Modified Fc fragments, antibodies containing same, and uses thereof

Abstract

The present invention relates to modified Fc fragments, antibodies comprising the same, and uses thereof. The Fc fragment is derived from human IgG1, and the CH2 domain of the constant region of the Fc fragment contains multiple substitutions. These substitutions can reduce the binding ability of the Fc fragment to Fcγ receptors (FcγRs) and reduce nonspecific T cell activation by antibodies (e.g., anti-CD3 antibodies).

Description

[Technical field]

[0001] The present invention relates to antibodies, and in particular to modified Fc fragments and antibodies comprising same. [Background technology]

[0002] Human natural antibodies, such as IgG1, IgG2, IgG3 and IgG4, all have the ability to bind to FcγR. Human FcγR is divided into three types: FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16). Here, each type of receptor is expressed correspondingly on the surface of different monocytes, and each type of receptor is further divided into various subtypes such as a, b and c. Natural antibodies bind to FcγR with their own Fc and play immunoeffector roles such as antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP) and complement-mediated cytotoxicity (CDC). In the process of developing antibody drugs, it is necessary to reduce the binding ability of some types of antibodies to FcγR in order to reduce the production of ADCC, ADCP and CDC and to reduce the toxicity and side effects of antibodies. In some cases, anti-CD3 antibodies, particularly multifunctional antibodies, may simultaneously target tumor cells and CD3-expressing T lymphocytes, and therefore it is necessary to remove FcγR binding as much as possible to reduce toxic side effects caused by the release of many cytokines resulting from non-specific T cell activation.

[0003] A multifunctional antibody is an antibody or antibody-class molecule with multiple different binding specificities. Multifunctional antibodies are widely used in biomedicine, particularly in immunotherapy for tumors. One of the current issues in immunotherapy is how to utilize the cytotoxic activity of multifunctional antibodies to kill target cells. Multifunctional antibodies are designed to simultaneously target tumor cells and effector cells, and stimulate the effector cells to kill tumor cells.

[0004] Multifunctional antibodies are prepared, for example, by chemical, cell and genetic engineering methods. The advances in genetic engineering have made it possible to easily modify antibodies and design and produce various forms of multifunctional antibody fragments, including diabodies, tanderm ScFv and single-chain diabodies and their derivatives (see Jin and Zhu, "The design and engineering of IgG-Like bispecific antibodies", RE Kontermann (ed.), Bispecific antibodies). These multifunctional antibodies do not have the IgG Fc domain and are small in size, so they have a strong tumor penetration ability, a short half-life in the body, and no ADCC activity associated with the antibody constant region.

[0005] At present, there are several Fc modification technologies that can reduce the binding ability of Fc to FcγR. For example, (1) the literature "Curr Opin Biotechnol. 2011 Dec; 22(6): 858-67. Bypassing glycosylation engineering aglycosylated full-length IgG antibodies for human therapy" describes that the glycosylation of Fc can be efficiently removed by mutation at asparagine (N297) at position 297 of the Fc of human IgG1, thereby reducing the binding to FcγR; (2) the patent application "WO2009100309A2" describes that the glycosylation of Fc can be efficiently removed by mutation at positions 234 (leucine L234), 235 (leucine L235), and 331 (proline L236) of the Fc of human IgG1. (3) Patent application “US20130078249A1” describes that the binding to FcγR can be reduced by mutating the 234th position (leucine L234), the 235th position (leucine L235), and the 331st position (proline P329) of the Fc of human IgG1 to alanine, alanine, and glycine (L234A / L235A / P329G), respectively; (4) the paper “Protein Eng Des Sel. 2016 Oct;29(10):457-466. Novel human IgG1 and IgG4 Fc-engineered antibodies with completely abolished immune effector (5) Patent application WO2011066501A1 describes that the binding to FcγR can be reduced by mutating the leucine at position 234 (L234) and the leucine at position 235 (L235) of the Fc of human IgG1 to alanine and alanine (L234A / L235A), respectively; (6) patent application WO2011066501A1 describes that the binding to FcγR can be reduced by mutating the leucine at position 234 (valine V234), the leucine at position 237 (glycine G237), the proline at position 238 (proline P238), the histidine at position 268 (histidine H268), the valine at position 309 (valine V309), the alanine at position 330 (alanine A330), and the proline at position 331 (proline H268) of the Fc of human IgG2. It has been reported that the binding to FcγR can be reduced by mutating the leucine, leucine, and serine (V234A / G237A / P238S / H268A / V309L / A330S / P331S) to alanine, alanine, serine, alanine, leucine, serine, and serine, respectively.

[0006] However, when the above-mentioned Fc modification techniques are applied to the structure of a multifunctional antibody, it is not possible to completely eliminate non-specific T cell activation by anti-CD3 antibodies (e.g., L234F / L235E / P331S mutations, L234A / L235A / P329G mutations, and L234A / L235A mutations in human IgG1), and modifications at certain positions result in a decrease in antibody stability (e.g., elimination of glycosylation by mutation of asparagine at position 297 in human IgG1). Furthermore, the document "J Immunol 2003;170:3134-3138; Human IgG2 can form covalent dimers" describes that human IgG2 easily dimerizes to form tetravalent complexes with a molecular weight of up to 300 kD, and that making amino acid mutations at seven positions in human IgG2 (e.g., V234A / G237A / P238S / H268A / V309L / A330S / P331S) carries the risk of increasing immunogenicity.

[0007] To solve the above problems, it is necessary to prevent non-specific T cell activation and reduce the binding ability of an antibody (e.g., an anti-CD3 antibody) between Fc and FcγR, thereby avoiding a decrease in antibody stability and an increase in immunogenicity. The present invention provides a novel Fc modification method, in which the entire CH2 domain in the Fc of human IgG1 is replaced with the CH2 domain of human IgG2 and amino acid residues in the domain are mutated, thereby significantly reducing the binding ability of the antibody to FcγR, more effectively eliminating non-specific T cell activation by the antibody (e.g., an anti-CD3 antibody), and maintaining good antibody stability. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2007 / 024249 [Patent Document 2] WO2010 / 065578 [Patent Document 3] WO2013 / 096221 [Patent Document 4] WO2017 / 052321 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention provides a method for modifying Fc, in which the entire CH2 domain of the constant region of human IgG1 is replaced with the CH2 domain of human IgG2, and preferably several amino acid residues are further substituted in the substituted CH2 domain.

[0010] Substitution of residues in CH2 can effectively reduce the binding ability of an antibody to FcγR, and when one of the antibody-binding sites is CD3, substitution of CH2 can also significantly reduce nonspecific T cell activation by the antibody.

[0011] The present invention provides three types of multifunctional antibodies and methods for their production. The structures of specific multifunctional antibodies are shown in Figures 1-3. [Means for solving the problem]

[0012] Specifically, the present invention relates to the following aspects:

[0013] [1] A polypeptide comprising or consisting of a modified Fc fragment, wherein the Fc fragment is derived from human IgG1, the CH2 domain of the constant region of the Fc fragment is replaced with the CH2 domain of human IgG2, and the sequence of the CH2 domain of human IgG2 is represented by SEQ ID NO: 94. [2] The polypeptide of [1], wherein the CH2 domain of the Fc fragment further comprises a mutation at a position selected from the group consisting of: C229, D265, D270, or any combination thereof, as represented by the Kabat numbering. [3] The mutation is a mutation at position C229, preferably selected from the following group: C229A, C229G, C229P, C229S, C229V, C229L, C229I, C229T, C229M, C229N, C229Q, C229D, C229E, C229K, C229R, C229F, C229Y, C229W or C229H, Preferred are C229S, C229A, C229G, and C229P. The polypeptide described in [2]. [4] The mutation is a mutation at D265 position, preferably selected from the following group: D265A, D265G, D265P, D265S, D265V, D265L, D265I, D265T, D265M, D265N, D265Q, D265E, D265K, D265R, D265F, D265Y, D265W or D265H; Preferably, it is D265A. The polypeptide described in [2]. [5] The mutation is a mutation at position D270, preferably selected from the following group: D270A, D270G, D270P, D270S, D270V, D270L, D270I, D270T, D270M, D270N, D270Q, D270E, D270K, D270R, D270F, D270Y, D270W or D270H, Preferably, it is D270A. The polypeptide described in [2]. [6] The mutation is a combination of any two of the mutations at positions C229, D265, and D270, or a combination of mutations at positions C229, D265, and D270; Preferably, it is a combination of mutations at positions D265 and D270. The polypeptide according to any one of [2] to [5]. [7] The polypeptide according to any one of [2] to [5], wherein the CH2 domain of the Fc fragment further comprises a mutation at a position selected from the group consisting of: G327, T339 or a combination thereof. [8] The polypeptide according to [6], wherein the mutation is selected from G327A, G327V, G327L, G327I and / or T339A. [9] The polypeptide according to any one of [2] to [8], wherein the mutation is selected from C229P / G327A, C229P / T339A, D270A / G327A, D270A / T339A, D270A / G327A / T339A, or C229P / D265A / D270A.

[10] The polypeptide described in any one of [2] to [5], wherein the sequence of the CH2 domain of the Fc fragment is selected from SEQ ID NOs: 94 to 101 and 122 to 176.

[11] An antibody or an antigen-binding fragment thereof comprising the polypeptide according to any one of [1] to

[10] , the antibody is selected from a monospecific antibody, a multispecific antibody, more preferably a bispecific antibody; Preferably, the antigen-binding fragment is selected from a Fab, Fab', F(ab')2, Fd, Fv, dAb, Fab / c, a complementarity determining region (CDR) fragment, a single chain antibody (e.g., scFv), a bivalent antibody, or a domain antibody. An antibody or an antigen-binding fragment thereof.

[12] The antibody specifically binds to an antigen selected from the following antigens: Tumor antigens, viral or bacterial antigens, endotoxins, immune antigens, Preferably, the tumor antigen is selected from PD-L1 (preferably represented by SEQ ID NO: 118), B7-H3, SLAMF7 (preferably represented by SEQ ID NO: 119), CD38 (preferably represented by SEQ ID NO: 116), EpCAM, CEA (preferably represented by SEQ ID NO: 120), CD19 and BCMA (preferably represented by SEQ ID NO: 117), and the immune antigen is selected from CD3 (preferably represented by SEQ ID NO: 121), CD16A, CD47 and NKG2D; The antibody described in

[11] .

[13] The antibody is an asymmetric bispecific antibody comprising a light chain, a heavy chain, and a fusion peptide 1; The fusion peptide 1 comprises an scFv and an Fc fragment, The antibody according to

[12] , wherein the antibody has a light chain-heavy chain pairing and a heavy chain-fusion peptide 1 pairing, each of which forms an interchain disulfide bond, the light chain-heavy chain pairing targets a tumor antigen, and the ScFv of the fusion peptide 1 targets an immune cell antigen.

[14] The antibody is an asymmetric trivalent bispecific antibody comprising two light chains, one heavy chain, and one fusion peptide 2, and has light chain-heavy chain pairing, light chain-fusion peptide 2 pairing, and heavy chain-fusion peptide 2 pairing, each of which forms an interchain disulfide bond; the fusion peptide 2 comprises a heavy chain variable region VH, a heavy chain first constant region CH1, ScFv and Fc, the ScFv being located between CH1 and Fc and linked by a linker; The antibody described in

[12] , wherein the light chain-heavy chain pairing targets a tumor antigen, the VH-CH1 and light chain pairing in fusion peptide 2 targets the same tumor antigen, and the ScFv targets an immune cell antigen.

[15] The antibody is an asymmetric trivalent bispecific antibody, comprising a fused heavy chain, a cross light chain, a heavy chain and a light chain, and having light chain-heavy chain pairing, light chain-fused heavy chain pairing, cross light chain-fused heavy chain pairing, and fused heavy chain-heavy chain pairing, each of which forms an interchain disulfide bond; the light chain comprises a first light chain variable domain VLm and a light chain constant domain CL; the fusion heavy chain comprises a first heavy chain variable domain VHm, a first heavy chain constant domain CH1, a second heavy chain variable domain VHs, a light chain constant domain CL, and an Fc; wherein VHs and CL are linked by a linker to form a peptide fragment "VHs-linker-CL", and the "VHs-linker-CL" is located between CH1 and Fc and linked by a linker / hinge; The antibody described in

[12] , wherein the cross light chain comprises a second light chain variable region VLs and CH1, the VLs and CH1 are linked by a linker, the VLm-VHm pairing targets a tumor antigen, and the VLs-VHs pairing targets an immune cell antigen.

[16] The antibody has two different CH3s and forms a heterodimer between the two CH3s through "projection-cavity structure pairing" and / or "salt bridge pairing"; Preferably, the sequence of the CH3 domain is represented by SEQ ID NO: 102-115. The antibody according to any one of

[13] to

[15] .

[17] The antibody according to any one of

[13] to

[16] , wherein the light chain-heavy chain pairing or VLm-VHm pairing sequence is selected from the following group: (1) SEQ ID NO: 12 and SEQ ID NO: 11, which target the tumor antigen B7-H3; (2) SEQ ID NO: 14 and SEQ ID NO: 13, which target the tumor antigen B7-H3; (3) SEQ ID NO: 16 and SEQ ID NO: 15, which target the tumor antigen CD38; (4) SEQ ID NO: 18 and SEQ ID NO: 17, which target the tumor antigen CD38; (5) SEQ ID NO: 20 and SEQ ID NO: 19, which target the tumor antigen CD38; (6) SEQ ID NO: 22 and SEQ ID NO: 21, targeting the tumor antigen EpCAM; (7) SEQ ID NO: 24 and SEQ ID NO: 23, which target the tumor antigen EpCAM; (8) SEQ ID NO: 26 and SEQ ID NO: 25, targeting the tumor antigen BCMA; (9) SEQ ID NO: 28 and SEQ ID NO: 27, targeting the tumor antigen BCMA; (10) SEQ ID NO: 30 and SEQ ID NO: 29, targeting the tumor antigen BCMA; (11) SEQ ID NO: 32 and SEQ ID NO: 31, which target the tumor antigen PD-L1; (12) SEQ ID NO: 34 and SEQ ID NO: 33, which target the tumor antigen PD-L1; (13) SEQ ID NO: 36 and SEQ ID NO: 35, which target the tumor antigen PD-L1; (14) SEQ ID NO: 38 and SEQ ID NO: 37, targeting the tumor antigen CD19; (15) SEQ ID NO: 40 and SEQ ID NO: 39, targeting the tumor antigen SLAMF7; (16) SEQ ID NO: 42 and SEQ ID NO: 41, targeting the tumor antigen CEA; (17) SEQ ID NO: 2 and SEQ ID NO: 1, targeting the immune antigen CD3; (18) SEQ ID NO: 4 and SEQ ID NO: 3, targeting the immune antigen CD3; (19) SEQ ID NO: 6 and SEQ ID NO: 5, targeting the immune antigen CD3; (20) SEQ ID NO: 8 and SEQ ID NO: 7, which target the immune antigen CD3; and (21) Sequences represented by SEQ ID NO: 10 and SEQ ID NO: 9, which target the immune antigen CD3.

