WO2022156739A1

WO2022156739A1 - Antigen binding molecule specifically binding to bcma and cd3, and medical use thereof

Info

Publication number: WO2022156739A1
Application number: PCT/CN2022/072945
Authority: WO
Inventors: 石金平; 应华; 黎婷婷; 陶维康
Original assignee: 江苏恒瑞医药股份有限公司; 上海恒瑞医药有限公司
Priority date: 2021-01-20
Filing date: 2022-01-20
Publication date: 2022-07-28
Also published as: CN116917333A; TW202246333A

Abstract

An antigen binding molecule specifically binding to BCMA and CD3, and a medical use thereof.

Description

Antigen-binding molecule that specifically binds BCMA and CD3 and its medical use

This application claims the priority of the Chinese patent application (application number CN202110076465.5) filed on January 20, 2021 and the Chinese patent application (application number CN202110795647.8) filed on July 14, 2021.

technical field

The present disclosure belongs to the field of biotechnology, and more particularly, the present disclosure relates to antigen binding molecules and their applications.

Background technique

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

Multiple myeloma (MM) is the second most common blood cancer in the world. It is mainly characterized by the uncontrolled proliferation of plasma cells in the bone marrow. Cancerous plasma cells rapidly proliferate and spread, resulting in the production of monoclonal immunoglobulins. This in turn causes immunosuppression, osteolysis, and end-organ damage.

Globally, there are more than 138,500 newly diagnosed MM patients. In the past few decades, the emergence of new treatments such as proteasome inhibitors, immunomodulators, and CD38 antibodies has greatly improved the clinical outcomes of MM patients, increasing the life expectancy of patients from 3 to 4 years to 7 years. to 8 years. But most patients still relapse due to drug resistance. Even patients with negative MRD (minimal residual disease) will still relapse. Therefore, there is an urgent need for more effective new treatments. Immunotherapy that reshapes the antitumor activity of immune cells while targeting MM cells would be an excellent therapy for MM.

BCMA (tumor necrosis factor receptor superfamily member 17, TNFRS17) is a type 3 transmembrane protein without a signal peptide, its extracellular domain is rich in cysteine, and TNFR superfamily factor B cell activating factor receptor (BAFF-R ) and TACI work together to regulate B cell proliferation and differentiate B cells into plasma cells. These functional receptors support the long-term survival of B cells by binding to the ligands BAFF or APRIL. BCMA is induced to express only when memory B cells differentiate into plasma cells at a later stage, and is only expressed on the surface of plasmablasts and differentiated plasma cells; while in memory B cells,

B cells or CD34+ hematopoietic stem cells, and other normal tissues are not expressed. Studies on BCMA knockout mice have shown that BCMA only has an important effect on long-term survival plasma cells, and does not regulate B cell homeostasis. Therefore, BCMA is a very specific MM antigen compared to CD38, which is widely expressed in normal tissues (especially immune cells and immune organs).

Bispecific antibodies targeting CD3/TAA are a novel immunotherapy that can simultaneously bind T cells and tumor cells, mimicking the interaction of MHC and TCR, enabling T cells to form lytic synapse and release perforin and granzyme, thereby specifically killing tumor cells. Activated T cells can release cytokines, initiate other immune cells and amplify the immune response against the tumor, ultimately leading to a cascade of T cell proliferation and tumor cell killing. Therefore, CD3/BCMA bispecific antibody can be used as a very excellent immunotherapy against MM.

SUMMARY OF THE INVENTION

The present disclosure provides an antigen binding molecule. It comprises at least one first antigen binding domain that specifically binds BCMA and at least one second antigen binding domain that specifically binds CD3. These antigen binding molecules can provide 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 and at least one second antigen-binding domain that specifically binds CD3, the first antigen-binding domain that specifically binds BCMA An antigen binding domain 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 the BCMA-VH comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 29, respectively; and the BCMA- BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in VL comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 30, respectively; or

(ii) the BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in the BCMA-VH respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 31; and the BCMA- BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in VL comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 32, respectively; or

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

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

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

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 respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 29; and the BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 respectively comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 30; or

(ii) the BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 31; and the BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 respectively comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 32; or

(iii) the BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 33; and the BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 respectively comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 34; or

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

The BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering conventions.

(i) the heavy chain variable region BCMA-VH comprises the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 29, and the light chain variable region BCMA-VL comprises SEQ ID NO: 29 The amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in NO: 30; or

(ii) the heavy chain variable region BCMA-VH comprises the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 31, and the light chain variable region BCMA-VL comprises SEQ ID The amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in NO: 32; or

(iii) the heavy chain variable region BCMA-VH comprises the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 33, and the light chain variable region BCMA-VL comprises SEQ ID NO: 33 The amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in NO: 34; or

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

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

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

(iii) the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 19 and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 22; or

(iv) The heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:23, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:24, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:25 and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 26, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 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, the antigen binding molecule as previously described, wherein:

(i) The heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:5, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:7 and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:8, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:9, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL are murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or the 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*01, and it is unsubstituted or has a structure selected from the group consisting of 48I, 67A, 71A, 73K, One or more amino acid substitutions in the group consisting of 76T and 93V; and/or the light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-39*01 and is unsubstituted or with 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*01 and FR4 derived from IGHJ6*01, and is unsubstituted or has a selected from 1E, 48I, 67A, one or more amino acid substitutions in the group consisting of 71A, 73K, 76T and 93V; and/or said BCMA-VL has FR1-3 derived from IGKV1-39*01 and FR4 derived from IGKJ2*01, and It 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) said heavy chain variable region BCMA-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 29, and said 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. In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:29, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:30.

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

The heavy chain variable region BCMA-VH comprises the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 13 and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 16. In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL are murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or the 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*02, and it is unsubstituted or has a structure selected from the group consisting of 2I, 44V, 45F, One or more amino acid substitutions in the group consisting of 46K, 75A, 76N and 93L; and/or the light chain variable region BCMA-VL has a light chain derived from IGKV1-8*01 or IGKV1-27*01 framework regions, and which are unsubstituted or have 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*02 and FR4 derived from IGHJ1*01, and is unsubstituted or has a selected from 1E, 21I, One or more amino acid substitutions in the group consisting of 44V, 45F, 46K, 75A, 76N and 93L; and/or the BCMA-VL has FR1-3 derived from IGKV1-8*01 or IGKV1-27*01 and FR4 derived from IGKJ4*01 and which are unsubstituted or have 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) said heavy chain variable region BCMA-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 31, and said 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. In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:31, and the light chain variable region BCMA-VL comprises the amino acid sequence of SEQ ID NO:32.

(ii) the heavy chain variable region BCMA-VH comprises at least 90%, 95%, 96% of the amino acid sequence selected from the group consisting of SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46, respectively an amino acid sequence of %, 97%, 98% or 99% sequence identity, and the 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 Amino acid sequences having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity, respectively. In some embodiments, the heavy chain variable region BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, and the light chain variable region Region BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:47 and SEQ ID NO:48. In some embodiments:

The heavy chain variable region BCMA-VH comprises the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 19 and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 22; in some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL are murine or humanized of. In some embodiments, the heavy chain variable region BCMA-VH and/or the 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*01 and is unsubstituted or has a composition selected from the group consisting of 30R, 47R, 49A and 93T one or more amino acid substitutions in the group of Amino acid substitution. In some embodiments, the BCMA-VH has FR1-3 derived from IGHV3-11*01 and FR4 derived from IGHJ1*01, and is unsubstituted or has a selected from 1E, 30R, 47R, One or more amino acid substitutions in the group consisting of 49A and 93T; and/or the BCMA-VL has FR1-3 derived from IGKV1-39*01 and FR4 derived from IGKJ2*01, and which are not Substituted or amino acid substitutions with 44V. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.

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

(iii) the heavy chain variable region BCMA-VH comprises at least 90%, 95%, 96%, 97%, 98% of the amino acid sequence selected from the group consisting of SEQ ID NO: 49 and SEQ ID NO: 50, respectively % or 99% sequence identity, and the light chain variable region BCMA-VL comprises at least 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO:51 Sequence identity of amino acid sequences. In some embodiments, the heavy chain variable region BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 49 and SEQ ID NO: 50, and the light chain variable region BCMA-VL comprises SEQ ID NO: 50 Amino acid sequence of ID NO:51. In some embodiments,

The heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:49, and the 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 the amino acid sequence of 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: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:23, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:24, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:25 and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 26, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 28; in some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL are murine or humanized of. In some embodiments, the heavy chain variable region BCMA-VH and/or the 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*01, and it is unsubstituted or has a structure selected from the group consisting of 24V, 30T, 37V, 49G, One or more amino acid substitutions in the group consisting of 73N, 80F and 89R; and/or the light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-33*01. In some embodiments, the BCMA-VH has FR1-3 derived from IGHV2-70*01 and FR4 derived from IGHJ6*01, and is unsubstituted or has a selected from 1E, 24V, 30T, One or more amino acid substitutions in the group consisting of 37V, 49G, 73N, 80F and 89R; and/or the BCMA-VL has FR1-3 derived from IGKV1-33*01 and FR4 derived from IGKJ2*01 . In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.

