Multispecific antigen-binding proteins that bind to CD3 and methods of using the same

Abstract

The present application provides multispecific antigen binding proteins that include a binding moiety for CD3. The present application also provides methods of using the multispecific antigen binding proteins in the manufacture of T cell activation and / or tumor therapy medicaments.

Description

Background Art

[0001] Bispecific antibodies can recognize two different targets simultaneously and activate more cellular control mechanisms, thus holding great potential for the treatment of tumors and other diseases. T cell engager bispecific antibodies are typical bispecific antibodies. They may form an immune synapse by simultaneously binding to T cell surface antigens (such as CD3) and tumor cell surface antigens (such as CD19). The distance between tumor cells and T cells is shortened by the bispecific antibody, and then the T cells are directly activated and proliferated. The activated T cells may directly kill tumor cells or release cytotoxins to kill tumor cells. Therefore, the process of activating T cells by bispecific antibodies does not include the presentation of tumor antigens to T cells to generate specific T lymphocyte clones. Therefore, this process is not restricted by MHC or HLA and is clinically applicable.

[0002] CD3 (cluster of differentiation 3) is a protein complex expressed on the surface of most T cells. Since it can transmit MHC-TCR binding signals and activate T cells, it is an ideal binding site for T cell engager bispecific antibodies. The first-generation anti-CD3 drugs have achieved great success. However, in order to improve safety (for example, to avoid cytokine release syndrome (CRS)), improvement is needed in the rational control of T cell activation and / or cytokine release. A decrease in the incidence of CRS will not only promote the clinical application of T cell engager bispecific antibodies in hematological cancers but also bring epoch-making progress in the application to solid tumors.

[0003] Therefore, there is an urgent need to provide more effective and safe multispecific antibodies against CD3 and corresponding humanized anti-CD3 antibodies.

Summary of the Invention

[0004] This application provides a multispecific antigen-binding protein comprising a binding moiety for CD3, wherein the binding moiety for CD3 comprises the HCDR1, HCDR2, and HCDR3 regions from the heavy chain variable region VH of SEQ ID NO: 8 and / or the LCDR1, LCDR2, and LCDR3 regions from the light chain variable region VL of SEQ ID NO: 7. The multispecific antigen-binding protein may further comprise a binding moiety for a tumor-associated antigen (TAA). The multispecific antigen-binding protein of the present application can be used in the manufacture of prophylactic, palliative, and / or therapeutic agents for tumors. The multispecific antigen-binding protein of the present application can exhibit a more effective killing effect on tumor cells. The multispecific antigen-binding protein of the present application shows an improvement in the safety profile for the administered subject and can, for example, induce less release of T cell-mediated cytokines. The multispecific antigen-binding protein and / or the binding moiety for CD3 of the present application exhibit a significantly enhanced binding affinity for CD3.

[0005] In one aspect, this application provides a multispecific antigen-binding protein comprising a CD3-binding moiety, wherein the CD3-binding moiety comprises an amino acid sequence having at least 95% identity with the heavy chain variable region HCDR1, HCDR2, and HCDR3. HCDR1 has the amino acid sequence set forth in any one of SEQ ID NOs: 4, 37, 45, and 53, HCDR2 has the amino acid sequence set forth in any one of SEQ ID NOs: 5, 38, 46, and 54, and HCDR3 has the amino acid sequence set forth in any one of SEQ ID NOs: and 6, 39, 47, and 55. And / or, the CD3-binding moiety comprises an amino acid sequence having at least 95% identity with the light chain variable region LCDR1, LCDR2, and LCDR3. LCDR1 has the amino acid sequence set forth in any one of SEQ ID NOs: 1, 40, 48, and 56. LCDR2 has the amino acid sequence set forth in any one of SEQ ID NOs: 2, 41, 49, and 57; LCDR3 has the amino acid sequence set forth in any one of SEQ ID NOs: 3, 42, 50, and 58, and the CD3-binding moiety induces activation of T cells.

[0006] In some embodiments, the CD3-binding portion comprises heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3. HCDR1 has the amino acid sequence set forth in any one of SEQ ID NOs: 4, 37, 45, and 53; HCDR2 has the amino acid sequence set forth in any one of SEQ ID NOs: 5, 38, 46, and 54; and HCDR3 has the amino acid sequence set forth in any one of SEQ ID NOs: 6, 39, 47, and 55. And / or the CD3-binding portion comprises light chain complementarity determining regions LCDR1, LCDR2, and LCDR3. LCDR1 has the amino acid sequence set forth in any one of SEQ ID NOs: 1, 40, 48, and 56; LCDR2 has the amino acid sequence set forth in any one of SEQ ID NOs: 2, 41, 49, and 57; and LCDR3 has the amino acid sequence set forth in any one of SEQ ID NOs: 3, 42, 50, and 58.

[0007] In some embodiments, the heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 have the sequences set forth in SEQ ID NOs: 4, 5, and 6, respectively, and / or the light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 have the sequences set forth in SEQ ID NOs: 1, 2, and 3. The heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 have the sequences set forth in SEQ ID NOs: 37, 38, and 39, and / or the light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 have the sequences set forth in SEQ ID NOs: 40, 41, and 42. The heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 have the sequences set forth in SEQ ID NOs: 45, 46, and 47, and / or the light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 have the sequences set forth in SEQ ID NOs: 48, 49, and 50. Or the heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 have the sequences set forth in SEQ ID NOs: 53, 54, and 55, and / or the light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 have the sequences set forth in SEQ ID NOs: 56, 57, and 58.

[0008] In some embodiments, the CD3 binding portion comprises an amino acid sequence having at least 80% identity with the heavy chain variable region, and the heavy chain variable region has the amino acid sequence set forth in any one of SEQ ID NOs: 8, 35, 43, and 51. And / or, the CD3 binding portion comprises an amino acid sequence having at least 80% identity with the light chain variable region, and the light chain variable region has the amino acid sequence set forth in any one of SEQ ID NOs: 7, 36, 44, and 52.

[0009] In some embodiments, the heavy chain variable region has the amino acid sequence set forth in any one of SEQ ID NOs: 8, 35, 43, and 51. And / or, the light chain variable region has the amino acid sequence set forth in any one of SEQ ID NOs: 7, 36, 44, and 52.

[0010] In some embodiments, the CD3 binding portion comprises an amino acid sequence having at least 80% identity to the heavy chain variable region of SEQ ID NO: 8 and / or the light chain variable region of SEQ ID NO: 7. The CD3 binding portion comprises an amino acid sequence having at least 80% identity to the heavy chain variable region of SEQ ID NO: 35 and / or the light chain variable region of SEQ ID NO: 36. The CD3 binding portion comprises an amino acid sequence having at least 80% identity to the heavy chain variable region of SEQ ID NO: 43 and / or the light chain variable region of SEQ ID NO: 44. Or, the CD3 binding portion comprises an amino acid sequence having at least 80% identity to the heavy chain variable region of SEQ ID NO: 51 and / or the light chain variable region of SEQ ID NO: 52.

[0011] In some embodiments, the CD3 binding portion comprises the heavy chain variable region of SEQ ID NO: 8 and / or the light chain variable region of SEQ ID NO: 7. The CD3 binding portion comprises the heavy chain variable region of SEQ ID NO: 35 and / or the light chain variable region of SEQ ID NO: 36. The CD3 binding portion comprises the heavy chain variable region of SEQ ID NO: 43 and / or the light chain variable region of SEQ ID NO: 44. Or, the CD3 binding portion comprises the heavy chain variable region of SEQ ID NO: 51 and / or the light chain variable region of SEQ ID NO: 52.

[0012] In some embodiments, VH includes framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0013] In some embodiments, the binding moiety to CD3 includes an antibody heavy chain constant region.

[0014] In some embodiments, the binding moiety to CD3 includes an antibody heavy chain constant region derived from an IgA, IgG, IgD, IgE, or IgM antibody.

[0015] In some embodiments, the antibody heavy chain constant region includes an IgG constant region.

[0016] In some embodiments, the antibody heavy chain constant region includes SEQ ID NO: 18 or SEQ ID NO: 26.

[0017] In some embodiments, VL includes framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0018] In some embodiments, the binding moiety to CD3 includes an antibody light chain constant region.

[0019] In some embodiments, the binding moiety to CD3 includes an antibody light chain constant region derived from an IgA, IgG, IgD, IgE, or IgM antibody.

[0020] In some embodiments, the antibody light chain constant region includes an Igκ constant region.

[0021] In some embodiments, the antibody light chain constant region includes SEQ ID NO: 17 or SEQ ID NO: 25.

[0022] In some embodiments, the binding moiety to CD3 includes an antibody or an antigen-binding fragment thereof.

[0023] In one aspect, the CD3 binding portion of the multispecific antigen-binding protein is humanized.

[0024] In some embodiments, the humanized binding portion to CD3 comprises amino acids having at least 80% identity to the heavy chain variable region of SEQ ID NO: 27 and / or the light chain variable region of SEQ ID NO: 31.

[0025] In some embodiments, the humanized binding portion to CD3 comprises amino acids having at least 80% identity to the heavy chain variable region of SEQ ID NO: 28 and / or the light chain variable region of SEQ ID NO: 31.

[0026] In some embodiments, the humanized binding portion to CD3 comprises the heavy chain variable region of SEQ ID NO: 27, and / or the light chain variable region of SEQ ID NO: 31.

[0027] In some embodiments, the humanized binding portion to CD3 comprises the heavy chain variable region of SEQ ID NO: 28, and / or the light chain variable region of SEQ ID NO: 31.

[0028] In some embodiments, the antigen-binding fragment comprises Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv and / or dAb.

[0029] In some embodiments, the antibody is selected from monoclonal antibody, chimeric antibody, humanized antibody, and fully human antibody.

[0030] In some embodiments, the multispecific antigen-binding protein is produced by recombinant expression in a host cell.

[0031] In some embodiments, the host cell expressing the multispecific antigen-binding protein is selected from bacterial cells, fungal cells, plant cells, mammalian cells or viruses.

[0032] In some embodiments, the bacterial cell is Escherichia coli.

[0033] In some embodiments, the fungal cell is a yeast cell.

[0034] In some embodiments, the mammalian cell is selected from CHO, NSO, BHK, or HEK293 cells.

[0035] In some embodiments, the multispecific antigen-binding protein is produced by hybridoma cells.

[0036] In some embodiments, the hybridoma cells are selected from mice, rats, or rabbits.

[0037] In some embodiments, the format of the multispecific antigen-binding protein is selected from bispecific antibodies, bispecific diabodies, bispecific scFvs, TandAbs, trivalent binding molecules, or tetravalent binding molecules.

[0038] In some embodiments, the multispecific antigen-binding protein comprises a binding moiety to at least one tumor-associated antigen (TAA).

[0039] In some embodiments, the TAAs of the multispecific antigen-binding protein are selected from the following group: EPCAM, CCR5, CD19, HER2, HER3neu, HER3, HER4, EGFR, PSMA, CEA, MUC1, MUC2, MUC3, MUC4, MUC5, MUC7, βhCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3, 9-0-Acetyl-GD3, GM2, globoH, fucosylGM1, PolySA, GD2, RON, c-Met, CEACAM-6, PCTA-1, PSA, PAP, ALCAM(CD166), PECAM-1, CD151, MAGE-1, TROP2, IGF1R, TGFBR2, GHRHR, GHR, IL-6R, gpl30, TNFR2, OSMRp, Patched-1, Frizzled.Robol, LTpR, CD26, CD27, CD44, CD80, CD81, CD86, CD100, CXCR4, SAS, BCMA, TWEAKR / Fnl4, FGFR4, VEGFR1, VEGFR2, SSX1, and SSX2, Carboanhydrase IX (MN / CAIX), CD44v6, Sonic Hedgehog (Shh), Wue-1, plasma cell antigen, (membrane-bound) IgE, melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, TNF-α precursor, STRAP, mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6; desmoglein 4, E-cadherin neoepitope, fetal acetylcholine receptor, CD25, CA19-9 marker, CA-125 marker and Müllerian inhibiting substance (MIS) receptor type II, sTn (sialylated Tn antigen, TAG72), FAP (fibroblast activation antigen), endothelin, EGFRvIII, L6, SAS, CD63, TF antigen, Cora antigen, CD7, CD79b, CD22, Igα, Igβ, gp100, MT-MMPs, F19-antigen, CO-29 and EphA2.

[0040] In some embodiments, the multispecific antigen-binding protein comprises a binding moiety for HER2.

[0041] In some embodiments, the binding portion of the multispecific antigen-binding protein to HER2 comprises an amino acid sequence having at least 95% identity to the heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 having the sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and / or the light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 having the sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively.

[0042] In some embodiments, the binding portion of the multispecific antigen-binding protein to HER2 comprises the heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 having the sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and / or the light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 having the sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively. The CD3 binding portion induces activation of T cells.

[0043] In some embodiments, the binding portion of the multispecific antigen-binding protein to HER2 comprises an amino acid sequence having at least 80% identity to the heavy chain variable region of SEQ ID NO: 16 and / or the light chain variable region of SEQ ID NO: 15.

[0044] In some embodiments, the binding portion of the multispecific antigen-binding protein to HER2 comprises the heavy chain variable region of SEQ ID NO: 16 and / or the light chain variable region of SEQ ID NO: 15.

[0045] In some embodiments, the binding portion to HER2 comprises an antibody heavy chain constant region.

[0046] In some embodiments, the binding portion to HER2 comprises an antibody heavy chain constant region derived from an IgA, IgG, IgD, IgE, or IgM antibody.

[0047] In some embodiments, the antibody heavy chain constant region comprises an IgG constant region.

[0048] In some embodiments, the antibody heavy chain constant region comprises SEQ ID NO: 18 or SEQ ID NO: 26.

[0049] In some embodiments, the binding moiety to HER2 comprises an antibody light chain constant region.

[0050] In some embodiments, the binding moiety to HER2 comprises an antibody light chain constant region derived from an IgA, IgG, IgD, IgE, or IgM antibody.

[0051] In some embodiments, the HER2 antibody light chain constant region comprises an IgK constant region.

[0052] In some embodiments, the HER2 antibody light chain constant region comprises the amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 25.

[0053] In some embodiments, the binding moiety to HER2 comprises an antibody or an antigen-binding fragment thereof.

[0054] In some embodiments, the antigen-binding fragment to HER2 comprises a Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv and / or dAb.

[0055] In some embodiments, the antibody to HER2 is selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0056] In some embodiments, the multispecific antigen-binding protein comprises a binding moiety to CD20.

[0057] In some embodiments, the heavy chain variable region VH of the binding moiety to CD20 comprises the amino acid sequence set forth in SEQ ID NO: 24.

[0058] In some embodiments, the light chain variable region VL of the binding moiety to CD20 comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0059] In some embodiments, the binding moiety to CD3 comprises a first polypeptide chain and a second polypeptide chain. The first polypeptide chain comprises the heavy chain variable region VH of an antibody that binds to CD3, and the second polypeptide chain comprises the light chain variable region VL of an antibody that binds to CD3.

[0060] In some embodiments, the binding moiety to TAA comprises a third polypeptide chain and a fourth polypeptide chain. The third polypeptide chain comprises the heavy chain variable region VH of an antibody that binds to TAA, and the fourth polypeptide chain comprises the light chain variable region VL of an antibody that binds to TAA.

[0061] In some embodiments, the third polypeptide chain comprises the heavy chain variable region VH of an antibody that binds to HER2, and the fourth polypeptide chain comprises the light chain variable region VL of an antibody that binds to HER2.