[18] The antibody according to any one of

[13] to

[17] , wherein the antibody is selected from the following group: (1) An antibody PDL1-M1-G2 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:69, SEQ ID NO:94 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:31, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:94 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:32 and SEQ ID NO:75; (2) an antibody PDL1-M1-SG2 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:69, SEQ ID NO:95 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:31, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:95 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:32 and SEQ ID NO:75; (3) an antibody CD38-M1-G2 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:71, SEQ ID NO:94 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:15, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:94 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:16 and SEQ ID NO:75; (4) An antibody CD38-M1-SG2 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:71, SEQ ID NO:95 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:15, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:95 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:16 and SEQ ID NO:75; (5) Antibody CD38-M1-SG2-1 comprising or consisting of fusion peptide 1, heavy chain and light chain; wherein fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:71, SEQ ID NO:95 and SEQ ID NO:109; wherein the heavy chain comprises or consists of SEQ ID NO:19, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:95 and SEQ ID NO:108; and wherein the light chain comprises or consists of SEQ ID NO:20 and SEQ ID NO:75; (6) An antibody CD38-M1-G2-3 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:71, SEQ ID NO:94 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:19, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:94 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:20 and SEQ ID NO:75; (7) Antibody CD38-M1-SG2-2 comprising or consisting of fusion peptide 1, heavy chain and light chain; wherein fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:71, SEQ ID NO:95 and SEQ ID NO:109; wherein the heavy chain comprises or consists of SEQ ID NO:17, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:95 and SEQ ID NO:108; and wherein the light chain comprises or consists of SEQ ID NO:18 and SEQ ID NO:75; (8) Antibody CD38-M1-G2-2 comprising or consisting of fusion peptide 1, heavy chain and light chain; wherein fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:71, SEQ ID NO:94 and SEQ ID NO:109; wherein the heavy chain comprises or consists of SEQ ID NO:17, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:94 and SEQ ID NO:108; and wherein the light chain comprises or consists of SEQ ID NO:18 and SEQ ID NO:75; (9) Antibody CD38-M1-G2-1 comprising or consisting of fusion peptide 1, heavy chain and light chain; wherein fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:94 and SEQ ID NO:109; wherein the heavy chain comprises or consists of SEQ ID NO:15, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:94 and SEQ ID NO:108; and wherein the light chain comprises or consists of SEQ ID NO:16 and SEQ ID NO:75; (10) Antibody CD38-M1-SG2-3 comprising or consisting of fusion peptide 1, heavy chain and light chain; wherein fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:95 and SEQ ID NO:109; wherein the heavy chain comprises or consists of SEQ ID NO:15, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:95 and SEQ ID NO:108; and wherein the light chain comprises or consists of SEQ ID NO:16 and SEQ ID NO:75; (11) An antibody CD38-M1-AG2 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:96 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:15, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:96 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:16 and SEQ ID NO:75; (12) Antibody CD38-M1-GG2 comprising or consisting of fusion peptide 1, heavy chain and light chain; wherein fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:97 and SEQ ID NO:109; wherein the heavy chain comprises or consists of SEQ ID NO:15, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:97 and SEQ ID NO:108; and wherein the light chain comprises or consists of SEQ ID NO:16 and SEQ ID NO:75; (13) Antibody CD38-M1-PG2 comprising or consisting of fusion peptide 1, heavy chain and light chain; wherein fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:98 and SEQ ID NO:109; wherein the heavy chain comprises or consists of SEQ ID NO:15, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:98 and SEQ ID NO:108; and wherein the light chain comprises or consists of SEQ ID NO:16 and SEQ ID NO:75; (14) An antibody CD38-M1-DG2 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:99 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:15, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:99 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:16 and SEQ ID NO:75; (15) Antibody CD38-M1-G2D comprising or consisting of fusion peptide 1, heavy chain and light chain; wherein fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:100 and SEQ ID NO:109; wherein the heavy chain comprises or consists of SEQ ID NO:15, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:100 and SEQ ID NO:108; and wherein the light chain comprises or consists of SEQ ID NO:16 and SEQ ID NO:75; (16) Antibody CD38-M1-DG2D comprising or consisting of fusion peptide 1, heavy chain and light chain; wherein fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:101 and SEQ ID NO:109; wherein the heavy chain comprises or consists of SEQ ID NO:15, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:101 and SEQ ID NO:108; and wherein the light chain comprises or consists of SEQ ID NO:16 and SEQ ID NO:75; (17) Antibody CD38-M1-G2D-1 comprising or consisting of fusion peptide 1, heavy chain and light chain; wherein fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:71, SEQ ID NO:100 and SEQ ID NO:109; wherein the heavy chain comprises or consists of SEQ ID NO:17, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:100 and SEQ ID NO:108; and wherein the light chain comprises or consists of SEQ ID NO:18 and SEQ ID NO:75; (18) An antibody M1IC-SG2 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:95 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:95 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (19) An antibody M1IC-G2 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:94 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:94 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (20) An antibody M1IC-AG2 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:96 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:96 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (21) An antibody M1IC-GG2 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:97 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:97 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (22) An antibody M1IC-PG2 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:98 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:98 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (23) An antibody M1IC-DG2 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:99 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:99 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (24) An antibody M1IC-G2D comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:100 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:100 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (25) An antibody M1IC-DG2D comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:101 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:101 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (26) An antibody M1IC-G2-1 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:94 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:94 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (27) An antibody M1IC-SG2-1 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:95 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:95 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (28) An antibody M1IC-AG2-1 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:96 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:96 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (29) An antibody M1IC-GG2-1 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:97 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:97 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (30) An antibody M1IC-PG2-1 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:98 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:98 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (31) An antibody M1IC-DG2-1 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:99 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:99 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (32) An antibody M1IC-G2D-1 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:100 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:100 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (33) An antibody M1IC-DG2D-1 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:101 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:101 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (34) An antibody M1IC-DG2D-1A comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:101 and SEQ ID NO:107; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:101 and SEQ ID NO:106; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (35) An antibody M1IC-DG2D-1B comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:101 and SEQ ID NO:115; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:101 and SEQ ID NO:114; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (36) An antibody BCMA-M2-G2 comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:74, SEQ ID NO:48, SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:94 and SEQ ID NO:107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:94 and SEQ ID NO:106; and an antibody wherein the light chain comprises or consists of SEQ ID NO:30 and SEQ ID NO:75; (37) An antibody BCMA-M2-SG2 comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:74, SEQ ID NO:48, SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:95 and SEQ ID NO:107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:95 and SEQ ID NO:106; and an antibody wherein the light chain comprises or consists of SEQ ID NO:30 and SEQ ID NO:75; (38) An antibody BCMA-M2-AG2 comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:74, SEQ ID NO:48, SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:96 and SEQ ID NO:107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:96 and SEQ ID NO:106; and an antibody wherein the light chain comprises or consists of SEQ ID NO:30 and SEQ ID NO:75; (39) An antibody BCMA-M2-GG2 comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:74, SEQ ID NO:48, SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:97 and SEQ ID NO:107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:97 and SEQ ID NO:106; and an antibody wherein the light chain comprises or consists of SEQ ID NO:30 and SEQ ID NO:75; (40) An antibody BCMA-M2-PG2 comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:74, SEQ ID NO:48, SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:98 and SEQ ID NO:107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:98 and SEQ ID NO:106; and an antibody wherein the light chain comprises or consists of SEQ ID NO:30 and SEQ ID NO:75; (41) An antibody BCMA-M2-DG2 comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:74, SEQ ID NO:48, SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:99 and SEQ ID NO:107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:99 and SEQ ID NO:106; and an antibody wherein the light chain comprises or consists of SEQ ID NO:30 and SEQ ID NO:75; (42) An antibody BCMA-M2-G2D comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:74, SEQ ID NO:48, SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:100 and SEQ ID NO:107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:100 and SEQ ID NO:106; and an antibody wherein the light chain comprises or consists of SEQ ID NO:30 and SEQ ID NO:75; (43) An antibody BCMA-M2-DG2D comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:74, SEQ ID NO:48, SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:101 and SEQ ID NO:107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:101 and SEQ ID NO:106; and an antibody wherein the light chain comprises or consists of SEQ ID NO:30 and SEQ ID NO:75; (44) An antibody M2IC-G2 comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 94 and SEQ ID NO: 107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 106; and an antibody wherein the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (45) An antibody M2IC-SG2 comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 95 and SEQ ID NO: 107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 106; and an antibody wherein the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (46) An antibody M2IC-AG2 comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 96 and SEQ ID NO: 107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 96 and SEQ ID NO: 106; and an antibody wherein the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (47) An antibody M2IC-GG2 comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 97 and SEQ ID NO: 107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 97 and SEQ ID NO: 106; and an antibody wherein the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (48) An antibody M2IC-PG2 comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 98 and SEQ ID NO: 107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 98 and SEQ ID NO: 106; and an antibody wherein the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (49) An antibody M2IC-DG2 comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 99 and SEQ ID NO: 107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 99 and SEQ ID NO: 106; and an antibody wherein the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (50) An antibody M2IC-G2D comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 100 and SEQ ID NO: 107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 106; and an antibody wherein the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (51) An antibody M2IC-DG2D comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 101 and SEQ ID NO: 107; an antibody wherein the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 106; and an antibody wherein the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (52) An antibody CEA-M3-G2 comprising or consisting of a fusion heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fusion heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75; (53) An antibody CEA-M3-SG2 comprising or consisting of a fused heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fused heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75; (54) An antibody CEA-M3-AG2 comprising or consisting of a fusion heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fusion heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 96 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 96 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75; (55) An antibody CEA-M3-GG2 comprising or consisting of a fusion heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fusion heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 97 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 97 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75; (56) Antibody CEA-M3-PG2 comprising or consisting of a fused heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fused heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 98 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 98 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75; (57) An antibody CEA-M3-DG2 comprising or consisting of a fused heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fused heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 99 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 99 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75; (58) An antibody CEA-M3-G2D comprising or consisting of a fusion heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fusion heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75; (59) An antibody CEA-M3-DG2D comprising or consisting of a fused heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fused heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75; (60) Antibody M3IC-G2 comprising or consisting of a fused heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fused heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 94 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (61) Antibody M3IC-SG2 comprising or consisting of a fused heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fused heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 95 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (62) Antibody M3IC-AG2 comprising or consisting of a fused heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fused heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 96 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 96 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (63) Antibody M3IC-GG2 comprising or consisting of a fused heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fused heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 97 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 97 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (64) Antibody M3IC-PG2 comprising or consisting of a fused heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fused heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 98 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 98 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (65) Antibody M3IC-DG2 comprising or consisting of a fused heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fused heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 99 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 99 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (66) Antibody M3IC-G2D comprising or consisting of a fused heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fused heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (67) Antibody M3IC-DG2D comprising or consisting of a fused heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fused heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (68) An antibody CD3mAb-G2 comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:94 and SEQ ID NO:102; (69) An antibody CD3mAb-SG2 comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:95 and SEQ ID NO:102; (70) An antibody CD3mAb-AG2 comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:96 and SEQ ID NO:102; (71) An antibody CD3mAb-GG2 comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:97 and SEQ ID NO:102; (72) An antibody CD3mAb-PG2 comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:98 and SEQ ID NO:102; (73) An antibody CD3mAb-G2-C229L comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:128 and SEQ ID NO:102; (74) An antibody CD3mAb-G2-C229F comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:129 and SEQ ID NO:102; (75) An antibody CD3mAb-G2-C229R comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:130 and SEQ ID NO:102; (76) An antibody CD3mAb-G2-C229V comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:131 and SEQ ID NO:102; (77) An antibody CD3mAb-G2-C229Q comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:132 and SEQ ID NO:102; (78) An antibody CD3mAb-G2-C229K comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:133 and SEQ ID NO:102; (79) An antibody CD3mAb-G2-C229D comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:134 and SEQ ID NO:102; (80) An antibody CD3mAb-G2-C229I comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:135 and SEQ ID NO:102; (81) An antibody CD3mAb-G2-C229Y comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:136 and SEQ ID NO:102; (82) An antibody CD3mAb-G2-C229N comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:137 and SEQ ID NO:102; (83) An antibody CD3mAb-G2-C229M comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:138 and SEQ ID NO:102; (84) An antibody CD3mAb-G2-C229T comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:139 and SEQ ID NO:102; (85) An antibody CD3mAb-G2-C229H comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:140 and SEQ ID NO:102; (86) An antibody CD3mAb-G2-C229E comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:141 and SEQ ID NO:102; (87) An antibody CD3mAb-G2-C229W comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:142 and SEQ ID NO:102; (88) An antibody CD3mAb-G2-C229L comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:128 and SEQ ID NO:102; (89) An antibody CD3mAb-DG2 comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:99 and SEQ ID NO:102; (90) An antibody CD3mAb-G2-D265P comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:143 and SEQ ID NO:102; (91) An antibody CD3mAb-G2-D265K comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:144 and SEQ ID NO:102; (92) An antibody CD3mAb-G2-D265S comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:145 and SEQ ID NO:102; (93) An antibody CD3mAb-G2-D265F comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:146 and SEQ ID NO:102; (94) An antibody CD3mAb-G2-D265R comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:147 and SEQ ID NO:102; (95) An antibody CD3mAb-G2-D265L comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:148 and SEQ ID NO:102; (96) An antibody CD3mAb-G2-D265G comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:149 and SEQ ID NO:102; (97) An antibody CD3mAb-G2-D265T comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:150 and SEQ ID NO:102; (98) An antibody CD3mAb-G2-D265Y comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:151 and SEQ ID NO:102; (99) An antibody CD3mAb-G2-D265W comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:152 and SEQ ID NO:102; (100) An antibody CD3mAb-G2-D265H comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:153 and SEQ ID NO:102; (101) An antibody CD3mAb-G2-D265V comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:154 and SEQ ID NO:102; (102) An antibody CD3mAb-G2-D265Q comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:155 and SEQ ID NO:102; (103) An antibody CD3mAb-G2-D265E comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:156 and SEQ ID NO:102; (104) An antibody CD3mAb-G2-D265M comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:157 and SEQ ID NO:102; (105) An antibody CD3mAb-G2-D265N comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:158 and SEQ ID NO:102; (106) An antibody CD3mAb-G2-D265I comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:159 and SEQ ID NO:102; (107) An antibody CD3mAb-G2D comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:100 and SEQ ID NO:102; (108) An antibody CD3mAb-G2-D270L comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:160 and SEQ ID NO:102; (109) An antibody CD3mAb-G2-D270R comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:161 and SEQ ID NO:102; (110) An antibody CD3mAb-G2-D270P comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:162 and SEQ ID NO:102; (111) An antibody CD3mAb-G2-D270G comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:163 and SEQ ID NO:102; (112) An antibody CD3mAb-G2-D270V comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:164 and SEQ ID NO:102; (113) An antibody CD3mAb-G2-D270H comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:165 and SEQ ID NO:102; (114) An antibody CD3mAb-G2-D270Y comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:166 and SEQ ID NO:102; (115) An antibody CD3mAb-G2-D270I comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:167 and SEQ ID NO:102; (116) An antibody CD3mAb-G2-D270E comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:168 and SEQ ID NO:102; (117) An antibody CD3mAb-G2-D270F comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:169 and SEQ ID NO:102; (118) An antibody CD3mAb-G2-D270K comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:170 and SEQ ID NO:102; (119) An antibody CD3mAb-G2-D270W comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:171 and SEQ ID NO:102; (120) An antibody CD3mAb-G2-D270S comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:172 and SEQ ID NO:102; (121) An antibody CD3mAb-G2-D270T comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:173 and SEQ ID NO:102; (122) An antibody CD3mAb-G2-D270Q comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:174 and SEQ ID NO:102; (123) An antibody CD3mAb-G2-D270M comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:175 and SEQ ID NO:102; (124) An antibody CD3mAb-G2-D270N comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:176 and SEQ ID NO:102; (125) An antibody CD3mAb-PG2-GA comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:122 and SEQ ID NO:102; (126) An antibody CD3mAb-PG2-TA comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and wherein the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:123 and SEQ ID NO:102; (127) An antibody CD3mAb-G2D-GA comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:124 and SEQ ID NO:102; (128) An antibody CD3mAb-G2D-TA comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:125 and SEQ ID NO:102; (129) An antibody CD3mAb-G2D-GATA comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO: 2 and SEQ ID NO: 75; and the heavy chain comprises or consists of SEQ ID NO: 1, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 126 and SEQ ID NO: 102; or (130) An antibody CD3mAb-PDG2D comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:127 and SEQ ID NO:102.

[19] A conjugate of the antibody according to any one of

[11] to

[18] , comprising a substance A conjugated or fused to the antibody, the substance A being selected from a therapeutic agent, a prodrug, a protein (e.g., an enzyme), a virus, a lipid, a biological response modifier (e.g., an immunomodulator), PEG, a hormone, an oligonucleotide, a diagnostic agent, a cytotoxic agent (e.g., a drug or a toxin), an ultrasound enhancing agent, a non-radioactive label, a detectable label, for example, a chemiluminescent labeling compound (e.g., luminol, isoluminol, an acridinium ester, an imidazole, an acridinium salt and an oxalate ester), or a fluorescent metal (e.g., 152Eu, or a lanthanum-based label).

[20] A polynucleotide encoding the polypeptide according to any one of [1] to

[10] .

[21] A cell comprising the polynucleotide described in

[20] .

[22] A composition, preferably a pharmaceutical composition, comprising the polypeptide according to any one of [1] to

[10] , or the antibody according to any one of

[11] to

[18] .

