔U型板，300g离心5分钟，弃上清，每孔加入100μL梯度稀释的抗体(起始浓度1800nM，3倍稀释，10个梯度)，4℃孵育60分钟。二抗加入50μL/孔Alexa Fluro647标记的山羊抗人IgG Fc(1:300稀释)，冰上孵育20分钟，洗1次后加入50μL/孔碘化丙啶(PI)溶液(1:300)，孵育5分钟，用流式细胞仪进行检测。图9和表5显示，CD20×CD3κλ双特异抗体高亲和力结合细胞CD20受体，在人和食蟹猴CD20稳转细胞上的亲合力相当。
表5、CD20×CD3κλ双特异抗体和CD20稳转细胞的结合
EC 50 人CD20-CHO 食蟹猴CD20-CHO
CD20×CD3κλ001 10nM 12nM
CD20×CD3κλ002 9nM 8nM
CD20×CD3κλ003 7nM 8nM
CD20×CD3κλ004 8nM 10nM
CD20×CD3κλ005 9nM 14nM
KLH×CD3 - -
“-”：不结合
(2)CD20×CD3κλ双特异抗体与人CD20+肿瘤细胞的结合
取对数生长期的SU-DHL-4、Raji和NALM-6细胞，加入200μg/mL鼠IgG(Jackson ImmunoResearch，115-005-03)冰浴封闭30分钟，用4％小牛血清调整细胞至5×10 5细胞/mL，每孔100μL加入96孔U型板，300g离心5分钟，弃上清，每孔加入100μL梯度稀释的抗体(起始浓度1800nM，3倍稀释，10个梯度)，4℃孵育60分钟。洗去一抗，加入50μL/孔Alexa Fluro647标记的山羊抗人IgG Fc(1:300稀释)，冰上孵育20分钟，洗1次后加入50μL/孔PI，孵育5分钟，用流式细胞仪进行检测。检测结果见图10和表6，CD20×CD3κλ双特异抗体高亲和力结合CD20+肿瘤细胞SU-DHL-4、Raji和NALM-6。
(3)CD20×CD3κλ双特异抗体与Jurkat细胞的结合
取对数生长期的Jurkat细胞，加入200μg/mL鼠IgG(Jackson ImmunoResearch，115-005-03)，冰浴30分钟。用4％小牛血清调整细胞至5×10 5细胞/mL，每孔100μL加入96孔U型板，300g离心去上清，每孔加入100μL梯度稀释的抗体(起始浓度1800nM，3倍稀释，10个梯度)，4℃孵育60分钟。二抗加入50μL/孔Alexa Fluro647标记的山羊抗人IgG Fc(1:300稀释)，冰上孵育20分钟，洗1次后加入50μL/孔PI，孵育5分钟，用流式细胞仪(BD C6)进行检测。检测结果如图11和表6，CD20×CD3κλ双特异抗体以中等亲和力结合人白血病T细胞系Jurkat细胞，EC 50约71-120nM，低于CD20抗原臂与CD20受体的结合力约10倍。
(4)CD20×CD3κλ双特异抗体与人外周血T细胞的结合
取人新鲜外周血，通过Ficoll.Paque Plu(GE，17-1440-03)分离得到PBMC。将PBMC用4％小牛血清(Hyclone，SH30626.06)调整细胞至5×10 5个细胞/mL，100μL/孔加入96孔U型板，离心弃上清，每孔加入100μL梯度稀释的抗体(起始浓度1800nM，3倍稀释，10个梯度)，4℃孵育60分钟。二抗加入50μL/孔Alexa Fluro647标记的山羊抗人IgG Fc(1:300稀释)，冰浴20分钟，洗1次后加入50μL/孔PI，孵育5分钟，用流式细胞仪(BD C6)进行检测。检测结果见图12和表6，CD20×CD3κλ双特异抗体识别人外周血CD4 +T和CD8 +T细胞，与人T细胞亲和力约65-98nM，弱于CD20抗原臂与CD20受体的结合力约10倍，有利于双特异抗体优先富集到肿瘤细胞。
表6、CD20×CD3κλ双特异抗体与人细胞的结合力(nM)
EC 50(nM) SU-DHL-4 Raji NALM-6 Jurkat CD4T CD8T
CD20×CD3κλ002 7 23 10 71 73 98
CD20×CD3κλ003 6 25 8 120 69 65
bsAB1 19 115 48 ND 25 22
KLH×CD3 - - - 33 62 65
ND：高浓度条件下未达到饱和
4.CD20×CD3κλ双特异抗体介导的TDCC作用
取新鲜分离得到的PBMC分别与处于对数生长期的靶细胞NALM-6、TMD-8和Toledo细胞混合，效应/靶细胞＝8:1，每孔加入50μL梯度稀释的抗体(抗体浓度从66.7nM起，10倍稀释，7个梯度)，5％CO 2 37℃培养24小时。培养结束后，将50μL上清转移至新的黑色酶标板，加入50μL/孔LDH检测底物，10分钟后终止反应并检测LDH释放；孔内剩余细胞用4％小牛血清洗2次后，加入100μg/mL人IgG孵育10分钟，再加入T细胞活化检测抗体(CD25-PE，CD4-APC，CD69-FITC和CD8-APC)，冰上孵育20分钟。洗涤弃上清，加入60μL/孔PI，置于冰上孵育5分钟，用流式细胞仪进行检测。图13A、13B分别显示CD20×CD3κλ双特异抗体对人B淋巴白血病细胞Nalm-6的杀伤和对T细胞的活化。图14A、14B分别显示CD20×CD3κλ双特异抗体对TMD-8细胞的杀伤和对T细胞的活化。图15A、15B分别显示CD20×CD3κλ双特异抗体对Toledo细胞的杀伤和对T细胞的活化。对于CD20表达水平不同的肿瘤细胞Nalm-6、TMD-8和Toledo，CD20×CD3κλ002和CD20×CD3κλ003均可介导T细胞产生有效杀伤作用，杀伤活性与对照抗体bsAB1相当或略强，且对T细胞的激活较后者更为温和。
5.CD20×CD3κλ双特异抗体对T细胞活化通路的激活
取对数生长期的Jurkat-NFAT-luc报告细胞和CD20阳性靶细胞(SU-DHL-4、Raji和NALM-6细胞)，离心弃上清，重悬至2×10 6个细胞/ml。50μl/孔接种靶细胞至96孔板，300g离心5分钟弃上清，50μl/孔接种Jurkat-NFAT-luc报告细胞至96孔板，每孔加入50μL梯度稀释的CD20×CD3κλ双特异抗体或对照抗体KLH×CD3(起始浓度20μg/ml，10倍稀释，10个梯度)，5％CO 2，37℃培养6小时。培养结束后，按照ONE-Glo Luciferase Assay System说明书，每孔加入100μL检测试剂，室温放置3分钟，于酶标仪(Biotek Synergy HT)检测。检测结果见图16和表7，CD20×CD3κλ双特异抗体以不同CD20表达水平的肿瘤细胞为靶细胞时，均可激活T细胞的NFAT信号通路。
表7、CD20×CD3κλ双特异抗体对T细胞NFAT通路的激活
EC 50(nM) SU-DHL-4 Raji NALM-6
CD20×CD3κλ002 0.10 0.02 0.14
CD20×CD3κλ003 0.11 0.02 0.14
KLH×CD3 - - -
6.CD20×CD3κλ双特异抗体与Fcγ受体的结合
将50μg/ml His-Tag抗体通过氨基偶联到CM5芯片，分别捕获带有His标签的FcγRI、FcγRIIA H131和FcγRIIIA V158重组蛋白(Sino Biological，#10256-H08H/10374-H08H1/10389-H08H1)，捕获时间40秒，流速10μL/min，基线平稳后，将梯度稀释的抗体(起始浓度37.5μg/mL，2倍稀释)以30μL/min的流速流过芯片，结合时间120秒，解离时间200秒，用Biacore evaluation软件拟合得到亲和力常数。由表8可见，CD20×CD3κλ双特异抗体与FcγRI、FcγRIIA H131和FcγRIIIA V158均无结合；野生型IgG4对照抗体以较强亲和力结合FcγRI，与FcγRIIA H131有微弱结合。
表8、CD20×CD3κλ双特异抗体与Fcγ受体的亲和力
KD FcγRI FcγRIIA H131 FcγRIIIA V158
CD20×CD3κλ002 不结合 不结合 不结合
CD20×CD3κλ003 不结合 不结合 不结合
IgG4同型对照 14nM 微弱 不结合
7.免疫重建小鼠皮下Raji移植瘤模型
选择6～8周雌性B-NGD小鼠(百奥赛图生物科技有限公司)，皮下接种3×10 6Raji细胞，待肿瘤长至60mm 3时，随机分组，分别设定为给药组3.0mg/kg、给药组0.6mg/kg、给药组0.12mg/kg和阴性对照组KLH×CD3 3mg/kg。每只小鼠尾静脉注射1×10 7个PBMC细胞，3天后开始第一次小鼠给药，给药间隔为5天一次，共给药3次。监测小鼠肿瘤体积及小鼠体重，实验结束后断颈处死小鼠，取瘤称重并记录。结果见图17，CD20×CD3κλ双特异抗体的体内药效呈现剂量相关性，中剂量和高剂量抑瘤率分别为82％和89％。荷瘤小鼠对以上剂量耐受良好，无体重减轻等不良反应。
8.免疫缺陷型小鼠皮下Raji与人PBMC混合接瘤模型