[0062] In some embodiments, the heavy chain variable region VH of the antibody that binds to HER2 comprises the amino acid sequence set forth in SEQ ID NO: 16, and the light chain variable region VL of the antibody that binds to HER2 comprises the amino acid sequence set forth in SEQ ID NO: 15. Alternatively, the heavy chain variable region VH of the antibody that binds to HER2 comprises the amino acid sequence set forth in SEQ ID NO: 20, and the light chain variable region VL of the antibody that binds to HER2 comprises the amino acid sequence set forth in SEQ ID NO: 19.

[0063] In some embodiments, the third polypeptide chain comprises the heavy chain variable region VH of an antibody that binds to CD20, and the fourth polypeptide chain comprises the light chain variable region VL of an antibody that binds to CD20.

[0064] In some embodiments, the heavy chain variable region VH of the antibody that binds to CD20 comprises the amino acid sequence set forth in SEQ ID NO: 24, and the light chain variable region VL of the antibody that binds to CD20 comprises the amino acid sequence set forth in SEQ ID NO: 23.

[0065] In another aspect, the present application provides one or more isolated nucleic acid molecules encoding the multispecific antigen-binding protein of the present application.

[0066] In another aspect, the present application provides a vector comprising the isolated nucleic acid molecule of the present application.

[0067] In another aspect, the present application provides a cell comprising the isolated nucleic acid molecule of the present application and / or the vector of the present application.

[0068] In another aspect, the present application provides a method for preparing the multispecific antigen-binding protein of the present application, which includes culturing the cells of the present application under conditions that enable the expression of the multispecific antigen-binding protein of the present application.

[0069] In another aspect, the present application provides a pharmaceutical composition comprising the multispecific antigen-binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, and / or the cell of the present application, and optionally a pharmaceutically acceptable adjuvant.

[0070] In another aspect, the present application provides a method of using the multispecific antigen-binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, and / or the pharmaceutical composition of the present application in the manufacture of a prophylactic, palliative and / or therapeutic agent for tumors.

[0071] In another aspect, the present application provides the multispecific antigen-binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, and / or the pharmaceutical composition of the present application for use in the prevention, alleviation and / or treatment of tumors.

[0072] In another aspect, the present application provides a method for preventing, alleviating and / or treating tumors, which includes administering the multispecific antigen-binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, and / or the pharmaceutical composition of the present application to a subject in need thereof.

[0073] In another aspect, the present application provides a method for preventing, alleviating, and / or treating tumors in a subject. Here, the tumors refer to glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine body cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, urinary tract cancer, hepatocellular carcinoma (HCC), anal cancer, penile cancer, head and neck cancer, or other cancers, including squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, squamous cell lung cancer, peritoneal cancer, hepatocellular cancer, gastric cancer, gastrointestinal cancer, esophageal cancer, pancreatic cancer.

[0074] In another aspect, the present application provides a method for activating T cells, a method for targeting CD3 expressed on T cells, and / or a method for promoting the interaction between T cells and tumor cells, including administering the multispecific antigen-binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, and / or the pharmaceutical composition of the present application.

[0075] Additional aspects and advantages of the present application will become readily apparent to those skilled in the art from the following detailed description, which illustrates and describes only exemplary embodiments of the present application. As will be understood, the present application is capable of other different embodiments, and some details thereof are capable of modification in various obvious respects without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature and not restrictive.

[0076] Cross-reference to related applications All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0077] The novel features of the present invention are described in detail in the appended claims. The features and advantages of the present invention will be understood by reference to the following detailed description, which illustrates exemplary embodiments in which the principles of the present invention are used, and the accompanying drawings (referred to as "FIGURES" and "DRAWINGS").

Brief Description of the Drawings

[0078]

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DETAILED DESCRIPTION OF THE INVENTION

[0079] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, numerous variations, modifications, and substitutions may occur to those skilled in the art. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0080] As used herein, the singular forms "a", "an", and "the" generally include plural references unless the context clearly indicates otherwise.

[0081] In the present application, the term "antigen-binding protein" generally refers to a protein that includes a portion that binds to an antigen and, optionally, a scaffold or framework portion that causes a conformation that promotes the binding of the antigen-binding protein to the antigen to be adopted by the antigen-binding portion. Examples of antigen-binding proteins can include antibodies, antibody fragments (e.g., the antigen-binding portion of an antibody), antibody derivatives, and antibody analogs. An antigen-binding protein can, for example, have the structure of a naturally occurring immunoglobulin. An "immunoglobulin" is a tetrameric molecule. In a naturally occurring immunoglobulin, each tetramer is composed of two pairs of identical polypeptide chains, and each pair has one light chain (LC) and one heavy chain (HC). The amino-terminal portion of each chain can include a variable region that contains about 100 to 110 or more amino acids that are mainly responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region that is mainly responsible for effector functions. Human light chains are classified as either or light chains. Heavy chains are classified as μ, δ, γ, α, ε, or κ λ, and the isotypes of the antibodies are defined as IgM, IgG, IgD, IgA, and IgE, respectively. The variable regions of naturally occurring immunoglobulin chains can exhibit the same general structure as a relatively conserved framework region (FR) linked by three hypervariable regions also called complementarity-determining regions or CDRs. From the N-terminus to the C-terminus, both the light chain and the heavy chain can include domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0082] As used herein, the term "CDR" generally refers to the amino acid sequences of the complementarity determining regions of an antigen-binding protein. These are the hypervariable regions of the immunoglobulin heavy and light chains. The variable portion of an immunoglobulin has three heavy chain and three light chain CDRs (or CDR regions). It will be apparent to those skilled in the art that there are various numbering systems for CDR sequences. For example, Chothia (Chothia et al. (1989) Nature 342:877-883), Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Maryland (1987 and 1991)). The Chothia numbering system is used to number the residues in the antibodies of the present disclosure.

[0083] As used in this application, the term "antigen-binding fragment" as used herein generally refers to a portion of an immunoglobulin molecule. An antigen-binding fragment can include one light chain and a portion of a heavy chain having a single antigen-binding site. Antigen-binding fragments can be obtained by enzymatic digestion of immunoglobulin molecules. For example, an antigen-binding fragment can include one constant domain and one variable domain of each of the heavy and light chains. The variable domain may include a paratope (antigen-binding site) containing a set of complementarity determining regions at the amino terminus of the immunoglobulin molecule.

[0084] As used in this application, the term "Fv fragment" as used herein generally refers to all or a portion of the variable regions of the heavy and light chains and does not include the constant regions of the heavy and light chains. For example, the variable regions of the heavy and light chains can include CDRs.

[0085] As used herein, the term "ScFv" generally refers to a single-chain antibody fragment. An ScFv can be a recombinant single-chain polypeptide molecule in which the variable regions of the light and heavy chains of the antibody are connected directly or via a peptide linker.

[0086] In the present application, the term "Fab" generally refers to an antibody fragment that includes two N-terminal portions of a heavy chain polypeptide linked by at least one disulfide bridge in the hinge region and two complete light chain polypeptides. For example, each light chain can be complexed with one N-terminal portion of the heavy chain. Fab can also include a Fab fragment that includes all or most of a light chain polypeptide (e.g., VLCL) complexed with an N-terminal portion of a heavy chain polypeptide (e.g., VHCH1).

[0087] In the present application, the term "Fab", as used herein, generally refers to an antibody fragment that is a reduction product of an F(ab)2 fragment. The Fab' fragment may differ from the Fab fragment by having several additional residues at the carboxy terminus of the CHI domain that includes one or more cysteines from the antibody hinge region. Fab'-SH can be, in the present specification, a designation for a Fab' in which one or more cysteine residues of a certain domain have a free thiol group.

[0088] In the present application, the term "F(ab)2", as used herein, generally refers to a monovalent antibody structure that can bind to an antigen but does not have an Fc portion. F(ab)2 can be an antibody that is digested by the enzyme papain to yield two F(ab) fragments, each of approximately 50 kDa, and an Fc fragment.

[0089] In the present application, the term "F(ab')2", as used herein, generally refers to an antibody fragment that includes two Fab fragments and a portion of the hinge region linked by a disulfide bond. F(ab')2 can be produced by pepsin digestion of an intact antibody. The F(ab')2 fragment has bivalent antigen-binding activity and may be able to cross-link antigens.

[0090] In the specification of the present application, the term "fully human antibody" generally refers to an antibody derived from a fully human amino acid sequence whose antigen specificity has been selected by an in vivo method using a genetically modified mouse or by an antibody engineering process combined with screening.

[0091] In the present application, the term "monoclonal antibody" generally refers to a group of antibodies that are substantially homologous. That is, each antibody contained in the antibody group is identical except for the possibility of minor naturally occurring variations. Monoclonal antibodies are understood to be antibodies produced by the same cell, which are clones of a unique parental cell, in contrast to polyclonal antibodies made from multiple different immune cells. For example, monoclonal antibodies can be prepared by hybridoma technology or produced in bacteria, eukaryotes, or plant cells by recombinant DNA methods. Alternatively, monoclonal antibodies can be prepared using, for example, the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., Mol. Biol., 222:581-597 (1991).

[0092] In the present application, the term "chimeric antibody" generally refers to an antibody in which a portion of the amino acid sequence of each heavy or light chain is homologous to or belongs to a specific class of the corresponding amino acid sequence of an antibody derived from a particular animal species, and the remaining portion of the chain is homologous to the corresponding sequence of another animal species. For example, the variable regions of both the light and heavy chains are derived from the variable regions of an antibody of a particular animal species (e.g., mouse, rat, etc.), and the constant region is homologous to the antibody sequence of another animal species (e.g., human). For example, to obtain a chimeric antibody, variable regions can be prepared using non-human B cells or hybridoma cells, and the constant region that binds thereto can be of human origin. The variable regions have the advantage of being easy to prepare, and their specificity is not affected by the origin of the constant region to which they bind. At the same time, since the constant region of a chimeric antibody may be of human origin, it is considered that the possibility of inducing an immune reaction when injecting a chimeric antibody is lower than that of an antibody with a non-human-derived constant region.

[0093] In the present application, the term "humanized antibody" generally refers to an antibody that has been engineered to reduce its immunogenicity in humans while retaining the antigen-binding properties of the original antibody, which is derived from non-human species (e.g., mouse or rat), immunoglobulin-binding proteins, and polypeptides. For example, techniques such as CDR grafting (Jones et al., Nature 321:522 (1986)), and "reshaping" (Verhoeyen et al., 1988 Science 239:1534-1536; Riechmann et al., 1988 Nature 332:323-337; Tempest et al., 1991 9:266-271) Bio / Technol 1991 9:266-271), "hyperchimerization" (Queen et al., 1989 Proc Natl Acad Sci USA 86:10029-10033; Co et al., 1991 Proc Natl Acad Sci USA 88:2869-2873; Co et al., 1992 J Immunol 148:1149-1154), "veneering" (Mark et al., "Derivation of therapeutically active humanized and veneered anti-CD 18 antibodies" and its variations can be used. See the above reference: Metcalf BW, Dalton BJ, eds. Cell Adhesion: From Molecular Definition to Therapeutic Potential. New York: Plenum Press, 1994:291-312). Alternatively, non-human binding domains can be humanized. Other regions such as the hinge region and the constant region domain can also be humanized if they are of non-human origin.

[0094] In the present application, the term "CD3" generally refers to the TCR complex containing CD3. CD3 is a protein complex containing four chains (i.e., CD3γ, CD3δ, and two CD3ε chains) in mammals, which associates with molecules known as T cell receptors (TCRs) and (ζ-chains and η-chains, as (homo- or hetero-dimers)) to generate activation signals in T lymphocytes. The intracellular tails of these CD3 molecules contain a conserved motif known as the "immunoreceptor tyrosine-based activation motif", abbreviated as ITAM, which is essential for the signaling ability of TCRs. The CD3γ-chain, δ-chain, ε-chain, and ζ-η chain are also called CD3-chains (the ζ- and CD3η chains are known as the T cell receptor complex together with the TCR).

[0095] In the present application, the term "HER2" generally refers to ErbB-2, NEU, HER-2, or CD340, and may refer to epidermal growth factor receptor 2 (e.g., human HER2, SwissProt P04626), and any variants, isoforms, and species homologs thereof. HER2 may be expressed natively in cells including tumor cells. It may be expressed by transfection of the HER2 gene or cDNA in cells.

[0096] In the present application, the term "CD20" generally refers to the bone marrow-derived lymphocyte antigen CD20, also known as the bone marrow-derived lymphocyte surface antigen B1 or the leukocyte surface antigen anti-FormerLeu-16, and may include native CD20 from any vertebrate, including mammals such as primates, humans, non-human primates, and rodents (such as mice and rats). The Uniprot accession number PI1836 of the amino acid sequence of human CD20 is shown. CD20 is a kind of hydrophobic transmembrane protein with a molecular weight of about 35 kD and can be expressed in precursor B lymphocytes and mature bone marrow-derived lymphocytes.

[0097] In the present application, the term "tumor-associated antigen" generally refers to a molecule or complex that can be expressed at a higher frequency or density by tumor cells than by non-tumor cells of the same tissue type. A tumor-associated antigen can be an antigen that is not normally expressed by the host, can be a mutation, cleavage, misfolding, or other abnormal expression of a molecule normally expressed by the host, can be the same as a normally expressed molecule but expressed at an abnormally high level, or can be expressed in an abnormal situation or environment. Tumor-associated antigens are, for example, proteins or protein fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, or combinations thereof, or other biological molecules. For example, tumor-associated antigens can include HER2 and / or CD20.

[0098] In the present application, the term "activation" generally means the primary response induced by the binding of a stimulatory molecule (e.g., the TCR / CD3 complex) to its cognate ligand, whereby signal transduction events are mediated. For example, activation can include signal transduction via the TCR / CD3 complex.

[0099] In the present application, the term "interaction between T cells and tumor cells" generally means the reaction between T cells and tumor cells that exerts a negative selective pressure on tumor cells recognized by the immune system (e.g., T cells). Therefore, in tumor cells, recognition by T cells may be reduced by down-regulation of molecules important for antigen processing and presentation and co-stimulation.

[0100] In the present application, the term "treat" as used herein generally refers to a clinical intervention that attempts to change the natural course of an individual or cell being treated. Treatment can be carried out for prevention or during the course of clinical pathology. Desirable effects can include prevention of the occurrence or recurrence of a disease, alleviation of symptoms, reduction of the direct or indirect pathological consequences of a disease, prevention of metastasis, reduction of the rate of progression of a disease, improvement or alleviation of a disease state, remission, or improvement of the prognosis, and the like.

[0101] In this application, the term "subject" as used herein generally refers to a mammal. For example, the subject can be a human. For example, the subject can be a non-human mammal. Non-human mammals can include livestock, sport animals, and pets.

[0102] In this application, the term "multispecificity" generally refers to the antigen-binding characteristics of bispecificity, trispecificity, or multispecificity. A multispecific antigen-binding protein can be specific for different epitopes of one target polypeptide or can include antigen-binding domains specific for epitopes of two or more target polypeptides. The multispecific antigen-binding protein can be a single multifunctional polypeptide or can be a multimeric complex of two or more polypeptides covalently or non-covalently linked to each other. The term "multispecific antigen-binding protein" can include the antibodies of this application and / or antigen-binding fragments of this application. For example, the antibodies and / or antigen-binding fragments of this application can be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent binding, or otherwise) to one or more other molecular entities such as a protein or a fragment thereof. Generate a bispecific or multispecific antigen-binding protein having a second binding specificity. According to this application, the term "multispecific antigen-binding protein" may also include bispecific, trispecific or multispecific antibodies or antigen-binding fragments thereof.

[0103] In this application, the term "isolated nucleic acid molecule or molecule" generally refers to a polymer of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or analogs thereof, that has been isolated from its native environment or artificially synthesized.

