CN116917333A

CN116917333A - Antigen binding molecules that specifically bind BCMA and CD3 and medical uses thereof

Info

Publication number: CN116917333A
Application number: CN202280010911.5A
Authority: CN
Inventors: 石金平; 应华; 黎婷婷; 陶维康
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2021-01-20
Filing date: 2022-01-20
Publication date: 2023-10-20
Also published as: WO2022156739A1; TW202246333A

Abstract

An antigen binding molecule that specifically binds BCMA and CD3 and its medical use.

Description

Antigen binding molecules that specifically bind BCMA and CD3 and medical uses thereof

The application claims priority from the Chinese patent application (application No. CN 202110076465.5) filed on 20 months of 2021 and the Chinese patent application (application No. CN 202110795647.8) filed on 14 months of 2021.

Technical Field

The present disclosure is in the field of biotechnology, more specifically, the disclosure relates to antigen binding molecules and uses thereof.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

Multiple Myeloma (MM) is the second most common hematological cancer worldwide, which is characterized by uncontrolled proliferation of plasma cells in the bone marrow, rapid proliferation and diffusion of cancerous plasma cells, which in turn leads to the massive production of monoclonal immunoglobulins which in turn cause immunosuppression, osteolysis and end organ damage.

Worldwide, there are over 13.85 tens of thousands of newly diagnosed MM patients. In the last decades, due to the emergence of new therapeutic means such as proteasome inhibitors, immunomodulators and CD38 antibodies, the clinical therapeutic effect of MM patients is greatly improved, and the life expectancy of patients is improved from 3 to 4 years to 7 to 8 years. Most patients still relapse due to drug resistance. Even MRD (minimal residual disease) negative patients relapse. Thus, there is a great clinical need for new treatments that are more effective. While targeting MM cells, immunotherapy that remodels the antitumor activity of immune cells would be a very excellent therapy for treating MM.

BCMA (tumor necrosis factor receptor superfamily member, tnfrs17) is a type 3 transmembrane protein without a signal peptide whose extracellular region is rich in cysteine, which works together with TNFR superfamily factor B cell activator receptor (BAFF-R) and TACI to regulate B cell proliferation and differentiate B cells into plasma cells. These functional receptors support long-term survival of B cells by binding to the ligand BAFF or APRIL. BCMA begins to be induced to express only when memory B cells later differentiate into plasma cells, and only on the surface of plasmablasts and differentiated plasma cells; and in memory B cells,B cells or cd34+ hematopoietic stem cells, as well as other normal tissues, and the like. Studies in BCMA knockout mice demonstrate that BCMA has an important effect only on long-term survival of plasma cells and does not regulate B cell homeostasis. Thus, BCMA is a very specific MM antigen compared to CD38, which is widely expressed in normal tissues (especially immune cells and immune organs).

The bispecific antibody targeting CD3/TAA is a novel immunotherapy, can combine T cells and tumor cells simultaneously, simulate the interaction of MHC and TCR, and release perforin and granzyme after the T cells form lytic synapse, thereby specifically killing the tumor cells. The activated T cells can release cytokines, initiate other immune cells and amplify immune responses against the tumor, ultimately leading to proliferation of T cells and a cascade of killing tumor cells. Thus, CD3/BCMA bispecific antibodies can be used as a very excellent immunotherapeutic approach to MM.

Disclosure of Invention

The present disclosure provides an antigen binding molecule. Comprising at least one first antigen binding domain that specifically binds BCMA and at least one second antigen binding domain that specifically binds CD 3. These antigen binding molecules are capable of providing better therapeutic activity than BCMA antibodies and CD3 antibodies.

In one aspect, the present disclosure provides an antigen binding molecule comprising at least one first antigen binding domain that specifically binds BCMA comprising heavy chain variable region BCMA-VH and light chain variable region BCMA-VL, and at least one second antigen binding domain that specifically binds CD3, wherein:

(i) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in said BCMA-VH comprise the amino acid sequence of SEQ ID NO:29, BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR 3; and BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 in said BCMA-VL comprise SEQ ID NO:30, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(ii) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in said BCMA-VH comprise the amino acid sequence of SEQ ID NO:31, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR 3; and BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 in said BCMA-VL comprise SEQ ID NO:32, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(iii) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in said BCMA-VH comprise the amino acid sequence of SEQ ID NO:33, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR 3; and BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 in said BCMA-VL comprise SEQ ID NO:34, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(iv) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in said BCMA-VH comprise the amino acid sequence of SEQ ID NO:35, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR 3; and BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 in said BCMA-VL comprise SEQ ID NO:36, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3.

In some embodiments, the BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 are defined according to Kabat, IMGT, chothia, abM or Contact numbering rules.

The heavy chain variable region BCMA-VH has BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3, and the light chain variable region BCMA-VL has BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3, wherein:

(i) The BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 comprise SEQ ID NO:29, BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR 3; and the BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 comprise SEQ ID NO:30, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(ii) The BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 comprise SEQ ID NO:31, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR 3; and the BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 comprise SEQ ID NO:32, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(iii) The BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 comprise SEQ ID NO:33, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR 3; and the BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 comprise SEQ ID NO:34, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(iv) The BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 comprise SEQ ID NO:35, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR 3; and the BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 comprise SEQ ID NO:36, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3;

The BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 are defined according to Kabat, IMGT, chothia, abM or Contact numbering rules.

(i) The heavy chain variable region BCMA-VH comprises SEQ ID NO:29, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:30, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(ii) The heavy chain variable region BCMA-VH comprises SEQ ID NO:31, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:32, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(iii) The heavy chain variable region BCMA-VH comprises SEQ ID NO:33, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:34, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(iv) The heavy chain variable region BCMA-VH comprises SEQ ID NO:35, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:36, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3;

(i) The heavy chain variable region BCMA-VH has: comprising SEQ ID NO:5, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:6, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:7, BCMA-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region BCMA-VL has: comprising SEQ ID NO:8, a BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:9, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:10, BCMA-LCDR3 of the amino acid sequence of 10; or (b)

(ii) The heavy chain variable region BCMA-VH has: comprising SEQ ID NO:11, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:12, and a BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:13, BCMA-HCDR3 of the amino acid sequence of 13; and the light chain variable region BCMA-VL has: comprising SEQ ID NO:14, a BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:15, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:16, BCMA-LCDR3 of the amino acid sequence of 16; or (b)

(iii) The heavy chain variable region BCMA-VH has: comprising SEQ ID NO:17, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:18, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:19, BCMA-HCDR3 of the amino acid sequence of 19; and the light chain variable region BCMA-VL has: comprising SEQ ID NO:20, comprising the amino acid sequence of SEQ ID NO:21, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:22, BCMA-LCDR3 of the amino acid sequence of 22; or (b)

(iv) The heavy chain variable region BCMA-VH has: comprising SEQ ID NO:23, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:24, and a BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:25, BCMA-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region BCMA-VL has: comprising SEQ ID NO:26, comprising the amino acid sequence of SEQ ID NO:27, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:28, BCMA-LCDR3 of the amino acid sequence of 28;

The BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 are defined according to the Kabat numbering convention.

In some embodiments, an antigen binding molecule as described previously, wherein:

(i) The heavy chain variable region BCMA-VH has: comprising SEQ ID NO:5, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:6, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:7, BCMA-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region BCMA-VL has: comprising SEQ ID NO:8, a BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:9, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:10, and BCMA-LCDR3 of the amino acid sequence of seq id no. In some embodiments, the heavy chain variable region BCMA-VH and/or light chain variable region BCMA-VL is murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or light chain variable region BCMA-VL are humanized.

In some embodiments, the heavy chain variable region BCMA-VH has a heavy chain framework region derived from IGHV1-46 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 48I, 67A, 71A, 73K, 76T, and 93V; and/or said light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-39 x 01 and which is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 43S, 45Q, 48V and 71Y. In some embodiments, the BCMA-VH has FR1-3 derived from IGHV1-46 x 01 and FR4 derived from IGHJ6 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 1E, 48I, 67A, 71A, 73K, 76T, and 93V; and/or said BCMA-VL has FR1-3 derived from IGKV1-39 x 01 and FR4 derived from IGKJ2 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 43S, 45Q, 48V and 71Y. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.

(i) The heavy chain variable region BCMA-VH comprises a sequence identical to SEQ ID NO:29, and the light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:30, an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:29, and said light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:30, and a sequence of amino acids.

(i) The heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 37. SEQ ID NO:38 and SEQ ID NO:39, and the light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 40. SEQ ID NO: 41. SEQ ID NO:42 and SEQ ID NO:43, respectively having an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 37. SEQ ID NO:38 and SEQ ID NO:39, and said light chain variable region BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO:42 and SEQ ID NO:43, and a sequence of amino acids of the group consisting of seq id no. In some embodiments:

The heavy chain variable region BCMA-VH comprises SEQ ID NO:37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:40, or

The heavy chain variable region BCMA-VH comprises SEQ ID NO:38, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:40, or

The heavy chain variable region BCMA-VH comprises SEQ ID NO:39, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:40, or

The heavy chain variable region BCMA-VH comprises SEQ ID NO:37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:41, or

The heavy chain variable region BCMA-VH comprises SEQ ID NO:37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:42, or

The heavy chain variable region BCMA-VH comprises SEQ ID NO:37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 43.

(ii) The heavy chain variable region BCMA-VH has: comprising SEQ ID NO:11, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:12, and a BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:13, BCMA-HCDR3 of the amino acid sequence of 13; and the light chain variable region BCMA-VL has: comprising SEQ ID NO:14, a BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:15, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:16, and a BCMA-LCDR3 of the amino acid sequence of 16. In some embodiments, the heavy chain variable region BCMA-VH and/or light chain variable region BCMA-VL is murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or light chain variable region BCMA-VL are humanized. In some embodiments, the heavy chain variable region BCMA-VH has a heavy chain framework region derived from IGHV7-4-1 x 02 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 2I, 44V, 45F, 46K, 75A, 76N, and 93L; and/or said light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-8 x 01 or IGKV1-27 x 01 and which is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 43S and 66D. In some embodiments, the BCMA-VH has FR1-3 derived from IGHV7-4-1 x 02 and FR4 derived from IGHJ1 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 1E, 21I, 44V, 45F, 46K, 75A, 76N, and 93L; and/or said BCMA-VL has FR1-3 derived from IGKV1-8 x 01 or IGKV1-27 x 01 and FR4 derived from IGKJ4 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 43S and 66D. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.

(ii) The heavy chain variable region BCMA-VH comprises a sequence identical to SEQ ID NO:31, and the light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:32 has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:31, and said light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:32, and a sequence of amino acids.

(ii) The heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 44. SEQ ID NO:45 and SEQ ID NO:46, and the light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 47 and SEQ ID NO:48 has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity, respectively. In some embodiments, the heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 44. SEQ ID NO:45 and SEQ ID NO:46, and said light chain variable region BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:47 and SEQ ID NO:48, and a sequence of amino acids of the group consisting of seq id no. In some embodiments:

The heavy chain variable region BCMA-VH comprises SEQ ID NO:44, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:47, or

The heavy chain variable region BCMA-VH comprises SEQ ID NO:45, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:47, or

The heavy chain variable region BCMA-VH comprises SEQ ID NO:46, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:47, or

The heavy chain variable region BCMA-VH comprises SEQ ID NO:44, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:48, or

The heavy chain variable region BCMA-VH comprises SEQ ID NO:45, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:48, or

The heavy chain variable region BCMA-VH comprises SEQ ID NO:46, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 48.

(iii) The heavy chain variable region BCMA-VH has: comprising SEQ ID NO:17, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:18, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:19, BCMA-HCDR3 of the amino acid sequence of 19; and the light chain variable region BCMA-VL has: comprising SEQ ID NO:20, comprising the amino acid sequence of SEQ ID NO:21, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:22, BCMA-LCDR3 of the amino acid sequence of 22; in some embodiments, the heavy chain variable region BCMA-VH and/or light chain variable region BCMA-VL is murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or light chain variable region BCMA-VL are humanized. In some embodiments, the heavy chain variable region BCMA-VH has a heavy chain framework region derived from IGHV3-11 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 30R, 47R, 49A, and 93T; and/or said light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-39 x 01 and which is unsubstituted or has an amino acid substitution of 44V. In some embodiments, the BCMA-VH has FR1-3 derived from IGHV3-11 x 01 and FR4 derived from IGHJ1 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 1E, 30R, 47R, 49A, and 93T; and/or said BCMA-VL has FR1-3 derived from IGKV1-39 x 01 and FR4 derived from IGKJ2 x 01 and is unsubstituted or has an amino acid substitution of 44V. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.

(iii) The heavy chain variable region BCMA-VH comprises a sequence identical to SEQ ID NO:33, and the light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:34, an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:33, and said light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:34, and a sequence of amino acids.

(iii) The heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 49 and SEQ ID NO:50, and the light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:51 has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 49 and SEQ ID NO:50, and said light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments of the present invention, in some embodiments,

The heavy chain variable region BCMA-VH comprises SEQ ID NO:49, and said light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:51, or

The heavy chain variable region BCMA-VH comprises SEQ ID NO:50, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 51.

(iv) The heavy chain variable region BCMA-VH has: comprising SEQ ID NO:23, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:24, and a BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:25, BCMA-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region BCMA-VL has: comprising SEQ ID NO:26, comprising the amino acid sequence of SEQ ID NO:27, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:28, BCMA-LCDR3 of the amino acid sequence of 28; in some embodiments, the heavy chain variable region BCMA-VH and/or light chain variable region BCMA-VL is murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or light chain variable region BCMA-VL are humanized. In some embodiments, the heavy chain variable region BCMA-VH has a heavy chain framework region derived from IGHV2-70 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 24V, 30T, 37V, 49G, 73N, 80F, and 89R; and/or said light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-33 x 01. In some embodiments, the BCMA-VH has FR1-3 derived from IGHV2-70 x 01 and FR4 derived from IGHJ6 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 1E, 24V, 30T, 37V, 49G, 73N, 80F, and 89R; and/or said BCMA-VL has FR1-3 derived from IGKV1-33 x 01 and FR4 derived from IGKJ2 x 01. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.

(iv) The heavy chain variable region BCMA-VH comprises a sequence identical to SEQ ID NO:35, and the light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:36, an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:35, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:36, and a nucleotide sequence of 36.

(iv) The heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 52 and SEQ ID NO:53, and the light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:54 have an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity, respectively. In some embodiments, the heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 52 and SEQ ID NO:53, and said light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:54, an amino acid sequence of seq id no. In some embodiments of the present invention, in some embodiments,

The heavy chain variable region BCMA-VH comprises SEQ ID NO:52, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:54, or

The heavy chain variable region BCMA-VH comprises SEQ ID NO:53, and said light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:54, an amino acid sequence of seq id no.

In some embodiments, the antigen-binding molecule of any one of the preceding claims, wherein the second antigen-binding domain that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein:

(i) The CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in the CD3-VH comprise the sequences of SEQ ID NOs: 63, CD3-HCDR1, CD3-HCDR2 and CD3-HCDR 3; and the CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3 in the CD3-VL comprise the amino acid sequences of SEQ ID NOs: 64, the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR 3; or (b)

(ii) The CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in the CD3-VH comprise the sequences of SEQ ID NOs: 65, CD3-HCDR1, CD3-HCDR2 and CD3-HCDR 3; and the CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3 in the CD3-VL comprise the amino acid sequences of SEQ ID NOs: 66, CD3-LCDR1, CD3-LCDR2 and CD3-LCDR 3.

In some embodiments, the CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3 are defined according to Kabat, IMGT, chothia, abM or Contact numbering rules.

the heavy chain variable region CD3-VH has CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3, and the light chain variable region CD3-VL has CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3, wherein:

(i) The CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 comprise the amino acid sequences of SEQ ID NO:63, CD3-HCDR1, CD3-HCDR2 and CD3-HCDR 3; and the CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3 comprise the amino acid sequences of SEQ ID NO:64, the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR 3; or (b)

(ii) The CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 comprise the amino acid sequences of SEQ ID NO:65, CD3-HCDR1, CD3-HCDR2 and CD3-HCDR 3; and the CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3 comprise the amino acid sequences of SEQ ID NO:66, the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR 3;

The CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 are defined according to Kabat, IMGT, chothia, abM or Contact numbering rules.

(i) The heavy chain variable region CD3-VH comprises SEQ ID NO:63, CD3-HCDR1, CD3-HCDR2, and CD3-HCDR3, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64, the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR 3; or (b)

(ii) The heavy chain variable region CD3-VH comprises SEQ ID NO:65, CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:66, the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR 3;

(i) The heavy chain variable region CD3-VH has: comprising SEQ ID NO:55, comprising the amino acid sequence of SEQ ID NO:56, and a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:57, CD3-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region CD3-VL has: comprising SEQ ID NO:58, a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:59, and a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:60, a CD3-LCDR3 of the amino acid sequence of 60; or (b)

(ii) The heavy chain variable region CD3-VH has: comprising SEQ ID NO:55, comprising the amino acid sequence of SEQ ID NO:61, and a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:62, CD3-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region CD3-VL has: comprising SEQ ID NO:58, a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:59, and a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:60, a CD3-LCDR3 of the amino acid sequence of 60;

the CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 are defined according to the Kabat numbering convention.

In some embodiments, the antigen binding molecule of any one of the preceding claims, wherein: the heavy chain variable region CD3-VH comprises a sequence identical to SEQ ID NO:63, and said light chain variable region CD3-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:64 has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity; in some embodiments, the heavy chain variable region CD3-VH comprises SEQ ID NO:63, and said light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64, and a sequence of amino acids.

In some embodiments, the antigen binding molecule of any one of the preceding claims, wherein: the heavy chain variable region CD3-VH comprises a sequence identical to SEQ ID NO:65, and said light chain variable region CD3-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:66 having an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity; in some embodiments, the heavy chain variable region CD3-VH comprises SEQ ID NO:65, and said light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO: 66.

In some embodiments, the antigen binding molecule of any one of the preceding claims, wherein the heavy chain variable region BCMA-VH has: comprising SEQ ID NO:5, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:6, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:7, BCMA-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region BCMA-VL has: comprising SEQ ID NO:8, a BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:9, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:10, and

The heavy chain variable region CD3-VH has: comprising SEQ ID NO:55, comprising the amino acid sequence of SEQ ID NO:56, and a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:57, CD3-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region CD3-VL has: comprising SEQ ID NO:58, a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:59, and a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:60, and a CD3-LCDR3 of the amino acid sequence of 60. In some embodiments, the CDRs are defined according to Kabat numbering convention.

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:40, and

the heavy chain variable region CD3-VH comprises SEQ ID NO:63, and said light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64, and a sequence of amino acids.

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:38, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:40, and

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:39, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:40, and

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:41, and

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:42, and

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:37, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:43, and

the heavy chain variable region CD3-VH has: comprising SEQ ID NO:55, comprising the amino acid sequence of SEQ ID NO:61, and a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:62, CD3-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region CD3-VL has: comprising SEQ ID NO:58, a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:59, and a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:60, and a CD3-LCDR3 of the amino acid sequence of 60. In some embodiments, the CDRs are defined according to Kabat numbering convention.

the heavy chain variable region CD3-VH comprises SEQ ID NO:65, and said light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO: 66.

In some embodiments, the antigen binding molecule of any one of the preceding claims, wherein the heavy chain variable region BCMA-VH has: comprising SEQ ID NO:11, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:12, and a BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:13, BCMA-HCDR3 of the amino acid sequence of 13; and the light chain variable region BCMA-VL has: comprising SEQ ID NO:14, a BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:15, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:16, and

the heavy chain variable region CD3-VH has: comprising SEQ ID NO:55, comprising the amino acid sequence of SEQ ID NO:56, and a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:57, CD3-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region CD3-VL has: comprising SEQ ID NO:58, a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:59, and a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:60, and a CD3-LCDR3 of the amino acid sequence of 60.

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:44, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:47, and

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:45, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:47, and

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:46, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:47, and

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:44, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:48, and

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:45, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:48, and

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:46, and said light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:48, and

In some embodiments, the antigen binding molecule of any one of the preceding claims, wherein the heavy chain variable region BCMA-VH has: comprising SEQ ID NO:17, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:18, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:19, BCMA-HCDR3 of the amino acid sequence of 19; and the light chain variable region BCMA-VL has: comprising SEQ ID NO:20, comprising the amino acid sequence of SEQ ID NO:21, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:22, and

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:49, and said light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:51, and

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:50, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:51, and

In some embodiments, the antigen binding molecule of any one of the preceding claims, wherein the heavy chain variable region BCMA-VH has: the heavy chain variable region BCMA-VH has: comprising SEQ ID NO:17, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:18, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:19, BCMA-HCDR3 of the amino acid sequence of 19; and the light chain variable region BCMA-VL has: comprising SEQ ID NO:20, comprising the amino acid sequence of SEQ ID NO:21, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:22, and

the heavy chain variable region CD3-VH has: comprising SEQ ID NO:55, comprising the amino acid sequence of SEQ ID NO:61, and a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:62, CD3-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region CD3-VL has: comprising SEQ ID NO:58, a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:59, and a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:60, and a CD3-LCDR3 of the amino acid sequence of 60.

In some embodiments, the antigen binding molecule of any one of the preceding claims, wherein the heavy chain variable region BCMA-VH has: the heavy chain variable region BCMA-VH has: comprising SEQ ID NO:23, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:24, and a BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:25, BCMA-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region BCMA-VL has: comprising SEQ ID NO:26, comprising the amino acid sequence of SEQ ID NO:27, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:28, and

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:52, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:54, and

In some embodiments, the heavy chain variable region BCMA-VH comprises SEQ ID NO:53, and said light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:54, and

In some embodiments, the antigen binding molecule of any one of the preceding claims, wherein the antigen binding molecule further comprises an Fc region comprising two subunits capable of associating. In some embodiments, the two subunits are the same or different first and second subunits. In some embodiments, the Fc region is an IgG Fc region, particularly IgG ₁ An Fc region. In some embodiments, the Fc region comprises one or more amino acid substitutions that reduce binding to Fc receptors. In particular, the amino acid substitutions reduce binding to fcγ receptors. In some embodiments, the Fc region comprises one or more amino acid substitutions that reduce binding to Fc receptors, particularly fcγ receptors, as compared to the wild-type Fc region. In some embodiments, the Fc region is a human IgG ₁ The Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. In some embodiments, the Fc region comprises SEQ ID NO: 69.