选择6～8周雌性B-NGD小鼠(百奥赛图生物科技有限公司)，将Raji(3×10 6个)和人PBMC(5×10 6个)混合后接种于小鼠皮下，待肿瘤成瘤体积到达60-100mm 3时，随机分组。分别设定为给药组3.0mg/mL、给药组0.6mg/mL、给药组0.12mg/mL和阴性对照组KLH×CD3 3mg/kg。给药间隔为5天一次，共给药2次。监测小鼠肿瘤体积及小鼠体重，实验结束后断颈处死小鼠，取瘤称重并记录。结果见图18，CD20×CD3κλ双特异抗体的体内药效呈现剂量相关性，低、中和高剂量抑瘤率分别为65％、98％和162％，高、中剂量组肿瘤完全抑制或消退。
9.CD20×CD3κλ双特异抗体在食蟹猴中的药效
将8只食蟹猴分配至4个剂量组，每个剂量组由2只猴子组成，雌雄各半。各剂量组接受的给药剂量分别为0.3、1、3mg/kg(每周1次，给药3周，共4次给药)和1mg/kg(单次给药)CD20×CD3κλ002双特异抗体，给药方案见表9。给药期及恢复期，各组所有猴一般状况良好，未见毒性反应，未见死亡或濒死。各剂量组体温未见明显异常改变，II导联心电图波形正常，其心率、R-R间期、P-R间期、QT间期、QRS时限、收缩压、舒张压等指标未见明显异常。在给药后不同时间点，通过流式细胞技术分析外周血中B细胞和T细胞群体数量的变化，B细胞使用细胞表面标记物CD20鉴定(CD20+细胞)，T细胞使用CD3鉴定(CD3+细胞)。给药后8小时，外周血中B细胞被快速清除，24小时已低于检测下限(图19)。
表9、CD20×CD3κλ双特异抗体给药方案
Figure PCTCN2021132195-appb-000093
实施例3：构建不同类型轻链形成的BCMA×CD3κλ双特异抗体
1.BCMA×CD3κλ双特异抗体的构建
以含κ轻链的BCMA人源化抗体，与含λ轻链的人源化CD3抗体，参考实施例2构建具有天然IgG构型的新型BCMA-CD3κλ人源化双特异抗体，同时在BCMA抗原臂和CD3臂引入电荷变异体(Vκ BCMA:Gln 42Lys；VH BCMA:Gln 39Glu；Vλ CD3:Gln 40Glu；VH CD3:Gln 39Lys)(序列见表10)；双特异抗体的Fc部分采用人IgG4knob-into-hole结构，以实现异源二聚体配对，并且通过突变Ser 228Pro、Leu 235Glu和Pro 329Ala，保持铰链区稳定并减弱与FcγR受体和C1q的相互作用。
表10、BCMA×CD3κλ双特异抗体
蛋白序列 BCMA臂轻链 BCMA臂重链 CD3臂轻链 CD3臂重链
BCMA×CD3κλ003 SEQ ID NO.80 SEQ ID NO.82 SEQ ID NO.66 SEQ ID NO.68
BCMA×CD3κλ004 SEQ ID NO.84 SEQ ID NO.82 SEQ ID NO.66 SEQ ID NO.68
BCMA×CD3κλ005 SEQ ID NO.80 SEQ ID NO.86 SEQ ID NO.66 SEQ ID NO.68
BCMA×CD3κλ006 SEQ ID NO.84 SEQ ID NO.86 SEQ ID NO.66 SEQ ID NO.68
BCMA×CD3κλ003
BCMA臂κ轻链：SEQ ID NO.80
Figure PCTCN2021132195-appb-000094
核苷酸序列：SEQ ID NO.81
Figure PCTCN2021132195-appb-000095
BCMA臂重链(重链1)：SEQ ID NO.82
Figure PCTCN2021132195-appb-000096
核苷酸序列：SEQ ID NO.83
Figure PCTCN2021132195-appb-000097
CD3臂λ轻链：SEQ ID NO.66
Figure PCTCN2021132195-appb-000098
核苷酸序列：SEQ ID NO.67
Figure PCTCN2021132195-appb-000099
CD3臂重链(重链2)：SEQ ID NO.68
Figure PCTCN2021132195-appb-000100
核苷酸序列：SEQ ID NO.69
Figure PCTCN2021132195-appb-000101
BCMA×CD3κλ004
BCMA臂κ轻链：SEQ ID NO.84
Figure PCTCN2021132195-appb-000102
核苷酸序列：SEQ ID NO.85
Figure PCTCN2021132195-appb-000103
BCMA臂重链(重链1)：SEQ ID NO.86
Figure PCTCN2021132195-appb-000104
核苷酸序列：SEQ ID NO.87
Figure PCTCN2021132195-appb-000105
CD3臂λ轻链：SEQ ID NO.66
Figure PCTCN2021132195-appb-000106
核苷酸序列：SEQ ID NO.67
Figure PCTCN2021132195-appb-000107
CD3臂重链(重链2)：SEQ ID NO.68
Figure PCTCN2021132195-appb-000108
核苷酸序列：SEQ ID NO.69
Figure PCTCN2021132195-appb-000109
BCMA×CD3κλ005
BCMA臂κ轻链：SEQ ID NO.80
Figure PCTCN2021132195-appb-000110
核苷酸序列：SEQ ID NO.81
Figure PCTCN2021132195-appb-000111
BCMA臂重链(重链1)：SEQ ID NO.86
Figure PCTCN2021132195-appb-000112
核苷酸序列：SEQ ID NO.87
Figure PCTCN2021132195-appb-000113
CD3臂λ轻链：SEQ ID NO.66
Figure PCTCN2021132195-appb-000114
核苷酸序列：SEQ ID NO.67
Figure PCTCN2021132195-appb-000115
CD3臂重链(重链2)：SEQ ID NO.68
Figure PCTCN2021132195-appb-000116
Figure PCTCN2021132195-appb-000117
核苷酸序列：SEQ ID NO.69
Figure PCTCN2021132195-appb-000118
BCMA×CD3κλ006
BCMA臂κ轻链：SEQ ID NO.84
Figure PCTCN2021132195-appb-000119
核苷酸序列：SEQ ID NO.85
Figure PCTCN2021132195-appb-000120
BCMA臂重链(重链1)：SEQ ID NO.86
Figure PCTCN2021132195-appb-000121
核苷酸序列：SEQ ID NO.87
Figure PCTCN2021132195-appb-000122
Figure PCTCN2021132195-appb-000123
CD3臂λ轻链：SEQ ID NO.66
Figure PCTCN2021132195-appb-000124
核苷酸序列：SEQ ID NO.67
Figure PCTCN2021132195-appb-000125
CD3臂重链(重链2)：SEQ ID NO.68
Figure PCTCN2021132195-appb-000126
核苷酸序列：SEQ ID NO.69
Figure PCTCN2021132195-appb-000127
2.BCMA×CD3κλ双特异抗体的表达和纯化
将编码相应抗体片段的质粒，按BCMA臂轻链(κ轻链)：CD3臂轻链(λ轻链)：BCMA臂重链(重链1)：CD3臂重链(重链2)＝2:2:1:1的比率混合，与3mg/mL PEI混合后，共同转染CHO-S细胞，在500mL CD CHO AGT培养基(Gibco#12490-001)37℃5％CO 2 150rpm培养，分别在瞬转第2、4和6天，加入4％CHO Feed C+补料(Gibco#A25031-05)。当细胞活率降至85％左右，收获发酵液，过滤后经Protein A亲和层析初步纯化，SEC-HPLC单体含量显示接近或高于92％；经Capto S ImpAct离子交换层析，单体含量进一步提高至98-99％以上(表11)。
表11、BCMA×CD3κλ双特异抗体的纯度
Figure PCTCN2021132195-appb-000128
3.BCMA×CD3κλ双特异抗体的结合活性
(1)BCMA×CD3κλ双特异抗体与抗原的亲和力测定
将10μg/mL人或食蟹猴BCMA或CD3εγ重组抗原通过氨基偶联结合到CM5芯片(GE healthcare)，控制抗原结合量约200RU。基线平稳后，将梯度稀释的抗体(从10μg/mL起，2倍稀释7个梯度)，以30μL/min的流速流过芯片，结合时间350秒，解离时间600秒。用Biacore T200evaluation软件以1:1结合模型，拟合得到动力学常数。亲和力测定结果见表12。
表12、BCMA×CD3κλ双特异性抗体与抗原的亲和力
KD(nM) 人BCMA 食蟹猴BCMA 人CD3εγ 食蟹猴CD3εγ
BCMA×CD3κλ003 1.3 0.16 31 43
BCMA×CD3κλ004 1.0 0.12 32 43
BCMA×CD3κλ005 1.1 0.09 28 20