[0104] In this application, the term "vector or vectors" generally refers to a nucleic acid vehicle into which a polynucleotide encoding a protein can be inserted and expressed. The genetic material element carried by the vector can be expressed in a host cell by transforming, transducing, or transfecting the host cell with the vector. Specific examples of vectors include artificial chromosomes such as plasmids, phagemids, cosmids, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), PI-derived artificial chromosomes (PACs); phages such as λ phage and M13 phage, and animal viruses. Vectors may contain various elements for controlling expression, such as promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, reporter genes, etc. Furthermore, vectors may also contain an origin of replication. In addition, vectors may contain components that assist in cell entry, such as virus particles, liposomes, protein shells, etc., in addition to these substances.

[0105] In this application, the term "cell" as used herein generally refers to a cell that can be used to carry the vector or vectors of the present disclosure, or to express or produce the antibodies, antigen-binding fragments, or variants of the present disclosure. The cells of the present disclosure can be host cells. The cells can be prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungal cells such as yeast cells and Aspergillus cells, insect cells such as S2 Drosophila cells and Sf9, and other cells suitable for antibody expression such as CHO cells, COS cells, NSO cells, HEK293 cells.

[0106] In this application, the term "conditions enabling expression" as used herein generally refers to the conditions enabling the expression of the multispecific antigen-binding proteins of the present disclosure. For example, the conditions may include incubation time, temperature, culture medium, may depend on the cell type, and can be readily determined by those skilled in the art.

[0107] In this application, the term "T cell engager" generally refers to a bispecific or multispecific antibody against a constant component of the T cell / CD3 complex and a tumor-associated antigen (TAA). By bypassing the normal TCR-MHC interaction that triggers T cell activation, T cell engagers exhibit two advantages: 1) the potential to induce a polyclonal T cell response; and 2) the potential to be unaffected by escape mechanisms involving downregulation of antigen presentation. T cell engagers may be referred to Methods 154(2019)102-117.

[0108] In this application, the term "about" generally refers to a variation in the range of ±0.5% to ±10% of a given value. For example, it can be ±0.5%, ±1%, ±1.5%, ±2%, ±2.5%, ±3%, ±3.5%, ±4%, ±4.5%, ±5%, ±5.5%, ±6%, ±6.5%, ±7%, ±7.5%, ±8%, ±8.5%, ±9%, ±9.5%, or ±10% of the given value.

[0109] Binding moiety to CD3 In this application, the binding moiety to CD3 may include an antibody or an antigen-binding fragment thereof. In this application, the binding moiety to CD3 may be specific for CD3. For example, the binding moiety to CD3 may be an anti-CD3 antibody or an antigen-binding fragment to CD3. The terms "binding moiety to CD3" and "CD3 binding moiety" are used interchangeably in this application.

[0110] In this application, the antibody may be selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0111] In this application, the antigen-binding fragment may include Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv and / or dAb.

[0112] In the present application, the binding moiety to CD3 comprises the amino acid sequence described in any of the HCDR1, HCDR2 and / or HCDR3 regions from the heavy chain variable region VH of SEQ ID NO: 8.

[0113] In the present application, the binding moiety to CD3 may comprise HCDR1-3. HCDR1 may comprise the amino acid sequence described in SEQ ID NO: 4. HCDR2 may comprise the amino acid sequence described in SEQ ID NO: 5. HCDR3 may comprise the amino acid sequence described in SEQ ID NO: 6.

[0114] In the present application, the binding moiety to CD3 comprises the amino acid sequence described in any of the HCDR1, HCDR2 and / or HCDR3 regions from the heavy chain variable region VH of SEQ ID NO: 35.

[0115] In the present application, the binding moiety to CD3 may comprise HCDR1-3. HCDR1 may comprise the amino acid sequence described in SEQ ID NO: 37. HCDR2 may comprise the amino acid sequence described in SEQ ID NO: 38. HCDR3 may comprise the amino acid sequence described in SEQ ID NO: 39.

[0116] In the present application, the binding moiety to CD3 comprises the amino acid sequence described in any of the HCDR1, HCDR2 and / or HCDR3 regions from the heavy chain variable region VH of SEQ ID NO: 43.

[0117] In the present application, the binding moiety to CD3 may comprise HCDR1-3. HCDR1 may comprise the amino acid sequence described in SEQ ID NO: 45. HCDR2 may comprise the amino acid sequence described in SEQ ID NO: 46. HCDR3 may comprise the amino acid sequence described in SEQ ID NO: 47.

[0118] In the present application, the binding moiety to CD3 comprises the amino acid sequence described in any of the HCDR1, HCDR2 and / or HCDR3 regions from the heavy chain variable region VH of SEQ ID NO: 51.

[0119] In the present application, the binding portion to CD3 may include HCDR1-3. HCDR1 may include the amino acid sequence set forth in SEQ ID NO: 53. HCDR2 may include the amino acid sequence set forth in SEQ ID NO: 54. HCDR3 may include the amino acid sequence set forth in SEQ ID NO: 55.

[0120] In the present application, the binding portion to CD3 may include VH. For example, VH of the binding portion to CD3 may include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0121] In the present application, VH of the binding portion to CD3 includes the amino acid sequence set forth in SEQ ID NO: 8. In the present application, VH of the binding portion to CD3 includes the amino acid sequence set forth in SEQ ID NO: 35. In the present application, VH of the binding portion to CD3 includes the amino acid sequence set forth in SEQ ID NO: 43. In the present application, VH of the binding portion to CD3 includes the amino acid sequence set forth in SEQ ID NO: 51.

[0122] In the present application, the binding portion to CD3 is humanized. In the present application, VH of the binding portion to CD3 includes the amino acid sequence set forth in SEQ ID NO: 27. In the present application, VH of the binding portion to CD3 includes the amino acid sequence set forth in SEQ ID NO: 28.

[0123] In the present application, the binding portion to CD3 may include an antibody heavy chain constant region.

[0124] In the present application, the antibody heavy chain constant region includes an IgG constant region.

[0125] In the present application, the antibody heavy chain constant region may include the amino acid sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 26.

[0126] In the present application, the binding portion to CD3 includes the amino acid sequence set forth in any one of the LCDR1, LCDR2, and / or LCDR3 regions from the light chain variable region VL of SEQ ID NO: 7.

[0127] In the present application, the binding moiety to CD3 may include LCDR1 to 3. LCDR1 may include the amino acid sequence set forth in SEQ ID NO: 1. LCDR2 may include the amino acid sequence set forth in SEQ ID NO: 2. LCDR3 may include the amino acid sequence set forth in SEQ ID NO: 3.

[0128] In the present application, the binding moiety to CD3 includes the amino acid sequence described in any of the LCDR1, LCDR2, and / or LCDR3 regions from the light chain variable region VL of SEQ ID NO: 36.

[0129] In the present application, the binding moiety to CD3 may include LCDR1 to 3. LCDR1 may include the amino acid sequence set forth in SEQ ID NO: 40. LCDR2 may include the amino acid sequence set forth in SEQ ID NO: 41. LCDR3 may include the amino acid sequence set forth in SEQ ID NO: 42.

[0130] In the present application, the binding moiety to CD3 includes the amino acid sequence described in any of the LCDR1, LCDR2, and / or LCDR3 regions from the light chain variable region VL of SEQ ID NO: 44.

[0131] In the present application, the binding moiety to CD3 may include LCDR1 to 3. LCDR1 may include the amino acid sequence set forth in SEQ ID NO: 48. LCDR2 may include the amino acid sequence set forth in SEQ ID NO: 49. LCDR3 may include the amino acid sequence set forth in SEQ ID NO: 50.

[0132] In the present application, the binding moiety to CD3 includes the amino acid sequence described in any of the LCDR1, LCDR2, and / or LCDR3 regions from the light chain variable region VL of SEQ ID NO: 52.

[0133] In the present application, the binding moiety to CD3 may include LCDR1 to 3. LCDR1 may include the amino acid sequence set forth in SEQ ID NO: 56. LCDR2 may include the amino acid sequence set forth in SEQ ID NO: 57. LCDR3 may include the amino acid sequence set forth in SEQ ID NO: 58.

[0134] In the present application, the binding portion to CD3 may include VL. For example, the VL of the binding portion to CD3 may include framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0135] In the present application, the VL of the binding portion to CD3 includes the amino acid sequence set forth in SEQ ID NO: 7. In the present application, the VL of the binding portion to CD3 includes the amino acid sequence set forth in SEQ ID NO: 36. In the present application, the VL of the binding portion to CD3 includes the amino acid sequence set forth in SEQ ID NO: 44. In the present application, the VL of the binding portion to CD3 includes the amino acid sequence set forth in SEQ ID NO: 52.

[0136] In the present application, the binding portion to CD3 is humanized. In the present application, the VL of the binding portion to CD3 includes the amino acid sequence set forth in SEQ ID NO: 31.

[0137] In the present application, the binding portion to CD3 may include an antibody light chain constant region.

[0138] In the present application, the antibody light chain constant region may include an IgK constant region.

[0139] In the present application, the antibody light chain constant region includes the amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 25.

[0140] In the present application, the binding portion to CD3 may include HCDR1-3 and LCDR1-3. HCDR1 may include the amino acid sequence set forth in SEQ ID NO: 4. HCDR2 may include the amino acid sequence set forth in SEQ ID NO: 5. HCDR3 may include the amino acid sequence set forth in SEQ ID NO: 6. LCDR1 may include the amino acid sequence set forth in SEQ ID NO: 1. LCDR2 may include the amino acid sequence shown in SEQ ID NO: 2, and LCDR3 may include the amino acid sequence set forth in SEQ ID NO: 3.

[0141] In the present application, the binding portion to CD3 may include HCDR1-3 and LCDR1-3. HCDR1 may include the amino acid sequence set forth in SEQ ID NO: 37. HCDR2 may include the amino acid sequence set forth in SEQ ID NO: 38. HCDR3 may include the amino acid sequence set forth in SEQ ID NO: 39. LCDR1 may include the amino acid sequence set forth in SEQ ID NO: 40. LCDR2 may include the amino acid sequence set forth in SEQ ID NO: 41. LCDR3 may include the amino acid sequence set forth in SEQ ID NO: 42.

[0142] In the present application, the binding portion to CD3 may include HCDR1-3 and LCDR1-3. HCDR1 may include the amino acid sequence set forth in SEQ ID NO: 45. HCDR2 may include the amino acid sequence set forth in SEQ ID NO: 46. HCDR3 may include the amino acid sequence set forth in SEQ ID NO: 47. LCDR1 may include the amino acid sequence set forth in SEQ ID NO: 48. LCDR2 may include the amino acid sequence set forth in SEQ ID NO: 59. LCDR3 may include the amino acid sequence set forth in SEQ ID NO: 50.

[0143] In the present application, the binding portion to CD3 may include HCDR1-3 and LCDR1-3. HCDR1 may include the amino acid sequence set forth in SEQ ID NO: 53. HCDR2 may include the amino acid sequence set forth in SEQ ID NO: 54. HCDR3 may include the amino acid sequence set forth in SEQ ID NO: 55. LCDR1 may include the amino acid sequence set forth in SEQ ID NO: 56. LCDR2 may include the amino acid sequence shown in SEQ ID NO: 57, and LCDR3 may include the amino acid sequence shown in SEQ ID NO: 48.

[0144] In the present application, the binding moiety to CD3 may include VH and VL. VH may include the amino acid sequence set forth in SEQ ID NO: 8. And VL may include the amino acid sequence set forth in SEQ ID NO: 7. VH may include the amino acid sequence shown in SEQ ID NO: 35, and VL may include the amino acid sequence shown in SEQ ID NO: 36. VH may include the amino acid sequence shown in SEQ ID NO: 43, and VL may include the amino acid sequence shown in SEQ ID NO: 44. VH may include the amino acid sequence shown in SEQ ID NO: 51, and VL may include the amino acid sequence shown in SEQ ID NO: 52.

[0145] In the present application, the binding moiety to CD3 may include humanized VH and humanized VL. VH may include the amino acid sequence shown in SEQ ID NO: 27, and VL may include the amino acid sequence shown in SEQ ID NO: 31. VH may include the amino acid sequence shown in SEQ ID NO: 28, and VL may include the amino acid sequence shown in SEQ ID NO: 31.

[0146] In the present application, the multispecific antigen-binding protein may include a constant region derived from an IgA, IgG, IgD, IgE, or IgM antibody.

[0147] Reference antibody In the present disclosure, the binding moiety to CD3 may compete with a reference antibody with respect to binding to CD3.

[0148] In some cases, the reference antibody may include at least one HCDR region from the heavy chain variable region VH of SEQ ID NO: 8 and at least one LCDR region from the light chain variable region VL of SEQ ID NO: 7. In some cases, the reference antibody may include at least one HCDR region from the heavy chain variable region VH of SEQ ID NO: 35 and at least one LCDR region from the light chain variable region VL of SEQ ID NO: 36. In some cases, the reference antibody may include at least one HCDR region from the heavy chain variable region VH of SEQ ID NO: 43 and at least one LCDR region from the light chain variable region VL of SEQ ID NO: 44. In some cases, the reference antibody may include at least one HCDR region from the heavy chain variable region VH of SEQ ID NO: 51 and at least one LCDR region from the light chain variable region VL of SEQ ID NO: 52.

[0149] In the present disclosure, the reference antibody may include HCDR1-3. HCDR1 may include the amino acid sequence set forth in SEQ ID NO: 4. HCDR2 may include the amino acid sequence set forth in SEQ ID NO: 5. HCDR3 may include the amino acid sequence set forth in SEQ ID NO: 6. In some cases, the reference antibody may include LCDR1-3. LCDR1 may include the amino acid sequence set forth in SEQ ID NO: 1. LCDR2 may include the amino acid sequence set forth in SEQ ID NO: 2. LCDR3 may include the amino acid sequence set forth in SEQ ID NO: 3.

[0150] In the present disclosure, the reference antibody may include HCDR1-3. HCDR1 may include the amino acid sequence set forth in SEQ ID NO: 37. HCDR2 may include the amino acid sequence set forth in SEQ ID NO: 38. HCDR3 may include the amino acid sequence set forth in SEQ ID NO: 39. In some cases, the reference antibody may include LCDR1-3. LCDR1 may include the amino acid sequence set forth in SEQ ID NO: 40. LCDR2 may include the amino acid sequence set forth in SEQ ID NO: 41. LCDR3 may include the amino acid sequence set forth in SEQ ID NO: 42.

[0151] In the present disclosure, the reference antibody may include HCDR1-3. HCDR1 may include the amino acid sequence set forth in SEQ ID NO: 45. HCDR2 may include the amino acid sequence set forth in SEQ ID NO: 46. HCDR3 may include the amino acid sequence set forth in SEQ ID NO: 47. Optionally, the reference antibody may include LCDR1-3. LCDR1 may include the amino acid sequence set forth in SEQ ID NO: 48. LCDR2 may include the amino acid sequence set forth in SEQ ID NO: 49. LCDR3 may include the amino acid sequence set forth in SEQ ID NO: 50.

[0152] In the present disclosure, the reference antibody may include HCDR1-3. HCDR1 may include the amino acid sequence set forth in SEQ ID NO: 53. HCDR2 may include the amino acid sequence set forth in SEQ ID NO: 54. HCDR3 may include the amino acid sequence set forth in SEQ ID NO: 55. Optionally, the reference antibody may include LCDR1-3. LCDR1 may include the amino acid sequence set forth in SEQ ID NO: 56. LCDR2 may include the amino acid sequence set forth in SEQ ID NO: 57. LCDR3 may include the amino acid sequence set forth in SEQ ID NO: 58.

