WO2025131075A1 - Anti-cd3 and anti-cd3 multispecific antibodies, and use - Google Patents

Abstract

Provided are an antibody specifically binding to human CD3 or an antigen-binding fragment thereof, and a multispecific antibody targeting CD3 and other antigens (e.g. tumor-associated antigens). Also provided are nucleic acid molecules encoding the antibodies, vectors, and host cells for expressing the antibodies, and treatment and diagnosis methods and uses of the antibodies or the antigen-binding fragment thereof.

Description

Anti-CD3 and anti-CD3 multispecific antibodies and uses thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese patent application 202311778073.9 filed on December 21, 2023, the entire contents of which are incorporated herein by reference in their entirety.

Technical Field

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

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 T cell receptor (T cell receptor, TCR), CD3 and CD28. TCR is responsible for receiving stimulation from antigens, but lacks the functional structure to transmit signals into the cell. TCR needs to form a complex (TCR/CD3 complex) with CD3 through non-covalent binding to complete the activation process of T cells. The intracellular region of the CD3 molecule has an immunoreceptor tyrosine-based activation motif (ITAM), which can mediate the activation, proliferation and survival of T cells.

Bispecific antibodies (BsAb) are a class of bifunctional antibodies that can specifically bind to two antigens or different epitopes of the same antigen. Bispecific T-cell engagers (BiTE) are representative non-IgG-like bispecific antibodies, which are formed by connecting two single-chain variable fragments (scFv) fragments that recognize T cell CD3 molecules and target cell antigens. They recruit T cells near tumor tissues, form immune synapses between tumor cells and T cells, and mediate T cell killing of tumor cells. Based on this drug design concept, Amgen of the United States 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.

Although CD3 bispecific antibodies have shown very good therapeutic effects on blood tumors, the incidence of cytokine storm (CRS) is too high, and sometimes even a serious side effect. The high incidence of CRS is related to the excessive activity of CD3 antibodies, so it is necessary to choose a CD3 antibody of appropriate strength for the development of bispecific antibodies.

Summary of the invention

The purpose of the present application is to provide an antibody or an antigen-binding fragment thereof that can specifically bind to CD3. Based on this, a multispecific antibody targeting CD3 and another antigen (such as CD19) is further provided, an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof or multispecific antibody of the present application, an expression vector and a host cell for expressing the antibody or antigen-binding fragment thereof or multispecific antibody of the present application, and the use of the antibody or antigen-binding fragment thereof or multispecific antibody of the present application.

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.

In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to CD3, wherein the antibody or the 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.

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:

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, respectively;

(II) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 16, SEQ ID NO: 56 and SEQ ID NO: 18, respectively; or HCDR1, HCDR2 and HCDR3 whose amino acid sequences differ from those of SEQ ID NO: 16, SEQ ID NO: 56 and SEQ ID NO: 18 by 1, 2 or 3 amino acids;

(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 whose amino acid sequences are shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; or HCDR1, HCDR2 and HCDR3 whose amino acid sequences differ from those of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 by 1, 2 or 3 amino acids;

(V) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; or HCDR1, HCDR2 and HCDR3 having 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 having 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 having 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, respectively;

(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 having 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 having 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 having amino acid sequences as shown in SEQ ID NO:42, SEQ ID NO:21 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:42, SEQ ID NO:21 and SEQ ID NO:22;

(II) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are as 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 having amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:22;

(IV) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are as 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 as 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 having amino acid sequences as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7;

(VII) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 7, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 7;

(VIII) 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; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:27 and SEQ ID NO:7;

(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 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35; 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 having 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:6 and SEQ ID NO:40.

In some embodiments, the antibody or antigen-binding fragment thereof of the present invention 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 having amino acid sequences 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 having amino acid sequences 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 having amino acid sequences 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 having amino acid sequences 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 with amino acid sequences 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 with amino acid sequences 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 with amino acid sequences 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 with amino acid sequences 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 with amino acid sequences as shown in 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 with amino acid sequences as shown in 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 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.

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

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

(I) a heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NO: 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 NO: 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 NO: 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 NO: 23, 8, 15, 28, 36 or 41.

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:

(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 having 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 having 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 shown in SEQ ID NO:12, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:12; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:15, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:15;

(IV) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:26, 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:26; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:28, 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:28;

(V) the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:32, or comprises 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:32; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:36, or comprises 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:36; or

(VI) the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:39, or comprises an amino acid sequence having 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 comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:41.

In some embodiments, the antibodies described herein are humanized antibodies.

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

(I) a heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NO: 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 NO: 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 NO: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 NO:47, 48, 49, 50, 51, 52 or 53; or

(III) a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 57 or 58 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO: 47;

The heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 58 and the light chain variable region having an amino acid sequence as shown in SEQ ID NO: 51;

The heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 60 and the light chain variable region having an amino acid sequence as shown in SEQ ID NO: 52 or 53; or

The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 61 and the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 52 or 53.

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.

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

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.

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

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 other antigens, 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).

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

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.

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.

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

In some embodiments, the multispecific antibody described in 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.

In some embodiments, 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.

In some embodiments, the multispecific antibody of the present invention is a bispecific antibody in the form of a common light chain. In some embodiments, the common light chain comprises LCDR1, LCDR2, LCDR3 with amino acid sequences as shown in SEQ ID NO: 42, SEQ ID NO: 21, SEQ ID NO: 22, respectively; or LCDR1, LCDR2, LCDR3 with amino acid sequences as shown in SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 22, respectively; or LCDR1, LCDR2, LCDR3 with amino acid sequences as shown in SEQ ID NO: 43, SEQ ID NO: 46, 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, SEQ ID NO: 22, respectively. In some embodiments, the common light chain comprises the sequence shown in SEQ ID NO: 47, 51, 52 or 53, for example, comprises the sequence shown in SEQ ID NO: 53.

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

i) peptide chain I-A, 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) peptide chain I-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;

iii) a peptide chain I-C 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 I-D, which comprises 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.

In some embodiments, the Fc domains contained in the peptide chains I-B and I-C respectively contain modifications (e.g., knob-into-hole modifications) to promote dimerization of I-B and I-C.

In some embodiments, the peptide chain I-C comprises the VH sequence shown in SEQ ID NO: 70 or a variant thereof, and the peptide chain I-D 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.

In some embodiments, the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 58, 57, 60 or 61 or a variant thereof, and the peptide chain I-A 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.

In some embodiments, the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 58 or 57 or a variant thereof, and the peptide chain I-A 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.

In some embodiments, the multispecific antibody of the present invention 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.

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.

In some embodiments, in the common light chain bispecific antibody: (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; or (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; or (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; or (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: 53 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; or (1) 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; or (2) 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 variants in any one of (1) to (7) have at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which they are derived.

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.

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.

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

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

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.

