Anti-cd3 and anti-cd3 multispecific antibodies and uses

Abstract

Provided are antibodies or antigen-binding fragments thereof that specifically bind to human CD3 and multispecific antibodies that target CD3 and an additional antigen, e.g., a tumor-associated antigen. Also provided are nucleic acid molecules encoding the antibodies, vectors and host cells for expressing the antibodies, and therapeutic and diagnostic methods and uses of the antibodies or antigen-binding fragments thereof.

Description

Anti-CD3 and anti-CD3 multispecific antibodies and uses

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311778073.9 filed on December 21, 2023, the entire contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present invention relates to a class of antibodies that specifically bind to human CD3, and multispecific antibodies that simultaneously bind to CD3 and tumor-associated antigens. Technical Background

[0004] T cells are a very important type of immune cell in the human body and play a key role in anti-tumor immunity. The activation of T cells requires the participation of a series of cell membrane surface molecules, such as the T cell receptor (TCR), CD3, and CD28. The TCR is responsible for receiving stimulation from antigens, but lacks the functional structure to transmit signals into the cell. The TCR needs to form a complex (TCR / CD3 complex) with CD3 through non-covalent binding to complete the T cell activation process. The intracellular region of the CD3 molecule contains an immunoreceptor tyrosine-based activation motif (ITAM), which can mediate the activation, proliferation, and survival of T cells.

[0005] Bispecific antibodies (BsAbs) are a class of bifunctional antibodies that can specifically bind to two antigens or different epitopes of the same antigen. Bispecific T-cell engagers (BiTEs) are representative non-IgG-like bispecific antibodies, formed by linking two single-chain variable fragments (scFv) that recognize T cell CD3 molecules and target cell antigens. They recruit T cells near tumor tissue, form immune synapses between tumor cells and T cells, and mediate T cell killing of tumor cells. Based on this drug design concept, Amgen developed blinatumomab, the world's first dual-target antibody drug for the treatment of acute lymphoblastic leukemia (ALL). Since its launch in 2014, the drug has achieved very good clinical results. Subsequently, a number of CD3-based bispecific antibodies were approved for marketing, including Roche's CD3xCD20 bispecific antibody Mosunetuzumab, Johnson & Johnson's CD3xBCMA bispecific antibody Teclistamab, and CD3xGPRC5D bispecific antibody Talquetamab.

[0006] Although CD3 bispecific antibodies have demonstrated promising therapeutic effects in hematologic malignancies, the incidence of cytokine release syndrome (CRS) is high, sometimes leading to serious side effects. This high incidence of CRS is associated with the overactivity of CD3 antibodies, making it essential to select a CD3 antibody of appropriate strength for bispecific antibody development. Summary of the Invention

[0007] The present application aims to provide an antibody or antigen-binding fragment thereof that can specifically bind to CD3. Furthermore, the present application provides multispecific antibodies that target CD3 and another antigen (such as CD19), isolated nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof or multispecific antibodies of the present application, expression vectors and host cells for expressing the antibodies or antigen-binding fragments thereof or multispecific antibodies of the present application, and uses of the antibodies or antigen-binding fragments thereof or multispecific antibodies of the present application.

[0008] The multispecific antibodies (eg, bispecific antibodies) of the present application activate T cells, thereby specifically killing tumor cells, and have significantly reduced cytokine release, thereby having significantly improved safety.

[0009] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to CD3, wherein the antibody or antigen-binding fragment thereof comprises: a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 16, 54, 1, 9 or 29; a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 55, 56, 17, 2, 10, 24, 30 or 37; a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 18, 3, 11, 25, 31 or 38; a LCDR1 with an amino acid sequence as shown in SEQ ID NO: 42, 43, 20, 5, 13 or 33; a LCDR2 with an amino acid sequence as shown in SEQ ID NO: 21, 45, 46, 6, 14, 27, 34 or 44; and a LCDR3 with an amino acid sequence as shown in SEQ ID NO: 22, 7, 35 or 40.

[0010] In one aspect, the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to CD3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region:

[0011] The heavy chain variable region comprises:

[0012] (I) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 16, SEQ ID NO: 55, and SEQ ID NO: 18, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 55, and SEQ ID NO: 18, respectively;

[0013] (II) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 16, SEQ ID NO: 56, and SEQ ID NO: 18, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 56, and SEQ ID NO: 18, respectively;

[0014] (III) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:18, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:18, respectively;

[0015] (IV) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively;

[0016] (V) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3;

[0017] (VI) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11;

[0018] (VII) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:9, SEQ ID NO:24, and SEQ ID NO:25, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:9, SEQ ID NO:24, and SEQ ID NO:25, respectively;

[0019] (VIII) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31; or

[0020] (IX) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:9, SEQ ID NO:37, and SEQ ID NO:38, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO:9, SEQ ID NO:37, and SEQ ID NO:38, respectively;

[0021] The light chain variable region comprises:

[0022] (I) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:42, SEQ ID NO:21, and SEQ ID NO:22, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:42, SEQ ID NO:21, and SEQ ID NO:22;

[0023] (II) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:42, SEQ ID NO:44, and SEQ ID NO:22, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:42, SEQ ID NO:44, and SEQ ID NO:22;

[0024] (III) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:22, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:22;

[0025] (IV) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:46, and SEQ ID NO:22, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:46, and SEQ ID NO:22;

[0026] (V) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22;

[0027] (VI) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7;

[0028] (VII) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 7, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 7;

[0029] (VIII) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO: 13, SEQ ID NO: 27, and SEQ ID NO: 7, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 13, SEQ ID NO: 27, and SEQ ID NO: 7;

[0030] (IX) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35; or

[0031] (X) LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:6 and SEQ ID NO:40, respectively; or LCDR1, LCDR2 and LCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:6 and SEQ ID NO:40.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0033] (I) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:16, SEQ ID NO:55, and SEQ ID NO:18, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:42, SEQ ID NO:21, and SEQ ID NO:22, respectively;

[0034] (II) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:16, SEQ ID NO:56, and SEQ ID NO:18, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:22, respectively;

[0035] (III) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 56, and SEQ ID NO: 18, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 43, SEQ ID NO: 46, and SEQ ID NO: 22, respectively;

[0036] (IV) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively;

[0037] (V) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively;

[0038] (VI) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:7, respectively;

[0039] (VII) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO:9, SEQ ID NO:24, and SEQ ID NO:25, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO:13, SEQ ID NO:27, and SEQ ID NO:7, respectively;

[0040] (VIII) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequences of SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively; or

[0041] (IX) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:37, and SEQ ID NO:38, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:6, and SEQ ID NO:40, respectively.

[0042] In some embodiments, the antibodies of the present invention are murine antibodies or chimeric antibodies.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0044] (I) a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 19, 4, 12, 26, 32, or 39; and a light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 23, 8, 15, 28, 36, or 41; or

[0045] (II) a heavy chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 19, 4, 12, 26, 32 or 39; and a light chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 23, 8, 15, 28, 36 or 41.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region:

[0047] (I) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:19, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:19; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:23, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:23;

[0048] (II) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:8;

[0049] (III) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15;

[0050] (IV) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:26, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:26; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:28, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:28;

[0051] (V) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:32, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:32; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:36, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:36; or

[0052] (VI) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:39, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:39; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:41, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:41.

[0053] In some embodiments, the antibodies of the present invention are humanized antibodies.

[0054] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0055] (I) a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NO: 57, 58, 59, 60 or 61; and

[0056] a light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NO: 47, 48, 49, 50, 51, 52 or 53;

[0057] (II) a heavy chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 57, 58, 59, 60 or 61; and

[0058] A light chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 47, 48, 49, 50, 51, 52 or 53; or

[0059] (III) a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 57 or 58 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO: 47;

[0060] a heavy chain variable region with the amino acid sequence set forth in SEQ ID NO: 58 and a light chain variable region with the amino acid sequence set forth in SEQ ID NO: 51;

[0061] The heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 60 and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 52 or 53; or

[0062] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 61, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 52 or 53.

[0063] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention, wherein the antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

[0064] In some embodiments, the antigen-binding fragment of the present invention is selected from Fab, Fab', F(ab')2, Fv, scFv or sdAb.

[0065] In some embodiments, the antibody or antigen-binding fragment thereof described in the present invention, wherein the antibody or antigen-binding fragment thereof is of any IgG subtype, such as IgG1, IgG2, IgG3 or IgG4, preferably IgG4 subtype.

[0066] In some embodiments, the antibody or antigen-binding fragment thereof described in the present invention comprises a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 63 and / or a light chain constant region as shown in SEQ ID NO: 62.

[0067] In another aspect, the present invention provides a multispecific antibody comprising an antigen-binding domain targeting CD3 and at least one antigen-binding domain targeting another antigen, wherein the antigen-binding domain targeting CD3 is selected from the antibodies or antigen-binding fragments thereof described herein, and the other antigen is selected from tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs).

[0068] In some embodiments, the multispecific antibody of the present invention is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0069] In some embodiments, the multispecific antibody of the present invention comprises the antigen-binding domain targeting CD3 and the antigen-binding domain targeting the tumor-associated antigen or tumor-specific antigen.

[0070] In another aspect, the multispecific antibody of the present invention, wherein the tumor-associated antigen or tumor-specific antigen is selected from CD19, CD20, CD22, EGFR, HER2, HER3, PSMA, EpCAM, EphA2, CD30, CD33, CD38, CD79b, CD123, CLDN18.2, MSLN, GUCY2C, AFP, PAP, TROP2, LRRC15, gp100, 5T4, CEA, UPK2, DLL3, PRAM E, CDH17, CDH19, GPA33, FAP, GPRC5D, GPC3, B7-H3, CLL-1, LIV-1, ACPP, CLDN6, PSCA, ENPP3, PRLR, MUC16, MUC17, CCR5, GD2, GD3, Ras, LewisY, BORIS, NY-ESO-1, OY-TES1, TSHR, LY6K, FLt3, IGFR-1, CAIX, c-MET, TROP2, or any combination thereof.

[0071] In some embodiments, in the multispecific antibody of the present invention, the tumor-associated antigen is selected from CD19.

[0072] In some embodiments, the multispecific antibody of the present invention comprises the antigen-binding domain targeting CD3 and the antigen-binding domain targeting CD19; the antigen-binding domain targeting CD19 comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprises the sequence shown in SEQ ID NO: 70 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO: 71 or a variant thereof; preferably, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.

[0073] In some embodiments, the antigen binding domain targeting CD3 comprises HCDR1, HCDR2, HCDR3 with amino acid sequences as shown in SEQ ID NO: 16, SEQ ID NO: 56, and SEQ ID NO: 18, respectively, and LCDR1, LCDR2, LCDR3 with amino acid sequences as shown in SEQ ID NO: 43, SEQ ID NO: 46, and SEQ ID NO: 22, respectively.

[0074] In some embodiments, the multispecific antibodies described herein are bispecific antibodies in the form of a common light chain. In some embodiments, the common light chain comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:42, SEQ ID NO:21, and SEQ ID NO:22, respectively; or LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:22, respectively; or LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:46, and SEQ ID NO:22, respectively. Preferably, the common light chain comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:46, and SEQ ID NO:22, respectively. In some embodiments, the common light chain comprises the sequence set forth in SEQ ID NO:47, 51, 52, or 53, for example, the sequence set forth in SEQ ID NO:53.

[0075] In some embodiments, the multispecific antibody of the present invention is a bispecific antibody in the form of DuoBody, comprising:

[0076] i) peptide chain IA, which comprises the VL of the antigen-binding domain targeting CD3 and the first light chain constant region (CL); preferably, the first light chain constant region is kappa;

[0077] ii) peptide chain IB, which comprises the VH and first heavy chain constant region (CH) of the CD3-targeting antigen-binding domain; preferably, the first heavy chain constant region is IgG, such as IgG1, IgG2, IgG3 or IgG4;

[0078] iii) a peptide chain IC comprising the VH of the antigen-binding domain targeting TAA or TSA and a second heavy chain constant region; preferably, the second heavy chain constant region is IgG, such as IgG1, IgG2, IgG3 or IgG4; and

[0079] iv) a peptide chain ID comprising the VL of the antigen-binding domain targeting TAA or TSA and a second light chain constant region; preferably, the second light chain constant region is kappa.

[0080] In some embodiments, the Fc domains contained in the peptide chains IB and IC each contain a modification (eg, a knob-into-hole modification) to promote dimerization of IB and IC.

[0081] In some embodiments, the peptide chain IC comprises the VH sequence shown in SEQ ID NO: 70 or a variant thereof, and the peptide chain ID comprises the VL sequence shown in SEQ ID NO: 71 or a variant thereof; preferably, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.

[0082] In some embodiments, the peptide chain IB comprises the VH sequence shown in SEQ ID NO: 58, 57, 60 or 61 or a variant thereof, and the peptide chain IA comprises the VL sequence shown in SEQ ID NO: 51, 47, 52 or 53 or a variant thereof; preferably, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.

[0083] In some embodiments, the peptide chain IB comprises the VH sequence shown in SEQ ID NO: 58 or 57 or a variant thereof, and the peptide chain IA comprises the VL sequence shown in SEQ ID NO: 51 or 47 or a variant thereof; preferably, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.