[23] A method for administering a polypeptide or antibody to a mammalian (preferably human) subject without inducing antibody-dependent cellular cytotoxicity, comprising administering to the subject a polypeptide described in any one of [1] to

[10] or an antibody described in any one of

[11] to

[18] .

[24] Use of a polypeptide according to any one of [1] to

[10] or an antibody according to any one of

[11] to

[18] in the preparation of a drug that does not induce antibody-dependent cellular cytotoxicity after administration to a mammalian (preferably human) subject.

[0014] The antibody comprises an Fc fragment of a heavy chain constant region, where Fc comprises a hinge, CH2 and CH3, and where the CH2 domain comprises one or more substitutions that can significantly reduce the binding ability of the Fc fragment to an Fcγ receptor (FcγR) and reduce non-specific T cell activation by the antibody (e.g., an anti-CD3 antibody).

[0015] In some embodiments, the Fc of the antibody comprises a CH3 domain, and the CH3 domain contains one or more substitutions that form knobs-into-holes pairings between two different CH3 domains.

[0016] After simultaneously carrying out both the above-mentioned CH2 and CH3 substitutions, the Fc fragment has a significantly reduced ability to bind to FcγR, and can reduce nonspecific T cell activation by antibodies (e.g., anti-CD3 antibodies).

[0017] In some embodiments, the antibody has a light chain-heavy chain pairing or a VLm-VHm pairing with specificity for a tumor antigen, in one embodiment, the tumor antigen is selected from PD-L1, SLAMF7, B7-H3, CEA, CD38, EpCAM, CD19, BCMA, etc. In one embodiment, the light chain-heavy chain pairing or the VLm-VHm pairing has specificity for a protein that is overexpressed in tumor cells compared to corresponding non-tumor cells.

[0018] In some embodiments, the antibody light chain-heavy chain pairing or VLm-VHm pairing has specificity for viruses or bacteria. In one embodiment, the antibody light chain-heavy chain pairing or VLm-VHm pairing has specificity for endotoxin.

[0019] In some aspects, the antibody comprises a fusion peptide or a pairing of light and heavy chain variable regions (VLs-VHs pairing), which has specificity for an immune cell surface antigen, such as CD3, CD16A, CD47, or NKG2D.

[0020] In some aspects, the immune cells are selected from T cells, CIK cells, NKT cells, B cells, monocytes, macrophages, neutrophils, dendritic cells, macrophages, natural killer cells, eosinophils, basophils, and mast cells.

[0021] In some embodiments, the heavy chain, fusion heavy chain and / or Fc fragment of the fusion peptide comprises one or more substitutions compared to a wild-type antibody fragment that form a protuberance-cavity pairing between the heavy chain and the fusion peptide or between the heavy chain and the fusion heavy chain, which can significantly improve the pairing efficiency of the heterodimer of the heavy chain and the fusion peptide.

[0022] In some embodiments, the heavy chain, the fusion heavy chain and / or the Fc fragment of the fusion peptide comprises one or more substitutions that form a salt-bridge between the heavy chain and the fusion peptide or between the heavy chain and the fusion heavy chain, which can significantly improve the pairing efficiency of the heterodimer of the heavy chain and the fusion peptide.

[0023] In some cases, the CH2 domain of the fusion peptide is located between the scFv fragment and the CH3 domain. In one embodiment, the fusion peptide does not include a CH1 domain.

[0024] In some embodiments, between the VH and hinge region of the fused heavy chain is a light chain constant region.

[0025] In some embodiments, the fusion heavy chain and the cross light chain are paired, and the cross light chain is in a VL-CH1 configuration.

[0026] In one embodiment, the invention further provides a composition comprising the antibody of any of the above embodiments. In one aspect, the composition further comprises a carrier, wherein the carrier is a pharmaceutical carrier.

[0027] In another aspect, there is provided a conjugate comprising any of the above antibodies that bind one or more antigens.

[0028] The present application further provides a method for preparing the above-mentioned antibody.

[0029] definition In addition, when there is no clear limit to the number, an entity is understood to refer to one or more (species) of that entity. For example, a "multifunctional antibody" is understood to refer to one or more (species) of multifunctional antibodies. Similarly, when there is no clear limit to the number, the terms "one or more" and "at least one" can be used interchangeably in this application.

[0030] The term "polypeptide" as used herein includes the singular "polypeptide" as well as the plural "polypeptides" and refers to a molecule having monomers (amino acids) linearly linked through amide bonds (also called peptide bonds). The term "polypeptide" refers to a chain or chains of amino acids or amino acids, and does not refer to a specific length. Thus, peptide, dipeptide, terpeptide, oligopeptide, "protein", "chain of amino acids", or other terms referring to a chain or chains of any amino acids or amino acids are all included in the definition of "polypeptide". The term "polypeptide" may also be used in place of or interchangeably with these terms. The term "polypeptide" also refers to the product of post-expression modifications of a polypeptide, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, hydrolytic dissolution of proteins, or modifications of non-naturally occurring amino acids. A polypeptide may be derived from a naturally occurring organism or produced by homologous recombination techniques, but is not necessarily translated from a specified nucleic acid sequence. They can be made in any manner, including chemical synthesis.

[0031] As used herein, the term "recombinant" when referring to a polypeptide or polynucleotide refers to a form of a polypeptide or polynucleotide that does not exist in nature, where one non-limiting example can be achieved by combining polynucleotides or polypeptides that do not normally exist together.

[0032] "Homology" or "identity" or "similarity" refers to the degree of sequence similarity between two peptide chain molecules or between two nucleic acid molecules. Homology can be measured by comparing the positions of each sequence. Comparison can be performed by alignment. If the same base or amino acid is present at a position in the compared sequences, the molecules at that position are considered to be homologous. The degree of homology between multiple sequences is a function of the number of pairings or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence has less than 40% homology, preferably less than 25% homology, with one of the sequences of the present invention.

[0033] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a "sequence identity" with another sequence at a certain percentile (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) means that the two sequences have similar bases (or amino acids) at that percentile when aligned and compared. Such alignment, percentile homology or sequence identity can be measured by software programs known in the art, such as those described in Current Protocols in Molecular Biology, edited by Ausubel et al. (2007). Preferably, the alignment is performed using default parameters. BLAST is one alignment program that uses default parameters. Specifically, the programs BLASTN and BLASTP are used, and the following initial parameters are used: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Detailed information on these programs is available at the following website: http: / / www.ncbi.nlm.nih.gov / blast / Blast.cgi, last accessed on May 21, 2008. Biologically equivalent polynucleotides refer to polynucleotides that have the specific percentile of homology described above and encode polypeptides having the same or similar biological activity.

[0034] The term "encode" when applied to a polynucleotide refers to a polynucleotide that is believed to "code" a particular polypeptide. When the polynucleotide is in its natural state or manipulated by methods known to those of skill in the art, it can be transcriptome and / or translated to produce the mRNA of the polypeptide and / or fragments thereof. The antisense strand is the complement of such a nucleic acid. The coding sequence can be derived from the antisense strand.

[0035] As used herein, an "antibody" or "antigen-binding polypeptide" refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody may be a complete antibody or any antigen-binding fragment or single chain thereof. Thus, the term "antibody" includes any protein or peptide containing a specific molecule that includes at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Examples of such embodiments include, but are not limited to, at least a portion of the complementarity determining regions (CDRs) of a heavy or light chain or a ligand binding portion thereof, the variable region of a heavy or light chain, the constant region of a heavy or light chain, the framework (FR) region or any portion thereof, or a binding protein.

[0036] The term "antibody fragment" or "antigen-binding fragment" as used herein refers to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, Fd, dAb, Fab / c, scFv, etc. Antibody fragments that bind to the same antigen, regardless of structure, are considered to be intact antibodies. The term "antibody fragment" includes aptamers, enantiomers of aptamers (spiegelmers) and diabodies. The term "antibody fragment" also includes synthetic or genetically engineered proteins that, like antibodies, are capable of binding to a specific antigen to form a complex.

[0037] "Single chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some cases, these regions are linked by a short linker peptide of 10 to about 25 amino acids. The linker may be glycine-rich for flexibility or may contain serine or threonine for solubility. The N-terminus of VH may be linked to the C-terminus of VL, or vice versa. The protein retains the properties of the original immunoglobulin, only the constant regions have been removed and the linker introduced. ScFv molecules are known and are described in U.S. Pat. No. 5,892,019.

[0038] The term "antibody" includes a wide variety of polypeptides capable of biochemical recognition. As will be understood by those skilled in the art, heavy chains are divided into gamma, mu, alpha, delta, and epsilon (γ, μ, α, δ, ε) with several subclasses (e.g., γ1-4). The "classes" of antibodies, e.g., IgG, IgM, IgA, IgD, IgE, or IgY, are determined by the properties of the chains. Immunoglobulin subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and functionally specific. In view of the present invention, the skilled artisan can readily recognize all variations of these classes and isotypes, and therefore these forms are within the scope of the present invention. All immunoglobulin classes are clearly included within the scope of the present invention, and the following description will generally refer to the IgG class of immunoglobulin molecules. A standard immunoglobulin molecule of IgG contains two identical light chain polypeptides (having a molecular weight of about 23,000 Da) and two identical heavy chain polypeptides (having a molecular weight of 53,000-70,000). These four chains are typically linked by disulfide bonds in a "Y" configuration, where the light chains support the heavy chains from the mouth of the "Y" structure and extend beyond the variable region.

[0039] Antibodies, antigen-binding polypeptides, variants or derivatives thereof of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized or chimeric antibodies, single chain antibodies, antigen-binding fragments, such as Fab, Fab' and F(ab')2, Fd, Fvs, single chain Fvs (scFv), single chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising a VL domain or a VH domain, fragments generated by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, the anti-Id antibodies or LIGHT antibodies disclosed herein). The immunoglobulin or antibody molecules of the present invention may be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), any class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.

[0040] Light chains are classified as kappa or lambda (κ, λ). Each type of heavy chain can be bound to a kappa or lambda light chain. Generally, the light and heavy chains are covalently bound. Furthermore, when these immunoglobulins are produced by hybridomas, B cells or genetically modified host cells, the "tail" portions of the two heavy chains are bound by covalent disulfide bonds or non-covalent bonds. In the heavy chains, the amino acid sequence extends from the N-terminus at the fork of the Y-shaped structure to the C-terminus at the bottom of each chain.

[0041] Both the light and heavy chains are divided into structural and functional homolog regions. The terms "constant" and "variable" are used for functional purposes. It should be recognized here that the light chain variable domain (VL) together with the heavy chain variable domain (VH) determine antigen recognition and specificity. Meanwhile, the light chain constant domain (CL) and the heavy chain constant domain (CH1, CH2 or CH3) provide important biological properties such as secretion, mobility across the placenta, binding to Fc receptors, binding to complement, etc. In general, the number of constant region domains increases with distance from the antigen-binding site or amino-terminal position of the antibody. The N-terminal portion is the variable region, the C-terminal portion is the constant region, and the CH3 and CL domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0042] As described above, the variable region allows the antibody to selectively recognize and specifically bind to an antigen epitope of an antigen. That is, the variable region that defines the three-dimensional antigen-binding site is composed of the VL domain and VH domain of the antibody, or a combination of subtypes of complementarity determining regions (CDRs). The tetravalent antibody structure forms an antigen-binding site. The antigen-binding site is present at the end of each arm of the Y-shape. More specifically, the antigen-binding site is defined by three CDRs of all VH and VL chains (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In some cases, for example, immunoglobulins derived from a certain species or modified based on immunoglobulins, the complete immunoglobulin molecule may be composed of only heavy chains and may not include light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993).

[0043] Assuming that the three-dimensional structure of an antibody is in an aqueous environment, naturally occurring antibodies have six "complementarity determining regions" or "CDRs" present in each antigen-binding domain, which are short, non-contiguous amino acid sequences that are specifically positioned to form the antigen-binding domain. The remaining amino acids of the antigen-binding domain are called "framework" regions and have fewer intramolecular variabilities. The framework regions mainly adopt a β-folded sheet structure, and the CDRs form a ring. The ring may be connected to and become part of the β-folded sheet structure. Thus, the framework regions form a scaffold for correctly orienting the CDRs with intrachain non-covalent interactions. The positioned CDRs form the antigen-binding domain and define a surface complementary to the immunoreactive antigen epitope. The complementary surface contributes to non-covalent binding between the antibody and its homologous epitope. The amino acids belonging to the CDR and framework regions, respectively, have already been clearly defined and can be readily recognized by one of skill in the art from any given heavy or light chain variable region (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., USDepartment of Health and Human Services, (1983); Chothia and Lesk, J. MoI. Biol., 196:901-917 (1987), which are incorporated herein by reference).

[0044] To the extent available and / or acceptable within the art, when a term has two or more definitions, the definition of the term as used herein includes all meanings thereof, unless specifically stated to the contrary. In one specific example, the term "complementarity determining region" ("CDR") is used to describe the non-contiguous antigen-binding sites present in the variable regions of both the heavy and light chain polypeptides. Such specific regions are described by Kabat et al., U.S. Department of Health and Public Service, "Sequences of Proteins of Immunological Interest" (1983), and by Chothia et al., J.MoI.Biol. 196:901-917 (1987), both of which are incorporated herein by reference. As defined by Kabat and Chothia, CDRs include amino acid residues or amino acid substructures that overlap when compared to each other. However, all applications of the definitions with respect to CDRs of an antibody or variants thereof are within the scope of the term as defined and used in this application. For comparison, the appropriate amino acid residues which comprise the CDRs as defined by all of the references cited above are set forth in the table below. The exact residue numbers which comprise a particular CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of the antibody, one of skill in the art can generally determine which residues comprise a particular CDR.

[0045] [Table 1]

[0046] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. One skilled in the art can use the "Kabat numbering" system for any variable domain sequence without any doubt and without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., the contents of which are set forth in the U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0047] Except for the contents of the table above, the CDR areas described by the Kabat numbering system are as follows: CDR-H1 begins at about amino acid 31 (i.e., about 9 residues from the cysteine ​​residue at position 1) and ends at the next tryptophan residue and includes about 5-7 amino acids. CDR-H2 begins at about amino acid 15 from the end of CDR-H1 and ends at the next arginine or lysine residue and includes about 16-19 amino acids. CDR-H3 begins at about amino acid 33 after the end of CDR-H2 and ends at the sequence WGXG and includes 3-25 amino acids, where X is any amino acid. CDR-L1 begins at about amino acid 24 (i.e., after the cysteine ​​residue) and ends at the next tryptophan residue and includes about 10-17 residues. CDR-L2 begins at about amino acid 16 after the end of CDR-L1 and includes about 7 residues. CDR-L3 begins approximately 33 residues after the end of CDR-L2 (i.e., after the cysteine ​​residue) and includes about 7-11 residues, ending with the sequence F or WGXG, where X is any amino acid.

[0048] The antibodies described in this application may be from any animal, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, alpaca, horse or chicken. In another embodiment, the variable region is from a condricthoid (e.g., from a shark).

[0049] The term "heavy chain constant region" as used herein includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region includes at least a CH1 domain, a hinge (e.g., an upper hinge region, a middle hinge region, and / or a lower hinge region), a CH2 domain, a CH3 domain, or any variant or fragment thereof. For example, an antigen-binding polypeptide for use in the present invention may include a polypeptide chain having a CH1 domain; a polypeptide having a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain having a CH1 domain and a CH3 domain; a polypeptide chain having a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain having a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the present invention includes a polypeptide chain having a CH3 domain. An antibody for use in the present invention may also lack at least a portion of a CH2 domain (e.g., all or a portion of a CH2 domain). As mentioned above, it should be understood by those of ordinary skill in the art that the heavy chain constant region may be altered so that its amino acid sequence differs from that of a naturally occurring immunoglobulin molecule.

[0050] The heavy chain constant regions of the antibodies described in this application may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another embodiment, the heavy chain constant region may comprise a hinge region that is partially derived from an IgG1 fragment and partially derived from an IgG3 molecule. In another embodiment, the heavy chain portion may comprise a chimeric hinge that is partially derived from an IgG1 fragment and partially derived from an IgG4 molecule.

[0051] As used herein, the term "light chain constant region" includes an amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region includes at least one of a kappa constant domain and a lambda constant domain.

[0052] "Light chain-heavy chain pairing" refers to the assembly of a light chain and a heavy chain that can form a dimer via disulfide bonds between the CL and CH1 domains of the light chain.