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

(iv) the heavy chain variable region BCMA-VH comprises at least 90%, 95%, 96%, 97%, 98% of the amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 53, respectively % or 99% sequence identity to the amino acid sequence, and the light chain variable region BCMA-VL comprises at least 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO: 54, respectively The sequence identity of the amino acid sequence. In some embodiments, the heavy chain variable region BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 53, and the light chain variable region BCMA-VL comprises SEQ ID NO: 53 Amino acid sequence of ID NO:54. In some embodiments,

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

In some embodiments, the antigen-binding molecule of any preceding item, 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) CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in said CD3-VH comprise the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 63, respectively; and said CD3-HCDR1 CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in VL comprise the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in SEQ ID NO: 64, respectively; or

(ii) CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in said CD3-VH comprise the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 65, respectively; and said CD3-HCDR1 CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in VL comprise the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in SEQ ID NO:66, respectively.

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

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 CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 63, respectively; and the CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 comprise the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in SEQ ID NO: 64, respectively; or

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

The CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering conventions.

(i) the heavy chain variable region CD3-VH comprises the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 63, and the light chain variable region CD3-VL comprises SEQ ID NO: 63 The amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in NO:64; or

(ii) the heavy chain variable region CD3-VH comprises the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 65, and the light chain variable region CD3-VL comprises SEQ ID NO: 65 Amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in NO: 66;

(i) the heavy chain variable region CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:56, and CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:57 and the light chain variable region CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:58, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 60; or

(ii) the heavy chain variable region CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:61, and CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:62 and the light chain variable region CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:58, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO: 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 preceding item, wherein: the heavy chain variable region CD3-VH comprises at least 90%, 95%, 96%, 97%, SEQ ID NO:63, An amino acid sequence of 98% or 99% sequence identity, and said light chain variable region CD3-VL comprises at least 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO:64 amino acid sequence of sequence identity; in some embodiments, the heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO: 63, and the light chain variable region CD3-VL comprises SEQ ID NO: 64 amino acid sequence.

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

In some embodiments, the antigen binding molecule of any preceding item, wherein the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:5, comprising SEQ ID NO:6 and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 8, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:9, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:10, and

The heavy chain variable region CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:56, and the amino acid sequence comprising SEQ ID NO:57 and the light chain variable region CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:58, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59, and CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59 The amino acid sequence of NO: 60 of CD3-LCDR3. In some embodiments, the above CDRs are defined according to the Kabat numbering convention.

In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of 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 the amino acid sequence of SEQ ID NO:63, and the light chain variable region CD3-VL comprises the amino acid sequence of SEQ ID NO:64.

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

In some embodiments, the antigen binding molecule of any one of the preceding items, wherein the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:5, comprising SEQ ID NO:6 and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 8, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:9, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO:10, and

The heavy chain variable region CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:61, and the amino acid sequence comprising SEQ ID NO:62 and the light chain variable region CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:58, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59, and CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59 The amino acid sequence of NO: 60 of CD3-LCDR3. In some embodiments, the above CDRs are defined according to the Kabat numbering convention.

The heavy chain variable region CD3-VH comprises the amino acid sequence of SEQ ID NO:65, and the 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 preceding item, wherein the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:11, comprising SEQ ID NO:12 and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 16, and

The heavy chain variable region CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:56, and the amino acid sequence comprising SEQ ID NO:57 and the light chain variable region CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:58, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59, and CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59 The amino acid sequence of NO: 60 of CD3-LCDR3.

In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of 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 the amino acid sequence of SEQ ID NO:46, and the 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 preceding item, wherein the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, comprising SEQ ID NO: 18 and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 22, and

In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of SEQ ID NO:49, and the 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 the amino acid sequence of 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 preceding item, wherein the heavy chain variable region BCMA-VH has: the heavy chain variable region BCMA-VH has: the amino acid comprising SEQ ID NO: 17 The sequence of BCMA-HCDR1, the BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and the BCMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 19; and the light chain variable region BCMA-VL having: comprising SEQ ID NO: 19 BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 22, and

The heavy chain variable region CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:61, and the amino acid sequence comprising SEQ ID NO:62 and the light chain variable region CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:58, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59, and CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59 The amino acid sequence of NO: 60 of CD3-LCDR3.

In some embodiments, the antigen binding molecule of any preceding item, wherein the heavy chain variable region BCMA-VH has: the heavy chain variable region BCMA-VH has: the amino acid comprising SEQ ID NO:23 The sequence of BCMA-HCDR1, the BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 24, and the BCMA-HCDR3 comprising the amino acid sequence of SEQ ID NO: 25; and the light chain variable region BCMA-VL having: comprising SEQ ID NO: 25 BCMA-LCDR1 comprising the amino acid sequence of ID NO: 26, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 27, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 28, and

In some embodiments, the heavy chain variable region BCMA-VH comprises the amino acid sequence of 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 the amino acid sequence of SEQ ID NO:53, and the 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 preceding item, wherein the antigen-binding molecule further comprises an Fc region comprising two subunits capable of association. 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 _an IgGi Fc region. In some embodiments, the Fc region comprises one or more amino acid substitutions that reduce its binding to an Fc receptor. In particular, the amino acid substitutions described can 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 to Fey receptors, compared to the wild-type Fc region. In some embodiments, the Fc region is _a human IgGi 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 the amino acid sequence of SEQ ID NO:69.

In some embodiments, the antigen-binding molecule of any preceding item, 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 an 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 a 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 a 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 a subunit of the Fc region, in N-terminal to C-terminal order, either directly or by linkage child connection. In some embodiments, the second antigen-binding domain that specifically binds CD3, the first antigen-binding domain that specifically binds BCMA, and a subunit of the Fc region, in N-terminal to C-terminal order, either directly or by linkage child connection. In some embodiments, the first antigen-binding domain that specifically binds BCMA, a subunit of the Fc region, and the second antigen-binding domain that specifically binds CD3, in N-terminal to C-terminal order, either directly or through a linker connect. In some embodiments, the second antigen-binding domain that specifically binds CD3, a subunit of the Fc region, the first antigen-binding domain that specifically binds BCMA, in N-terminal to C-terminal order, either directly or through a linker connect.

In some embodiments, the antigen-binding molecule of any preceding item, 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 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 a subunit of the Fc region, in N-terminal to C-terminal order, either directly or through a linker connect. In some embodiments, the C-terminus of the Fab heavy chain is fused to the N-terminus of the scFv, either directly or through a linker. In some embodiments, the heavy chain of the Fab, the scFv, and one subunit of the Fc region are linked in N-terminal to C-terminal order, either directly or through a linker.

In some embodiments, the antigen-binding molecule of any preceding item, wherein the antigen-binding molecule comprises two first strands having the structure of formula (a) and two first strands having the structure of formula (b) second chain,

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

(b) [BCMA-VL]-[CL].

In some embodiments, the formula (a) and formula (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 the structure L1-(GGGGS) _nL2 , wherein L1 is _a bond, _A , GS, GGS or GGGS (SEQ ID NO: 177) and n is 0 , 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, 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 following structure:

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

In some embodiments, the antigen binding molecule of any preceding item has:

a first strand comprising the amino acid sequence of SEQ ID NO:76, and a second strand comprising the amino acid sequence of SEQ ID NO:72; or

a first strand comprising the amino acid sequence of SEQ ID NO:77, and a second strand comprising the amino acid sequence of SEQ ID NO:72; or

a first strand comprising the amino acid sequence of SEQ ID NO:78, and a second strand comprising the amino acid sequence of SEQ ID NO:72; or

a first strand comprising the amino acid sequence of SEQ ID NO:76, and a second strand comprising the amino acid sequence of SEQ ID NO:73; or

a first strand comprising the amino acid sequence of SEQ ID NO:76, and a second strand comprising the amino acid sequence of SEQ ID NO:74; or

a first strand comprising the amino acid sequence of SEQ ID NO:76, and a second strand comprising the amino acid sequence of SEQ ID NO:75; or

a first strand comprising the amino acid sequence of SEQ ID NO:80, and a second strand comprising the amino acid sequence of SEQ ID NO:72; or

a first strand comprising the amino acid sequence of SEQ ID NO:81, and a second strand comprising the amino acid sequence of SEQ ID NO:72; or

a first strand comprising the amino acid sequence of SEQ ID NO:79, and a second strand comprising the amino acid sequence of SEQ ID NO:73; or

a first strand comprising the amino acid sequence of SEQ ID NO:79, and a second strand comprising the amino acid sequence of SEQ ID NO:74; or

a first strand comprising the amino acid sequence of SEQ ID NO:79, and a second strand comprising the amino acid sequence of SEQ ID NO:75; or

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

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

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

a first strand comprising the amino acid sequence of SEQ ID NO:84, and a second strand comprising the amino acid sequence of SEQ ID NO:83; or

a first strand comprising the amino acid sequence of SEQ ID NO:85, and a second strand comprising the amino acid sequence of SEQ ID NO:83; or

a first strand comprising the amino acid sequence of SEQ ID NO:86, and a second strand comprising the amino acid sequence of SEQ ID NO:83; or

a first strand comprising the amino acid sequence of SEQ ID NO:88, and a second strand comprising the amino acid sequence of SEQ ID NO:87; or

a first strand comprising the amino acid sequence of SEQ ID NO:89, and a second strand comprising the amino acid sequence of SEQ ID NO:87; or

a first strand comprising the amino acid sequence of SEQ ID NO:90, and a second strand comprising the amino acid sequence of SEQ ID NO:87; or

a first strand comprising the amino acid sequence of SEQ ID NO:91, and a second strand comprising the amino acid sequence of SEQ ID NO:87; or

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

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

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

In some embodiments, the antigen binding molecule of any preceding item has:

A first strand comprising the amino acid sequence of SEQ ID NO:79, and a second strand comprising the amino acid sequence of SEQ ID NO:75.