[0084] In some embodiments, the multispecific antibody of the present invention is a common light chain bispecific antibody comprising:

[0085] i) peptide chain II-A, which is a common light chain and comprises the VL and light chain constant region (CL) of the CD3-targeting antigen-binding domain; preferably, the light chain constant region is kappa;

[0086] ii) peptide chain II-B, which comprises the VH and first heavy chain constant region (CH) of the antigen binding domain targeting CD3; preferably, the first heavy chain constant region is IgG, such as IgG1, IgG2, IgG3 or IgG4; and

[0087] iii) peptide chain II-C, which comprises the VH of the antigen-binding domain targeting TAA or TSA and a second CH; preferably, the second CH is IgG, such as IgG1, IgG2, IgG3 or IgG4.

[0088] In some embodiments, the Fc domains comprised by the peptide chains II-B and II-C respectively comprise modifications to promote dimerization of II-B and II-C.

[0089] In some embodiments, in the common light chain bispecific antibody: (1) the peptide chain II-A comprises the VL sequence of SEQ ID NO: 47 or a variant thereof, and the peptide chain II-B comprises the VH sequence of SEQ ID NO: 57 or a variant thereof; or (2) the peptide chain II-A comprises the VL sequence of SEQ ID NO: 47 or a variant thereof, and the peptide chain II-B comprises the VH sequence of SEQ ID NO: 58 or a variant thereof; or (3) the peptide chain II-A comprises the VL sequence of SEQ ID NO: 51 or a variant thereof, and the peptide chain II-B comprises the VH sequence of SEQ ID NO: 58 or a variant thereof; or (4) the peptide chain II-A comprises the VL sequence of SEQ ID NO: 52 or a variant thereof, and the peptide chain II-B comprises the VH sequence of SEQ ID NO: 60 or a variant thereof; or (5) the peptide chain II-A comprises the VL sequence of SEQ ID NO: 53 or a variant thereof, and the peptide chain II-B comprises the VH sequence of SEQ ID NO: 60 or a variant thereof; or (6) the peptide chain II-A comprises the VL sequence of SEQ ID NO: 54 or a variant thereof, and the peptide chain II-B comprises the VH sequence of SEQ ID NO: 61 or a variant thereof. NO: 52 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 61 or a variant thereof; or (7) the peptide chain II-A comprises the VL sequence shown in SEQ ID NO: 53 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 61 or a variant thereof; the variant in any one of (1) to (7) has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.

[0090] In some embodiments, the peptide chain II-C comprises the VH sequence shown in SEQ ID NO: 70 or a variant thereof; the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.

[0091] In yet another aspect, the present invention provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof, or a heavy chain variable region and / or a light chain variable region thereof, as described herein.

[0092] In some embodiments, the present invention provides isolated nucleic acid molecules encoding the multispecific antibodies or polypeptide chains thereof described herein.

[0093] In yet another aspect, the present invention provides an expression vector comprising the isolated nucleic acid molecule described herein.

[0094] In yet another aspect, the present invention provides a host cell comprising an isolated nucleic acid molecule described herein or a vector (eg, an expression vector) described herein.

[0095] In another aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof, or the multispecific antibody described herein, which method comprises culturing the host cell described herein under conditions that allow protein expression, and re-collecting the antibody or antigen-binding fragment thereof, or the multispecific antibody from the culture of the cultured host cell.

[0096] In another aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein, a multispecific antibody described herein, an isolated nucleic acid molecule described herein, a vector described herein, or a host cell described herein, and a pharmaceutically acceptable carrier and / or excipient.

[0097] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof described herein, the isolated nucleic acid molecule described herein, the vector described herein, the host cell described herein, or the pharmaceutical composition described herein in the preparation of a medicament for preventing and / or treating a disease.

[0098] In another aspect, the present invention provides a method for preventing and / or treating a disease, comprising administering to a subject in need thereof an effective dose of an antibody or antigen-binding fragment thereof described herein, a multispecific antibody described herein, an isolated nucleic acid molecule described herein, a vector described herein, or a host cell described herein, or a pharmaceutical composition described herein.

[0099] In some embodiments, in the uses or methods described in the present invention, the disease is a tumor, an inflammatory disease or an autoimmune disease; preferably, the tumor is selected from ovarian cancer, breast cancer, gastric cancer, bile duct cancer, colorectal cancer, lung cancer, acute myeloid leukemia, chronic lymphocytic leukemia, melanoma or colorectal cancer.

[0100] Detailed Description of the Invention

[0101] CD3 binding portion

[0102] The present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to CD3. The terms "anti-CD3 antibody," "anti-CD3," "CD3 antibody," or "binding to CD3" refer to antibodies that can bind to the CD3 protein or fragment thereof with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent targeting CD3.

[0103] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to CD3, wherein the antibody or antigen-binding fragment thereof comprises: a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 16, 54, 1, 9 or 29; a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 55, 56, 17, 2, 10, 24, 30 or 37; a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 18, 3, 11, 25, 31 or 38; a LCDR1 with an amino acid sequence as shown in SEQ ID NO: 42, 43, 20, 5, 13 or 33; a LCDR2 with an amino acid sequence as shown in SEQ ID NO: 21, 45, 46, 6, 14, 27, 34 or 44; and a LCDR3 with an amino acid sequence as shown in SEQ ID NO: 22, 7, 35 or 40.

[0104] In some embodiments, the present invention provides an antibody or antigen-binding fragment thereof that can specifically bind to CD3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region:

[0105] The heavy chain variable region comprises:

[0106] (I) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 16, SEQ ID NO: 55, and SEQ ID NO: 18, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 55, and SEQ ID NO: 18, respectively;

[0107] (II) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 16, SEQ ID NO: 56, and SEQ ID NO: 18, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 56, and SEQ ID NO: 18, respectively;

[0108] (III) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:18, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:54, SEQ ID NO:56, and SEQ ID NO:18, respectively;

[0109] (IV) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively;

[0110] (V) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3;

[0111] (VI) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11;

[0112] (VII) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:9, SEQ ID NO:24, and SEQ ID NO:25, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:9, SEQ ID NO:24, and SEQ ID NO:25, respectively;

[0113] (VIII) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31; or

[0114] (IX) HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:9, SEQ ID NO:37, and SEQ ID NO:38, respectively; or HCDR1, HCDR2, and HCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences of SEQ ID NO:9, SEQ ID NO:37, and SEQ ID NO:38, respectively;

[0115] The light chain variable region comprises:

[0116] (I) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:42, SEQ ID NO:21, and SEQ ID NO:22, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:42, SEQ ID NO:21, and SEQ ID NO:22;

[0117] (II) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:42, SEQ ID NO:44, and SEQ ID NO:22, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:42, SEQ ID NO:44, and SEQ ID NO:22;

[0118] (III) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:22, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:22;

[0119] (IV) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:46, and SEQ ID NO:22, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:46, and SEQ ID NO:22;

[0120] (V) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22;

[0121] (VI) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7;

[0122] (VII) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 7, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 7;

[0123] (VIII) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO: 13, SEQ ID NO: 27, and SEQ ID NO: 7, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO: 13, SEQ ID NO: 27, and SEQ ID NO: 7;

[0124] (IX) LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35, respectively; or LCDR1, LCDR2, and LCDR3 with 1, 2, or 3 amino acid differences from the amino acid sequences as set forth in SEQ ID NO:33, SEQ ID NO:34, and SEQ ID NO:35; or

[0125] (X) LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:6 and SEQ ID NO:40, respectively; or LCDR1, LCDR2 and LCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:6 and SEQ ID NO:40.

[0126] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0127] (I) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:16, SEQ ID NO:55, and SEQ ID NO:18, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:42, SEQ ID NO:21, and SEQ ID NO:22, respectively;

[0128] (II) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 with amino acid sequences as set forth in SEQ ID NO:16, SEQ ID NO:56, and SEQ ID NO:18, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 with amino acid sequences as set forth in SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:22, respectively;

[0129] (III) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 56, and SEQ ID NO: 18, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 43, SEQ ID NO: 46, and SEQ ID NO: 22, respectively;

[0130] (IV) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22, respectively;

[0131] (V) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively;

[0132] (VI) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:7, respectively;

[0133] (VII) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 having amino acid sequences as set forth in SEQ ID NO:9, SEQ ID NO:24, and SEQ ID NO:25, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 having amino acid sequences as set forth in SEQ ID NO:13, SEQ ID NO:27, and SEQ ID NO:7, respectively;

[0134] (VIII) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 as shown in the amino acid sequences of SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 as shown in the amino acid sequences of SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively; or

[0135] (IX) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:37, and SEQ ID NO:38, respectively; and a light chain variable region comprising LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:6, and SEQ ID NO:40, respectively.

[0136] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0137] (I) a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 19, 4, 12, 26, 32, or 39; and a light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NOs: 23, 8, 15, 28, 36, or 41; or

[0138] (II) a heavy chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 19, 4, 12, 26, 32 or 39; and a light chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 23, 8, 15, 28, 36 or 41.

[0139] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region:

[0140] (I) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:19, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:19; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:23, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:23;

[0141] (II) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:4, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:4; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:8, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:8;

[0142] (III) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 12, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 12; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 15, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 15;

[0143] (IV) the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:26, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:26; and the light chain variable region comprises the amino acid sequence of SEQ ID NO:28, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:28;

[0144] (V) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:32, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:32; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:36, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:36; or

[0145] (VI) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:39, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:39; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:41, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:41.

[0146] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0147] a heavy chain variable region comprising the amino acid sequence shown in any one of SEQ ID NO: 57, 58, 59, 60 or 61; and

[0148] A light chain variable region comprising the amino acid sequence shown in any one of SEQ ID NO: 47, 48, 49, 50, 51, 52 or 53.

[0149] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0150] a heavy chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 57, 58, 59, 60 or 61; and

[0151] A light chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 47, 48, 49, 50, 51, 52 or 53.

[0152] In some embodiments, the antibody or antigen-binding fragment thereof of the present invention comprises:

[0153] a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 57 or 58 and a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 47;

[0154] a heavy chain variable region with the amino acid sequence set forth in SEQ ID NO: 58 and a light chain variable region with the amino acid sequence set forth in SEQ ID NO: 51;

[0155] The heavy chain variable region with the amino acid sequence shown in SEQ ID NO: 60 and the light chain variable region with the amino acid sequence shown in SEQ ID NO: 52 or 53; or

[0156] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 61, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 52 or 53.

[0157] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are selected from antibodies having the CDR and / or variable region sequences of antibody NP010-018 or humanized antibodies 18, 18-5, 18-8, 18-11, 18-12, 18-13 or 18-14 thereof, antibodies having at least 80% (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identity with their CDR or variable region sequences, and antibodies whose difference from their CDR or variable region sequences lies only in the deletion, addition or substitution of one or several amino acids.

[0158] In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise a constant region, such as a heavy chain constant region (e.g., IgG, such as IgG1, IgG2, IgG3, or IgG4) and / or a light chain constant region (e.g., κ or λ) derived from a human immunoglobulin. In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise a heavy chain constant region as set forth in SEQ ID NO: 63 and / or a light chain constant region as set forth in SEQ ID NO: 62.

[0159] In some embodiments, the antibody or antigen-binding fragment thereof described in the present invention is a murine antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0160] In some embodiments, the antibody molecules of the present invention are humanized. The different methods for making antibody humanization are known to technicians, as reviewed by Almagro & Fransson, the contents of which are fully incorporated herein by reference (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13: 1619-1633). In some embodiments, the antibody of the present invention comprises a heavy chain framework region (e.g., a heavy chain framework region included in the amino acid sequence encoded by human heavy chain germline genes) derived from human immunoglobulin, and / or a light chain framework region (e.g., a light chain framework region included in the amino acid sequence encoded by human light chain germline genes). The heavy chain framework region and / or light chain framework region optionally include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to mouse residues.

[0161] In some embodiments, the antibody molecules of the present invention are human antibodies or humanized antibodies. Human antibodies or humanized antibodies can be prepared using various techniques known in the art.

[0162] In one embodiment, the antibody molecules of the present invention also encompass antigen-binding fragments thereof, such as the following antibody fragments: Fab, Fab', F(ab')2, Fv, scFv or sdAb.

[0163] The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia (Chothia et al. (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), based on the three-dimensional structure of the antibody and the topology of the CDR loops; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., US Department of Health and Human Services, National Institutes of Health (1987)), based on the variability of antibody sequences; AbM (University of Bath); Contact (University College London); the international ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0164] Unless otherwise indicated, the CDR of an antibody of the present invention can be determined by those skilled in the art according to any scheme (e.g., different assignment systems or combinations) of this area. In some embodiments, the CDR of an antibody of the present invention is defined by Kabat, Chothia, AbM, Contact, North, or IMGT. In some embodiments, the CDR of an antibody of the present invention is defined by Kabat.

[0165] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment systems or combinations).

[0166] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDR is different between antibodies, only a limited number of amino acid positions in the CDR are directly involved in antigen binding. Using at least two of Kabat, Chothia, AbM, Contact and North methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of a CDR. As those skilled in the art will appreciate, by the structure of the antibody and protein folding, the residues of the rest of the CDR sequence can be determined. Therefore, the present invention also contemplates variants of any CDR provided herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, and the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.

[0167] The humanized antibodies of the present invention can be prepared by inserting murine CDR regions into human germline framework regions using methods known in the art, such as those described in U.S. Patent Nos. 5,225,539 to Winter et al. and 5,530,101, 5,585,089, 5,693,762, and 6,180,370 to Queen et al.