[0053] As also mentioned above, the subunit structures and three-dimensional structures of the constant regions of various immunoglobulins are known. As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "CH1 domain" includes the first (often amino-terminal) constant region of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino-terminal to the hinge region of the immunoglobulin heavy chain molecule.

[0054] The term "CH2 domain" as used herein includes a portion of a heavy chain molecule, for example ranging from approximately residue 244 to residue 360 ​​of an antibody in the common numbering scheme (corresponding to residues 244 to 360 in the Kabat numbering system and residues 231-340 in the EU numbering system, see Kabat et al., U.S. Department of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983)). The CH2 domain is unique in that it is not tightly paired with the other domain. Instead, two N-linked side chain carbohydrate chains are inserted between the two CH2 domains in an intact native IgG molecule. The literature describes the CH3 domain as extending from the CH2 domain to the C-terminus of the IgG molecule and containing approximately 108 residues.

[0055] As used herein, the term "hinge region" includes the portion of a heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region contains approximately 25 residues and is flexible, allowing the two N-terminal antigen-binding regions to move independently. The hinge region can be divided into three distinct domains: the upper, middle, and lower hinge domains (Roux et al., J. Immunol, 161:4083 (1998)).

[0056] The term "disulfide bond" as used herein includes a covalent bond formed between two sulfur atoms. Cysteine ​​contains a sulfhydryl group that can form or bridge a disulfide bond with another sulfhydryl group. In the majority of naturally occurring IgG molecules, the CH1 and CL areas are linked by a disulfide bond, and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 according to the Kabat numbering system (positions 226 or 229 according to the EU numbering system).

[0057] As used herein, the term "chimeric antibody" refers to any antibody in which the immune reactive region or site is from or obtainable from a first species and the constant region (which may be complete, partial or modified according to the present invention) is from a second species. In some embodiments, the target binding region or site is of non-human (e.g., murine or primate) origin and the constant region is of human origin.

[0058] As used herein, the "humanized percentile" is calculated by determining the number of in-frame amino acid differences (i.e., non-CDR differences) between the humanized domain and its species domain, subtracting that number from the total number of amino acids, dividing by the total number of amino acids and multiplying by 100.

[0059] The term "specifically binds" or "has specificity for" generally means that an antibody binds to an antigen epitope through its antigen-binding domain such that the binding has some complementarity between the antigen-binding domain and the antigen epitope. By this definition, an antibody is deemed to "specifically bind" to an antigen epitope if the antibody binds to the antigen epitope more readily through its antigen-binding domain than it would to a random, unrelated antigen epitope. In this application, the term "specificity" is used to determine the affinity with which an antibody binds to a particular antigen epitope. For example, antibody "A" may be considered to have a higher specificity for a particular antigen epitope than antibody "B". Or, antibody "A" may be said to have a higher specificity for binding to correlated antigen epitope "D" compared to antigen epitope "C".

[0060] As used herein, the term "treatment" refers to therapeutic treatment and prophylaxis, or prophylactic and curative measures to prevent or slow (alleviate) the progression of an undesirable physiological change or disease in a subject, such as cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or not, reduction in the stage of a disease, stabilization of a disease state (e.g., preventing progression), delay or slowing of disease progression, improvement or palliation of a disease state, and remission (whether partial or total). "Treatment" may also refer to a prolongation of survival compared to expected survival in the absence of treatment. Conditions requiring treatment include those already suffering from a disease or condition, those predisposed to a disease or condition, or those preventing a disease or condition.

[0061] multifunctional antibody An embodiment of the present invention provides various multifunctional antibodies having two different antigen-binding polypeptide elements. The antibody domains that bind to the antigens are Fab, or ScFv, or non-covalent pairing between the heavy chain variable region (VH) and the light chain variable region (VL). In particular, these multifunctional antibodies all have an Fc fragment of an antibody heavy chain constant region, the Fc including: (1) a hinge, (2) a heavy chain second constant region (CH2) and / or a heavy chain third constant region (CH3), where the hinge and CH3 are both domains corresponding to human IgG1 type, and CH3 is modified to have a "protuberance-cavity pairing" mutation, and CH2 is a CH2 domain corresponding to human IgG2 type.

[0062] Any of the above antibodies or polypeptides may also comprise additional polypeptides to form a fusion protein or fusion peptide, such as the encoded polypeptides described herein, a signal peptide of an antibody constant region to direct secretion, or other heterologous polypeptides described herein.

[0063] As will be appreciated by those of ordinary skill in the art, antibodies derived from naturally occurring binding polypeptides described herein may be modified to differ in amino acid sequence from these naturally occurring binding polypeptides. For example, a polypeptide or amino acid sequence derived from a particular protein may be similar to the starting sequence, e.g., have a certain percentage of identity with the starting sequence. For example, it may have 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identity with the starting sequence.

[0064] Nucleotide or amino acid substitutions, deletions, or insertions may also be made to make conservative substitutions or changes to "non-essential" amino acid regions. For example, a polypeptide or amino acid sequence derived from a particular protein can be identical to a starting sequence except for one or more independent amino acid substitutions, insertions, or deletions, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more independent amino acid substitutions, insertions, or deletions. In some embodiments, a polypeptide or amino acid sequence derived from a particular protein has 1-5, 1-10, 1-15, or 1-20 independent amino acid substitutions, insertions, or deletions from the starting sequence.

[0065] In other embodiments, the antigen-binding polypeptides of the invention may contain conservative amino acid substitutions.

[0066] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Groups of amino acid residues having similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred that non-essential amino acid residues in immunoglobulin polypeptides are replaced with other amino acid residues from the same side chain group. In another embodiment, the amino acid sequence of one stretch can be replaced with structurally similar amino acids of another stretch, which differ in the order and / or composition of side chain groups.

[0067] Non-limiting examples of conservative amino acid substitutions are provided in the table below, where a similarity score of 0 or greater indicates a conservative substitution between the two amino acids.

[0068] [Table 2A] [Table 2B]

[0069] In some embodiments, the antibody conjugate may be conjugated to a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a virus, a lipid, a biological response modifier, a drug, or PEG.

[0070] The antibody may be linked or fused to a therapeutic agent, which may include a detectable label such as a radiolabel, an immunomodulatory agent, a hormone, an enzyme, an oligonucleotide, a photoactive therapeutic or diagnostic agent, a cytotoxic agent (which may be a drug or a chemokine), an ultrasound enhancing agent, a non-radioactive label, combinations thereof, and other similar moieties known in the art.

[0071] The antibody is detectably labeled by conjugation with a chemiluminescent compound, and the presence of the chemiluminescent-labeled antigen-binding polypeptide is determined by detecting the luminescence generated during the course of a chemical reaction. Particularly useful chemiluminescent labeling compounds include luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate.

[0072] The antibody may be detectably labeled with a fluorescent metal, such as 152Eu, or other lanthanum-based labels. These metals may be linked to the antibody with a metal chelating group, such as diethylene-triaminepentaacetic acid (DTPA), ethylenediaminetetraacetic acid (EDTA). Techniques for linking various groups to antibodies are known and include those described, for example, in Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., "Antibodies For Drug Delivery," in Controlled Drug Delivery (2nd Edition), Robinson et al. (eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibodies In See, for example, "Antibody-Toxin Conjugates," in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates," Immunol. Rev. (52:119-58 (1982)).

[0073] Methods for producing antibodies Methods for producing antibodies are known in the art and are described herein. In some embodiments, the variable and constant regions of the antigen-binding polypeptides of the invention are fully human. Fully human antibodies can be prepared by techniques described in the prior art and as described herein. For example, fully human antibodies against a particular antigen can be prepared by administering the antigen to a genetically modified animal whose endogenous locus has been inhibited and which has been modified to produce antibodies that respond to antigenic stimulation of this type. Exemplary techniques that can be used to prepare such antibodies are described in U.S. Patents: 6,150,584, 6,458,592, and 6,420,140, ​​which are incorporated herein by reference in their entireties.

[0074] The binding specificity of an antigen-binding polypeptide of the invention can be determined by in vitro experiments, such as immunoprecipitation, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0075] Alternatively, the single chain units of the present invention can be prepared using techniques for preparing single chain units described in (U.S. Pat. No. 4,694,778; Bird, Science 242:423-442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 55:5879-5883 (1988); Ward et al., Nature 334:544-554 (1989)). Single chain fusion peptides can be obtained by linking heavy and light chain fragments of the Fv region via an amino acid bridge to form a single chain unit. Also, techniques for synthesizing functional Fv fragments in E. coli (Skerra et al., Science 242:1038-1041 (1988)) can be used.

[0076] Examples of techniques used to produce single chain Fvs (scFvs) and antibodies include those described in U.S. Patent Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For certain applications, including the use of antibodies in humans and in vitro assays, chimeric, humanized or human antibodies are preferred. Chimeric antibodies are molecules in which different portions of the antibody are derived from different animal species, e.g., antibodies having a variable region from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol. Methods 125:191-202 (1989); U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816,397, each of which is incorporated by reference in its entirety.

[0077] A humanized antibody is an antibody molecule from a non-human species that binds to a desired antigen, the antibody molecule having one or more complementarity determining regions (CDRs) from the non-human species and a framework region from a human immunoglobulin molecule. In general, frame residues in the human framework regions are modified by substitution of corresponding residues from the CDR donor antibody, preferably to increase antigen binding capacity. These frame substitutions can be recognized by methods known in the art. For example, by establishing an interaction model of the CDR and frame residues, the relatively important frame residues for antigen binding and sequence are identified, and unusual frame residues at specific positions are sought (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties). Several techniques known in the art are used to humanize antibodies. Such techniques include, for example, CDR-grafting (EP 239,400; PCT Publication No. WO 91 / 09967; U.S. Patent Nos. 5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., Proc. Natl. Sci. USA 91:969-973 (1994)), and chain shuffling (U.S. Patent No. 5,565,332, incorporated herein by reference in its entirety).

[0078] By conventional recombinant DNA techniques, one or more CDRs of the antigen-binding polypeptide of the invention can be inserted into framework regions, e.g., into human framework regions to humanize a non-human antibody. The framework regions may be naturally occurring or shared framework regions, preferably human framework regions (see, e.g., Chothia et al., J. Mol. Biol. 278:457-479 (1998), for a list of human framework regions). Preferably, the polynucleotide generated by combining the framework regions and the CDRs encodes a polypeptide that specifically binds to at least one antigen epitope (e.g., LIGHT) of the desired polypeptide. Preferably, one or more amino acid substitutions may be made in the framework regions, where the amino acid substitutions are preferably substitutions that improve the binding ability of the antibody to the antigen. The method also allows for amino acid substitutions or deletions of one or more cysteine ​​residues (cysteine ​​residues involved in the formation of intrachain disulfide bonds) in the variable regions. Thus, an antibody molecule lacking one or more intrachain disulfide bonds can be obtained. Any other alterations made to the polynucleotide are within the scope of the present invention, as well as within the prior art.

[0079] Also available is the technique of combining a mouse-derived antibody molecule having the appropriate antigen specificity with a human antibody molecule gene having the appropriate biological activity, followed by gene splicing to produce a "chimeric antibody" (Morrison et al., Proc. Natl. Acad. Sci. USA:851-855 (1984); Neuberger et al., Nature 372:604-608 (1984); Takeda et al., Nature 314:452-454 (1985)). As used herein, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.

[0080] However, another efficient method used for the production of recombinant antibodies is disclosed by Newman, Biotechnology 10:1455-1460 (1992). Specifically, the technique produces primatized antibodies containing monkey variable domains and human constant sequences, which document is incorporated herein by reference. The technique is also described in coassigned U.S. Patent Nos. 5,658,570, 5,693,780, and 5,756,096, each of which is incorporated herein by reference.

[0081] Also, antibody-producing cell lines may be selected and cultured using techniques familiar to those skilled in the art. Such techniques are described in a variety of laboratory manuals and major publications. In this regard, techniques suitable for use herein are described in Current Protocols in Immunology, Coligan et al., eds., Green Publishing Associates And Wiley-Interscience, John Wiley and Sons, New York (1991), which is incorporated herein by reference, including supplementary references, as follows:

[0082] Additionally, standard techniques known to those skilled in the art can be used to introduce mutations into the nucleotide sequence encoding the antibody of the invention. Such mutations include, but are not limited to, site-directed mutagenesis and PCR-induced mutations to generate amino acid substitutions. Preferably, the variants (including derivatives) of the CDR-H1, CDR-H2, CDR-H3, light chain variable region, CDR-L1, CDR-L2 or CDR-L3 encode fewer than 50 amino acid substitutions, fewer than 40 amino acid substitutions, fewer than 30 amino acid substitutions, fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, or fewer than 2 amino acid substitutions compared to the reference variable heavy chain region. Alternatively, mutations may be introduced randomly along all or part of the coding sequence. For example, if saturation mutagenesis is used, the resulting variants can be screened for biological activity to ensure that they carry active mutations.

[0083] Structural information of antibodies A monospecific antibody is an antibody that has two identical light chains and two identical heavy chains linked by disulfide bonds between the light and heavy chains to target a corresponding antigen, and a disulfide bond between the heavy and heavy chains, making the whole antibody a "Y" shaped structure. The light chain has a light chain variable region (VL) and a light chain constant region (Lc), the heavy chain has a heavy chain variable region (VH) and a heavy chain constant region, the heavy chain constant region includes CH1 and Fc, and Fc includes a hinge, CH2 and CH3.

[0084] Multifunctional antibody structure 1 is an asymmetric bispecific antibody comprising a light chain, a heavy chain, and a fusion peptide 1, the fusion peptide 1 comprising an scFv and an Fc fragment, having light-heavy chain pairing and heavy chain-fusion peptide 1 pairing, each of which forms an interchain disulfide bond, the light-heavy chain pairing targets a tumor antigen, and the ScFv in the fusion peptide 1 targets an immune cell antigen.

[0085] FIG. 1A is a schematic diagram of a structure having a multifunctional antibody structure 1, and FIG. 1B is a schematic diagram of the primary protein structures of each component of the antibody.

[0086] Multifunctional antibody structure 2 is an asymmetric trivalent bispecific antibody comprising two light chains, one heavy chain and one fusion peptide 2, comprising light-heavy chain pairing, light chain-fusion peptide 2 pairing and heavy chain-fusion peptide 2 pairing, each of which forms an interchain disulfide bond, fusion peptide 2 comprising a heavy chain variable region (VH), a heavy chain first constant region (CH1), an ScFv and an Fc, wherein the ScFv is located between the CH1 and the Fc and linked by a linker, the light-heavy chain pairing targets a tumor antigen, the VH-CH1 and light chain pairing of the fusion peptide 2 targets the same tumor antigen, and the ScFv targets an immune cell antigen.

[0087] FIG. 2A is a schematic diagram of the structure of the multifunctional antibody structure 2, and FIG. 2B is a schematic diagram of the primary protein structure of each component of the antibody.

[0088] Multifunctional antibody structure 3 is an asymmetric trivalent bispecific antibody comprising: a fused heavy chain, a cross light chain, a heavy chain and a light chain, and has light chain-heavy chain pairing, light chain-fused heavy chain pairing, cross light chain-fused heavy chain pairing, and fused heavy chain-heavy chain pairing, each of which forms an interchain disulfide bond, and the light chain comprises a first light chain variable region (VLm) and a light chain constant region (CL); the fused heavy chain comprises a first heavy chain variable region (VHm), a first heavy chain constant region (CH1), a second heavy chain variable region (VHs) a cross light chain comprising a second light chain variable region (VLs) and CH1, wherein the VLs and CH1 are linked by a linker, and wherein the VLm-VHm pairing targets a tumor antigen and the VLs-VHs pairing targets an immune cell antigen.

[0089] FIG. 3A is a schematic diagram of the structure of the multifunctional antibody structure 3, and FIG. 3B is a schematic diagram of the primary protein structure of each component of the antibody.

[0090] The three types of multifunctional antibody structures all have an Fc fragment containing CH2 and / or CH3, in which the CH2 is the native sequence of CH2 of human IgG2 or a modified sequence containing amino acid point mutations, some specific sequences are shown in SEQ ID NOs: 83-101, 122-176, and each multifunctional antibody has two different CH3s, and a heterodimer is formed between the two CH3s by "protrusion-cavity structure pairing" and / or "salt bridge pairing", the specific sequences are shown in SEQ ID NOs: 102-115.