BCMA×CD3κλ006 1.2 0.10 27 33
(2)BCMA×CD3κλ双特异抗体与BCMA+细胞的结合
分别取对数生长期的CHO-人BCMA稳转细胞(CHO-hBCMA)和CHO-食蟹猴BCMA稳转细胞(CHO-cynoBCMA)，和肿瘤细胞NCI-H929和RPMI-8226，封闭后每孔加入100μL梯度稀释的抗体(起始浓度1800nM，3倍稀释，10个梯度)，4℃孵育60分钟。二抗加入50μL/孔Alexa Fluro647标记的山羊抗人IgG Fc(1:300稀释)，冰上孵育20分钟，洗1次后加入50μL/孔PI溶液(1:300)，孵育5分钟，用流式细胞仪进行检测。图20显示，BCMA×CD3κλ双特异抗体高亲和力结合人、食蟹猴BCMA稳转细胞；图21显示，BCMA×CD3κλ双特异抗体高亲和力结合BCMA+肿瘤细胞NCI-H929和RPMI-8226。BCMA×CD3κλ双特异抗体与细胞的结合常数EC 50见表13。
表13、BCMA×CD3κλ双特异性抗体和BCMA稳转细胞的结合
EC 50(nM) 人BCMA-CHO 食蟹猴BCMA-CHO NCI-H929 RPMI-8226
BCMA×CD3κλ003 16 3 34 34
BCMA×CD3κλ004 22 3 36 39
BCMA×CD3κλ005 24 3 33 38
BCMA×CD3κλ006 16 3 32 19
KLH×CD3 - - - -
(3)BCMA×CD3κλ双特异抗体与Jurkat细胞的结合
取对数生长期的Jurkat细胞，加入200μg/mL鼠IgG(Jackson ImmunoResearch，115-005-03)，冰浴30分钟。用4％小牛血清调整细胞至5×10 5细胞/mL，每孔100μL加入96孔U型板，300g离心去上清，每孔加入100μL梯度稀释的抗体(起始浓度1800nM，3倍稀释，10个梯度)，4℃孵育60分钟。二抗加入50μL/孔Alexa Fluro647标记的山羊抗人IgG Fc(1:300稀释)，冰上孵育20分钟，洗1次后加入50μL/孔PI，孵育5分钟，用流式细胞仪(BD C6)进行检测。检测结果如图22和表14，BCMA×CD3κλ双特异抗体以中亲和力结合人白血病T细胞系Jurkat细胞。
(4)BCMA×CD3κλ双特异抗体与外周血T细胞的结合
取新鲜人外周血，通过Ficoll.Paque Plu(GE，17-1440-03)分离得到PBMC。将PBMC用4％小牛血清(Hyclone，SH30626.06)调整细胞至5×10 5个细胞/mL，100μL/孔加入96孔U型板，离心弃上清，每孔加入100μL梯度稀释的抗体(起始浓度1800nM，3倍稀释，10个梯度)，4℃孵育60分钟。二抗加入50μL/孔Alexa Fluro647标记的山羊抗人IgG Fc(1:300稀释)，冰浴20分钟，洗1次后加入50μL/孔PI，孵育5分钟，用流式细胞仪(BD C6)进行检测。对照抗体REGN5458参照US20200024356合成并制备。检测结果见图23和表14所示，BCMA×CD3κλ双特异抗体识别人外周血CD4+T和CD8+T细胞，与人T细胞亲和力约60-97nM，均弱于BCMA抗原臂与BCMA受体的结合力，有利于双特异抗体优先富集到肿瘤细胞。
表14、BCMA×CD3κλ双特异抗体与Jurkat细胞的结合
EC 50(nM) Jurkat CD4+T CD8+T
BCMA×CD3κλ003 66 89 97
BCMA×CD3κλ004 61 79 67
BCMA×CD3κλ005 51 66 60
BCMA×CD3κλ006 56 91 93
KLH×CD3 76 57 62
4.BCMA×CD3κλ双特异抗体介导的TDCC作用
取新鲜分离得到的PBMC分别与处于对数生长期的靶细胞NCI-H929和RPMI-8226细胞混合，效应/靶细胞＝8:1，每孔加入50μL梯度稀释的抗体(抗体浓度从66.7nM起，10倍稀释，7个梯度)，5％CO 2 37℃培养24小时。培养结束后，将50μL上清转移至新的黑色酶标板，加入50μL/孔LDH检测底物，10分钟后终止反应并检测LDH释放；孔内剩余细胞用4％小牛血清洗2次后，加入100μg/mL人IgG孵育10分钟，再加入T细胞活化检测抗体(CD25-PE，CD4-APC，CD69-FITC和CD8-APC)，冰上孵育20分钟。洗涤弃上清，加入60μL/孔PI，置于冰上孵育5分钟，用流式细胞仪进行检测。图24A、24B分别显示BCMA×CD3κλ双特异抗体对NCI-H929细胞的杀伤和对T细胞的活化。图25A、25B分别显示BCMA×CD3κλ双特异抗体对RPMI-8226细胞的杀伤和对T细胞的活化。对于BCMA 表达水平不同的肿瘤细胞NCI-H929和RPMI-8226，BCMA×CD3κλ双特异抗体均可介导T细胞产生有效杀伤作用，杀伤活性与对照抗体REGN5458相当。
5.BCMA×CD3κλ双特异抗体对T细胞活化通路的激活
取对数生长期的Jurkat-NFAT-luc报告细胞和BCMA阳性靶细胞RPMI-8226，离心弃上清，重悬至2×10 6个细胞/ml。50μl/孔接种靶细胞至96孔板，300g离心5分钟弃上清，50μl/孔接种Jurkat-NFAT-luc报告细胞至96孔板，每孔加入50μL梯度稀释的BCMA×CD3κλ双特异抗体或对照抗体KLH×CD3(起始浓度20μg/ml，10倍稀释，10个梯度)，5％CO 2，37℃培养6小时。培养结束后，按照ONE-Glo Luciferase Assay System说明书，每孔加入100μL检测试剂，室温放置3分钟，于酶标仪(Biotek Synergy HT)检测。检测结果见图26，BCMA×CD3κλ双特异抗体以RPMI-8226肿瘤细胞为靶细胞时，可激活T细胞的NFAT信号通路；无靶细胞时，不激活NFAT信号通路。
6.BCMA×CD3κλ双特异抗体对PBMC的非特异激活
取新鲜分离得到的PBMC，每孔加入50μL梯度稀释的抗体(抗体浓度从66.7nM起，10倍稀释，7个梯度)，5％CO 2 37℃培养24小时。培养结束后，将50μL上清转移至新的黑色酶标板，加入50μL/孔LDH检测底物，10分钟后终止反应并检测LDH释放；孔内剩余细胞用4％小牛血清洗2次后，加入100μg/mL人IgG孵育10分钟，再加入T细胞活化检测抗体(CD25-PE，CD4-APC，CD69-FITC和CD8-APC)，冰上孵育20分钟。洗涤弃上清，加入60μL/孔PI，置于冰上孵育5分钟，用流式细胞仪进行检测。检测结果见图27，在缺乏靶细胞的条件下，BCMA×CD3κλ双特异抗体对外周血T细胞无激活作用，与阴性对照KLH×CD3相当。
7.BCMA×CD3κλ人源化双特异抗体与Fc受体的结合
将50μg/ml His-Tag抗体通过氨基偶联到CM5芯片，分别捕获带有His6标签的FcγRI、FcγRIIA H131和FcγRIIIA V158重组蛋白，捕获时间40秒，流速10μL/min，基线平稳后，将梯度稀释的抗体(起始浓度37.5μg/mL，2倍稀释)以30μL/min的流速流过芯片，结合时间120秒，解离时间200秒，用Biacore evaluation软件拟合得到亲和力常数。由图28可见，BCMA×CD3κλ双特异抗体与FcγRI、FcγRIIA H131和FcγRIIIA V158均无结合；野生型IgG4对照抗体以较强亲和力结合FcγRI，与FcγRIIA H131有弱结合。
8.免疫缺陷型小鼠皮下NCI-H929移植瘤模型
选择6～8周雌性B-NGD小鼠(百奥赛图生物科技有限公司)，皮下接种2×10 6NCI-H929细胞(与基质胶(Matrigel)1:1混合)，待肿瘤长至60mm 3时，随机分组，分别为给药组3.0mg/kg、给药组0.6mg/kg和阴性对照组KLH×CD33mg/kg。每只小鼠尾静脉注射1×10 7个PBMC细胞，3天后开始第一次小鼠给药，给药间隔为5天一次，共给药2次。每2天监测一次小鼠肿瘤体积及小鼠体重，实验结束后断颈处死小鼠，取瘤称重并记录。结果见图29，BCMA×CD3κλ双特异抗体的体内药效呈现剂量相关性，3.0mg/kg给药组和0.6mg/kg给药组抑瘤率分别为95％和108％(BCMA×CD3κλ005)以及94％和108％(BCMA×CD3κλ006)。荷瘤小鼠对以上剂量耐受良好，无体重减轻等不良反应。
实施例4：构建不同类型轻链形成的GPC3×CD3κλ双特异抗体
1.GPC3×CD3κλ双特异抗体的构建
将含κ轻链的人源化GPC3抗体，与含λ轻链的人源化CD3抗体，参考实施例2构建具有天然IgG构型的新型GPC3-CD3κλ人源化双特异抗体，同时在GPC3抗原臂和CD3臂引入电荷变异体(Vκ GPC3:Gln 43Lys；VH GPC3:Gln 39Glu；Vλ CD3:Gln 40Glu；VH CD3:Gln 39Lys)(序列见表15)。双特异抗体的Fc部分采用人IgG4knob-into-hole结构，以实现异源二聚体配对，并且通过突变Ser 228Pro、Leu 235Glu和Pro 329Ala，保持铰链区稳定并减弱与Fcγ受体和C1q的相互作用。
表15、GPC3×CD3κλ人源化双特异抗体