In some embodiments, the antigen binding molecule of any one of the preceding claims, comprising at least two first antigen binding domains that specifically bind BCMA, at least two second antigen binding domains that specifically bind CD3, and an Fc region. In some embodiments, it comprises two first antigen binding domains that specifically bind BCMA, two second antigen binding domains that specifically bind CD3, and one Fc region.

In some embodiments, the first antigen binding domain that specifically binds BCMA is a Fab and/or the second antigen binding domain that specifically binds CD3 is a scFv. In some embodiments, the first antigen binding domain that specifically binds BCMA is Fab and/or the second antigen binding domain that specifically binds CD3 is Fab. In some embodiments, the first antigen binding domain that specifically binds BCMA is an scFv and/or the second antigen binding domain that specifically binds CD3 is an scFv. In some embodiments, the first antigen binding domain that specifically binds BCMA is an scFv and/or the second antigen binding domain that specifically binds CD3 is a Fab.

In some embodiments, the first antigen binding domain that specifically binds BCMA, the second antigen binding domain that specifically binds CD3, and one subunit of the Fc region are coupled in any order. In some embodiments, the first antigen binding domain that specifically binds BCMA, the second antigen binding domain that specifically binds CD3, and one subunit of the Fc region are linked directly or through a linker in the order of N-terminus to C-terminus. In some embodiments, the second antigen binding domain that specifically binds CD3, the first antigen binding domain that specifically binds BCMA, and one subunit of the Fc region are linked directly or through a linker in the order of N-terminus to C-terminus. In some embodiments, the first antigen binding domain that specifically binds BCMA, one subunit of the Fc region, the second antigen binding domain that specifically binds CD3 are linked directly or through a linker in the order of N-terminus to C-terminus. In some embodiments, the second antigen binding domain that specifically binds CD3, one subunit of the Fc region, the first antigen binding domain that specifically binds BCMA is linked directly or through a linker in the order of N-terminus to C-terminus.

In some embodiments, the antigen binding molecule of any one of the preceding claims, comprising two first antigen binding domains that specifically bind BCMA, two second antigen binding domains that specifically bind CD3, and an Fc region; wherein the first antigen binding domain that specifically binds BCMA is a Fab and the second antigen binding domain that specifically binds CD3 is a scFv. In some embodiments, the first antigen binding domain that specifically binds BCMA, the second antigen binding domain that specifically binds CD3, and one subunit of the Fc region are linked directly or through a linker in the order of N-terminus to C-terminus. In some embodiments, the C-terminus of the Fab heavy chain is fused to the N-terminus of the scFv directly or through a linker. In some embodiments, the heavy chain, the scFv, and one subunit of the Fc region of the Fab are linked directly or through a linker in the order of N-terminus to C-terminus.

In some embodiments, the antigen binding molecule of any one of the preceding claims, wherein the antigen binding molecule comprises two first chains having the structure of formula (a) and two second chains having the structure of formula (b),

(a) [ BCMA-VH ] - [ CH1] - [ CD3-VH ] - [ linker ] - [ CD3-VL ] - [ linker ] - [ subunit of Fc region ]; the linkers in formula (a) are preferably identical or different peptide linkers;

(b)[BCMA-VL]-[CL]。

in some embodiments, the formulas (a) and (b) are each arranged from the N-terminus to the C-terminus.

In some embodiments, the peptide linker is a flexible peptide linker. In some embodiments, the peptide linker is 3-15 amino acid residues in length. In some embodiments, the peptide linker independently has L ₁ -(GGGGS)n-L ₂ Wherein L is ₁ Is a bond, A, GS, GGS or GGGS (SEQ ID NO: 177), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and L2 is a bond, G, GG, GGG or GGGG (SEQ ID NO: 177). In some embodiments, the peptide linker is not a bond. In some embodiments, the peptide linker is GGGGSGGGGSGGGGS (SEQ ID NO: 155) or GGG (SEQ ID NO: 156). In some embodiments, the first strand has the structure:

[ BCMA-VH ] - [ CH1] - [ CD3-VH ] - [ GGGGSGGGGSGGGGS ] - [ CD3-VL ] - [ GGG ] - [ one subunit of the Fc region ].

In some embodiments, the antigen binding molecule of any one of the preceding claims, having:

comprising SEQ ID NO:76, and a first strand comprising the amino acid sequence of SEQ ID NO:72, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:77, and a first strand comprising the amino acid sequence of SEQ ID NO:72, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:78, and a first strand comprising the amino acid sequence of SEQ ID NO:72, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:76, and a first strand comprising the amino acid sequence of SEQ ID NO:73, a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:76, and a first strand comprising the amino acid sequence of SEQ ID NO:74, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:76, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:80, and a first strand comprising the amino acid sequence of SEQ ID NO:72, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:81, and a first strand comprising the amino acid sequence of SEQ ID NO:72, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:79, and a first strand comprising the amino acid sequence of SEQ ID NO:73, a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:79, and a first strand comprising the amino acid sequence of SEQ ID NO:74, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:79, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:84, and a first strand comprising the amino acid sequence of SEQ ID NO:82, a second strand of an amino acid sequence; or (b)

Comprising SEQ ID NO:85, and a first strand comprising the amino acid sequence of SEQ ID NO:82, a second strand of an amino acid sequence; or (b)

Comprising SEQ ID NO:86, and a first strand comprising the amino acid sequence of SEQ ID NO:82, a second strand of an amino acid sequence; or (b)

Comprising SEQ ID NO:84, and a first strand comprising the amino acid sequence of SEQ ID NO:83, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:85, and a first strand comprising the amino acid sequence of SEQ ID NO:83, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:86, and a first strand comprising the amino acid sequence of SEQ ID NO:83, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:88, and a first strand comprising the amino acid sequence of SEQ ID NO:87, a second strand of the amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:89, and a first strand comprising the amino acid sequence of SEQ ID NO:87, a second strand of the amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:90, and a first strand comprising the amino acid sequence of SEQ ID NO:87, a second strand of the amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:91, and a first strand comprising the amino acid sequence of SEQ ID NO:87, a second strand of the amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:95, and a first strand comprising the amino acid sequence of SEQ ID NO:94, and a second strand of an amino acid sequence of 94; or (b)

Comprising SEQ ID NO:96, and a first strand comprising the amino acid sequence of SEQ ID NO:94, and a second strand of an amino acid sequence of 94; or (b)

Comprising SEQ ID NO:97, and a first strand comprising the amino acid sequence of SEQ ID NO:94, and a second strand of an amino acid sequence of 94; or (b)

Comprising SEQ ID NO:79, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no.

In some embodiments, the antigen binding molecule as described previously, wherein the first antigen binding domain that specifically binds BCMA is a Fab and the second antigen binding domain that specifically binds CD3 is a substituted Fab comprising a tin chain and an Obscurin chain linked to two polypeptide chains of the variable region, either directly or through a linker, respectively, excluding the light chain constant region and the heavy chain constant region CH1; or the second antigen binding domain that specifically binds CD3 is a Fab, and the first antigen binding domain that specifically binds BCMA is a substituted Fab comprising a tin chain and an Obscurin chain linked to two polypeptide chains of the variable region, respectively, either directly or through a linker, excluding the light chain constant region and the heavy chain constant region CH1.

In some embodiments, the antigen binding molecule as described previously, wherein the first antigen binding domain that specifically binds BCMA is a Fab and the second antigen binding domain that specifically binds CD3 is a substituted Fab in which the heavy chain constant region CH1 and the light chain constant region are replaced with a tin chain and an Obscurin chain; or the second antigen binding domain that specifically binds CD3 is a Fab and the first antigen binding domain that specifically binds BCMA is a substituted Fab in which the heavy chain constant region CH1 and the light chain constant region are replaced with a tin chain and an Obscurin chain.

In some embodiments, the first antigen binding domain that specifically binds BCMA is Fab; the second antigen binding domain that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the C-terminus of the heavy chain variable region CD3-VH is fused to the N-terminus of the tin chain directly or through a linker, and the C-terminus of the light chain variable region CD3-VL is fused to the N-terminus of the Obscurin chain directly or through a linker. In some embodiments, the first antigen binding domain that specifically binds BCMA is Fab; the second antigen binding domain that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the C-terminus of the heavy chain variable region CD3-VL is fused to the N-terminus of the tin chain directly or through a linker, and the C-terminus of the light chain variable region CD3-VH is fused to the N-terminus of the Obscurin chain directly or through a linker. In some embodiments, the second antigen binding domain that specifically binds CD3 is a Fab; the first antigen binding domain that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, and the C-terminus of the heavy chain variable region BCMA-VH is fused to the N-terminus of the tin chain directly or through a linker, and the C-terminus of the light chain variable region BCMA-VL is fused to the N-terminus of the Obscurin chain directly or through a linker. In some embodiments, the second antigen binding domain that specifically binds CD3 is a Fab; the first antigen binding domain that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, and the C-terminus of the heavy chain variable region BCMA-VL is fused to the N-terminus of the tin chain directly or through a linker, and the C-terminus of the light chain variable region BCMA-VH is fused to the N-terminus of the Obscurin chain directly or through a linker.

In some embodiments, the antigen binding molecule further comprises an Fc region comprising a first subunit capable of associating with a second subunit, the first and second subunits having one or more amino acid substitutions that reduce homodimerization of the Fc region. In some embodiments, the antigen binding molecule further comprises an Fc region comprising a first subunit capable of associating with a second subunit, the first and second subunits having one or more amino acid substitutions that reduce homodimerization of the Fc region, as compared to the wild-type Fc region. In some embodiments, the first subunit has a raised structure according to the knob and socket technique and the second subunit has a hole structure according to the knob and socket technique. In some embodiments, the C-terminus of the tin chain is fused to the N-terminus of the first subunit directly or through a linker, and the C-terminus of CH1 of the Fab is fused to the N-terminus of the second subunit directly or through a linker. In some embodiments, the C-terminus of the tin chain is fused to the N-terminus of the second subunit directly or through a linker, and the C-terminus of CH1 of the Fab is fused to the N-terminus of the first subunit directly or through a linker. In some embodiments, the C-terminus of the Obscurin chain is fused to the N-terminus of the first subunit directly or through a linker, and the C-terminus of CH1 of the Fab is fused to the N-terminus of the second subunit directly or through a linker. In some embodiments, the C-terminus of the Obscurin chain is fused to the N-terminus of the second subunit directly or through a linker, and the C-terminus of CH1 of the Fab is fused to the N-terminus of the first subunit directly or through a linker.

In some embodiments, the antigen binding molecule as described previously, the amino acid residue substitution of the first subunit comprises one or more amino acid substitutions at position 366 and the amino acid residue of the second subunit comprises one or more amino acid substitutions at positions selected from the group consisting of 366, 368 and 407. In some embodiments, the first subunit comprises one or more amino acid substitutions of 366W and the second subunit comprises one or more amino acid substitutions selected from 366S, 368A, and 407V. In some embodiments, the first subunit comprises amino acid substitutions of 366W and the second subunit comprises amino acid substitutions of 366S, 368A, and 407V.

In some embodiments, an antigen binding molecule as described previously, comprising a first chain having a structure represented by formula (c), a second chain having a structure represented by formula (b), a third chain having a structure represented by formula (d), and a fourth chain having a structure represented by formula (e),

(c) A second subunit of [ BCMA-VH ] - [ CH1] - [ Fc region ];

(b)[BCMA-VL]-[CL]；

(d) A first subunit of a [ CD3-VH ] - [ linker ] - [ Titin chain ] - [ Fc region ];

(e) [ CD3-VL ] - [ linker ] - [ Obscurin strand ].

In some embodiments, the first subunit of the Fc region has a raised structure according to the knob-to-socket technique, and the second subunit of the Fc region has a pore structure according to the knob-to-socket technique. In some embodiments, the linkers in formulas (d) and (e) are preferably identical or different peptide linkers.

In some embodiments, the formula (C), formula (b), formula (d), and formula (e) are each arranged from the N-terminus to the C-terminus.

In some embodiments, an antigen binding molecule as described previously comprising a first chain having a structure represented by formula (f), a second chain having a structure represented by formula (b), a third chain having a structure represented by formula (g), and a fourth chain having a structure represented by formula (h),

(f) [ BCMA-VH ] - [ CH1] - [ first subunit of Fc region ];

(b)[BCMA-VL]-[CL]；

(g) A second subunit of [ CD3-VH ] - [ linker ] - [ Obscurin chain ] - [ Fc region ];

(h) [ CD3-VL ] - [ linker ] - [ tin chain ];

wherein a first subunit of the Fc region has a raised structure according to the knob and socket technique and a second subunit of the Fc region has a pore structure according to the knob and socket technique; the linkers in formulae (g) and (h) are preferably identical or different peptide linkers.

In some embodiments, the formula (f), formula (b), formula (g), and formula (h) are each arranged from the N-terminus to the C-terminus.

In some embodiments, the peptide linker is a flexible peptide linker. In some embodiments, the peptide linker is 3-15 amino acid residues in length. In some embodiments of the present invention, in some embodiments,the peptide linker independently has L ₁ -(GGGGS)n-L ₂ Wherein L is ₁ Is a bond, A, GS, GGS or GGGS, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and L2 is a bond, G, GG, GGG or GGGG. In some embodiments, the peptide linker is not a bond. In some embodiments, the peptide linker is GGGGS (SEQ ID NO: 157).

In some embodiments, the tin chain comprises a sequence selected from the group consisting of SEQ ID NOs: 98 to SEQ ID NO:116, said Obscurin chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:117 to SEQ ID NO:152 and SEQ ID NO:162 to SEQ ID NO:166, and a sequence of amino acids of the group consisting of seq id no. In some embodiments, the tin chain comprises SEQ ID NO:114, said Obscurin chain comprising the amino acid sequence of SEQ ID NO: 152.

In some embodiments, the antigen binding molecule has:

comprising SEQ ID NO:92, comprising the amino acid sequence of SEQ ID NO:87, comprising the amino acid sequence of SEQ ID NO:70, and a third strand comprising the amino acid sequence of SEQ ID NO:71, and a fourth strand of the amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:93, comprising the amino acid sequence of SEQ ID NO:87, comprising the amino acid sequence of SEQ ID NO:70, and a third strand comprising the amino acid sequence of SEQ ID NO:71, and a fourth strand of the amino acid sequence of seq id no.

In some embodiments, the antigen binding molecule has:

comprising SEQ ID NO:171, comprising the amino acid sequence of SEQ ID NO:83, comprising the amino acid sequence of SEQ ID NO:167, and a third strand comprising the amino acid sequence of SEQ ID NO:168, a fourth strand of an amino acid sequence of 168; or (b)

Comprising SEQ ID NO:172, comprising the amino acid sequence of SEQ ID NO:87, comprising the amino acid sequence of SEQ ID NO:167, and a third strand comprising the amino acid sequence of SEQ ID NO:168, and a fourth strand of the amino acid sequence of 168.

In another aspect, the present disclosure provides an antigen binding molecule that competes with the antigen binding molecule of any one of the preceding claims for binding to human BCMA and human CD3.

In some embodiments, the antigen binding molecule of any one of the preceding claims, which is preferably a bispecific antibody.

In another aspect, the present disclosure provides an anti-BCMA antibody comprising a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL wherein:

(iv) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in said BCMA-VH comprise the amino acid sequence of SEQ ID NO:35, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR 3; and BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 in said BCMA-VL comprise SEQ ID NO:36, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3. In some embodiments, the BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 are defined according to Kabat, IMGT, chothia, abM or Contact numbering rules.

In another aspect, the present disclosure provides an anti-BCMA antibody comprising a heavy chain variable region BCMA-VH having BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3 and a light chain variable region BCMA-VL having BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3, wherein:

In some embodiments, an anti-BCMA antibody as described previously wherein:

the heavy chain variable region BCMA-VH has: comprising SEQ ID NO:5, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:6, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:7, BCMA-HCDR3 of the amino acid sequence of seq id no; and the light chain variable region BCMA-VL has: comprising SEQ ID NO:8, a BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:9, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:10, BCMA-LCDR3 of the amino acid sequence of 10; in some embodiments, the heavy chain variable region BCMA-VH and/or light chain variable region BCMA-VL is murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or light chain variable region BCMA-VL are humanized. In some embodiments, the heavy chain variable region BCMA-VH has a heavy chain framework region derived from IGHV1-46 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 48I, 67A, 71A, 73K, 76T, and 93V; and/or said light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-39 x 01 and which is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 43S, 45Q, 48V and 71Y. In some embodiments, the BCMA-VH has FR1-3 derived from IGHV1-46 x 01 and FR4 derived from IGHJ6 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 1E, 48I, 67A, 71A, 73K, 76T, and 93V; and/or said BCMA-VL has FR1-3 derived from IGKV1-39 x 01 and FR4 derived from IGKJ2 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 43S, 45Q, 48V and 71Y. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.

In some embodiments, an anti-BCMA antibody as described previously wherein:

(i) The heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 37. SEQ ID NO:38 and SEQ ID NO:39, and the light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 40. SEQ ID NO: 41. SEQ ID NO:42 and SEQ ID NO:43 has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 37. SEQ ID NO:38 and SEQ ID NO:39, and said light chain variable region BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO:42 and SEQ ID NO:43, and a sequence of amino acids of the group consisting of seq id no. In some embodiments of the present invention, in some embodiments,

In some embodiments, an anti-BCMA antibody as described previously wherein:

(ii) The heavy chain variable region BCMA-VH has: comprising SEQ ID NO:11, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:12, and a BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:13, BCMA-HCDR3 of the amino acid sequence of 13; and the light chain variable region BCMA-VL has: comprising SEQ ID NO:14, a BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:15, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:16, BCMA-LCDR3 of the amino acid sequence of 16; in some embodiments, the heavy chain variable region BCMA-VH and/or light chain variable region BCMA-VL is murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or light chain variable region BCMA-VL are humanized. In some embodiments, the heavy chain variable region BCMA-VH has a heavy chain framework region derived from IGHV7-4-1 x 02 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 21I, 44V, 45F, 46K, 75A, 76N, and 93L; and/or said light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-8 x 01 or IGKV1-27 x 01 and which is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 43S and 66D. In some embodiments, the BCMA-VH has FR1-3 derived from IGHV7-4-1 x 02 and FR4 derived from IGHJ1 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 1E, 2I, 44V, 45F, 46K, 75A, 76N, and 93L; and/or said BCMA-VL has FR1-3 derived from IGKV1-8 x 01 or IGKV1-27 x 01 and FR4 derived from IGKJ4 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 43S and 66D. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.

In some embodiments, an anti-BCMA antibody as described previously wherein:

(ii) The heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 44. SEQ ID NO:45 and SEQ ID NO:46, and the light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 47 and SEQ ID NO:48 has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 44. SEQ ID NO:45 and SEQ ID NO:46, and said light chain variable region BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:47 and SEQ ID NO:48, and a sequence of amino acids of the group consisting of seq id no. In some embodiments of the present invention, in some embodiments,

In some embodiments, an anti-BCMA antibody as described previously wherein:

(iii) The heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 49 and SEQ ID NO:50, and the light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:51 has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 49 and SEQ ID NO:50, and said light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO: 51. In some embodiments of the present invention, in some embodiments,

In some embodiments, an anti-BCMA antibody as described previously wherein:

(iv) The heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 52 and SEQ ID NO:53, and the light chain variable region BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the amino acid sequence of SEQ ID NO:54 has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 52 and SEQ ID NO:53, and said light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:54, an amino acid sequence of seq id no. In some embodiments of the present invention, in some embodiments,

In another aspect, the present disclosure provides a pharmaceutical composition comprising: a therapeutically effective amount of an antigen binding molecule or an anti-BCMA antibody of any one of the preceding claims, and one or more pharmaceutically acceptable carriers, diluents, buffers, or excipients.

In some embodiments, the pharmaceutical composition further comprises at least one second therapeutic agent.

In another aspect, the disclosure also provides an isolated nucleic acid encoding the antigen binding molecule or anti-BCMA antibody of any one of the preceding claims.

In another aspect, the disclosure also provides a host cell comprising the aforementioned nucleic acid.

In another aspect, the disclosure also provides a method of treating a disease, the method comprising administering to a subject a therapeutically effective amount of an antigen binding molecule, anti-BCMA antibody, nucleic acid or composition of any one of the preceding claims.

In some embodiments, the disease is a B cell disorder and an autoimmune disease. In some embodiments, the disease is a B cell disorder associated with BCMA expression and autoimmune disease. In other embodiments, the B cell disorder associated with BCMA expression is a plasma cell disorder; the autoimmune disease is systemic lupus erythematosus. In some embodiments thereof, the plasma cell disorder is selected from the group consisting of: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, fahrenheit macroglobulinemia, isolated bone plasmacytoma, extramedullary plasmacytoma, bone sclerosis myeloma, heavy chain disease, unknown monoclonal gammaglobulopathy, and stasis type multiple myeloma.

In another aspect, the disclosure also provides the use of an antigen binding molecule, anti-BCMA antibody, nucleic acid or composition of any one of the preceding claims in the manufacture of a medicament for treating or preventing a disease. In some embodiments, the disease is a B cell disorder and an autoimmune disease. In some embodiments, the disease is a B cell disorder associated with BCMA expression and autoimmune disease. In other embodiments, the B cell disorder associated with BCMA expression is a plasma cell disorder; the autoimmune disease is systemic lupus erythematosus. In some embodiments thereof, the plasma cell disorder is selected from the group consisting of: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, fahrenheit macroglobulinemia, isolated bone plasmacytoma, extramedullary plasmacytoma, bone sclerosis myeloma, heavy chain disease, unknown monoclonal gammaglobulopathy, and stasis type multiple myeloma.

In another aspect, the disclosure also provides an antigen binding molecule, anti-BCMA antibody, nucleic acid or composition of any one of the preceding claims for use as a medicament. In some embodiments, the disease is a B cell disorder and an autoimmune disease. In some embodiments, the medicament is for treating or preventing B cell disorders and autoimmune diseases associated with BCMA expression. In other embodiments, the B cell disorder associated with BCMA expression is a plasma cell disorder; the autoimmune disease is systemic lupus erythematosus. In some embodiments thereof, the plasma cell disorder is selected from the group consisting of: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, fahrenheit macroglobulinemia, isolated bone plasmacytoma, extramedullary plasmacytoma, bone sclerosis myeloma, heavy chain disease, unknown monoclonal gammaglobulopathy, and stasis type multiple myeloma.