[0168] In some embodiments, the amino acid differences comprise amino acid deletions, insertions, or substitutions. In some embodiments, the anti-CD3 antibodies or antigen-binding fragments thereof of the invention include those having amino acid sequences that have been mutated by amino acid deletions, insertions, or substitutions, but still have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the above-described antibodies (particularly in the CDR regions depicted in the above sequences). In some embodiments, the antibodies of the invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the CDR regions when compared to the CDR regions depicted in the specific sequences. In some embodiments, the antibodies of the invention have no more than 1, 2, 3, 4, or 5 amino acid mutations in the framework regions when compared to the framework regions in the specific sequences.

[0169] In some embodiments, the polynucleotide molecules encoding the antibodies of the present invention include polynucleotide molecules that have been mutated by nucleotide deletion, insertion or substitution, but still have at least about 60, 70, 80, 90, 95 or 100% identity with the CDR corresponding coding regions depicted in the sequences described above.

[0170] In some embodiments, the CD3 antibody is an IgG antibody, such as an IgG1, IgG2, IgG3, or IgG4 antibody, or a modified form thereof, as described in the following sections.

[0171] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0172] In some embodiments, it may be desirable to generate cysteine ​​engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues.

[0173] In some embodiments, the antibodies provided herein can be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0174] Reference Antibodies

[0175] In some embodiments, the reference antibody SP34 (Teclistamab-CD3) is selected from the heavy chain set forth in SEQ ID NO: 66 and the light chain set forth in SEQ ID NO: 67.

[0176] In some embodiments, the reference antibody blinatumumab is selected from the heavy chain set forth in SEQ ID NO:68 and the light chain set forth in SEQ ID NO:69.

[0177] Multispecific antibodies and tumor-associated antigen (TAA) or tumor-specific antigen (TSA) binding portions

[0178] The present invention provides a multispecific antibody comprising an antigen-binding domain targeting CD3 and at least one antigen-binding domain targeting another antigen, wherein the antigen-binding domain targeting CD3 is selected from the antibodies or antigen-binding fragments thereof described herein, and the other antigen is selected from tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs).

[0179] In some embodiments, the multispecific antibody of the present invention is a bispecific antibody, a trispecific antibody, or a tetraspecific antibody.

[0180] In some embodiments, the multispecific antibody of the present invention comprises the antigen-binding domain targeting CD3 and the antigen-binding domain targeting the tumor-associated antigen or tumor-specific antigen.

[0181] The multispecific antibody of the present invention, wherein the tumor-associated antigen or tumor-specific antigen is selected from CD19, CD20, CD22, EGFR, HER2, HER3, PSMA, EpCAM, EphA2, CD30, CD33, CD38, CD79b, CD123, CLDN18.2, MSLN, GUCY2C, AFP, PAP, TROP2, LRRC15, gp100, 5T4, CEA, UPK2, DLL3, PRAME, CD H17, CDH19, GPA33, FAP, GPRC5D, GPC3, B7-H3, CLL-1, LIV-1, ACPP, CLDN6, PSCA, ENPP3, PRLR, MUC16, MUC17, CCR5, GD2, GD3, Ras, LewisY, BORIS, NY-ESO-1, OY-TES1, TSHR, LY6K, FLt3, IGFR-1, CAIX, c-MET, TROP2, or any combination thereof.

[0182] In some embodiments, in the multispecific antibody of the present invention, the tumor-associated antigen is selected from CD19.

[0183] In some embodiments, the multispecific antibody of the present invention comprises the antigen-binding domain targeting CD3 and the antigen-binding domain targeting CD19; the antigen-binding domain targeting CD19 comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprises the sequence shown in SEQ ID NO: 70 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO: 71 or a variant thereof; preferably, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.

[0184] In some embodiments, the CD3-targeting antigen-binding domain of such multispecific antibodies comprises HCDR1, HCDR2, HCDR3 with amino acid sequences as shown in SEQ ID NO: 16, SEQ ID NO: 56, and SEQ ID NO: 18, and LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 43, SEQ ID NO: 46, and SEQ ID NO: 22, respectively.

[0185] In some embodiments, such multispecific antibodies are bispecific antibodies in the form of a common light chain.

[0186] In some embodiments, the common light chain of such multispecific antibodies comprises LCDR1, LCDR2, LCDR3 with amino acid sequences as shown in SEQ ID NO:42, SEQ ID NO:21, and SEQ ID NO:22, respectively; or LCDR1, LCDR2, LCDR3 with amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:22, respectively; or LCDR1, LCDR2, LCDR3 with amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:46, and SEQ ID NO:22, respectively; preferably, the common light chain comprises LCDR1, LCDR2, LCDR3 with amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:46, and SEQ ID NO:22, respectively.

[0187] In some embodiments, the common light chain of such multispecific antibodies comprises the sequence set forth in SEQ ID NO: 47, 51, 52, or 53.

[0188] In some embodiments, the CD3-targeting antigen-binding domain of the multispecific antibody of the present invention is Fab.

[0189] In some embodiments, the antigen-binding domain targeting TAA or TSA of the multispecific antibody of the present invention is Fab or scFv.

[0190] In some embodiments, the multispecific antibodies of the present invention further comprise an Fc domain. In some embodiments, the Fc domain is an IgG, such as IgG1, IgG2, IgG3, or IgG4. In some embodiments, the Fc domain comprises modifications to promote dimerization of the first Fc monomer and the second Fc monomer.

[0191] In some embodiments, the modification is selected from mutations that promote Fab arm exchange. In some embodiments, the modification includes a K409R mutation in one of the two monomers and a F405L mutation in the other of the two monomers to promote heterodimerization of the Fab arms. In some embodiments, the heavy chain constant region sequence comprising K409R is shown in SEQ ID NO: 63. In some embodiments, the heavy chain constant region sequence comprising F405L is shown in SEQ ID NO: 72.

[0192] In some embodiments, the modification comprises a knob modification in one of the two monomers and a hole modification in the other of the two monomers to form a knob-into-hole modification.

[0193] In some embodiments, the Fc domain may also be a native Fc sequence.

[0194] In some embodiments, the multispecific antibody of the present invention is a DuoBody or a common light chain bispecific antibody.

[0195] In some embodiments, the multispecific antibody of the present invention is a DuoBody, which comprises:

[0196] (i) peptide chain IA, which comprises the VL of the CD3-targeting antigen-binding domain and the first light chain constant region (CL); preferably, the first CL is kappa;

[0197] (ii) a peptide chain IB comprising the VH and first heavy chain constant region (CH) of the CD3-targeting antigen-binding domain; preferably, the first CH is IgG, such as IgG1, IgG2, IgG3 or IgG4;

[0198] (iii) a peptide chain IC comprising the VH and a second CH of the antigen-binding domain targeting TAA or TSA; preferably, the second CH is IgG, such as IgG1, IgG2, IgG3 or IgG4; and

[0199] (iv) a peptide chain ID comprising the VL of the antigen-binding domain targeting TAA or TSA and a second CL; preferably, the second CL is kappa.

[0200] In some embodiments, the Fc domains comprised by the peptide chains IB and IC respectively comprise modifications to promote dimerization of IB and IC.

[0201] In some embodiments, the Fc domains comprised by peptide chains IB and IC comprise mutations that promote Fab arm exchange, such as a K409R mutation in the CH3 region of one Fc domain and a F405L mutation in the CH3 region of the other Fc domain. In some embodiments, the peptide chains IB and IC comprise the heavy chain constant region sequences set forth in SEQ ID NOs: 63 and 72, respectively. In some embodiments, the peptide chains IB and IC comprise the heavy chain constant region sequences set forth in SEQ ID NOs: 72 and 63, respectively.

[0202] In some embodiments, the peptide chains IA and ID comprise the light chain constant region sequence shown in SEQ ID NO:62.

[0203] In some embodiments, the antigen binding domain targeting TAA or TSA is an antigen binding domain targeting CD19, the peptide chain IC comprises the VH sequence shown in SEQ ID NO: 70 or a variant thereof, and the peptide chain ID comprises the VL sequence shown in SEQ ID NO: 71 or a variant thereof; preferably, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.

[0204] In some embodiments, the peptide chain IA comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO:42, SEQ ID NO:21 and SEQ ID NO:22, respectively, and the peptide chain IB comprises HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:16, SEQ ID NO:55 and SEQ ID NO:18, respectively.

[0205] In some embodiments, the peptide chain IB comprises the VH sequence shown in SEQ ID NO: 58, 57, 60 or 61 or a variant thereof, and the peptide chain IA comprises the VL sequence shown in SEQ ID NO: 51, 47, 52 or 53 or a variant thereof; preferably, the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.

[0206] In some embodiments, the peptide chain IA comprises the VL sequence set forth in SEQ ID NO: 51 or 47, or a variant thereof; the peptide chain IB comprises the VH sequence set forth in SEQ ID NO: 58 or 57, or a variant thereof. In some embodiments, the peptide chain IA comprises the VL sequence set forth in SEQ ID NO: 51, or a variant thereof; the peptide chain IB comprises the VH sequence set forth in SEQ ID NO: 58, or a variant thereof. In some embodiments, the peptide chain IA comprises the VL sequence set forth in SEQ ID NO: 47, or a variant thereof; the peptide chain IB comprises the VH sequence set forth in SEQ ID NO: 57, or a variant thereof. Preferably, the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence from which it is derived.

[0207] In some embodiments, the peptide chain IC comprises the VH sequence shown in SEQ ID NO: 70, the peptide chain ID comprises the VL sequence shown in SEQ ID NO: 71, the peptide chain IA comprises the VL sequence shown in SEQ ID NO: 51, and the peptide chain IB comprises the VH sequence shown in SEQ ID NO: 58.

[0208] In some embodiments, the peptide chain IC comprises the VH sequence shown in SEQ ID NO: 70, the peptide chain ID comprises the VL sequence shown in SEQ ID NO: 71, the peptide chain IA comprises the VL sequence shown in SEQ ID NO: 47, and the peptide chain IB comprises the VH sequence shown in SEQ ID NO: 57.

[0209] In some embodiments, the present invention provides the following exemplary DuoBody:

[0210] (1) the peptide chain IA comprises the light chain sequence shown in SEQ ID NO: 65; the peptide chain IB comprises the heavy chain sequence shown in SEQ ID NO: 64; the peptide chain IC comprises the heavy chain sequence shown in SEQ ID NO: 68; the peptide chain ID comprises the light chain sequence shown in SEQ ID NO: 69; or,

[0211] (2) The peptide chain IA comprises the light chain sequence shown in SEQ ID NO:84; the peptide chain IB comprises the heavy chain sequence shown in SEQ ID NO:83; the peptide chain IC comprises the heavy chain sequence shown in SEQ ID NO:68; and the peptide chain ID comprises the light chain sequence shown in SEQ ID NO:69.

[0212] In some embodiments, the multispecific antibody of the present invention is a common light chain bispecific antibody comprising:

[0213] (i) peptide chain II-A, which is a common light chain and comprises the VL and light chain constant region (CL) of the CD3-targeting antigen-binding domain; preferably, the CL is kappa;

[0214] (ii) peptide chain II-B, which comprises the VH and first heavy chain constant region (CH) of the CD3-targeting antigen-binding domain; preferably, the first CH is IgG, such as IgG1, IgG2, IgG3 or IgG4; and

[0215] (iii) peptide chain II-C, which comprises the VH and the second CH of the antigen-binding domain targeting TAA or TSA; preferably, the second CH is IgG, such as IgG1, IgG2, IgG3 or IgG4;

[0216] Among them, the VL of the peptide chain II-A and the VH of the peptide chain II-B form a first binding site targeting CD3, and the VL of the peptide chain II-A and the VH of the peptide chain II-C form a second binding site targeting TAA or TSA.

[0217] In some embodiments, the VH of peptide chain II-B and the VL of peptide chain II-A of the common light chain bispecific antibody respectively comprise:

[0218] (I) HCDR1, HCDR2, and HCDR3 with amino acid sequences set forth in SEQ ID NO: 16, SEQ ID NO: 55, and SEQ ID NO: 18, respectively, and LCDR1, LCDR2, and LCDR3 with amino acid sequences set forth in SEQ ID NO: 42, SEQ ID NO: 21, and SEQ ID NO: 22, respectively;

[0219] (II) HCDR1, HCDR2, and HCDR3 with amino acid sequences as shown in SEQ ID NO: 16, SEQ ID NO: 56, and SEQ ID NO: 18, respectively, and LCDR1, LCDR2, and LCDR3 with amino acid sequences as shown in SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 22, respectively; or

[0220] (III) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 16, SEQ ID NO: 56 and SEQ ID NO: 18, respectively, and LCDR1, LCDR2 and LCDR3 with amino acid sequences as shown in SEQ ID NO: 43, SEQ ID NO: 46 and SEQ ID NO: 22, respectively.

[0221] In some embodiments, the VH of peptide chain II-B and the VL of peptide chain II-A of the common light chain bispecific antibody respectively comprise: HCDR1, HCDR2, HCDR3 with amino acid sequences as shown in SEQ ID NO: 16, SEQ ID NO: 56, and SEQ ID NO: 18, respectively, and LCDR1, LCDR2, LCDR3 with amino acid sequences as shown in SEQ ID NO: 43, SEQ ID NO: 46, and SEQ ID NO: 22, respectively.

[0222] In some embodiments, peptide chain II-A of the common light chain bispecific antibody comprises a VL sequence as set forth in SEQ ID NO: 47, 51, 52, or 53, or a variant thereof, preferably wherein the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence from which it is derived. In some embodiments, peptide chain II-B of the common light chain bispecific antibody comprises a VH sequence as set forth in SEQ ID NO: 57, 58, 60, or 61, or a variant thereof, preferably wherein the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence from which it is derived.