  CD20臂轻链 CD20臂重链 CD3臂轻链 CD3臂重链
GPC3×CD3κλ002 SEQ ID NO.88 SEQ ID NO.90 SEQ ID NO.66 SEQ ID NO.68
GPC3×CD3κλ003 SEQ ID NO.92 SEQ ID NO.94 SEQ ID NO.66 SEQ ID NO.68
GPC3×CD3κλ002：
GPC3臂κ轻链SEQ ID NO.88
Figure PCTCN2021132195-appb-000129
核苷酸序列SEQ ID NO.89
Figure PCTCN2021132195-appb-000130
GPC3臂重链(重链1)SEQ ID NO.90
Figure PCTCN2021132195-appb-000131
核苷酸序列SEQ ID NO.91
Figure PCTCN2021132195-appb-000132
CD3臂λ轻链SEQ ID NO.66
Figure PCTCN2021132195-appb-000133
核苷酸序列SEQ ID NO.67
Figure PCTCN2021132195-appb-000134
CD3臂重链(重链2)SEQ ID NO.68
Figure PCTCN2021132195-appb-000135
核苷酸序列SEQ ID NO.69
Figure PCTCN2021132195-appb-000136
Figure PCTCN2021132195-appb-000137
GPC3×CD3κλ003：
GPC3臂κ轻链SEQ ID NO.92
Figure PCTCN2021132195-appb-000138
核苷酸序列SEQ ID NO.93
Figure PCTCN2021132195-appb-000139
GPC3臂重链(重链1)SEQ ID NO.94
Figure PCTCN2021132195-appb-000140
核苷酸序列SEQ ID NO.95
Figure PCTCN2021132195-appb-000141
CD3臂λ轻链SEQ ID NO.66
Figure PCTCN2021132195-appb-000142
核苷酸序列SEQ ID NO.67
Figure PCTCN2021132195-appb-000143
Figure PCTCN2021132195-appb-000144
CD3臂重链(重链2)SEQ ID NO.68
Figure PCTCN2021132195-appb-000145
核苷酸序列SEQ ID NO.69
Figure PCTCN2021132195-appb-000146
将编码相应抗体片段的质粒，按GPC3臂轻链(κ轻链)：CD3臂轻链(λ轻链)：GPC3臂重链(重链1)：CD3臂重链(重链2)＝2:2:1:1的比率混合，与3mg/mL PEI混合后，共同转染CHO-S细胞，在500mL CD CHO AGT培养基(Gibco#12490-001)37℃5％CO 2 150rpm培养，分别在瞬转第2、4和6天，加入4％CHO Feed C+补料(Gibco#A25031-05)。当细胞活率降至85％左右，收获发酵液，过滤后经Protein A亲和层析初步纯化，SEC-HPLC单体含量显示高于92％；经Butyl HP疏水层析和Capto Q阴离子层析，单体含量进一步提高至99.5％以上(表16)。
表16、GPC3-CD3人源化双特异抗体的纯化
Figure PCTCN2021132195-appb-000147
2.GPC3×CD3κλ双特异抗体与GPC3稳转细胞的结合
取对数生长期的CHO-人GPC3、CHO-食蟹猴GPC3稳转细胞或人肝细胞癌HepG2肿瘤细胞，封闭后调整细胞至5×10 5个细胞/ml，加入100μl/孔细胞悬液至96孔U型板，300g离心5分钟，弃上清，每孔加入100μL梯度稀释的抗体(起始浓度1800nM，3倍稀释，10个梯度)，4℃孵育60分钟。二抗加入50μL/孔Alexa Fluro647标记的山羊抗人IgG Fc(1:300稀释)，冰上孵育20分钟，洗1次后加入50μL/孔PI溶液(1:300)，孵育5分钟，用流式细胞仪进行检测。结果见图30-31和表17，GPC3×CD3κλ双特异抗体高亲和力结合GPC3+细胞。
表17、GPC3×CD3κλ双特异性抗体和GPC3+细胞的结合
EC 50 人GPC3-CHO 食蟹猴GPC3-CHO HepG2
GPC3×CD3κλ002 2.8nM 3.0nM 2.1nM
GPC3×CD3κλ003 2.9nM 3.0nM 2.5nM
KLH×CD3 不结合 不结合 不结合
3.GPC3×CD3κλ双特异抗体与Jurkat细胞的结合
取对数生长期的Jurkat细胞，加入200μg/mL鼠IgG(Jackson ImmunoResearch，115-005-03)，冰浴30分钟。用4％小牛血清调整细胞至5×10 5细胞/mL，每孔100μL加入96孔U型板，300g离心去上清，每孔加入100μL梯度稀释的抗体(起始浓度1800nM，3倍稀释，10个梯度)，4℃孵育60分钟。二抗加入50μL/孔Alexa Fluro647标记 的山羊抗人IgG Fc(1:300稀释)，冰上孵育20分钟，洗1次后加入50μL/孔PI，孵育5分钟，用流式细胞仪(BD C6)进行检测。检测结果如图32和表18，GPC3×CD3κλ双特异抗体以中等亲和力结合人白血病T细胞系Jurkat细胞，EC 50约20-40nM。
4.GPC3×CD3κλ双特异抗体与外周血T细胞的结合
取新鲜人或食蟹猴外周血，通过Ficoll.Paque Plus(GE，17-1440-03)分离得到PBMC。将PBMC用4％小牛血清(Hyclone，SH30626.06)调整细胞至5×10 5个细胞/mL，100μL/孔加入96孔U型板，离心弃上清，每孔加入100μL梯度稀释的抗体(起始浓度1800nM，3倍稀释，10个梯度)，4℃孵育60分钟。二抗加入50μL/孔Alexa Fluro647标记的山羊抗人IgG Fc(1:300稀释)，冰浴20分钟，洗1次后加入50μL/孔PI，孵育5分钟，用流式细胞仪(BD Celesta)进行检测。检测结果见图33和表18，GPC3×CD3κλ双特异抗体以低亲和力结合人外周血T细胞。
表18、GPC3×CD3κλ双特异抗体与T细胞的结合
EC 50 Jurkat CD4+T CD8+T
GPC3×CD3κλ002 36nM 65nM 65nM
GPC3×CD3κλ003 26nM 56nM 69nM
KLH×CD3 27nM 77nM 84nM
5.GPC3×CD3κλ双特异抗体介导的TDCC作用
取新鲜分离得到的PBMC分别与处于对数生长期的靶细胞HepG2细胞混合，效应/靶细胞＝10:1，每孔加入50μL梯度稀释的抗体(抗体浓度从66.7nM起，10倍稀释，7个梯度)，5％CO 2 37℃培养24小时。培养结束后，将50μL上清转移至新的黑色酶标板，加入50μL/孔LDH检测底物，10分钟后终止反应并检测LDH释放；孔内剩余细胞用4％小牛血清洗2次后，加入100μg/mL人IgG孵育10分钟，再加入T细胞活化检测抗体(CD25-BV421，CD4-FITC，CD69-BV605和CD8-APC)，冰上孵育20分钟。洗涤弃上清，加入60μL/孔PI，置于冰上孵育5分钟，用流式细胞仪进行检测。图34A、34B分别显示GPC3×CD3κλ双特异抗体对HepG2细胞的杀伤和对T细胞的活化。
6.GPC3×CD3κλ双特异抗体对T细胞活化通路的激活
取对数生长期的靶细胞CHO-人GPC3，离心弃上清，重悬至2×10 5个细胞/ml。50μL/孔接种靶细胞至96孔板，5％CO 2，37℃培养过夜。取对数生长期的Jurkat-NFAT-luc报告细胞300g离心5分钟弃上清，重悬至4×10 6个细胞/ml，取出96孔板，弃上清，25μL/孔接种Jurkat-NFAT-luc报告细胞至96孔板，每孔加入25μL梯度稀释的GPC3×CD3κλ双特异抗体或对照抗体KLH×CD3(起始浓度20μg/ml，3倍稀释，10个梯度)，5％CO 2，37℃培养6小时。培养结束后，按照ONE-Glo Luciferase Assay System说明书，每孔加入100μL检测试剂，于酶标仪(MD SpectraMax i3x)检测。检测结果见图35，GPC3×CD3κλ双特异抗体以CHO-人GPC3为靶细胞时，可激活T细胞的NFAT信号通路。
7.GPC3×CD3κλ双特异抗体PBMC的非特异激活
取新鲜分离得到的PBMC，加入100μL抗体(10μg/mL)，5％CO 2 37℃培养24小时。孔内细胞用4％小牛血清洗2次后，加入100μg/mL人IgG孵育10分钟，再加入T细胞活化检测抗体(CD25-BV421，CD4-FITC，CD69-BV605和CD8-APC)，冰上孵育20分钟。洗涤弃上清，加入60μL/孔PI，置于冰上孵育5分钟，用流式细胞仪进行检测。阳性对照抗体ERY974参照US20170267783制备。检测结果见图36，在缺乏靶细胞的条件下，GPC3×CD3κλ双特异抗体对外周血T细胞无激活作用，与阴性对照KLH×CD3相当。