In some embodiments, the antigen-binding molecule as previously described, wherein the first antigen-binding domain that specifically binds BCMA is a Fab, the second antigen-binding domain that specifically binds CD3 is a substituted Fab, and The substituted Fab comprises a Titin chain and an Obscurin chain connected directly or through a linker to the two polypeptide chains of the variable region, respectively, and does not comprise the light chain constant region and the heavy chain constant region CH1; or the specific binding CD3. The second antigen-binding domain is a Fab, and the first antigen-binding domain that specifically binds BCMA is a substituted Fab comprising the two polypeptide chains of the variable region linked directly or via a linker, respectively Titin chain and Obscurin chain do not contain light chain constant region and heavy chain constant region CH1.

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

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

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

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

In some embodiments, the aforementioned antigen-binding molecule comprises a first chain having a structure represented by formula (c), a second chain having a structure represented by formula (b), and a second chain having a structure represented by formula (d) the third strand of the structure and the fourth strand having the structure represented by formula (e),

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

(b) [BCMA-VL]-[CL];

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

(e) [CD3-VL]-[Linker]-[Obscurin chain].

In some embodiments, the first subunit of the Fc region has a raised structure according to the knob and hole technology, and the second subunit of the Fc region has a pore structure according to the knob and hole technology. In some embodiments, the linkers in formula (d) and (e) are preferably the same 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, the aforementioned antigen-binding molecule comprises a first chain having a structure represented by formula (f), a second chain having a structure represented by formula (b), and a second chain having a structure represented by formula (g) the third strand of the structure and the fourth strand having the structure represented by formula (h),

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

(b) [BCMA-VL]-[CL];

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

(h) [CD3-VL]-[Linker]-[Titin chain];

wherein the first subunit of the Fc region has a raised structure according to the knob and hole technology, and the second subunit of the Fc region has a pore structure according to the knob and hole technology; linkers in formulas (g) and (h) The same or different peptide linkers are preferred.

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

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 the structure L1-(GGGGS) _nL2 , wherein L1 is _a bond, A, GS, GGS or GGGS, and n is 0, ₁ , 2, 3, 4, 5, 6, 7, 8, 9 or 10, 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 Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 98 to SEQ ID NO: 116 and the Obscurin chain comprises an amino acid sequence selected from SEQ ID NO: 117 to SEQ ID NO: 152 and Amino acid sequences of the group consisting of SEQ ID NO: 162 to SEQ ID NO: 166. In some embodiments, the Titin chain comprises the amino acid sequence of SEQ ID NO: 114 and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 152.

In some embodiments, the antigen binding molecule has:

The first strand comprising the amino acid sequence of SEQ ID NO:92, the second strand comprising the amino acid sequence of SEQ ID NO:87, the third strand comprising the amino acid sequence of SEQ ID NO:70, and the third strand comprising the amino acid sequence of SEQ ID NO:71 the fourth strand of the amino acid sequence; or

The first strand comprising the amino acid sequence of SEQ ID NO:93, the second strand comprising the amino acid sequence of SEQ ID NO:87, the third strand comprising the amino acid sequence of SEQ ID NO:70, and the third strand comprising the amino acid sequence of SEQ ID NO:71 The fourth strand of the amino acid sequence.

In some embodiments, the antigen binding molecule has:

The first strand comprising the amino acid sequence of SEQ ID NO: 171, the second strand comprising the amino acid sequence of SEQ ID NO: 83, the third strand comprising the amino acid sequence of SEQ ID NO: 167, and the third strand comprising the amino acid sequence of SEQ ID NO: 168 the fourth strand of the amino acid sequence; or

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

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

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

In another aspect, the 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 sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 35, respectively; and said BCMA- BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in VL comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 36, respectively. In some embodiments, the BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 are defined according to the Kabat, IMGT, Chothia, AbM or Contact numbering conventions.

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

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

The heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:5, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:7 and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:8, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:9, and BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:9 BCMA-LCDR3 of the amino acid sequence of NO: 10; In some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL are murine or humanized. In some embodiments, the heavy chain variable region BCMA-VH and/or the 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*01, and it is unsubstituted or has a structure selected from the group consisting of 48I, 67A, 71A, 73K, One or more amino acid substitutions in the group consisting of 76T and 93V; and/or the light chain variable region BCMA-VL has a light chain framework region derived from IGKV1-39*01 and is unsubstituted or with 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*01 and FR4 derived from IGHJ6*01, and is unsubstituted or has a selected from 1E, 48I, 67A, one or more amino acid substitutions in the group consisting of 71A, 73K, 76T and 93V; and/or said BCMA-VL has FR1-3 derived from IGKV1-39*01 and FR4 derived from IGKJ2*01, and It 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, the anti-BCMA antibody as previously described, wherein:

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

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

(ii) the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 11, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 12, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 13 and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 14, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 16; in some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL are murine or humanized of. In some embodiments, the heavy chain variable region BCMA-VH and/or the 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*02, and it is unsubstituted or has a structure selected from 21I, 44V, 45F, One or more amino acid substitutions in the group consisting of 46K, 75A, 76N and 93L; and/or the light chain variable region BCMA-VL has a light chain derived from IGKV1-8*01 or IGKV1-27*01 framework regions, and which are unsubstituted or have 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*02 and FR4 derived from IGHJ1*01, and is unsubstituted or has a selected from 1E, 2I, One or more amino acid substitutions in the group consisting of 44V, 45F, 46K, 75A, 76N and 93L; and/or the BCMA-VL has FR1-3 derived from IGKV1-8*01 or IGKV1-27*01 and FR4 derived from IGKJ4*01 and which are unsubstituted or have 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, the anti-BCMA antibody as previously described, wherein:

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

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

(ii) the heavy chain variable region BCMA-VH comprises at least 90%, 95%, 96% of the amino acid sequence selected from the group consisting of SEQ ID NO:44, SEQ ID NO:45 and SEQ ID NO:46 , an amino acid sequence of 97%, 98% or 99% sequence identity, and the 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 having Amino acid sequences of at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the heavy chain variable region BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46, and the light chain variable region Region BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:47 and SEQ ID NO:48. In some embodiments,

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

(iii) the heavy chain variable region BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 19 and the light chain variable region BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO: 20, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO: 21, and BCMA-LCDR3 comprising the amino acid sequence of SEQ ID NO: 22; in some embodiments, the heavy chain variable region BCMA-VH and/or the light chain variable region BCMA-VL are murine or humanized of. In some embodiments, the heavy chain variable region BCMA-VH and/or the 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*01, and it is unsubstituted or has a composition selected from 30R, 47R, 49A and 93T one or more amino acid substitutions in the group of Amino acid substitution. In some embodiments, the BCMA-VH has FR1-3 derived from IGHV3-11*01 and FR4 derived from IGHJ1*01, and is unsubstituted or has a selected from 1E, 30R, 47R, One or more amino acid substitutions in the group consisting of 49A and 93T; and/or the BCMA-VL has FR1-3 derived from IGKV1-39*01 and FR4 derived from IGKJ2*01, and which are not Substituted or amino acid substitutions with 44V. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.

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

(iii) the heavy chain variable region BCMA-VH comprises at least 90%, 95%, 96%, 97%, 98% of the amino acid sequence selected from the group consisting of SEQ ID NO:49 and SEQ ID NO:50 or an amino acid sequence of 99% sequence identity, and the light chain variable region BCMA-VL comprises at least 90%, 95%, 96%, 97%, 98% or 99% sequence with SEQ ID NO:51 identical amino acid sequences. In some embodiments, the heavy chain variable region BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 49 and SEQ ID NO: 50, and the light chain variable region BCMA-VL comprises SEQ ID NO: 50 Amino acid sequence of ID NO:51. In some embodiments,

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

(iv) the heavy chain variable region BCMA-VH comprises at least 90%, 95%, 96%, 97%, 98% of the amino acid sequence selected from the group consisting of SEQ ID NO:52 and SEQ ID NO:53 or an amino acid sequence of 99% sequence identity, and the light chain variable region BCMA-VL comprises at least 90%, 95%, 96%, 97%, 98% or 99% sequence with SEQ ID NO: 54 identical amino acid sequences. In some embodiments, the heavy chain variable region BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 52 and SEQ ID NO: 53, and the light chain variable region BCMA-VL comprises SEQ ID NO: 53 Amino acid sequence of ID NO:54. In some embodiments,

In another aspect, the present disclosure provides a pharmaceutical composition comprising: a therapeutically effective amount of the antigen binding molecule or anti-BCMA antibody of any one of the foregoing, 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 present disclosure also provides an isolated nucleic acid encoding the antigen binding molecule or anti-BCMA antibody of any of the foregoing.