[0091] Variable region of antibody sequence

[0092] [Table 3]

[0093] (1) Antibodies targeting tumor-associated antigens

[0094] [Table 4]

[0095] [Table 5]

[0096] [Table 6]

[0097] [Table 7]

[0098] [Table 8]

[0099] [Table 9]

[0100] [Table 10]

[0101] [Table 11]

[0102] [Table 12]

[0103] Sequences of other domains (1) Amino acid sequence of the linker domain

[0104] [Table 13]

[0105] (2) Amino acid sequence of the hinge domain

[0106] [Table 14]

[0107] (3) Amino acid sequence of the light chain constant region CL domain

[0108] [Table 15]

[0109] (4) Amino acid sequence of the heavy chain constant region CH1 domain

[0110] [Table 16]

[0111] Specific information on Fc modifications The numbering of Fc amino acids follows the Kabat numbering system. "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Department of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The specific numbers are shown in the table below.

[0112] [Table 17]

[0113] Where: Amino acids 221-227 are the hinge domain; Amino acids 228-340 are the CH2 domain of the second constant region of the heavy chain; Amino acids 341-447 are the CH3 domain of the third heavy chain constant region.

[0114] In one embodiment, the CH2 domain contains one or more substitutions to reduce the binding ability to FcFcγR. Optionally substituted amino acid residues include, but are not limited to, E233, L234, L235, G236, D265, D270, K274, Y296, N297, Y300, L309, A327, P329, P331, A339. Non-limiting examples of these substitution combinations are listed in the following table:

[0115] [Table 18]

[0116] [Table 19]

[0117] In the above table, glycine at position 236 (G236) is deleted, but the amino acid number "236" is kept and indicated by "-".

[0118] The CH3 domain of the antibody can also be altered to improve the pairing efficiency of the heterodimer. For example, in some cases, the Fc fragment of the monovalent unit heavy chain and / or the Fc fragment of the fusion peptide may contain one or more substitutions (knobs-into-holes pairing structures are formed between these substitutions) compared to the wild-type antibody fragment. Knobs-into-holes pairing structures are known in the art. See, for example, Ridgway et al., "'Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization," Protein Engineering 9(7):617-21 (1996).

[0119] In one embodiment, T366 in one CH3 domain is replaced with a relatively large amino acid residue, such as tyrosine (Y) or tryptophan (W), and Y407 in the other CH3 domain may be replaced with a relatively small amino acid residue, such as threonine (T), alanine (A), or valine (V). Some non-limiting examples of these substitution combinations are shown in Table 20.

[0120] [Table 20]

[0121] In one embodiment, one of the CH3 domains contains one or more substitutions and is substituted with an amino acid residue that is positively charged under physiological conditions, and the other CH3 domain contains one or more substitutions and is substituted with one or more amino acid residues that are negatively charged under physiological conditions. In one embodiment, the positively charged amino acid residue may be arginine (R), histidine (H) or lysine (K). Meanwhile, the negatively charged amino acid residue may be aspartic acid (D) or glutamic acid (E). Optionally substituted amino acid residues include, but are not limited to, D356, L368, K392, D399 and K409. Non-limiting examples of these substitution combinations are listed in Table 21 below.

[0122] [Table 21]

[0123] In one embodiment, one CH3 domain has S354 substituted with cysteine ​​and the other CH3 domain has Y349 substituted with cysteine, and the residues at the two substitution positions form a disulfide bond. An example of such a combination of substitutions is shown in the following table.

[0124] [Table 22]

[0125] In some aspects, the antibody may comprise a CH2 that reduces FcγR binding, or a CH3 that improves heterodimer pairing, or both. In one embodiment, substitution of H435 and Y436 in one CH3 domain with arginine and phenylalanine, respectively, significantly reduced the binding ability of Fc to Protein A. This results in different Protein A binding activities between the heterodimer and the homodimer, making it easier to separate the two during affinity chromatography. An example of such a combination of substitutions is shown in the following table.

[0126] [Table 23] In Tables 18 to 23 above, any combination of amino acid substitutions in different domains can be constructed and formed into a complete Fc fragment according to a connection mode such as "hinge-CH2-CH3", and the Fc fragment satisfies the following: (1) reduces the binding ability to FcγR, (2) contributes to the formation of heterodimers, and (3) changes the binding ability to protein A.

[0127] Some examples of specific Fc sequences

[0128] [Table 24-1] [Table 24-2] [Table 24-3] [Table 24-4] [Table 24-5] [Table 24-6]

[0129] [Table 25]

[0130] The specific sequence of the antigen

[0131] [Table 26]

[0132] [Table 27] [Brief description of the drawings]

[0133] [Figure 1] 1A is a schematic diagram of the structure of multifunctional antibody 1, and FIG. 1B is a schematic diagram of the primary protein structure of each component of multifunctional antibody 1. FIG. [Diagram 2] 2A is a schematic diagram of the structure of multifunctional antibody 2, and FIG. 2B is a schematic diagram of the primary protein structure of each component of multifunctional antibody 2. FIG. [Diagram 3] 3A is a schematic diagram of the structure of multifunctional antibody 3, and FIG. 3B is a schematic diagram of the primary protein structure of each component of multifunctional antibody 3. FIG. [Figure 4] 1 shows the in vitro toxicity of different multifunctional antibodies having multifunctional antibody structure 1 against non-small cell lung cancer cells H358. [Diagram 5] 1 shows the in vitro toxicity of different isotype control antibodies having multifunctional antibody structure 1 against non-small cell lung cancer cells H358. [Figure 6] This shows the in vitro cytotoxicity of CD38×CD3 antibody with multifunctional antibody structure 1 against multiple myeloma cells MC / CAR. [Figure 7] In vitro non-specific cytotoxicity of isotype control antibody with multifunctional antibody structure 1 against multiple myeloma cells MC / CAR. [Figure 8] Shows T cell activation in PBMCs by isotype control antibody with multifunctional antibody structure 1, and the percentage of CD3+CD69+ T cells. [Figure 9] Shows T cell activation in PBMCs by isotype control antibody with multifunctional antibody structure 1, and the percentage of CD3+CD25+ T cells. [Figure 10] 1 shows the FcγR1 binding ability of an isotype control antibody having multifunctional antibody structure 1, and detection of a fluorescent signal due to activation of Jurkat-luciferase cells by the antibody. [Figure 11] 1 shows the FcγR2 binding ability of an isotype control antibody having multifunctional antibody structure 1, and detection of a fluorescent signal due to activation of Jurkat-luciferase cells by the antibody. [Figure 12] 1 shows the FcγR3A binding ability of an isotype control antibody having multifunctional antibody structure 1, and detection of a fluorescent signal due to activation of Jurkat-luciferase cells by the antibody. [Figure 13] 1 shows the in vitro cytotoxicity of different multifunctional antibodies having multifunctional antibody structure 2 against myeloma cells U266B1. [Figure 14] 1 shows in vitro cytotoxicity of different isotype control antibodies having multifunctional antibody structure 2 against myeloma cells U266B1. [Figure 15] Shows T cell activation in PBMCs by isotype control antibody with multifunctional antibody structure 2, and the percentage of CD3+CD69+ T cells. [Figure 16] Shows T cell activation in PBMCs and the percentage of CD3+CD25+ T cells by isotype control antibody with multifunctional antibody structure 2. [Figure 17] This shows the in vitro cytotoxicity of CEA×CD3 antibody with multifunctional antibody structure 3 against gastric cancer cell MKN-45. [Figure 18] 1 shows the in vitro toxicity of an isotype control antibody having multifunctional antibody structure 3 against gastric cancer cells MKN-45. [Figure 19] Shows T cell activation in PBMCs and the percentage of CD3+CD69+ T cells by isotype control antibody with multifunctional antibody structure 3. [Figure 20] Shows T cell activation in PBMCs by isotype control antibody with multifunctional antibody structure 3, and the percentage of CD3+CD25+ T cells. [Figure 21] FIG. 1 shows an accelerated thermal stability test of different antibodies with multifunctional antibody structure 1 at 40° C. for 14 days. [Figure 22]1 shows acid resistance tests of different antibodies having multifunctional antibody structure 1. [Figure 23] 1 shows an accelerated thermal stability test of different antibodies with multifunctional antibody structure 2 at 40° C. for 14 days. [Figure 24] 1 shows acid resistance tests of different antibodies with multifunctional antibody structure 2. [Diagram 25] 1 shows an accelerated thermal stability test of different antibodies with multifunctional antibody structure 3 at 40° C. for 14 days. [Figure 26] 1 shows acid resistance tests of different antibodies with multifunctional antibody structure 3. [Figure 27] FIG. 1 is a schematic diagram of the structure of a monoclonal antibody. [Figure 28] Binding of different Fc-modified 4420 monoclonal antibodies to macrophages detected and analyzed by flow cytometry. [Figure 29] Binding of different Fc-modified 4420 monoclonal antibodies to B cells detected and analyzed by flow cytometry. [Diagram 30] NK cell binding of different Fc-modified 4420 monoclonal antibodies detected and analyzed by flow cytometry. [Figure 31A] The figure shows the degree of activation of T cells in PBMCs by a CD3 monoclonal antibody in which the CH2 domain of the Fc of an IGG1 type CD3 monoclonal antibody was replaced with the CH2 domain of IGG2, and then the residue at position 229 of the Fc was replaced with another amino acid residue, as detected by flow cytometry. (A) is the percentage of CD69+ T cells, and (B) is the percentage of CD25+ T cells. [Figure 31B] The figure shows the degree of activation of T cells in PBMCs by a CD3 monoclonal antibody in which the CH2 domain of the Fc of an IGG1 type CD3 monoclonal antibody was replaced with the CH2 domain of IGG2, and then the residue at position 229 of the Fc was replaced with another amino acid residue, as detected by flow cytometry. (A) is the percentage of CD69+ T cells, and (B) is the percentage of CD25+ T cells. [Diagram 32]The figure shows the degree of activation of T cells in PBMCs by a CD3 monoclonal antibody in which the CH2 domain of the Fc of an IGG1 type CD3 monoclonal antibody was replaced with the CH2 of IGG2 and then the 265th amino acid residue of the Fc was replaced with another amino acid residue, as detected by flow cytometry. (A) is the percentage of CD69+ T cells, and (B) is the percentage of CD25+ T cells. [Diagram 33] The graph shows the degree of activation of T cells in PBMCs by a CD3 monoclonal antibody in which the CH2 domain of the Fc of an IGG1 type CD3 monoclonal antibody was replaced with the CH2 of IGG2, and then the 270th amino acid residue in the Fc was replaced with another amino acid residue, as detected by flow cytometry. (A) is the percentage of CD69+ T cells, and (B) is the percentage of CD25+ T cells. [Diagram 34] The graph shows the degree of activation of T cells in PBMCs by a CD3 monoclonal antibody in which the CH2 domain of the Fc of an IGG1 type CD3 monoclonal antibody was replaced with the CH2 domain of IGG2, and then multiple mutations were made to the Fc, as detected by flow cytometry. (A) is the percentage of CD69+ T cells, and (B) is the percentage of CD25+ T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0134] Hereinafter, specific embodiments of the present invention will be described in detail with reference to the drawings. The present invention can be implemented in various forms other than those described in this specification, and those skilled in the art can make similar modifications without departing from the spirit of the present invention, so the scope of protection of the present invention is based on the scope of the claims and is not limited to the specific embodiments disclosed below. EXAMPLES

[0135] Example 1. Preparation of Antibodies A. Construction of Antibody Expression Plasmids Based on the sequences in Tables 28-30, the DNA of the coding sequence was synthesized by Wuhan genecreate Biological Engineering Co., Ltd. and cloned into the vector pcDNA 3.1 (purchased from Invitrogen). Then, each was transformed into Trans10 competent cells (purchased from TransGen Biotech, Beijing). After identification by a sequencer, expression plasmids were obtained.

[0136] Specifically, the following expression plasmid constructions were utilized: 1) The multifunctional antibody construct 1 in Figure 1 was constructed using three plasmids. The three plasmids were a light chain expression plasmid (pL), a heavy chain expression plasmid (pH) and a fusion peptide 1 expression plasmid (pF1), respectively. 2) The multifunctional antibody construct 2 in Figure 2 utilized the construction of three plasmids. The three plasmids were a light chain expression plasmid (pL), a heavy chain expression plasmid (pH) and a fusion peptide 2 expression plasmid (pF2), respectively. 3) The multifunctional antibody construct 3 in Figure 3 utilized the construction of four plasmids, which were a light chain expression plasmid (pL), a heavy chain expression plasmid (pH), a cross light chain expression plasmid (pcL) and a fusion heavy chain expression plasmid (pFH), respectively.

[0137] B. Methods for expressing multifunctional antibodies For transfection, two transient transfection expression systems, CHO-S (purchased from Gibco) or 293E (purchased from ATCC), were used.

[0138] Specifically, the co-transfected plasmid DNA is: 1) Expression of multifunctional antibody 1 shown in FIG. 1 requires co-transfection of plasmids pL, pH and pF1 into CHO-S or 293E cells. 2) Expression of multifunctional antibody 2 shown in FIG. 2 requires co-transfection of plasmids pL, pH and pF2 into CHO-S or 293E cells. 3) To express the multifunctional antibody 3 shown in FIG. 3, it is necessary to co-transfect and express the plasmids pL, pH, pcL and pFH into CHO-S or 293E cells. In general, when two plasmids are co-transfected and expressed, the molar ratio of the two plasmids may be 1:1 or any other ratio. When three plasmids are co-transfected and expressed, the molar ratio of the three plasmids may be 1:1:1 or any other ratio. When four plasmids are co-transfected and expressed, the molar ratio of the four plasmids may be 1:1:1:1 or any other ratio.

[0139] C. Methods for Purifying Multifunctional Antibodies The purification of antibodies mainly uses affinity chromatography, ion exchange chromatography, hydrophobic chromatography and molecular sieves. These are common technical means in this field. For details, please refer to the molecular cloning experiment guide. In this embodiment, step 1 of the purification method is protein A affinity chromatography, and then ion exchange chromatography is used to remove aggregates, so that the final protein purity is 95% or more.

[0140] The partial abbreviations of specific expressed antibodies, and the corresponding antibody variable region amino acid sequences, are shown in the table below.

[0141] [Table 28-1] [Table 28-2] [Table 28-3] [Table 28-4]

Table 28-5

Table 28-6

Table 28-7

Table 28-8

Table 28-9

Table 28-10

Table 28-11

Table 28-12

Table 28-13

Table 28-14

Table 28-15

Table 28-16

Table 28-17

Table 28-18

Table 28-19

Table 28-20

Table 28-21

Table 28-22

Table 28-23

Table 28-24

Table 28-25

Table 28-26

Table 28-27

Table 28-28

Table 28-29

Table 28-30

Table 28-31

Table 28-32

Table 28-33

Table 28-34

Table 28-35

Table 28-36

Table 28-37

Table 28-38

Table 28-39

Table 28-40

Table 28-41

Table 28-42

[0142]

Table 29-1

Table 29-2

Table 29-3

Table 29-4

Table 29-5

Table 29-6

Table 29-7

Table 29-8

Table 29-9

Table 29-10

Table 29-11

Table 29-12

Table 29-13

Table 29-14

Table 29-15

Table 29-16

Table 29-17

Table 29-18

Table 29-19

Table 29-20

Table 29-21

Table 29-22

Table 29-23

[0143]

Table 30-1

Table 30-2

Table 30-3

Table 30-4

Table 30-5

Table 30-6

Table 30-7

Table 30-8

Table 30-9

Table 30-10

Table 30-11

Table 30-12

Table 30-13

Table 30-14

Table 30-15

Table 30-16

Table 30-17

Table 30-18

Table 30-19

Table 30-20

Table 30-21

[0144] Example 2: Antibody Biological Activity Assay 1. Cell affinity 1) Cell preparation: CD3-positive T cells isolated from human whole blood were used to perform a CD3-terminal affinity test of multifunctional antibody molecules, and tumor antigen affinity tests were performed using tumor cells positive for the corresponding antigen. For example, CD38-positive MM.1S cells (purchased from the Cell Resource Center of Shanghai Institute for Life Sciences, Chinese Academy of Sciences) or RPMI 8226 cells (purchased from the Cell Resource Center of Shanghai Institute for Life Sciences, Chinese Academy of Sciences) were used for the CD38 antigen test, and PD-L1-positive H358 cells (purchased from the Cell Resource Center of Shanghai Institute for Life Sciences, Chinese Academy of Sciences) were used for the PD-L1 antigen test. The cells were resuspended in 1% FBS-PBS to a density of 4 × 10 6 50 μl was added per well, and 2 × 10 5 The cells were seeded on a plate so that each cell number was 1. The cells were seeded on ice. 2) Addition of antibody: The antibody was diluted stepwise according to the experimental design. The antibody was diluted on ice. For example, the initial concentration of the antibody was 3000 nM, and it was diluted 3-fold to 11 concentration steps. 50 μl of the diluted antibody was added to each well containing cells, mixed well, and incubated at 4°C and 1100 rpm / min for 2 h. After washing, the cells were resuspended in 1% FBS-PBS, and diluted secondary antibody PE anti-human IgG FC (Biolegend, 409304) was added so that the final concentration of the secondary antibody was 8 μg / ml and the volume was 50 μl / well. Along with this, wells to which only cells and the secondary antibody were added were set up as controls, mixed well, and incubated at 4°C under light protection and with shaking at 1100 rpm / min for 1 h. 3) Washing and fixation: The cells were resuspended and washed in 1% FBS-PBS, and then 2% paraformaldehyde was added to the wells to fix the cells at room temperature. 4) Flow cytometer detection: The cells were resuspended in 1% FBS-PBS and then loaded into a flow cytometer for testing. 5) Data analysis: Data was analyzed using FlowJo 7.6, a flow cytometer analysis software, and the mean fluorescence intensity at a specific antibody concentration was obtained. A graph was created using Graphpad Prism 5, with the horizontal axis representing the antibody concentration (nM) and the vertical axis representing the mean fluorescence intensity, and the EC50 value was calculated using the one-site-specific binding method. This value was the cell affinity between the antibody and the corresponding target antigen.