The antigen binding molecules provided by the present disclosure have the characteristics of therapeutic activity, safety, pharmacokinetic properties, and good patentability (e.g., stability).

Drawings

Fig. 1A: structural schematic diagrams of Format-11 and Format-11-6164;

Fig. 1B: a structural schematic diagram of Format-14;

fig. 1C: structural schematic diagram of Format-15.

Detailed Description

Terminology

The terminology used herein is for the purpose of describing embodiments only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

As used in the specification and in the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

Throughout the specification and claims, the words "comprise," "have," "include," and the like are to be construed as having an inclusive, rather than an exclusive or exhaustive, meaning unless the context clearly requires otherwise; that is, the meaning of "including but not limited to". Unless otherwise indicated, "comprising" includes "consisting of … …. For example, for a polypeptide comprising SEQ ID NO:5, which specifically comprises the amino acid sequence of BCMA-HCDR1 as set forth in SEQ ID NO:5, BCMA-HCDR1.

The amino acid three-letter codes and one-letter codes used in the present disclosure are as described in j.biol. Chem,243, p3558 (1968).

The term "and/or", e.g. "X and/or Y", should be understood to mean "X and Y" or "X or Y" and should be used to provide explicit support for both meanings or either meaning.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (i.e., an alpha carbon to which hydrogen, carboxyl, amino, and R groups are bound), e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to naturally occurring amino acids.

The term "amino acid mutation" includes amino acid substitutions, deletions, insertions and modifications. Any combination of substitutions, deletions, insertions, and modifications may be made to achieve the final construct, provided the final construct possesses the desired properties, such as reduced or binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino-and/or carboxy-terminus of the polypeptide chain. The specific amino acid mutation may be an amino acid substitution. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, i.e., the substitution of one amino acid with another amino acid having a different structure and/or chemical property. Amino acid substitutions include substitutions by non-naturally occurring amino acids or by derivatives of 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine). Genetic or chemical methods known in the art may be used to generate amino acid mutations. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is contemplated that other methods than genetic engineering, such as chemical modification, may be useful to alter the amino acid side chain groups. Various names may be used herein to indicate the same amino acid mutation. Herein, the amino acid residue at a particular position may be represented by position +amino acid residue, e.g., 366W, and then the amino acid residue at position 366 is represented as W. T366W indicates that the amino acid residue at position 366 is mutated from the original T to W.

The term "antigen binding molecule" is used in its broadest sense to encompass a variety of molecules that specifically bind to an antigen, including but not limited to antibodies, other polypeptides having antigen binding activity, and antibody fusion proteins fused to both. Illustratively, the antigen binding molecules herein are bispecific antigen binding molecules (e.g., bispecific antibodies), which may comprise two identical first chains and two identical second chains; or first, second, third and fourth chains that are different from each other. Illustratively, the chain is a polypeptide chain. Illustratively, the first or third polypeptide chain may be a heavy chain of an antibody or a polypeptide comprising an Fc region, and the second or fourth polypeptide chain may be a light chain of an antibody or an engineered antibody light chain.

The term "bispecific antigen binding molecule" refers to an antigen binding molecule capable of specifically binding to two different antigens or at least two different epitopes of the same antigen.

The term "antibody" is used in the broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions) so long as they exhibit the desired antigen-binding activity. "Natural antibody" refers to a naturally occurring immunoglobulin molecule. For example, a natural IgG antibody is a heterotetrameric protein of about 150,000 daltons, consisting of two light chains and two heavy chains that are disulfide bonded. From N to C-terminal, each heavy chain has one variable region (VH, also called variable heavy domain, heavy chain variable region) followed by three constant domains (CH 1, CH2 and CH 3). Similarly, from N-to C-terminus, each light chain has a variable region (VL, also known as a variable light domain, or light chain variable domain) followed by a constant light domain (light chain constant region, CL). The terms "full length antibody", "whole antibody" and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to the structure of a natural antibody or having a heavy chain comprising an Fc region.

The term "bispecific antibody" refers to an antibody (including an antibody or antigen binding fragment thereof, such as a single chain antibody) capable of specifically binding to two different antigens or two different epitopes of the same antigen. Bispecific antibodies of various structures have been disclosed in the prior art, and can be classified into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies according to the integrity of IgG molecules; bispecific antibodies that can be classified as bivalent, trivalent, tetravalent, or more multivalent depending on the number of antigen binding regions; bispecific antibodies of symmetrical structure and those of asymmetrical structure can be classified according to whether the structures are symmetrical or not. Wherein a fragment-type bispecific antibody, such as a Fab fragment lacking an Fc fragment, forms a bispecific antibody by binding 2 or more Fab fragments in one molecule, which has lower immunogenicity, and has a small molecular weight, and higher tumor tissue permeability; typical antibody structures of this type are e.g.F (ab) 2, scFv-Fab, (scFv) 2-Fab, etc. IgG-like bispecific antibodies (e.g., having Fc fragments) of relatively large molecular weight, which facilitate purification of the antibody and increase its solubility, stability, and which may also bind to the receptor FcRn, increasing antibody serum half-life, typical bispecific antibody structural models such as KiH, crossMAb, triomab quadrama, fcΔAdp, ART-Ig, biMAb, biclonics, BEAT, duoBody, azymetric, xmAb, 2:1TCBs, 1Fab-IgG TDB, fynoMAb, two-in-one/DAF, scFv-Fab-IgG, DART-Fc, LP-DART, CODV-Fab-TL, HLE-BiTE, F (Ab) 2-CrossMAb, igG- (scFv) 2, bs4Ab, DVD-Ig, tetravalent-DART-Fc, (scFv) 4-Fc, CODV-Ig, mAb2, F (Ab) 4-Crossb, etc. (see ArF.Labjn et al, nature Reviews Drug Discovery volume, page201608; immunol, 2019; 20141, etc.).

The term "variable region" or "variable domain" refers to the domain of an antibody that is involved in binding an antigen by an antibody in the heavy or light chain. Herein, the heavy chain variable region in the first antigen-binding domain that specifically binds BCMA is designated BCMA-VH and the light chain variable region is designated BCMA-VL; the heavy chain variable region in the second antigen-binding domain that specifically binds CD3 is designated CD3-VH and the light chain variable region is designated CD3-VL. VH and VL each comprise four conserved Framework Regions (FR) and three Complementarity Determining Regions (CDRs). Wherein the term "complementarity determining region" or "CDR" refers to a region within the variable domain that primarily contributes to binding to an antigen; "framework" or "FR" refers to variable domain residues other than CDR residues. VH comprises 3 CDR regions: HCDR1, HCDR2 and HCDR3; VL comprises 3 CDR regions: LCDR1, LCDR2, and LCDR3. Herein, 3 CDR regions in BCMA-VH are denoted as BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3, respectively; the 3 CDRs in BCMA-VL are labeled BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3, respectively; the 3 CDRs in the CD3-VH are designated CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3, respectively; the 3 CDRs in CD3-VL are designated as CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3, respectively. Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen binding specificity.

The amino acid sequence boundaries of the CDRs can be determined by various well-known schemes, such as: "Kabat" numbering convention (see Kabat et al (1991), "Sequences of Proteins of Immunological Interest", 5 th edition, public Health Service, national Institutes of Health, bethesda, MD), "Chothia" numbering convention, "ABM" numbering convention, "contact" numbering convention (see Martin, ACR.protein Sequence and Structure Analysis of Antibody Variable Domains [ J ]. 2001) and ImMunoGenTics (IMGT) numbering convention (Lefranc, M.P. et al, dev.Comp. Immunol.,27, 55-77 (2003); front immunol.2018Oct 16; 9:2278), etc.; the correspondence between the various numbering systems is well known to those skilled in the art, and is exemplary, as shown in table 1 below.

TABLE 1 relationship between CDR numbering systems

CDR	IMGT	Kabat	AbM	Chothia	Contact
HCDR1	27-38	31-35	26-35	26-32	30-35
HCDR2	56-65	50-65	50-58	52-56	47-58
HCDR3	105-117	95-102	95-102	95-102	93-101
LCDR1	27-38	24-34	24-34	24-34	30-36
LCDR2	56-65	50-56	50-56	50-56	46-55
LCDR3	105-117	89-97	89-97	89-97	89-96

Unless otherwise indicated, the variable region and CDR sequences in the examples of the present disclosure apply the "Kabat" numbering convention.

The term "antibody fragment" refers to a molecule other than an intact antibody that comprises the intact antibodyA portion of an antibody that retains the antigen binding capacity of the intact antibody. Examples of antibody fragments include, but are not limited to Fv, fab, fab ', fab ' -SH, F (ab ') ₂ Single domain antibodies, single chain Fab (scFab), diabodies, linear antibodies, single chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of the antibody heavy chain, including the native Fc region and engineered Fc region. In some embodiments, the Fc region comprises two subunits, which may be the same or different. In some embodiments, the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. Suitable native sequence Fc regions for antibodies described herein include human IgG1, igG2 (IgG 2A, igG 2B), igG3, and IgG4. The numbering convention for the Fc region is EU numbering convention, unless otherwise indicated.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chains are derived from a particular source or species, while the remainder of the heavy and/or light chains are derived from a different source or species.

The term "humanized" antibody is an antibody that retains the reactivity of a non-human antibody while having lower immunogenicity in humans. For example, humanization may be achieved by retaining non-human CDR regions and replacing the remainder of the antibody with human counterparts (i.e., framework regions portions of the constant and variable regions).

The term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding ligand (e.g., an antigen). As used herein, unless otherwise indicated, "binding affinity" refers to an internal binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its ligand Y can generally be expressed by a dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. The term "kasloc" or "ka" refers to the rate of association of a particular antibody-antigen interaction, while the term "kdis" or "kd" as used herein is intended to refer to the rate of dissociation of a particular antibody-antigen interaction. As used herein, the term "KD" refers to the dissociation constant, which is obtained from the ratio of KD to ka (i.e., KD/ka) and is expressed as molar concentration (M). The KD values of antibodies can be determined using methods known in the art, for example: methods for determining antibody KD include measuring surface plasmon resonance using a biosensing system such as a system, or measuring affinity in solution by Solution Equilibrium Titration (SET).

The term "effector functions" refers to those biological activities attributable to the Fc region of an antibody (native sequence Fc region or amino acid sequence variant Fc region) and which vary with the antibody isotype. Examples of antibody effector functions include: c1q binding and complement dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

The term "monoclonal antibody" refers to a population of substantially homogeneous antibodies or members thereof, i.e., the amino acid sequences of the antibody molecules comprised in the population are identical, except for natural mutations that may be present in minor amounts. In contrast, "polyclonal antibody" preparations typically include a plurality of different antibodies having different amino acid sequences in their variable domains, which are typically specific for different epitopes. "monoclonal" refers to the characteristics of the antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by a particular method. In some embodiments, the antibodies provided by the present disclosure are monoclonal antibodies.

The term "antigen" refers to a molecule or portion of a molecule that is capable of being selectively recognized or bound by an antigen binding protein (including, for example, an antibody). An antigen may have one or more epitopes that are capable of interacting with different antigen binding proteins (e.g., antibodies).

The term "epitope" refers to a region (area or region) on an antigen that is capable of specifically binding to an antibody or antigen binding fragment thereof. Epitopes can be formed by consecutive amino acids (linear epitopes); or comprise non-contiguous amino acids (conformational epitopes), e.g. spatially accessed by folding of the antigen (i.e. tertiary folding of the antigen by proteinaceous nature). Conformational epitopes differ from linear epitopes in that: in the presence of denaturing solvents, binding of the antibody to conformational epitopes is lost. An epitope comprises at least 3, at least 4, at least 5, at least 6, at least 7, or 8-10 amino acids in a unique spatial conformation. Antibodies that bind to a particular epitope (i.e., those that bind to the same epitope) can be screened using routine methods in the art, such as, but not limited to, alanine scanning, peptide blotting (see meth.mol.biol.248 (2004) 443-463), peptide cleavage analysis, epitope excision, epitope extraction, chemical modification of the antigen (see prot.sci.9 (2000) 487-496), and cross-blocking (see "Antibodies", harlow and Lane (Cold Spring Harbor Press, cold Spring harbor., NY)).

An antibody that "competes for binding with" a reference antibody refers to an antibody that blocks the binding of the reference antibody to an antigen by 50% or more, or an antibody whose binding to an antigen is blocked by 50% or more by the reference antibody in a competition assay.

The term "capable of specifically binding", "specifically binding" or "binding" refers to an antibody that is capable of binding with higher affinity to an antigen or epitope within the antigen than other antigens or epitopes. Typically, the antibodies are present in an amount of about 1X 10 ^-7 M or less (e.g., about 1X 10) ^-8 M or less) binds to an antigen or an epitope within an antigen. In some embodiments, the antibody binds to an antigen with a KD of 10% or less (e.g., 1%) of the KD of the antibody to a non-specific antigen (e.g., BSA, casein). KD can be measured using known methods, for example, byAs measured by surface plasmon resonance. However, antibodies that specifically bind to an antigen or an epitope within an antigen may be cross-reactive to other related antigens, e.g., to antibodies derived from other species (homologous), such as humans or monkeys, e.g., cynomolgus macaque (Macaca fascicularis) (cynomolgus, cyno), chimpanzee (Pan troglymes) (chimpanzee, chimp)), or marmoset (Callithrix jacchus) (common marmoset, marmos)et) are cross-reactive.

The terms "anti-BCMA antibody" and "antigen binding domain that specifically binds BCMA" refer to an antibody or domain that is capable of binding BCMA with sufficient affinity such that the anti-BCMA antibody or a molecule containing the domain can be used as a diagnostic and/or therapeutic agent for targeting BCMA. In certain embodiments, an antibody or domain that binds BCMA has a dissociation constant (KD) of < about 1 μm, < about 100nM, < about 10nM, as measured by FACS method. In certain embodiments, an anti-BCMA antibody or antigen binding domain that specifically binds BCMA binds to a BCMA epitope that is conserved in BCMA from different species.

The term "antigen binding domain that specifically binds CD 3" refers to a domain that is capable of binding CD3 with sufficient affinity such that molecules containing the domain can be used as diagnostic and/or therapeutic agents for targeting CD 3. In certain embodiments, the antigen binding domain that specifically binds CD3 binds a CD3 epitope that is conserved in CD3 from a different species. Herein, an antigen binding domain may refer to an antigen binding moiety, such as a Fab, a substituted Fab, or a scFv.

The term "linker" refers to a linking unit that links two polypeptide fragments. In this context, the linkers present in the same structure may be the same or different. The linker may be a peptide linker comprising one or more amino acids, typically about 1-30, 2-24 or 3-15 amino acids. The linkers used herein may be the same or different. When "-" appears in the formula, it means that the units on both sides are directly linked by covalent bonds. When the term "bond" is present in a structural unit, it means that the unit has no amino acid and the units on either side of the unit are directly linked.

The terms "cytotoxicity of antibody-dependent cells", "antibody-dependent cell-mediated cytotoxicity" or "ADCC" are mechanisms that induce cell death, which depend on the interaction of antibody-coated target cells with effector cells having lytic activity, such as natural killer cells (NK), monocytes, macrophages and neutrophils, via fcγr expressed on the effector cells. For example, NK cells express fcyriiia, whereas monocytes express fcyri, fcyrii, and fcyriiia. ADCC activity of the antibodies provided herein can be assessed using in vitro assays using cells expressing the antigen as target cells and NK cells as effector cells. Cell lysis is detected based on a label (e.g., a radioactive substrate, fluorescent dye, or native intracellular protein) released from the lysed cells.

The term "antibody-dependent cellular phagocytosis" ("ADCP") refers to the mechanism by which antibody-coated target cells are eliminated by internalization of phagocytes, such as macrophages or dendritic cells.

The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism that induces cell death in which the Fc-effector domain of a target binding antibody binds to and activates complement components C1q, C1q in turn activating the complement cascade, resulting in target cell death. Activation of complement can also result in deposition of complement components on the target cell surface that promote CDC by binding to complement receptors (e.g., CR 3) on leukocytes.

The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, have similar binding properties as the reference nucleic acid, and are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location different from its natural chromosomal location. An isolated nucleic acid encoding an anti-BCMA antibody or the antigen binding molecule refers to one or more nucleic acid molecules encoding the heavy and light chains of the antibody (or fragments thereof), including such one or more nucleic acid molecules in a single vector or separate vectors, and such one or more nucleic acid molecules present at one or more positions in a host cell. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be obtained by generating sequences in which the third position of one or more selected (or all) codons is substituted with degenerate bases and/or deoxyinosine residues, as described in detail below.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are corresponding artificial chemical mimics of a naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

The term "sequence identity" refers to the degree (percent) to which the amino acids/nucleic acids of two sequences are identical at equivalent positions when optimally aligned; wherein gaps are allowed to be introduced as necessary during the alignment to obtain the maximum percent sequence identity, but any conservative substitutions are not considered to form part of the sequence identity. To determine percent sequence identity, the alignment may be accomplished by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN-2, or Megalign (DNASTAR) software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences compared.

The term "fusion" or "linkage" refers to the attachment of components (e.g., antigen binding domain and Fc domain) by covalent bonds, either directly or via one or more linkers. When the linker is a peptide linker, the covalent bond is a peptide bond.

The term "vector" means a polynucleotide molecule capable of delivering another polynucleotide to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop in which additional DNA segments may be ligated. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV 2), wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating with the host genome. The term "expression vector" or "expression construct" refers to a vector suitable for transforming a host cell and containing a nucleic acid sequence that directs and/or controls (along with the host cell) the expression of one or more heterologous coding regions to which it is operably linked. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, when present, RNA splicing of the coding region to which they are operably linked.

The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells" which include primary (primary) transformed cells and progeny derived therefrom, irrespective of the number of passages (passages). The progeny may not be exactly identical in nucleic acid content to the parent cell, but may contain a mutation. Herein, the term includes mutant progeny that have the same function or biological activity as the cells selected or selected for in the primary transformed cell. Host cells include prokaryotic and eukaryotic host cells, where eukaryotic host cells include, but are not limited to, mammalian cells, insect cell line plant cells, and fungal cells. Mammalian host cells include human, mouse, rat, canine, monkey, pig, goat, bovine, equine, and hamster cells, including, but not limited to, chinese Hamster Ovary (CHO) cells, NSO, SP2 cells, heLa cells, baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), human hepatocellular carcinoma cells (e.g., hep G2), a549 cells, 3T3 cells, and HEK-293 cells. Fungal cells include yeast and filamentous fungal cells, including, for example, pichia pastoris (Pichia pastoris), pichia finland (Pichia finlandica), pichia pastoris (Pichia trehalophila), colorado Ma Bichi yeast (Pichia koalae), pichia membranaceus (Pichia membranaefaciens), pichia pastoris (Pichia minuta) (Ogataea minuta, pichia lindneri), pichia pastoris (Aspergillus nidulans), pichia pastoris (Pichia thermotolerans), liu Bichi yeast (Pichia salictaria), pichia guerbeta, pi Jiepu Pichia pastoris (Pichia pijperi), pichia pastoris (Pichia stiptis), methanol yeast (Pichia methanolica), pichia, saccharomyces cerevisiae (Saccharomyces cerevisiae), saccharomyces, hansenula polymorpha (Hansenula polymorpha), kluyveromyces lactis (Kluyveromyces lactis), candida albicans (Candida albicans), aspergillus nidulans (3232), aspergillus niger (Aspergillus niger), aspergillus niger (86), aspergillus oryzae (Fusarium venenatum), fusarium roseum (Fusarium venenatum). Pichia, any Saccharomyces, hansenula polymorpha (Hansenula polymorpha), any Kluyveromyces, candida albicans (Candida albicans), any Aspergillus, trichoderma reesei (Trichoderma reesei), lekkera (Chrysosporium lucknowense), any Fusarium, yarrowia lipolytica (Yarrowia lipolytica), and Neurospora crassa (Neurospora crassa).

As used in the present application, the expressions "cell", "cell line" and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include primary subject cells and cultures derived therefrom, regardless of the number of passages. It is also understood that not all progeny will have the exact same DNA content, either due to deliberate or unintentional mutation. Including mutant progeny that have the same function or biological activity as the original transformed cells from which they were selected.

"optional" or "optionally" means that the subsequently described feature may, but need not, occur, and that the inclusion of the feature may or may not be taken.

The term "pharmaceutical composition" means a mixture comprising one or more antibodies or antigen binding molecules described herein and other chemical components, such as physiological/pharmaceutically acceptable carriers and excipients.

The term "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation (formulation) that is different from the active ingredient and is non-toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

The term "subject" or "individual" includes both human and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as non-human primates (e.g., cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. The terms "patient" or "subject" are used interchangeably herein unless specifically indicated. As used herein, the term "cynomolgus monkey (cyno)" or "cynomolgus monkey (cynomolgus)" refers to cynomolgus monkey (Macaca fascicularis). In certain embodiments, the individual or subject is a human.

"administering" or "administering," when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contacting an exogenous pharmaceutical, therapeutic, diagnostic, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid.

The term "sample" refers to a collection (e.g., fluid, cell, or tissue) isolated from a subject, as well as fluid, cell, or tissue present in the subject. Exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph, urine, saliva, cyst fluid, tears, fecal matter, sputum, mucous secretions of tissues or organs, vaginal secretions, ascites, pleura, pericardium, peritoneal and other body cavity fluids, fluids collected by bronchial lavage, synovial fluid, liquid solutions in contact with a subject or biological source, such as culture media (including conditioned media), lavage fluid, and the like, tissue biopsy samples, fine needle punctures, surgically excised tissue, organ cultures, or cell cultures.

"treatment" and "treatment" (and grammatical variations thereof) refer to a clinical intervention intended to be administered to an individual being treated, and may be performed for prophylactic purposes, or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and regression or improved prognosis. In some embodiments, the molecules of the present disclosure are used to delay the formation of a disease or to slow the progression of a disease.