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

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

In some embodiments, the disease is a B cell disorder and an autoimmune disease. In some embodiments, the disease is a B cell disorder and autoimmune disease 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 of these embodiments, the plasma cell disorder is selected from the group consisting of: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, isolated bone plasma cells tumor, extramedullary plasmacytoma, sclerosing myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma.

In another aspect, the present disclosure also provides use of the antigen-binding molecule, anti-BCMA antibody, nucleic acid or composition of any one of the foregoing 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 and autoimmune disease 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 of these embodiments, the plasma cell disorder is selected from the group consisting of: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, isolated bone plasma cells tumor, extramedullary plasmacytoma, sclerosing myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma.

In another aspect, the present disclosure also provides the antigen binding molecule, anti-BCMA antibody, nucleic acid, or composition of any of the foregoing 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 used to treat or prevent 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 of these embodiments, the plasma cell disorder is selected from the group consisting of: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, isolated bone plasma cells tumor, extramedullary plasmacytoma, sclerosing myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma.

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

Description of drawings

Figure 1A: Schematic diagram of the structure of Format-11 and Format-11-6164;

Figure 1B: Schematic diagram of the structure of Format-14;

Figure 1C: Schematic diagram of the structure of Format-15.

Detailed ways

the term

The terminology used herein is for the purpose of describing the embodiments only and is not intended to be limiting. Unless otherwise defined, 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 the claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprising," "having," "including," and the like, should be construed in an inclusive rather than an exclusive or exhaustive sense; that is, " including but not limited to". Unless stated otherwise, "comprising" includes "consisting of." For example, for BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:5, it specifically comprises BCMA-HCDR1 having the amino acid sequence shown in SEQ ID NO:5.

The three-letter and one-letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

The terms "and/or" such as "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 either or both meanings.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that have been modified later, such as hydroxyproline, gamma-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., alpha carbon bound to hydrogen, carboxyl, amino and R groups) as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide , methionine methyl sulfonium. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have structures that differ from the general chemical structure of amino acids, but function in a similar manner 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 can be made to achieve the final construct, so long as the final construct possesses the desired properties, such as reduction or binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino-terminus and/or the carboxy-terminus of the polypeptide chain. Particular amino acid mutations can be amino acid substitutions. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, ie, replacing one amino acid with another amino acid with different structural and/or chemical properties. Amino acid substitutions include substitutions by non-naturally occurring amino acids or by derivatives of the 20 naturally occurring amino acids (eg, 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5-hydroxylysine) . Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, and the like. It is anticipated that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modifications, may also be available. Various names may be used herein to refer to the same amino acid mutation. Herein, the amino acid residue at a specific position may be expressed in the form of position + amino acid residue, for example, 366W, the amino acid residue at position 366 is W. T366W means that the amino acid residue at position 366 is mutated from the original T to W.

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

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

The term "antibody" is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (eg, bispecific antibodies), full length antibodies, and antibodies fragments (or antigen-binding fragments, or antigen-binding portions) so long as they exhibit the desired antigen-binding activity. "Native antibody" refers to a naturally occurring immunoglobulin molecule. For example, native IgG antibodies are heterotetrameric proteins of about 150,000 Daltons, composed of two light and two heavy chains that are disulfide-bonded. From N to C-terminus, each heavy chain has one variable domain (VH, also known as variable heavy domain, heavy chain variable domain) followed by three constant domains (CH1, CH2 and CH3). Similarly, from N to C-terminus, each light chain has a variable domain (VL, also known as variable light domain, or light chain variable domain) followed by a constant light domain (light chain constant region, CL ). The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to that of a native antibody or having a heavy chain containing an Fc region.

The term "bispecific antibody" refers to an antibody (including antibodies or antigen-binding fragments thereof, such as single chain antibodies) capable of specifically binding to two different antigens or to two different epitopes of the same antigen. Bispecific antibodies of various structures have been disclosed in the prior art, which can be divided into IgG-like bispecific antibodies and antibody fragment-type bispecific antibodies according to the integrity of the IgG molecule; according to the number of antigen-binding regions, they can be divided into bivalent antibodies , trivalent, tetravalent or more bispecific antibodies; according to whether the structure is symmetrical, it can be divided into symmetrical structure bispecific antibodies and asymmetric structure bispecific antibodies. Among them, fragmented bispecific antibodies, such as Fab fragments lacking Fc fragments, which form bispecific antibodies by combining 2 or more Fab fragments in one molecule, have lower immunogenicity and small molecular weight , with high tumor tissue permeability; typical antibody structures of this type such as F(ab)2, scFv-Fab, (scFv)2-Fab, etc. IgG-like bispecific antibodies (for example, with Fc fragments), such antibodies have relatively large molecular weights. The Fc fragments help the purification of the antibody and improve its solubility and stability. The Fc part may also bind to the receptor FcRn, Increase antibody serum half-life, typical bispecific antibody structural models such as KiH, CrossMAb, Triomab quadroma, 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-CrossMAb, etc. (see Aran F. Labrijn et al., Nature Reviews Drug Discovery volume 18, pages 585–608 (2019) ); Chen S1 et al., J Immunol Res. 2019 Feb 11; 2019:4516041).

The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding an antibody to an antigen. Herein, the heavy chain variable region in the first antigen-binding domain that specifically binds BCMA is designated as BCMA-VH, and the light chain variable region is designated as BCMA-VL; the heavy chain variable region in the second antigen-binding domain that specifically binds to CD3 is designated as BCMA-VH. The chain variable region is designated as CD3-VH and the light chain variable region is designated as CD3-VL. VH and VL each contain four conserved framework regions (FRs) and three complementarity determining regions (CDRs). Among them, the term "complementarity determining region" or "CDR" refers to the region within the variable domain that mainly contributes to antigen binding; "framework" or "FR" refers to variable domain residues other than CDR residues. VH contains 3 CDR regions: HCDR1, HCDR2 and HCDR3; VL contains 3 CDR regions: LCDR1, LCDR2 and LCDR3. Herein, the three CDR regions in BCMA-VH are designated as BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3, respectively; the three CDR regions in BCMA-VL are designated as BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3, respectively The three CDR regions in CD3-VH are marked as CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 respectively; the three CDR regions in CD3-VL are marked as CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 respectively. Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the 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 CDRs can be determined by various well-known schemes, for example: the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed., Public Health Service, National Institutes of Health , Bethesda, MD), "Chothia" numbering scheme, "ABM" numbering scheme, "contact" numbering scheme (see Martin, ACR. Protein Sequence and Structure Analysis of Antibody Variable Domains [J]. 2001) and ImMunoGenTics (IMGT) numbering Rules (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9:2278), etc.; the correspondence between various numbering systems is well known to those skilled in the art , exemplary, as shown in Table 1 below.

Table 1. Relationship between CDR numbering systems

CDRCDRs	IMGTIMGT	KabatKabat	AbMAbM	ChothiaChothia	ContactContact
HCDR1HCDR1	27-3827-38	31-3531-35	26-3526-35	26-3226-32	30-3530-35
HCDR2HCDR2	56-6556-65	50-6550-65	50-5850-58	52-5652-56	47-5847-58
HCDR3HCDR3	105-117105-117	95-10295-102	95-10295-102	95-10295-102	93-10193-101
LCDR1LCDR1	27-3827-38	24-3424-34	24-3424-34	24-3424-34	30-3630-36
LCDR2LCDR2	56-6556-65	50-5650-56	50-5650-56	50-5650-56	46-5546-55
LCDR3LCDR3	105-117105-117	89-9789-97	89-9789-97	89-9789-97	89-9689-96

Unless otherwise stated, the "Kabat" numbering convention applies to the variable regions and CDR sequences in the examples of the present disclosure.

The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that retains the antigen-binding ability 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 an antibody heavy chain, including native and engineered Fc regions. In some embodiments, the Fc region comprises two subunits that are 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 the antibodies described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3, and IgG4. Unless otherwise stated, the numbering scheme for the Fc region is the EU numbering scheme.

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 and 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 being less immunogenic in humans. For example, humanization can be achieved by retaining the non-human CDR regions and replacing the remainder of the antibody with human counterparts (ie, the constant regions and the framework region portions of the variable regions).

The term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to an internal binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of a molecule X for its ligand Y can generally be represented by the dissociation constant (KD). Affinity can be measured by conventional methods known in the art, including those described herein. The term "kassoc" or "ka" refers to the on-rate of a particular antibody-antigen interaction, whereas 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 (ie, kd/ka) and expressed as molar concentration (M). The KD value of an antibody can be determined using methods known in the art, e.g., methods for determining the KD of an antibody include measuring surface plasmon resonance using a biosensing system such as a system, or measuring KD in solution by solution equilibrium titration (SET). Affinity.