[0153] In the present disclosure, the reference antibody may include VH and VL. VH may include the amino acid sequence set forth in SEQ ID NO: 8. And VL may include the amino acid sequence set forth in SEQ ID NO: 7. VH may include the amino acid sequence shown in SEQ ID NO: 35. VL may include the amino acid sequence shown in SEQ ID NO: 36. VH may include the amino acid sequence shown in SEQ ID NO: 43. L may include the amino acid sequence shown in SEQ ID NO: 44. VH may include the amino acid sequence shown in SEQ ID NO: 51. VL may include the amino acid sequence shown in SEQ ID NO: 52.

[0154] In the present disclosure, the reference antibody may include a heavy chain constant region and a light chain constant region. The heavy chain constant region may include the amino acid sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 26, and the light chain constant region may include the amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 25.

[0155] The binding moiety to CD3 may also include homologs or variants thereof that have substantially the same function / characteristics. In some cases, the homolog or variant may be a polypeptide that differs from the antigen-binding protein by at least one amino acid. For example, the homolog or variant may be a polypeptide different from the antigen-binding protein and may differ by addition, deletion, or substitution of one or more amino acids, such as 1-50, 1-40, 1-30, 1-20, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, or 1-2 amino acids.

[0156] Binding moiety to tumor-associated antigen (TAA) In the present application, the multispecific antigen-binding protein may include a binding moiety to tumor-associated antigen (TAA).

[0157] In this application, the TAAs are selected from the following target groups: EPCAM, CCR5, CD19, HER2, HER3neu, HER3, HER4, EGFR, PSMA, CEA, MUC1, MUC2, MUC3, MUC4, MUC5, MUC7, βhCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3, 9-O-Acetyl-GD3, GM2, globoH, fucosylGM1, PolySA, GD2, RON, c-Met, CEACAM-6, PCTA-1, PSA, PAP, ALCAM (CD166), PECAM-1, CD151, MAGE-1, TROP2, IGF1R, TGFBR2, GHRHR, GHR, IL-6R, gp130, TNFR2, OSMRp, Patched-1, Frizzled.Robol, LTpR, CD26, CD27, CD44, CD80, CD81, CD86, CD100, CXCR4, SAS, BCMA, TWEAKR / Fnl4, FGFR4, VEGFR1, VEGFR2, SSX1, and SSX2, carbonic anhydrase IX (MN / CAIX), CD44v6, sonic hedgehog (Shh), Wue-1, plasma cell antigen, (membrane-bound) IgE, melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, TNFα precursor, STEAP, mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6; desmoglein 4, E-cadherin neoepitope, fetal acetylcholine receptor, CD25, CA19-9 marker, CA-125 marker, Mullerian inhibitor substance (MIS) receptor type II, sTn (sialylated Tn antigen, TAG72), FAP (fibroblast activation antigen), endosialin, EGFRvIII, L6, SAS, CD63, TF antigen, Cora antigen, CD7, CD79b, CD22, Igα, Igβ, gp100, MT-MMPs, FI9 antigen, CO-29, EphA2.

[0158] In this application, the multispecific antigen-binding protein may comprise a binding moiety for HER2.

[0159] For example, the binding moiety for HER2 can be an anti-HER2 antibody or an antigen-binding fragment for HER2.

[0160] In the present application, the antibody can be selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0161] In the present application, the antigen-binding fragment can include Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv and / or dAb.

[0162] In the present application, the binding portion to HER2 can include the amino acid sequence described in any of the HCDR1, HCDR2 and / or HCDR3 regions from the heavy chain variable region VH of SEQ ID NO: 16 or SEQ ID NO: 20.

[0163] In the present application, the binding portion to HER2 can include HCDR1-3. HCDR1 can include the amino acid sequence described in SEQ ID NO: 12. HCDR2 can include the amino acid sequence described in SEQ ID NO: 13. HCDR3 can include the amino acid sequence described in SEQ ID NO: 14.

[0164] In the present application, the binding portion to HER2 can include VH. For example, the VH of the binding portion to HER2 can include framework regions H-FR1, H-FR2, H-FR3 and H-FR4.

[0165] In the present application, the VH of the binding portion to HER2 can include the amino acid sequence described in SEQ ID NO: 16 or SEQ ID NO: 20.

[0166] In the present application, the binding portion to HER2 can include the constant region of the antibody heavy chain.

[0167] In the present application, the constant region of the antibody heavy chain includes the IgG constant region.

[0168] In the present application, the constant region of the antibody heavy chain can include the amino acid sequence described in SEQ ID NO: 18 or SEQ ID NO: 26.

[0169] In the present application, the binding portion to HER2 may include the amino acid sequences described in any of the LCDR1, LCDR2, and / or LCDR3 regions from the variable light chain region VL of SEQ ID NO: 15 or SEQ ID NO: 19.

[0170] In the present application, the binding portion to HER2 may include LCDR1-3. LCDR1 may include the amino acid sequence described in SEQ ID NO: 9. LCDR2 may include the amino acid sequence described in SEQ ID NO: 10. LCDR3 may include the amino acid sequence described in SEQ ID NO: 11.

[0171] In the present application, the binding portion to HER2 may include VL. For example, the VL of the binding portion to HER2 may include the framework regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0172] In the present application, the binding portion to HER2 may include the antibody light chain constant region.

[0173] In the present application, the antibody light chain constant region may include the lgk constant region.

[0174] In the present application, the antibody light chain constant region includes the amino acid sequence described in SEQ ID NO: 17 or SEQ ID NO: 25.

[0175] In the present application, the binding portion to HER2 may include HCDR1-3 and LCDR1-3. HCDR1 may include the amino acid sequence described in SEQ ID NO: 12. HCDR2 may include the amino acid sequence described in SEQ ID NO: 13. HCDR3 may include the amino acid sequence described in SEQ ID NO: 14. LCDR1 may include the amino acid sequence described in SEQ ID NO: 9. LCDR2 may include the amino acid sequence described in SEQ ID NO: 10. LCDR3 may include the amino acid sequence described in SEQ ID NO: 11.

[0176] In the present application, the binding portion to HER2 may include VH and VL. VH may include the amino acid sequence described in SEQ ID NO: 16, and VL may include the amino acid sequence described in SEQ ID NO: 15.

[0177] In this application, the binding portion to HER2 may include VH and VL. VH may include the amino acid sequence set forth in SEQ ID NO: 20, and VL may include the amino acid sequence set forth in SEQ ID NO: 19.

[0178] In this application, the multi-specific antigen-binding protein may include a binding portion to CD20.

[0179] For example, the binding portion to CD20 may be an anti-CD20 antibody or an antigen-binding fragment to CD20.

[0180] In this application, the antibody may be selected from monoclonal antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies.

[0181] In this application, the antigen-binding fragment may include Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv and / or dAb.

[0182] In this application, the binding portion to CD20 includes the amino acid sequence set forth in any of the HCDR1, HCDR2 and / or HCDR3 regions from the heavy chain variable region VH of SEQ ID NO: 24.

[0183] In this application, VH of the binding portion to CD20 includes the amino acid sequence set forth in SEQ ID NO: 24.

[0184] In this application, the binding portion to CD20 may include an antibody heavy chain constant region.

[0185] In this application, the antibody heavy chain constant region includes an IgG constant region.

[0186] In this application, the antibody heavy chain constant region may include the amino acid sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 26.

[0187] In the present application, the binding moiety to CD20 includes the amino acid sequence described in any of the LCDR1, LCDR2, and / or LCDR3 regions from the variable light chain region VL of SEQ ID NO: 23.

[0188] In the present application, the binding moiety to CD20 may include the constant region of the antibody light chain.

[0189] In the present application, the constant region of the antibody light chain may include the lgK constant region.

[0190] In the present application, the constant region of the antibody light chain includes the amino acid sequence described in SEQ ID NO: 17 or SEQ ID NO: 25.

[0191] In the present application, the binding moiety to CD20 may include VH and VL. VH may include the amino acid sequence described in SEQ ID NO: 24, and VL may include the amino acid sequence shown in SEQ ID NO: 23.

[0192] Multi-specific antigen-binding protein In the present application, the multi-specific antigen-binding protein can be an antibody or its antigen-binding fragment. For example, the multi-specific antigen-binding protein can be an intact antibody (e.g., monoclonal antibody, chimeric antibody, humanized antibody, and / or fully human antibody). For example, the multi-specific antigen-binding protein can be an IgG antibody. For example, the multi-specific antigen-binding protein can be its antigen-binding fragment. For example, the multi-specific antigen-binding protein can include Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv, and / or dAb. For example, the multi-specific antigen-binding protein can be F(ab’)2.

[0193] In the present application, the binding moiety to CD3 may include a first polypeptide chain and a second polypeptide chain. The first polypeptide chain may include the variable heavy chain region VH of an antibody that binds to CD3, and the second polypeptide chain may include the variable light chain region VL of an antibody that binds to CD3.

[0194] In the present application, the heavy chain variable region VH of the antibody binding to CD3 may contain the amino acid sequence set forth in SEQ ID NO: 8. The light chain variable region VL of the antibody binding to CD3 may contain the amino acid sequence set forth in SEQ ID NO: 7.

[0195] In the present application, the first polypeptide chain may contain the light chain of the antibody binding to CD3. Also, in the present application, the second polypeptide chain may contain the heavy chain of the antibody binding to CD3.

[0196] In the present application, the first polypeptide chain may contain VL of the antibody binding to CD3 and the light chain constant region. Also, in the present application, the second polypeptide chain may contain VH of the antibody binding to CD3, CHI of the heavy chain constant region, and the hinge.

[0197] For example, the antibody binding to CD3 may include the CD3 antibody of the present application.

[0198] In the present application, the light chain constant region may be derived from IgG. For example, it may be derived from IgG2 or murine IgG2 (for example, it may be derived from IgG2a).

[0199] In the present application, the heavy chain constant region (for example, CHI) may be derived from IgG. For example, it may be derived from IgG2 or rat IgG2 (for example, it may be derived from IgG2b). In the present application, the hinge may be derived from IgG. For example, it may be derived from IgG2 or rat IgG2 (for example, it may be derived from IgG2b).

[0200] In the present application, the second polypeptide chain may contain a linker at the C-terminus of the hinge. For example, the linker may be a flexible linker. For example, the linker is GGGGS.

[0201] In the present application, the second polypeptide chain may include a tag at its C-terminus. For example, the tag can be any tag that can be used for isolation and / or purification of a protein (e.g., the second polypeptide chain and / or the binding moiety to CD3). For example, the tag can be a His tag.

[0202] In the present application, the binding moiety to TAA may include a third polypeptide chain and a fourth polypeptide chain. The third polypeptide chain may constitute the heavy chain variable region VH of an antibody that binds to TAA, and the fourth polypeptide chain may constitute the light chain variable region VL of an antibody that binds to TAA.

[0203] In the present application, the third polypeptide chain constitutes the heavy chain variable region VH of an antibody that binds to HER2, and the fourth polypeptide chain may include the light chain variable region VL of an antibody that binds to HER2.

[0204] In the present application, the heavy chain variable region VH of an antibody that binds to HER2 may include the amino acid sequence set forth in SEQ ID NO: 16, and the light chain variable region VL of an antibody that binds to HER2 may include the amino acid sequence set forth in SEQ ID NO: 15. Alternatively, the heavy chain variable region VH of an antibody that binds to HER2 may include the amino acid sequence set forth in SEQ ID NO: 20, and the light chain variable region VL of an antibody that binds to HER2 may include the amino acid sequence set forth in SEQ ID NO: 19.

[0205] In the present application, the third polypeptide chain constitutes the heavy chain variable region VH of an antibody that binds to CD20, and the fourth polypeptide chain may include the light chain variable region VL of an antibody that binds to CD20.

[0206] In the present application, the heavy chain variable region VH of an antibody that binds to CD20 may include the amino acid sequence set forth in SEQ ID NO: 24, and the light chain variable region VL of an antibody that binds to CD20 may include the amino acid sequence set forth in SEQ ID NO: 23.

[0207] In the present application, the third polypeptide chain may include the light chain of an antibody that binds to HER2. Also, in the present application, the fourth polypeptide chain may include the heavy chain of an antibody that binds to HER2.

[0208] Alternatively, in the present application, the third polypeptide chain may include the VL and the light chain constant region of an antibody that binds to HER2. And, in the present application, the fourth polypeptide chain may include the VH of an antibody that binds to HER2, CHI of the heavy chain constant region, and the hinge.

[0209] For example, the antibody that binds to HER2 may include the HER2 antibody of the present application.

[0210] In the present application, the third polypeptide chain may include the light chain of an antibody that binds to CD20. Also, in the present application, the fourth polypeptide chain may include the heavy chain of an antibody that binds to CD20.

[0211] Alternatively, in the present application, the third polypeptide chain may include the VL and the light chain constant region of an antibody that binds to CD20. And, in the present application, the fourth polypeptide chain may include the VH of an antibody that binds to CD20, CHI of the heavy chain constant region, and the hinge.

[0212] For example, the antibody that binds to CD20 may include the CD20 antibody of the present application.

[0213] In the present application, the light chain constant region may be derived from IgG. For example, it may be derived from IgG2 or murine IgG2 (for example, it may be derived from IgG2a).

[0214] In the present application, the heavy chain constant region (for example, CHI) may be derived from IgG. For example, it may be derived from IgG2 or murine IgG2 (for example, it may be derived from IgG2a). In the present application, the hinge may be derived from IgG. For example, it may be derived from IgG2 or murine IgG2 (for example, it may be derived from IgG2a).

[0215] In the present application, the fourth polypeptide chain may include a linker at the C-terminus of the hinge. For example, the linker may be a flexible linker. For example, the linker is GGGGS.

[0216] In the present application, the fourth polypeptide chain may include a tag at its C-terminus. For example, the tag may be any tag that can be used for the isolation and / or purification of a protein (e.g., the fourth polypeptide chain, and / or the binding moiety to HER2 or CD20). For example, the tag may be strep-tagII.

[0217] In the present application, the multispecific antigen-binding protein may include one first polypeptide chain, one second polypeptide chain, one third polypeptide chain, and one fourth polypeptide chain. In the multispecific antigen-binding protein, the fourth polypeptide chain may correspond to the third polypeptide chain, and the third polypeptide chain and the fourth polypeptide chain may be capable of binding to the same TAA.

[0218] In the present application, the multispecific antigen-binding protein may be produced by recombinant expression in a host cell.

[0219] In the present application, the host cell producing the multispecific antigen-binding protein may be a bacterial cell, a fungal cell, a plant cell, a mammalian cell, or a virus.

[0220] In the present application, the bacterial cell may be Escherichia coli.

[0221] In the present application, the fungal cell may be a yeast cell.

[0222] In the present application, the mammalian cell may be a CHO, NSO, BHK, or HEK293 cell.

[0223] In the present application, the multispecific antigen-binding protein may be produced by hybridoma cells.

[0224] In the present application, the hybridoma cells can be derived from mice, rats or rabbits.

[0225] In the present application, the format of the multispecific antigen-binding protein can be a bispecific antibody, a bispecific diabody, a bispecific scFv, a TandAb, a trivalent binding molecule, or a tetravalent binding molecule.

[0226] In the present application, the multispecific antigen-binding protein can include a constant region derived from an IgA, IgG, IgD, IgE, or IgM antibody.

[0227] In the present application, the protein can also include a polypeptide having at least 80% sequence identity with the protein. For example, the homolog or variant can be a polypeptide having 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) sequence identity with the protein.