An "effective amount" is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate such symptoms and/or underlying etiology, prevent the appearance of symptoms and/or underlying etiology, and/or ameliorate or improve the damage caused by or associated with a disease state. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or condition, particularly a state or condition associated with the disease state, or otherwise hinder, delay or reverse the progression of the disease state, or any undesirable condition associated with the disease. A "prophylactically effective amount" is an amount that, when administered to a subject, will have a predetermined prophylactic effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or related symptoms. The complete therapeutic or prophylactic effect does not necessarily occur immediately after administration of one dose, but may occur after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount may be administered in one or more administrations. The "therapeutically effective amount" and "prophylactically effective amount" may vary depending on a variety of factors: such as the disease state, age, sex, and weight of the individual, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in the individual. Exemplary indicators of effective therapeutic agents or combinations of therapeutic agents include, for example, improved health of a patient.

Antigen binding molecules of the disclosure

The present disclosure provides antigen binding molecules having a number of advantageous properties, such as affinity, specificity for cell surface BCMA, activity to specifically activate T cells in the presence of BCMA, therapeutic activity, safety (e.g., lower cytokine release), pharmacokinetic properties, and patentability (e.g., yield, purity, stability, etc.).

Exemplary antigen binding molecules

In one aspect, the present disclosure provides an antigen binding molecule comprising at least one first antigen binding domain that specifically binds BCMA and at least one second antigen binding domain that specifically binds CD 3. In particular, the antigen binding molecules of the present disclosure have:

a. high affinity for membrane surface BCMA. In some embodiments, the antigen binding molecule binds to BCMA protein expressed by stably transformed cell line K562 overexpressing human BCMA with an affinity EC50 of less than 20nM, as determined by FACS method. In some embodiments, the antigen binding molecule binds to BCMA protein expressed by steady cell line CHOK1 that overexpresses cynomolgus BCMA with an affinity EC50 of less than 100nM, as determined by FACS method. Specific test methods are shown in test example 1.

b. Ability to preferentially bind membrane surface BCMA without being affected by high concentrations of soluble BCMA, in some embodiments, 200ng/ml soluble BCMA in the presence of said antigen binding molecule does not change by more than 30% in affinity EC50 for binding to BCMA protein expressed by endogenous BCMA expressing myeloma cell line NCIH929, said affinity EC ₅₀ Is determined by FACS method. Specific test methods are shown in test example 2.

c. Specifically activate T cell activity in vitro. In some embodiments, the antigen binding molecule activates T cells when BCMA expressing cells, but does not activate T cells in the presence of cells that do not express BCMA. Specific test methods are shown in test example 3.

d. Specific killing activity against BCMA expressing cells in vitro. In some embodiments, the antigen binding molecule can specifically kill cells that express BCMA, but cannot kill cells that do not express BCMA. Specific test methods are shown in test example 4.

e. Low levels of cytokine (IL 6 and ifnγ) release are induced. In some embodiments, the antigen binding molecule produces no more than 2ng/mL IL-6 when killing BCMA expressing cell line U266B at a concentration of 100nM, and the cytokine release is detected by ELISA. In some embodiments, the antigen binding molecule produces no more than 10ng/mL ifnγ when killing BCMA expressing cell line U266B at a concentration of 20nM or less, and the cytokine release is detected by HTRF method. Specific test methods are shown in test example 6.

f. Stronger therapeutic activity in vivo. (test examples 7 and 8).

(i) The heavy chain variable region BCMA-VH has: the amino acid sequence is shown in SEQ ID NO:5, and the amino acid sequence of the BCMA-HCDR1 is shown as SEQ ID NO:6, and the amino acid sequence of BCMA-HCDR2 is shown as SEQ ID NO: BCMA-HCDR3 shown in fig. 7; and the light chain variable region BCMA-VL has: the amino acid sequence is shown in SEQ ID NO:8, and the amino acid sequence of the BCMA-LCDR1 is shown as SEQ ID NO:9, and a BCMA-LCDR2 having an amino acid sequence as set forth in SEQ ID NO: BCMA-LCDR3 shown in fig. 10; or (b)

(ii) The heavy chain variable region BCMA-VH has: the amino acid sequence is shown in SEQ ID NO:11, and the amino acid sequence of the BCMA-HCDR1 is shown as SEQ ID NO:12, and a BCMA-HCDR2 having an amino acid sequence as set forth in SEQ ID NO:13, BCMA-HCDR3; and the light chain variable region BCMA-VL has: the amino acid sequence is shown in SEQ ID NO:14, and the amino acid sequence of the BCMA-LCDR1 is shown in SEQ ID NO:15, and a BCMA-LCDR2 having an amino acid sequence as set forth in SEQ ID NO:16, BCMA-LCDR3; or (b)

(iii) The heavy chain variable region BCMA-VH has: the amino acid sequence is shown in SEQ ID NO:17, and the amino acid sequence of the BCMA-HCDR1 is shown as SEQ ID NO:18, and a BCMA-HCDR2 having an amino acid sequence as set forth in SEQ ID NO:19 a BCMA-HCDR3; and the light chain variable region BCMA-VL has: the amino acid sequence is shown in SEQ ID NO:20, and the amino acid sequence of the BCMA-LCDR1 is shown in SEQ ID NO:21, and a BCMA-LCDR2 having an amino acid sequence as set forth in SEQ ID NO: BCMA-LCDR3 shown at 22; or (b)

(iv) The heavy chain variable region BCMA-VH has: the amino acid sequence is shown in SEQ ID NO:23, and the amino acid sequence of the BCMA-HCDR1 is shown as SEQ ID NO:24, and a BCMA-HCDR2 having an amino acid sequence as set forth in SEQ ID NO:25, BCMA-HCDR3; and the light chain variable region BCMA-VL has: the amino acid sequence is shown in SEQ ID NO:26, and the amino acid sequence of the BCMA-LCDR1 is shown in SEQ ID NO:27, and a BCMA-LCDR2 having the amino acid sequence shown in SEQ ID NO:28, BCMA-LCDR3.

In some embodiments, an antigen binding molecule as described previously, wherein

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:29 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:30, or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:37 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:40, or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:38 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:40, or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:39 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:40, or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:37 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:41 or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:37 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:42, or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:37 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:43 or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:31 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:32 or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:44 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:47, or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:45 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:47, or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:46 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:47, or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:44 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:48, or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:45 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:48, or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:46 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:48, or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:33 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:34 or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:49 and the light chain variable region BCMA-VL has the amino acid sequence shown in SEQ ID NO:51, or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:50 and the amino acid sequence of the light chain variable region BCMA-VL is shown as SEQ ID NO:51, or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:35 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:36 or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:52 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:54 or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:53 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: indicated at 54.

(i) The heavy chain variable region CD3-VH has: the amino acid sequence is shown in SEQ ID NO:55, and the amino acid sequence of the CD3-HCDR1 is shown as SEQ ID NO:56, and a CD3-HCDR2 having an amino acid sequence as set forth in SEQ ID NO: 57-CD 3-HCDR3; and the light chain variable region CD3-VL has: the amino acid sequence is shown in SEQ ID NO:58, and the amino acid sequence of the CD3-LCDR1 is shown in SEQ ID NO: 59, and a CD3-LCDR2 having an amino acid sequence as set forth in SEQ ID NO:60, CD3-LCDR3; or (b)

(ii) The heavy chain variable region CD3-VH has: the amino acid sequence is shown in SEQ ID NO:55, and the amino acid sequence of the CD3-HCDR1 is shown as SEQ ID NO:61, and a CD3-HCDR2 having an amino acid sequence as set forth in SEQ ID NO:62 CD3-HCDR3; and the light chain variable region CD3-VL has: the amino acid sequence is shown in SEQ ID NO:58, and the amino acid sequence of the CD3-LCDR1 is shown in SEQ ID NO:59, and a CD3-LCDR2 having an amino acid sequence as set forth in SEQ ID NO:60, CD3-LCDR3.

In some embodiments, the CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3 are defined according to the Kabat numbering convention.

In some embodiments, the antigen binding molecule of any one of the preceding claims, the heavy chain variable region CD3-VH has an amino acid sequence set forth in SEQ ID NO:63 and the light chain variable region CD3-VL has the amino acid sequence set forth in SEQ ID NO: indicated at 64; or (b)

The amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO:65 and the light chain variable region CD3-VL having the amino acid sequence set forth in SEQ ID NO: shown at 66.

In some embodiments, the antigen binding molecule of any one of the preceding claims, wherein the heavy chain variable region BCMA-VH has: the amino acid sequence is shown in SEQ ID NO:5, and the amino acid sequence of the BCMA-HCDR1 is shown as SEQ ID NO:6, and the amino acid sequence of BCMA-HCDR2 is shown as SEQ ID NO: BCMA-HCDR3 shown in fig. 7; and the light chain variable region BCMA-VL has: the amino acid sequence is shown in SEQ ID NO:8, and the amino acid sequence of the BCMA-LCDR1 is shown as SEQ ID NO:9, and a BCMA-LCDR2 having an amino acid sequence as set forth in SEQ ID NO:10, and

The heavy chain variable region CD3-VH has: the amino acid sequence is shown in SEQ ID NO:55, and the amino acid sequence of the CD3-HCDR1 is shown as SEQ ID NO:56, and a polypeptide comprising the amino acid sequence of SEQ ID NO: the amino acid sequence is shown in SEQ ID NO: 57-CD 3-HCDR3; and the light chain variable region CD3-VL has: the amino acid sequence is shown in SEQ ID NO:58, and the amino acid sequence of the CD3-LCDR1 is shown in SEQ ID NO:59, and a CD3-LCDR2 having an amino acid sequence as set forth in SEQ ID NO:60, CD3-LCDR3.

In some embodiments, the amino acid sequence of the heavy chain variable region BCMA-VH is set forth in SEQ ID NO:37 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:40, and

the amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO:63 and the light chain variable region CD3-VL has the amino acid sequence set forth in SEQ ID NO: shown at 64.

The heavy chain variable region CD3-VH has: the amino acid sequence is shown in SEQ ID NO:55, and the amino acid sequence of the CD3-HCDR1 is shown as SEQ ID NO:61, and a CD3-HCDR2 having an amino acid sequence as set forth in SEQ ID NO:62 CD3-HCDR3; and the light chain variable region CD3-VL has: the amino acid sequence is shown in SEQ ID NO:58, and the amino acid sequence of the CD3-LCDR1 is shown in SEQ ID NO:59, and a CD3-LCDR2 having an amino acid sequence as set forth in SEQ ID NO:60, CD3-LCDR3.

In some embodiments, the amino acid sequence of the heavy chain variable region BCMA-VH is set forth in SEQ ID NO:37 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:43, and

the amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO:65 and the light chain variable region CD3-VL having the amino acid sequence set forth in SEQ ID NO:66, shown at 66

In some embodiments, the antigen binding molecule of any one of the preceding claims, wherein the antigen binding molecule further comprises an Fc region comprising two subunits capable of associating. In some embodiments, the two subunits are the same or different first and second subunits. In some embodiments, the Fc region is an IgG Fc region, particularly IgG ₁ An Fc region.

Structure of antigen binding molecule

The present disclosure provides a bivalent antigen binding molecule (2+2format) that specifically binds BCMA and bivalent specifically binds CD 3.

Illustratively, the antigen binding molecule comprises two first chains having a structure represented by formula (a) and two second chains having a structure represented by formula (b),

(b)[BCMA-VL]-[CL]。

exemplary molecules include, an antigen binding molecule having:

Illustratively, the antigen binding molecule comprises two first chains having the structure of formula (a-1) and two second chains having the structure of formula (b-1),

(a-1) [ CD3-VH ] - [ CH1] - [ BCMA-VH ] - [ linker ] - [ BCMA-VL ] - [ linker ] - [ one subunit of Fc region ]; the linkers in formula (a) are preferably identical or different peptide linkers;

(b-1)[CD3-VL]-[CL]。

the present disclosure also provides a monovalent antigen binding molecule (1+1format) that specifically binds BCMA and monovalent specifically binds CD 3.

Illustratively, it comprises a first chain having a structure represented by formula (c), a second chain having a structure represented by formula (b), a third chain having a structure represented by formula (d), and a fourth chain having a structure represented by formula (e),

(c) A second subunit of [ BCMA-VH ] - [ CH1] - [ Fc region ];

(b)[BCMA-VL]-[CL]；

(e) [ CD3-VL ] - [ linker ] - [ Obscurin strand ];

the linkers in formulae (d) and (e) are preferably identical or different peptide linkers.

Illustratively, the antigen binding molecule comprises a first chain having a structure represented by formula (c), a second chain having a structure represented by formula (b), a third chain having a structure represented by formula (d-1), and a fourth chain having a structure represented by formula (e-1),

(c) A second subunit of [ BCMA-VH ] - [ CH1] - [ Fc region ];

(b)[BCMA-VL]-[CL]；

(d-1) [ CD3-VH ] - [ linker ] - [ Obscurin chain ] - [ first subunit of Fc region ];

(e-1) [ CD3-VL ] - [ linker ] - [ tin chain ];

the linkers in formulas (d-1) and (e-1) are preferably identical or different peptide linkers.

Illustratively, the antigen binding molecule comprises a first chain having a structure represented by formula (c-1), a second chain having a structure represented by formula (b-1), a third chain having a structure represented by formula (d-2), and a fourth chain having a structure represented by formula (e-2),

(c-1) [ CD3-VH ] - [ CH1] - [ second subunit of Fc region ];

(b-1)[CD3-VL]-[CL]；

(d-2) [ BCMA-VH ] - [ linker ] - [ tin chain ] - [ first subunit of Fc region ];

(e-2) [ BCMA-VL ] - [ linker ] - [ Obscurin strand ];

the linkers in formulas (d-2) and (e-2) are preferably identical or different peptide linkers.

Illustratively, the antigen binding molecule comprises a first chain having a structure represented by formula (c-1), a second chain having a structure represented by formula (b-1), a third chain having a structure represented by formula (d-3), and a fourth chain having a structure represented by formula (e-3),

(c-1) [ CD3-VH ] - [ CH1] - [ second subunit of Fc region ];

(b-1)[CD3-VL]-[CL]；

(d-3) [ BCMA-VH ] - [ linker ] - [ Obscurin chain ] - [ first subunit of Fc region ];

(e-3) [ BCMA-VL ] - [ linker ] - [ tin chain ];

the linkers in formulas (d-3) and (e-3) are preferably identical or different peptide linkers.

Illustratively, the antigen binding molecule comprises a first chain having a structure represented by formula (f), a second chain having a structure represented by formula (b), a third chain having a structure represented by formula (g), and a fourth chain having a structure represented by formula (h),

(f) [ BCMA-VH ] - [ CH1] - [ first subunit of Fc region ];

(b)[BCMA-VL]-[CL]；

(h) [ CD3-VL ] - [ linker ] - [ tin chain ];

the linkers in formulae (g) and (h) are preferably identical or different peptide linkers.

Illustratively, the antigen binding molecule comprises a first chain having a structure represented by formula (f), a second chain having a structure represented by formula (b), a third chain having a structure represented by formula (g-1), and a fourth chain having a structure represented by formula (h-1),

(f) [ BCMA-VH ] - [ CH1] - [ first subunit of Fc region ];

(b)[BCMA-VL]-[CL]；

(g-1) [ CD3-VH ] - [ linker ] - [ tin chain ] - [ second subunit of Fc region ];

(h-1) [ CD3-VL ] - [ linker ] - [ Obscurin strand ];

the linkers in formulas (g-1) and (h-1) are preferably identical or different peptide linkers.

Illustratively, the antigen binding molecule comprises a first chain having a structure represented by formula (c-2), a second chain having a structure represented by formula (b-1), a third chain having a structure represented by formula (d-4), and a fourth chain having a structure represented by formula (e-2),

(c-2) [ CD3-VH ] - [ CH1] - [ first subunit of Fc region ];

(b-1)[CD3-VL]-[CL]；

(d-4) [ BCMA-VH ] - [ linker ] - [ tin chain ] - [ second subunit of Fc region ];

(e-2) [ BCMA-VL ] - [ linker ] - [ Obscurin strand ];

the linkers in formulas (d-4) and (e-2) are preferably identical or different peptide linkers.

Illustratively, the antigen binding molecule comprises a first chain having a structure represented by formula (c-2), a second chain having a structure represented by formula (b-1), a third chain having a structure represented by formula (d-5), and a fourth chain having a structure represented by formula (e-3),

(c-2) [ CD3-VH ] - [ CH1] - [ first subunit of Fc region ];

(b-1)[CD3-VL]-[CL]；

(d-5) [ BCMA-VH ] - [ linker ] - [ Obscurin chain ] - [ second subunit of Fc region ];

(e-3) [ BCMA-VL ] - [ linker ] - [ tin chain ];

the linkers in formulas (d-5) and (e-3) are preferably identical or different peptide linkers.

Exemplary anti-BCMA antibodies

In one aspect, the present disclosure provides an anti-BCMA antibody comprising a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL wherein:

(ii) The heavy chain variable region BCMA-VH has: the amino acid sequence is shown in SEQ ID NO:11, and the amino acid sequence of the BCMA-HCDR1 is shown as SEQ ID NO:12, and the amino acid sequence of BCMA-HCDR2 shown in SEQ ID NO:13, BCMA-HCDR3; and the light chain variable region BCMA-VL has: the amino acid sequence is shown in SEQ ID NO:14, and the amino acid sequence of the BCMA-LCDR1 is shown in SEQ ID NO:15, and a BCMA-LCDR2 having an amino acid sequence as set forth in SEQ ID NO:16, BCMA-LCDR3; or (b)

In some embodiments, an anti-BCMA antibody as described previously comprising:

Variants of antigen binding molecules or anti-BCMA antibodies

In certain embodiments, amino acid sequence variants of the antigen binding molecules provided herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of antibodies. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into, and/or substitutions of residues within the amino acid sequence of the antigen binding molecule. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding properties.

Substitution, insertion, and deletion variants

In certain embodiments, variants of antigen binding molecules having one or more amino acid substitutions are provided. Substitutions are made at sites of interest, including CDRs and FR. Conservative substitutions are shown in table 2 under the heading of "preferred substitutions". More substantial variations are provided in table 2 under the heading of "exemplary substitutions" and are described further below with reference to the amino acid side chain class. Amino acid substitutions may be introduced into the antibody of interest and the product screened for a desired activity, such as retention/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

TABLE 2 substitution of amino acids

Original residue	Exemplary substitution	Preferred substitution
Ala(A)	Val；Leu；Ile	Val
Arg(R)	Lys；Gln；Asn	Lys
Asn(N)	Gln；His；Asp,Lys；Arg	Gln
Asp(D)	Glu；Asn	Glu
Cys(C)	Ser；Ala	Ser
Gln(Q)	Asn；Glu	Asn
Glu(E)	Asp；Gln	Asp
Gly(G)	Ala	Ala
His(H)	Asn；Gln；Lys；Arg	Arg
Ile(I)	Leu; val; met; ala; phe; norleucine (N-leucine)	Leu
Leu(L)	Norleucine; ile; val; met; ala; phe (Phe)	Ile
Lys(K)	Arg；Gln；Asn	Arg
Met(M)	Leu；Phe；Ile	Leu
Phe(F)	Trp；Leu；Val；Ile；Ala；Tyr	Tyr
Pro(P)	Ala	Ala
Ser(S)	Thr	Thr
Thr(T)	Ser	Ser
Trp(W)	Tyr；Phe	Tyr
Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile；Leu; met; phe; ala; norleucine (N-leucine)	Leu

Amino acids can be grouped according to common side chain characteristics as follows:

(1) Hydrophobic: nle, met, ala, val, leu, ile;

(2) Neutral, hydrophilic: cys, ser, thr, asn, gin;

(3) Acidic: asp, glu;

(4) Alkaline: his, lys, arg;

(5) Residues that affect chain orientation (orientation): gly, pro;

(6) Aromatic: trp, tyr, phe.

Non-conservative substitutions refer to the replacement of a member of one class with a member of another class.

One class of substitution variants involves substitution of one or more CDR residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further investigation will have alterations (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody, and/or will substantially retain certain biological properties of the parent antibody. One exemplary substitution variant is an affinity matured antibody, which can be conveniently produced, for example, using phage display-based affinity maturation techniques (such as those described herein). Briefly, one or more CDR residues are mutated and the variant antibody is displayed on phage and screened for a particular biological activity (e.g., binding affinity). Changes (e.g., substitutions) may be made to the CDRs, for example, to improve antibody affinity. Such changes may be made to CDR "hot spots" (i.e., residues encoded by codons that undergo mutations at high frequencies, and/or residues that contact an antigen during the somatic maturation process) while testing binding affinity to the resulting variant VH or VL. In some embodiments of affinity maturation, diversity is introduced into the variable gene selected for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). Then, a secondary library is created. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves CDR-directed approaches in which several CDR residues (e.g., 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. In particular, HCDR3 and LCDR3 are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, provided that such changes do not substantially reduce the ability of the antibody to bind to an antigen. For example, conservative changes (e.g., conservative substitutions, as provided herein) may be made to the CDRs that do not substantially reduce binding affinity. Such changes do not occur at antigen-contacting residues. In certain embodiments of the variant VH and VL sequences provided above, each CDR is unchanged or contains no more than 1, 2, or 3 amino acid substitutions.

One method that may be used to identify residues or regions in an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis". In this method, a residue or group of residues (e.g., charged residues such as Arg, asp, his, lys and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., ala or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. In addition, the contact point between the antibody and the antigen can be identified by studying the crystal structure of the antigen-antibody complex. These contact residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants may be screened to determine whether they contain the desired property.

Amino acid sequence insertions include: a polypeptide fused at the amino and/or carboxy terminus to 1 residue or 100 or more residues in length; and single or multiple amino acid residues. Examples of insertions at the ends include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusions of an enzyme (or a polypeptide that extends the serum half-life of an antibody) fused to the N-or C-terminus of the antibody.

Modification of Fab

In one aspect, the first antigen binding domain that specifically binds BCMA in the antigen binding molecule of the present disclosure is a Fab, and the second antigen binding domain that specifically binds CD3 is a substituted Fab comprising a tin chain and an Obscurin chain linked to a variable region, i.e., the original CH1 and CL of the Fab are replaced with the tin chain and the Obscurin chain; or (b)

The second antigen binding domain that specifically binds CD3 is a Fab and the first antigen binding domain that specifically binds BCMA is a substituted Fab comprising a tin chain and an Obscurin chain linked to a variable region, i.e. the original CH1 and CL of the Fab are replaced by the tin chain and the Obscurin chain.