The term "effector function" refers to those biological activities attributable to an antibody Fc region (either a native sequence Fc region or an amino acid sequence variant Fc region) and which vary with antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (eg, B cell receptors) ; and B cell activation.

The term "monoclonal antibody" refers to a substantially homogeneous population of antibodies or members thereof, ie, the amino acid sequences of antibody molecules contained in the population are identical, except for natural mutations that may be present in minor amounts. In contrast, "polyclonal antibody" preparations typically contain multiple different antibodies with different amino acid sequences in their variable domains, which are typically specific for different epitopes. "Monoclonal" refers to a characteristic of an antibody obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring that the antibody be produced 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 molecular portion capable of being selectively recognized or bound by an antigen-binding protein, including, for example, an antibody. Antigens can have one or more epitopes capable of interacting with different antigen binding proteins (eg, antibodies).

The term "epitope" refers to an area or region on an antigen that is capable of specific binding by an antibody or antigen-binding fragment thereof. Epitopes may be formed from contiguous amino acids (linear epitopes); or comprise non-contiguous amino acids (conformational epitopes), for example, such that the non-contiguous amino acids are sterically due to folding of the antigen (ie, tertiary folding of the antigen by proteinaceous nature) be approached. A conformational epitope differs from a linear epitope in that in the presence of a denaturing solvent, binding of the antibody to the conformational epitope 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 specific epitopes (ie, those that bind 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 antigens (see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see "Antibodies", Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY)).

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

The terms "capable of specifically binding", "specifically binding" or "binding" mean that an antibody is capable of binding to an antigen or an epitope within that antigen with a higher affinity than other antigens or epitopes. Typically, an antibody binds an antigen or an epitope within an antigen with an equilibrium dissociation constant (KD) of about 1×10 ⁻⁷ M or less (eg, about 1×10 ⁻⁸ M or less). In some embodiments, the KD for binding of an antibody to an antigen is 10% or less (eg, 1%) of the KD of the antibody for binding to a non-specific antigen (eg, BSA, casein). KD can be measured using known methods, such as by

measured by surface plasmon resonance assay. However, antibodies that specifically bind to an antigen or to an epitope within an antigen may be cross-reactive to other related antigens, eg, to those from other species (homologous) such as humans or monkeys, eg, Macaca fascicularis Corresponding antigens (cynomolgus, cyno), chimpanzees (Pan troglodytes) (chimpanzee, chimp)) or marmosets (Callithrix jacchus) (common marmoset, marmoset) are cross-reactive.

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

The term "antigen-binding domain that specifically binds CD3" refers to a domain capable of binding CD3 with sufficient affinity such that molecules containing this domain are useful as diagnostic and/or therapeutic agents targeting CD3. In certain embodiments, the antigen binding domain that specifically binds CD3 binds a CD3 epitope that is conserved among CD3 from different species. Herein, an antigen binding domain may refer to an antigen binding moiety, eg, Fab, substituted Fab, or scFv.

The term "linker" refers to a linking unit that connects two polypeptide fragments. In this context, the linkers appearing 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 a structural formula, it indicates that the units on both sides are directly connected by covalent bonds. When the term "bond" appears in a structural unit, it means that the unit has no amino acids and the units on either side of the unit are directly connected.

The terms "antibody-dependent cellular cytotoxicity", "antibody-dependent cell-mediated cytotoxicity" or "ADCC" are mechanisms for inducing cell death that rely on antibody-coated target cells and effector cells with lytic activity (such as natural killer cells (NK), monocytes, macrophages and neutrophils) via Fcγ receptors (FcγRs) expressed on effector cells. For example, NK cells express FcyRIIIa, while monocytes express FcyRI, FcyRII, and FcyRIIIa. ADCC activity of the antibodies provided herein can be assessed using an in vitro assay using antigen-expressing cells as target cells and NK cells as effector cells. Cell lysis is detected based on the release of labels (eg, radioactive substrates, fluorescent dyes, or native intracellular proteins) from lysed cells.

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

The term "complement-dependent cytotoxicity" or "CDC" refers to a mechanism of inducing cell death in which the Fc effector domain of a target-binding antibody binds and activates the complement component C1q, which in turn activates the complement cascade leading to target cell death. Activation of complement can also lead to deposition of complement components on the surface of target cells that promote CDC by binding to complement receptors (eg, CR3) on leukocytes.

The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, naturally occurring and non-naturally occurring, have binding properties similar to the reference nucleic acid, and are Metabolized in a manner similar to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acid (PNA) ). 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 said 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, and such one or more nucleic acid molecules present in one or more locations in the host cell. Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences as well as explicitly indicated sequences. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is replaced by a degenerate base and/or Deoxyinosine residue substitution.

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 artificial chemical mimetics of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Conservatively modified variants thereof are also implicitly encompassed by a particular polypeptide sequence unless otherwise stated.

The term sequence "identity" refers to the degree (percentage) to which the amino acids/nucleic acids of two sequences are identical at equivalent positions when optimally aligned; where gaps are allowed to be introduced if necessary during the alignment process 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, alignment can be accomplished by techniques known to those skilled in the art, eg, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

The terms "fused" or "linked" refer to the attachment of components (eg, an antigen binding domain and an 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 into which additional DNA segments can be ligated. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can integrate into the genome of the host cell upon introduction into the host cell, thereby replicating together with the host genome. The term "expression vector" or "expression construct" refers to a device suitable for transforming a host cell and containing one or more heterologous coding regions to direct and/or control (along with the host cell) the expression of which is operably linked to it. Vectors of nucleic acid sequences. Expression constructs can include, but are not limited to, sequences that affect or control transcription, translation, and when introns are 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 transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Herein, the term includes mutant progeny that have the same function or biological activity as the cell screened or selected in the primary transformed cell. Host cells include prokaryotic and eukaryotic host cells, wherein 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, porcine, goat, bovine, equine and hamster cells, including but not limited to Chinese hamster ovary (CHO) cells, NSO, SP2 cells, HeLa cells, baby hamsters Kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, Hep G2), A549 cells, 3T3 cells and HEK-293 cells. Fungal cells include yeast and filamentous fungal cells, including, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia puntiae, Pichia thermolerans, Pichia salictaria), Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia, Saccharomyces cerevisiae, Saccharomyces cerevisiae , Hansenula polymorpha, Kluyveromyces, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum , Physcomitrella patens and Neurospora crassa. Pichia, any Saccharomyces cerevisiae, Hansenula polymorpha, any Kluyveromyces, Candida albicans, any Aspergillus, Trichoderma reesei, Luc Mold (Chrysosporium lucknowense), any Fusarium spp., Yarrowia lipolytica and Neurospora crassa.

As used in this application, the expressions "cell", "cell line" and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include primary subject cells and cultures derived therefrom, regardless of the number of passages. It will also be appreciated that not all progeny will have exactly the same DNA content due to intentional or unintentional mutations. Mutant progeny that have the same function or biological activity as the original transformed cells from which they were screened are included.

"Optional" or "optionally" means that the subsequently described feature may, but need not, occur and is intended to include instances where the feature occurs or does not occur.

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

The term "pharmaceutically acceptable vehicle" refers to an ingredient in a pharmaceutical formulation that is distinct from the active ingredient and that is not 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 (eg, mammals and non-mammals) such as non-human primates (eg, cynomolgus monkeys), sheep, dogs, cows, chickens, amphibians, and reptiles. Unless expressly stated otherwise, the terms "patient" or "subject" are used interchangeably herein. As used herein, the term "cyno" or "cynomolgus" refers to a cynomolgus monkey (Macaca fascicularis). In certain embodiments, the individual or subject is a human.

"Administering" or "administering," as it applies to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, means that an exogenous drug, therapeutic agent, diagnostic agent, or composition interacts with the animal, human, or biological fluid. , subject, cell, tissue, organ or biological fluid contact.

The term "sample" refers to a collection (eg, fluid, cells, or tissue) isolated from a subject, as well as fluids, cells, or tissue present in a subject. Exemplary samples are biological fluids such as blood, serum and serous fluid, plasma, lymph, urine, saliva, cystic fluid, tears, feces, sputum, mucosal secretions of secretory tissues or secretory organs, vaginal secretions , fluids in ascites, pleura, pericardium, peritoneum, peritoneal cavity and other body cavities, fluids collected from bronchial lavage, synovial fluid, liquid solutions in contact with subjects or biological sources, such as culture media (including conditioned media), Lavage fluid, etc., tissue biopsy samples, fine needle aspiration, surgically resected tissue, organ cultures or cell cultures.

"Treatment or treat" and "treatment" (and grammatical variants thereof) refer to a clinical intervention intended to be applied to a treated individual, and may be performed for prophylactic purposes, or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating disease state, and remission or improved prognosis . In some embodiments, the molecules of the present disclosure are used to delay the development of a disease or slow the progression of a disease.