[0228] The term "percent (%) sequence identity" as used in the context of the polypeptide sequences identified in this application generally can refer to "the percentage of amino acid residues or nucleotides in a query sequence that are identical to those of a second reference polypeptide sequence or a portion thereof, after aligning the sequences and introducing gaps as necessary". Optionally, the maximum percent of sequence identity is achieved without considering conservative substitutions as part of the sequence identity. Alignments for the purpose of determining percent amino acid / nucleotide sequence identity can be achieved in a variety of ways within the skill of the art using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software. One of ordinary skill in the art can determine appropriate parameters for measuring the alignment, including any algorithm necessary to achieve the maximum alignment over the entire length of the sequences being compared. The percent identity can be measured over the length of the defined polypeptide / polynucleotide sequence as a whole or over a shorter length, for example, over the length of a fragment taken from a larger defined polypeptide / polynucleotide sequence. It is understood that any fragment length supported by the sequences shown in this specification, tables, figures or sequence listings can be used to describe the length over which the percent identity can be measured.

[0229] Any of the amino acid sequences of 1 to 16 (and / or any one or more fragments and / or derivatives thereof) can also be replaced with other amino acids as desired by one of ordinary skill in the art. For example, one of ordinary skill in the art can make conservative substitutions by replacing a particular amino acid with another amino acid as shown in Table 1 below. The particular amino acid substitutions selected may depend on the position of the site selected. Such conservative amino acid substitutions may involve replacing a natural amino acid residue with a non-natural residue such that there is little or no effect on the size, polarity, charge, hydrophobicity or hydrophilicity of the amino acid residue at that position.

[0230]

Table 1

[0231] Nucleic acids, vectors, cells In another aspect, the present application provides one or more isolated nucleic acid molecules encoding the multispecific antigen-binding protein of the present application.

[0232] In another aspect, the present application provides a vector that may contain the isolated nucleic acid molecule of the present application.

[0233] In another aspect, the present application provides a cell that may contain the isolated nucleic acid molecule of the present application and / or the vector of the present application.

[0234] In another aspect, the present application provides a method for preparing the multispecific antigen-binding protein of the present application, which method may include culturing the cells of the present application under conditions that allow the expression of the multispecific antigen-binding protein of the present application.

[0235] The isolated nucleic acid may comprise one or more nucleic acid molecules each encoding an antigen-binding protein (e.g., a binding moiety to CD3 and / or a binding moiety to a TAA). For example, the isolated nucleic acid may comprise at least two nucleic acid molecules. One encodes an antibody heavy chain or a fragment thereof, and the other encodes an antibody light chain or a fragment thereof.

[0236] Isolated nucleic acid or nucleic acids can be synthesized using recombinant techniques well known in the art. For example, isolated nucleic acid or nucleic acids can be synthesized using an automated DNA synthesizer. Standard recombinant DNA and molecular cloning techniques include those described by Sambrook, J., Fritsch, E.F., and Maniatis, T. in Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis), T.J., Silhavy, M.L., Bennan, L.W., and Enquist, L.W. in "Experiments with Gene Fusions", Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1984), and Ausubel, F.M. et al. in Current Protocols in Molecular Biology, Greene Publishing Assoc., and Wiley-Interscience (1987). Briefly, the nucleic acids of interest can be prepared from genomic DNA fragments, cDNA, and RNA, all of which can be directly extracted from cells or recombinantly generated by various amplification processes including but not limited to PCR and RT-PCR.

[0237] In the direct chemical synthesis of nucleic acids, it generally involves the sequential addition of 3'- and 5'-blocked nucleotide monomers to the 5'-hydroxyl group at the end of the growing nucleotide polymer chain, and each addition can be affected by a nucleophilic attack at the 5' end. The hydroxyl group of the growing chain at the 3' position of the added monomer. Typically, they are phosphorus derivatives such as phosphotriesters and phosphoramidites. For example, Matteuci et al., Tet. Lett. 521:719 (1980); U.S. Patent No. 4,500,707 to Caruthers et al.; and U.S. Patent Nos. 5,436,327 and 5,700,637 to Southern et al.

[0238] The vector can be any linear nucleic acid, plasmid, phagemid, cosmid, RNA vector, viral vector, etc. Non-limiting examples of viral vectors can include retroviruses, adenoviruses, and adeno-associated viruses. In the present application, the vector can be an expression vector, such as a plasmid.

[0239] An expression vector may be suitable for use in a particular type of host cell but may not be suitable for use in other types of host cells. For example, an expression vector can be introduced into a host organism, and then the survival rate and expression of any gene / polynucleotide contained in the vector can be monitored.

[0240] The expression vector may also contain one or more selectable marker genes that, upon expression, confer one or more phenotypic traits useful for selecting or identifying host cells carrying the expression vector. Non-limiting examples of selectable markers suitable for eukaryotic cells include dihydrofolate reductase and neomycin resistance.

[0241] The subject vector can be stably or transiently introduced into host cells by various established techniques. For example, one method can involve calcium chloride treatment where the expression vector is introduced via calcium precipitates. Other salts, such as calcium phosphate, can be used following a similar procedure. Additionally, electroporation (i.e., the application of an electric current to increase the permeability of cells to nucleic acids) can also be used. Other examples of transformation methods include microinjection, DEAE dextran-mediated transformation, heat shock in the presence of lithium acetate, etc. Lipid complexes, liposomes, dendrimers can also be used to transfect host cells.

[0242] In the present application, a cell can express the multispecific antigen-binding protein of the present disclosure. The cell can be a eukaryotic cell or a prokaryotic cell. Appropriate cells can be transformed or transfected with the nucleic acid or vector of the present application and used for the expression and / or secretion of the multispecific antigen-binding protein of the present application. For example, the cell can be a HEK293 cell, other bacterial host cells, yeast cells, or various higher eukaryotic cells.

[0243] In the present application, the method may further optionally include harvesting the multispecific antigen-binding protein of the present application.

[0244] Pharmaceutical Compositions, Uses and Methods In another aspect, the present application provides a pharmaceutical composition comprising the multispecific antigen-binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, and / or the cell of the present application, and optionally a pharmaceutically acceptable adjuvant.

[0245] In another aspect, the present application provides a method of using the vector of the present application, the cell of the present application, and / or the pharmaceutical composition of the present application in the manufacture of a medicament for preventing, alleviating and / or treating a tumor; a method of using the vector of the present application, the cell of the present application, and / or the pharmaceutical composition of the present application in the manufacture of a medicament for preventing, alleviating and / or treating a tumor.

[0246] In another aspect, the present application provides the multispecific antigen-binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, and / or the pharmaceutical composition of the present application for use in preventing, alleviating and / or treating a tumor.

[0247] In another aspect, the present application provides a method for preventing, alleviating and / or treating a tumor, comprising administering to a subject in need thereof the multispecific antigen-binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, and / or the pharmaceutical composition of the present application.

[0248] In this application, "pharmaceutically acceptable adjuvant" may include any preservative, wetting agent, emulsifying agent, and dispersing agent. Prevention of the presence of microorganisms can be ensured by both the sterilization procedures described above and the inclusion of various antibacterial and antifungal agents. It may also be desirable to include isotonic agents in the composition. Furthermore, by including an agent that delays absorption, the absorption of an injectable pharmaceutical form can be prolonged.

[0249] The pharmaceutical composition is typically sterile and can be stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier can be, for example, a solvent or dispersion medium including water, ethanol, and their appropriate mixtures. Appropriate fluidity can be maintained, for example, by the use of a coating. Sustained absorption of an injectable composition can be brought about by including an agent that delays absorption in the composition.

[0250] The pharmaceutical composition of this application may include other compounds, drugs, and / or agents used in the treatment of tumors.

[0251] In this application, tumors can include solid tumors and / or b100d tumors. For example, b100d tumors can include B-cell lymphoma and / or leukemia, such as acute T-cell leukemia, or human B-cell lymphoma. For example, solid tumors include breast cancer.

[0252] In this application, preventing, alleviating, and / or treating a tumor can include inhibiting and / or reducing the growth of the tumor, and inhibiting the progression of the tumor. For example, preventing, alleviating, and / or treating a tumor can also include administering another agent, such as any agent known in the art for treating tumors.

[0253] The pharmaceutical composition of the present application and / or the multispecific antigen-binding protein of the present application can be administered to a subject in need in any reasonable dosage, regimen, and / or manner, and all these administration conditions can be adjusted according to the type of tumor, the condition of the subject, etc.

[0254] In another aspect, the present application provides the use of the binding moiety to CD3 of the present application and / or the multispecific antigen-binding protein of the present application in the manufacture of a T cell engager.

[0255] In the present application, the T cell engager can comprise bispecific T cell engagers (BiTEs). The T cell engager can induce the host immune system and may exhibit cytotoxic activity of T cells more specific to tumor cells. The T cell engager may be safer as a pharmaceutical for treating tumors. And the T cell engager may exhibit more effective killing against tumor cells.

[0256] In another aspect, the present application provides a method of activating T cells, which comprises administering the multispecific antigen-binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, and / or the pharmaceutical composition of the present application to a subject in need.

[0257] In another aspect, the present application provides a method of targeting CD3 expressed on T cells, which comprises administering the multispecific antigen-binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, and / or the pharmaceutical composition of the present application.

[0258] In another aspect, the present application provides a method of promoting the interaction between T cells and tumor cells, which comprises administering the multispecific antigen-binding protein of the present application, the isolated nucleic acid molecule of the present application, the vector of the present application, the cell of the present application, and / or the pharmaceutical composition of the present application.

[0259] In another aspect, the present application provides methods for treating squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer (NSCLC), lung adenocarcinoma, squamous cell lung cancer, peritoneal cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal cancer, esophageal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, urinary tract cancer, hepatocellular carcinoma (HCC), anal cancer, penile cancer, head and neck cancer, etc.

Examples

[0260] The following examples are described to provide a complete disclosure and description of the manufacturing and usage methods of the present invention to those skilled in the art, and are not intended to limit the scope of what the inventors regard as their invention. They are not intended to represent that they are all the experiments or the only experiments in which the following experiments were performed. Efforts have been made to ensure the accuracy of the numerical values (amounts, temperatures, etc.) used, but some experimental errors and deviations need to be taken into account. Unless otherwise specified, parts are parts by weight, molecular weight is weight-average molecular weight, temperature is in degrees Celsius, pressure is atmospheric pressure or near atmospheric pressure. Standard abbreviations may be used. For example, bp, base pair(s). kb, kilobase(s). pL, picoliter(s). s or sec, second(s). min, minute(s). h or hr, hour(s). aa, amino acid(s). nt, nucleotide(s). i.m., intramuscularly. i.p., intraperitoneally. s.c., subcutaneously, etc.

[0261] Example 1 Preparation and Screening of Hybridoma HER2-CD3 Bispecific Antibody A composition containing the HER2 antigen and the CD3 antigen was injected into mice and rats respectively, stimulating the proliferation of B lymphocytes in the spleen and specifically secreting antibodies against HER2 and CD3. The spleens of mice and rats were surgically removed after multiple immunizations, and polyethylene glycol (PEG) was used for cell fusion with myeloma cells. Subsequently, the fused cells were screened through HAT selection medium. The HAT selection medium is composed of three components: hypoxanthine (H), aminopterin (A), and thymine (T). Aminopterin in the HAT selection medium is an inhibitor of dihydrofolate reductase and can effectively block the endogenous pathway of DNA synthesis. Myeloma cells before fusion cannot produce antibodies and lack the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) gene, so they are highly sensitive to the HAT selection medium and thus cannot survive. However, fused cells can synthesize the HGPRT enzyme and inherit the dual characteristics of B lymphocytes and myeloma cells, so they can proliferate infinitely in the HAT selection medium.

[0262] The fused cells were incubated in HAT selection medium for about 10 - 14 days, and then the medium was diluted in a multi-well plate so that each well contained only one hybridoma cell. Hybrid cell lines secreting a predetermined specific antibody were selected from these single cell clones by ELISA.

[0263] One of the selected hybridomas was a hybridoma expressing a mouse-derived anti-HER2 antibody (abbreviated as HER2 antibody).

[0264] The other four were hybridomas expressing rat-derived anti-CD3 antibodies (abbreviated as CD3A antibody, CD3B antibody, CD3C antibody, and CD3D antibody).

[0265] Hybridoma cells expressing the HER2 antibody were fused with hybridoma cells expressing the CD3D antibody to obtain a bispecific hybridoma expressing an anti-HER2 × anti-CD3 bispecific antibody, namely hybridoma HER2-CD3D bispecific antibody (hereinafter referred to as hHER2×CD3D).

[0266] Example 2 Binding Activity of Various CD3 Antibodies to Human T Cell Lines a) Reagents and Consumables: 1. Human T cell line Jurkat (clone E6-1, ATCC♯TIB-152) 2. Anti-CD3 antibodies: (i) Anti-CD3A antibody (having VH containing the amino acids described in SEQ ID NO: 35 and VL containing the amino acids described in SEQ ID NO: 36) (ii) Anti-CD3B antibody (having VH containing the amino acids described in SEQ ID NO: 43 and VL containing the amino acids described in SEQ ID NO: 44) (iii) Anti-CD3C antibody (having VH containing the amino acids described in SEQ ID NO: 51 and VL containing the amino acids described in SEQ ID NO: 52) (iv) Anti-CD3D antibody (having VH containing the amino acids described in SEQ ID NO: 8 and VL containing the amino acids described in SEQ ID NO: 7) (v) UCHT-l (eBioscience, 2100293) 3. Secondary antibodies: FITC-labeled AffmiPure Rat Anti-Mouse IgG (H+L) (Jackson Imm. Res., 415-095-166), FITC-labeled AffmiPure Mouse Anti-Rat IgG (H+L) (Jackson Imm. Res., 212-095-168) 4. PBS (St. Louis, CR20012) 5. 96-well plate with U-bottom plate (Jetbio, TCP002096) 6. Flow cytometer (Beckmen, A00-1-1102)

[0267] b) Method: To compare the binding activities of the CD3 antibodies of the present application and commercially available UCHT-1 to human CD3 antigen, the binding activity of the antibodies was detected using in vitro cell experiments.

[0268] First, the concentration of Jurkat cells was adjusted to 1×10 with PBS 7Adjusted to cells / ml. The concentrations of these two antibodies were adjusted to 80 μg / ml, 40 μg / ml, 20 μg / ml, 10 μg / ml, 5 μg / ml, 2.5 μg / ml, 1.25 μg / ml, 0.625 μg / ml, 0.3125 μg / ml, 0.15625 μg / ml, 78.125 ng / ml, 39.0625 ng / ml, 19.53125 ng / ml, and 0 ng / ml. Next, a suspension of Jurkat cells (50 μl / well) was added, followed by adding these five types of CD3 antibodies (50 μl / well) at different concentrations to a 96-well plate equipped with a U-bottom plate. Finally, the final concentrations of these five CD3 antibodies in the mixture were 40 μg / ml, 20 μg / ml, 10 μg / ml, 5 μg / ml, 2.5 μg / ml, 1.25 μg / ml, 0.625 μg / ml, 0.3125 μg / ml, 0.15625 μg / ml, 78.125 ng / ml, 39.0625 ng / ml, 19.53125 ng / ml, 9.765625 ng / ml, and 0 ng / ml, respectively, and the mixture was incubated in a refrigerator at 4°C for 1 hour.

[0269] After incubation, the mixture was centrifuged at 300 g for 5 minutes to remove the supernatant and washed twice with PBS (200 μl / well). Finally, a secondary antibody, FITC-labeled AffmiPure Mouse Anti-Rat IgG (H+L) or FITC-labeled AffmiPure Rat Anti-Mouse IgG (H+L), was used to bind to the anti-CD3 antibody-Jurkat cell complex or UCHT-1-Jurkat cell complex, respectively, and flow cytometry was performed. To evaluate the binding activity of the antibody to Jurkat cells, FITC-positive Jurkat cells were counted (Figure 8).