In another aspect, the present invention provides a method for preparing the antibody or antigen-binding fragment thereof, or the multispecific antibody described herein, the method comprising culturing the host cell described herein under conditions allowing protein expression, and recollecting the antibody or antigen-binding fragment thereof, or the multispecific antibody from the culture of the cultured host cell.

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

In yet 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.

In yet 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 the antibody or antigen-binding fragment thereof described herein, the multispecific antibody described herein, the isolated nucleic acid molecule described herein, the vector described herein, or the host cell described herein, or the pharmaceutical composition described herein.

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.

DETAILED DESCRIPTION OF THE INVENTION

CD3 binding part

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

In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to CD3, wherein the antibody or the 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.

In some embodiments, the present invention provides an antibody or an antigen-binding fragment thereof that can specifically bind to CD3, wherein the antibody or the 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, respectively;

(II) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 16, SEQ ID NO: 56 and SEQ ID NO: 18, respectively; or HCDR1, HCDR2 and HCDR3 whose amino acid sequences differ from those of SEQ ID NO: 16, SEQ ID NO: 56 and SEQ ID NO: 18 by 1, 2 or 3 amino acids;

(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 whose amino acid sequences are shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; or HCDR1, HCDR2 and HCDR3 whose amino acid sequences differ from those of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 by 1, 2 or 3 amino acids;

(V) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3, respectively; or HCDR1, HCDR2 and HCDR3 having 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 having 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 having 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, respectively;

(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 having 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 having 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, respectively;

The light chain variable region comprises:

(I) 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; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:42, SEQ ID NO:21 and SEQ ID NO:22;

(II) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are as 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 having amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:22;

(IV) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are as 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 as 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 having amino acid sequences as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7;

(VII) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 7, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 7;

(VIII) 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; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:27 and SEQ ID NO:7;

(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 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35; 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 having 1, 2 or 3 amino acid differences from the amino acid sequences shown in SEQ ID NO:13, SEQ ID NO:6 and SEQ ID NO:40.

In some embodiments, the antibody or antigen-binding fragment thereof of the present invention 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 having amino acid sequences 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 having amino acid sequences 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 having amino acid sequences 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 having amino acid sequences 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 with amino acid sequences 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 with amino acid sequences 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 with amino acid sequences 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 with amino acid sequences 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 with amino acid sequences as shown in 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 with amino acid sequences as shown in 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 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.

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

(I) a heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NO: 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 NO: 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 NO: 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 NO: 23, 8, 15, 28, 36 or 41.

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:

(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 having 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 having 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 shown in SEQ ID NO:12, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:12; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:15, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:15;

(IV) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:26, 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:26; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:28, 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:28;

(V) the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:32, or comprises 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:32; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:36, or comprises 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:36; or

(VI) the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:39, or comprises an amino acid sequence having 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 comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:41.

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

A heavy chain variable region comprising an amino acid sequence as shown in any one of SEQ ID NO: 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 NO:47, 48, 49, 50, 51, 52 or 53.

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

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 NO: 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 NO:47, 48, 49, 50, 51, 52 or 53.

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

The heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 57 or 58 and the light chain variable region having an amino acid sequence as shown in SEQ ID NO: 47;

The heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 58 and the light chain variable region having an amino acid sequence as shown in SEQ ID NO: 51;

The heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 60 and the light chain variable region having an amino acid sequence as shown in SEQ ID NO: 52 or 53; or

The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 61 and the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 52 or 53.

In some embodiments, the antibodies or antigen-binding fragments thereof described in the present invention 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 compared with their CDR or variable region sequences is only the deletion, addition or substitution of one or several amino acids.

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

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.

In some embodiments, the antibody molecules of the present invention are humanized. Different methods for humanizing antibodies are known to the skilled person, as reviewed by Almagro & Fransson, the contents of which are incorporated herein by reference in their entirety (Almagro JC and Fransson J (2008) Frontiers in Bioscience 13: 1619-1633). In some embodiments, the antibodies of the present invention comprise a heavy chain framework region derived from a human immunoglobulin (e.g., a heavy chain framework region contained in an amino acid sequence encoded by a human heavy chain germline gene), and/or a light chain framework region (e.g., a light chain framework region contained in an amino acid sequence encoded by a human light chain germline gene). The heavy chain framework region and/or the light chain framework region optionally comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) back mutations from human residues to mouse residues.

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.

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.

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)), which are based on the three-dimensional structure of the antibody and the topology of the CDR loops; and Kabat (Kabat et al., Sequences of Proteins of Immunoglobulins), which are based on the variability of antibody sequences. logical Interest, 4th edition, U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), international ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and North CDR definition based on affinity propagation clustering using a large number of crystal structures.

Unless otherwise indicated, the CDR of antibodies 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 antibodies of the present invention is defined by Kabat, Chothia, AbM, Contact, North or IMGT. In some embodiments, the CDR of antibodies of the present invention is defined by Kabat.

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 are different. Therefore, when it comes to defining antibodies using 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).

Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs. However, although CDRs are different between antibodies, only a limited number of amino acid positions in CDRs 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 given 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.

The humanized antibodies of the present invention can be prepared by inserting mouse CDR regions into human germline framework regions using methods known in the art, such as Winter et al. U.S. Pat. No. 5,225,539 and Queen et al. U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370.

In some embodiments, the amino acid difference comprises an amino acid deletion, insertion or substitution. 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 deletion, insertion or substitution, but are still at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the above 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 by amino acid deletion, insertion or substitution 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 by amino acid deletion, insertion or substitution when compared to the framework regions in the specific sequences.

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 region depicted in the sequence described above.

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.

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.

In some embodiments, it may be desirable to generate cysteine engineered antibodies, such as "thioMAbs," in which one or more residues of an antibody are replaced with cysteine residues.

In some embodiments, the antibodies provided herein can be further modified to contain other non-protein 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.

Reference Antibodies

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

In some embodiments, the reference antibody blinatumumab is selected from the heavy chain shown in SEQ ID NO: 68 and the light chain shown in SEQ ID NO: 69.

Multispecific antibodies and tumor-associated antigen (TAA) or tumor-specific antigen (TSA) binding moieties

The present invention provides a multispecific antibody, which comprises 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 antibodies or antigen binding fragments thereof described herein, and the other antigen is selected from tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs).

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

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.

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.

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

In some embodiments, the multispecific antibody described in 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.

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

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

In some embodiments, the common light chain of such multispecific antibodies comprises LCDR1, LCDR2, LCDR3 whose amino acid sequences are shown in SEQ ID NO: 42, SEQ ID NO: 21, and SEQ ID NO: 22, respectively; or LCDR1, LCDR2, 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, LCDR3 whose amino acid sequences are 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 whose amino acid sequences are shown in SEQ ID NO: 43, SEQ ID NO: 46, and SEQ ID NO: 22, respectively.