8.免疫重建小鼠皮下HepG2移植瘤模型
选择6～8周雌性B-NGD小鼠(百奥赛图生物科技有限公司)，皮下接种HepG2细胞(7×10 6/只)，待肿瘤长至60-100mm 3时，随机分组，分别设定为高剂量给药组3.0mg/kg、中剂量给药组1.0mg/kg、低剂量给药组0.3mg/kg、阳性对照组ERY974和阴性对照组KLH×CD3 3mg/kg。每只小鼠尾静脉注射1×10 7PBMC细胞，3天后开始第一次小鼠给药，给药间隔为5天一次，共给药2次。监测小鼠肿瘤体积及小鼠体重，实验结束后断颈处死小鼠，取瘤称重并记录。结果见图37，GPC3×CD3κλ双特异抗体的体内药效呈现剂量相关性，抑瘤率(低剂量到高剂量)分别为：76.7％、81.3％和95.9％。荷瘤小鼠对以上剂量耐受良好，无体重减轻等不良反应。
9.CD3人源化鼠Hepa1-6/人GPC3移植瘤模型
选择6周雌性C57/BL6-hCD3小鼠(百奥赛图生物科技有限公司)，将Hepa1-6/人GPC3(6×10 6/只)接种于小鼠皮下，待肿瘤成瘤体积到达60-100mm 3时，随机分组。分别设定为高剂量给药组10mg/mL、中剂量给药组3mg/mL、低剂量给药组1mg/mL、阳性对照组ERY974和阴性对照组KLH×CD3 10mg/kg。给药间隔为3天一次，共给药3次。监测小鼠肿瘤体积及小鼠体重，实验结束后断颈处死小鼠，取瘤称重并记录。结果见图38，GPC3×CD3κλ双特异抗体可显著介导免疫细胞杀死肿瘤细胞，减小荷瘤体积；其10mg/kg剂量与ERY974药效相当。

Claims (17)

  1. 一种双特异性抗体或其抗原结合片段，其包含：
    (a)第一抗原结合部分或其抗原结合片段，所述第一抗原结合部分包含第一轻链和第一重链，所述第一轻链为κ型轻链，所述第一抗原结合部分包含与第一抗原结合的第一结合结构域；和
    (b)第二抗原结合部分或其抗原结合片段，所述第二抗原结合部分包含第二轻链和第二重链，所述第二轻链为λ型轻链，所述第二抗原结合部分包含与第二抗原结合的第二结合结构域。
  2. 根据权利要求1所述的双特异性抗体或其抗原结合片段，其中，所述第二抗原为CD3抗原；
    优选地，所述第二抗原结合部分的第二轻链可变区具有Gln 40Glu突变(Vλ CD3:Gln 40Glu)；所述第二抗原结合部分的第二重链可变区具有Gln 39Lys突变(VH CD3:Gln 39Lys)；
    优选地，所述第二结合结构域包含选自氨基酸序列SEQ ID NO:7-9、14、15、20、21或其任何变体的第二轻链CDR；和/或选自氨基酸序列SEQ ID NO:26-28、31、34、40、43、46、47或其任何变体的第二重链CDR；
    优选地，所述第二结合结构域包含选自氨基酸序列SEQ ID NO:7、14或其任何变体的第二轻链CDR1，选自氨基酸序列SEQ ID NO:8、15、20或其任何变体的第二轻链CDR2，选自氨基酸序列SEQ ID NO:9、21或其任何变体的第二轻链CDR3；和/或选自氨基酸序列SEQ ID NO:26、31、46或其任何变体的第二重链CDR1，选自氨基酸序列SEQ ID NO:27、47或其任何变体的第二重链CDR2，选自氨基酸序列SEQ ID NO:28、34、37、40、43或其任何变体的第二重链CDR3；
    优选地，所述第二结合结构域的第二轻链CDR选自：分别包含氨基酸序列SEQ ID NO:7、8、9的第二轻链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:14、15、9的第二轻链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:7、8、21的第二轻链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:7、20、21的第二轻链CDR1、CDR2及CDR3序列；和/或所述第二结合结构域的重链CDR选自：分别包含氨基酸序列SEQ ID NO:26、27、28的第二重链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:31、27、28的第二重链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:31、27、34的第二重链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:31、27、37的第二重链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:31、27、40的第二重链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:31、27、43的第二重链CDR1、CDR2及CDR3序列；分别包含氨基酸序列SEQ ID NO:46、47、28的第二重链CDR1、CDR2及CDR3序列；
    优选地，所述第二结合结构域包含选自氨基酸序列SEQ ID NO:5、10、12、16、18、22或其任何变体的第二轻链可变区；和/或选自氨基酸序列SEQ ID NO:24、29、32、35、38、41、44、48、50、52或其任何变体的第二重链可变区；
    优选地，所述第二结合结构域包含氨基酸序列SEQ ID NO:18或其任何变体的第二轻链可变区；和氨基酸序列SEQ ID NO:24或其任何变体的第二重链可变区；
    优选地，所述第二结合结构域包含氨基酸序列SEQ ID NO:5或其任何变体的第二轻链可变区；和氨基酸序列SEQ ID NO:48或其任何变体的第二重链可变区；
    优选地，所述第二结合结构域包含氨基酸序列SEQ ID NO:18或其任何变体的第二轻链可变区；和氨基酸序列SEQ ID NO:48或其任何变体的第二重链可变区；
    优选地，所述第二结合结构域包含氨基酸序列SEQ ID NO:5或其任何变体的第二轻链可变区；和氨基酸序列SEQ ID NO:50或其任何变体的第二重链可变区；
    优选地，所述第二结合结构域包含氨基酸序列SEQ ID NO:10或其任何变体的第二轻链可变区；和氨基酸序列SEQ ID NO:50或其任何变体的第二重链可变区；
    优选地，所述第二结合结构域包含氨基酸序列SEQ ID NO:12或其任何变体的第二轻链可变区；和氨基酸序列SEQ ID NO:50或其任何变体的第二重链可变区；
    优选地，所述第二结合结构域包含氨基酸序列SEQ ID NO:18或其任何变体的第二轻链可变区；和氨基酸序列SEQ ID NO:50或其任何变体的第二重链可变区；
    优选地，所述第二抗原结合部分的第二轻链选自氨基酸序列SEQ ID NO:58和66；和/或所述第二抗原结合部分的第二重链选自氨基酸序列SEQ ID NO:60和68；更优选地，所述第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:58；和所述第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:60；更优选地，所述第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66；和所述第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68。
  3. 根据权利要求1或2所述的双特异性抗体或其抗原结合片段，其中，所述第一抗原为肿瘤抗原；
    优选地，所述肿瘤抗原选自：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、间皮素、CD33/IL3Ra、c-Met、PSCA、PSMA、糖脂F77、EGFRvIII、GD-2、MY-ESO-1、Her2、Her3、MUC1、MUC17、Claudin18或MAGEA3，
    更优选地，所述肿瘤相关抗原选自CD20、BCMA和GPC3。
  4. 根据权利要求1-3任一项所述的双特异性抗体或其抗原结合片段，其中，所述第一抗原为CD20抗原；
    优选地，所述第一抗原结合部分的第一轻链可变区具有Gln 38Lys突变(Vκ CD20:Gln 38Lys)；更优选地，所述第一抗原结合部分的第一重链可变区具有Gln 39Glu突变(VH CD20:Gln 39Glu)；
    优选地，所述第一抗原结合部分的第一轻链可变区具有Gln 38Lys突变(Vκ CD20:Gln 38Lys)，且第一轻链恒定区具有Glu 123Lys和Gln 124Lys突变(Vκ-Ck CD20:Gln 38Lys\Glu 123Lys\Gln 124Lys)；更优选地，所述第一抗原结合部分第一抗原结合部分的第一重链可变区具有Gln 39Glu突变(VH CD20:Gln 39Glu)，且第一重链恒定区具有Lys 152Glu和Lys 218Glu突变(V H-C H1 CD20:Gln 39Glu\Lys 152Glu\Lys 218Glu)。