[0145] [Table 31A] [Table 31B]

[0146] Note: The PDL1-M1 series of molecules are all antibodies with multifunctional antibody structure 1 that dual target PD-L1 and CD3, the CD38-M1 series of molecules are all antibodies with multifunctional antibody structure 1 that dual target CD38 and CD3, and the M1IC series of molecules are all isotype control antibodies with multifunctional antibody structure 1 that target CD3 and luciferase (no tumor targeting).

[0147] [Table 32]

[0148] Note: The BCMA-M2 series are antibodies with multifunctional antibody structure 2 that dual target BCMA and CD3, and the M2IC series are isotype control antibodies with multifunctional antibody structure 2 that target CD3 and luciferase and have no tumor targeting properties.

[0149] [Table 33]

[0150] Note: The CEA-M3 series are antibodies with multifunctional antibody structure 3 that dual target CEA and CD3, and the M3IC series are isotype control antibodies with multifunctional antibody structure 3 that target CD3 and luciferase and have no tumor targeting properties.

[0151] As can be seen from the above data, when the same antibody structure and the same antibody variable region sequence were used but the Fc modifications were different, there was no significant difference in the binding activity of the antibodies to the target, nor was there any significant difference in the expression level of each antibody.

[0152] 2. In Vitro Cytotoxicity 1) A sufficient amount of tumor cells was taken to prepare a single cell suspension. 2) CFSE staining of tumor cells: A certain amount of cell suspension was centrifuged (300×g, 5 min) and the supernatant was removed. CFSE solution prepared in 2 ml PBS was added so that the final concentration of CFSE was 5 μM. The cells were placed in a 5% CO2, 37°C incubator and incubated for 15 min. The cells were removed from the incubator, washed with PBS, and centrifuged (300×g, 5 min) to remove the supernatant. The washing was repeated three times, and the cells were resuspended in complete medium and the suspension was taken and counted. 3) Tumor cell seeding: Cell density was 2 × 10 in complete medium. 5 Resuspend the cells at 2 × 10 / ml. 4 The mixture was added to a 96-well plate at 100 μl / well. 4) Addition of effector cells, PBMC (peripheral blood mononuclear cells, isolated from human whole blood): Effector cells were resuspended in complete medium used for tumor cells in the experimental system, and the corresponding effector cells were added to the above plate in an amount calculated according to the E:T ratio of the experimental design. The addition volume was 50 μl / well. 5) Addition of diluted antibody: As per the experimental design, the highest concentration of antibody is 10μg / ml. If 50μl of antibody is added, it corresponds to 1 / 4 of the total volume of 200μl, so it is necessary to prepare 4 times the above concentration before adding the antibody. That is, the antibody is first diluted to 40μg / ml, and then 10-fold dilution (9 steps, volume 50μl / well) is started from 40μg / ml. 6) Observe the 96-well plate under a microscope to confirm that the cells are uniformly distributed in the culture wells, and then place in a cell incubator at 5% CO2 and 37°C for culture in preparation for detection. 7) After the detection time was reached, the treatment for adherent cells was aspirated: the cell supernatant was aspirated, washed with 30 μl / well of PBS, the washing solution was aspirated, and combined with the previously aspirated supernatant. Trypsin was added to the cell wells at 30 μl / well, digested for 3-5 min in a cell incubator at 5% CO2 and 37°C, and the previously collected supernatant was added, and the cells in each well were pipetted until a single cell suspension was obtained. The treatment for suspended cells was: pipetted multiple times to mix evenly. 8) 10-15 min before loading into the flow cytometer, PI (final concentration 10 μg / ml) was added to each sample at 10 ul / well. 9) Flow cytometer detection: The flow cytometer detection results were analyzed using FlowJo software, and the data was exported to Microsoft Excel and a form was created using GraphPad for analysis. Formula for injury calculation: The percentage of CFSE and PI double positive cells among CFSE positive cells was used as the mortality rate of target cells.

[0153] The calculation formula is as follows:

[0154]

number

[0155] 10) Calculation of antibody damage to tumor cells: The target cell death rate for all antibody concentrations was calculated using the target cell death rate calculation formula, and a curve was created with the antibody concentration on the horizontal axis and the target cell death rate on the vertical axis. Graphpad Prism 5 was used as data analysis software, and the EC50 value was calculated using the log(agonist) vs. response -- variable slope method, which was used to determine the cytotoxicity of the antibody.

[0156] 3. T cell activation (antibody + PBMC co-culture system) PBMCs were isolated from whole blood of healthy volunteers by density gradient centrifugation and added to tumor cell-seeded plates according to the E:T ratio (effector cells:target cells) of the experimental design.

[0157] According to the experimental design, the antibody was diluted in a series of 3-fold concentration steps and each concentration of antibody was added.

[0158] The 96-well plate was incubated in a 37°C, 5% CO2 incubator until the test time. The suspended PBMC cells were pooled together, and the corresponding CD3, CD69, and CD25 detection antibodies (all purchased from BD) were added and incubated for 1 h, after which excess antibodies were washed away. The cells were resuspended and the percentage of CD3 / CD69 double positive cells or CD3 / CD25 double positive cells was detected using a flow cytometer, which was taken as the percentage of T cell activation in PBMC induced by the antibodies. Specifically, it was calculated using the following formula:

[0159]

number

[0160] Using the "GraphPad Prism 5" software, nonlinear fitting (log(agonist) vs. response--Variable slope) was performed with the bibody concentration on the horizontal axis and the CD3&CD69 (CD3&CD25)% value on the vertical axis to calculate the T cell activation curve and EC50 value.

[0161] 4. T cell activation (antibody + tumor cell + PBMC co-culture system) Take well-cultured tumor cells, prepare a single cell suspension, and then culture 2 x 10 cells. 4 / well in a 96-well plate.

[0162] PBMCs were isolated from whole blood of healthy volunteers by density gradient centrifugation and added to tumor cell-seeded plates according to the E:T ratio (effector cells:target cells) of the experimental design.

[0163] According to the experimental design, the antibody was diluted in a series of concentration steps and each concentration of the antibody was added.

[0164] The 96-well plate was incubated in a 37°C, 5% CO2 incubator until the test time. The suspended PBMC cells were pooled together, and the corresponding CD3, CD69, and CD25 detection antibodies were added and incubated for 1 h, after which the excess antibodies were washed away. The cells were resuspended and the percentage of CD3 / CD69 double positive cells or CD3 / CD25 double positive cells was detected by a flow cytometer, which was taken as the percentage of T cell activation in PBMC induced by the antibodies. Specifically, it was calculated using the following formula:

[0165]

number

[0166] Using the "GraphPad Prism 5" software, nonlinear fitting (log(agonist) vs. response--Variable slope) was performed with the bibody concentration on the horizontal axis and the CD3&CD69 (CD3&CD25)% value on the vertical axis to calculate the T cell activation curve and EC50 value.

[0167] 5. Jurkat-luciferase cell activation Different FcγRs (FcγR1, FcγR2, and FcγR3A, all purchased from ACRO Biosystems) were coated onto a microplate at 100 μl / well with a protein concentration of 1 ug / ml. o The plate was incubated at C overnight. The coating solution was discarded, and the plate was washed once with PBS.

[0168] Each well of the microplate was filled with 40 ul of antibody at different concentrations (1 to 10,000 ng / ml) and a fixed amount of Jurkat-luciferase cells (2 × 10 6 / ml, 40ul / well) was added and incubated for 37 o C for 6 h.

[0169] Fluorescent staining was performed according to the instructions in the Bio-Glo Luciferase Assay System kit (product number G7940, Promega), and the fluorescent signal values ​​were detected under a fluorescent microscope (LUX 3020, manufacturer: Thermo).

[0170] Result part: 1. The detection results of the damage of PD-L1 × CD3 antibody with multifunctional antibody structure 1 to non-small cell lung cancer cell H358 (PD-L1 positive expression, Cell Resource Center, Shanghai Institutes for Life Sciences, Chinese Academy of Sciences) are shown in Figure 4 (here, the ratio of the number of human PBMC as effector cells to the number of H358 as target cells: 10:1, treatment time: 48h, PD-L1 mAb purchased from Roche). hIgG is the negative control group using IgG antibody isolated from human serum, E+T is the negative control group of no added antibody, and the same below.

[0171] [Table 34]

[0172] As can be seen from Figure 4 and Table 34, when the antibodies having the multifunctional antibody structure 1 have the same sequence but different Fc modifications, there is no significant difference in the cytotoxicity of the tumor cell H358. This proves that the Fc modification does not affect the cytotoxic activity of the antibody with dual targeting function, i.e., does not impair the efficacy of the antibody.

[0173] 2. Figure 5 shows the results of detection of the damage of isotype control antibody (4420×CD3) having multifunctional antibody structure 1 against non-small cell lung cancer cells H358 (here, the ratio of the number of effector cells, human PBMC, to the number of target cells, H358, was 10:1, and treatment time was 48 h).

[0174] [Table 35]

[0175] As can be seen from FIG. 5 and Table 35, the isotype control antibodies with different Fc modifications also had different cytotoxicity against tumor cells H358. This is because the isotype control antibody itself targets only CD3 and does not target tumor antigens. The cytotoxicity caused by the isotype control antibody was due to CD3-positive nonspecific T cell activation. Here, M1IC-WT showed significant cytotoxicity, proving that the Fc of the antibody (corresponding CH2 is WT, SEQ ID NO: 83) can cause significant nonspecific activation of T cells. M1IC-NQ showed significantly high maximum cytotoxicity, proving that in the Fc, when the corresponding CH2 is N297Q (SEQ ID NO: 89), it can cause specific activation of T cells. The other antibodies did not activate T cells, proving that the nonspecific T cell activation is significantly reduced by Fc modification. 3. The detection results of the cytotoxicity of the CD38×CD3 antibody having the multifunctional antibody structure 1 against multiple myeloma cells MC / CAR (CD38 positive expression, purchased from ATCC) are shown in FIG. 6 (here, the ratio of the number of human PBMC as effector cells to the number of MC / CAR as target cells: 5:1, treatment time: 48 hours, CD38 mAb is the CD38 monoclonal antibody control group, where the sequences of CD38 mAb are VL as sequence number 16, CL as sequence number 75, VH as sequence number 15, CH1 as sequence number 82, hinge as sequence number 66, CH2 as sequence number 83, and CH3 as sequence number 102).

[0176] [Table 36]

[0177] As can be seen from Figure 6 and Table 36, the multifunctional antibodies with the same antibody variable region sequence have no significant difference in their ability to kill MC / CAR tumor cells even though they have different Fc modifications. This proves that the Fc modifications do not affect the cytotoxic activity of the antibody with dual targeting function, i.e., do not impair the efficacy of the antibody.

[0178] 4. The results of detection of the cytotoxicity of an isotype control antibody (4420×CD3) having multifunctional antibody structure 1 against multiple myeloma cells MC / CAR (positive expression of CD38) are shown in Figure 7 (here, the ratio of the number of human PBMCs as effector cells to the number of MC / CARs as target cells: 5:1, treatment time: 48 h).

[0179] [Table 37]

[0180] As can be seen from Figure 7 and Table 37, the isotype control antibodies with different Fc modifications have different damage to tumor cells MC / CAR. This is because the isotype control antibody itself only targets CD3 and does not target tumor antigens. And the damage caused by the isotype control antibody was caused by CD3-positive non-specific T cell activation. Here, M1IC-WT showed significant damage, proving that the Fc of the antibody (the corresponding CH2 is WT) can cause significant non-specific activation of T cells. In contrast, the isotype control antibody with the modified Fc of the present invention can only cause weak non-specific activation of T cells or completely avoid non-specific T cell activation.

[0181] 5. This was a test of T cell activation among peripheral blood monocytes using an isotype control antibody (4420×CD3) having multifunctional antibody structure 1. The detection results are shown in Figures 8 and 9.

[0182] [Table 38]

[0183] As can be seen from Figures 8, 9 and Table 38, the above isotype control antibodies induced the strongest non-specific T cell activation in PBMCs with M1IC-WT, weak non-specific T cell activation with M1IC-SG2, M1IC-G2, M1IC-SG2-1 and M1IC-G2-1, and no T cell activation with M1IC-AG2 / GG2 / DG2 / G2D / DG2D. This demonstrates that the antibody having multifunctional antibody structure 1 targets CD3 and attenuates non-specific T cell activation when CH2 is SG2CH2 (SEQ ID NO:95) or G2CH2 (SEQ ID NO:94). When the isotype control antibody having the multifunctional antibody structure 1 was AG2CH2 (SEQ ID NO:96), GG2CH2 (SEQ ID NO:97), PG2CH2 (SEQ ID NO:98), DG2CH2 (SEQ ID NO:99), G2DCH2 (SEQ ID NO:100) or DG2DCH2 (SEQ ID NO:101), non-specific T cell activation was more effectively avoided.

[0184] 6. Jurkat-luciferase cell activation test using isotype control antibody (4420×CD3) with multifunctional antibody structure 1, and the detection results are shown in Figures 10, 11 and 12.

[0185] When the anti-CD3 antibody binds to the immobilized Fc receptor and then binds to Jurkat-luciferase cells bearing the CD3 surface antigen, the cells are activated and a fluorescent signal can be detected. The stronger the fluorescent signal, the higher the degree of activation of the cells, and the stronger the antibody binds to the Fc receptor. As can be seen from Figures 10-12, among the above isotype control antibodies, M1IC-WT-1 activated Jurkat-luciferase cells most strongly, and when the immobilized antigen was FcγR2, M1IC-FES-1 activated Jurkat-luciferase cells relatively significantly, while M1IC-AG2-1, M1IC-GG2-1, M1IC-PG2-1, M1IC-DG2-1, M1IC-G2D-1, M1IC-DG2D-1, M1IC-DG2D-1A or M1IC-DG2D-1B activated Jurkat-luciferase cells very weakly, with no significant difference between them. This proves that when the isotype control antibody CH2 having the multifunctional antibody structure 1 is AG2CH2 (SEQ ID NO:96), GG2CH2 (SEQ ID NO:97), PG2CH2 (SEQ ID NO:98), DG2CH2 (SEQ ID NO:99), G2DCH2 (SEQ ID NO:100) or DG2D (SEQ ID NO:101), non-specific T cell activation can be effectively avoided by FES (SEQ ID NO:85).