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

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

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

In some embodiments, the multispecific antibody of the present invention further comprises an Fc domain. In some embodiments, the Fc domain is IgG, such as IgG1, IgG2, IgG3 or IgG4. In some embodiments, the Fc domain comprises a modification to promote dimerization of the first Fc monomer and the second Fc monomer.

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 arm. 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.

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.

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

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

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

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

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

(iii) a peptide chain I-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; and

(iv) a peptide chain I-D, which comprises the VL of the antigen-binding domain targeting TAA or TSA and a second CL; preferably, the second CL is kappa.

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

In some embodiments, the Fc domains contained in the peptide chains I-B and I-C contain mutations that promote Fab arm exchange, such as a K409R mutation in the CH3 region of one of the Fc domains and a F405L mutation in the CH3 region of the other Fc domain. In some embodiments, the peptide chains I-B and I-C contain the heavy chain constant region sequences shown in SEQ ID NOs: 63 and 72, respectively. In some embodiments, the peptide chains I-B and I-C contain the heavy chain constant region sequences shown in SEQ ID NOs: 72 and 63, respectively.

In some embodiments, the peptide chains I-A and I-D contain the light chain constant region sequence shown in SEQ ID NO:62.

In some embodiments, the antigen binding domain targeting TAA or TSA is an antigen binding domain targeting CD19, the peptide chain I-C comprises the VH sequence shown in SEQ ID NO: 70 or a variant thereof, and the peptide chain I-D 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.

In some embodiments, the peptide chain I-A comprises LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:42, SEQ ID NO:21 and SEQ ID NO:22, respectively, and the peptide chain I-B comprises HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:16, SEQ ID NO:55 and SEQ ID NO:18, respectively.

In some embodiments, the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 58, 57, 60 or 61 or a variant thereof, and the peptide chain I-A 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.

In some embodiments, the peptide chain I-A comprises the VL sequence shown in SEQ ID NO: 51 or 47 or a variant thereof; the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 58 or 57 or a variant thereof. In some embodiments, the peptide chain I-A comprises the VL sequence shown in SEQ ID NO: 51 or a variant thereof; the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 58 or a variant thereof. In some embodiments, the peptide chain I-A comprises the VL sequence shown in SEQ ID NO: 47 or a variant thereof; the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 57 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.

In some embodiments, the peptide chain I-C comprises the VH sequence shown in SEQ ID NO: 70, the peptide chain I-D comprises the VL sequence shown in SEQ ID NO: 71, the peptide chain I-A comprises the VL sequence shown in SEQ ID NO: 51, and the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 58.

In some embodiments, the peptide chain I-C comprises the VH sequence shown in SEQ ID NO: 70, the peptide chain I-D comprises the VL sequence shown in SEQ ID NO: 71, the peptide chain I-A comprises the VL sequence shown in SEQ ID NO: 47, and the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 57.

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

(1) the peptide chain I-A comprises the light chain sequence shown in SEQ ID NO:65; the peptide chain I-B comprises the heavy chain sequence shown in SEQ ID NO:64; the peptide chain I-C comprises the heavy chain sequence shown in SEQ ID NO:68; the peptide chain I-D comprises the light chain sequence shown in SEQ ID NO:69; or,

(2) The peptide chain I-A comprises the light chain sequence shown in SEQ ID NO:84; the peptide chain I-B comprises the heavy chain sequence shown in SEQ ID NO:83; the peptide chain I-C comprises the heavy chain sequence shown in SEQ ID NO:68; and the peptide chain I-D comprises the light chain sequence shown in SEQ ID NO:69.

In some embodiments, the multispecific antibody of the present invention 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 CL 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 CH is IgG, such as IgG1, IgG2, IgG3 or IgG4; and

(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;

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.

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:

(I) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO:16, SEQ ID NO:55 and SEQ ID NO:18, respectively, and LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:42, SEQ ID NO:21 and SEQ ID NO:22, respectively;

(II) HCDR1, HCDR2 and 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 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:22, respectively; or

(III) HCDR1, HCDR2 and 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 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 43, SEQ ID NO: 46 and SEQ ID NO: 22 respectively.

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 whose amino acid sequences are shown in SEQ ID NO: 16, SEQ ID NO: 56, and SEQ ID NO: 18, 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.

In some embodiments, peptide chain II-A of the common light chain bispecific antibody comprises a VL sequence shown in SEQ ID NO: 47, 51, 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. In some embodiments, peptide chain II-B of the common light chain bispecific antibody comprises a VH sequence shown in SEQ ID NO: 57, 58, 60 or 61 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.

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 described in any of (1) to (7) has at least 95%, 96%, 97%, 98% or 99% sequence identity compared to the sequence from which it is derived.

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 contain: the VH sequence shown in SEQ ID NO: 61, and the VL sequence shown in SEQ ID NO: 53.

In some embodiments, peptide chain II-C of the common light chain bispecific antibody comprises a VH of an antigen binding domain targeting CD19. In some embodiments, VH of peptide chain II-C of the common light chain bispecific antibody comprises a sequence shown 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.

In some embodiments, the Fc domains contained in peptide chains II-B and II-C of the common light chain bispecific antibody optionally contain modifications to promote dimerization of I-B and I-C, respectively.

In some embodiments, the Fc domains contained in peptide chains II-B and II-C contain a K409R mutation in the CH3 region of one of the Fc domains and a F405L mutation in the CH3 region of the other Fc domain. In some embodiments, the peptide chains II-B and II-C contain the heavy chain constant region sequences shown in SEQ ID NOs: 63 and 72, respectively. In some embodiments, the peptide chains II-B and II-C contain the heavy chain constant region sequences shown in SEQ ID NOs: 72 and 63, respectively.

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

Antibody expression

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.

In yet another aspect, the present invention provides a vector (e.g., an expression vector) comprising an isolated nucleic acid molecule as described herein, preferably, the vector is 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).

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.

In one embodiment, the invention provides a method for preparing an antibody of the invention, wherein the method comprises, when the expression vector is introduced into a mammalian host cell, producing the antibody by culturing the host cell for a sufficient period of time to allow the antibody to be expressed in the host cell, or more preferably, the antibody to be secreted into the culture medium in which the host cell is grown. The antibody can be recovered from the culture medium using standard protein purification methods.

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 include Chinese hamster ovary (CHO) cells, NS0, 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 a high expression level.

It is likely that antibodies expressed by different cell lines or expressed in transgenic animals 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 unlikely to be immunogenic because their sugar structures are normal components of native human serum IgG.

Pharmaceutical compositions and pharmaceutical preparations

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

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

Pharmaceutical preparations containing the antibodies described herein can be prepared by mixing the antibodies of the present invention having the desired purity with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of an aqueous solution or a lyophilized preparation.

Medical uses and treatments

Any antibody provided herein can be used in a method of treatment. 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 a therapeutically effective amount or a prophylactically effective amount in a method of treatment or prevention described in any embodiment of the present invention.