[0223] In some embodiments, the VH of peptide chain II-B and the VL of peptide chain II-A of the common light chain bispecific antibody respectively comprise: (1) the VH sequence shown in SEQ ID NO: 57 or a variant thereof, the VL sequence shown in SEQ ID NO: 47 or a variant thereof; (2) the VH sequence shown in SEQ ID NO: 58 or a variant thereof, the VL sequence shown in SEQ ID NO: 47 or a variant thereof; (3) the VH sequence shown in SEQ ID NO: 58 or a variant thereof, the VL sequence shown in SEQ ID NO: 51 or a variant thereof; (4) the VH sequence shown in SEQ ID NO: 60 or a variant thereof, the VL sequence shown in SEQ ID NO: 52 or a variant thereof; (5) the VH sequence shown in SEQ ID NO: 60 or a variant thereof, the VL sequence shown in SEQ ID NO: 53 or a variant thereof; (6) the VH sequence shown in SEQ ID NO: 61 or a variant thereof, the VL sequence shown in SEQ ID NO: 52 or a variant thereof; or (7) the VH sequence shown in SEQ ID NO: 61 or a variant thereof, the VL sequence shown in SEQ ID NO: 53 or a variant thereof. The variant of any one of (1) to (7) has at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence from which it is derived.

[0224] In some embodiments, the VH of peptide chain II-B and the VL of peptide chain II-A of the common light chain bispecific antibody respectively comprise: the VH sequence shown in SEQ ID NO: 61 and the VL sequence shown in SEQ ID NO: 53.

[0225] In some embodiments, peptide chain II-C of the common light chain bispecific antibody comprises a VH of an antigen-binding domain targeting CD 19. In some embodiments, the VH of peptide chain II-C of the common light chain bispecific antibody comprises the sequence set forth in SEQ ID NO: 70 or a variant thereof; preferably, the variant has at least 95%, 96%, 97%, 98%, or 99% sequence identity compared to the sequence from which it is derived.

[0226] In some embodiments, the Fc domains comprised by peptide chains II-B and II-C of the common light chain bispecific antibody optionally comprise modifications to promote dimerization of IB and IC, respectively.

[0227] In some embodiments, the Fc domains comprised by peptide chains II-B and II-C comprise a K409R mutation in the CH3 region of one Fc domain and a F405L mutation in the CH3 region of the other Fc domain. In some embodiments, peptide chains II-B and II-C comprise the heavy chain constant region sequences set forth in SEQ ID NOs: 63 and 72, respectively. In some embodiments, peptide chains II-B and II-C comprise the heavy chain constant region sequences set forth in SEQ ID NOs: 72 and 63, respectively.

[0228] In some embodiments, peptide chain II-A of the common light chain bispecific antibody comprises the light chain constant region sequence shown in SEQ ID NO:62.

[0229] Antibody expression

[0230] In yet another aspect, the present invention provides an isolated nucleic acid molecule encoding any of the above antibodies or fragments thereof or any of their chains. In one embodiment, the isolated nucleic acid molecule may comprise a polynucleotide encoding the amino acid sequence of the light chain variable region and / or the heavy chain variable region of the antibody, or a polynucleotide encoding the amino acid sequence of the light chain and / or the heavy chain of the antibody. In some embodiments, the isolated nucleic acid molecule comprises a polynucleotide encoding each peptide chain of the multispecific antibody of the present invention.

[0231] In yet another aspect, the present invention provides vectors (e.g., expression vectors) comprising an isolated nucleic acid molecule as described herein, preferably, a eukaryotic expression vector. In some embodiments, the isolated nucleic acid molecule as described herein is contained in one or more vectors. Vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0232] In yet another aspect, the present invention provides a host cell comprising an isolated nucleic acid molecule as described herein or an expression vector as described herein, preferably, the host cell is a eukaryotic cell, more preferably a mammalian cell (e.g., a CHO cell or a 293 cell). In another embodiment, the host cell is prokaryotic.

[0233] In one embodiment, the present invention provides a method for preparing an antibody of the present invention, wherein the method comprises introducing an expression vector into a mammalian host cell and then culturing the host cell for a sufficient period of time to allow expression of the antibody in the host cell, or more preferably, secretion of the antibody into the culture medium in which the host cell is grown, to produce the antibody. The antibody can be recovered from the culture medium using standard protein purification methods.

[0234] The present invention provides mammalian host cells for expressing the recombinant antibodies of the present invention, including many immortalized cell lines available from the American Type Culture Collection (ATCC). These especially include Chinese hamster ovary (CHO) cells, NSO, SP2 / 0 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells, A549 cells, 293T cells and many other cell lines. Mammalian host cells include humans, mice, rats, dogs, monkeys, pigs, goats, cattle, horses and hamster cells. Particularly preferred cell lines are selected by determining which cell line has high expression levels.

[0235] It is likely that antibodies expressed by different cell lines or in transgenic animals will have different glycosylation from one another. However, all antibodies encoded by the nucleic acid molecules provided herein or comprising the amino acid sequences provided herein are part of the present invention, regardless of the glycosylation of the antibodies. Likewise, in certain embodiments, non-fucosylated antibodies are advantageous because they generally have more potent efficacy in vitro and in vivo than their fucosylated counterparts and are less likely to be immunogenic because their carbohydrate structures are normal components of natural human serum IgG.

[0236] Pharmaceutical compositions and pharmaceutical preparations

[0237] In another aspect, the present invention provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof described herein, a multispecific antibody described herein, an isolated nucleic acid molecule described herein, a vector described herein, or a host cell described herein, and a pharmaceutically acceptable carrier and / or excipient.

[0238] The term "pharmaceutical composition" refers to a preparation that permits the active ingredient contained therein to exist in biologically effective form, and that contains no additional ingredients that are unacceptably toxic to a subject to which the preparation would be administered.

[0239] Pharmaceutical formulations containing the antibodies described herein can be prepared by mixing the antibodies of the invention having the desired degree of purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of aqueous solutions or lyophilized formulations.

[0240] Medical uses and treatments

[0241] Any of the antibodies provided herein can be used in therapeutic methods. It should also be understood that when discussing "antibodies," compositions comprising antibodies are also included. The antibodies of the present invention can be used in therapeutically effective amounts or prophylactically effective amounts in the treatment or prevention methods described in any embodiment of the present invention.

[0242] In another aspect, the present invention provides use of the antibody or antigen-binding fragment thereof described herein, the isolated nucleic acid molecule described herein, the vector described herein, the host cell described herein, or the pharmaceutical composition described herein in the preparation of a medicament for preventing and / or treating a disease.

[0243] In another aspect, the present invention provides a method for preventing and / or treating a disease, comprising administering to a subject in need thereof an effective dose of an antibody or antigen-binding fragment thereof described herein, a multispecific antibody described herein, an isolated nucleic acid molecule described herein, a vector described herein, or a host cell described herein, or a pharmaceutical composition described herein.

[0244] In some embodiments, in the uses or methods provided above, the disease is a tumor, an inflammatory disease or an autoimmune disease; preferably, the tumor is selected from ovarian cancer, breast cancer, gastric cancer, bile duct cancer, colorectal cancer, lung cancer, acute myeloid leukemia, chronic lymphocytic leukemia, melanoma or colorectal cancer.

[0245] In some embodiments, the administration of the present invention includes, but is not limited to, oral, intravenous, subcutaneous, intramuscular, intraarterial, intraarticular (e.g., in arthritic joints), by inhalation, aerosol delivery, or intratumoral administration.

[0246] Definition of terms

[0247] In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, procedures in cell culture, molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, nucleic acid chemistry, immunology, and the like used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0248] In order to make it easier to understand the present invention, certain scientific and technological terms are specifically defined below. Unless otherwise clearly defined elsewhere in this article, the scientific and technological terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. Regarding the definitions and terms in this area, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. The singular form used herein (including the claims) includes its corresponding plural form unless otherwise clearly provided in the text.

[0249] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit of 5% less than the specified numerical value and an upper limit of 5% greater than the specified numerical value, including but not limited to ±5%, ±2%, ±1%, and ±0.1%, as such variations are appropriate for performing the disclosed methods.

[0250] The term "and / or" should be understood to mean any one of the alternatives or a combination of any two or more of the alternatives.

[0251] The term "CD3" refers to a part of the T cell receptor complex, which consists of three different chains, CD3ε, CD3δ and CD3γ. The concentration of CD3 on T cells, for example, by the fixation of anti-CD3 antibodies, leads to the activation of T cells, similar to T cell receptor-mediated activation, but independent of the specificity of the TCR clone. The vast majority of anti-CD3 antibodies recognize the CD3ε chain. The term refers to any natural CD3 from any vertebrate (including mammals such as primates (e.g., humans)) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full-length" unprocessed CD3 and any form of CD3 or any fragment thereof produced by intracellular processing. The term also includes naturally occurring variants of CD3, for example, splice variants or allelic variants. In a preferred embodiment, CD3 refers to the full length or fragments thereof from humans and cynomolgus monkeys (such as mature fragments thereof lacking a signal peptide). In a preferred embodiment, CD3 refers to the full length or fragments thereof from mice / rat (such as mature fragments thereof lacking a signal peptide).

[0252] The term "CD19" refers to the Cluster of Differentiation 19 protein (CD19), which is an antigenic determinant detectable on leukemia precursor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found in UniProt / Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 can be found in accession number NM_001178098. CD19 is expressed in most B-lineage cancers, such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma.

[0253] As used herein, the term "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one of the numbers or elements in the list, but also including more than one, and optionally, additional unlisted items. Only when explicitly stated to the contrary, such as "only one" or "exactly one" or when used in a claim, "consisting of..." will refer to only one of the listed numbers or one of the elements of the list.

[0254] Unless expressly stated otherwise, as used herein, the words "a" and "an" should be understood to mean "at least one."

[0255] The term "percent (%) amino acid sequence identity," or simply "identity," is defined as the percentage of amino acid residues in a candidate amino acid sequence that are identical to the amino acid residues in a reference amino acid sequence, after aligning the amino acid sequences (and introducing gaps, if necessary) to achieve maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence alignment to determine percent amino acid sequence identity can be performed using various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or MEGALIGN (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximum alignment over the full length of the compared sequences.

[0256] The term "immune response" refers to the actions of, for example, lymphocytes, antigen-presenting cells, phagocytes, granulocytes, and soluble macromolecules (including antibodies, cytokines, and complement) produced by the above cells or the liver, which result in the selective damage, destruction, or elimination from the body of invading pathogens, cells or tissues infected with pathogens, cancer cells, or, in the case of autoimmunity or pathological inflammation, normal human cells or tissues.

[0257] The term "signal transduction pathway" or "signal transduction activity" refers to a biochemical cause-and-effect relationship, typically initiated by protein-protein interactions such as the binding of a growth factor to a receptor, that results in the transmission of a signal from one part of a cell to another. Typically, the transmission involves specific phosphorylation of one or more tyrosine, serine, or threonine residues on one or more proteins in a series of reactions that lead to signal transduction. The penultimate process typically involves nuclear events that result in changes in gene expression.

[0258] The terms "activity" or "biological activity", or the terms "biological property" or "biological characteristic" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize CD3 activity in vivo or in vitro, IC 50 , the in vivo stability of the antibody and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies well known in the art include, for example, cross-reactivity (i.e., usually with non-human homologs of the target peptide, or with cross-reactivity of other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics can be observed, measured or assessed using techniques well known in the art, including but not limited to ELISA, FACS or BIACORE plasma resonance analysis, unrestricted in vitro or in vivo neutralization assays, receptor binding, production and / or secretion of cytokines or growth factors, signal transduction, and immunohistochemistry of tissue sections from different sources (including humans, primates, or any other source).

[0259] The term "antibody" refers to any form of antibody having the desired biological activity. Therefore, it is used in the broadest sense and specifically includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, fully human antibodies, chimeric antibodies, and camelized single domain antibodies.

[0260] The term "isolated antibody" refers to the purified state of the binding compound, and in this case means that the molecule is substantially free of other biomolecules, such as nucleic acids, proteins, lipids, sugars, or other substances such as cell debris and growth medium. The term "isolated" does not imply the complete absence of such substances or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with experimental or therapeutic applications of the binding compounds described herein.

[0261] The term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic epitope. In contrast, conventional (polyclonal) antibody preparations typically include a large number of antibodies directed against (or specific for) different epitopes. The modifier "monoclonal" indicates the character of the antibody as being obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method.

[0262] The term "full-length antibody" refers to an immunoglobulin molecule that, when naturally present, comprises at least four peptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain: CL. The VH and VL regions can be further subdivided into highly variable complementarity determining regions (CDRs) separated by more conserved regions called framework regions (FRs). Each VH or VL region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domain that interacts with the antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.

[0263] The term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of an antibody, typically including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody that retains at least some of the binding specificity of the parent antibody. Examples of antibody binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments. When the binding activity to the antigen is expressed on a molar basis, the binding fragment or derivative typically retains at least 10% of its antigen-binding activity. Preferably, the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-binding affinity of the parent antibody. It is also contemplated that antigen-binding fragments of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as "conservative variants" or "function-conservative variants" of the antibody). The term "binding compound" refers to both antibodies and their binding fragments.

[0264] The term "single-chain Fv" or "scFv" antibody refers to an antibody fragment comprising the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide generally further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0265] The term "domain antibody" refers to an immunologically functional immunoglobulin fragment containing only the variable region of a heavy chain or a light chain. In some cases, two or more VH regions are covalently linked with a peptide linker to form a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0266] The term "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen-specificity. However, a bivalent antibody can be bispecific.