[0270] GraphPad Prism 7 software was used to visualize the graph and calculate the half-maximal effective concentration (EC50) of the binding activity of the antibody to Jurkat cells. The EC50 values of the CD3A, CD3B, CD3C, CD3D, and UCHT-1 antibodies were 25.87 pM, 9.56 pM, 15.23 pM, 10.71 pM, and 22.79 pM, respectively.

[0271] Example 3 Expression of Recombinant HER2-CD3 Bispecific Antibody in CHO In this example, CHO cells were genetically modified to express and produce a recombinant HER2×CD3D bispecific antibody (hereinafter abbreviated as rHER2×CD3D).

[0272] 1. Synthesis of Protein Sequence The structure of the rHER2-CD3D bispecific antibody is shown in Figure 1. The antibody contains two different antigen-binding parts, namely the HER2-binding part and the CD3-binding part. The HER2-binding part is derived from the mouse anti-HER2 antibody of Example 1, and the CD3-binding part is derived from the rat anti-CD3D antibody of Example 1.

[0273] The nucleic acid sequences encoding the heavy and light chains of the anti-HER2 antibody of Example 1 were inserted into two different expression cassettes of p2MPT (containing the puromycin resistance gene) respectively to obtain the recombinant plasmid p2MPT-EHEL. The nucleic acid sequences encoding the heavy and light chains of the anti-CD3D antibody of Example 1 were inserted into two different expression cassettes of p2MPT (containing the puromycin resistance gene) respectively to obtain the recombinant plasmid p2MPT-DHDL.

[0274] [Table 2]

[0275] To express the rHER2-CD3D bispecific antibody, two types of recombinant plasmids were stably co-introduced into CHO cells at a ratio of 1:1. The recombinant plasmid pHT-PBase-ori containing the transposase gene was used as a reagent for stable transfection. During transfection, the cell density was adjusted to 4.0×10 6 cells / ml, and the medium was replaced with fresh proCH05 medium. The transfection reagent was polyethyleneimine (PEI), and the ratio of total DNA to PEI was 1:5.

[0276] According to Table 3, DNA and PEI were sequentially added to CHO cells suspended in fresh medium, quickly mixed, and then the mixture was placed on a shaker at 37°C for transfection.

[0277]

Table 3

[0278] 2. Cell pool screening of rHER2-CD3D bispecific antibody 72 hours after transfection, 10 μg / ml puromycin was added to 10 ml of 5×10 6 cells, and the cells were cultured on a shaker at 37°C. The medium was refreshed every 2 - 3 days, and the cells were passaged at a density of 0.5×10 6 cells / ml and maintained in 10 μg / ml puromycin. After 10 days of screening, a positive cell pool with a viability of 97% or more was obtained.

[0279] 3. Expression of rHER2-CD3D bispecific antibody 5.0×10 6 cells / ml of positive cells were cultured in medium supplemented with 2 mM sodium butyrate at 31°C for 4 days. The supernatant was collected by centrifugation at 1500 rpm for 5 minutes.

[0280] 4. Purification of rHER2-CD3D bispecific antibody The supernatant obtained in Step 3 was centrifuged at 2000 rpm to remove cell debris, and then filtered through a 0.45 μm microporous membrane. The antibody with an Fc portion was concentrated from the supernatant using a MabselectSure affinity chromatography column, and the anti-CD3D monoclonal antibody was eluted using a sodium citrate buffer at pH 5.8. Then, a gradient linear elution from pH 5.8 to pH 3.0 was performed for 20 CV.

[0281] The samples taken were adjusted to pH 5.4 with Tris-HCl and then the rFIER2-CD3D bispecific antibody and anti-HER2 monoclonal antibody were separated using a Capto S strong anion exchange chromatography column. The purified rHER2-CD3D bispecific antibody is a recombinant HER2-CD3D bispecific antibody and is hereinafter referred to as rHER2×CD3D.

[0282] Ultrafiltration with a 30 Kd cut-off was performed to replace the elution buffer with PBS solution. The purity of rHER2×CD3D was confirmed by Western blot and SDS-PAGE. Figure 2 shows the results of the Western blot. In Figure 2, M represents the marker, the first and second rows represent rHER2×CD3D, the third row represents the anti-HER2 monoclonal antibody, and the fourth row represents the anti-CD3D monoclonal antibody. Figure 3 shows the results of SDS-PAGE. Line 1 represents the marker and line 2 represents rHER2×CD3D.

[0283] Example 4 Preparation of F(ab)’2 Fragment of Recombinant Anti-CD3 Bispecific Antibody The heavy chain variable region VH and the light chain variable region VL targeting HER2 and CD20 respectively were obtained from commercially available Herceptin and Rituximab. The heavy chain constant region CHI, the light chain constant region CL and its hinge region were derived from mouse IgG2a. For purification, Strep-tagII was ligated to the C-terminus of CHI.

[0284] The heavy chain variable region VH and the light chain variable region VL targeting CD3 were derived from the anti-CD3D antibody of Example 1 or commercially available UCHT1. The heavy chain constant region CHI, the light chain constant region CL and its hinge region were derived from rat IgG2b. His tag was ligated to the C-terminus of CHI for purification.

[0285] The two parts of the rat-derived Fab and the mouse-derived Fab were joined via disulfide bonds in the hinge region to form the F(ab)’2 structure of the recombinant anti-CD3 bispecific antibody.

[0286] The F(ab)'2 fragment of the recombinant anti-CD3 bispecific antibody was obtained by co-expressing two different Fab antibody fragments in host cells. A cell pool containing the F(ab)'2 fragment of the recombinant anti-CD3 bispecific antibody was obtained by stable gene expression (SGE). High molecular weight polyethyleneimine (PEI) mediated DNA introduction into host cells, and a stable cell pool was obtained using the piggyBac transposon system. The complete F(ab)'2 fragment of the recombinant anti-CD3 bispecific antibody was obtained by co-introducing two different plasmids into CHO cells.

[0287] 1. Protein sequence analysis and synthesis Eight genes were synthesized as follows: 1) Gene 1, encoding: Herceptin VH (having the amino acids described in SEQ ID NO: 20) - CH1 of mouse IgG2a - hinge of mouse IgG2a - flexible linker G4S - strep - tag II 2) Gene 2, encoding: Herceptin VL (having the amino acids described in SEQ ID NO: 19) - CL of mouse IgG2a 3) Gene 3, encoding: Rituximab VH (having the amino acids described in SEQ ID NO: 24) - CH1 of mouse IgG2a - hinge of mouse IgG2a - flexible linker G4S - strep - tag II 4) Gene 4, encoding: Rituximab VL (having the amino acids described in SEQ ID NO: 23) - CL of mouse IgG2a 5) Gene 5, encoding: anti - CD3DVH (having the amino acids described in SEQ ID NO: 8) - CHI of rat IgG2b - hinge of rat IgG2b - flexible linker G4S - His - tag 6) Gene 6, encoding: anti - CD3DVL (having the amino acids described in SEQ ID NO: 7) - CL of mouse IgG2a 7) Gene 7, encoding: UCHT1VH (having the amino acids described in SEQ ID NO: 22) - CHI of rat IgG2b - hinge of rat IgG2b - flexible linker G4S - His - tag, and, 8) Gene 8, encoding: UCHT1VL (having the amino acids described in SEQ ID NO: 21) - CL of mouse IgG2a

[0288] Genes 1-8 were ligated into an expression vector and transfected into a host cell line to produce a recombinant anti-CD3 bispecific antibody of two different F(ab)'2 fragments, and its structure is shown in Figures 4-5. The structure of the antibody does not contain an Fc region. The antibody contains two different antigen-binding parts, namely a HER2-binding part and a CD3-binding part, or a CD20-binding part and a CD3-binding part. Due to this structure, the antibody can bind simultaneously to tumor-associated antigens (TAAs) expressed on tumor cells and CD3 molecules on T cells. And the F(ab)'2 fragment of the recombinant anti-CD3 bispecific antibody has both Strep-II and His peptides for purification.

[0289] 2. Plasmid construction Genes 1-2 or genes 3-4 were separately inserted into two different expression cassettes of p2MPT (containing a puromycin resistance gene) to obtain recombinant plasmids named p2MPT-HH-HL and p2MPT-RH-RL. Genes 5-6 or genes 7-8 were separately inserted into two different expression cassettes of p2MPT (containing a puromycin resistance gene) to obtain recombinant plasmids named p2MPT-CH-CL and p2MPT-UH-UL.

[0290]

Table 4

[0291] Using the recombinant plasmids in Table 4, F(ab)'2 fragments of a total of four recombinant anti-CD3 bispecific antibodies were constructed: (1) Herceptin×CD3DF(ab)'2; (2) Herceptin×UCHT1F(ab)'2; (3) Rituximab×CD3DF(ab)'2; and (4) Rituximab×UCHT1F(ab)'2.

[0292] 3. Cell transfection CHO-DG44 cells were stably transfected to express the above four F(ab)'2 fragments of the recombinant anti-CD3 bispecific antibody.

[0293] During transfection, the cell density was adjusted to 3.0×10 6 cells / ml, and the medium was replaced with fresh proCH05 medium. The transfection reagent was polyethyleneimine (PEI), and the ratio of total DNA to PEI was 1:3.

[0294] Plasmids p2MPT-HH-HL and p2MPT-CH-CL, p2MPT-RH-RL and p2MPT-CH-CL, p2MPT-HH-HL and p2MPT-UH-UL, p2MPT-RH-RL and p2MPT-UH-UL were added, and their mass ratio was 1:1. At the same time, the recombinant plasmid pHT-PBase-ori containing the transposase gene was added, and the addition amount was 1 / 10 of the total DNA amount. According to Table 5, DNA and PEI were sequentially added to CHO-DG44 cells suspended in fresh medium, quickly mixed, and then the mixture was placed in a shaker at 37°C for transfection.

[0295]

Table 5

[0296] 4. Screening of cell pools for F(ab)'2 fragments of recombinant anti-CD3 bispecific antibody 96 hours after transfection, 10 μg / ml of puromycin was added to 1.0×10 7 cells in a 10 ml volume, and cultured in a shaker at 37°C. The medium was refreshed every 2 days, and the cells were passaged at a density of 1.0×10 6 cells / ml and maintained in 10 μg / ml of puromycin. After 12 days of screening, a positive cell pool with a survival rate of 95% or more was obtained.

[0297] 5. Expression of F(ab)'2 fragments of recombinant anti-CD3 bispecific antibody 4.0×10 6Positive cells per ml were cultured in a medium supplemented with 2 mM sodium butyrate at 31 °C for 7 days. The supernatant was collected by centrifugation at 1000 rpm for 5 minutes.

[0298] 6. Purification of the F(ab)’2 fragment of the recombinant CD3 bispecific antibody The supernatant obtained in Step 5 was centrifuged at 2000 rpm to remove cell debris and filtered through a 0.45 μm microporous membrane. His-tagged F(ab’)2 was concentrated from the CHO expression supernatant using a Histrapexcel affinity chromatography column, and subsequently, the F(ab’)2 fragment of the recombinant anti-CD3bi specific antibody concentrated from a Histrap affinity column was eluted with a 300 mM imidazole solution. The eluted sample was concentrated using a StrepTrapHP affinity chromatography column. After concentration, it was eluted with 5 mM desthiobiotin to obtain the target product with both His-tag and Strep-tagII. Ultrafiltration with a 30Kd cut-off was performed to replace the elution buffer with a PBS solution. The results of detecting the purified anti-CD3 bispecific antibody F(ab’)2 fragment by SDS-PAGE are shown in Figures 6-7. In Figure 6, line 1 represents Herceptin×CD3DF(ab)’2, line 2 represents Herceptin×UCHT1F(ab)’2, and line 3 represents the marker. In Figure 7, the first row represents the marker, the second row represents Rituximab×CD3DF(ab)’2, and the third row represents Rituximab×UCHT1F(ab)’2.

[0299] The structures of HER2×CDF(ab)’2 and CD20×CDF(ab)’2 are shown in Figures 4 and 5.

[0300] Example 5 In vitro SK-BR3 cell killing assay with rHER2×CD3D and hHER2×CD3D a) Reagents and consumables: 1. Cell line: SK-BR-3 (ATCC♯HTB-30) 2. Peripheral b100d mononuclear cells (PBMC) (Lead, PBMC-2020110403) 3. HER2×CD3 bispecific antibody: (i) rHER2×CD3D (generated in Example 3) (ii) hHER2×CD3D (prepared in Example 1) 4. PBS (St. Louis, CR20012) 5. RPMI 1640 (Gibco, 22400089) 6. FBS (HyClone, SH30406.05) 7. CellTrace Far Red Cell Proliferation Kit (Invitrogen, C34572) 8. Propidium Iodide (PI) (Sigma, P4170) 9. 96-well plate with U-bottom plate (Jetbio, TCP002096) 10. Flow cytometer (Beckmen, A00-1-1102)

[0301] b) Method: To compare the pharmacodynamic activities of bispecific antibodies prepared by different antibody production methods, an in vitro experiment was conducted to detect the killing effects of rHER2×CD3D and hHER2×CD3D on SK-BR-3 cells.

[0302] First, SK-BR-3, which highly expresses HER2 on the surface, was stained with cell staining (FarRed). Then, the cells were resuspended in RPMI-1640 medium containing 10% FBS. Subsequently, the cell concentration was adjusted to 8×10 5 cells / ml. PBMC from healthy donors were resuspended in RPMI-1640 medium containing 10% FBS, and the cell concentration was adjusted to 8×10 6 cells / ml. Finally, PBMC and SK-BR-3 were well mixed at a volume ratio of 1:1.

[0303] The concentrations of rHER2×CD3D or hHER2×CD3D were 0, 2×10 -4 , 2×10 -3 , 2×10 -2 , 2×10 -1 , 2×10 0 , 2×10 1 , 2×10 2 , 2×10 3 and 2×104 It was serially diluted. A cell mixture of PBMC and SK-BR-3 (50 μl / well) was added to a 96-well plate, and then serially diluted rHER2×CD3D or hHER2×CD3D (50 μl / well) was added to the cell mixture. The final concentrations of rHER2×CD3D or hHER2×CD3D were 0, 10 -4 、10 -3 、10 -2 、10 -1 、10 0 、10 1 、10 2 、10 3 and 10 4 pM.

[0304] After incubating the mixture of the bispecific antibody and cells in an incubator at 37 °C for 24 hours, it was centrifuged at 400 g for 5 minutes to remove the supernatant. The cells were digested with pancreatin for 10 minutes and washed twice with PBS (200 μl / well).

[0305] Finally, dead cells were stained with PI, and the amount of remaining SK-BR-3 cells was analyzed by flow cytometry to determine the killing effect of antibody-mediated cytotoxicity on SK-BR-3 cells.

[0306] When performing flow cytometry, the APC channel was used to label SK-BR-3 cells in the cell suspension (far red staining), and the PE channel was used to distinguish live cells from dead cells (PI staining). Since PI can stain dead cells, the number of APC-positive cells and Pi-negative cells (APC+PT) analyzed by flow cytometry was finally used to quantify the remaining SK-BR-3 cells and calculate the killing rate.