In yet 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.

In yet 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 the antibody or antigen-binding fragment thereof described herein, the multispecific antibody described herein, the isolated nucleic acid molecule described herein, the vector described herein, or the host cell described herein, or the pharmaceutical composition described herein.

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.

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, etc.

Definition of terms

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular biology (including recombinant technology), microbiology, cell biology, biochemistry, nucleic acid chemistry, immunology and other operating steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

In order to make the present invention more easily understood, certain scientific and technical terms are specifically defined as follows. Unless otherwise expressly defined in other parts of this document, the scientific and technical terms used in this document have the meanings commonly understood by ordinary technicians in the field to which the present invention belongs. Regarding the definitions and terms in this field, professionals can specifically refer to Current Protocols in Molecular Biology (Ausubel). The abbreviations of 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 expressly provided in the text.

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 that is 5% less than the specified numerical value and an upper limit that is 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.

The term "and/or" should be understood to mean any one of the optional items or a combination of any two or more of the optional items.

The term "CD3" refers to a part of the T cell receptor complex, which is composed of three different chains CD3ε, CD3δ and CD3γ. The concentration of CD3 on T cells, such as by the fixation of anti-CD3 antibodies, leads to the activation of T cells, similar to the activation mediated by the T cell receptor, 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 specified. The term encompasses "full-length" unprocessed CD3 and any form of CD3 or any fragment thereof produced by intracellular processing. The term also includes variants of naturally occurring CD3, such as 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 lacking signal peptides). In a preferred embodiment, CD3 refers to the full length or fragments thereof from mice/rat (such as mature fragments lacking signal peptides).

The term "CD19" refers to the Cluster of Differentiation 19 protein (CD19), which is an antigenic determinant detectable on leukemic 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 No. P15391, and the nucleotide sequence encoding human CD19 can be found in Accession No. NM_001178098. CD19 is expressed on most B-lineage cancers, such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma.

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, that is, 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 terms are clearly indicated to the contrary, such as "only one" or "exactly one" or when "consisting of..." is used in the claims, it will refer to only one number listed or one element of the list.

As used herein, the words "a" and "an" should be understood to mean "at least one" unless expressly stated to the contrary.

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 the amino acid sequences are aligned (and gaps introduced where necessary) to obtain the maximum percent sequence identity, and any conservative substitutions are not considered part of the sequence identity. Sequence alignments can be performed using various methods in the art to determine percent amino acid sequence identity, 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 alignments, including any algorithm required to achieve maximum alignment over the entire length of the compared sequences.

The term "immune response" refers to the actions of, for example, lymphocytes, antigen presenting cells, phagocytes, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines and complement), which result in the selective damage, destruction or elimination from the human 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.

The term "signal transduction pathway" or "signal transduction activity" refers to a biochemical cause-effect relationship, usually 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 part of the cell. 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 usually involves nuclear events, resulting in changes in gene expression.

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, _IC50 , the in vivo stability of the antibody, and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the targeted peptide, or with other proteins or tissues in general), and the ability to maintain high expression levels of the protein in mammalian cells. The aforementioned properties or characteristics are observed, measured or evaluated using techniques known in the art, including but not limited to ELISA, FACS or BIACORE plasma resonance analysis, in vitro or in vivo neutralization assays, receptor binding, cytokine or growth factor production and/or secretion, signal transduction, and immunohistochemistry of tissue sections from different sources (including human, primate or any other source).

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

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 the experimental or therapeutic use of the binding compound described herein.

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.

The term "full-length antibody" refers to an immunoglobulin molecule that contains at least four peptide chains when naturally present: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists 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 consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into highly variable complementary determining regions (CDRs) and regions separated by more conservative framework regions (FRs). Each VH or VL region consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. 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.

The term "antigen-binding fragment" of an antibody ("parent antibody") includes fragments or derivatives of an antibody, typically including at least one fragment of an antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody, which 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 of the antigen is expressed on a molar concentration basis, the binding fragment or derivative generally 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 the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly change its biological activity (referred to as "conservative variants" or "functional conservative variants" of antibodies). The term "binding compound" refers to both antibodies and their binding fragments.

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 also comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

The term "domain antibody" is an immunologically functional immunoglobulin fragment containing only the variable region of the heavy chain or the variable region of the 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.

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.

The term "diabody" refers to a small antibody fragment with two antigen binding sites, which comprises 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.

The term "murine antibody" or "hybridoma antibody" in the present disclosure refers to a monoclonal antibody against human CD3 prepared according to the knowledge and skills in the art. During preparation, a test subject is injected with CD3 antigen, and then a hybridoma expressing an antibody with the desired sequence or functional characteristics is isolated.

The term "chimeric antibody" is 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 antibodies such as rodents ("parent antibodies"), while the constant domain sequences are obtained from human antibodies, such that the resulting chimeric antibodies are less likely to induce adverse immune responses in human subjects than the parent rodent antibodies.

The term "humanized antibody" refers to an antibody form containing sequences from 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 the hypervariable loops of non-human immunoglobulins, and all or substantially all of the framework (FR) regions are framework regions of human immunoglobulin sequences. A humanized antibody may optionally comprise at least a portion of a human immunoglobulin constant region (Fc).

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 sugar 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 sugar chains. Similarly, a "rat antibody" refers to an antibody that contains only rat immunoglobulin sequences.

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, such as to enhance or reduce effector function or binding to Fc receptors.

The term "Fc region" is used herein 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, such as Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

The term "Knob into Hole structure" refers to the mutation of the hydrophobic amino acids of CH3 of antibody Fc. The side chain amino acids of one CH3 chain are mutated to form a relatively large hydrophobic amino acid (knob) to strengthen the hydrophobic force; the side chain amino acids of another CH3 are mutated to form a small amino acid (hole) to reduce steric hindrance; after the mutation, the CH3 with Knob and the CH3 with Hole form a Knob into Hole structure (KiH) in the form of hydrophobic interaction, which is conducive to the formation of heavy chain heterodimers; the KiH mutation mainly occurs in the internal hydrophobic amino acids of the spatial structure of the CH3 domain, and the amino acids exposed outside are almost unchanged after the mutation, so it does not affect the effector function of Fc and the immunogenicity caused.

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

The term "common light chain" refers to a light chain comprising the same light chain variable region and light chain constant region, which can be paired 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 can also be paired 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. Further, 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.

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, 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 interaction between the CH3 domains in the resulting bispecific antibody to be stronger than the interaction 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, a single point mutation is located at different residues in a monospecific antibody relative to another monospecific antibody. For example, one monospecific antibody may comprise the mutation F405L (EU numbering) and another monospecific antibody may comprise the mutation K409R (EU numbering).