[0186] 7. The results of detection of the killing of BCMA × CD3 antibody with multifunctional antibody structure 2 and isotype control antibody against myeloma cells U266B1 (BCMA positive expression, China Typical Culture Depository Center) are shown in Figures 13 and 14 (here, the ratio of effector cells (human PBMC) to target cells (U266B1): 5:1, treatment time: 48 h).

[0187] [Table 39]

[0188] As can be seen from Figure 13 and Table 39, the BCMA x CD3 antibodies with multifunctional antibody structure 2 having different Fc modifications can all significantly damage tumor cells U266B1, and there is no significant difference between the damage effects. This proves that the Fc modifications do not affect the cytotoxic activity of the antibody with dual targeting function, i.e., do not impair the efficacy of the antibody.

[0189] [Table 40]

[0190] As can be seen from Figure 14 and Table 40, when the antibodies having multifunctional antibody structure 2 have the same variable region sequence but different modifications to Fc, the isotype control antibody M2IC-WT significantly killed U266B1, proving that the Fc can induce non-specific T cell activation. When CH2 of Fc is G2CH2 (SEQ ID NO:94), a weak cytotoxic effect was observed, proving that the Fc can induce weak non-specific T cell activation. When the CH2 of the Fc was SG2CH2 (SEQ ID NO:95), AG2CH2 (SEQ ID NO:96), GG2CH2 (SEQ ID NO:97), PG2CH2 (SEQ ID NO:98), DG2CH2 (SEQ ID NO:99), G2DCH2 (SEQ ID NO:100) or DG2DCH2 (SEQ ID NO:101), there was no cytotoxicity, demonstrating that these five Fc's do not cause nonspecific T cell activation.

[0191] 8. Activation test of T cells in peripheral blood mononuclear cells (PBMC) using an isotype control antibody having multifunctional antibody structure 2 (treatment time: 48 hours). The detection results are shown in Figures 15 and 16.

[0192] [Table 41]

[0193] As can be seen from Figure 15, Figure 16 and Table 41, in the absence of correlated tumor cells, the analysis of the maximum activation level showed that M2IC-WT significantly activated nonspecific T cells, M2IC-G2 or M2IC-SG2 reduced nonspecific T cell activation, and other antibodies did not significantly activate T cells. When CH2 of Fc was AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) or DG2DCH2 (SEQ ID NO: 101), there was no cytotoxicity, proving that these five Fc did not cause nonspecific T cell activation.

[0194] 9. The detection results of the damage of CEA×CD3 antibody with multifunctional antibody structure 3 to gastric cancer cells MKN-45 (CEA positive expression, Basic Medical Cell Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) are shown in Figures 17 and 18 (here, the ratio of the number of effector cells (human PBMC) to target cells (MKN-45): 5:1, treatment time: 48 h).

[0195] [Table 42]

[0196] As can be seen from Figure 17 and Table 42, the CEA x CD3 antibody having the multifunctional antibody structure 3 had no significant difference in cytotoxicity against tumor cells MKN-45 when the antibody had the same sequence but different Fc modifications, proving that the Fc modifications did not affect the cytotoxic activity of the antibody with dual targeting function, i.e., did not impair the efficacy of the antibody.

[0197] [Table 43]

[0198] As can be seen from FIG. 18 and Table 43, the maximum cytotoxicity rate indicates that antibody M3IC-WT caused significant cytotoxicity. M3IC-G2 or M3IC-SG2 caused weak cytotoxicity, and all other antibodies did not cause significant cytotoxicity. It was also demonstrated that the Fc of multifunctional antibody structure 3 has a weakened non-specific T cell activation effect after CH2 is modified to G2CH2 (SEQ ID NO: 94) and SG2CH2 (SEQ ID NO: 95) compared with the control M3IC-WT, and five Fcs of AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) or DG2DCH2 (SEQ ID NO: 101) do not cause non-specific T cell activation.

[0199] 10. Activation test of T cells in peripheral blood mononuclear cells (PBMC) using an isotype control antibody (4420×CD3) having multifunctional antibody structure 3 (treatment time: 48 h). The detection results are shown in Figures 19 and 20.

[0200] [Table 44]

[0201] As can be seen from Figure 19, Figure 20 and Table 44, in the isotype control antibodies having multifunctional antibody structure 3, M3IC-WT activated T cells the strongest, and M3IC-G2 activated T cells weakly, which shows that when CH2 is G2CH2 (SEQ ID NO: 94), it showed weak non-specific T cell activation compared to M2IC-WT. Compared to M2IC-WT, all other antibodies activated T cells weakly, and CH2 is G2DCH2 (SEQ ID NO: 100) was the best.

[0202] Example 3: Antibody stability testing Experimental work: A.40 o The thermal acceleration stability test of C was carried out as follows: 1) The sample was substituted into a buffer having a composition of 20 mM citric acid, pH 5.5, and the sample concentration was adjusted to 1 mg / mL. 2) Put 500μL of each sample into each tube (total of 6 tubes), seal them, and store for 40 o C water bath, and samples were taken on days 0, 3, 5, 7, 10, and 14 for HPLC-SEC detection, for a total water bath time of 14 days.

[0203] B. Acid resistance test, also known as stability at low pH, is an index to investigate whether antibody molecules can maintain their original state when neutralized to physiological conditions after being treated in an acidic environment for a certain period of time. The specific work procedure was as follows:

[0204] When performing Protein A affinity chromatography on antibody molecules, the antibody solution eluted in the acid elution process (using citrate buffer of pH 3.5) was kept in the buffer for a certain period of time without being neutralized, and then samples were taken at 30 and 60 minutes and neutralized by adding 1 / 10 of the volume of 1 M Tris-HCl (pH 8.0), and this sample was subjected to HPLC-SEC test.

[0205] Result part: 1. Multifunctional antibody structure 1 of 40 o The results of the C accelerated thermal stability detection are shown in FIG. As can be seen from FIG. 21, different antibodies having multifunctional antibody structure 1 have 40 oAfter 14 days of treatment with 500 mM NaCl, the purity of all antibodies was maintained at 90% or more, and no significant aggregation or degradation was observed. In this case, the purity of the antibody whose CH2 of Fc is N297Q was significantly decreased compared with other antibodies, and there was no significant difference between the antibodies whose CH2 was modified. It was demonstrated that the multifunctional antibody structure 1 has good thermal stability when CH2 is G2CH2 (SEQ ID NO: 94), SG2CH2 (SEQ ID NO: 95), AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) and DG2DCH2 (SEQ ID NO: 101).

[0206] 2. The results of detection of the acid resistance of multifunctional antibody structure 1 are shown in FIG. As can be seen from FIG. 22, after the antibodies having the multifunctional antibody structure 1 with different Fc modifications were treated under low pH conditions for 60 minutes, there was no obvious modification in the purity of all the antibodies, and there was no significant difference between the different antibodies, which demonstrated that the above-mentioned Fc-modified multifunctional antibody structure 1 has good acid resistance.

[0207] Taking Figures 21 and 22 together, it can be seen that antibodies having multifunctional antibody structure 1 in which CH2 is G2CH2 (SEQ ID NO:94), SG2CH2 (SEQ ID NO:95), AG2CH2 (SEQ ID NO:96), GG2CH2 (SEQ ID NO:97), PG2CH2 (SEQ ID NO:98), DG2CH2 (SEQ ID NO:99), G2DCH2 (SEQ ID NO:100) and DG2DCH2 (SEQ ID NO:101) have good thermal stability and acid resistance.

[0208] 3. Multifunctional antibody structure 2 of 40 o The results of the C accelerated thermal stability detection are shown in FIG. As can be seen from FIG. 23, when CH2 of multifunctional antibody structure 2 is N297A (SEQ ID NO:87), 40 oThe purity of the antibodies after treatment with C for 14 days was clearly decreased. The purity of the other antibodies was not obviously altered, and there was no significant difference between the antibodies, demonstrating that the antibodies having the multifunctional antibody structure 2, except for M2IC-NA, had good accelerated thermal stability.

[0209] 4. The results of detection of the acid resistance of multifunctional antibody structure 2 are shown in FIG. As can be seen from Figure 24, after the antibodies having the multifunctional antibody structure 1 with different Fc modifications were treated under low pH conditions for 60 minutes, there was no obvious modification in the purity of all the antibodies, and there was no significant difference between the different antibodies, which demonstrated that the above Fc-modified multifunctional antibody structure 2 has good acid resistance.

[0210] Taking Figures 23 and 24 together, it can be seen that antibodies having multifunctional antibody structure 2 in which CH2 is G2CH2 (SEQ ID NO:94), SG2CH2 (SEQ ID NO:95), AG2CH2 (SEQ ID NO:96), GG2CH2 (SEQ ID NO:97), PG2CH2 (SEQ ID NO:98), DG2CH2 (SEQ ID NO:99), G2DCH2 (SEQ ID NO:100) and DG2DCH2 (SEQ ID NO:101) have good thermal stability and acid resistance.

[0211] 5. Multifunctional antibody structure 3 of 40 o The results of the C accelerated thermal stability detection are shown in FIG. As can be seen from FIG. 25, antibodies having different multifunctional antibody structures 3 have a 40 o After treatment with C for 14 days, there was no obvious change in purity and no significant difference between the antibodies, demonstrating that the antibody having the above-mentioned multifunctional antibody structure 3 has good accelerated thermal stability.

[0212] 6. The results of detection of the acid resistance of multifunctional antibody structure 3 are shown in FIG. As can be seen from Figure 26, after the antibodies having the multifunctional antibody structure 3 with different Fc modifications were treated under low pH conditions for 60 minutes, there was no obvious modification in the purity of all the antibodies, and there was no significant difference between the different antibodies, which demonstrated that the above Fc-modified multifunctional antibody structure 3 has good acid resistance.

[0213] Taking Figures 25 and 26 together, it can be seen that antibodies having multifunctional antibody structure 3 in which CH2 is G2CH2 (SEQ ID NO:94), SG2CH2 (SEQ ID NO:95), AG2CH2 (SEQ ID NO:96), GG2CH2 (SEQ ID NO:97), PG2CH2 (SEQ ID NO:98), DG2CH2 (SEQ ID NO:99), G2DCH2 (SEQ ID NO:100) and DG2DCH2 (SEQ ID NO:101) have good thermal stability and acid resistance.

[0214] Example 4: Fc modification of monoclonal antibodies and binding experiments with cells expressing FcγR 1. Preparation of Monoclonal Antibodies (1) In the present invention, the prepared monoclonal antibody has a structure identical to or similar to that of a natural antibody, for example, a human IgG1, IgG2, IgG3, or IgG4 subtype, exhibits a symmetric "Y" structure, functions by binding to the same target antigen in a bivalent manner, and has an Fc domain of the corresponding subtype.

[0215] A schematic diagram of a specific structure is shown in FIG. (2) The construction of expression plasmid, transfection expression and purification methods of monoclonal antibodies were consistent with the related methods of multifunctional antibodies described in Example 1 of the present invention, and the sequences used are shown in Table 45.

[0216] [Table 45-1] [Table 45-2] [Table 45-3] [Table 45-4]

[0217] 2. Binding experiment between monoclonal antibodies and cells expressing FcγR (1) FcγR that binds to human IgG antibody Fc is divided into three types: FcγRI, FcγRII, and FcγRIII. Here, FcγRI (also called CD64) and FcγRIIA are expressed on macrophages and neutrophils, FcγRIIB is expressed on B cells, and FcγRIIIA (also called CD16A) is expressed on natural killer (NK) cells. The surface marker of dendritic cells is CD83, the surface marker of macrophages is CD14, the surface marker of B cells is CD20, and the surface marker of NK cells is CD56.

[0218] Nine monoclonal antibodies in Table 45 were prepared and then labeled with biotin, respectively. The negative control was antibody 4420 (VL is SEQ ID NO: 44, CL is SEQ ID NO: 75, VH is SEQ ID NO: 43, and CH1 is SEQ ID NO: 82), a Fab structure without Fc, labeled with biotin.

[0219] PBMCs were isolated from blood samples of healthy donors and divided into three groups:

[0220] Group 1 was used to examine the binding ability of the Fc of 4420 monoclonal antibody to macrophages. Here, PBMCs were co-incubated with PE-labeled anti-CD14 antibody (purchased from Thermofisher) and the nine 4420 monoclonal antibodies labeled with biotin (each antibody was used at three concentrations: 500 μg / ml, 50 μg / ml, and 5 μg / ml) for 2 h (room temperature), washed three times, and incubated with FITC-labeled avidin for another 30 min (room temperature). After five washes, the cells were detected by a flow cytometer. The PE-fluorescent cell population was surrounded and analyzed for FITC mean fluorescence intensity.

[0221] The second group investigated the binding ability of the Fc of 4420 monoclonal antibody to B cells. The steps were the same as in a), except that PE-labeled anti-CD14 antibody was used instead of PE-labeled anti-CD20 antibody (purchased from Thermofisher).

[0222] Group 3 investigated the binding ability of 4420 monoclonal antibody to Fc and NK cells. The steps were the same as in a), except that PE-labeled anti-CD14 antibody was used instead of anti-CD56 antibody (purchased from Thermofisher).

[0223] The results of cell detection by flow cytometry are shown in Figures 28-30. As can be seen from Figure 28, 4420mAb-WT significantly bound to macrophages, and when CH2 was G2CH2 (SEQ ID NO: 94), SG2CH2 (SEQ ID NO: 95), AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) and DG2DCH2 (SEQ ID NO: 101), the function of binding to FcγRI or FcγRIIA was significantly reduced or eliminated.

[0224] As can be seen from Figure 29, 4420mAb-WT significantly bound to B cells, and compared to 4420mAb-WT, when Fc CH2 was SG2CH2 (SEQ ID NO:95), G2CH2 (SEQ ID NO:94), AG2CH2 (SEQ ID NO:96), GG2CH2 (SEQ ID NO:97), PG2CH2 (SEQ ID NO:98), DG2CH2 (SEQ ID NO:99), G2DCH2 (SEQ ID NO:100) and DG2DCH2 (SEQ ID NO:101), the antibody had significantly reduced or eliminated function of binding to FcγRIIB.

[0225] As can be seen from Figure 30, only 4420mAb-WT significantly bound to NK, demonstrating that when the CH2 of Fc is SG2CH2 (sequence number 95), G2CH2 (sequence number 94), AG2CH2 (sequence number 96), GG2CH2 (sequence number 97), PG2CH2 (sequence number 98), DG2CH2 (sequence number 99), G2DCH2 (sequence number 100) and DG2DCH2 (sequence number 101), the function of binding to FcγRIIIA is all significantly reduced or eliminated.

[0226] 28-30 taken together, it is clear that by changing Fc and changing CH2 to G2CH2 (SEQ ID NO: 94), SG2CH2 (SEQ ID NO: 95), AG2CH2 (SEQ ID NO: 96), GG2CH2 (SEQ ID NO: 97), PG2CH2 (SEQ ID NO: 98), DG2CH2 (SEQ ID NO: 99), G2DCH2 (SEQ ID NO: 100) and DG2DCH2 (SEQ ID NO: 101), the function of the antibody Fc binding to its receptors FcγRI, FcγRIIA, FcγRIIB and FcγRIIIA can be significantly reduced or eliminated. It is known that the activation of T cells by CD3 antibodies is largely due to the binding of the antibody Fc to FcγR on the surface of other cells. The present invention involves innovative Fc alterations, replacing the CH2 domain of human IgG1 Fc with the CH2 domain of human IgG2, preferably involving substitution of several amino acid residues, resulting in antibodies or fusion proteins with significantly reduced or eliminated FcγR binding ability, where these Fc modifications have no effect on the stability and biological activity of the antibody itself.

[0227] Example 5: T cell activation test using anti-CD3 monoclonal antibody 1. Preparation of Monoclonal Antibodies See Example 4, part 1, and Table 46 for the sequences used.

[0228] [Table 46-1] [Table 46-2] [Table 46-3] [Table 46-4] [Table 46-5] [Table 46-6]

Table 46-7

Table 46-8

Table 46-9

Table 46-10

Table 46-11

Table 46-12

Table 46-13

Table 46-14

Table 46-15

Table 46-16

Table 46-17

Table 46-18

Table 46-19

Table 46-20

Table 46-21

Table 46-22

Table 46-23

[0229] 2. Activation test of T cells in PBMCs using CD3 monoclonal antibody The test method was the same as "3. T cell activation (antibody + PBMC co-culture system)" in "Example 2: Biological activity assay of antibody". The detection results are shown in Tables 47-48 and Figures 31-34.