[0307] To compare the effects of rHER2×CD3D and hHER2×CD3D that mediate the killing activity of PBMC against SK-BR-3 cells, GraphPad Prism 7 software was used for graph visualization, and non-linear regression of a four-parameter fitting curve was used to calculate the half-maximal effective concentration (EC50). In the case of rHER2×CD3D, the EC50 value was approximately 3.33 pM. In the case of hHER2×CD3D, the EC50 value was approximately 22.52 pM. Therefore, the surprising result was obtained that rHER2×CD3D produced from CHO cells has significantly superior killing activity against tumor cells than hHER2×CD3D produced from hybridomas. Figure 9 shows the killing effect of PBMC against SK-BR-3 cells via hHER2×CD3D and rHER2×CD3D.

[0308] Example 6 The CD3-binding portion contributes to the cytotoxic effect of rHER2×CD3D on SK-BR3 cell death a) Reagents and consumables: 1. Cell line: SK-BR-3 (ATCC♯HTB-30) 2. T cells purified from PBMC (healthy donor #1: Reid, PBMC-2021110902; healthy donor #2: Oricells, LP210818013, Z0231) 3. HER2×CD3 bispecific antibody: (i) rHER2×CD3D (generated in Example 3); (ii) hHER2×CD3D (prepared in Example 1) 4. PBS (Senlui, CR20012) 5. RPMI1640 (Gibco, 22400089) 6. FBS (HyClone, SH30406.05) 7. CellTrace Far Red Cell Proliferation Kit (Invitrogen, C34572) 8. Propidium iodide (PI) (Sigma, P4170) 9. 96-well plate with U-bottom plate (Jetbio, TCP002096) 10. Flow cytometer (Beckmen, A00-1-1102)

[0309] b) Method: Based on Example 6, to determine whether the CD3-binding portion contributes to the improvement of the cytotoxic effect of rHER2×CD3D on the killing of SK-BR3 cells, an in vitro experiment was conducted to detect the killing effect of rHER2×CD3D and hHER2×CD3D on SK-BR-3 cells. For the experiment, T cells were purified from the PBMC of healthy donors.

[0310] SK-BR-3, which highly expresses HER2 on the surface, was stained with cell staining (FarRed). Subsequently, the cells were resuspended in RPMI-1640 medium containing 10% FBS. Subsequently, the cell concentration was adjusted to 2×10 4 cells / ml. T cells purified from the PBMC of healthy donors were resuspended in RPMI-1640 medium containing 10% FBS, and the T cell concentration was adjusted to 2×10 5 cells / ml. Finally, the T cells purified from PBMC and SK-BR-3 were thoroughly mixed at a volume ratio of 1:1.

[0311] The concentrations of rHER2×CD3D or hHER2×CD3D were serially diluted to 0, 2×10 -4 、2×10 -3 、2×10 -2 、2×10 -1 、2×10 0 、2×10 1 、2×10 2 、2×10 3 and 2×10 4 pM. The cell mixture of T cells purified from PBMC and SK-BR-3 (50 μl / well) was added to a 96-well plate, and subsequently, the serially diluted rHER2×CD3D or hHER2×CD3D (50 μl / well) was added to the cell mixture. The final concentrations of rHER2×CD3D or hHER2×CD3D were 0, 10 -4 、10 -3 、10 -2 、10 -1 、10 0 、10 1 、10 2 、10 3 and 104 It was adjusted to pM.

[0312] The mixture of bispecific antibody and cells was incubated in an incubator at 37 °C for 24 hours, and then centrifuged at 400 g for 5 minutes to remove the supernatant. The cells were digested with pancreatin for 10 minutes and washed twice with PBS (200 μl / well).

[0313] Finally, dead cells were stained with PI, and the amount of remaining SK-BR-3 cells was analyzed by flow cytometry to determine the killing effect of antibody-mediated cytotoxicity on SK-BR-3 cells.

[0314] When performing flow cytometry, the APC channel was used to label SK-BR-3 cells in the cell suspension (far red staining), and the PE channel was used to distinguish live and dead cells (PI staining). Since PI can stain dead cells, the number of APC-positive and Pi-negative cells (APC+PI-) analyzed by flow cytometry was used to quantify the remaining SK-BR-3 cells and calculate the killing rate.

[0315] To compare the effects of rHER2×CD3D and hHER2C’D3D, which mediate the killing activity of T cells purified from PBMC against SK-BR-3 cells, GraphPad Prism 7 software was used for graph visualization, and nonlinear regression of a four-parameter fitting curve was used to calculate the half-maximal effective concentration (EC50). For rHER2CD3D, the EC50 value for donor 1 was approximately 7.65 pM, and for hHER2×CD3D, the EC50 value for donor 1 was approximately 12.75 pM. For rHER2×CD3D, the EC50 value for donor 2 was approximately 3.73 pM, and for hHER2×CD3D, the EC50 value for donor 2 was approximately 7.28 pM. Therefore, the CD3-binding portion contributes to a significantly better cytotoxic effect in killing SK-BR3 cells for rHER2×CD3D than for hHER2×CD3D. Figure 15 shows the killing effect of T cells on SK-BR-3 cells via hHER2×CD3D and rHER2×CD3D.

[0316] Release of IL-6 induced by hHER2×CD3D and rHER2×CD3D in Example 7 a) Reagents and consumables: 1. Kit: LEGENDMAXTM Human IL-6 ELISA (BioLegend, B329676) 2. Test samples: (i) Supernatant from hHER2×CD3D of Example 5; (ii) Supernatant from rHER2×CD3D of Example 5; 3. 96-well plate with U-bottom plate (Jetbio, TCP002096) 4. Flow cytometer (Beckmen, A00-1-1102)

[0317] b) Method: Since cytokine release syndrome (CRS) is a major safety concern for T cell-engaging bispecific antibodies used clinically, the release of cytokines in the supernatant is relevant to the safety when administering the anti-CD3 bispecific antibody of the present application. Cytokines that induce CRS include TNF-α, IFN-γ, IL-1β, IL-2, IL-6, IL-10, etc., and among them, IL-6 is the most important cytokine in the induction of CRS.

[0318] rHER2×CD3D showed the maximum cytotoxic effect at a concentration of 10 2 pM, and hHER2×CD3D showed a cytotoxic effect equivalent to that of rHER2×CD3D at a concentration of 10 4 pM (Figure 9). Therefore, the correlation between antibody-induced cytotoxicity and the release of cytokine IL-6 at antibody concentrations of 10 2 pM and 10 4 pM was analyzed. As shown in Table 6, compared with hHER2×CD3D, rHER2×CD3D at 10 2When the concentration of pM significantly increased tumor cell killing by PBMC (54.05% vs 16.18%), the level of IL-6 release was significantly lower (1795.5 pg / ml vs 2928.79 pg / ml); compared with hHER2×CD3D, rHER2×CD3D slightly increased tumor cell killing at a concentration of 104 pM (61.9% vs 58.35%), but the level of IL-6 release did not increase significantly (3837 pg / ml vs 3295.82 pg / ml). As a conclusion, compared with the hybridoma HER2×CD3D, recombinant HER2×CD3D enhances the anti-tumor effect through PBMC, but cytokine release is less, making it a safer option for clinical use.

[0319]

Table 6

[0320] Example 8 In vitro SK-BR3 Cell Killing Assay with Herceptin×CD3 Bispecific Antibody a) Reagents and Consumables: 1. Cell line: SK-BR-3 (ATCC♯HTB-30) 2. PBMC: Healthy donor #1 (Lead, PBMC-2021010602); Healthy donor #2 (Lead, PBMC-2021011802) 3. Herceptin×CD3 bispecific antibody: (i) Herceptin×CD3DF(ab)’2, generated in Example 4; (ii) Herceptin×UCHT1F(ab)’2, generated in Example 4; 4. PBS (St. Louis, CR20012) 5. RPMI1640 (Gibco, 22400089) 6. FBS (HyClone, SH30406.05) 7. CellTrace Far Red Cell Proliferation Kit (Invitrogen, C34572) 8. Propidium Iodide (PI) (Sigma, P4170) 9. 96-well plate with U-bottom plate (Jetbio, TCP002096) 10. Flow cytometer (Beckmen, A00-1-1102) b) Method: To compare the pharmacodynamic activities of bispecific antibodies produced by different CD3 antibodies, in vitro cell experiments were conducted to detect the killing effects of Herceptin × CD3DF(ab)'2 and Herceptin × UCHT1F(ab)'2 on SK-BR-3 cells.

[0321] First, SK-BR-3 cells with high expression of HER2 on the surface were stained with cell staining (FarRed). Then, the cells were resuspended in RPMI-1640 medium containing 10% FBS. Subsequently, the cell concentration was adjusted to 8×10 5 cells / ml. PBMC from healthy donors were resuspended in RPMI-1640 medium containing 10% FBS, and the cell concentration was adjusted to 8×10 6 cells / ml. Finally, PBMC and SK-BR-3 were well mixed at a volume ratio of 1:1. The concentrations of HerceptinCD3DF(ab)'2 or Herceptin × UCHT1F(ab)'2 were serially diluted to 0, 2×10 0 、2×10 1 、2×10 2 、2×10 3 、2×10 4 、10 5 and 2×10 5 pM. The cell mixture of PBMC and SK-BR-3 (50 μl / well) was added to a 96-well plate, and then serially diluted Herceptin × CD3DF(ab)'2 or Herceptin × UCHT1F(ab)'2 (50 μl / well) was added. The final concentrations of Herceptin × CD3DF(ab)'2 or Herceptin × UCHT1F(ab)'2 were adjusted to 0, 10 0 、10 1 、10 2 、10 3 、10 4 、5×10 4 、10 5 pM.

[0322] After incubating the mixture of bispecific antibody and cells in an incubator at 37°C for 24 hours, the supernatant was removed by centrifugation at 400 g for 5 minutes. The cells were digested with pancreatin for 10 minutes and washed twice with PBS (200 μl / well). Finally, dead cells were stained with PI, and the amount of remaining SK-BR-3 cells was analyzed by flow cytometry to determine the killing effect of antibody-mediated cytotoxicity on SK-BR-3 cells.

[0323] When performing flow cytometry, the APC channel was used to label SK-BR-3 cells in the cell suspension (far red staining), and the PE channel was used to distinguish live cells from dead cells (PI staining). Since PI can stain dead cells, the number of APC-positive and Pi-negative (APC+PT-) cells analyzed by flow cytometry was finally used to quantify the remaining SK-BR-3 cells and calculate the killing rate.

[0324] To compare the effects of Herceptin×CD3DF(ab)'2 and Herceptin×UCHT1F(ab)'2 on mediating the killing activity of PBMC against SK-BR-3 cells, GraphPad Prism 7 software was used to visualize the graph and perform non-linear regression. A four-parameter fitting curve was used to calculate the half-maximal effective concentration (EC50). For Herceptin×CD3DF(ab)'2, the EC50 value for the first donor was approximately 680.7 pM, and for the second donor, it was approximately 82.58 pM. For Herceptin×UCHT1F(ab)'2, the EC50 value for the first donor was approximately 1003 pM, and for the second donor, it was approximately 941.2 pM. These results showed that Herceptin×CD3DF(ab)'2 had a significantly better killing effect on tumor cells compared to Herceptin×UCHT1F(ab)'2. Figure 10 shows the killing effects of Herceptin×CD3DF(ab)'2 and Herceptin×UCHT1F(ab)'2 on SK-BR-3 cells.

[0325] Example 9 In vitro SU-DHL-8 cell killing assay with rituximab×CD3 bispecific antibody a) Reagents and Consumables: 1. Cell line: SU-DHL-8 (ATCC♯CRL-2961) 2. PBMC: Healthy donor #1 (Lead, PBMC-2020122101); Healthy donor #2 (Lead, PBMC-2021010602) 3. Rituximab×CD3 bispecific antibody F(ab’)2: (i) Rituximab×CD3DF(ab)’2, produced in Example 4. (ii) Rituximab×UCHT1F(ab)’2, produced in Example 4. 4. PBS (St. Louis, CR20012) 5. RPMI1640 (Gibco, 22400089) 6. FBS (HyClone, SH30406.05) 7. CellTrace Far Red Cell Proliferation Kit (Invitrogen, C34572) 8. Propidium Iodide (PI) (Sigma, P4170) 9. 96-well plate with U-bottom plate (Jetbio, TCP002096) 10. Flow cytometer (Beckmen, A00-1-1102)

[0326] b) Methods: To compare the pharmacodynamic activities of bispecific antibodies produced by different CD3 antibodies, in vitro cell experiments were performed to detect the killing effects of rituximab×CD3DF(ab)’2 and rituximab×UCHT1F(ab)’2 on SU-DHL-8 cells. First, SU-DHL-8 cells expressing CD20 on the surface were stained with cell staining (FarRed). Then, the cells were resuspended in RPMI-1640 medium containing 10% FBS. Subsequently, the cell concentration was adjusted to 8×10 5 cells / ml. PBMCs from healthy donors were resuspended in RPMI-1640 medium containing 10% FBS, and the cell concentration was adjusted to 8×10 6 cells / ml. Finally, PBMCs and SU-DHL-8 were well mixed at a volume ratio of 1:1.

[0327] The concentrations of rituximab×CD3DF(ab)'2 or rituximab×UCHT1F(ab)'2 were serially diluted. A cell mixture of PBMC and SU-DHL-8 (50 μl / well) was added to a 96-well plate, and then the serially diluted rituximab×CD3DF(ab)'2 or rituximab×UCHT1F(ab)'2 (50 μl / well) was added to the cell mixture. After incubating the mixture of the bispecific antibody and cells in an incubator at 37 °C for 24 hours, it was centrifuged at 400 g for 5 minutes to remove the supernatant. The cells were digested with pancreatin for 10 minutes and washed twice with PBS (200 μl / well). The U-DHL-8 cells were analyzed by flow cytometry to measure the killing effect of antibody-mediated cytotoxicity on SU-DHL-8 cells. When performing flow cytometry, the SU-DHL-8 cells in the cell suspension were labeled using the APC channel (far red staining), and live and dead cells were distinguished using the PE channel (PI staining). Since PI can stain dead cells, the number of APC-positive and Pi-negative (APC+PT-) cells analyzed by flow cytometry was finally used to quantify the remaining SU-DHL-8 cells and calculate the killing rate. To compare the mediating effects of the killing activities of PBMC on SU-DHL-8 cells by rituximab×CD3DF(ab)'2 and rituximab×UCHT1F(ab)'2, GraphPad Prism 7 software was used for graph visualization, and non-linear regression of a four-parameter fitting curve was used to calculate the half-maximal effective concentration (EC50). For rituximab×CD3DF(ab)'2, the EC50 value of the first donor was approximately 72.83 pM, and the EC50 value of the second donor was approximately 59.98 pM. For rituximab×UCHT1F(ab)'2, the EC50 value of the first donor was approximately 605.5 pM, and the EC50 value of the second donor was approximately 274.4 pM. From these results, it was shown that rituximab×CD3DF(ab)'2 had a significantly better killing effect on tumor cells compared to rituximab×UCHT1F(ab)'2. Figure 11 shows the killing effects of rituximab×CD3DF(ab)'2 and rituximab×UCHT1F(ab)'2 on SU-DHL-8 cells.

[0328] Example 10 Release of Granzyme B and Perforin a) Reagents and Consumables: 1. Detection Kit: (i) Granzyme B ELISA Kit (Dayou, Lot: 2007-1); (ii) Perforin ELISA Kit (Dayou, Lot: 2008-1) 2. Test Samples: (i) Supernatant from Example 8 (from healthy donor #1). (ii) Supernatant from Example 9 (from healthy donor #2). 3. PBS (Senlui, CR20012) 4. 96-well Plate with U-bottom Plate (Jetbio, TCP002096) 5. Microplate Reader (Thermo, Multiskan FC)

[0329] b) Method: The granzyme B / perforin pathway was considered the main mechanism by which cytotoxic lymphocytes kill tumor cells. To confirm that the F(ab)'2 fragment of the recombinant CD3 bispecific antibody can kill target cells mediated by PBMC and that the cytotoxic effect is due to the interaction between CD3 and T cells, the release of granzyme B and perforin was quantified.