Alternative techniques for the tin and Obscurin chains are described in detail in PCT/CN2021/070832 and CN202110527339.7 and patents that take them as priority documents, which are incorporated herein by reference in their entirety. An alternative tin chain and an Obscurin chain are shown in the following table, wherein the tin chain comprises a sequence selected from the group consisting of SEQ ID NOs: 98. to SEQ ID NO:116, the Obscurin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO:117 to SEQ ID NO:152 and SEQ ID NO:162 to SEQ ID NO:166, and a sequence of amino acids of the group consisting of seq id no.

TABLE 3 amino acid sequence of Titin chain

TABLE 3 amino acid sequence of Obscurin chain

Engineering of the Fc region

In one aspect, the Fc region of an antigen binding molecule of the present disclosure comprises one or more amino acid substitutions that reduce its binding to an Fc receptor, e.g., its binding to an fcγ receptor, and reduce or eliminate effector function. Native IgG Fc region, in particular IgG ₁ Fc region or IgG ₄ The Fc region, may result in the antigen binding molecules of the present disclosure targeting cells expressing Fc receptors, rather than cells expressing antigens. The engineered Fc regions of the present disclosure exhibit reduced binding affinity to Fc receptors and/or reduced effector function. In some embodiments, the engineered Fc region has a 50%, 80%, 90%, or more than 95% decrease in binding affinity to Fc receptors as compared to the native Fc region. In some embodiments, the Fc receptor is an fcγ receptor. In some embodiments, the Fc receptor is a human fcγ receptor, e.g., fcγri, fcγriia, fcγriib, fcγriiia. In some embodiments, the engineered Fc region has reduced binding affinity for complement (e.g., C1 q) as compared to the native Fc region. In some embodiments, the engineered Fc region has no reduced binding affinity for neonatal Fc receptor (FcRn) compared to the native Fc region. In some embodiments, the engineered Fc region has reduced effector functions, which may include, but are not limited to, one or more of the following: reduced Complement Dependent Cytotoxicity (CDC), reduced antibody dependent cell mediated cytotoxicity (ADCC), reduced Antibody Dependent Cellular Phagocytosis (ADCP), reduced cytokine secretion, reduced antigen uptake by immune complex mediated antigen presenting cells, reduced Reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling induced apoptosis, reduced dendritic cell maturation or reduced T cell priming.

For IgG ₁ Amino acid residue substitutions at positions 238, 265, 269, 270, 297, 327 and 329 etc. of the Fc region may reduce effector function. In some embodiments, the Fc region is a human IgG ₁ The Fc region, and the amino acid residues at positions 234 and 235 are a, numbering according to EU numbering rules. For IgG ₄ The Fc region, amino acid residue substitution at position 228, etc., can reduce effector function.

The antigen binding molecule may also comprise disulfide alterations such as 354C for the first subunit and 349C for the second subunit. Based on different sources, the 356 th amino acid residue of the Fc region may be E or D, and the 358 th amino acid residue may be M or L. In some embodiments, the 356 th amino acid residue of the Fc region may be E and the 358 th amino acid residue is M. In some embodiments, the 356 th amino acid residue of the Fc region may be D and the 358 th amino acid residue L.

When the antigen binding molecule comprises a binding domain fused to two subunits of the Fc region, undesired homodimerization may result. To increase yield and purity, it would therefore be advantageous to introduce modifications in the Fc region of the antigen binding molecules of the present disclosure that promote heterodimerization. In some embodiments, the Fc region of the present disclosure comprises modifications according to the knob-in-hole (KIH) technique involving the introduction of a raised structure (knob) at the interface of a first subunit and a hole structure (hole) at the interface of a second subunit. So that the protruding structures can be positioned in the pore structure, promoting the formation of heterodimers and inhibiting the production of homodimers. Raised structures are constructed by substituting small amino acid side chains from the interface of the first subunit with larger side chains (e.g., tyrosine or tryptophan). Whereas the pore structure is created in the interface of the second subunit by replacing the large amino acid side chain with a smaller amino acid side chain (e.g., alanine or threonine). The raised structures and pore structures were prepared by altering the nucleic acid encoding the polypeptide, with optional amino acid substitutions as shown in the following table:

TABLE 4 KIH mutant combinations

In addition to the mortar and pestle technique, other techniques for modifying heavy chain CH3 domains to achieve heterodimerization are also known in the art, such as WO96/27011, WO98/050431, EP1870459, WO2007/110205, WO 007/147901, WO2009/089004, WO2010/129304, WO2011/90754, WO2011/143545, WO2012/058768, WO2013/157954 and WO 013/096291.

The C-terminus of the Fc region may be the complete C-terminus ending with the amino acid residue PGK; or truncated (truncated) C-terminal, e.g. with one or two C-terminal amino acid residues removed in the truncated C-terminal. In a preferred aspect, the C-terminus of the Fc region is a shortened C-terminus ending with PG. Thus, in some embodiments, the composition of an intact antibody may include a population of antibodies that have all K447 residues and/or g446+k447 residues removed. In some embodiments, the composition of intact antibodies may include a population of antibodies that do not remove the K447 residue and/or the g446+k447 residue. In some embodiments, the composition of intact antibodies has an antibody population with and without a mixture of antibodies with the K447 residue and/or the g446+k447 residue.

Recombination method

Antibodies can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding an antibody are provided.

In the case of a natural antibody, a fragment of a natural antibody or a bispecific antibody with homodimeric heavy chains, two nucleic acids are required, one for the light chain or fragment thereof and one for the heavy chain or fragment thereof. Such nucleic acids encode amino acid sequences comprising an antibody VL and/or amino acid sequences comprising an antibody VH (e.g., light chain and/or heavy chain of an antibody). These nucleic acids may be on the same expression vector or on different expression vectors.

In the case of bispecific antibodies with heterodimeric heavy chains, four nucleic acids are required, one for the first light chain, one for the first heavy chain comprising a first heteromonomer Fc region polypeptide, one for the second light chain, and one for the second heavy chain comprising a second heteromonomer Fc region polypeptide. These four nucleic acids may be contained in one or more nucleic acid molecules or expression vectors, typically the nucleic acids are located on two or three expression vectors, i.e., one vector may contain more than one of the nucleic acids.

In one embodiment, the disclosure provides isolated nucleic acids encoding antibodies as described above. Such nucleic acids may independently encode any of the polypeptide chains described above. In another aspect, the disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acids.

In another aspect, the disclosure provides host cells comprising such nucleic acids. In one embodiment, a method of making an antigen binding molecule, an anti-BCMA antibody is provided; wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

For recombinant production of antigen binding molecules or anti-BCMA antibodies, nucleic acids encoding the proteins are isolated and inserted into one or more vectors for further cloning and/or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to genes encoding heavy and light chains of the antibody), or produced by recombinant methods or obtained by chemical synthesis.

Suitable host cells for cloning or expressing the antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies may be produced in bacteria, particularly when the antibody does not require glycosylation and Fc effector function. After expression, the antibodies may be isolated from the bacterial cell paste in a soluble fraction and may be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding antibodies, including fungal and yeast strains, whose glycosylation pathways have been "humanized" resulting in the production of antibodies with a partially or fully human glycosylation pattern. Suitable host cells suitable for expression (glycosylation) of antibodies may also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified which can be used in combination with insect cells, in particular for transfection of Spodoptera frugiperda (Spodoptera frugiperda) cells; plant cell cultures may also be used as hosts, for example US5959177, US 6040498, US6420548, US7125978 and US6417429; vertebrate cells can also be used as hosts, for example mammalian cell lines adapted to grow in suspension. Other examples of suitable mammalian host cell lines are the SV40 transformed monkey kidney CVl line (COS-7); human embryonic kidney lines (293 or 293T cells); baby hamster kidney cells (BHK); mouse sertoli (sertoli) cells (TM 4 cells); monkey kidney cells (CV 1); african green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat (buffalo rate) hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines, such as Y0, NS0, and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production see, e.g., yazaki, p. And Wu, a.m., methods in Molecular Biology, vol.248, lo, b.k.c. (ed), humana Press, totowa, NJ (2004), pages 255-268.

Measurement

The antigen binding molecules or anti-BCMA antibodies provided herein can be identified, screened, or characterized for their physical/chemical characteristics and/or biological activity by a variety of assays known in the art.

In one aspect, antibodies of the disclosure are tested for antigen binding activity, e.g., by known methods such as ELISA, western blot, and the like.

In another aspect, competition assays can be used to identify antibodies that compete for binding to BCMA. In certain embodiments, the competitive antibody binds to the same epitope (e.g., a linear or conformational epitope) as the antigen binding molecule or anti-BCMA antibody in an exemplary competition assay, the immobilized BCMA is incubated in a solution comprising a first labeled antibody that binds BCMA and a second unlabeled antibody that is tested for its ability to compete with the first labeled antibody for binding to BCMA. The second unlabeled antibody may be present in the hybridoma supernatant. As a control, the immobilized BCMA was incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody, excess unbound antibody was removed, and the amount of label associated with the immobilized BCMA was measured. If the amount of label associated with the immobilized BCMA is substantially reduced in the test sample relative to the control sample, the second antibody is indicated to compete with the first antibody for binding to BCMA.

In one aspect, an assay for identifying an anti-BCMA antibody having biological activity is provided. See the test examples of the disclosure for details.

Immunoconjugates

The present disclosure also provides immunoconjugates comprising an antigen binding molecule or an anti-BCMA antibody conjugated to one or more cytotoxic agents such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., bacterial, fungal, plant, or animal-derived protein toxins, enzymatically active toxins, or fragments thereof), or radioisotopes.

Diagnostic and therapeutic compositions

In certain embodiments, the antigen binding molecules provided by the present disclosure can be used to detect the presence of BCMA or CD3 in a biological sample, and the anti-BCMA antibodies provided by the present disclosure can be used to detect the presence of BCMA in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises a cell or tissue, such as a tumor tissue.

In one embodiment, an antigen binding molecule or an anti-BCMA antibody for use in a diagnostic or detection method is provided. In yet another aspect, a method of detecting the presence of BCMA or CD3 in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an antigen binding molecule or an anti-BCMA antibody under suitable conditions and detecting whether a complex is formed between the detection reagent and the antigen. Such methods may be in vitro or in vivo. In one embodiment, the use of an antigen binding molecule or an anti-BCMA antibody to select a subject suitable for treatment, e.g., BCMA or CD3, is a biomarker for selecting patients.

Exemplary conditions that can be diagnosed using the antibodies of the disclosure, such as B cell disorders associated with BCMA expression, plasma cell disorders, multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, fahrenheit macroglobulinemia, isolated myeloma, extramedullary plasmacytoma, bone sclerosis myeloma, heavy chain disease, meaningless monoclonal gammaglobulinopathy, and stasis-type multiple myeloma; autoimmune diseases, systemic lupus erythematosus.

In certain embodiments, a labeled antigen binding molecule or an anti-BCMA antibody is provided. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent, chromogenic, electron dense, chemiluminescent, and radioactive labels), and indirectly detected moieties (e.g., indirectly detected via enzymatic reactions or molecular interactions, such as enzymes or ligands).

In a further aspect, a pharmaceutical composition comprising the antigen binding molecule or anti-BCMA antibody is provided, e.g., for use in any of the following methods of treatment. In one aspect, the pharmaceutical composition comprises any of the antibodies provided herein and a pharmaceutically acceptable carrier. In another aspect, the pharmaceutical composition comprises any of the antibodies provided herein and at least one additional therapeutic agent.

The pharmaceutical composition of the antigen binding molecules or anti-BCMA antibodies described in the present disclosure is prepared by: such antibodies of the desired purity are contacted with one or more optional pharmaceutically acceptable carriers, in the form of a lyophilized composition or an aqueous solution. Formulations for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

Methods of treatment and routes of administration

Any of the antigen binding molecules or anti-BCMA antibodies provided herein can be used in a method of treatment.

In yet another aspect, the present disclosure provides the use of an antigen binding molecule or an anti-BCMA antibody in the manufacture or preparation of a medicament. In one embodiment, the medicament is for the treatment of BCMA-related diseases, such as B-cell disorders, plasma cell disorders, multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, fahrenheit macroglobulinemia, isolated osteosarcoma, extramedullary plasma cell tumor, bone sclerosis myeloma, heavy chain disease, meaningless monoclonal gammaglobulinopathy, and stasis-type multiple myeloma associated with BCMA expression; autoimmune diseases, systemic lupus erythematosus. And the medicament is in a form effective for the above-mentioned diseases. In some embodiments, the effective amount is a unit dose (e.g., a daily unit dose or a weekly unit dose). In one such embodiment, the use further comprises administering to the subject an effective amount of at least one additional therapeutic agent (e.g., one, two, three, four, five, or six additional therapeutic agents). The "subject" according to any of the above embodiments may be a human.

In a further aspect, a pharmaceutical composition comprising the antigen binding molecule or anti-BCMA antibody is provided, e.g., for use in any of the above pharmaceutical uses or methods of treatment. In one embodiment, the pharmaceutical composition comprises any of the anti-BCMA antibodies provided herein and a pharmaceutically acceptable carrier. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

The antigen binding molecules or anti-BCMA antibodies of the present disclosure may be used alone or in combination with other agents for therapy. For example, an antibody of the disclosure may be administered in combination (simultaneously, or sequentially) with at least one additional therapeutic agent.

The antigen binding molecules of the present disclosure or anti-BCMA antibodies (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and if topical treatment is desired, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration may be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration and pulse infusion.

The antigen binding molecules or anti-BCMA antibodies of the present disclosure will be formulated, administered and administered in a manner that complies with the Goose MEDICAL PRACTICE (GMP) guide line. Factors considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the medical practitioner. The antigen binding molecule or anti-BCMA antibody is optionally formulated with one or more agents currently used to prevent or treat the disorder. The effective amount of such other agents depends on the amount of antigen binding molecule or anti-BCMA antibody present in the pharmaceutical composition, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as described herein, or at about 1 to 99% of the dosages described herein, or at any dosage, and by any route of empirical/clinical determination as appropriate.

For the prevention or treatment of a disease, the appropriate dosage of the antigen binding molecules or anti-BCMA antibodies of the present disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the type of therapeutic molecule, the severity and course of the disease, whether administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the therapeutic molecule, and the discretion of the attending physician. The therapeutic molecule is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg of antigen binding molecule or anti-BCMA antibody may be the initial candidate dose for administration to a patient, whether by one or more separate administrations or by continuous infusion, for example. A typical daily dose may be in the range of about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. Accordingly, an exemplary unit daily dose is 50 μg to 5g, for example, a 50kg body weight.

Article of manufacture

In another aspect of the present disclosure, an article of manufacture is provided that comprises a material useful for treating, preventing, and/or diagnosing the above-described conditions. The article comprises a container, and a label or package insert (package insert) on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, and the like. The container may be formed from a variety of materials such as glass or plastic. The container contains a composition effective to treat, prevent and/or diagnose the condition, alone or in combination with another composition, and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an antigen binding molecule or an anti-BCMA antibody of the present disclosure. The label or package insert indicates that the composition is to be used to treat the selected condition. Further, the article may comprise: (a) A first container having a composition contained therein, wherein the composition comprises an antigen binding molecule or an anti-BCMA antibody of the present disclosure; and (b) a second container having a composition contained therein, wherein the composition comprises an additional cytotoxic agent or other therapeutic agent. The article of manufacture in this embodiment of the disclosure may further comprise a package insert indicating that the composition may be used to treat a particular condition. Alternatively, or in addition, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer. From a commercial and user standpoint, it may further include other materials as desired, including other buffers, diluents, filters, needles and syringes.

Examples and test examples

The present disclosure is further described below in conjunction with examples and test examples, which are not intended to limit the scope of the present disclosure. The experimental methods of the examples and test examples of the present disclosure, in which specific conditions are not noted, are generally according to conventional conditions, such as an antibody technical laboratory manual of cold spring harbor, a molecular cloning manual; or according to the conditions recommended by the manufacturer of the raw materials or goods. The reagents of specific origin are not noted and are commercially available conventional reagents.

Example 1 expression of human BCMA and cynomolgus monkey BCMA

The sequences encoding human BCMA with the human IgG1-Fc tag and the cynomolgus BCMA extracellular domain were inserted into phr vector, constructed into expression plasmids, and then transfected with HEK293. The sequence encoding the His-tagged human BCMA extracellular domain was inserted into the phr vector, constructed as an expression plasmid, and then HEK293 was transfected. The specific transfection steps are as follows: HEK293E cells were grown at 1X 10 ⁶ the/mL was inoculated into fresh expression medium (containing 1% FBS, gibco, 12338-026) and placed on a 37℃constant temperature shaker (120 rpm) for further culture for 24 hours. After 24 hours, the transfected plasmid and the transfection reagent PEI were sterilized with a 0.22 μm filter, and then the transfected plasmid was adjusted to 100. Mu.g/100 mL cells, the mass ratio of PEI (1 mg/mL) to plasmid was 3:1, 10mL of Opti-MEM and 200. Mu.g of plasmid were mixed evenly and allowed to stand for 5min; another 10mL of Opti-MEM was mixed with 400. Mu.g PEI and allowed to stand for 5min. Mixing the plasmid and PEI, and standing for 15min. The plasmid and PEI mixture was slowly added to 200mL HEK293E cells and 8% CO was added ₂ Culturing in a shaker at 120rpm and 37 ℃. Day 3 of transfection, 10% volume of feed medium (20 mM glucose+2 mM L-glutamic acid) was supplemented. On day 6 of transfection, the cell supernatant was collected by centrifugation at 4500rpm for 10min and purified according to the method of test example 2. The purified proteins can be used in the various examples or test case experiments described below.

Wherein the amino acid sequences of the humanBCMA-ECD-Fc, the humanBCMA-ECD-his, the cynoBCMA-ECD-his and the cynoBCMA-ECD-Fc are shown below. CynoBCMA-ECD-his was purchased from ACRO company.

(1) Human BCMA with human Fc tag: humanBCMA-ECD-Fc (SEQ ID NO: 1)

Note that: underlined is the signal peptide sequence; the italic part is a human Fc-linker-tag;

(2) ECD of His-tagged human BCMA: humanBCMA-ECD-his (SEQ ID NO: 2)

Note that: underlined is the signal peptide sequence; the italic part is His6-linker-tag;

(3) ECD of His-tagged cynomolgus BCMA: cynoBCMA-ECD-his (BCA-C4E 3H7-1 mg) (SEQ ID NO: 3)

Note that: the italic part is linker-10his-tag;

(4) Cynomolgus BCMA with human Fc tag: cynoBCMA-ECD-Fc (SEQ ID NO: 4)

Note that: underlined is the signal peptide sequence; double underlines are linker sequences; the italic part is human Fc-tag.

EXAMPLE 2 purification of protein A affinity chromatography of Fc tagged recombinant proteins and Nickel column purification of his tagged recombinant proteins

Purification of human Fc tagged proteins: cell expressed BCMA supernatant samples were high-speed centrifuged to remove impurities and purified by Protein a column. The column was washed with PBS until the a280 reading dropped to baseline. The target protein was eluted with 100mM acetic acid pH3.5 and neutralized with 1M Tris-HCl, pH 8.0. The eluted sample is properly concentrated and then is changed into PBS, and the obtained protein is split into separate packages for standby after electrophoresis, peptide mapping and LC-MS identification.

Purification of human his-tagged proteins: and centrifuging the BCMA supernatant sample expressed by the cells at a high speed to remove impurities. The nickel column was equilibrated with buffer containing PBS, the column volume was 2-5 times greater, and the supernatant sample was applied to Ni Sepharose excel column at a flow rate. Washing the column with PBS buffer until the reading of A280 is reduced to a baseline, washing the chromatographic column with PBS+10mM imidazole to remove non-specifically bound impurity proteins, collecting effluent, eluting target proteins with 300mM imidazole-containing PBS solution, collecting elution peaks, concentrating and changing liquid, and packaging the obtained proteins for later use after electrophoresis, peptide mapping and LC-MS identification as correct.

EXAMPLE 3 construction and identification of cell lines expressing recombinant human BCMA and cynomolgus monkey BCMA

To screen for antibodies that bind well to cell surface BCMA, the present disclosure constructs K562-BCMA cell lines expressing human BCMA. Cloning human BCMA full-length gene onto mammal cell expression vector pCDH, co-transfecting HEK293T cell with three plasmids of pVSV-G, pCMV-dR8.91 and pCDH-humanBCMA CRL-11268) to package virus, and collecting virus-infected K562 cells after 48 hours of transfectionCCL-243). The K562 monoclonal cells highly expressing humanBCMA were obtained by flow sorting 72 hours after infection. The present disclosure also constructs a cynomolgus monkey expressing CHO-K1-cyno bcma cell line. Cloning full-length gene of cynomolgus monkey BCMA onto mammal cell expression vector pCDH, co-transfecting HEK293T cell with three plasmids of pVSV-G, pCMV-dR8.91 and pCDH-cynoBCMACRL-11268) to package the virus, 48 hours of transfectionAfter that, virus-infected CHOK1 cells are collectedCCL-61). CHOK1 monoclonal cells highly expressing cynoBCMA were obtained by flow sorting 72 hours after infection.

Example 4 screening and identification of anti-human BCMA hybridoma antibodies

The present disclosure prepares monoclonal antibodies against human BCMA by hybridoma technology. The obtained antibody specifically binds with high affinity to human BCMA and can cross react with cynomolgus monkey BCMA; the obtained antibody has better binding activity with human BCMA and cynomolgus BCMA on the cell surface, and the binding activity is not interfered by soluble BCMA.

humanBCMA-ECD-his and cynoBCMA-ECD-his were used as cross-immunizing agents,gold Adjuvant (Sigma Cat No. T2684) and Thermo Alum (Thermo Cat No. 77161) was used as an adjuvant to cross immunize mice. Mice with high antibody titers in serum 10-6# (titer 625K) were selected for spleen cell fusion after primary and 7 booster immunizations. After fusion, hybridoma culture supernatants were assayed for cell growth density and screened for antibodies that specifically bind cell surface BCMA.