[0267] The term "diabody" refers to small antibody fragments with two antigen-binding sites, which contain a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL or VL-VH). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and generate two antigen-binding sites.

[0268] The term "murine antibody" or "hybridoma antibody" as used herein refers to a monoclonal antibody against human CD3 prepared according to the knowledge and skills in the art. A subject is injected with a CD3 antigen, and then a hybridoma expressing an antibody with the desired sequence or functional properties is isolated.

[0269] The term "chimeric antibody" refers to an antibody having the variable domains of a first antibody and the constant domains of a second antibody, wherein the first antibody and the second antibody are from different species. Typically, the variable domains are obtained from an antibody such as a rodent ("parent antibody"), while the constant domain sequences are obtained from a human antibody, such that the resulting chimeric antibody is less likely to induce an adverse immune response in a human subject than the parent rodent antibody.

[0270] The term "humanized antibody" refers to an antibody form containing sequences from both human and non-human (e.g., mouse, rat) antibodies. In general, a humanized antibody comprises substantially all of at least one, usually two, variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. Optionally, the humanized antibody may comprise at least a portion of a human immunoglobulin constant region (Fc).

[0271] The term "fully human antibody" refers to an antibody that contains only human immunoglobulin protein sequences. If produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "mouse antibody" refers to an antibody that contains only mouse immunoglobulin sequences. Alternatively, if produced in a rat, in a rat cell, or in a hybridoma derived from a rat cell, a fully human antibody may contain rat carbohydrate chains. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

[0272] An "isotype" antibody refers to the class of antibody provided by the heavy chain constant region genes (e.g., IgM, IgE, IgG such as IgG1, IgG2, or IgG4). Isotypes also include modified forms of one of these classes, where the modifications have been made to alter Fc function, for example to enhance or diminish effector function or binding to an Fc receptor.

[0273] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain that includes at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In some embodiments, the human IgG heavy chain Fc region extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present (the numbering in this paragraph is according to the EU numbering system, also known as the EU index, as in Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0274] The term "knob-into-hole structure" refers to the mutation of hydrophobic amino acids in the CH3 region of an antibody Fc. The side chain amino acids of one CH3 chain are mutated to form a larger hydrophobic amino acid (knob) to enhance hydrophobic interactions; the side chain amino acids of another CH3 chain are mutated to form a smaller amino acid (hole) to reduce steric hindrance. After the mutation, the CH3 with the knob and the CH3 with the hole interact hydrophobically to form a knob-into-hole structure (KiH), which facilitates the formation of heavy chain heterodimers. The KiH mutation primarily occurs in the internal hydrophobic amino acids of the CH3 domain's spatial structure, leaving the exposed amino acids largely unchanged, thus having no impact on the Fc's effector functions or immunogenicity.

[0275] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes are typically composed of various chemically active surface molecules such as amino acids or sugar side chains and generally have specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0276] The term "common light chain" refers to a light chain comprising the same light chain variable region and light chain constant region, which is capable of pairing with the heavy chain of a first antibody that binds to a first antigen to form a binding site that specifically binds to the first antigen, and is also capable of pairing with the heavy chain of a second antibody that binds to a second antigen to form a binding site that specifically binds to the second antigen. Furthermore, the light chain variable region of the common light chain forms a first antigen-binding site with the heavy chain variable region of the first antibody, and the light chain variable region of the common light chain forms a second antigen-binding site with the heavy chain variable region of the second antibody.

[0277] The term "Duobody" refers to a heterodimer comprising a heavy chain and a light chain from a first antibody and a heavy chain and a light chain from a second antibody paired therewith. Typically, a Duobody can be prepared by combining half of a first monospecific antibody comprising two heavy chains and two light chains with half of a second monospecific antibody comprising two heavy chains and two light chains. When two monospecific antibodies recognize different epitopes on different antigens, the resulting heterodimer is a bispecific antibody. In some embodiments, each monospecific antibody includes a heavy chain constant region with a single point mutation in the CH3 domain. These point mutations allow the resulting bispecific antibodies to have stronger interactions between the CH3 domains than between the CH3 domains in any monospecific antibody without the mutation. In some embodiments, the single point mutation in each monospecific antibody can be located at residues 366, 368, 370, 399, 405, 407, or 409 (according to EU numbering) in the CH3 domain of the heavy chain constant region. In some embodiments, the single point mutation is located at a different residue in one monospecific antibody relative to another monospecific antibody. For example, one monospecific antibody may comprise the mutation F405L (EU numbering), while another monospecific antibody may comprise the mutation K409R (EU numbering).

[0278] As used herein, the term "cross-reactivity" refers to binding to antigenic fragments of the same target molecule of human, monkey, and / or murine (mouse or rat) origin. Thus, "cross-reactivity" should be understood as interspecies reactivity with the same molecule X expressed in different species. The cross-reactivity specificity of monoclonal antibodies recognizing human CD3, monkey, and / or murine CD3 (mouse or rat) can be determined by FACS analysis.

[0279] "Affinity" or "binding affinity" refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D ) represents the equilibrium dissociation constant, which is the dissociation rate constant and the association rate constant (k dis and k on Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

[0280] The term "not binding" to a protein or cell means that the protein or cell does not bind to the protein or cell, or does not bind to the protein or cell with high affinity, i.e., the KD of the binding protein or cell is 1.0×10 -6 M or higher, more preferably 1.0×10 -5 M or higher, more preferably 1.0×10 -4 M or higher, 1.0×10-3 M or higher, more preferably 1.0×10 -2 M or higher.

[0281] The term "high affinity" for IgG antibodies refers to a KD of 1.0 × 10 -6 M or less, preferably 5.0×10 -8 M or less, more preferably 1.0×10 -8 M or lower, 5.0×10 -9 M or less, more preferably 1.0×10 -9 M or lower. For other antibody subtypes, “high affinity” binding may vary. For example, “high affinity” binding for the IgM subtype is defined as a KD of 10 -6 M or less, preferably 10 -7 M or less, more preferably 10 -8 M or lower.

[0282] The ability to "compete for binding" refers to the ability of an antibody or antigen-binding fragment to inhibit the binding interaction between two molecules (e.g., human CD3 and an anti-CD3 antibody) to any detectable extent (e.g., inhibition of at least 85%, or at least 90%, or at least 95%).

[0283] The terms "antibody-dependent cellular cytotoxicity," "antibody-dependent cell-mediated cytotoxicity," or "ADCC" refer to a cell-mediated immune defense in which immune system effector cells actively bind cell membrane surface antigens to antibodies.

[0284] The term "complement-dependent cytotoxicity" or "CDC" refers to the effector function of IgG and IgM antibodies, which, when bound to surface antigens, trigger the classic complement pathway, including formation of the membrane attack complex and target cell lysis.

[0285] The terms "nucleic acid," "polynucleotide," "nucleic acid molecule," and "polynucleotide molecule" refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless expressly limited otherwise, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing the mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0286] "Construct" refers to any recombinant polynucleotide molecule (such as a plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecule), derived from any source, capable of integrating with a genome or autonomously replicating, constituting a polynucleotide molecule in which one or more polynucleotide molecules have been linked (i.e., operably linked) in a functionally operable manner. Recombinant constructs will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences that direct transcription of the polynucleotide in a host cell. Both heterologous and non-heterologous (i.e., endogenous) promoters can be used to direct expression of the nucleic acids of the present invention.

[0287] "Vector" refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial origins of replication). After being introduced into the host cell, other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell and are therefore replicated together with the host genome. In addition, certain vectors are capable of directing the expression of operatively connected genes. Such vectors are referred to herein as "expression vectors."

[0288] As used herein, the term "expression vector" refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected, or transduced into a host cell. Expression vectors contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, to provide for amplification within the host.

[0289] "Activation," "stimulation," and "treatment" as applied to a cell or receptor may have the same meaning, e.g., a cell or receptor is activated, stimulated, or treated with a ligand, unless the context dictates otherwise or explicitly. "Ligand" includes natural and synthetic ligands, e.g., cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies. "Ligand" also includes small molecules, e.g., peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" may refer to cell activation regulated by internal mechanisms as well as external or environmental factors. "Response," e.g., a response of a cell, tissue, organ, or organism, includes a change in biochemical or physiological behavior (e.g., concentration, density, adhesion or migration within a biological compartment, rate of gene expression, or differentiation state), where the change is related to activation, stimulation, or treatment, or to internal mechanisms such as genetic programming.

[0290] As used herein, the terms "treatment" or "treating" of any disease or condition refer, in one embodiment, to ameliorating the disease or condition (i.e., slowing or preventing or reducing the progression of the disease or at least one of its clinical symptoms). In another embodiment, "treatment" or "treating" refers to alleviating or improving at least one physical parameter, including those physical parameters that may not be discerned by the patient. In another embodiment, "treatment" or "treating" refers to regulating the disease or condition physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of physical parameters), or both. Unless expressly described herein, methods for assessing the treatment and / or prevention of a disease are generally known in the art.

[0291] "Subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc. As used herein, the term "cyno" or "cynomolgus monkey" refers to a cynomolgus monkey.

[0292] Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in either order.

[0293] "Therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of a CD3 antibody or antigen-binding fragment thereof of the present invention that, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, is effective to prevent or ameliorate the symptoms of one or more diseases or conditions, or the progression of such diseases or conditions. A therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in amelioration of symptoms, e.g., to treat, cure, prevent, or ameliorate the relevant medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of the active ingredients that results in a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter by at least 10%, typically by at least 20%, preferably by at least about 30%, more preferably by at least 40%, and most preferably by at least 50%.

[0294] "Pharmaceutically acceptable carrier" refers to a component of a pharmaceutical preparation or composition other than the active ingredient that is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0295] The term "cancer" is used herein to refer to a group of cells that exhibit an abnormally high level of proliferation and growth. Cancer may be benign (also known as a benign tumor), pre-malignant, or malignant. Cancer cells may be solid cancer cells or leukemia cancer cells. The term "tumor" as used herein refers to one or more cells comprising a cancer. The term "tumor growth" is used herein to refer to the proliferation or growth of one or more cells comprising a cancer, which results in a corresponding increase in the size or extent of the cancer.

[0296] Advantageous Effects of the Invention

[0297] The present invention conducts in-depth analysis and comparison of multiple anti-human CD3 antibodies, optimizes sequences and performs humanized transformation to obtain humanized CD3 antibodies.

[0298] The humanized anti-CD3 antibody described in this invention exhibits enhanced tumor-killing activity and safety when used in the preparation of bispecific antibodies. This humanized sequence disclosed in this invention offers advantages and features over humanized sequences from other companies. When used in the preparation of new anti-tumor drugs, it exhibits enhanced tumor-killing activity and significantly improved physicochemical stability. Furthermore, it significantly reduces cytokine release, resulting in significantly improved safety, making it more suitable for the screening and development of anti-tumor drugs. This innovative achievement contributes to the beneficial effects and extremely high medical application value of this invention.

[0299] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only intended to illustrate the present invention and are not intended to limit the scope of the invention. Based on the following detailed description of the drawings and preferred embodiments, the various objects and advantages of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0300] Figure 1A-C: Binding experiments of anti-human CD3E antibodies to Jurkat cells.

[0301] Figure 2A-E: Detection of activation activity of Jurkat-NFAT cells by anti-human CD3E antibody.

[0302] Figure 3A-B: Binding experiments of anti-CD3 humanized antibodies on Jurkat cells.

[0303] Figure 4: Binding experiment of anti-CD3 humanized antibody to cynomolgus monkey CD3.

[0304] Figure 5A-E: Validation of the CD3xCD19 bispecific antibody in Duobody format and its in vitro killing experiment.

[0305] Figure 6A-F: Validation of the CD3xBCMA bispecific antibody in Duobody format and its in vitro killing assay.

[0306] Figure 7A-E: Cytokine release assay of CD3 bispecific antibodies in Duobody format.

[0307] 8A-B : In vitro binding experiments of CD3xBCMA common light chain bispecific antibodies.

[0308] Figure 9A-E: In vitro killing experiments of CD3xBCMA common light chain bispecific antibodies.

[0309] Figure 10: In vivo anti-tumor activity experiment of CD3xBCMA common light chain bispecific antibody.

[0310] Sequence information

[0311] Table 1: The information of the sequences involved in the present invention is described in the table below: DETAILED DESCRIPTION

[0312] The present invention includes all combinations of the specific embodiments described. Further embodiments of the present invention and the full scope of its applicability will become apparent from the detailed description provided below. However, it should be understood that although the detailed description and specific examples indicate preferred embodiments of the present invention, these descriptions and examples are provided by way of illustration only, as various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from this detailed description. All publications, patents, and patent applications cited herein, including citations, are incorporated herein by reference in their entirety for all purposes.

[0313] The antibodies of the present invention can be prepared by a variety of methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other methods, and equivalent replacement methods well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0314] The present invention is illustrated by the following examples, but is not intended to be limiting thereof. The present invention has been described in detail herein, and specific embodiments thereof are disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope of the invention.