[0330] The supernatant collected in Example 8 or 9 was used as a sample, and the release of granzyme B and perforin was quantified using a granzyme B / perforin ELISA kit.

[0331] The results showed that for the supernatant of Herceptin × CD3DF(ab)'2 in Example 8, the contents of granzyme B and perforin were 21233.57 pg / ml and 1597.45 pg / ml respectively. For the supernatant of Rituximab × CD3DF(ab)'2 in Example 9, the contents of granzyme B and perforin were 24410.04 pg / ml and 1675.97 pg / ml respectively. In the supernatant of Rituximab × UCHT1 F(ab)'2, the contents of granzyme B and perforin were 48863.12 pg / ml and 2183.46 pg / ml respectively.

[0332] The above results indicated that the F(ab)'2 fragment of the recombinant CD3 bispecific antibody in this application mediated the death of tumor cells through the release pathways of granzyme B and perforin.

[0333] Example 11 Release of cytokine IL-6 induced by Herceptin × CD3DF(ab)'2 and Herceptin × UCHT1F(ab)'2 a) Reagents and consumables: 1. Kit: LEGENDplexTM Human CD8 / NK Panel (Biolegend, 740267) 2. Test samples: (i) The supernatant of Example 8 (derived from healthy donor #1); (ii) The supernatant of Example 9 (derived from healthy donor #2); 3. 96-well plate with U-bottom plate (Jetbio, TCP002096) 4. Flow cytometer (Beckmen, A00-1-1102)

[0334] b) Method: Cytokine release syndrome (CRS) is a major safety concern for T cell-engaging bispecific antibodies used clinically. Therefore, the release of cytokines in the supernatant is relevant to the safety when administering the anti-CD3 bispecific antibody of the present application. Cytokines that induce CRS include TNF-α, IFN-γ, IL-1β, IL-2, IL-6, IL-10, etc. Among them, IL-6 is the most important cytokine in the induction of CRS.

[0335] The IL-6 levels in the supernatants collected from Example 8 and Example 9 were measured using the LEGENDplexTM Human CD8 / NK Pane Panel kit. The IL-6 content in the supernatant was measured at an anti-CD3 antibody concentration of 10 5 pM. The results are shown in Table 7.

[0336] As a result, in the supernatant of Example 8, when Herceptin × CD3DF(ab)’2 showed a significantly better antitumor effect than Herceptin × UCHT1F(ab)’2, the IL-6 content in the supernatant was almost the same. On the other hand, in the supernatant of Example 9 where Rituximab × CD3DF(ab)’2 showed a relatively similar antitumor effect as Rituximab × UCHT1F(ab)’2, the IL-6 content in the supernatant of Rituximab × UCHT1F(ab)’2 was significantly higher than that in the supernatant of Rituximab × CD3DF(ab)’2 and exceeded the detection upper limit value of the kit.

[0337] In conclusion, compared with UCHT-1, the anti-CD3D bispecific antibody had lower cytokine release and was safer when mediating the death of tumor cells.

[0338]

Table 7

[0339] Example 12 Humanized anti-CD3 monoclonal antibody 12.1 Preparation of plasmid constructs encoding anti-CD3 monoclonal antibodies The heavy chain vector insert or light chain vector insert was ligated into the expression cassette in the pTT5 vector as depicted in Figure 12 (named pTT5-HC or pTT5-LC). The nucleotide sequence of the vector + insert containing the resulting signal peptide was also determined to confirm the correct reading frame and nucleotide sequence of the coding DNA. The amino acid sequences inserted into each plasmid are shown in Table 8.

[0340]

Table 8

[0341] According to Table 8, C3000 is a rat-derived anti-CD3 antibody, while C3002-1 and C3002-2 are humanized anti-CD3 antibodies.

[0342] 12.2 Transfection method Plasmids pTT5-HC and pTT5-LC were transiently transfected into ExpiCHO®-S cells to obtain anti-CD3 monoclonal antibodies. ExpiCHO®-S cells were cultured at 37 °C in ExpiCHO® Expression Medium at a density of 6.0×10 6 cells / ml. Two solutions were prepared as follows: 1) Solution 1: 64 μl of Expifectamine CHO reagent was mixed with 800 μl of OptiPRO® medium. 2) Solution 2: A mixture of 8 μg of pTT5-HC and 8 μg of pTT5-LC was diluted with 800 μl of Expifectamine® CHO reagent. Solution 1 and Solution 2 were mixed to form an ExpiFectamine® CHO / plasmid DNA mixture. After a 5-minute incubation, the ExpiFectamine® CHO / plasmid DNA mixture was added to the ExpiCHO®-S cell culture to form a 20 ml culture system. The cell culture mixture was cultured at 37 °C and 8% CO2.

[0343] 12.3 Protein expression Twenty-four hours after transfection, 120 μL of ExpiCHO® Enhancer and 4.8 mL of ExpiCHO® Feed solution were added to the cell culture from 11.2. The culture was further incubated at 31 °C for 7 days. The supernatant was collected by centrifugation.

[0344] 12.4 Purification The supernatant obtained in Step 11.3 was purified using a MabSelect SuReLx column. The supernatant was washed with PBS at pH 7.4 and eluted with 0.1 M glycine at pH 3.0. The collected samples were neutralized with 1 M Tris buffer at pH 8.0 and then ultrafiltered to replace the elution buffer with PBS solution. The purified samples were further identified by SDS-PAGE and Western blot. Figures 13A - 13F show the results of Western blot and SDS-PAGE of anti-CD3 mAb. Figures 13A, 13C, and 13E show the results of Western blot of C3000, C3002-1C, and 3002-2, respectively. The first row of Figure 13B, Figure 13D, and Figure 13F show the results of reduced SDS-PAGE of C3000, C3002-1C, and 3002-2, respectively. The second row of Figure 13B, Figure 13D, and Figure 13F show the results of non-reduced SDS-PAGE of C3000, C3002-1C, and 3002-2, respectively.

[0345] 12.5 Binding affinity of CD3e and CD3γ heterodimer proteins from anti-CD3 mAb C3000, C3002-1C, and 3002-2 The ELISA plate was coated with 100 ng / well of CD3ε&CD3γ Heterodimer Protein at 4°C overnight, and after further blocking with 3% BSA in PBS for 2 hours, serially diluted anti-CD3 mAbs C3000, C3002-1C, and 3002-2 were added to the plate and incubated at 37°C for the following times. After washing and drying the plate, 100 μL / well of the detection antibody THE His Tag Antibody [HRP] was added and incubated at 37°C for 1 hour. After washing and drying the plate, 100 μL / well of the TMB chromogenic solution was added and incubated at 37°C for 10 minutes. After color development, 50 μL / well of the stop solution was added to stop the color reaction. The optimal density of the samples was measured at 450 nm (OD450). As shown in Figure 14, using GraphPad Prism 8 software, a graph of the binding affinity of anti-CD3 mAbs C3000, C3002-1C, and 3002-2 for the CD3ε and CD3γ heterodimer proteins was visualized. According to Figure 14, compared with rat-derived C3000, the humanized anti-CD3 antibodies C3002-1C and 3002-2 showed equivalent binding affinity for the CD3ε and CD3γ heterodimer proteins.

[0346] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. Although the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not intended to be construed in a limiting sense. Those skilled in the art will envision numerous variations, modifications, and substitutions without departing from the present invention. Further, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative ratios described herein, which depend on various conditions and variables. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in practicing the present invention. Accordingly, the present invention is considered to cover such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

Claims

1. A multispecific antigen binding protein comprising a CD3 binding portion, wherein the CD3 binding portion comprises heavy chain variable regions HCDR1, HCDR2 and HCDR3, the heavy chain variable regions HCDR1, HCDR2 and HCDR3 have the sequences set forth in SEQ ID NOs: 4, 5 and 6, respectively, the CD3 binding portion comprises light chain variable regions LCDR1, LCDR2 and LCDR3, and the light chain variable regions LCDR1, LCDR2 and LCDR3 have the sequences set forth in SEQ ID NOs: 1, 2 and 3, respectively. A multispecific antigen binding protein.

2. The multispecific antigen binding protein according to claim 1, wherein the CD3 binding portion comprises a heavy chain variable region of SEQ ID NO: 8, or a heavy chain variable region having at least 90% identity to the heavy chain variable region of SEQ ID NO: 8, and a light chain variable region of SEQ ID NO: 7, or a light chain variable region having at least 90% identity to the light chain variable region of SEQ ID NO:

7.

3. The multispecific antigen binding protein according to claim 1 or 2, wherein the CD3 binding portion is humanized.

4. The humanized CD3 binding portion is (i) a heavy chain variable region of SEQ ID NO: 27 and a light chain variable region of SEQ ID NO: 31, (ii) or an amino acid sequence comprising a heavy chain variable region and a light chain variable region selected from the heavy chain variable region of SEQ ID NO: 28 and the light chain variable region of SEQ ID NO: 31 and having at least 90% identity thereto. The multispecific antigen binding protein according to claim 3.

5. The humanized CD3 binding portion has an amino acid sequence of the heavy chain variable region and the light chain variable region that is (i) a heavy chain variable region of SEQ ID NO: 27 and a light chain variable region of SEQ ID NO: 31, (ii) or selected from the heavy chain variable region of SEQ ID NO: 28 and the light chain variable region of SEQ ID NO:

31. The multispecific antigen binding protein according to claim 4.

6. The multispecific antigen binding protein according to any one of claims 1 to 5, produced by recombinant expression from a host cell.

7. The multispecific antigen binding protein according to claim 6, wherein the host cell is selected from bacterial cells, fungal cells, plant cells, mammalian cells or viruses.

8. The multispecific antigen binding protein according to claim 7, wherein the bacterial cell is Escherichia coli.

9. The multispecific antigen binding protein according to claim 7, wherein the fungal cell is a yeast cell.

10. The mammalian cell is selected from CHO, NSO, BHK, or HEK293 cells, and the multispecific antigen-binding protein according to claim 7.

11. The multispecific antigen-binding protein according to any one of claims 1 to 5, produced by hybridoma cells.

12. The hybridoma cells are selected from mouse, rat, and rabbit, and the multispecific antigen-binding protein according to claim 11.

13. The form of the multispecific antigen-binding protein is selected from bispecific antibody, bispecific diabody, bispecific scFv, tandAb, trivalent binding molecule, or tetravalent binding molecule, and the multispecific antigen-binding protein according to any one of claims 1 to 12.

14. The multispecific antigen-binding protein according to any one of claims 1 to 13, which is a humanized antibody.

15. The multispecific antigen-binding protein according to any one of claims 1 to 13, which is a chimeric antibody.

16. The multispecific antigen-binding protein according to any one of claims 1 to 13, which is a monoclonal antibody.

17. The multispecific antigen-binding protein according to any one of claims 1 to 16, comprising a constant region derived from an IgA, IgG, IgD, IgE, or IgM antibody.

18. The antigen-binding protein comprises Fab, Fab’, F(ab)2, Fv fragment, F(ab’)2, scFv, di-scFv, and / or dAb, and the multispecific antigen-binding protein according to one of claims 1 to 17.

19. The multispecific antigen-binding protein according to one of claims 1 to 18, comprising at least one tumor-associated antigen (TAA) binding portion.

20. The TAA is selected from the group consisting of EP-CAM, CCR5, CD19, HER2, HER3neu, HER3, HER4, EGFR, PSMA, CEA, MUC1, MUC2, MUC3, MUC4, MUC5, MUC7, bhCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3, 9-O-Acetyl-GD3, GM2, globo H, fucosyl GM1, PolySA, GD2, RON, c-Met, CEACAM-6, PCTA-1, PSA, PAP, ALCAM (CD166), PECAM-1, CD151, MAGE-1, TROP2, IGF1R, TGFBR2, GHRHR, GHR, IL-6R, gp130, TNFR2, OSMRp, Patched-1, Frizzled, Robol, LTpR, CD26, CD27, CD44, CD80, CD81, CD86, CD100, CXCR4, SAS, BCMA, TWEAKR / Fnl4, FGFR4, VEGFR1, VEGFR2, SSX1, and SSX2, carbonic anhydrase IX (MN / CAIX), CD44v6, sonic hedgehog (Shh), Wue-1, plasma cell antigen, (membrane-bound) IgE, melanoma chondroitin sulfate proteoglycan (MCSP), CCR8, TNF-α precursor, STRAP, mesothelin, A33 antigen, prostate stem cell antigen (PSCA), Ly-6; desmoglein 4, E-cadherin neoepitope, fetal acetylcholine receptor, CD25, CA19-9 marker, CA-125 marker and Mullerian inhibiting substance (MIS) receptor type II, sTn (sialylated Tn antigen, TAG72), FAP (fibroblast activation antigen)), endosialin, EGFRvIII, L6, SAS, CD63, TF antigen, Cora antigen, CD7, CD79b, CD22, Igα, Igβ, gp100, MT-MMPs, F19-antigen, CO-29 and EphA2, the multispecific antigen-binding protein according to claim 19.

21. The TAA is HER2 or CD20, the multispecific antigen-binding protein according to claim 19.

22. The HER2-binding portion comprises an amino acid sequence having heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) having the sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) having the sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively, of the multispecific antigen-binding protein according to claim 21.

23. The HER2-binding portion comprises heavy chain complementarity-determining regions (HCDR1, HCDR2, and HCDR3) having the sequences set forth in SEQ ID NOs: 12, 13, and 14, respectively, and light chain complementarity-determining regions (LCDR1, LCDR2, and LCDR3) having the sequences set forth in SEQ ID NOs: 9, 10, and 11, respectively, and the CD3-binding portion induces T cell activation, of the multispecific antigen-binding protein according to claim 22.

24. The HER2-binding portion comprises an amino acid sequence having at least 90% identity with the heavy chain variable region of SEQ ID NO: 16 and the light chain variable region of SEQ ID NO: 15, of the multispecific antigen-binding protein according to claim 22.

25. The HER2-binding portion comprises the heavy chain variable region of SEQ ID NO: 16 and the light chain variable region of SEQ ID NO: 15, of the multispecific antigen-binding protein according to claim 24.

26. An isolated nucleic acid molecule encoding the multispecific antigen-binding protein according to any one of claims 1 to 25.

27. A vector comprising the isolated nucleic acid molecule according to claim 26.

28. A host cell comprising the vector according to claim 27.

29. A method for producing the multispecific antigen-binding protein according to any one of claims 1 to 25, comprising culturing the host cell according to claim 28 under conditions capable of expressing the multispecific antigen-binding protein.

30. A pharmaceutical composition comprising the multispecific antigen-binding protein according to any one of claims 1 to 25, the isolated nucleic acid molecule according to claim 26, the vector according to claim 27, and / or the host cell according to claim 28, and optionally a pharmaceutically acceptable adjuvant.

31. Use of a multispecific antigen-binding protein according to any one of claims 1 to 25, an isolated nucleic acid molecule according to claim 26, a vector according to claim 27, a host cell according to claim 28, or a pharmaceutical composition according to claim 30 in the manufacture of a medicament for activating T cells, targeting CD3 expressed on T cells, or promoting the interaction between T cells and tumor cells.

32. Use of a multispecific antigen-binding protein according to any one of claims 1 to 25, an isolated nucleic acid molecule according to claim 26, a vector according to claim 27, a cell according to claim 28, or a pharmaceutical composition according to claim 30 in the manufacture of a medicament for preventing, reducing and / or treating tumors.

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