The monoclonal hybridoma cell strains 4E3, 33H4, 3B2 and 78 with good activity are obtained by screening. Collecting hybridoma cells in logarithmic growth phase, extracting RNA with NucleoZol (MN) (according to the procedure of the kit instructions), and performing reverse transcription (PrimeScript) ^TM Reverse Transcriptase, takara, cat# 2680A). The cDNA obtained by reverse transcription was subjected to PCR amplification using mouse Ig-Primer Set (Novagen, TB326Rev. B0503) and then sequenced. Amino acid sequences of CDRs and variable regions of 4E3, 33H4, 3B2 and 78 such asThe following steps:

TABLE 5 BCMA antibody CDR

4E3 murine heavy chain variable region (SEQ ID NO: 29)

4E3 murine light chain variable region (SEQ ID NO: 30)

33H4 murine heavy chain variable region (SEQ ID NO: 31)

33H4 murine light chain variable region (SEQ ID NO: 32)

3B2 murine heavy chain variable region (SEQ ID NO: 33)

3B2 murine light chain variable region (SEQ ID NO: 34)

78 murine heavy chain variable region (SEQ ID NO: 35)

78 murine light chain variable region (SEQ ID NO: 36)

Note that: the underlined tag region is the CDR region obtained according to the Kabat numbering convention.

Example 5 humanized design of anti-human BCMA monoclonal antibodies

Humanization of murine monoclonal antibodies was performed according to methods well known in the art from a number of literature. Briefly, based on the obtained murine antibody VH/VL CDR-typical structure, homologous sequences of the light chain variable region (VL) and the heavy chain variable region (VH) are searched from a humanized germline database, the CDR regions of the murine antibody are grafted onto a humanized template, and partial residues of VL and VH are mutated to replace the constant regions of the murine antibody with human constant regions, resulting in the final humanized molecule.

Selection and back-mutation of the human FR region of 1.4E3. Wherein, FR1-3 of the heavy chain variable region adopts IGHV1-46×01, FR4 adopts IGHJ6×01; FR1-3 of the light chain variable region adopts IGKV1-39 x 01, FR4 adopts IGKJ2 x 01. The origin and back mutations of FR1-3 are shown in the following table.

Human FR region selection and back mutation of table 6.4E3

Note that: graft represents murine antibody CDR implantation human germline FR region sequences. Illustratively, R71A represents mutating R at position 71 back to A according to the Kabat numbering system. The following is the same.

In addition, the amino acid residue at position 1 of the antibody heavy chain variable region was mutated from Q to E, and the specific sequence of the antibody variable region obtained by humanizing the murine antibody 4E3 was as follows (the underlined represents the CDR, the following are given):

>hu4E3H1 Graft(IGHV1-46*01)+R71A,A93V(SEQ ID NO：37)

>hu4E3H2 Graft(IGHV1-46*01)+R71A,T73K,S76T,A93V(SEQ ID NO：38)

>hu4E3H3 Graft(IGHV1-46*01)+M48I,V67A,R71A,T73K,S76T,A93V(SEQ ID NO：39)

>hu4E3L1 Graft(IGKV1-39*01)(SEQ ID NO：40)

>hu4E3L2 Graft(IGKV1-39*01)+I48V(SEQ ID NO：41)

>hu4E3L3 Graft(IGKV1-39*01)+I48V,F71Y(SEQ ID NO：42)

>hu4E3L4 Graft(IGKV1-39*01)+I48V,F71Y,A43S,K45Q(SEQ ID NO：43)

Selection and back-mutation of the human FR region of 2.33H4. Wherein, FR1-3 of the heavy chain variable region adopts IGHV7-4-1 x 02, FR4 adopts IGHJ1 x 01; FR1-3 of the light chain variable region adopts IGKV1-8×01 or IGKV1-27×01, FR4 adopts IGKJ4×01. The origin and back mutations of FR1-3 are shown in the following table.

Human FR region selection and back mutation of table 7.33H4

In addition, the amino acid residue at position 1 of the antibody heavy chain variable region was mutated from Q to E, and the specific sequence of the antibody variable region obtained by humanizing the murine antibody 33H4 was as follows:

>hu33H4H1 Graft(IGHV7-4-1*02)+A93L(SEQ ID NO：44)

>hu33H4H2 Graft(IGHV7-4-1*02)+V2I,G44V,L45F,A93L(SEQ ID NO：45)

>hu33H4H3 Graft(IGHV7-4-1*02)+V2I,G44V,L45F,E46K,V75A,S76N,A93L(SEQ ID NO：46)

>hu33H4L1 Graft(IGKV1-8*01)+A43S,G66D(SEQ ID NO：47)

>hu33H4L2 Graft(IGKV1-27*01)+V43S,G66D(SEQ ID NO：48)

selection and back-mutation of the human FR region of 3.3B2. Wherein, FR1-3 of the heavy chain variable region adopts IGHV3-11×01, FR4 adopts IGHJ1×01; FR1-3 of the light chain variable region adopts IGKV1-39 x 01, FR4 adopts IGKJ2 x 01. The origin and back mutations of FR1-3 are shown in the following table.

Human FR region selection and back mutation of table 8.3B2

The specific sequence of the antibody variable region obtained by humanizing the murine antibody 3B2 is as follows:

>hu3B2H1 Graft(IGHV3-11*01)+Q1E,S30R,A93T(SEQ ID NO：49)

>hu3B2H2 Graft(IGHV3-11*01)+Q1E,S30R,W47R,S49A,A93T(SEQ ID NO：50)

>hu3B2L1 Graft(IGKV1-39*01)+P44V(SEQ ID NO：51)

4.78, selection and back-mutation of the human FR region. Wherein, FR1-3 of the heavy chain variable region adopts IGHV2-70×01, FR4 adopts IGHJ6×01; FR1-3 of the light chain variable region adopts IGKV1-33×01, FR4 adopts IGKJ2×01. The origin and back mutations of FR1-3 are shown in the following table.

Human FR region selection and back mutation of Table 9.78

In addition, the amino acid residue at position 1 of the antibody heavy chain variable region was mutated from Q to E, and the specific sequence of the antibody variable region obtained by humanizing the murine antibody 78 was as follows:

>hu78H1 Graft(IGHV2-70*01)+F24V,S30T,T73N(SEQ ID NO：52)

>hu78H2 Graft(IGHV2-70*01)+F24V,S30T,I37V,A49G,T73N,L80F,T89R(SEQ ID NO：53)

>hu78L1 Graft(IGKV1-33*01)(SEQ ID NO：54)

example 6 preparation and identification of anti-BCMA/CD 3 bispecific antibodies

The CD3 binding molecules of the present disclosure may be derived from any suitable antibody. Particularly suitable antibodies are described, for example, in International application No. PCT/CN2019/123548 (incorporated herein by reference in its entirety).

The CDR and variable region sequences of the anti-CD 3 arm in the bispecific antibodies of the present disclosure are shown below:

TABLE 10 CDR of CD3 arm

The specific sequence of the variable region is as follows:

>S107E-VH(SEQ ID NO：63)

>S107E-VL(SEQ ID NO：64)

>6164-VH(SEQ ID NO：65)

>6164-VL(SEQ ID NO：66)

humanized anti-BCMA antibody variable regions were separated from the variable regions of S107E and 6164 of anti-CD 3 antibody, respectively, and IgG ₁ Mutant IgG ₁ (AA) (L234A/L235A) combinations. Each forms several different forms of bispecific antibodies, format-11-6164, format-14 and Format-15, respectively.

Format-11 is a symmetrical structural molecule comprising two identical heavy chains (chain 1) and two identical light chains (chain 2).

The heavy chain is: VH (anti-BCMA) -IgG ₁ (CH1)-VH(S107E)-linker1-VL(S107E)-linker2-IgG ₁ (AA)Fc；

The light chain is VL (anti-BCMA) -CL, and the schematic diagram is shown in FIG. 1A.

Format-11-6164 is a symmetrical structural molecule comprising two identical heavy chains and two identical light chains.

The heavy chain is: VH (anti-BCMA) -IgG ₁ (CH1)-VH(6164)-linker1-VL(6164)-linker2-IgG ₁ (AA)Fc；

Wherein:

>IgG ₁ (CH1)(SEQ ID NO：67)

>CL(SEQ ID NO：68)

>IgG ₁ (AA)Fc(SEQ ID NO：69)

format-14 is an asymmetric structural molecule, the complete molecule has four chains, the four chains are different, and chain 1 is: VH (anti-BCMA) -IgG ₁ (CH1)-IgG ₁ Fc (Hole), chain 2 is: VL (anti-BCMA) -CL, chain 3 is T-Knob: VH (S107E) -linker3-Titin-IgG ₁ Fc (Knob); chain 4 is VL-Ob: VL (S107E) -linker3-Obscurin, as shown in FIG. 1B (Ob stands for Obscurin).

Wherein:

>T-Knob：VH(S107E)-linker3-Titin-IgG ₁ Fc(Knob)(SEQ ID NO：70)

>VL-Ob：VL(S107E)-linker3-Obscurin(SEQ ID NO：71)

format-15 is an asymmetric structural molecule, the complete molecule has four chains, the four chains are different, and chain 1 is: VH (anti-BCMA) -IgG ₁ (CH1)-IgG ₁ Fc (Knob, E356D, M358L), chain 2 is: VL (anti-BCMA) -CL, chain 3 is Ob-Hole; chain 4 is VL-tin, schematically shown in FIG. 1C (Ob stands for Obscurin).

>Ob-Hole：VH(S107E)-linker3-Obscurin-IgG ₁ Fc(Hole，E356D，M358L)(SEQ ID NO：167)

>VL-Titin：VL(S107E)-linker3-Titin(SEQ ID NO：168)

Note that: the single underlined tag region is the CDR region of the CD3 binding domain obtained according to Kabat numbering convention, the double underlined tag region is the tin or Obscurin sequence, italics is the constant region.

>IgG ₁ Fc(Knob)(SEQ ID NO：153)

>IgG ₁ Fc(Hole)(SEQ ID NO：154)

>IgG ₁ Fc-1(Knob，E356D，M358L)(SEQ ID NO：169)

>IgG ₁ Fc-1(Hole，E356D，M358L)(SEQ ID NO：170)

Based on the humanized antibody amino acid sequences of 4E3, 33H4, 3B2 and 78, bispecific antibodies as shown below were respectively constructed.

TABLE 11 bispecific antibodies of the disclosure

Numbering device	Chain 1	Chain 2	Chain 3	Chain 4
4E3-11-1	hu4E3H1-11	hu4E3L1-11
4E3-11-2	hu4E3H2-11	hu4E3L1-11
4E3-11-3	hu4E3H3-11	hu4E3L1-11
4E3-11-4	hu4E3H1-11	hu4E3L2-11
4E3-11-5	hu4E3H1-11	hu4E3L3-11
4E3-11-6	hu4E3H1-11	hu4E3L4-11
4E3-11-6164-1	hu4E3H2-11-6164	hu4E3L1-11
4E3-11-6164-2	hu4E3H3-11-6164	hu4E3L1-11
4E3-11-6164-3	hu4E3H1-11-6164	hu4E3L2-11
4E3-11-6164-4	hu4E3H1-11-6164	hu4E3L3-11
4E3-11-6164-5	hu4E3H1-11-6164	hu4E3L4-11
33H4-11-1	hu33H4H1-11	hu33H4L1-11
33H4-11-2	hu33H4H2-11	hu33H4L1-11
33H4-11-3	hu33H4H3-11	hu33H4L1-11
33H4-11-4	hu33H4H1-11	hu33H4L2-11
33H4-11-5	hu33H4H2-11	hu33H4L2-11
33H4-11-6	hu33H4H3-11	hu33H4L2-11
33H4-15-1	hu33H4H2-15	hu33H4L2-11	Ob-Hole	VL-Titin
3B2-11-1	hu3B2H1-11	hu3B2L1-11
3B2-11-2	hu3B2H2-11	hu3B2L1-11
3B2-11-6164-1	hu3B2H1-11-6164	hu3B2L1-11
3B2-11-6164-2	hu3B2H2-11-6164	hu3B2L1-11
3B2-14-1	hu3B2H1-14	hu3B2L1-11	T-Knob	VL-Ob
3B2-14-2	hu3B2H2-14	hu3B2L1-11	T-Knob	VL-Ob
3B2-15-1	hu3B2H2-15	hu3B2L1-11	Ob-Hole	VL-Titin
78-11-1	hu78H1-11	hu78L1-11
78-11-6164-1	hu78H1-11-6164	hu78L1-11
78-11-6164-2	hu78H2-11-6164	hu78L1-11

In Table 11, hu4E3H1-11 in 4E3-11-1 shows that hu4E3H1 was used as VH (anti-BCMA) and the structure was Format-11; the expression hu4E3H2-11-6164 in 4E3-11-6164-1 means that hu4E3H2 is used as VH (anti-BCMA), the structure is Format-11-6164, and so on. The specific amino acid sequence is as follows:

>hu4E3L1-11(SEQ ID NO：72)

>hu4E3L2-11(SEQ ID NO：73)

>hu4E3L3-11(SEQ ID NO：74)

>hu4E3L4-11(SEQ ID NO：75)

>hu4E3H1-11(SEQ ID NO：76)

>hu4E3H2-11(SEQ ID NO：77)

>hu4E3H3-11(SEQ ID NO：78)

>hu4E3H1-11-6164(SEQ ID NO：79)

>hu4E3H2-11-6164(SEQ ID NO：80)

>hu4E3H3-11-6164(SEQ ID NO：81)

>hu33H4L1-11(SEQ ID NO：82)

>hu33H4L2-11(SEQ ID NO：83)

>hu33H4H1-11(SEQ ID NO：84)

>hu33H4H2-11(SEQ ID NO：85)

>hu33H4H3-11(SEQ ID NO：86)

>hu33H4H2-15(SEQ ID NO：171)

>hu3B2L1-11(SEQ ID NO：87)

>hu3B2H1-11(SEQ ID NO：88)

>hu3B2H2-11(SEQ ID NO：89)

>hu3B2H1-11-6164(SEQ ID NO：90)

>hu3B2H2-11-6164(SEQ ID NO：91)

>hu3B2H1-14(SEQ ID NO：92)

>hu3B2H2-14(SEQ ID NO：93)

>hu3B2H2-15(SEQ ID NO：172)

>hu78L1-11(SEQ ID NO：94)

>hu78H1-11(SEQ ID NO：95)

>hu78H1-11-6164(SEQ ID NO：96)

>hu78H2-11-6164(SEQ ID NO：97)

Positive control molecules AMGEN701 and REGN5458 for use in the present disclosure.

AMGEN701 (SEQ ID NO: 158), source of sequence: WO2017134134

REGN5458 sequence source: patent WO2020018830

Chain 1: BCMA heavy chain (SEQ ID NO: 159)

Chain 2: CD3 heavy chain (SEQ ID NO: 160)

Strand 3/strand 4: BCMA light/CD 3 light chain (SEQ ID NO: 161)

EXAMPLE 7 antigen binding molecules containing Titin-T chain/Obscurin-O chain

The tin/Obscurin chains of the present disclosure may be derived from any suitable polypeptide, including polypeptides derived from PCT/CN2021/070832 and its publications (incorporated herein by reference in their entirety) and CN202110527339.7 and patents (incorporated herein by reference in their entirety) for priority documents. Bispecific antibodies were constructed in which CL is the kappa light chain constant region in PCT/CN2021/070832, the amino acid sequences of the tin chain and the Obscurin chain are shown in tables 3-1 and 3-2, and the linker sequences include GGGGS (SEQ ID NO: 157), ASTKG (SEQ ID NO: 173) or RTVAS (SEQ ID NO: 174), and the amino acid sequences of Fc1, fc2 and CH1 in this example are shown in SEQ ID NO: 153. SEQ ID NO:154 and SEQ ID NO: 67.

7.1 DI bispecific antibodies

Referring to PCT/CN2021/070832 example 5, DI bispecific antibodies against hNGF and hRANKL were constructed: DI-2 to DI-20 comprising a first heavy chain, a second heavy chain, a first light chain and a second light chain as described below:

First heavy chain: the sequence from the N end to the C end is as follows: [ VH1-I ] - [ linker 1] - [ Obscurin chain ] - [ Fc2],

first light chain: the sequence from the N end to the C end is as follows: [ VL1-I ] - [ linker 2] - [ tin chain ],

second heavy chain: the sequence from the N end to the C end is as follows: [ VH2-D ] - [ CH1] - [ Fc1], and

second light chain: the sequence from the N end to the C end is as follows: [ VL2-D ] - [ CL ];

wherein VH1-I and VL1-I are the heavy chain variable region and the light chain variable region of I0 in PCT/CN2021/070832, respectively, and VH2-D and VL2-D are the heavy chain variable region and the light chain variable region of D0 in PCT/CN2021/070832, respectively. The structures of the Obscurin-O chain, the Titin-T chain, the linker 1 and the linker 2 in the DI bispecific antibody in this example are shown in the following Table.

Table i-1. Table of the correspondence of the Obscurin-O chain/tin-T chain and linker in di bispecific antibodies

Note that: the numbers of the Titin chain and the Obscurin chain in the tables are shown in tables 3-1 and 3-2.

The binding activity of the DI-2 to DI-20 bispecific antibodies to their antigens was tested using the method of test example 4 of PCT/CN 2021/070832. Antibodies were subjected to a heat stability study. The research method comprises the following steps: the concentration of the antibody was diluted to 5mg/mL with PBS and the thermal stability (loading amount 9. Mu.L; parameter setting: start Temp 20 ℃ C.; incubation 0s; rate 0.3 ℃ C./min; plate Hold 5s; end Temp 95 ℃ C.) was measured using a high throughput differential scanning fluorescent apparatus (UNCHAINED, specification: unit). Experimental results show that the binding activity of the modified bispecific antibody to the antigen is not changed remarkably; and compared with DI-2, the Tm1 (DEG C) and Tonset (DEG C) of DI-4 to DI-8, DI-10 to DI-16 and DI-20 are obviously improved, and the thermal stability of the bispecific antibody is better.

Table i-2. Detection of binding Activity of DI bispecific antibodies

Table i-3. Results of thermal stability experiments for DI bispecific antibodies

Numbering device	Tm1(℃)	Tonset(℃)	Numbering device	Tm1(℃)	Tonset(℃)
DI-2	55.6	48.3	DI-11	57.35	-
DI-4	60.1	52.493	DI-12	59.9	51.726
DI-5	61	51.967	DI-13	61	50.988
DI-6	60.8	53.012	DI-14	61.2	52.191
DI-7	60.34	52.003	DI-15	60.41	50.558
DI-8	60.61	50.425	DI-16	61.5	50.691
DI-10	60.2	52.766	DI-20	60.7	51.859

The solution containing DI bispecific antibody was prepared with 10mM acetic acid, ph5.5,9% sucrose buffer, placed in a 40 ℃ incubator for four weeks of incubation, after the end of incubation, the antibody concentration was concentrated to the concentration at the beginning of incubation, and the solution was observed for precipitation. Experimental results show that the DI-2 bispecific antibody group solution precipitates, and DI-3 to DI-7 have better stability than DI-2.

Precipitation of di bispecific antibody

Numbering device	Initial concentration	Concentrating to concentration at week 4	Solution precipitation conditions
DI-2	20mg/ml	20mg/ml	Precipitation occurs
DI-3	20mg/ml	20mg/ml	No precipitate
DI-4	60mg/ml	60mg/ml	No precipitate
DI-5	25mg/ml	25mg/ml	No precipitate
DI-6	60mg/ml	60mg/ml	No precipitate
DI-7	16mg/ml	16mg/ml	No precipitate

7.2 PL bispecific antibodies

Construction of PL bispecific antibodies against hPDL1 and hCTLA 4: PL-1 to PL-19 comprising a first heavy chain, a second heavy chain, a first light chain and a second light chain as described below:

first heavy chain: the sequence from the N end to the C end is as follows: [ VH1-P ] - [ linker 1] - [ Obscurin chain ] - [ Fc1],

first light chain: the sequence from the N end to the C end is as follows: [ VL1-P ] - [ linker 2] - [ tin chain ],

second heavy chain: the sequence from the N end to the C end is as follows: [ VH2-L ] - [ CH1] - [ Fc2], and

second light chain: the sequence from the N end to the C end is as follows: [ VL2-L ] - [ CL ];

Wherein VH1-P and VL1-P are the heavy chain variable region and the light chain variable region of the h1831K antibody of WO2020177733A1, respectively, and the amino acid sequences of VH1-L and VL1-L are shown below.

>VH2-L(SEQ ID NO：175)

>VL2-L(SEQ ID NO：176)

The structures of the Obscurin-O chain, the Titin-T chain, the linker 1 and the linker 2 in the PL bispecific antibody in this example are shown in the following table.

Table i-4. Correspondence table of Obscurin-O chain/Titin-T chain and linker in PL bispecific antibody

Binding activity of the PL bispecific antibodies was tested by reference to the ELISA method in test example 4 in PCT/CN2021/070832, where hPDL1, hCTLA4 antigens were purchased from: sino biology. Antibodies were subjected to a heat stability study. The method comprises the following steps: the concentration of the antibody was diluted to 1.4-3mg/mL with PBS and the thermal stability was measured using a high throughput differential scanning fluorescent apparatus (UNCHAINED, specification: unit) with a loading of 9. Mu.L, a parameter setting of Start Temp 20 ℃ C.; infusion 0s; rate 0.3 ℃ C./min; plate Hold 5s; end Temp95 ℃ C.). Experimental results show that the PL bispecific antibody still has good binding activity to antigen; and compared with PL-1, tm1 (DEG C), tagg 266 (DEG C) and Tonset (DEG C) of PL-2 to PL-19 are obviously improved, and the heat stability of the bispecific antibody is better.

Table i-5. Detection of binding Activity of PL bispecific antibodies

Table i-6. Results of thermal stability experiments for PL bispecific antibodies

7.3 HJ bispecific antibodies

Construction of HJ bispecific antibodies against hIL5 and hTSLP: HJ-3 to HJ11 comprising a first heavy chain, a second heavy chain, a first light chain and a second light chain as described below:

first heavy chain: the sequence from the N end to the C end is as follows: [ VH1-H ] - [ linker 1] - [ Titin chain ] - [ Fc1],

first light chain: the sequence from the N end to the C end is as follows: [ VL1-H ] - [ linker 2] - [ Obscurin strand ],

second heavy chain: the sequence from the N end to the C end is as follows: [ VH2-J ] - [ CH1] - [ Fc2], and

second light chain: the sequence from the N end to the C end is as follows: [ VL2-J ] - [ CL ];

wherein VH1-H and VL1-H are the heavy chain variable region and the light chain variable region of H0 in PCT/CN2021/070832, respectively, and VH2-J and VL2-J are the heavy chain variable region and the light chain variable region of J1 in PCT/CN2021/070832, respectively. In this example, the structures of the Obscurin-O chain, titin-T chain, linker 1 and linker 2 in the HJ bispecific antibody are shown in the following Table.