The term "cross-reactivity" as described herein refers to the binding of antigenic fragments of the same target molecule of human, monkey, and/or murine origin (mouse or rat). Therefore, "cross-reactivity" should be understood as an interspecies reaction 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.

"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 be generally represented by the equilibrium dissociation constant ( _KD ), which is the ratio of the dissociation rate constant and the association rate constant ( _kdis and _kon , respectively). Affinity can be measured by common methods known in the art. One specific method for measuring affinity is the ForteBio kinetic binding assay herein.

The term "does not bind" to a protein or cell means that it does not bind to the protein or cell, or does not bind to the protein or cell with high affinity, i.e., the KD for binding to the 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.

The term "high affinity" for IgG antibodies refers to a KD for an antigen of 1.0× ^10-6 M or less, preferably 5.0× ^10-8 M or less, more preferably 1.0× ^10-8 M or less, 5.0× ^10-9 M or less, and more preferably 1.0× ^10-9 M or less. For other antibody subtypes, "high affinity" binding may vary. For example, "high affinity" binding for the IgM subtype refers to a KD of ^10-6 M or less, preferably ^10-7 M or less, and more preferably ^10-8 M or less.

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 anti-CD3 antibody) to any detectable extent (e.g., inhibition of at least 85%, or at least 90%, or at least 95%).

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.

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

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, 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. Pat. 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 specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as 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 bases 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)).

"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 include a polynucleotide of the invention operably linked to a transcription initiation regulatory sequence that directs 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 invention.

"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 with bacterial replication origins and episomal mammalian vectors). After introduction 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".

The term "expression vector" as used herein refers to a nucleic acid molecule that can replicate and express a target gene when transformed, transfected or transduced into a host cell. The expression vector comprises one or more phenotypic selection markers and an origin of replication to ensure maintenance of the vector and to provide amplification in the host when necessary.

"Activation," "stimulation," and "treatment" as used for 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 otherwise or clearly dictates. "Ligand" includes natural and synthetic ligands, such as cytokines, cytokine variants, analogs, muteins, and binding compounds derived from antibodies. "Ligand" also includes small molecules, such as 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/reaction," such as the response of a cell, tissue, organ, or organism, includes changes in biochemical or physiological behavior (e.g., concentration, density, adhesion or migration within a biological compartment, gene expression rate, or differentiation state), where the change is related to activation, stimulation, or treatment, or to internal mechanisms such as genetic programming.

As used herein, the term "treatment" or "treating" of any disease or condition refers to improving the disease or condition (i.e., slowing down or preventing or reducing the progression of the disease or at least one of its clinical symptoms) in one embodiment. 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 in both aspects. Unless explicitly described herein, methods for assessing the treatment and/or prevention of a disease are generally known in the art.

"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 cynomolgus monkeys.

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

"Therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to an amount of the 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 improve the symptoms of one or more diseases or conditions or the development of the disease or condition. A therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to cause symptomatic improvement, such as an amount to treat, cure, prevent or improve a related medical condition or to increase the rate of treatment, cure, prevention or improvement 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 active ingredients that cause a therapeutic effect, whether administered in combination, sequentially or simultaneously. An effective amount of a therapeutic agent will result in an increase of at least 10%, typically at least 20%, preferably at least about 30%, more preferably at least 40%, and most preferably at least 50% in a diagnostic criterion or parameter.

"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.

The term "cancer" is used herein to refer to a group of cells that exhibit abnormally high levels 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.

Advantageous Effects of the Invention

The present invention conducts in-depth analysis and comparison on multiple strains of anti-human CD3 antibodies, optimizes the sequences and performs humanization transformation to obtain humanized CD3 antibodies.

The humanized anti-CD3 antibody of the present invention shows stronger tumor killing activity and safety when used for the preparation of bispecific antibodies. The humanized sequence disclosed by the present invention has more advantages and characteristics than the humanized sequences of other companies. It shows better tumor killing activity when used for the preparation of new anti-tumor drugs, and the physical and chemical stability is also greatly improved. Not only that, it has significantly reduced cytokine release, thus having significantly improved safety, and is more suitable for the screening and development of anti-tumor drugs. This creative achievement constitutes the beneficial effects and extremely high medical application value of the present invention.

Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the following detailed description of the accompanying drawings and preferred embodiments, various objects and advantages of the present invention will become practicable to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A-C: Binding experiment of anti-human CD3E antibody to Jurkat cells.

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

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

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

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

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

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

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

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

FIG. 10 : In vivo anti-tumor activity experiment of CD3xBCMA common light chain bispecific antibody.

Sequence information

Table 1: The information of the sequences involved in the present invention is described in the table below:

DETAILED DESCRIPTION

The present invention includes all combinations of the specific embodiments described. Further embodiments of the present invention and the full scope of 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 only by way of illustration, 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. For all purposes, all publications, patents and patent applications cited herein, including citations, will be incorporated herein by reference in their entirety.

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.

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

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

The heterodimeric protein CD3E&G (Uniprot database, P07766-1&P09693) of the extracellular region of the recombinant human CD3 antigen protein was purchased from Acro Biosystems (Cat. No. CDG-H52W5) or the KLH-coupled CD3E peptide (sequence DGNEEMGGITQTPYKVSISGTTVILTC-KLH) was used to immunize 6- to 8-week-old Balb/C mice (purchased from Vital River) or Sprague-Dawley rats (purchased from Vital River). The first immunization was performed with complete Freund's adjuvant at a dose of 50 μg antigen per mouse (or rat), and the second and subsequent immunizations were performed with incomplete Freund's adjuvant mixed with 25 μg protein antigen per mouse (or rat). The mouse immunization time points were day 0, day 14, day 28, and day 49, respectively.

After immunization for 4 times, the tail blood of mice was collected and the titer of CD3E (Acro Biosystems, catalog number H5223) antibody in the serum was detected. The mouse (or rat) with the highest CD3E antibody titer in the serum was selected, and its spleen cells were isolated and then fused with mouse myeloma cells SP2/0. After plating and culturing in HAT selection medium for 10 to 14 days, the wells with positive binding of culture supernatant to human CD3E were selected for subcloning, and then the monoclones that maintained positive binding to human CD3E after subcloning were selected, and the variable region sequence of anti-human CD3E antibody was obtained by Sanger sequencing. The sequence composition of the light chain and heavy chain variable region and CDR (KABAT numbering rule) of anti-human CD3E antibody is shown in Table 2 below, and the specific amino acid sequence is shown in Table 1.

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

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

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.

The binding of anti-human CD3E antibody 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 (Duluth's phosphate buffered saline), centrifuge at 1000rpm and harvest the cells, and adjust the cell density to 50,000 per well with FACS solution buffer (DPBS (Duluth's phosphate buffered saline) + 2% FBS (fetal bovine serum)). Incubate the chimeric antibody of the anti-human CD3E antibody with gradient dilution (starting concentration 15 μg/mL, 3-fold gradient dilution) with Jurkat cells on ice for 60 minutes. Wash the cells twice with pre-cooled FACS buffer, and then incubate 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 The fluorescence signal was detected by iQue3 flow cytometer.