  5. 根据权利要求4所述的双特异性抗体或其抗原结合片段，其中，所述第一抗原结合部分的第一轻链选自氨基酸序列SEQ ID NO:54、62和70；和/或所述第一抗原结合部分的第一重链选自氨基酸序列SEQ ID NO:56、64和72；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:54，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:56；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:62，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:64；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:70，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:72；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:54，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:56，所述第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:58，和所述第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:60；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:62，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:64，所述第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和所述第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68，所述第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:58，和所述第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:60；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:70，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:72，所述第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和所述第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:62，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:64，所述第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:58，和所述第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:60；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:54，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:56，所述第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和所述第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68。
  6. 根据权利要求1-3任一项所述的双特异性抗体或其抗原结合片段，其中，所述第一抗原为BCMA抗原；
    优选地，所述第一抗原结合部分第一抗原结合部分的第一轻链可变区具有Gln 42Lys突变(Vκ BCMA:Gln 42Lys)；更优选地，所述第一抗原结合部分第一抗原结合部分的第一重链可变区具有Gln 39Glu突变(VH BCMA:Gln 39Glu)。
  7. 根据权利要求6所述的双特异性抗体或其抗原结合片段，其中，所述第一抗原结合部分的第一轻链选自氨基酸序列SEQ ID NO:80和84；和/或所述第一抗原结合部分的第一重链选自氨基酸序列SEQ ID NO:82和86；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:80；和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:82；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:84；和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:82；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:80；和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:86；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:84；和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:86；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:80，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:82，所述第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和所述第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:84，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:82，所述第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和所述第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:80，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:86，所述第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和所述第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:84，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:86，所述第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和所述第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68。
  8. 根据权利要求1-3任一项所述的双特异性抗体或其抗原结合片段，其中，所述第一抗原为GPC3抗原；
    优选地，所述第一抗原结合部分的第一轻链可变区具有Gln 43Lys和Gln 39Glu突变(Vκ GPC3:Gln 43Lys；VH GPC3: Gln 39Glu)。
  9. 根据权利要求8所述的双特异性抗体或其抗原结合片段，其中，所述第一抗原结合部分的第一轻链选自氨基酸序列SEQ ID NO:88和92；和/或所述第一抗原结合部分的第一重链选自氨基酸序列SEQ ID NO:90和94；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:88，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:90；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:92，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:94；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:88，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:90，所述第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和所述第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68；
    优选地，所述第一抗原结合部分的第一轻链为氨基酸序列SEQ ID NO:92，和所述第一抗原结合部分的第一重链为氨基酸序列SEQ ID NO:94，所述第二抗原结合部分的第二轻链为氨基酸序列SEQ ID NO:66，和所述第二抗原结合部分的第二重链为氨基酸序列SEQ ID NO:68。