Table i-7. Table of the correspondence of Obscurin-O chain/Titin-T chain and linker in HJ bispecific antibody

The antigen binding activity of the HJ bispecific antibody was tested by the method described in test example 4 in PCT/CN 2021/070832. The thermostability of the antibodies was studied by: the HJ bispecific antibody diluted solution was prepared with 10mM acetic acid ph5.5, 9% sucrose buffer, then the bispecific antibody was concentrated by ultrafiltration concentration to obtain HJ bispecific antibody solutions of different concentrations (see table 13-2 for HJ bispecific antibody concentration), then the concentrated solutions were incubated in a 40 ℃ incubator, on day 0 (i.e. before 40 ℃ incubation was started, D0), on day 7 (40 ℃ incubation, day 7, D7), on day 14 (40 ℃ incubation, day 14, D14), on day 21 (40 ℃ incubation, day 21, D21) and on day 28 (40 ℃ incubation, day 28, D28) to detect SEC purity of the samples, and immediately after 40 ℃ incubation, samples were sampled to detect CE-SDS purity. Experimental results show that the binding activity of the HJ bispecific antibody constructed by the present disclosure to antigen is not significantly changed; and, compared to HJ-3, the thermal stability of the HJ-5 to HJ-11 bispecific antibodies is better.

Table i-8. Detection of binding Activity of PL bispecific antibodies

Table i-9. Results of the accelerated stability test of HJ bispecific antibody

Test case

Test example 1 FACS determination of the affinity of BCMA/CD3 bispecific antibodies for BCMA

To test the binding capacity of the BCMA/CD3 bispecific antibodies of the present disclosure to BCMA, the present test example examined the binding capacity of BCMA/CD3 bispecific antibodies to stably transgenic cell line K562-humanbma BCMA-High (K562) overexpressing human BCMA and stably transgenic cell line CHOK1-cyno BCMA-High (CHOK 1) overexpressing cynomolgus BCMA by flow cytometry.

The method comprises the following steps: the cells were cultured in IMDM medium containing 10% FBS, and placed at 37℃in 5% CO ₂ Culturing in incubator for 2 days, wherein the number of cells per well is 1×10 ⁵ Cells were added to the cell plates, centrifuged at 300g for 5min and washed once with 1% BSA. Antibody was diluted in a gradient of 8 concentrations, 100. Mu.L per well was added to the cell plate, incubated at 4℃for 1 hour, washed once with 1% BSA, and 100. Mu.L of APC-coat Anti-human IgG Fc fluorescent secondary antibody diluent (1:400) per well was added and incubated at 4℃for 1 hour. Plates were washed three times with 1% BSA and 100. Mu.L PBS read plate was added to each well. The EC50 (nM) of each antibody binding to the corresponding cell is shown in the table below.

TABLE 12 determination of antibodies binding to BCMA

The results show that the BCMA antibodies screened using the present disclosure are constructed as different BCMA-CD3 diabodies that all have good ability to bind membrane surface BCMA and have good cross-binding activity with CynoBCMA.

Test example 2 degree of influence of soluble BCMA on antibody binding Membrane BCMA of the present disclosure

The presence of high concentrations of soluble BCMA (30 ng/mL on average) in the blood of patients with multiple myeloma interferes with the specific binding of BCMA-CD3 bispecific antibodies to membrane surface BCMA. This test example tests whether the presence of high concentration of soluble BCMA affects the BCMA antigen on the surface of the bispecific antibody binding membrane of the present disclosure.

The endogenous BCMA-expressing myeloma cell line NCIH929 with a viability (viability) of greater than or equal to 90% was added to 3795 cell plates with a cell count of 1X 10 per well ⁵ 300g were centrifuged for 5min, washed once with 1% BSA, 50. Mu.L of a solution containing 1% BSA and 200ng/mL soluble human BCMA was added, followed by gradient dilution of the bispecific antibody and incubation at 4℃for 1 hour. After washing the plate once with 1% BSA, 100. Mu.L of APC gold Anti-Mouse Ig dilution (1:400) was added to each well, incubated at 4℃for 1 hour, and after washing the plate three times with 1% BSA, 100. Mu.L of PBS was added to each well and the plate was read. The results are shown in the following table. The affinity of the antibody for cell surface BCMA in the presence of 200ng/mL of soluble BCMA solution was not statistically significantly changed, had very excellent binding ability to membrane surface BCMA, and was not affected by soluble BCMA. However, the control antibody amben 701 was significantly affected by soluble BCMA.

TABLE 13 determination of antibody binding to Membrane surface BCMA

Test example 3 bispecific antibodies of the present disclosure activate T cells in vitro

This test example investigated the ability of antibodies of the present disclosure to activate T cells, using BCMA-expressing cell line U266B and CHOK1, which do not express BCMA, as target cells to detect bispecific antibody pairsJurkat-Lucia ^TM Activation ability of NFAT cells.

Jurkat-Lucia ^TM NFAT cell collection centrifugation, medium resuspension count using 1640+10% fbs, cell count was adjusted to 1×10 ⁶ mu.L (5X 10) of each well was added to each well at a concentration of 50. Mu.L/mL ⁴ /hole). After target cells U266B or CHOK1 were collected and centrifuged, the cells were resuspended in medium 1640+10% FBS and the number of cells was adjusted to 8X 10 ⁵ mu.L/mL, E: t is 2.5 respectively: 1. bispecific antibody was diluted in 1640+10% FBS medium at an initial concentration of 80nM, and after 5-fold dilution to generate 9 gradients, 25. Mu.L of solution was added to each well. The treated cells were then placed at 37℃with 5% CO ₂ After 5h of incubation in the incubator of (C), the plates were removed and 100. Mu.L of QUANTI-Luc was added at a ratio of 1:1 ^TM Gold, incubated at room temperature for 5min, read values of luminescence were detected with an enzyme-labeled instrument, and data were analyzed by treatment using Graphpad Prism 5.

The results show that the bispecific antibodies of the present disclosure can specifically activate Jurkat-Lucia in the presence of target cell U266B ^TM NFAT cells with significantly increased activation capacity compared to amben 701; whereas on CHOK1 cells that do not express BCMA, the bispecific antibodies of the present disclosure do not activate Jurkat-Lucia ^TM NFAT cells, demonstrating that the ability of bispecific antibodies of the present disclosure to activate T cells is BCMA dependent on their target.

TABLE 14 Activity of antibodies to activate T cells in vitro

Note that: "-" indicates negative.

Test example 4 in vitro cytotoxic Activity of bispecific antibodies of the present disclosure

This test example investigated the killing activity of bispecific antibodies of the present disclosure as T cell adapter molecules (adaptors) against tumor cells. The target specific cytotoxic activity of the bispecific antibodies of the present disclosure was tested using the BCMA expressing cell lines RPMI8226, H929, and CHOK1 that did not express BCMA as target cells.

300g of fresh PBMC (from Xuanfeng Bio Inc.) was centrifuged for 10min, the supernatant was discarded, resuspended in phenol red-free 1640+4% FBS, centrifuged again, resuspended and counted to adjust the cell number to 1.5X10) ⁶ mu.L/mL was added to each well. Collecting target cells, centrifuging at 1000rpm for 3min, re-suspending, counting, and adjusting cell number to 3×10 ⁵ mu.L of E/T was added per well at 10:1. The antibodies were diluted in phenol red free complete medium at an initial concentration of 400nM (4 Xfinal concentration), 9 gradients diluted 10-fold and 25. Mu.L added per well. The cells were then placed at 37℃with 5% CO ₂ Is cultured for 48 hours. Before detection, 10. Mu.L of medium was aspirated into two of the wells of only target cells, then 10. Mu.L of Lysis Solution (10X) was added, after Lysis for 45min, the plates were removed, centrifuged at 1000rpm for 3min, 50. Mu.L of supernatant was aspirated into a new 96-well plate (# 3590), and 50. Mu.L of dissolved Cytotox was added at a 1:1 ratioReagent, after incubation at room temperature for 0.5h, 50. Mu.L of stop solution was added, and absorbance (490 nm) was measured with FlexStation 3 (Molecular Devices).

TABLE 15-1 cytotoxic Activity of antibodies against RPMI8266

TABLE 15-2 cytotoxic Activity of antibodies against H929

Antibodies to	EC50(pM)	Cleavage Rate%
3B2-15-1	46.38	85.01
33H4-15-1	58.27	80.38
AMGEN701	88.60	87.73

TABLE 15-3 cytotoxic Activity of antibodies against CHOK1

The results show that the bispecific antibodies of the present disclosure have greater cytotoxic activity than amben 701; also, the cytotoxic activity of the bispecific antibodies of the present disclosure is BCMA target specific, exhibiting weaker cytotoxic activity on CHOK1 cell lines without BCMA expression, suggesting better safety of the bispecific antibodies of the present disclosure.

Test example 5. Effect of soluble BCMA on in vitro cytotoxic Activity of antibodies of the present disclosure

The present disclosure investigated the effect of soluble BCMA on BCMA/CD3 bispecific antibody tumor cell killing activity in vitro.

Use of BCMA-expressing cell line U266B as target cell in cytotoxic Activity assays Soluble BCMA is additionally added. Fresh PBMC (available from Xuanfeng Bio Inc.) was taken, centrifuged at 300g for 10min, the supernatant was discarded, resuspended in phenol red-free 1640+4% FBS, centrifuged again and then resuspended, counted, and the cell count was adjusted to 1.5X10% ⁶ mu.L of cell suspension was added to each well at a volume of each mL. Collecting target cell U266B, centrifuging at 1000rpm for 3min, re-suspending, counting, and adjusting cell number to 3×10 ⁵ mu.L of cell suspension was added per well at a volume of each mL, ensuring that E:T was 10:1. The antibodies were diluted in phenol red free complete medium containing soluble BCMA at an initial concentration of 400nM (4 x final concentration), 10-fold diluted 9 gradients, 25 μl per well, and the final concentration of soluble BCMA was 30ng/mL. The cells were then placed at 37℃with 5% CO ₂ Is cultured for 48 hours. Before detection, 10. Mu.L of medium was aspirated into two wells containing only target cells, then 10. Mu.L of Lysis Solution (10X) was added, after Lysis for 45min, the plates were removed, centrifuged at 1000rpm for 3min, 50. Mu.L of supernatant was aspirated into a new 96-well plate (# 3590), and 50. Mu.L of dissolved Cytotox was added at a 1:1 ratioReagent, after incubation at room temperature for 0.5h, 50. Mu.L of Stop solution was added, and absorbance (490 nm) was measured with FlexStation 3 (Molecular Devices).

TABLE 16 Effect of soluble BCMA on antibody cell killing Activity

The results show that the cytotoxic activity of the bispecific antibodies of the present disclosure is less affected by soluble BCMA, and that 30ng/mL of soluble BCMA only increases the cytotoxic EC50 by a factor of 4-9 compared to 20.5 compared to the AMGEN701 in the absence of soluble BCMA; REGN5458 is affected by soluble BCMA comparable to the bispecific antibodies of the present disclosure, but cell killing activity is significantly lower than the bispecific antibodies of the present disclosure.

Test example 6 cytokine release level of BCMA/CD3 bispecific antibody

CD3T cell adaptor molecules can cause cytokine storms. Thus, in the development of CD3T cell adaptor molecules, it is desirable to keep cytokines (especially IL6, which is not related to potency but causes side effects) at a low level. This test example investigated the cytokine ifnγ and IL6 release levels of the bispecific antibodies of the present disclosure.

300g of fresh PBMC (from Xuanfeng Bio Inc.) was centrifuged for 10min, the supernatant was discarded, resuspended in phenol red-free 1640+4% FBS, centrifuged again, resuspended and counted to adjust the cell number to 1.5X10) ⁶ mu.L/mL was added to each well. Collecting target cell U266B or H929, centrifuging at 1000rpm for 3min, re-suspending, counting, and adjusting cell number to 3×10 ⁵ mu.L of E/T was added per well at 10:1. The antibodies were diluted in phenol red free complete medium at an initial concentration of 400nM (4 Xfinal concentration), diluted in a gradient and added at 25. Mu.L per well. The cells were then placed at 37℃with 5% CO ₂ Is cultured for 48 hours for standby.

IL6 was detected by ELISA. Centrifuging the cell sample at 1000rpm for 3min, collecting 50 mu L of cell supernatant, diluting with a general dilution of a specimen for 6 times, diluting with an IL6 standard for 6 times, adding diluted samples or standards with different concentrations (100 mu L/hole) into a detection plate, sealing a reaction hole with sealing plate gummed paper, incubating for 90min at 37 ℃ and then washing the plate for 5 times, adding a biotinylated antibody working solution (100 mu L/hole), sealing the reaction hole with new sealing plate gummed paper, incubating for 60 min at 37 ℃ and then washing the plate for 5 times, adding an enzyme conjugate working solution (100 mu L/hole), sealing the reaction hole with new sealing plate gummed paper, incubating for 30 min at 37 ℃ and then washing the plate for 5 times, adding a chromogenic substrate (TMB) for 100 mu L/hole, incubating for 8 min at 37 ℃ in a dark place, adding a reaction termination solution for 100 mu L/hole, mixing uniformly, and immediately measuring an OD450 value (within 3 min).

Ifnγ was detected using HTRF method. The cell samples were centrifuged at 1000rpm for 3min, 50. Mu.L of cell supernatant was collected, the assay kit was equilibrated to room temperature, and two detection antibodies in the kit were diluted with assay buffer (20-fold dilution). Taking 16 mu L of a 20-fold diluted sample and an IFN gamma standard substance in a 384-well plate, and adding 4 mu L of diluted corresponding detection antibody; the sealing material is attached, shake-mixed evenly, centrifuged at 1000rpm for 1 minute, after incubation at normal temperature and overnight, centrifuged at 1000rpm for 1 minute, the sealing material is removed, absorption values of 665nm and 620nm are read by using a PHERAstar multifunctional enzyme labeling instrument, and data are processed and analyzed by using Graphpad Prism 5.

TABLE 17-1U 266B cytotoxic Activity experiments IL-6 Release

Release of ifnγ in the u266b cytotoxic activity assay

TABLE 17-3H 929 Release of IFNgamma in cytotoxic Activity experiments

The results show that the bispecific antibodies of the present disclosure release IL6 and ifnγ at lower levels than amben 701, suggesting that the bispecific antibodies of the present disclosure have better safety.

Biological evaluation of in vivo Activity

Test example 7 efficacy of BCMA/CD3 bispecific antibodies in NCI-H929 subcutaneous transplantation tumor model

The present disclosure uses the human myeloma NCI-H929 cell human PBMC NOG mouse xenograft tumor model to evaluate BCMA & CD3 dual antibodies in terms of anti-tumor activity.

NOG mice, females, 8-10 weeks old, were purchased from beijing vitelli laboratory animal technologies limited.

Human multiple myeloma cell line NCI-H929 was purchased from Shanghai Ming Biotech Co.

NCI-H929 cells were added to RPMI-1640 medium containing 10% FBS and 0.05mM 2-mercaptoethanol and incubated in 5% CO ₂ Culturing in a saturated humidity incubator at 37 ℃. Collecting NCI-H929 cells in logarithmic growth phase, re-suspending in RPMI-1640 medium containing 50% matrigel, and adjusting cell concentration to 2×10 ⁷ /mL. Inoculating 0.1mL cell suspension under aseptic condition to the right back of mice, the inoculation concentration is 2×10 ⁶ 0.1 mL/mouse. 1 day after tumor cell inoculation, resuscitate hBMC frozen in liquid nitrogen, culture in PRMI-1640 medium containing 10% HIFBS (FBS, 56 ℃ for 30 min), and culture in medium containing 5% CO ₂ Incubate for 6h at 37 ℃. After incubation hBMC was collected, resuspended in PBS buffer and the cell concentration was adjusted to 2.5X10 ⁷ /mL. Under aseptic condition, injecting 0.2mL of cell suspension into mice in abdominal cavity with injection concentration of 5×10 ⁶ 0.2 mL/mouse. At an average tumor volume of up to 100mm ³ When left and right, animals are randomly grouped according to tumor volumes, so that the difference of the tumor volumes of each group is less than 10% of the average value. The group diary was D0 and dosing was started according to animal body weight. Immediately after grouping, 2 times per week and 4 times continuously (IP, BIW 2). Animals were sacrificed on day 17 (D17), tumor was taken, weighed and photographed for recording.

Tumor inhibition rate (%) =1-T/C (%). T/C (%) = (T-T0)/(C-C0) ×100%, where T, C is the tumor volume or tumor weight of the treatment and control groups at the end of the experiment; t0 and C0 are tumor volumes or tumor weights at the beginning of the experiment.

TABLE 18 drug efficacy of antibodies in NCI-H929 subcutaneous transplantation tumor model

Administration group	Dosage (mg/kg)	Tumor inhibition rate (volume)	Tumor inhibition rate (weight)%
Blank (PBS)
4E3-11-1	0.5	95.14	98.07
4E3-11-1	0.05	63.55	63.74
AMGEN701	0.5	47.39	42.71
AMGEN701	0.1	13.09	8.12
REGN5458	0.75	49.69	43.23

Experimental results show that 4E3-11-1 of the present disclosure has higher activity in inhibiting tumor growth in vivo than the corresponding doses of magen 701 and REGN5458 at lower dosing conditions. Drug-related animal death and other obvious drug-related adverse side effects were not observed during the experiment.

Test example 8 efficacy of BCMA/CD3 bispecific antibody in RPMI-8226 in situ tumor model

In the experiment, an in-situ tumor model is established by inoculating human myeloma RPMI-8226-lucG cells through tail veins of an NDG (tumor-associated mice), and the curative effect of the BCMA-CD3 bispecific antibody on in-situ transplantation tumor of the human myeloma RPMI-8226-lucG mice is evaluated by measuring the bioluminescence signal value (Total Flux) and the weight (BW) of the tumor-associated mice after the BCMA-CD3 bispecific antibody is respectively administered.

RPMI-8226-lucG cells were cultured at 5X 10 ⁶ The mice were inoculated intravenously at 200. Mu.L/rat tail in NDG (from Bai Osai laboratory animal Co., ltd.). Two donors were freshly isolated PBMCs mixed at a 1:1 ratio at 4X 10 14 days after tumor cell inoculation ⁶ mu.L/mouse was injected into the abdominal cavity of the mice. After 18 days of tumor cell inoculation, each mouse was intraperitoneally injected with a bioluminescent substrate (15 mg/mL), injected at a volume of 10mL/kg, anesthetized by isoflurane, and imaged by a small animal imaging system after 10 minutes of injection. Body weight was removed, bioluminescence signal values were too large and too small, mice were randomly grouped by bioluminescence signal, 6 mice per group, and the day of grouping was defined as the day of the experiment D0, D1 starting intraperitoneal injection of each antibody, 2 times per week, 6 times total (table 19). Imaging was performed 2 times per week, body weight was weighed, and data was recorded. The following tumor suppression rate was calculated as data of D21.

Tumor inhibition rate (%) =1-T/C (%). T/C (%) = (T-T0)/(C-C0) ×100%, where T, C is the bioluminescence signal value of the treatment group and control group at the end of the experiment; t0 and C0 are bioluminescence signal values at the beginning of the experiment.

TABLE 19 drug efficacy of antibodies in RPMI-8226 in situ tumor model

Administration group	Dosage (mg/kg)	Tumor inhibition rate%
Blank (PBS)	/	/
4E3-11-1	0.05	>100％
3B2-11-6164-2	0.2	69.22％
4E3-11-6164-5	0.2	>100％
REGN5458	0.15	-5.35％

Experimental results show that the tumor inhibition rate of 4E3-11-1 and 4E3-11-6164-5 reaches more than 100%, and 3B2-11-6164-2 also shows stronger tumor inhibition effect; and REGN5458 did not exhibit efficacy.

RPMI-8226-lucG cells were cultured at 5X 10 ⁶ The mice were inoculated with 200. Mu.L/rat tail vein. After 14 days of tumor cell inoculation, each mouse was intraperitoneally injected with a bioluminescent substrate (15 mg/mL), injected at a volume of 10mL/kg, anesthetized by isoflurane, and imaged by a small animal imaging system after 10 minutes of injection. The mice were removed with too little body weight, too large and too small bioluminescence signal values, and randomly divided into groups of Vehicle (PBS) and 33H4-15-1 (0.2 mpk) 2, 9 per group, by bioluminescence signal. On day 2 after grouping, one donor was freshly isolated PBMCs at 4.5×10 ⁶ The mice were injected into the abdominal cavity. Intraperitoneal injection of antibody was started on day 5 after the grouping, and this day was defined as experiment D0, administered 2 times a week 1 (table 20). Imaging was performed 2 times per week, body weight was weighed, and data was recorded. The following tumor suppression rates were calculated as data for D14.

TABLE 20 drug efficacy of antibodies in RPMI-8226 in situ tumor model

Administration group	Dosage (mg/kg)	Tumor inhibition rate%
Blank (PBS)	/	/
33H4-15-1	0.2	>100％

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, the description and examples should not be construed as limiting the scope of the disclosure. The disclosures of all patent and scientific literature cited herein are expressly incorporated by reference in their entirety.