An "effective amount" is generally sufficient to reduce the severity and/or frequency of symptoms, eliminate those symptoms and/or underlying causes, prevent the appearance of symptoms and/or underlying causes, and/or ameliorate or ameliorate the symptoms caused by or associated with a disease state amount of damage. 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 symptom, particularly a state or symptom associated with the disease state, or to otherwise hinder, delay or reverse the disease state, or any adverse effects associated with the disease Progression of ideal symptoms. A "prophylactically effective amount" is an amount that, when administered to a subject, will have a predetermined preventive effect, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the onset of the disease state or associated symptoms ( or recurrence). Full therapeutic or prophylactic effect does not necessarily occur immediately after the administration of one dose, but may occur after the administration of a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more administrations. A "therapeutically effective amount" and a "prophylactically effective amount" may vary depending on 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 an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status in a patient.

Antigen-binding molecules of the present disclosure

The present disclosure provides antigen-binding molecules with advantageous properties such as affinity, specificity for cell surface BCMA, activity to specifically activate T cells in the presence of BCMA, therapeutic activity, safety (eg, lower cytokines) release), pharmacokinetic properties and druggability (such as yield, purity and 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 CD3. In particular, the antigen binding molecules of the present disclosure have:

a. High affinity for BCMA on the membrane surface. In some embodiments, the antigen-binding molecule binds to the BCMA protein expressed by the stably transfected cell line K562 overexpressing human BCMA with an affinity EC50 of less than 20 nM, and the affinity EC50 is determined by FACS method. In some embodiments, the antigen-binding molecule binds to the BCMA protein expressed by the stably transfected cell line CHOK1 overexpressing cynomolgus monkey BCMA with an affinity EC50 of less than 100 nM, and the affinity EC50 is determined by a FACS method. For the specific test method, see Test Example 1.

b. The ability to preferentially bind to membrane surface BCMA unaffected by high concentrations of soluble BCMA, in some embodiments, the antigen binding molecule is associated with endogenous BCMA in the presence of 200 ng/ml soluble BCMA compared to the absence of soluble BCMA The BCMA-expressing myeloma cell line NCIH929 expressed BCMA protein binding with an _EC50 that did not vary by more than 30%, as determined by FACS. The specific test method is shown in Test Example 2.

c. In vitro specific activation of T cell activity. In some embodiments, the antigen binding molecule activates T cells in the presence of cells that express BCMA, but does not activate T cells in the presence of cells that do not express BCMA. The specific test method is shown in Test Example 3.

d. In vitro specific killing activity against BCMA expressing cells. In some embodiments, the antigen binding molecule can specifically kill cells that express BCMA, but not cells that do not express BCMA. The specific test method is shown in Test Example 4.

e. Induction of low levels of cytokine (IL6 and IFNy) release. In some embodiments, the antigen binding molecule produces no more than 2 ng/mL of IL-6 upon killing the BCMA-expressing cell line U266B at a concentration of 100 nM, and the cytokine release is detected by an ELISA method. In some embodiments, the antigen binding molecule produces no more than 10 ng/mL of IFNγ upon killing the BCMA-expressing cell line U266B at a concentration of 20 nM or less, and the cytokine release is detected by the HTRF method. The specific test method is shown in Test Example 6.

f. Greater in vivo therapeutic activity. (Test Examples 7, 8).

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

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

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

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

In some embodiments, the antigen binding molecule as previously described, 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 amino acid sequence of the light chain variable region BCMA-VL is 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 in 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:54.

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

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

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 items, 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 The amino acid sequence of SEQ ID NO: 64 is shown; or

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

In some embodiments, the antigen binding molecule of any one of the preceding items, wherein the heavy chain variable region BCMA-VH has: BCMA-HCDR1 having an amino acid sequence as set forth in SEQ ID NO: 5, and an amino acid sequence as set forth in SEQ ID BCMA-HCDR2 shown in NO: 6, and BCMA-HCDR3 with amino acid sequence shown in SEQ ID NO: 7; and the light chain variable region BCMA-VL has: amino acid sequence shown in SEQ ID NO: 8 BCMA-LCDR1, BCMA-LCDR2 whose amino acid sequence is shown in SEQ ID NO:9, and BCMA-LCDR3 whose amino acid sequence is shown in SEQ ID NO:10, and

The heavy chain variable region CD3-VH has: CD3-HCDR1 whose amino acid sequence is shown in SEQ ID NO: 55, CD3-HCDR2 whose amino acid sequence is shown in SEQ ID NO: 56, and comprises SEQ ID NO: amino acid sequence CD3-HCDR3 as shown in SEQ ID NO: 57; and the light chain variable region CD3-VL has: CD3-LCDR1 with amino acid sequence as shown in SEQ ID NO: 58, and amino acid sequence as shown in SEQ ID NO: 59 CD3-LCDR2 shown, and CD3-LCDR3 whose amino acid sequence is shown in SEQ ID NO:60.

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 set forth 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 amino acid sequence of the light chain variable region CD3-VL is shown in SEQ ID NO: 64.

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

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 set forth 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 amino acid sequence of the light chain variable region CD3-VL is shown in SEQ ID NO: 66

In some embodiments, the antigen-binding molecule of any preceding item, wherein the antigen-binding molecule further comprises an Fc region comprising two subunits capable of association. 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 _an IgGi Fc region.

Structure of an antigen-binding molecule

The present disclosure provides a bivalent antigen binding molecule that specifically binds BCMA and a bivalent antigen binding molecule that specifically binds CD3 (2+2 format).

Exemplarily, 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:

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

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

(b-1) [CD3-VL]-[CL].

The present disclosure also provides a monovalent antigen binding molecule (1+1 format) that specifically binds BCMA and a monovalent CD3.

Exemplarily, 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 having a structure represented by formula (e) The fourth strand of the structure shown,

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

(b) [BCMA-VL]-[CL];

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

(e) [CD3-VL]-[Linker]-[Obscurin chain];

The linkers in formula (d) and (e) are preferably the same or different peptide linkers.

Exemplarily, 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), and a third chain having a structure represented by formula (d-1) and the fourth chain having the structure shown in formula (e-1),

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

(b) [BCMA-VL]-[CL];

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

(e-1) [CD3-VL]-[Linker]-[Titin chain];

The linkers in formula (d-1) and (e-1) are preferably the same or different peptide linkers.

Exemplarily, 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), and a structure represented by formula (d-2) The third chain and the fourth chain having the structure shown in formula (e-2),

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

(b-1)[CD3-VL]-[CL];

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

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

The linkers in formula (d-2) and (e-2) are preferably the same or different peptide linkers.

Exemplarily, 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), and a structure represented by formula (d-3) The third chain and the fourth chain having the structure shown in 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]-[Titin chain];

The linkers in formula (d-3) and (e-3) are preferably the same or different peptide linkers.

Exemplarily, 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 the fourth chain of the structure represented by formula (h),

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

(b) [BCMA-VL]-[CL];

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

(h) [CD3-VL]-[Linker]-[Titin chain];

The linkers in formula (g) and (h) are preferably the same or different peptide linkers.

Exemplarily, 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), and a third chain having a structure represented by formula (g-1) and the fourth chain having the structure shown in formula (h-1),

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

(b) [BCMA-VL]-[CL];

(g-1) [CD3-VH]-[Linker]-[Titin chain]-[Second subunit of Fc region];

(h-1) [CD3-VL]-[Linker]-[Obscurin chain];

The linkers in formula (g-1) and (h-1) are preferably the same or different peptide linkers.

Exemplarily, 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), and a structure represented by formula (d-4) The third chain and the fourth chain having the structure shown in formula (e-2),

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

(b-1)[CD3-VL]-[CL];

(d-4) [BCMA-VH]-[Linker]-[Titin chain]-[Second subunit of Fc region];

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

The linkers in formula (d-4) and (e-2) are preferably the same or different peptide linkers.

Exemplarily, 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), and a structure represented by formula (d-5) The third chain and the fourth chain having the structure shown in 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]-[Titin chain];

The linkers in formula (d-5) and (e-3) are preferably the same 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:

In some embodiments, the anti-BCMA antibody as previously described, 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 encompassed. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. 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, and/or insertions, and/or substitutions of residues within the amino acid sequence of the antigen-binding molecule. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, so long as the final construct possesses the desired characteristics, such as antigen binding properties.