[0315] Example 1: Production of anti-human CD3E monoclonal antibodies using the hybridoma method

[0316] Balb / C mice (purchased from Vital River) or Sprague-Dawley rats (purchased from Vital River) aged 6 to 8 weeks were immunized with either the heterodimeric protein CD3E&G (Uniprot database, P07766-1 & P09693) containing the extracellular domain of the recombinant human CD3 antigen protein, purchased from Acro Biosystems (Cat. No. CDG-H52W5) or a KLH-conjugated CD3E peptide (sequence: DGNEEMGGITQTPYKVSISGTTVILTC-KLH). The first immunization was performed with complete Freund's adjuvant at a dose of 50 μg of antigen per mouse (or rat). The second and subsequent immunizations were performed with incomplete Freund's adjuvant mixed with 25 μg of protein antigen per mouse (or rat). Mice were immunized on days 0, 14, 28, and 49.

[0317] After four immunizations, tail blood was collected from mice and the serum CD3E (Acro Biosystems, Catalog No. H5223) antibody titers were measured. Mice (or rats) with the highest serum CD3E antibody titers were selected, and their spleen cells were isolated and fused with mouse myeloma SP2 / 0 cells. After plating and culturing in HAT selection medium for 10-14 days, wells containing culture supernatants that positively bound to human CD3E were selected for subcloning. Single clones that maintained positive human CD3E binding after subcloning were selected and the variable region sequences of the anti-human CD3E antibodies were sequenced using Sanger sequencing. The sequence compositions of the light and heavy chain variable regions and CDRs (KABAT numbering convention) of the anti-human CD3E antibodies are shown in Table 2 below. For detailed amino acid sequences, please refer to Table 1.

[0318] Table 2: Sequence composition of the light and heavy chain variable regions of anti-human CD3E antibodies

[0319] Example 2: Jurkat binding assay of anti-human CD3E antibodies

[0320] The variable region sequence of the anti-human CD3E antibody was subcloned into the human IgG4 (SEQ ID NO: 63) / Kappa (SEQ ID NO: 62) backbone, expressed in the HEK293 system and purified by Protein A to obtain the anti-human CD3E human-mouse chimeric antibodies NP010-007, NP010-012, NP010-018, NP010-023, NP010-025 and NP010-030.

[0321] The binding of anti-human CD3E antibodies to Jurkat cells was determined by flow cytometry. The specific method is as follows: After washing Jurkat cells (Clone E6-1, from ATCC, catalog number TIB-152) twice with DPBS (Dulbecco's phosphate buffered saline), centrifuge at 1000 rpm and harvest the cells. The cell density was adjusted to 50,000 per well using FACS solution buffer (DPBS (Dulbecco's phosphate buffered saline) + 2% FBS (fetal bovine serum)). The chimeric antibody of the anti-human CD3E antibody with gradient dilution (starting concentration 15 μg / mL, 3-fold gradient dilution) was incubated with Jurkat cells on ice for 60 minutes. The cells were washed twice with pre-cooled FACS lysis buffer and then incubated with 1:1000 diluted PE (fluorescein) labeled anti-human IgG secondary antibody on ice for half an hour. After washing twice, resuspend and use Sartorius Fluorescence signals were detected by iQue3 flow cytometer.

[0322] SP34 is a positive control antibody (its heavy chain sequence is shown in SEQ ID NO: 66, and its light chain sequence is shown in SEQ ID NO: 67), expressed in-house at Junshi Biosciences. Figures 1A-C show the binding of anti-human CD3E antibodies to Jurkat cells.

[0323] Example 3: Detection of activation activity of anti-human CD3E antibodies on Jurkat-NFAT cells

[0324] This experiment uses the principle of inducing the expression of the NFAT reporter gene in the cell line Jurkat-NFAT-Luc under anti-CD3 antibody stimulation to detect the degree of CD3 antibody activation of T cells. 40 μl of Jurkat-NFAT cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) (10,000 cells per well) were added to a 96-well cell culture plate (Corning, Cat. No. 3917), followed by 40 μl of serially diluted CD3 chimeric antibodies (starting at 10 μg / ml, 3-fold dilution, a total of 8 concentration gradients) and incubated at 37°C for 6 hours. Afterwards, 50 μl of luciferase substrate One-Lite (Novozymes, DD1203-01) was added, mixed, incubated at room temperature for 6 minutes, and read using an M5 microplate reader (Molecular Devices). As shown in Figures 2A-E, different CD3 chimeric antibodies all activated Jurkat-NFAT cells to a certain extent.

[0325] Example 4: Humanization of anti-human CD3E antibody

[0326] The anti-human CD3E antibody NP010-018 was selected for humanization. The CDR grafting method used for humanization is briefly described as follows: the variable regions of the heavy and light chains of the murine antibody are compared with the germline sequences, and sequences with high similarity are selected as humanization templates for the heavy and light chains, respectively, for CDR grafting. Simultaneously, homology modeling is performed on the VH and VL regions of known structural antibodies with high homology to identify key amino acids in the framework region that maintain the binding activity of the murine antibody, from which reference sites for reversion mutations are selected.

[0327] By searching and comparing NP010-018 with the human germline library, based on the degree of similarity of the antibody sequences, the light chain IGKV1-33*01 and IGKV1-39*02 were selected as humanized templates, with back mutations at positions 43P, 53N, 55Q, and 87S in the light chain. The heavy chain IGHV3-15*01 and IGHV3-72*01 were humanized templates, with back mutations at positions 35Y, 49A, and 81V in the heavy chain.

[0328] The humanized light chains after CDR transplantation are NP010-Hz018-VL (IGKV1-33*01), NP010-Hz018-VL1 (IGKV1-33*01), NP010-Hz018-VL2 (IGKV1-33*01), NP010-Hz018-VL3 (IGKV1-33*01), NP010-Hz018-VL4 (IGKV1-33*01), NP010-Hz018-VL5 (IGKV1-39*02), and NP010-Hz018-VL6 (IGKV1-39*02). Their sequence compositions are shown in Table 3 below.

[0329] Table 3: Light chain sequence composition of humanized anti-human CD3E antibodies

[0330] The humanized heavy chains after CDR transplantation are NP010-Hz018-VH (IGHV3-15*01), NP010-Hz018-VH1 (IGHV3-15*01), NP010-Hz018-VH3 (IGHV3-72*01), NP010-Hz018-VH4 (IGHV3-72*01), and NP010-Hz018-VH5 (IGHV3-72*01), and their sequence compositions are shown in Table 3 below.

[0331] Table 4: Heavy chain sequence composition of humanized anti-human CD3E antibodies

[0332] The humanized antibody was named NP010-Hz018, with the following number representing the sequence number of the humanized antibody, such as NP010-Hz018-5, NP010-Hz018-8, and NP010-Hz018-14. The humanized antibody sequence was cloned into the human IgG4 (SEQ ID NO: 63) / kappa (SEQ ID NO: 62) backbone (sequence shown below), and the humanized antibody was expressed and purified in a HEK293 expression system.

[0333] The light and heavy chain pairings used in a series of antibodies generated after humanization of the NP010-018 chimeric antibody are shown in Table 5 below.

[0334] Table 5: Light and heavy chain (variable region) pairings of humanized antibodies

[0335] The light and heavy chain sequence compositions of the humanized antibodies are shown in Tables 6 and 7 below.

[0336] Table 6: Heavy chain sequence composition of humanized antibodies

[0337] Table 7: Light chain sequence composition of humanized antibodies

[0338] Example 5: Binding activity of anti-CD3 humanized antibodies on Jurkat cells

[0339] In a 96-well U-bottom cell culture plate, 100 μl of Jurkat cells (total number of cells: 5 × 10 4 ), then 100 μl of serially diluted CD3 chimeric and humanized monoclonal antibodies (starting at 15 μg / ml, with 5-fold dilutions, totaling eight concentrations) were added to a 96-well plate and incubated at 4°C for 30 minutes. Following incubation, the plate was washed twice with flow cytometry staining buffer (BD, Catalog No. 554658) to remove unbound antibody. Then, 50 μl of Alexa Fluor 488-labeled Goat Anti-Human IgG secondary antibody (Jackson Lab, Catalog No. 109-546-098, 1:1000 dilution) was added, and the cells were incubated at 4°C for 1 hour. Finally, the plate was washed twice with flow cytometry staining buffer, and the cells were resuspended in 100 μl of staining buffer and analyzed on a flow cytometer. Data were analyzed using FlowJo software. As shown in Figures 3A and 3B, a series of antibodies generated from humanized NP010-018 chimeric antibody exhibited varying degrees of binding activity to Jurkat cells. Among them, 18-14 had the strongest cell binding activity, and 18-8 had the weakest binding activity.

[0340] Example 6: Binding activity of anti-CD3 humanized antibodies to cynomolgus monkey CD3

[0341] CHO-Cyno CD3e cells (CHO cells were obtained from Beijing Zhongyuan Heju Economic and Trade Co., Ltd., and overexpressed cynomolgus monkey CD3e on the CHO cell surface) were incubated with different concentrations of CD3 humanized antibodies 18 and 18-5 and the control molecule Sp34 (starting at 20 μg / ml, 3-fold dilution, a total of 8 concentration gradients) at 4°C for 30 minutes, then washed and incubated with fluorescently labeled secondary antibodies at 4°C in the dark for 30 minutes. Finally, the plate was washed twice with flow cytometry staining solution, and the cells were resuspended in 100 μl of staining solution and detected on a flow cytometer. FlowJo software analyzed the raw data to obtain the MFI value, and GraphPad was used to fit the antibody dose-dependent binding curve. As shown in Figure 4, both 18 and 18-5 can bind to cynomolgus monkey CD3.

[0342] Example 7: Preparation of CD3xCD19 bispecific antibodies using the Duobody method and verification of their in vitro cytotoxicity

[0343] The present invention utilizes the Duobody in vitro recombination method (see Example 1 of patent CN103097417B for details) to prepare CD3xCD19 bispecific antibodies. 18 and 18-8 clones were selected at the CD3 end, and the CD19 end was selected from the blinatumumab bispecific antibody already on the market by Amgen (the sequence of the CD19-end antibody is as follows, wherein the heavy chain variable region sequence is SEQ ID NO: 70, the light chain variable region sequence is SEQ ID NO: 71, the heavy chain constant region sequence is SEQ ID NO: 72, the light chain constant region sequence is SEQ ID NO: 62, the heavy chain sequence is SEQ ID NO: 68, and the light chain sequence is SEQ ID NO: 69). This produced two bispecific antibodies, 18xblinatumumab and 18-8xblinatumumab.

[0344] Human peripheral blood mononuclear cells (PBMC, Stemexpress, Cat. No. PBMNC50C) were resuscitated and isolated using a total T cell purification kit (Miltenyi Biotec, Cat. No. 130-096-535). Purified total human T cells (100,000 cells per well) were then incubated with Nalm-6 cells (20,000 cells per well, ATCC, Cat. No. CRL-3273) pre-labeled with Cell-Trace violet (Invitrogen, Cat. No. C34557) and a serially diluted CD3xCD19 antibody (starting at 10 μg / ml, followed by 3-fold dilutions for a total of eight concentrations) in a 96-well plate at 37°C for 48 h. After incubation, cells were harvested and stained with FITC-labeled anti-CD4 (BD, Catalog No. 550628), BV711-labeled anti-CD8 (BD, Catalog No. 563677), APC-labeled anti-CD69 (BD, Catalog No. 555533), and APC-cy7-labeled anti-CD25 (BD, Catalog No. 557753) flow cytometry antibodies for 30 minutes at 4°C. After incubation, cells were washed with flow cytometry buffer and then labeled with PI (Sigma, Catalog No. P4864) at a final concentration of 1 μg / ml for dead cells. Flow cytometry was performed on a BD, Fortessa flow cytometer. FlowJo software was used to analyze the percentage of target cell Nalm-6 cytotoxicity and the activation of CD4+ and CD8+ T cells.

[0345] As shown in Figure 5A, after 48 hours of co-culture, the two CD3xCD19 dual antibodies in the present invention can mediate the killing of target cells, with a maximum killing ratio of nearly 90%. Compared to 18xblinatumumab, the killing activity of 18-8 dual antibodies is significantly weaker, and the strength of its killing activity is related to the T cell binding activity at the CD3 end. CD3xCD19 dual antibodies can effectively stimulate CD4+ and CD8+ T cells to upregulate membrane surface activation molecules CD25 and CD69, among which 18xblinatumumab has a stronger activation of T cells (Figure 5B-E). The above results show that the Duobody form CD3 dual antibodies of the present invention have moderate activation activity on T cells, which can reduce the side effects of cytokine storm while ensuring efficient killing.

[0346] Example 8: Preparation of CD3xBCMA bispecific antibodies using the Duobody method and verification of their in vitro killing activity

[0347] To validate the biological activity of the CD3 antibody, the present invention utilized Genmab's Duobody in vitro recombination method (see Example 1 of patent CN103097417B for details) to generate a CD3xBCMA bispecific antibody. Clone 18-8 was selected for CD3, with Sp34 used as a strong control. Junshi's own antibody, numbered BCMA-005, was selected for BCMA (heavy chain variable region sequence of SEQ ID NO: 79, heavy chain variable region HCDR1 sequence of SEQ ID NO: 73, heavy chain variable region HCDR2 sequence of SEQ ID NO: 74, heavy chain variable region HCDR3 sequence of SEQ ID NO: 75, light chain variable region sequence of SEQ ID NO: 80, light chain variable region LCDR1 sequence of SEQ ID NO: 76, light chain variable region LCDR2 sequence of SEQ ID NO: 77, light chain variable region LCDR3 sequence of SEQ ID NO: 79, heavy chain constant region sequence of SEQ ID NO: 72, light chain constant region sequence of SEQ ID NO: 62). This resulted in two bispecific antibodies, 18-8xBCMA-005 and sp34xBCMA-005.