Claims

An antigen-binding molecule comprising at least one first antigen-binding domain that specifically binds BCMA comprising heavy chain variable region BCMA-VH and light chain variable region BCMA-VL, and at least one second antigen-binding domain that specifically binds CD3, wherein:

(i) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in said BCMA-VH comprise the amino acid sequence of SEQ ID NO:29, BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR 3; and BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 in said BCMA-VL comprise SEQ ID NO:30, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(ii) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in said BCMA-VH comprise the amino acid sequence of SEQ ID NO:31, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR 3; and BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 in said BCMA-VL comprise SEQ ID NO:32, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(iii) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in said BCMA-VH comprise the amino acid sequence of SEQ ID NO:33, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3; and BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 in said BCMA-VL comprise SEQ ID NO:34, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3; or (b)

(iv) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in said BCMA-VH comprise the amino acid sequence of SEQ ID NO:35, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3; and BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 in said BCMA-VL comprise SEQ ID NO:36, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3;

preferably:

(i) The BCMA-VH has: comprising SEQ ID NO:5, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:6, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:7, BCMA-HCDR3 of the amino acid sequence of seq id no; and the BCMA-VL has: comprising SEQ ID NO:8, a BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:9, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:10, BCMA-LCDR3 of the amino acid sequence of 10; or (b)

(ii) The BCMA-VH has: comprising SEQ ID NO:11, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:12, and a BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:13, BCMA-HCDR3 of the amino acid sequence of 13; and the BCMA-VL has: comprising SEQ ID NO:14, a BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:15, and BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:16, BCMA-LCDR3 of the amino acid sequence of 16; or (b)

(iii) The BCMA-VH has: comprising SEQ ID NO:17, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:18, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:19, BCMA-HCDR3 of the amino acid sequence of 19; and the BCMA-VL has: comprising SEQ ID NO:20, comprising the amino acid sequence of SEQ ID NO:21, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:22, BCMA-LCDR3 of the amino acid sequence of 22; or (b)

(iv) The BCMA-VH has: comprising SEQ ID NO:23, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:24, and a BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:25, BCMA-HCDR3 of the amino acid sequence of seq id no; and the BCMA-VL has: comprising SEQ ID NO:26, comprising the amino acid sequence of SEQ ID NO:27, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:28, and a BCMA-LCDR3 of the amino acid sequence of seq id no.
The antigen binding molecule of claim 1, wherein:

(i) The BCMA-VH comprises SEQ ID NO:29, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:30, an amino acid sequence of seq id no; or (b)

The BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 37. SEQ ID NO:38 and SEQ ID NO:39, and the BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO:42 and SEQ ID NO:43, and a sequence of amino acids of the group consisting of seq id no;

Preferably, the method comprises the steps of,

the BCMA-VH comprises SEQ ID NO:37, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:40, or

The BCMA-VH comprises SEQ ID NO:38, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:40, or

The BCMA-VH comprises SEQ ID NO:39, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:40, or

The BCMA-VH comprises SEQ ID NO:37, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:41, or

The BCMA-VH comprises SEQ ID NO:37, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:42, or

The BCMA-VH comprises SEQ ID NO:37, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:43, an amino acid sequence of seq id no; or (b)

(ii) The BCMA-VH comprises SEQ ID NO:31, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:32, an amino acid sequence of seq id no; or (b)

The BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 44. SEQ ID NO:45 and SEQ ID NO:46, and the BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:47 and SEQ ID NO:48, and a sequence of amino acids of the group consisting of seq id no;

Preferably, the method comprises the steps of,

the BCMA-VH comprises SEQ ID NO:44, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:47, or

The BCMA-VH comprises SEQ ID NO:45, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:47, or

The BCMA-VH comprises SEQ ID NO:46, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:47, or

The BCMA-VH comprises SEQ ID NO:44, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:48, or

The BCMA-VH comprises SEQ ID NO:45, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:48, or

The BCMA-VH comprises SEQ ID NO:46, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO: 48; or (b)

(iii) The BCMA-VH comprises SEQ ID NO:33, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:34, an amino acid sequence of seq id no; or (b)

The BCMA-VH comprises SEQ ID NO:49 or SEQ ID NO:50, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:51, an amino acid sequence of seq id no;

preferably, the method comprises the steps of,

the BCMA-VH comprises SEQ ID NO:49, and said BCMA-VL comprises the amino acid sequence of SEQ ID NO:51, or

The BCMA-VH comprises SEQ ID NO:50, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:51, an amino acid sequence of seq id no; or (b)

(iv) The BCMA-VH comprises SEQ ID NO:35, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:36, an amino acid sequence of seq id no; or (b)

The BCMA-VH comprises SEQ ID NO:52 or SEQ ID NO:53, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:54, an amino acid sequence of seq id no;

preferably, the method comprises the steps of,

the BCMA-VH comprises SEQ ID NO:52, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:54, or

The BCMA-VH comprises SEQ ID NO:53, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:54, an amino acid sequence of seq id no.
The antigen-binding molecule of claim 1 or 2, wherein the second antigen-binding domain that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein:

(i) The CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in the CD3-VH comprise the sequences of SEQ ID NOs: 63, CD3-HCDR1, CD3-HCDR2 and CD3-HCDR 3; and the CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3 in the CD3-VL comprise the amino acid sequences of SEQ ID NOs: 64, the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR 3; or (b)

(ii) The CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in the CD3-VH comprise the sequences of SEQ ID NOs: 65, CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3; and the CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3 in the CD3-VL comprise the amino acid sequences of SEQ ID NOs: 66, the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3;

preferably, the method comprises the steps of,

(i) The CD3-VH has: comprising SEQ ID NO:55, comprising the amino acid sequence of SEQ ID NO:56, and a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:57, CD3-HCDR3 of the amino acid sequence of seq id no; and the CD3-VL has: comprising SEQ ID NO:58, a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:59, and a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:60, a CD3-LCDR3 of the amino acid sequence of 60; or (b)

(ii) The CD3-VH has: comprising SEQ ID NO:55, comprising the amino acid sequence of SEQ ID NO:61, and a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:62, CD3-HCDR3 of the amino acid sequence of seq id no; and the CD3-VL has: comprising SEQ ID NO:58, a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:59, and a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:60, a CD3-LCDR3 of the amino acid sequence of 60;

More preferably, the CD3-VH comprises SEQ ID NO:63, and said CD3-VL comprises the amino acid sequence of SEQ ID NO:64, an amino acid sequence of 64; or (b)

The CD3-VH comprises SEQ ID NO:65, and said CD3-VL comprises the amino acid sequence of SEQ ID NO: 66.
The antigen-binding molecule of any one of claims 1 to 3, wherein the second antigen-binding domain that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein:

(i) The BCMA-VH has: comprising SEQ ID NO:5, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:6, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:7, BCMA-HCDR3 of the amino acid sequence of seq id no; and the BCMA-VL has: comprising SEQ ID NO:8, a BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:9, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:10, and

the CD3-VH has: comprising SEQ ID NO:55, comprising the amino acid sequence of SEQ ID NO:56, and a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:57, CD3-HCDR3 of the amino acid sequence of seq id no; and the CD3-VL has: comprising SEQ ID NO:58, a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:59, and a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:60, a CD3-LCDR3 of the amino acid sequence of 60; or (b)

(ii) The BCMA-VH has: comprising SEQ ID NO:5, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:6, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:7, BCMA-HCDR3 of the amino acid sequence of seq id no; and the BCMA-VL has: comprising SEQ ID NO:8, a BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:9, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:10, and

the CD3-VH has: comprising SEQ ID NO:55, comprising the amino acid sequence of SEQ ID NO:61, and a CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:62, CD3-HCDR3 of the amino acid sequence of seq id no; and the CD3-VL has: comprising SEQ ID NO:58, a CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:59, and a CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:60, a CD3-LCDR3 of the amino acid sequence of 60;

preferably, the method comprises the steps of,

(i) The BCMA-VH comprises SEQ ID NO:37, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:40, and

the CD3-VH comprises SEQ ID NO:63, and said CD3-VL comprises the amino acid sequence of SEQ ID NO:64, an amino acid sequence of 64; or (b)

(ii) The BCMA-VH comprises SEQ ID NO:37, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:43, and

The CD3-VH comprises SEQ ID NO:65, and said CD3-VL comprises the amino acid sequence of SEQ ID NO: 66.
The antigen binding molecule of any one of claims 1 to 4, wherein the antigen binding molecule further comprises an Fc region comprising two subunits capable of associating; the Fc region is preferably an IgG Fc region, more preferably an IgG ₁ An Fc region;

preferably, the Fc region comprises one or more amino acid substitutions that reduce binding to an Fc receptor, more preferably, the amino acid substitutions reduce binding to an fcγ receptor;

more preferably, the Fc region is a human IgG ₁ The Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index.
The antigen binding molecule of any one of claims 1 to 5, wherein the antigen binding molecule comprises two first antigen binding domains that specifically bind BCMA, two second antigen binding domains that specifically bind CD3, and an Fc region;

preferably, wherein the first antigen binding domain that specifically binds BCMA is Fab and/or the second antigen binding domain that specifically binds CD3 is scFv.
The antigen binding molecule of any one of claims 1 to 6, wherein the antigen binding molecule comprises two first chains having a structure of formula (a) and two second chains having a structure of formula (b);

(a) [ BCMA-VH ] - [ CH1] - [ CD3-VH ] - [ linker ] - [ CD3-VL ] - [ linker ] - [ subunit of Fc region ]; the linkers in formula (a) are preferably identical or different peptide linkers;

(b)[BCMA-VL]-[CL]；

preferably, the antigen binding molecule has:

comprising SEQ ID NO:76, and a first strand comprising the amino acid sequence of SEQ ID NO:72, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:77, and a first strand comprising the amino acid sequence of SEQ ID NO:72, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:78, and a first strand comprising the amino acid sequence of SEQ ID NO:72, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:76, and a first strand comprising the amino acid sequence of SEQ ID NO:73, a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:76, and a first strand comprising the amino acid sequence of SEQ ID NO:74, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:76, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:80, and a first strand comprising the amino acid sequence of SEQ ID NO:72, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:81, and a first strand comprising the amino acid sequence of SEQ ID NO:72, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:79, and a first strand comprising the amino acid sequence of SEQ ID NO:73, a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:79, and a first strand comprising the amino acid sequence of SEQ ID NO:74, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:79, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:84, and a first strand comprising the amino acid sequence of SEQ ID NO:82, a second strand of an amino acid sequence; or (b)

Comprising SEQ ID NO:85, and a first strand comprising the amino acid sequence of SEQ ID NO:82, a second strand of an amino acid sequence; or (b)

Comprising SEQ ID NO:86, and a first strand comprising the amino acid sequence of SEQ ID NO:82, a second strand of an amino acid sequence; or (b)

Comprising SEQ ID NO:84, and a first strand comprising the amino acid sequence of SEQ ID NO:83, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:85, and a first strand comprising the amino acid sequence of SEQ ID NO:83, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:86, and a first strand comprising the amino acid sequence of SEQ ID NO:83, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:88, and a first strand comprising the amino acid sequence of SEQ ID NO:87, a second strand of the amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:89, and a first strand comprising the amino acid sequence of SEQ ID NO:87, a second strand of the amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:90, and a first strand comprising the amino acid sequence of SEQ ID NO:87, a second strand of the amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:91, and a first strand comprising the amino acid sequence of SEQ ID NO:87, a second strand of the amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:95, and a first strand comprising the amino acid sequence of SEQ ID NO:94, and a second strand of an amino acid sequence of 94; or (b)

Comprising SEQ ID NO:96, and a first strand comprising the amino acid sequence of SEQ ID NO:94, and a second strand of an amino acid sequence of 94; or (b)

Comprising SEQ ID NO:97, and a first strand comprising the amino acid sequence of SEQ ID NO:94, and a second strand of an amino acid sequence of 94; or (b)

More preferably, the antigen binding molecule has:

comprising SEQ ID NO:76, and a first strand comprising the amino acid sequence of SEQ ID NO:72, and a second strand of an amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:79, and a first strand comprising the amino acid sequence of SEQ ID NO:75, and a second strand of an amino acid sequence of seq id no.
The antigen binding molecule of any one of claims 1 to 5, wherein the first antigen binding domain that specifically binds BCMA is Fab; the second antigen binding domain that specifically binds CD3 is a substituted Fab comprising a tin chain and an obscur chain linked directly or through a linker, respectively, to two polypeptide chains of the variable region, excluding the light chain constant region and the heavy chain constant region CH1; or (b)

The second antigen binding domain that specifically binds CD3 is Fab; the first antigen binding domain that specifically binds BCMA is a substituted Fab comprising a tin chain and an obscur chain linked directly or through a linker, respectively, to two polypeptide chains of the variable region, excluding the light chain constant region and the heavy chain constant region CH1;

preferably, the tin chain comprises a sequence selected from the group consisting of SEQ ID NOs: 98 to SEQ ID NO:116, said Obscurin chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO:117 to SEQ ID NO:152 and SEQ ID NO:162 to SEQ ID NO:166, an amino acid sequence of the group consisting of seq id no;

more preferably, the tin chain comprises SEQ ID NO:114, said Obscurin chain comprising the amino acid sequence of SEQ ID NO: 152.
The antigen binding molecule of claim 8, wherein the antigen binding molecule further comprises an Fc region comprising a first subunit and a second subunit capable of associating, the C-terminus of the tin chain is fused to the N-terminus of the first subunit directly or through a linker, and the C-terminus of CH1 of the Fab is fused to the N-terminus of the second subunit directly or through a linker; the first subunit and the second subunit have one or more amino acid substitutions that reduce homodimerization of the Fc region; or (b)

Wherein the antigen binding molecule further comprises an Fc region comprising a first subunit capable of associating with a second subunit, the C-terminus of the Obscurin chain is fused to the N-terminus of the second subunit directly or through a linker, and the C-terminus of CH1 of the Fab is fused to the N-terminus of the first subunit directly or through a linker; the first subunit and the second subunit have one or more amino acid substitutions that reduce homodimerization of the Fc region;

preferably, the antigen binding molecule comprises a first chain having a structure represented by formula (c), a second chain having a structure represented by formula (b), a third chain having a structure represented by formula (d), and a fourth chain having a structure represented by formula (e),

(c) A second subunit of [ BCMA-VH ] - [ CH1] - [ Fc region ];

(b)[BCMA-VL]-[CL]；

(d) A first subunit of a [ CD3-VH ] - [ linker ] - [ Titin chain ] - [ Fc region ];

(e) [ CD3-VL ] - [ linker ] - [ Obscurin strand ];

wherein a first subunit of the Fc region has a raised structure according to the knob and socket technique and a second subunit of the Fc region has a pore structure according to the knob and socket technique; the linkers in formulae (d) and (e) are preferably identical or different peptide linkers; or alternatively

The antigen binding molecule comprises a first chain having a structure represented by formula (f), a second chain having a structure represented by formula (b), a third chain having a structure represented by formula (g), and a fourth chain having a structure represented by formula (h),

(f) [ BCMA-VH ] - [ CH1] - [ first subunit of Fc region ];

(b)[BCMA-VL]-[CL]；

(g) A second subunit of [ CD3-VH ] - [ linker ] - [ Obscurin chain ] - [ Fc region ];

(h) [ CD3-VL ] - [ linker ] - [ tin chain ];

wherein a first subunit of the Fc region has a raised structure according to the knob and socket technique and a second subunit of the Fc region has a pore structure according to the knob and socket technique; the linkers in formulae (g) and (h) are preferably identical or different peptide linkers;

more preferably, the antigen binding molecule has:

comprising SEQ ID NO:92, comprising the amino acid sequence of SEQ ID NO:87, comprising the amino acid sequence of SEQ ID NO:70, and a third strand comprising the amino acid sequence of SEQ ID NO:71, and a fourth strand of the amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:93, comprising the amino acid sequence of SEQ ID NO:87, comprising the amino acid sequence of SEQ ID NO:70, and a third strand comprising the amino acid sequence of SEQ ID NO:71, and a fourth strand of the amino acid sequence of seq id no; or (b)

Comprising SEQ ID NO:171, comprising the amino acid sequence of SEQ ID NO:83, comprising the amino acid sequence of SEQ ID NO:167, and a third strand comprising the amino acid sequence of SEQ ID NO:168, a fourth strand of an amino acid sequence of 168; or (b)

Comprising SEQ ID NO:172, comprising the amino acid sequence of SEQ ID NO:87, comprising the amino acid sequence of SEQ ID NO:167, and a third strand comprising the amino acid sequence of SEQ ID NO:168, and a fourth strand of the amino acid sequence of 168.
An antigen binding molecule that competes with the antigen binding molecule of any one of claims 1 to 9 for binding to human BCMA and/or human CD3.
An anti-BCMA antibody comprising a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein:

(i) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in said BCMA-VH comprise the amino acid sequence of SEQ ID NO:29, BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR 3; and BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 in said BCMA-VL comprise SEQ ID NO:30, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(ii) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in said BCMA-VH comprise the amino acid sequence of SEQ ID NO:31, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR 3; and BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 in said BCMA-VL comprise SEQ ID NO:32, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(iii) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in said BCMA-VH comprise the amino acid sequence of SEQ ID NO:33, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3; and BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 in said BCMA-VL comprise SEQ ID NO:34, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3; or (b)

(iv) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in said BCMA-VH comprise the amino acid sequence of SEQ ID NO:35, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3; and BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 in said BCMA-VL comprise SEQ ID NO:36, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3;

preferably:

(i) The BCMA-VH has: comprising SEQ ID NO:5, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:6, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:7, BCMA-HCDR3 of the amino acid sequence of seq id no; and the BCMA-VL has: comprising SEQ ID NO:8, a BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:9, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:10, BCMA-LCDR3 of the amino acid sequence of 10; or (b)

(ii) The BCMA-VH has: comprising SEQ ID NO:11, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:12, and a BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:13, BCMA-HCDR3 of the amino acid sequence of 13; and the BCMA-VL has: comprising SEQ ID NO:14, a BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:15, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:16, BCMA-LCDR3 of the amino acid sequence of 16; or (b)

(iii) The BCMA-VH has: comprising SEQ ID NO:17, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:18, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:19, BCMA-HCDR3 of the amino acid sequence of 19; and the BCMA-VL has: comprising SEQ ID NO:20, comprising the amino acid sequence of SEQ ID NO:21, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:22, BCMA-LCDR3 of the amino acid sequence of 22; or (b)

(iv) The BCMA-VH has: comprising SEQ ID NO:23, a BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:24, and a BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:25, BCMA-HCDR3 of the amino acid sequence of seq id no; and the BCMA-VL has: comprising SEQ ID NO:26, comprising the amino acid sequence of SEQ ID NO:27, and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:28, and a BCMA-LCDR3 of the amino acid sequence of seq id no.
The anti-BCMA antibody according to claim 11 wherein:

(i) The BCMA-VH comprises SEQ ID NO:29, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:30, an amino acid sequence of seq id no; or (b)

The BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 37. SEQ ID NO:38 and SEQ ID NO:39, and the BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 40. SEQ ID NO: 41. SEQ ID NO:42 and SEQ ID NO:43, and a sequence of amino acids of the group consisting of seq id no;

Preferably, the method comprises the steps of,

the BCMA-VH comprises SEQ ID NO:37, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:40, or

The BCMA-VH comprises SEQ ID NO:38, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:40, or

The BCMA-VH comprises SEQ ID NO:39, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:40, or

The BCMA-VH comprises SEQ ID NO:37, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:41, or

The BCMA-VH comprises SEQ ID NO:37, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:42, or

The BCMA-VH comprises SEQ ID NO:37, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:43, an amino acid sequence of seq id no; or (b)

(ii) The BCMA-VH comprises SEQ ID NO:31, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:32, an amino acid sequence of seq id no; or (b)

The BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 44. SEQ ID NO:45 and SEQ ID NO:46, and the BCMA-VL comprises the amino acid sequence of the group consisting of SEQ ID NO:47 or SEQ ID NO: 48;

preferably, the method comprises the steps of,

the BCMA-VH comprises SEQ ID NO:44, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:47, or

The BCMA-VH comprises SEQ ID NO:45, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:47, or

The BCMA-VH comprises SEQ ID NO:46, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:47, or

The BCMA-VH comprises SEQ ID NO:44, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:48, or

The BCMA-VH comprises SEQ ID NO:45, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:48, or

The BCMA-VH comprises SEQ ID NO:46, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO: 48; or (b)

(iii) The BCMA-VH comprises SEQ ID NO:33, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:34, an amino acid sequence of seq id no; or (b)

The BCMA-VH comprises SEQ ID NO:49 or SEQ ID NO:50, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:51, an amino acid sequence of seq id no;

preferably, the method comprises the steps of,

the BCMA-VH comprises SEQ ID NO:49, and said BCMA-VL comprises the amino acid sequence of SEQ ID NO:51, or

The BCMA-VH comprises SEQ ID NO:50, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:51, an amino acid sequence of seq id no; or (b)

(iv) The BCMA-VH comprises SEQ ID NO:35, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:36, an amino acid sequence of seq id no; or (b)

The BCMA-VH comprises SEQ ID NO:52 or SEQ ID NO:53, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:54, an amino acid sequence of seq id no;

preferably, the method comprises the steps of,

the BCMA-VH comprises SEQ ID NO:52, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:54, or

The BCMA-VH comprises SEQ ID NO:53, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:54, an amino acid sequence of seq id no.
A pharmaceutical composition comprising:

a therapeutically effective amount of the antigen binding molecule of any one of claims 1 to 10 or the anti-BCMA antibody of claim 11 or 12, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients;

preferably, the pharmaceutical composition further comprises at least one second therapeutic agent.
An isolated nucleic acid encoding the antigen binding molecule of any one of claims 1 to 10 or the anti-BCMA antibody of claim 11 or 12.
A host cell comprising the isolated nucleic acid of claim 14.
A method of treating or ameliorating a B cell disorder or an autoimmune disease associated with BCMA expression, the method comprising the step of administering to a subject a therapeutically effective amount of the antigen binding molecule of any one of claims 1 to 10 or the anti-BCMA antibody of claim 11 or 12;

Preferably, the B cell disorder associated with BCMA expression is a plasma cell disorder; the autoimmune disease is systemic lupus erythematosus;

more preferably, the plasma cell disorder is selected from: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, fahrenheit macroglobulinemia, isolated bone plasmacytoma, extramedullary plasmacytoma, bone sclerosis myeloma, heavy chain disease, unknown monoclonal gammaglobulopathy, and stasis type multiple myeloma.
A method of treating or ameliorating a disease, the method comprising the step of administering to a subject a therapeutically effective amount of the antigen binding molecule of any one of claims 1 to 10 or the anti-BCMA antibody of claim 11 or 12;

preferably, the disease is a B cell disorder or an autoimmune disease;

more preferably, the B cell disorder is selected from: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, fahrenheit macroglobulinemia, isolated bone plasmacytoma, extramedullary plasmacytoma, bone sclerosis myeloma, heavy chain disease, meaningless monoclonal gammaglobulosis, and stasis type multiple myeloma; the autoimmune disease is systemic lupus erythematosus.