  10. 根据权利要求1-9任一项所述的双特异性抗体或其抗原结合片段，其中，所述双特异抗体的第一抗原结合部分和/或第二抗原结合部分的Fc部分采用knob-into-hole结构；优选地，采用人IgG4 knob-into-hole结构；
    优选地，所述双特异抗体的第一抗原结合部分和/或第二抗原结合部分还具有Ser 228Pro、Leu 235Glu和/或Pro 329Ala突变。
  11. 编码根据1-10任一项所述的双特异性抗体或其抗原结合部分的核酸；
    优选地，所述第二抗原结合部分结合CD3抗原，所述第二抗原结合部分的第二轻链可变区的编码核酸选自核苷酸序列SEQ ID NO:6、11、13、17、19和23；和/或所述第二抗原结合部分的第二重链可变区的编码核酸选自核苷酸序列SEQ ID NO:25、30、33、36、39、42、45、49、51和53；优选地，所述第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:59和67；和/或所述第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:61和69；更优选地，所述第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:59，和所述第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:61；更优选地，所述第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和所述第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69；
    优选地，所述第一抗原结合部分结合CD20抗原，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:55、63和71；和/或所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:57、65和73；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:55，和所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:57；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:63，和所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:65；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:71，和所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:73；
    优选地，所述第一抗原结合部分结合BCMA抗原，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:81和85；和/或所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:83和87；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:81，和所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:83；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:85，和所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:83；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:81，和所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:87；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:85，和所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:87；
    优选地，所述第一抗原结合部分结合GPC3抗原，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:89和93；和/或所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:91和95；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:89，和所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:91；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:93，和所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:95；
    更优选地，所述双特异性抗体的所述第一抗原结合部分结合CD20抗原，所述第二抗原结合CD3抗原，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:55，所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:57，所述第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:59，和所述第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:61；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:63，和所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:65，所述第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和所述第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69，所述第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:59，和所述第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID  NO:61；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:71，和所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:73，所述第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和所述第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:63，和所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:65，所述第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:59，和所述第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:61；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:55，所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:57，所述第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和所述第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69；