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), which is expressed in-house by Junshi Biosciences. Figures 1A-C show the binding of anti-human CD3E antibody to Jurkat cells.

Example 3: Detection of activation activity of anti-human CD3E antibody on Jurkat-NFAT cells

This experiment uses the principle that the cell line Jurkat-NFAT-Luc is induced to express the NFAT reporter gene under the stimulation of anti-CD3 antibodies to detect the degree of activation of T cells by CD3 antibodies. 40μl 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), and then 40μl of gradient diluted CD3 chimeric antibodies (starting concentration of 10μg/ml, 3-fold dilution, a total of 8 concentration gradients) were added and incubated at 37°C for 6 hours. After that, 50μl of luciferase substrate One-Lite (Novozyme, DD1203-01) was added, mixed and 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.

Example 4: Humanization of anti-human CD3E antibody

The anti-human CD3E antibody NP010-018 was selected for humanization. The CDR transplantation method used for humanization is briefly described as follows: the variable regions of the heavy and light chains of the mouse antibody are compared with the germline sequence, and the sequences with high similarity are selected as the humanization templates of the heavy and light chains, respectively, for CDR transplantation. At the same time, the VH and VL of known structural antibodies with high homology are homologously modeled to find the key amino acids of the Framework that maintain the binding activity of the mouse antibody, and the reference sites that need to restore the mutation are selected from them.

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, and the light chain back mutation sites were 43P, 53N, 55Q, and 87S. The heavy chain IGHV3-15*01 and IGHV3-72*01 were humanized templates, and the heavy chain back mutation sites were 35Y, 49A, and 81V.

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), and their sequence compositions are shown in Table 3 below.

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

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.

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

The humanized antibody was named NP010-Hz018, and the number behind it represents 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 (the sequence is shown below), and the humanized antibody was expressed and purified in the HEK293 expression system.

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.

Table 5: Light and heavy chain (variable region) pairing of humanized antibodies

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

Table 6: Heavy chain sequence composition of humanized antibodies

Table 7: Light chain sequence composition of humanized antibodies

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

In a 96-well U-bottom cell culture plate, 100 μl of Jurkat cells (total number of cells: 5x10 ⁴ ) were added, and then 100 μl of gradiently diluted CD3 chimeric and humanized monoclonal antibodies (starting and ending at 15 μg/ml, 5-fold dilution, a total of 8 concentration gradients) were added and incubated in a 96-well plate at 4°C for 30 minutes. After the incubation, the plate was washed twice with flow staining solution (BD, catalog number 554658) to remove antibodies that were not bound to cells. Then 50 μl of Alexa Fluor 488-labeled Goat Anti-Human IgG secondary antibody (Jackson Lab, catalog number 109-546-098, 1:1000 dilution) was added and incubated at 4°C for 1 hour. Finally, the plate was washed twice with flow staining solution, and the cells were resuspended in 100 μl staining solution and detected by flow cytometry. The data were analyzed using Flowjo software. As shown in Figures 3A and 3B, a series of antibodies generated by humanizing the chimeric antibody NP010-018 exhibited different strengths of binding activity to Jurkat cells, among which 18-14 had the strongest cell binding activity and 18-8 had the weakest binding activity.

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

CHO-Cyno CD3e cells (CHO cells were obtained from Beijing Zhongyuan Heju Economic and Trade Co., Ltd., and cynomolgus monkey CD3e was overexpressed on the surface of CHO cells) were incubated with different concentrations of CD3 humanized antibodies 18, 18-5 and control molecule Sp34 (starting concentration was 20μg/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 staining solution, the cells were resuspended in 100μl staining solution, and detected on the flow machine. The raw data were analyzed by FlowJo software to obtain the MFI value, and the antibody dose-dependent binding curve was fitted by GraphPad. As shown in Figure 4, both 18 and 18-5 can bind to cynomolgus monkey CD3.

Example 7: Preparation of CD3xCD19 bispecific antibodies using Duobody method and verification of their in vitro killing activity

The present invention utilizes the Duobody in vitro recombination method (see Example 1 of patent CN103097417B for details) to prepare CD3xCD19 bispecific antibodies. The 18 and 18-8 clones were selected at the CD3 end, and the CD19 end of the blinatumumab bispecific antibody that Amgen has launched was selected (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). Thus, two bispecific antibodies, 18xblinatumumab and 18-8xblinatumumab, were produced.

Human peripheral blood mononuclear cells (PBMC, Stemexpress, catalog number PBMNC50C) were resuscitated and purified using a total T cell purification kit (Miltenyi Biotec, catalog number 130-096-535). Purified total human T cells (100,000 cells per well) and Nalm-6 cells (20,000 cells per well, from ATCC, catalog number: CRL-3273) pre-labeled with cell-trace violet (Invitrogen, catalog number C34557) and gradiently diluted CD3xCD19 dual antibodies (starting concentration of 10 μg/ml, 3-fold dilution, a total of 8 concentration gradients) were incubated in a 96-well plate at 37°C for 48 h. 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 staining solution, and then dead cells were marked with PI staining solution (Sigma, Catalog No. P4864) at a final concentration of 1 μg/ml, and detected on a flow cytometer (BD, Fortessa). FlowJo software was used to analyze the killing ratio of target cell Nalm-6 and the activation of CD4+ and CD8+ T cells.

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

Example 8: Preparation of CD3xBCMA bispecific antibody using Duobody method and verification of its in vitro killing activity

In order to verify the biological activity of CD3 antibodies, the present invention utilizes the Duobody in vitro recombination method of Genmab (see Example 1 of patent CN103097417B for details) to prepare CD3xBCMA bispecific antibodies. The 18-8 clone was selected for CD3, Sp34 was used as a strong control, and Junshi's own antibody was selected for BCMA, numbered BCMA-005 (wherein the heavy chain variable region sequence is SEQ ID NO: 79, wherein the HCDR1 sequence of the heavy chain variable region is SEQ ID NO: 73, the HCDR2 sequence of the heavy chain variable region is SEQ ID NO: 74, the HCDR3 sequence of the heavy chain variable region is SEQ ID NO: 75, the light chain variable region sequence is SEQ ID NO: 80, wherein the LCDR1 sequence of the light chain variable region is SEQ ID NO: 76, the LCDR2 sequence of the light chain variable region is SEQ ID NO: 77, the LCDR3 sequence of the light chain variable region is SEQ ID NO: 79, the heavy chain constant region sequence is SEQ ID NO: 72, and the light chain constant region sequence is SEQ ID NO: 62). Thus, two bispecific antibodies, 18-8xBCMA-005 and sp34xBCMA-005, were generated.