[0348] Human peripheral blood mononuclear cells (PBMC, Stemexpress, Cat. No. PBMNC50C) were resuscitated and isolated and purified using a total T cell purification kit (Miltenyi Biotec, Cat. No. 130-096-535). Purified total human T cells (100,000 cells per well) were then incubated with NCI-H929 cells (obtained from ATCC, Cat. No. CRL-3580, 20,000 cells per well) pre-labeled with cell-trace violet (Invitrogen, Cat. No. C34557) and a serially diluted CD3xBCMA antibody (starting at 10 μg / ml, followed by 3-fold dilutions for a total of eight concentrations) in a 96-well plate at 37°C for 24 or 48 hours. Finally, the cells were collected and flow cytometry-stained with FITC-labeled anti-CD4 (BD, Catalog No. 550628), BV711-labeled anti-CD8 (BD, Catalog No. 563677), APC-labeled anti-CD69 (BD, Catalog No. 555533), and APC-cy7-labeled anti-CD25 (BD, Catalog No. 557753) for 30 minutes at 4°C. After incubation, the cells were washed with PBS and then labeled with PI stain (Sigma, Catalog No. P4864) at a final concentration of 1 μg / ml for dead cells. The cells were then analyzed on a flow cytometer (BD, Fortessa). FlowJo software was used to analyze the killing ratio of the target cell NCI-H929 and the activation of CD4+ and CD8+ T cells.

[0349] As shown in Figure 6A, after 24 hours of co-culture, 18-8xBCMA-005 of the present invention can mediate T cell killing of target cells NCI-H929, with a maximum killing ratio of 80%. Compared with the strong CD3 control molecule sp34xBCMA-005, the killing activity of 18-8xBCMA-005 is significantly weaker, EC 50 As shown in Figure 6B, after 48 h of co-culture, 18-8xBCMA-005 was able to mediate the same maximum killing ratio (>90%) as sp34xBCMA-005.

[0350] CD69 is a marker for the early stage of T cell activation, while CD25 is a marker for the late stage of T cell activation. By analyzing these two T cell activation markers, the degree of activation of T cells by CD3 dual antibodies can be obtained. As shown in Figures 6C and 6D, after 48 hours of co-culture with target cells, 18-8xBCMA-005 showed a dose-dependent upregulation of CD25 and CD69 expression on CD4+T cells. Similarly, for CD8+T cells, 18-8xBCMA-005 induced an upregulation of CD25 and CD69 expression (see Figures 6E and 6F). Compared with sp34xBCMA-005, the activation effect of the 18-8xBCMA-005 dual antibody of the present invention on T cells is significantly weaker. The above results show that the Duobody-form CD3 dual antibody of the present invention has moderate activation activity on T cells, which can reduce the side effects of cytokine storm while ensuring efficient killing.

[0351] Example 9: Cytokine Release by Duobody-Format CD3 Bispecific Antibodies

[0352] T cells were isolated and purified from PBMCs using a Miltenyi magnetic bead separation kit. T cells were then co-cultured with NCI-H929 target cells (50,000 and 10,000 cells per well, respectively) in a 96-well U-bottom plate at a 5:1 effector / target ratio. A serially diluted CD3xBCMA antibody (starting at 10 μg / ml, diluted three-fold over a total of eight concentrations) was added and incubated at 37°C for 24 hours. Cell culture supernatants were collected by centrifugation and cytokine analysis was performed using a CBA multifactor assay kit (BD, Cat. No. 551809). As shown in Figures 7A-E, compared with the sp34xBCMA-005 control molecule, 18-8xBCMA-005 in the present invention induced significantly lower levels of cytokine release, such as INF-γ, TNF-α, IL-2, IL-4 and IL-10. While achieving the same maximum killing ratio, 18-8xBCMA-005 has a significantly lower level of cytokine release, which may be safer in clinical practice.

[0353] Example 10: In vitro binding activity of common light chain CD3xBCMA bispecific antibodies

[0354] To prepare common light chain bispecific antibodies, the light chains of humanized CD3 molecules (18, 18-5, 18-8, 18-11, 18-12, 18-13, and 18-14) were paired with the heavy chain of an anti-BCMA antibody (BCMA-41) (whose sequence composition is shown in Table 8 below), resulting in seven anti-BCMA antibodies. The humanized CD3 molecules and anti-BCMA antibodies were recombined in vitro to ultimately produce seven common light chain CD3xBCMA bispecific antibodies. Since the light chains of 18 and 18-5 are identical, the light chains of 18-11 and 18-13 are identical, and the light chains of 18-12 and 18-14 are identical, one of these molecules was selected to test the binding activity of the common light chain CD3xBCMA bispecific antibody to the BCMA end.

[0355] Table 8: BCMA-41 heavy chain sequence composition

[0356] In a 96-well U-bottom cell culture plate, 50 μl of NCI-H929 cells (total cell number 1x10) that highly express BCMA molecules were added. 5), then added 50 μl of a serially diluted CD3xBCMA antibody (starting at 15 μg / ml, with 5-fold dilutions, for a total of eight concentrations) in a 96-well plate and incubated for 30 minutes at 4°C. Following incubation, the plate was washed twice with flow cytometry staining buffer (BD, Cat. No. 554658) to remove unbound antibody. Then, 50 μl of Alexa Fluor 488-labeled Goat Anti-Human IgG secondary antibody (Jackson Lab, Cat. No. 109-546-098, 1:1000 dilution) was added, and incubated at 4°C for 1 hour. Finally, the plate was washed twice with flow cytometry staining buffer, and the cells were resuspended in 150 μl of staining buffer and analyzed on a flow cytometer. Data were analyzed using FlowJo software. As shown in Figure 8A, after using the light chain of the CD3 antibody as the common light chain and preparing the CD3xBCMA common light chain bispecific antibody with the BCMA-41 antibody, its BCMA binding activity was well maintained, which was comparable to the binding activity of the bivalent BCMA-41 antibody.

[0357] Human peripheral blood mononuclear cells (PBMC, Stemexpress, catalog number PBMNC50C) were revived and total human T cells were isolated and purified using a total T cell purification kit (Miltenyi Biotec, catalog number 130-096-535). 50 μl of purified human T cells (1x10 per well) were then added to the culture medium. 5 Cells were added to a 96-well U-bottom cell culture plate. 50 μl of a serially diluted CD3xBCMA common light chain antibody (starting at 200 nM, with 3-fold dilutions, and a total of eight concentrations) was then added and incubated at 4°C for 30 minutes. Following incubation, the plate was washed twice with flow cytometry staining buffer (BD, Cat. No. 554658) to remove unbound antibody. 50 μl of PE-conjugated Goat Anti-Human IgG secondary antibody (Biolegend, Cat. No. 398004, 1:1000 dilution) was then added, and the cells were incubated at 4°C for 1 hour. Finally, the plate was washed twice with flow cytometry staining buffer, and the cells were resuspended in 150 μl of staining buffer and analyzed on a flow cytometer. Data were analyzed using FlowJo software. As shown in Figure 8B, compared with the marketed Teclistamab of Johnson & Johnson, the CD3xBCMA bispecific antibody of the present invention has relatively weak binding to T cells, and the binding strength is from strong to weak in the order of 18-14>18-12=18-13>18-11>18>18-5>18-8, which can effectively reduce the occurrence of cytokine storm.

[0358] Example 11: In vitro killing activity of common light chain CD3xBCMA bispecific antibody

[0359] Human peripheral blood mononuclear cells (PBMC, Stemexpress, Cat. No. PBMNC50C) were resuscitated and isolated using a total T cell purification kit (Miltenyi Biotec, Cat. No. 130-096-535). Purified total human T cells (100,000 cells per well) were then incubated with NCI-H929 cells (20,000 cells per well) pre-labeled with Cell-Trace violet (Invitrogen, Cat. No. C34557) and a serially diluted CD3xBCMA common light chain bispecific antibody (starting at 66.7 nM, followed by 3-fold dilutions over a total of eight concentrations) in a 96-well plate at 37°C for 24 hours. After incubation, cells were collected and stained with FITC-labeled anti-CD4 (BD, Catalog No. 550628), BV711-labeled anti-CD8 (BD, Catalog No. 563677), APC-labeled anti-CD69 (BD, Catalog No. 555533), and APC-cy7-labeled anti-CD25 (BD, Catalog No. 557753) flow cytometry antibodies for 30 minutes at 4°C. After incubation, cells were washed with flow cytometry buffer and then labeled with PI stain (Sigma, Catalog No. P4864) at a final concentration of 1 μg / ml for dead cells. The cells were analyzed on a flow cytometer (BD, Fortessa). FlowJo software was used to analyze the killing ratio of target cells NCI-H929 and the activation of CD4+ and CD8+ T cells.

[0360] As shown in Figure 9A, after 24 hours of co-culture, the CD3xBCMA common light chain bispecific antibody of the present invention was able to mediate killing of target NCI-H929 cells, with a maximum killing rate of nearly 90%. Compared with Teclistamab, the killing activity of the common light chain bispecific antibody was significantly weaker, and the strength of the killing activity was correlated with the T cell binding activity of the CD3 end. Among them, the bispecific antibody with the weakest activity was 18-8xBCMA, but it still achieved the same maximum killing rate after 24 hours under high concentration conditions.

[0361] The common light chain bispecific antibody can effectively stimulate CD4+ and CD8+ T cells to upregulate the membrane activation molecules CD25 and CD69. Among them, 18-8xBCMA has the weakest activation effect on T cells, and 18-14xBCMA has the strongest stimulation, but it is still weaker than the control molecule Teclistamab (Figure 9B-E). These results show that the common light chain CD3 bispecific antibody of the present invention has moderate activation activity on T cells, while ensuring efficient killing, it can reduce the side effects of cytokine storm.

[0362] Example 12: In vivo anti-tumor activity of CD3xBCMA common light chain bispecific antibody

[0363] Female immunodeficient NDG mice aged 6-8 weeks (purchased from Biocytogen) were injected with 5x10 6 NCI-H929-Luc cells (purchased from Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP30061L) were injected into the tail vein 13 days later. 7 PBMC cells (Stemexpress, Catalog No. PBMNC50C). Four days later, 3 mg of luciferase substrate D-luciferin potassium salt (Fubaike Bio, Catalog No. 12505) was injected into each mouse via the tail vein. Luciferase substrate signal was then detected on a small animal in vivo imaging device (Guangzhou Boluteng Biotechnology Co., Ltd., Model Aniview100) to determine tumor size. The mice were randomly divided into five groups based on tumor size, with five animals in each group.

[0364] G1: saline control group

[0365] G2: 18-8xBCMA41

[0366] G3: 18xBCMA41

[0367] G4: 18-14xBCMA41

[0368] G5: Teclistamab

[0369] The 18-8xBCMA41, 18xBCMA41, and 18-14xBCMA41 common light chain bispecific antibodies were injected into the tail vein at a dose of 10 μg per mouse, with Teclistamab used as the control antibody for this experiment. Thereafter, the drug was administered once every 5 days for a total of 4 doses. Tumor growth was assessed by tail vein injection of D-luciferin potassium salt (3 mg per animal), followed by detection of luciferase substrate signal on an AniView100 small animal in vivo imager. A higher signal value indicates faster tumor growth.

[0370] As shown in Figure 10, compared with the control antibody Teclistamab, the common light chain CD3xBCMA antibodies of the present invention all showed good anti-tumor efficacy, especially the two bispecific antibodies 18xBCMA41 and 18-14xBCMA41, with 4 and 3 tumors achieving or approaching complete tumor regression, respectively.

[0371] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and variations may be made to the details based on all the teachings disclosed, and that such modifications are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody or antigen-binding fragment thereof that can specifically bind to CD3, wherein the antibody or antigen-binding fragment thereof comprises: a HCDR1 with an amino acid sequence as shown in SEQ ID NO: 16, 54, 1, 9 or 29; a HCDR2 with an amino acid sequence as shown in SEQ ID NO: 55, 56, 17, 2, 10, 24, 30 or 37; a HCDR3 with an amino acid sequence as shown in SEQ ID NO: 18, 3, 11, 25, 31 or 38; a LCDR1 with an amino acid sequence as shown in SEQ ID NO: 42, 43, 20, 5, 13 or 33; a LCDR2 with an amino acid sequence as shown in SEQ ID NO: 21, 45, 46, 6, 14, 27, 34 or 44; and a LCDR3 with an amino acid sequence as shown in SEQ ID NO: 22, 7, 35 or 40.