    更优选地，所述双特异性抗体的所述第一抗原结合部分结合BCMA抗原，所述第二抗原结合CD3抗原，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:81，所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:83，所述第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和所述第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:85，所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:83，所述第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和所述第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:81，所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:87，所述第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和所述第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:85，所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:87，所述第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和所述第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69；
    更优选地，所述双特异性抗体的所述第一抗原结合部分结合GPC3抗原，所述第二抗原结合CD3抗原，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:89，所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:91，所述第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和所述第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69；更优选地，所述第一抗原结合部分的第一轻链的编码核酸选自核苷酸序列SEQ ID NO:93，所述第一抗原结合部分的第一重链的编码核酸选自核苷酸序列SEQ ID NO:95，所述第二抗原结合部分的第二轻链的编码核酸选自核苷酸序列SEQ ID NO:67，和所述第二抗原结合部分的第二重链的编码核酸选自核苷酸序列SEQ ID NO:69。
  12. 含有权利要求11所述核酸的载体。
  13. 含有权利要求11所述的核酸或权利要求12所述载体的细胞。
  14. 一种组合物，其包含权利要求1-10任一项所述的双特异性抗体或其抗原结合部分、权利要求11所述的核酸、权利要求12所述的载体和/或权利要求13所述的细胞。
  15. 一种抗体-药物偶联物，其包含共价附着至治疗部分的权利要求1-10任一项所述的双特异性抗体或其抗原结合部分；
    优选地，所述治疗部分选自细胞毒性部分、化疗剂、细胞因子、免疫抑制剂、免疫刺激剂、裂解肽或放射性同位素；
    优选地，所述细胞毒性部分选自以下：紫杉醇；细胞松弛素B；短杆菌肽D；溴化乙锭；吐根碱；丝裂霉素；依托泊苷；替尼泊苷；长春新碱；长春碱；秋水仙碱；多柔比星；柔红霉素；二羟基蒽二酮；微管蛋白抑制剂如美登素或其类似物或衍生物；抗有丝分裂剂如单甲基奥瑞他汀E或F或其类似物或衍生物；海兔毒素10或15或其类似物；伊立替康或其类似物；米托蒽醌；光辉霉素；放线菌素D；1-脱氢睾酮；糖皮质激素；普鲁卡因；丁卡因；利多卡因；普萘洛尔；嘌呤霉素；卡奇霉素或其类似物或衍生物；抗代谢药，如甲氨喋呤、6巯基嘌呤、6硫鸟嘌呤、阿糖胞苷、氟达拉滨、5氟尿嘧啶、癸二嗪、羟基脲、天冬酰胺酶、吉西他滨或克拉屈滨；烷化剂，如二氯甲基二乙胺、硫代嘌呤、苯丁酸氮芥、美法仑、卡莫司汀(BSNU)、洛莫司汀(CCNU)、环磷酰胺、白消安、二溴甘露醇、链脲佐菌素、达卡巴嗪(DTIC)、丙卡巴嗪、丝裂霉素C；铂类衍生物，如顺铂或卡铂；多卡霉素A、多卡霉素SA、雷切霉素(CC-1065)或其类似物或衍生物；抗生素，如放线菌素、博来霉素、柔红霉素、多柔比星、伊达比星、光霉素、丝裂霉素、米托蒽醌、普力霉素、安定霉素(AMC)；吡咯并[2,1-c][1,4]-苯并二氮杂卓(PDB)；白喉毒素及相关分子如白喉A链及其活性片段和杂合分子、蓖麻毒素如蓖麻毒素A或去糖基化蓖麻毒素A链毒素、霍乱毒素、志贺样毒素如SLT I、SLT II、SLT IIV、LT毒素、C3毒素、志贺毒素、百日咳毒素、破伤风毒素、大豆Bowman-Birk蛋白酶抑制剂、假单胞菌外毒素、阿罗林、皂草素、蒴莲根毒素、胶凝蛋白、相思豆毒素A链、蒴莲根毒素A链、α-sarcin、油桐(Aleurites fordii)蛋白、石竹素蛋白、美洲商陆蛋白如PAPI、PAPII和PAP-S、苦瓜(momordica charantia)抑制剂、泻果素、巴豆毒素、肥阜草(sapaonaria officinalis)抑制剂、白树毒素、丝裂霉素、局限曲菌素、酚霉素和依诺霉素毒素；核糖核酸酶(RNase)；DNase I、葡萄球菌内毒素A；商陆抗病毒蛋白；白喉毒素和假单胞菌内毒素；
    优选地，所述细胞因子选自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配体、干细胞因子、安西司亭和TNFa；
    优选地，所述放射性同位素选自 3H、 14C、 15N、 35S、 67Cu、 90Y、 99Tc、 125I、 131I、 186Re、 188Re、 211At、 212Bi、 212Pb、 213Bi、 225Ac和 227Th。
  16. 一种试剂盒，其含其包含权利要求1-10任一项所述的双特异性抗体或其抗原结合部分、权利要求11所述的核酸、权利要求12所述的载体、或权利要求13所述的细胞、权利要求14所述的组合物和/或权利要求15所述的抗体-药物偶联物。
  17. 含权利要求1-10任一项所述的双特异性抗体或其抗原结合部分、权利要求11所述的核酸、权利要求12所述的载体、或权利要求13所述的细胞、权利要求14所述的组合物和/或权利要求15所述的抗体-药物偶联物在制备用于诊断、治疗或预防与肿瘤抗原相关疾病药物或试剂盒中的用途；
    优选地，所述肿瘤抗原为CD20，所述肿瘤抗原相关疾病为与CD20相关疾病；优选地，与CD20相关的疾病包括B细胞疾病，例如B细胞增殖性病症，特别是CD20阳性B细胞病症；优选地，所述疾病选自非霍奇金淋巴瘤(NHL)，急性淋巴细胞性白血病(ALL)，慢性淋巴细胞性白血病(CLL)，弥漫性大B细胞淋巴瘤(DLBCL)，滤泡性淋巴瘤(FL)，套细胞淋巴瘤(MCL)，边缘区淋巴瘤(MZL)，以及多发性骨髓瘤(MM)和霍奇金淋巴瘤(HL)。
    优选地，所述肿瘤抗原为BCMA，所述肿瘤抗原相关疾病为与BCMA相关疾病；优选地，与BCMA相关的疾病包括B细胞疾病；优选地，所述疾病是癌症；更优选地，所述癌症是B-细胞相关癌症，其选自多发性骨髓瘤、恶性浆细胞瘤、霍奇金淋巴瘤、结节性淋巴细胞为主的霍奇金淋巴瘤、Kahler’s病和骨髓性白血病、浆细胞白血病、浆细胞瘤、B-细胞幼淋巴细胞白血病、毛细胞白血病、B-细胞非霍奇金淋巴瘤(NHL)、急性髓性白血病(AML)、慢性淋巴细胞白血病(CLL)、急性淋巴细胞白血病(ALL)、慢性髓性白血病(CML)、滤泡性淋巴瘤、伯基特淋巴瘤、边缘区淋巴瘤、套细胞淋巴瘤、大细胞淋巴瘤、前体B-淋巴细胞淋巴瘤、髓性白血病、瓦尔登斯特伦巨球蛋白血症、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤、边缘区淋巴瘤、粘膜相关淋巴组织淋巴瘤、小细胞淋巴细胞性淋巴瘤、套细胞淋巴瘤、伯基特淋巴瘤、原发性纵隔(胸腺)大B细胞淋巴瘤、淋巴浆细胞淋巴瘤、瓦尔登斯特伦巨球蛋白血症、淋巴结边缘区B细胞淋巴瘤、脾边缘区淋巴瘤、血管内大B-细胞淋巴瘤、原发性渗出性淋巴瘤、淋巴瘤样肉芽肿病、富含T细胞/组织细胞的大B-细胞淋巴瘤、原发性中枢神经系统淋巴瘤、原发性皮肤弥漫性大B-细胞淋巴瘤(腿型)、老年人EBV阳性弥漫性大B-细胞淋巴瘤、炎症相关的弥漫性大B-细胞淋巴瘤、血管内大B-细胞淋巴瘤、ALK阳性大B-细胞淋巴瘤、浆母细胞淋巴瘤、HHV8相关的多中心Castleman病中产生的大B-细胞淋巴瘤、未分类的具有弥漫性大B-细胞淋巴瘤和伯基特淋巴瘤中间特征的B-细胞淋巴瘤、未分类的具有弥漫性大B-细胞淋巴瘤和经典霍奇金淋巴瘤中间特征的B-细胞淋巴瘤以及其他B-细胞相关淋巴瘤；更优选地，所述B细胞疾病是B细胞障碍；优选地，浆细胞障碍选自：多发性骨髓瘤、浆细胞瘤、浆细胞白血病、巨球蛋白血症、淀粉样变性、华氏巨球蛋白血症、孤立性骨浆细胞瘤、髓外浆细胞瘤、骨硬化性骨髓瘤、重链病、意义不明确的单克隆丙种球蛋白病以及郁积型多发性骨髓瘤；优选地，所述疾病是自身免疫性病症，如系统性红斑性狼疮或类风湿性关节炎；
    优选地，所述肿瘤抗原为GPC3，所述肿瘤抗原相关疾病为与GPC3相关疾病；优选地，与GPC3相关的疾病包括肿瘤；优选地，所述肿瘤是癌症；更优选地，所述癌症是GPC3阳性癌症，例如，可以为GPC3阳性肝癌、GPC3阳性肝细胞癌、GPC3阳性胰腺癌、GPC3阳性肺癌、GPC3阳性结肠癌、GPC3阳性乳腺癌、GPC3阳性前列腺癌、GPC3阳性白血病或GPC3阳性淋巴瘤。
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