Human peripheral blood mononuclear cells (PBMC, Stemexpress, catalog number PBMNC50C) were resuscitated and purified using a total T cell purification kit (Miltenyi Biotec, catalog number 130-096-535). Purified total human T cells (100,000 cells per well) and NCI-H929 cells (obtained from ATCC, catalog number CRL-3580, 20,000 cells per well) pre-labeled with cell-trace violet (Invitrogen, catalog number C34557) and gradiently diluted CD3xBCMA dual antibodies (starting concentration of 10 μg/ml, 3-fold dilution, a total of 8 concentration gradients) were co-incubated in a 96-well plate at 37°C for 24 h or 48 h. Finally, the cells were collected and flow cytometry was performed on the cells for 30 minutes at 4°C 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). After incubation, the cells were washed with PBS, and then dead cells were marked with PI dye (Sigma, Catalog No. P4864) at a final concentration of 1 μg/ml, and detected 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.

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 cell 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, with EC ₅₀ of 9.6ng/mL and 780ng/mL, respectively. As shown in Figure 6B, after 48 hours of co-culture, 18-8xBCMA-005 can mediate the same maximum killing ratio (>90%) as sp34xBCMA-005.

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 18-8xBCMA-005 dual antibodies of the present invention on T cells is significantly weaker. 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 storms while ensuring efficient killing.

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

Using Miltenyi's magnetic bead sorting kit, T cells were separated and purified from PBMCs, and then co-cultured with T cells and NCI-H929 target cells (50,000 and 10,000 cells per well, respectively) in a 96-well U-bottom plate at an effector-target ratio of 5:1, and gradiently diluted CD3xBCMA dual antibodies (starting concentration of 10 μg/ml, 3-fold dilution, a total of 8 concentration gradients) were added and incubated at 37°C in a 96-well plate for 24 hours. Finally, the cell culture supernatant was collected by centrifugation, and cytokine detection was performed using the CBA multi-factor detection kit (BD, catalog number 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.

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

To prepare common light chain bispecific antibodies, the light chains of CD3 humanized molecules (18, 18-5, 18-8, 18-11, 18-12, 18-13, 18-14) were matched to the heavy chains of anti-BCMA antibodies (BCMA-41) (whose sequence composition is shown in Table 8 below), and 7 anti-BCMA antibodies were obtained. The CD3 humanized molecules and anti-BCMA antibodies were recombined in vitro to finally produce 7 common light chain CD3xBCMA bispecific antibodies. Since the light chains of 18 and 18-5 are exactly the same, the light chains of 18-11 and 18-13 are the same, and the light chains of 18-12 and 18-14 are the same, one of the molecules was selected to detect the binding activity of the common light chain CD3xBCMA bispecific antibody to the BCMA end.

Table 8: BCMA-41 heavy chain sequence composition

In a 96-well U-bottom cell culture plate, 50 μl of NCI-H929 cells (total number of cells 1x10 ⁵ ) highly expressing BCMA molecules were added, and then 50 μl of gradient diluted CD3xBCMA dual antibody (starting and ending concentration was 15 μg/ml, 5-fold dilution, a total of 8 concentration gradients) were added and incubated in the 96-well plate at 4°C for 30 minutes. After the incubation, the plate was washed twice with flow staining solution (BD, catalog number 554658) to remove antibodies that did not bind to cells. Then 50 μl of Alexa Fluor 488-labeled Goat Anti-Human IgG secondary antibody (Jackson Lab, catalog number 109-546-098, 1:1000 dilution) was added and incubated at 4°C for 1 hour. Finally, the plate was washed twice with flow staining solution, and the cells were resuspended in 150 μl staining solution and detected by flow cytometry. The 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 equivalent to the binding activity of the bivalent BCMA-41 antibody.

Human peripheral blood mononuclear cells (PBMC, Stemexpress, catalog number PBMNC50C) were resuscitated and human total T cells were isolated and purified using a total T cell purification kit (Miltenyi Biotec, catalog number 130-096-535). Then 50 μl of purified human T cells (1x10 ⁵ cells per well) were added to a 96-well U-bottom cell culture plate, and then 50 μl of gradient diluted CD3xBCMA common light chain dual antibody (starting and ending concentration was 200nM, 3-fold dilution, a total of 8 concentration gradients) was added and incubated in a 96-well plate at 4°C for 30 minutes. After the incubation, the plate was washed twice with flow staining solution (BD, catalog number 554658) to remove antibodies that were not bound to cells. Then 50 μl of PE-labeled Goat Anti-Human IgG secondary antibody (Biolegend, catalog number 398004, 1:1000 dilution) was added and incubated at 4°C for 1 hour. Finally, the plate was washed twice with flow cytometry staining solution, the cells were resuspended in 150 μl staining solution, and the flow cytometry was detected. The data were analyzed using Flowjo software. As shown in Figure 8B, compared with the Teclistamab of Johnson & Johnson, which has been on the market, the CD3xBCMA dual antibody in the present invention has a relatively weak binding to T cells, and its 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 generation of cytokine storm.

Example 11: In vitro killing activity of common light chain CD3xBCMA bispecific antibodies

Human peripheral blood mononuclear cells (PBMC, Stemexpress, catalog number PBMNC50C) were resuscitated and purified using a total T cell purification kit (Miltenyi Biotec, catalog number 130-096-535). Purified total human T cells (100,000 cells per well) and NCI-H929 cells (20,000 cells per well) pre-labeled with cell-trace violet (Invitrogen, catalog number C34557) and gradiently diluted CD3xBCMA common light chain bispecific antibody (starting concentration of 66.7 nM, 3-fold dilution, a total of 8 concentration gradients) were incubated in a 96-well plate at 37°C for 24 h. 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 staining solution, and then dead cells were marked with PI staining solution (Sigma, Catalog No. P4864) at a final concentration of 1 μg/ml, and detected 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.

As shown in Figure 9A, after 24 hours of co-culture, the CD3xBCMA common light chain dual antibody of the present invention can mediate the killing of target cells NCI-H929, and the maximum killing ratio is close to 90%. Compared with Teclistamab, the killing activity of the common light chain dual antibody is significantly weaker, and the strength of the killing activity is related to the T cell binding activity at the CD3 end. Among them, the weakest dual antibody is 18-8xBCMA, but it can still reach the same maximum killing ratio after 24 hours under high concentration conditions.

The common light chain bispecific antibody can effectively stimulate CD4+ and CD8+ T cells to upregulate the membrane surface activation molecules CD25 and CD69, among which 18-8xBCMA has the weakest activation on T cells, and 18-14xBCMA has the strongest stimulation on T cells, but it is still weaker than the control molecule Teclistamab (Figure 9B-E). The above results show that the common light chain form CD3 bispecific 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.