2. An antibody or antigen-binding fragment thereof capable of specifically binding to CD3, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and / or a light chain variable region: The heavy chain variable region comprises: (I) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:16, SEQ ID NO:55 and SEQ ID NO:18, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:16, SEQ ID NO:55 and SEQ ID NO:18; (II) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:16, SEQ ID NO:56 and SEQ ID NO:18, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:16, SEQ ID NO:56 and SEQ ID NO:18; (III) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:54, SEQ ID NO:56 and SEQ ID NO:18, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:54, SEQ ID NO:56 and SEQ ID NO:18; (IV) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18; (V) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; (VI) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; (VII) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:24 and SEQ ID NO:25, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:24 and SEQ ID NO:25; (VIII) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31; or (IX) HCDR1, HCDR2 and HCDR3 with amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:37 and SEQ ID NO:38, respectively; or HCDR1, HCDR2 and HCDR3 with 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:37 and SEQ ID NO:38; The light chain variable region comprises: (I) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are as shown in SEQ ID NO:42, SEQ ID NO:21 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 whose amino acid sequences differ from those of SEQ ID NO:42, SEQ ID NO:21 and SEQ ID NO:22 by 1, 2 or 3 amino acids; (II) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 whose amino acid sequences differ from those of SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:22 by 1, 2 or 3 amino acids; (III) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 whose amino acid sequences differ from those of SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:22 by 1, 2 or 3 amino acids; (IV) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:43, SEQ ID NO:46 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 whose amino acid sequences differ from those of SEQ ID NO:43, SEQ ID NO:46 and SEQ ID NO:22 by 1, 2 or 3 amino acids; (V) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 whose amino acid sequences differ from those of SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22 by 1, 2 or 3 amino acids; (VI) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively; or LCDR1, LCDR2 and LCDR3 whose amino acid sequences differ from those of SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 by 1, 2 or 3 amino acids; (VII) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 7, respectively; or LCDR1, LCDR2 and LCDR3 whose amino acid sequences differ from those of SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 7 by 1, 2 or 3 amino acids; (VIII) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:13, SEQ ID NO:27 and SEQ ID NO:7, respectively; or LCDR1, LCDR2 and LCDR3 whose amino acid sequences differ from those of SEQ ID NO:13, SEQ ID NO:27 and SEQ ID NO:7 by 1, 2 or 3 amino acids; (IX) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are as shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, respectively; or LCDR1, LCDR2 and LCDR3 whose amino acid sequences differ from those of SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35 by 1, 2 or 3 amino acids; or (X) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:13, SEQ ID NO:6 and SEQ ID NO:40, respectively; or LCDR1, LCDR2 and LCDR3 whose amino acid sequences differ from those of SEQ ID NO:13, SEQ ID NO:6 and SEQ ID NO:40 by 1, 2 or 3 amino acids; Preferably, the antibody or antigen-binding fragment thereof comprises: (I) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO:16, SEQ ID NO:55 and SEQ ID NO:18, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:42, SEQ ID NO:21 and SEQ ID NO:22, respectively; (II) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:16, SEQ ID NO:56 and SEQ ID NO:18, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:22, respectively; (III) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:16, SEQ ID NO:56 and SEQ ID NO:18, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:43, SEQ ID NO:46 and SEQ ID NO:22, respectively; (IV) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:20, SEQ ID NO:21 and SEQ ID NO:22, respectively; (V) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively; (VI) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:14 and SEQ ID NO:7, respectively; (VII) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 having amino acid sequences as shown in SEQ ID NO:9, SEQ ID NO:24 and SEQ ID NO:25, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:27 and SEQ ID NO:7, respectively; (VIII) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in the amino acid sequences of SEQ ID NO:29, SEQ ID NO:30 and SEQ ID NO:31, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in the amino acid sequences of SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35, respectively; or (IX) a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:9, SEQ ID NO:37 and SEQ ID NO:38, respectively; and a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:13, SEQ ID NO:6 and SEQ ID NO:40, respectively; Preferably, the antibody or antigen-binding fragment thereof comprises: (I) a heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 19, 4, 12, 26, 32 or 39; and a light chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 23, 8, 15, 28, 36 or 41; or (II) a heavy chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 19, 4, 12, 26, 32 or 39; and a light chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 23, 8, 15, 28, 36 or 41; Further preferably, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region: (I) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:19, or comprises an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:19; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:23, or comprises an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:23; (II) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:4, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO:4; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:8, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as set forth in SEQ ID NO:8; (III) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:12, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:12; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:15, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:15; (IV) the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO:26, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:26; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:28, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:28; (V) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:32, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:32; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:36, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence as set forth in SEQ ID NO:36; or (VI) the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:39, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:39; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:41, or an amino acid sequence that has at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:

41.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises: (I) a heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 57, 58, 59, 60 or 61; and a light chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NOs: 47, 48, 49, 50, 51, 52 or 53; (II) a heavy chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 57, 58, 59, 60 or 61; and a light chain variable region comprising an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence shown in any one of SEQ ID NOs: 47, 48, 49, 50, 51, 52 or 53; or (III) a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 57 or 58 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO: 47; a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 58 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO: 51; a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 60 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO: 52 or 53; or a heavy chain variable region with an amino acid sequence as set forth in SEQ ID NO: 61 and a light chain variable region with an amino acid sequence as set forth in SEQ ID NO: 52 or 53.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody or a fully human antibody, and the antigen-binding fragment is selected from a Fab, Fab', F(ab')2, Fv, scFv or sdAb.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is of any IgG subtype, such as IgG1, IgG2, IgG3 or IgG4, preferably IgG4 subtype.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody comprises a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 63 and / or a light chain constant region with an amino acid sequence as shown in SEQ ID NO:

62.

7. A multispecific antibody comprising an antigen binding domain targeting CD3 and at least one antigen binding domain targeting other antigens, wherein the antigen binding domain targeting CD3 is selected from the antibody or antigen binding fragment thereof according to any one of claims 1 to 6, and the other antigen is selected from a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).

8. The multispecific antibody of claim 7, wherein the multispecific antibody is a bispecific antibody, a trispecific antibody or a tetraspecific antibody; preferably, the multispecific antibody is a bispecific antibody, which comprises the antigen binding domain targeting CD3 and the antigen binding domain targeting the tumor-associated antigen or tumor-specific antigen.

9. The multispecific antibody of any one of claims 7-8, wherein the tumor-associated antigen or tumor-specific antigen is selected from CD19, CD20, CD22, EGFR, HER2, HER3, PSMA, EpCAM, EphA2, CD30, CD33, CD38, CD79b, CD123, CLDN18.2, MSLN, GUCY2C, AFP, PAP, TROP2, LRRC15, gp100, 5T4, CEA, UPK2, DLL3, PRAME, CDH17, C DH19, GPA33, FAP, GPRC5D, GPC3, B7-H3, CLL-1, LIV-1, ACPP, CLDN6, PSCA, ENPP3, PRLR, MUC16, MUC17, CCR5, GD2, GD3, Ras, LewisY, BORIS, NY-ESO-1, OY-TES1, TSHR, LY6K, FLt3, IGFR-1, CAIX, c-MET, TROP2 or any combination thereof; preferably, the tumor-associated antigen is selected from CD19.

10. The multispecific antibody of any one of claims 7-8, comprising the antigen binding domain targeting CD3 and the antigen binding domain targeting CD19; the antigen binding domain targeting CD19 comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprises the sequence shown in SEQ ID NO: 70 or a variant thereof, and the light chain variable region comprises the sequence shown in SEQ ID NO: 71 or a variant thereof.

11. The multispecific antibody according to any one of claims 7 to 10, wherein the multispecific antibody is a bispecific antibody in the form of DuoBody, comprising: i) a peptide chain IA, which comprises the VL of the antigen binding domain targeting CD3 and the first light chain constant region (CL); preferably, the first light chain constant region is kappa; ii) a peptide chain IB comprising the VH of the antigen binding domain targeting CD3 and a first heavy chain constant region (CH); preferably, the first heavy chain constant region is IgG, such as IgG1, IgG2, IgG3 or IgG4; iii) a peptide chain IC comprising the VH of the antigen-binding domain targeting TAA or TSA and a second heavy chain constant region; preferably, the second heavy chain constant region is IgG, such as IgG1, IgG2, IgG3 or IgG4; and iv) a peptide chain ID comprising the VL of the antigen binding domain targeting TAA or TSA and a second light chain constant region; preferably, the second light chain constant region is kappa; Preferably, the Fc domains comprised by the peptide chains IB and IC respectively comprise modifications to promote dimerization of IB and IC; Preferably, the peptide chain IC comprises the VH sequence shown in SEQ ID NO: 70 or a variant thereof, and the peptide chain ID comprises the VL sequence shown in SEQ ID NO: 71 or a variant thereof; Preferably, the peptide chain IB comprises the VH sequence shown in SEQ ID NO: 58, 57, 60 or 61 or a variant thereof, and the peptide chain IA comprises the VL sequence shown in SEQ ID NO: 51, 47, 52 or 53 or a variant thereof; Preferably, the peptide chain IB comprises the VH sequence shown in SEQ ID NO: 58 or 57 or a variant thereof, and the peptide chain IA comprises the VL sequence shown in SEQ ID NO: 51 or 47 or a variant thereof.

12. The multispecific antibody according to any one of claims 7 to 8, wherein the antigen binding domain targeting CD3 comprises HCDR1, HCDR2, HCDR3 whose amino acid sequences are shown in SEQ ID NO: 16, SEQ ID NO: 56, and SEQ ID NO: 18, respectively, and LCDR1, LCDR2, LCDR3 whose amino acid sequences are shown in SEQ ID NO: 43, SEQ ID NO: 46, and SEQ ID NO: 22, respectively. The multispecific antibody according to claim 12 , which is a bispecific antibody in the form of a common light chain.

14. The multispecific antibody of claim 13, wherein the common light chain comprises LCDR1, LCDR2, LCDR3 having amino acid sequences as shown in SEQ ID NO:42, SEQ ID NO:21, and SEQ ID NO:22, respectively; or LCDR1, LCDR2, LCDR3 having amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:45, and SEQ ID NO:22, respectively; or LCDR1, LCDR2, LCDR3 having amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:46, and SEQ ID NO:22, respectively; preferably, the common light chain comprises LCDR1, LCDR2, LCDR3 having amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:46, and SEQ ID NO:22, respectively.

15. The multispecific antibody of claim 13 or 14, wherein the common light chain comprises the sequence shown in SEQ ID NO: 47, 51, 52 or 53.

16. The multispecific antibody according to any one of claims 7 to 9, 12 to 15, wherein the multispecific antibody is a common light chain bispecific antibody comprising: i) peptide chain II-A, which is a common light chain and comprises the VL and light chain constant region (CL) of the antigen binding domain targeting CD3; preferably, the light chain constant region is kappa; ii) peptide chain II-B, which comprises the VH of the antigen binding domain targeting CD3 and the first heavy chain constant region (CH); preferably, the first heavy chain constant region is IgG, such as IgG1, IgG2, IgG3 or IgG4; and iii) peptide chain II-C, which comprises the VH of the antigen-binding domain targeting TAA or TSA and a second CH; preferably, the second CH is IgG, such as IgG1, IgG2, IgG3 or IgG4; Preferably, the Fc domains comprised by the peptide chains II-B and II-C respectively comprise modifications to promote dimerization of II-B and II-C; Preferably, (1) the peptide chain II-A comprises the VL sequence shown in SEQ ID NO: 47 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 57 or a variant thereof; (2) the peptide chain II-A comprises the VL sequence shown in SEQ ID NO: 47 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 58 or a variant thereof; (3) the peptide chain II-A comprises the VL sequence shown in SEQ ID NO: 51 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 58 or a variant thereof; (4) the peptide chain II-A comprises the VL sequence shown in SEQ ID NO: 52 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 60 or a variant thereof; (5) the peptide chain II-A comprises the VL sequence shown in SEQ ID NO: 53 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 60 or a variant thereof; (6) the peptide chain II-A comprises the VL sequence shown in SEQ ID NO: 52 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 61 or a variant thereof; or (7) the peptide chain II-A comprises the VL sequence shown in SEQ ID NO: 53 or a variant thereof, and the peptide chain II-B comprises the VH sequence shown in SEQ ID NO: 61 or a variant thereof; The variant of any one of (1) to (7) has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived; More preferably, the peptide chain II-C comprises the VH sequence shown in SEQ ID NO: 70 or a variant thereof; the variant has at least 95%, 96%, 97%, 98% or 99% sequence identity to the sequence from which it is derived.

17. An isolated nucleic acid molecule encoding: - the antibody or antigen-binding fragment thereof, or the heavy chain variable region and / or light chain variable region thereof according to any one of claims 1 to 6; or - The multispecific antibody or polypeptide chain thereof according to any one of claims 7 to 16.

18. A vector comprising the isolated nucleic acid molecule of claim 17.

19. A host cell comprising the isolated nucleic acid molecule of claim 17 or the vector of claim 18.

20. A method for preparing an antibody or an antigen-binding fragment thereof, or a multispecific antibody, comprising culturing the host cell of claim 19 under conditions that allow protein expression, and recollecting the antibody or an antigen-binding fragment thereof, or the multispecific antibody from the culture of the cultured host cell.

21. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the multispecific antibody according to any one of claims 7 to 16, the isolated nucleic acid molecule according to claim 17, the vector according to claim 18, or the host cell according to claim 19, and a pharmaceutically acceptable carrier and / or excipient.

22. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the multispecific antibody according to any one of claims 7 to 16, the isolated nucleic acid molecule according to claim 17, the vector according to claim 18, the host cell according to claim 19, or the pharmaceutical composition according to claim 21 in the preparation of a medicament for preventing and / or treating a disease.

23. A method for preventing and / or treating a disease, comprising administering to a subject in need thereof an effective dose of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the multispecific antibody according to any one of claims 7 to 16, the isolated nucleic acid molecule according to claim 17, the vector according to claim 18, or the host cell according to claim 19, or the pharmaceutical composition according to claim 21.

24. The use according to claim 22 or the method according to claim 23, wherein The disease is a tumor, an inflammatory disease or an autoimmune disease; preferably, the tumor is selected from ovarian cancer, breast cancer, gastric cancer, bile duct cancer, colorectal cancer, lung cancer, acute myeloid leukemia, chronic lymphocytic leukemia, melanoma or colorectal cancer.