[0230] [Table 47A] [Table 47B]

[0231] As can be seen from Table 47 and Figures 31-33, compared with the use of the Fc CH1 domain, when the Fc CH2 domain of an IgG1 type CD3 monoclonal antibody was replaced with the CH2 domain of IgG2, and then cysteine ​​(C) at position 229, aspartic acid (D) at position 265, or aspartic acid (D) at position 270 was replaced with other essential amino acids, T cell activation was significantly reduced in all cases.

[0232] [Table 48]

[0233] As can be seen from Table 48 and FIG. 34, the CH2 domain of Fc of IgG1 type CD3 monoclonal antibody was substituted with the CH2 of IgG2, and then the following substitutions were made to amino acids at some positions: (1) No substitution (2) Substitution at one position: C229S, C229A, C229G, C229P, D265A, D270A (3) Substitutions at two positions: C229P / G327A, C229P / T339A, D270A / G327A, D270A / T339A (4) Substitutions at three positions: D270A / G327A / T339A, C229P / D265A / D270A, etc. This confirmed that, compared with wild-type IgG1, all of the monoclonal antibodies with the above four types of substitutions had significantly reduced T cell activation, and even more significantly reduced T cell activation than FES.

Claims

1. 1. An antibody comprising a polypeptide, the antibody is a monospecific antibody or a multispecific antibody, an antibody, wherein the polypeptide comprises or consists of a modified Fc fragment, the Fc fragment comprising a hinge, a CH2 and a CH3 and derived from human IgG1, the CH2 domain of the constant region of the Fc fragment being replaced with the CH2 domain of human IgG2 represented by SEQ ID NO:94, and the modified Fc fragment further comprises an amino acid substitution mutation in the CH2 domain of human IgG2, the amino acid substitution mutation comprising a mutation at position D270 as represented by the Kabat numbering, the mutated CH2 domain having 90% or more identity to its starting sequence, SEQ ID NO:94, and the mutation at position D270 being selected from the group consisting of D270A, D270V, D270T, D270M, and D270N.

2. The antibody of claim 1, wherein the CH2 domain of the modified Fc fragment further comprises a mutation at a position selected from the group consisting of: C229, D265 or any combination thereof as represented by the Kabat numbering.

3. the mutation is at position C229; The antibody of claim 2.

4. The mutation is at position D265. The antibody of claim 2.

5. the mutations are at positions C229 and D270, or at positions D265 and D270, or at positions C229, D265, and D270; An antibody described in any one of claims 1 to 4.

6. The antibody of any one of claims 1 to 4, wherein the CH2 domain of the Fc fragment further comprises a mutation at a position selected from the group consisting of: G327, T339 or a combination thereof.

7. 6. The antibody of claim 5, wherein the mutation is selected from G327A, G327V, G327L, G327I and / or T339A.

8. The antibody of any one of claims 1 to 7, wherein the mutation is selected from D270A / G327A, D270A / T339A, D270A / G327A / T339A or C229P / D265A / D270A.

9. The antibody of any one of claims 1 to 4, wherein the sequence of the CH2 domain of the Fc fragment is selected from SEQ ID NOs: 100, 101, 124-127, 164, 173, 175 and 176.

10. The antibody according to any one of claims 1 to 9, which is a bispecific antibody.

11. Specifically binds to an antigen selected from the following antigens: Tumor antigens, viral or bacterial antigens, endotoxins, immunogens, An antibody described in any one of claims 1 to 10.

12. The antibody of claim 2, having any of the following characteristics: (1) The mutation at C229 is selected from the following group: C229A, C229G, C229P, C229S, C229V, C229L, C229I, C229T, C229M, C229N, C229Q, C229D, C229E, C229K, C229R, C229F, C229Y, C229W or C229H; (2) the mutation at C229 is C229S, C229A, C229G, or C229P; (3) The mutation at D265 is selected from the following group: D265A, D265G, D265P, D265S, D265V, D265L, D265I, D265T, D265M, D265N, D265Q, D265E, D265K, D265R, D265F, D265Y, D265W or D265H; (4) the mutation at D265 is D265A; (5) the mutation at D270 is D270A; (6) The mutation is a combination of D265 and D270 mutations; (7) The antibody specifically binds to an antigen selected from the following antigens: PD-L1, B7-H3, SLAMF7, CD38, EpCAM, CEA, CD19, BCMA, CD3, CD16A, CD47, and NKG2D; (8) The antibody specifically binds to an antigen selected from the following antigens: PD-L1 represented by SEQ ID NO: 118, SLAMF7 represented by SEQ ID NO: 119, CD38 represented by SEQ ID NO: 116, CEA represented by SEQ ID NO: 120, BCMA represented by SEQ ID NO: 117, and CD3 represented by SEQ ID NO:

121.

13. the antibody is an asymmetric bispecific antibody comprising a light chain, a heavy chain and a fusion peptide 1; The fusion peptide 1 comprises an scFv and an Fc fragment, The antibody of claim 11 or 12, wherein the antibody has a light chain-heavy chain pairing and a heavy chain-fusion peptide 1 pairing, each of which forms an interchain disulfide bond, the light chain-heavy chain pairing targets a tumor antigen, and the ScFv of the fusion peptide 1 targets an immune cell antigen.

14. the antibody is an asymmetric trivalent bispecific antibody comprising two light chains, one heavy chain and one fusion peptide 2, and has a light chain-heavy chain pairing, a light chain-fusion peptide 2 pairing and a heavy chain-fusion peptide 2 pairing, each of which forms an interchain disulfide bond; the fusion peptide 2 comprises a heavy chain variable region VH, a heavy chain first constant region CH1, ScFv and Fc, the ScFv being located between CH1 and Fc and linked by a linker; The antibody of claim 11 or 12, wherein the light chain-heavy chain pairing targets a tumor antigen, the VH-CH1 and light chain pairing of fusion peptide 2 targets the same tumor antigen, and the ScFv targets an immune cell antigen.

15. the antibody is an asymmetric trivalent bispecific antibody, comprising a fused heavy chain, a cross light chain, a heavy chain and a light chain, and having light chain-heavy chain pairing, light chain-fused heavy chain pairing, cross light chain-fused heavy chain pairing, and fused heavy chain-heavy chain pairing, each of which forms an interchain disulfide bond; the light chain comprises a first light chain variable domain VLm and a light chain constant domain CL; the fusion heavy chain comprises a first heavy chain variable domain VHm, a first heavy chain constant domain CH1, a second heavy chain variable domain VHs, a light chain constant domain CL, and an Fc; wherein VHs and CL are linked by a linker to form a peptide fragment "VHs-linker-CL", and the "VHs-linker-CL" is located between CH1 and Fc and linked by a linker / hinge; The antibody of claim 11 or 12, wherein the cross light chain comprises a second light chain variable region VLs and CH1, the VLs and CH1 being linked by a linker, and the VLm-VHm pairing targets a tumor antigen and the VLs-VHs pairing targets an immune cell antigen.

16. It has two different CH3s and forms a heterodimer by "protrusion-cavity structure pairing" and / or "salt bridge pairing" between the two CH3s. An antibody described in any one of claims 13 to 15.

17. The sequence of the CH3 domain is represented by SEQ ID NO: 102-115; The antibody of claim 16.

18. The antibody according to any one of claims 13 to 17, wherein the sequence of the light chain-heavy chain pairing or the VLm-VHm pairing is selected from the group consisting of: (1) SEQ ID NO: 12 and SEQ ID NO: 11, which target the tumor antigen B7-H3; (2) SEQ ID NO: 14 and SEQ ID NO: 13, which target the tumor antigen B7-H3; (3) SEQ ID NO: 16 and SEQ ID NO: 15, which target the tumor antigen CD38; (4) SEQ ID NO: 18 and SEQ ID NO: 17, which target the tumor antigen CD38; (5) SEQ ID NO: 20 and SEQ ID NO: 19, which target the tumor antigen CD38; (6) SEQ ID NO: 22 and SEQ ID NO: 21, targeting the tumor antigen EpCAM; (7) SEQ ID NO: 24 and SEQ ID NO: 23, which target the tumor antigen EpCAM; (8) SEQ ID NO: 26 and SEQ ID NO: 25, targeting the tumor antigen BCMA; (9) SEQ ID NO: 28 and SEQ ID NO: 27, targeting the tumor antigen BCMA; (10) SEQ ID NO: 30 and SEQ ID NO: 29, targeting the tumor antigen BCMA; (11) SEQ ID NO: 32 and SEQ ID NO: 31, which target the tumor antigen PD-L1; (12) SEQ ID NO: 34 and SEQ ID NO: 33, which target the tumor antigen PD-L1; (13) SEQ ID NO: 36 and SEQ ID NO: 35, which target the tumor antigen PD-L1; (14) SEQ ID NO: 38 and SEQ ID NO: 37, targeting the tumor antigen CD19; (15) SEQ ID NO: 40 and SEQ ID NO: 39, targeting the tumor antigen SLAMF7; (16) SEQ ID NO: 42 and SEQ ID NO: 41, targeting the tumor antigen CEA; (17) SEQ ID NO: 2 and SEQ ID NO: 1, targeting the immune antigen CD3; (18) SEQ ID NO: 4 and SEQ ID NO: 3, which target the immune antigen CD3; (19) SEQ ID NO: 6 and SEQ ID NO: 5, which target the immune antigen CD3; (20) SEQ ID NO: 8 and SEQ ID NO: 7, which target the immune antigen CD3; and (21) Sequences of SEQ ID NO: 10 and SEQ ID NO: 9, which target the immune antigen CD3.

19. The antibody according to any one of claims 13 to 18, selected from the group consisting of: (1) An antibody CD38-M1-G2D comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:100 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:15, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:100 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:16 and SEQ ID NO:75; (2) An antibody CD38-M1-DG2D comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:101 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:15, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:101 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:16 and SEQ ID NO:75; (3) An antibody CD38-M1-G2D-1 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:71, SEQ ID NO:100 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:17, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:100 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:18 and SEQ ID NO:75; (4) An antibody M1IC-G2D comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:100 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:100 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (5) An antibody M1IC-DG2D comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:9, SEQ ID NO:54, SEQ ID NO:10, SEQ ID NO:68, SEQ ID NO:101 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:101 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (6) An antibody M1IC-G2D-1 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:100 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:100 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (7) An antibody M1IC-DG2D-1 comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:101 and SEQ ID NO:109; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:101 and SEQ ID NO:108; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (8) An antibody M1IC-DG2D-1A comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:101 and SEQ ID NO:107; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:101 and SEQ ID NO:106; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (9) An antibody M1IC-DG2D-1B comprising or consisting of a fusion peptide 1, a heavy chain and a light chain; wherein the fusion peptide 1 comprises or consists of SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:101 and SEQ ID NO:115; the heavy chain comprises or consists of SEQ ID NO:43, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:101 and SEQ ID NO:114; and the light chain comprises or consists of SEQ ID NO:44 and SEQ ID NO:75; (10) An antibody BCMA-M2-G2D comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:74, SEQ ID NO:48, SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:100 and SEQ ID NO:107; the heavy chain comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:100 and SEQ ID NO:106; and the light chain comprises or consists of SEQ ID NO:30 and SEQ ID NO:75; (11) An antibody BCMA-M2-DG2D comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:74, SEQ ID NO:48, SEQ ID NO:1, SEQ ID NO:54, SEQ ID NO:2, SEQ ID NO:68, SEQ ID NO:101 and SEQ ID NO:107; the heavy chain comprises or consists of SEQ ID NO:29, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:101 and SEQ ID NO:106; and the light chain comprises or consists of SEQ ID NO:30 and SEQ ID NO:75; (12) An antibody M2IC-G2D comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 100 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 106; and the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (13) An antibody M2IC-DG2D comprising or consisting of a fusion peptide 2, a heavy chain and a light chain; wherein the fusion peptide 2 comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 74, SEQ ID NO: 48, SEQ ID NO: 1, SEQ ID NO: 54, SEQ ID NO: 2, SEQ ID NO: 68, SEQ ID NO: 101 and SEQ ID NO: 107; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 106; and the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (14) An antibody CEA-M3-G2D comprising or consisting of a fusion heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fusion heavy chain comprises or consists of SEQ ID NO:41, SEQ ID NO:82, SEQ ID NO:51, SEQ ID NO:1, SEQ ID NO:46, SEQ ID NO:81, SEQ ID NO:66, SEQ ID NO:100 and SEQ ID NO:107; the cross light chain comprises or consists of SEQ ID NO:2, SEQ ID NO:45 and SEQ ID NO:82; the heavy chain comprises or consists of SEQ ID NO:41, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:100 and SEQ ID NO:106; the light chain comprises or consists of SEQ ID NO:42 and SEQ ID NO:75; (15) An antibody CEA-M3-DG2D comprising or consisting of a fusion heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fusion heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 41, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 42 and SEQ ID NO: 75; (16) Antibody M3IC-G2D comprising or consisting of a fusion heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fusion heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 100 and SEQ ID NO: 106; and the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (17) Antibody M3IC-DG2D comprising or consisting of a fused heavy chain, a cross light chain, a heavy chain and a light chain; wherein the fused heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 51, SEQ ID NO: 1, SEQ ID NO: 46, SEQ ID NO: 81, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 107; the cross light chain comprises or consists of SEQ ID NO: 2, SEQ ID NO: 45 and SEQ ID NO: 82; the heavy chain comprises or consists of SEQ ID NO: 43, SEQ ID NO: 82, SEQ ID NO: 66, SEQ ID NO: 101 and SEQ ID NO: 106; the light chain comprises or consists of SEQ ID NO: 44 and SEQ ID NO: 75; (18) An antibody CD3mAb-G2D comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:100 and SEQ ID NO:102; (19) An antibody CD3mAb-G2-D270V comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:164 and SEQ ID NO:102; (20) An antibody CD3mAb-G2-D270T comprising or consisting of a light chain and a heavy chain; wherein said light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and said heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:173 and SEQ ID NO:102; (21) An antibody CD3mAb-G2-D270M comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:175 and SEQ ID NO:102; (22) An antibody CD3mAb-G2-D270N comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:176 and SEQ ID NO:102; (23) An antibody CD3mAb-G2D-GA comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:124 and SEQ ID NO:102; (24) An antibody CD3mAb-G2D-TA comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:125 and SEQ ID NO:102; (25) An antibody CD3mAb-G2D-GATA comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:126 and SEQ ID NO:102; or (26) An antibody CD3mAb-PDG2D comprising or consisting of a light chain and a heavy chain; wherein the light chain comprises or consists of SEQ ID NO:2 and SEQ ID NO:75; and the heavy chain comprises or consists of SEQ ID NO:1, SEQ ID NO:82, SEQ ID NO:66, SEQ ID NO:127 and SEQ ID NO:

102.

20. 20. A conjugate of an antibody according to any one of claims 11 to 19, comprising a substance A conjugated or fused to the antibody, said substance A being selected from a therapeutic agent, a prodrug, a protein, a virus, a lipid, a biological response modifier, PEG, a hormone, an oligonucleotide, a diagnostic agent, a cytotoxic agent, an ultrasound enhancing agent, a detectable label.

21. A conjugate according to claim 20, having any of the following characteristics: (1) The substance A is selected from an enzyme, an immunomodulator, a drug, a toxin, a non-radioactive label, a chemiluminescent label compound, or a fluorescent metal; (2) The substance A is selected from luminol, isoluminol, an acridinium ester, an imidazole, an acridinium salt, an oxalate ester, 152Eu, or a lanthanum label.

22. A polynucleotide encoding the antibody of any one of claims 1 to 19.

23. A cell comprising the polynucleotide of claim 20.

24. A composition comprising an antibody according to any one of claims 1 to 19.

25. The composition of claim 22, having any of the following characteristics: (1) The composition is a pharmaceutical composition; (2) The composition has antibody-dependent cellular cytotoxicity for administration to a mammalian subject. It is a non-inducing composition; (3) The composition has antibody-dependent cellular cytotoxicity for administration to a human subject. It is a non-inducing composition.

26. 20. Use of an antibody according to any one of claims 1 to 19 in the preparation of a medicament which does not induce antibody-dependent cellular cytotoxicity after administration to a mammalian subject.

27. 27. The use of claim 26, wherein the subject is a human.

Images