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

Female immunodeficient NDG mice aged 6-8 weeks (purchased from Biocytogen Biotech Co., Ltd.) were injected with 5x10 ⁶ NCI-H929-Luc cells (purchased from Nanjing Kebai Biotechnology Co., Ltd., catalog number CBP30061L) through the tail vein, and this time was recorded as day 0. 13 days later, 1x10 ⁷ PBMC cells (Stemexpress, catalog number PBMNC50C) were injected through the tail vein. 4 days later, 3 mg of luciferase substrate D-luciferin potassium salt (Fubaike Bio, catalog number 12505) was injected into each mouse through the tail vein, and then the luciferase substrate signal was detected on a small animal in vivo imager (Guangzhou Bolu Teng Biotechnology Co., Ltd., model Aniview100) to determine the size of the tumor, and the mice were randomly divided into 5 groups according to the size of the tumor, with 5 animals in each group. They were:

G1: saline control group

G2: 18-8xBCMA41

G3: 18xBCMA41

G4: 18-14xBCMA41

G5: Teclistamab

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, and Teclistamab was used as the control antibody for this experiment. After that, the drug was administered once every 5 days for a total of 4 times. The size of tumor growth was measured by tail vein injection of D-luciferin potassium salt (3 mg per animal), and then the luciferase substrate signal was detected on the AniView100 small animal in vivo imager. The larger the signal value, the faster the tumor growth.

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 mice achieving or approaching complete tumor regression, respectively.

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 changes may be made to the details according to all the teachings that have been published, and these changes are within the scope of protection of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims (24)

An antibody or antigen-binding fragment thereof capable of specifically binding 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. 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, respectively; (II) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are shown in SEQ ID NO: 16, SEQ ID NO: 56 and SEQ ID NO: 18, respectively; or HCDR1, HCDR2 and HCDR3 whose amino acid sequences differ from those of SEQ ID NO: 16, SEQ ID NO: 56 and SEQ ID NO: 18 by 1, 2 or 3 amino acids; (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 whose amino acid sequences are shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively; or HCDR1, HCDR2 and HCDR3 whose amino acid sequences differ from those of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18 by 1, 2 or 3 amino acids; (V) HCDR1, HCDR2 and HCDR3 whose amino acid sequences are as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; or HCDR1, HCDR2 and HCDR3 having 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 having 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 having 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, respectively; (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 having 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 having 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 having amino acid sequences as shown in SEQ ID NO:42, SEQ ID NO:21 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:42, SEQ ID NO:21 and SEQ ID NO:22; (II) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are as 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 having amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:22, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:22; (IV) LCDR1, LCDR2 and LCDR3 whose amino acid sequences are as 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 as 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 having amino acid sequences as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7; (VII) LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 7, respectively; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 7; (VIII) 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; or LCDR1, LCDR2 and LCDR3 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:13, SEQ ID NO:27 and SEQ ID NO:7; (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 having 1, 2 or 3 amino acid differences from the amino acid sequences as shown in SEQ ID NO:33, SEQ ID NO:34 and SEQ ID NO:35; or (Ⅹ) LCDR1, LCDR2 and LCDR3 having 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 having 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; 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 having amino acid sequences 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 having amino acid sequences 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 having amino acid sequences 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 having amino acid sequences 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 with amino acid sequences 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 with amino acid sequences 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 with amino acid sequences 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 with amino acid sequences 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 with amino acid sequences as shown in 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 with amino acid sequences as shown in 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 having 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 having amino acid sequences 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 NO: 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 NO: 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 having 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 having 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 shown in SEQ ID NO:12, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:12; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:15, or comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:15; (IV) the heavy chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:26, 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:26; and the light chain variable region comprises the amino acid sequence as set forth in SEQ ID NO:28, 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:28; (V) the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:32, or comprises 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:32; and the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:36, or comprises 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:36; or (VI) the heavy chain variable region comprises the amino acid sequence as shown in SEQ ID NO:39, or comprises an amino acid sequence having 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 comprises an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence as shown in SEQ ID NO:41. 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 shown in SEQ ID NO: 57 or 58 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 47; a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 58 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 51; a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 60 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 52 or 53; or a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 61 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 52 or 53. 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. 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. The antibody or antigen-binding fragment thereof as described in any one of claims 1 to 5, wherein the antibody comprises a heavy chain constant region as shown in the amino acid sequence of SEQ ID NO: 63 and/or a light chain constant region as shown in SEQ ID NO: 62. 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). The multispecific antibody according to 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 the tumor-specific antigen. The multispecific antibody according to any one of claims 7 to 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, CD H19, 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. The multispecific antibody as described in any one of claims 7-8, which 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. 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) peptide chain I-A, 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) peptide chain I-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; iii) peptide chain I-C, which comprises 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 I-D, which comprises 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 I-B and I-C respectively comprise modifications to promote dimerization of I-B and I-C; Preferably, the peptide chain I-C comprises the VH sequence shown in SEQ ID NO: 70 or a variant thereof, and the peptide chain I-D comprises the VL sequence shown in SEQ ID NO: 71 or a variant thereof; Preferably, the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 58, 57, 60 or 61 or a variant thereof, and the peptide chain I-A comprises the VL sequence shown in SEQ ID NO: 51, 47, 52 or 53 or a variant thereof; Preferably, the peptide chain I-B comprises the VH sequence shown in SEQ ID NO: 58 or 57 or a variant thereof, and the peptide chain I-A comprises the VL sequence shown in SEQ ID NO: 51 or 47 or a variant thereof. According to the multispecific antibody described in any one of claims 7-8, 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. The multispecific antibody of claim 12, which is a bispecific antibody in the form of a common light chain. The multi-specific antibody as described in claim 13, wherein the common light chain comprises LCDR1, LCDR2, LCDR3 whose amino acid sequences are respectively as shown in SEQ ID NO: 42, SEQ ID NO: 21, and SEQ ID NO: 22; or LCDR1, LCDR2, LCDR3 whose amino acid sequences are respectively as shown in SEQ ID NO: 43, SEQ ID NO: 45, and SEQ ID NO: 22; or LCDR1, LCDR2, LCDR3 whose amino acid sequences are respectively as shown in SEQ ID NO: 43, SEQ ID NO: 46, and SEQ ID NO: 22; preferably, the common light chain comprises LCDR1, LCDR2, LCDR3 whose amino acid sequences are respectively as shown in SEQ ID NO: 43, SEQ ID NO: 46, and SEQ ID NO: 22. The multispecific antibody as described in claim 13 or 14, wherein the common light chain comprises the sequence shown in SEQ ID NO: 47, 51, 52 or 53. The multispecific antibody according to any one of claims 7 to 9 and 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 compared to the sequence from which it is derived. 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. A vector comprising the isolated nucleic acid molecule of claim 17. A host cell comprising the isolated nucleic acid molecule of claim 17 or the vector of claim 18. 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. 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. 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. 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. The use of claim 22 or the method of 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.