Substitution, insertion, and deletion variants

In certain embodiments, antigen binding molecule variants with one or more amino acid substitutions are provided. Make substitutions at sites of interest, including CDRs and FRs. Conservative substitutions are shown in Table 2 under the heading "Preferred Substitutions". More substantial changes are provided in Table 2 under the heading "Exemplary Substitutions" and are described further below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the product screened for the desired activity, eg, retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

Table 2. Amino acid substitutions

原始残基original residue	示例性取代Exemplary substitution	优选的取代Preferred substitution
Ala(A)Ala(A)	Val；Leu；IleVal; Leu; Ile	ValVal
Arg(R)Arg(R)	Lys；Gln；AsnLys; Gln; Asn	LysLys
Asn(N)Asn(N)	Gln；His；Asp,Lys；ArgGln; His; Asp, Lys; Arg	GlnGln
Asp(D)Asp(D)	Glu；AsnGlu; Asn	GluGlu
Cys(C)Cys(C)	Ser；AlaSer; Ala	SerSer
Gln(Q)Gln(Q)	Asn；GluAsn;Glu	AsnAsn
Glu(E)Glu(E)	Asp；GlnAsp;Gln	AspAsp
Gly(G)Gly(G)	AlaAla	AlaAla
His(H)His(H)	Asn；Gln；Lys；ArgAsn; Gln; Lys; Arg	ArgArg
Ile(I)Ile(I)	Leu；Val；Met；Ala；Phe；正亮氨酸Leu; Val; Met; Ala; Phe; Norleucine	LeuLeu
Leu(L)Leu(L)	正亮氨酸；Ile；Val；Met；Ala；PheNorleucine; Ile; Val; Met; Ala; Phe	IleIle
Lys(K)Lys(K)	Arg；Gln；AsnArg; Gln; Asn	ArgArg
Met(M)Met(M)	Leu；Phe；IleLeu; Phe; Ile	LeuLeu
Phe(F)Phe(F)	Trp；Leu；Val；Ile；Ala；TyrTrp; Leu; Val; Ile; Ala; Tyr	TyrTyr
Pro(P)Pro(P)	AlaAla	AlaAla
Ser(S)Ser(S)	ThrThr	ThrThr
Thr(T)Thr(T)	SerSer	SerSer
Trp(W)Trp(W)	Tyr；PheTyr; Phe	TyrTyr
Tyr(Y)Tyr(Y)	Trp；Phe；Thr；SerTrp; Phe; Thr; Ser	PhePhe
Val(V)Val(V)	Ile；Leu；Met；Phe；Ala；正亮氨酸Ile; Leu; Met; Phe; Ala; Norleucine	LeuLeu

According to common side chain properties, amino acids can be grouped as follows:

(1) Hydrophobic: Nle, Met, Ala, Val, Leu, Ile;

(2) Neutral, hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) Acidic: Asp, Glu;

(4) Basic: His, Lys, Arg;

(5) Residues that affect strand orientation: Gly, Pro;

(6) Aromatic: Trp, Tyr, Phe.

A non-conservative substitution refers to the substitution of a member of one class for a member of another class.

One class of substitutional variants involves substituting one or more CDR residues of a parent antibody (eg, a humanized or human antibody). Generally, the resulting variant selected for further study will have an altered (eg, improved) certain biological property (eg, increased affinity, decreased immunogenicity) relative to the parent antibody, and/or will Certain biological properties of the parent antibody are substantially retained. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently produced, eg, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more CDR residues are mutated, and variant antibodies are displayed on phage and screened for specific biological activity (eg, binding affinity). Changes (eg, substitutions) can be made to the CDRs, eg, to improve antibody affinity. Such changes can be made to CDR "hot spots" (ie, residues encoded by codons that undergo mutation with high frequency during the somatic maturation process, and/or residues that contact antigen), while the resulting variants are VH or VL tests for binding affinity. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (eg, error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). middle. Then, create secondary libraries. The library is then screened to identify any antibody variants with the desired affinity. Another approach to introducing diversity involves a CDR-directed approach, in which several CDR residues (eg, 4-6 residues at a time) are randomized. CDR residues involved in antigen binding can be specifically identified, eg, using alanine scanning mutagenesis or modeling. In particular, HCDR3 and LCDR3 are frequently targeted.

In certain embodiments, substitutions, insertions or deletions may occur within one or more CDRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (eg, conservative substitutions, as provided herein) can 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 can be used to identify residues or regions of an antibody that can be targeted for mutagenesis is called "alanine scanning mutagenesis." In this method, a residue or group of residues (eg, charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced with neutral or negatively charged amino acids (eg, Ala or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions can be introduced at amino acid positions that show functional sensitivity to the initial substitution. Additionally, antibody-antigen contact points 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 can be screened to determine whether they contain desired properties.

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

Fab Retrofit

In one aspect, in the antigen-binding molecules of the present disclosure, 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, the substituted Fab The Fab comprises a Titin chain and an Obscurin chain linked to the variable region, that is, the original CH1 and CL of the Fab are replaced by a Titin 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 comprising a Titin chain and Obscurin linked to a variable region The chains, ie the original CH1 and CL of the Fab were replaced by Titin and Obscurin chains.

The replacement technology of the Titin chain and Obscurin chain is described in detail in PCT/CN2021/070832 and CN202110527339.7 and the patents which are used as priority documents, which are incorporated herein by reference in their entirety. An optional Titin chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 98 to SEQ ID NO: 116 and an Obscurin chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 117 to SEQ ID NO: 117 are shown in the table below. Amino acid sequences of the group consisting of ID NO: 152 and SEQ ID NO: 162 to SEQ ID NO: 166.

Table 3-1. Amino acid sequence of Titin chain

Table 3-2. Amino acid sequence of Obscurin chain

Modification 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, eg, its binding to an Fcγ receptor, and reduce or Remove the effector function. Native IgG Fc regions, particularly _IgGi Fc regions or _IgG4 Fc regions, may result in the antigen binding molecules of the present disclosure targeting Fc receptor-expressing cells, but not antigen-expressing cells. The engineered Fc regions of the present disclosure exhibit reduced binding affinity for Fc receptors and/or reduced effector function. In some embodiments, the engineered Fc region has a reduced binding affinity for an Fc receptor by more than 50%, 80%, 90%, or 95% 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, eg, FcγRI, FcγRIIa, FcγRIIB, FcγRIIIa. In some embodiments, the engineered Fc region has reduced binding affinity for complement (eg, C1q) compared to the native Fc region. In some embodiments, the engineered Fc region does not have reduced binding affinity to the neonatal Fc receptor (FcRn) compared to the native Fc region. In some embodiments, the engineered Fc region has reduced effector function, which can include, but is not limited to, one or more of the following: reduced complement dependent cytotoxicity (CDC), reduced effector function Antibody-dependent cell-mediated cytotoxicity (ADCC), reduced antibody-dependent cellular phagocytosis (ADCP), reduced cytokine secretion, reduced immune complex-mediated antigen uptake by antigen-presenting cells, reduced 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 cells primed.

For the IgGi Fc region, substitution _of amino acid residues at positions 238, 265, 269, 270, 297, 327, and 329 can reduce effector function. In some embodiments, the Fc region is _a human IgGi Fc region and the amino acid residues at positions 234 and 235 are A, numbered according to the EU numbering convention. For the _IgG4 Fc region, substitution of amino acid residues at positions such as 228 can reduce effector function.

Antigen binding molecules may also contain disulfide bond modifications, such as 354C of the first subunit and 349C of the second subunit. Based on different sources, the amino acid residue at position 356 of the Fc region may be E or D, and the amino acid residue at position 358 may be M or L. In some embodiments, amino acid residue 356 of the Fc region can be E and amino acid residue 358 is M. In some embodiments, amino acid residue 356 of the Fc region can be D and amino acid residue 358 is L.

Undesirable homodimerization may result when the antigen-binding molecule comprises a binding domain fused to two subunits of the Fc region. In order to improve 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 regions of the present disclosure comprise modifications according to the knob-into-hole (KIH) technique, which involves introducing a knob at the interface of the first subunit and a knob at the interface of the second subunit A hole is introduced at the interface of the base. The protruding structures can be positioned in the pore structures, promoting the formation of heterodimers and inhibiting the production of homodimers. The raised structure is constructed by replacing small amino acid side chains from the interface of the first subunit with larger side chains (eg, tyrosine or tryptophan). The pore structure is created in the interface of the second subunit by replacing the large amino acid side chains with smaller amino acid side chains, such as alanine or threonine. The bulge and pore structures are prepared by altering the nucleic acid encoding the polypeptide, with optional amino acid substitutions shown in the table below:

Table 4. KIH mutation combinations

In addition to the knob-hole technique, other techniques for modifying heavy chain CH3 domains to achieve heterodimerization are also known in the art, eg 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 a complete C-terminus ending with the amino acid residue PGK; it may also be a truncated C-terminus, for example, one or two C-terminal amino acid residues have been removed from the truncated C-terminus base. In a preferred aspect, the C-terminus of the Fc region is a shortened C-terminus terminated by a PG. Thus, in some embodiments, a composition of intact antibodies may include a population of antibodies with all K447 residues and/or G446+K447 residues removed. In some embodiments, a composition of intact antibodies may include a population of antibodies without removal of K447 residues and/or G446+K447 residues. In some embodiments, the composition of intact antibodies has a population of antibodies with and without a mixture of antibodies of K447 residues and/or G446+K447 residues.

Recombination method

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

In the case of native antibodies, native antibody fragments or bispecific antibodies 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 the VL of the antibody and/or amino acid sequences comprising the VH of the antibody (eg, the light and/or heavy chains of the antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

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

In one embodiment, the present disclosure provides an isolated nucleic acid encoding an antibody as previously described. Such nucleic acids may independently encode any of the aforementioned polypeptide chains. In another aspect, the disclosure provides one or more vectors (eg, expression vectors) comprising such nucleic acids.

In another aspect, the present disclosure provides host cells comprising such nucleic acids. In one embodiment, there is provided a method of making an antigen binding molecule, an anti-BCMA antibody; wherein the method comprises, under conditions suitable for expression of the antibody, culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, 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, the nucleic acid encoding the protein is 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 routine procedures (eg, by the use of oligonucleotide probes capable of binding specifically to genes encoding antibody heavy and light chains), or produced by recombinant methods or obtained by chemical synthesis.

Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies can be produced in bacteria, especially when glycosylation and Fc effector functions are not required for the antibody. After expression, the antibody can be isolated from the bacterial cell paste in the soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in Antibodies with partially or fully human glycosylation pattern. Suitable host cells for expression (glycosylated) antibodies can also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. A number of baculovirus strains have been identified that can be used in combination with insect cells, especially for transfection of Spodoptera frugiperda cells; plant cell cultures can also be used as hosts, e.g. US5959177, US6040498, US6420548, US7125978 and US6417429; vertebrate cells can also be used as hosts, eg mammalian cell lines adapted to grow in suspension. Other examples of suitable mammalian host cell lines are SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); sertoli) cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat (buffalo rat) hepatocytes ( BRL3A); human lung cells (W138); human hepatocytes (Hep G2); mouse breast 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, NSO, 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. (eds.), Humana Press, Totowa, NJ (2004) , pp. 255-268.

Determination

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, the antibodies of the present disclosure are tested for antigen-binding activity, eg, by known methods such as ELISA, Western blotting, and the like.

In another aspect, competition assays can be used to identify antibodies that compete with BCMA for binding. In certain embodiments, the competing antibody binds the same epitope (eg, a linear or conformational epitope) as the antigen binding molecule or anti-BCMA antibody. In an exemplary competition assay, immobilized BCMA is incubated in solution, The solution contains 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. A second unlabeled antibody can be present in the hybridoma supernatant. As a control, immobilized BCMA was incubated in a solution containing the first labeled antibody but not the second unlabeled antibody, excess unbound antibody was removed, and the amount of label associated with 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, this indicates that the secondary antibody competes with the primary antibody for binding to BCMA.

In one aspect, assays for identifying biologically active anti-BCMA antibodies are provided. See the test cases of this disclosure for details.

immunoconjugate

The present disclosure also provides immunoconjugates comprising antigen binding molecules or anti-BCMA antibodies conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth Inhibitors, toxins (eg, protein toxins, enzymatically active toxins, or fragments thereof of bacterial, fungal, plant or animal origin), 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 biological samples, and the anti-BCMA antibodies provided by the present disclosure can be used to detect the presence of BCMA in biological samples. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample comprises cells or tissue, such as tumor tissue.

In one embodiment, antigen binding molecules or anti-BCMA antibodies for use in a diagnostic or detection method are 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 a 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 methods. In one embodiment, antigen binding molecules or anti-BCMA antibodies are used to select subjects suitable for treatment, eg BCMA or CD3 are biomarkers for selecting patients.

Exemplary disorders that can be diagnosed using the antibodies of the present disclosure, such as B cell disorders associated with BCMA expression, plasma cell disorders, multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenström macroglobulinemia, solitary plasmacytoma of bone, extramedullary plasmacytoma, sclerosing myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma; autoimmune STDs, systemic lupus erythematosus.

In certain embodiments, labeled antigen binding molecules or anti-BCMA antibodies are provided. Labels include, but are not limited to, labels or moieties that are directly detected (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), and moieties that are indirectly detected (eg, indirectly detected via enzymatic reactions or molecular interactions). modules, such as enzymes or ligands).

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

Pharmaceutical compositions of the antigen binding molecules or anti-BCMA antibodies described in the present disclosure are prepared by contacting such antibodies of the desired purity with one or more optional pharmaceutically acceptable carriers, the drug The composition is 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 a sterile filter.

Methods of Treatment and Routes of Administration

Any of the antigen binding molecules or anti-BCMA antibodies provided herein can be used in methods of therapy.

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 associated with BCMA expression, plasma cell disorders, multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis degeneration, Waldenström macroglobulinemia, solitary plasmacytoma of bone, extramedullary plasmacytoma, sclerosing myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma ; Autoimmune disease, systemic lupus erythematosus. And the medicament is in the form of an effective amount for the above-mentioned diseases. In some embodiments, the effective amount is a unit dose (eg, 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 (eg, one, two, three, four, five, or six additional therapeutics) agent). A "subject" according to any of the above embodiments may be a human.

In yet another aspect, there is provided a pharmaceutical composition comprising the antigen binding molecule or anti-BCMA antibody, eg, for use in any of the above pharmaceutical uses or methods of treatment. In one embodiment, a 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 can be used alone or in combination with other agents for therapy. For example, the antibodies of the present disclosure can be administered in combination (simultaneously, or sequentially) with at least one additional therapeutic agent.

The antigen binding molecules or anti-BCMA antibodies of the present disclosure (and any additional therapeutic agents) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and if local treatment is desired, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be by any suitable route, eg, by injection, such as intravenous or subcutaneous injection, depending in part on whether the 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 consistent with the GOOD MEDICAL PRACTICE (GMP) Guideline. Factors considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and others known to the medical practitioner. factor. 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 doses and routes of administration as described herein, or at about 1 to 99% of the doses described herein, or at any dose and by whatever route is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of an antigen binding molecule or anti-BCMA antibody 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 it is administered for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecule is suitably administered to the patient in one or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg of the antigen binding molecule or anti-BCMA antibody may be an initial candidate dose for administration to a patient, whether, for example, by one or more separate administrations or by continuous infusion. A typical daily dose may range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. Accordingly, taking a body weight of 50 kg as an example, an exemplary unit daily dose is 50 μg-5 g.

product

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

Examples and Test Cases

The present disclosure is further described below in conjunction with the examples and test examples, but these examples and test examples do not limit the scope of the present disclosure. The experimental methods without specific conditions in the disclosed examples and test examples usually follow conventional conditions, such as Cold Spring Harbor Antibody Technology Experiment Manual, Molecular Cloning Manual; or conditions suggested by raw material or commodity manufacturers. Reagents with no specific source indicated are conventional reagents purchased in the market.

Example 1. Expression of human BCMA and cynomolgus BCMA

The sequences encoding the extracellular regions of human BCMA and cynomolgus monkey BCMA tagged with human IgG1-Fc were inserted into the phr vector, constructed into expression plasmids, and then transfected into HEK293. The His-tagged human BCMA extracellular domain sequence was inserted into the phr vector, constructed into an expression plasmid, and then transfected into HEK293. The specific transfection steps were as follows: HEK293E cells were inoculated into freestyle expression medium (containing 1% FBS, Gibco, 12338-026) at 1×10 ⁶ /mL, and placed in a constant temperature shaker (120 rpm) at 37 degrees for 24 hours. After 24 hours, the transfection plasmid and transfection reagent PEI were sterilized with a 0.22 μm filter, and then the transfected plasmid was adjusted to 100 μg/100 mL cells, and the mass ratio of PEI (1 mg/mL) and plasmid was 3:1. 10 mL of Opti-MEM and 200 μg of plasmid were mixed, and allowed to stand for 5 minutes; another 10 mL of Opti-MEM and 400 μg of PEI were taken and mixed, and allowed to stand for 5 minutes. The plasmid and PEI were mixed and allowed to stand for 15 min. The plasmid and PEI mixture was slowly added to 200 mL of HEK293E cells, and cultured in a shaker at 8% CO ₂ , 120 rpm, and 37°C. On day 3 of transfection, feed medium (20 mM glucose + 2 mM L-glutamate) was supplemented with 10% volume by volume. On the 6th day of transfection, samples were collected by centrifugation at 4500 rpm for 10 min to collect the cell supernatant, and purified according to the method of Test Example 2. The purified protein can be used in the experiments of the following examples or test examples.

Among them, the amino acid sequences of humanBCMA-ECD-Fc, humanBCMA-ECD-his, cynoBCMA-ECD-his and 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: The underline is the signal peptide sequence; the italicized part is human Fc-linker-tag;

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

Note: The underline is the signal peptide sequence; the italic part is His6-linker-tag;

(3) ECD of His-tagged cynomolgus monkey BCMA: cynoBCMA-ECD-his (BCA-C4E3H7-1 mg) (SEQ ID NO: 3)

Note: The italicized part is linker-10his-tag;

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

Note: The underline is the signal peptide sequence; the double underline is the linker sequence; the italicized part is human Fc-tag.

Embodiment 2.Protein A affinity chromatography purifies the recombinant protein with Fc tag and nickel column purifies the recombinant protein with his tag

Purification of human Fc-tagged protein: The supernatant sample of BCMA expressed by cells was centrifuged at high speed to remove impurities, and purified by Protein A column. The column was rinsed with PBS until the A280 reading dropped to baseline. The target protein was eluted with 100 mM acetic acid, pH 3.5, and neutralized with 1 M Tris-HCl, pH 8.0. The eluted samples were properly concentrated and then changed into PBS, and the obtained proteins were identified as correct by electrophoresis, peptide map and LC-MS, and then separated for use.

Purification of human his-tagged proteins: The supernatant samples of BCMA expressed by cells were centrifuged at high speed to remove impurities. Equilibrate the nickel column with buffer containing PBS, rinse 2-5 times the column volume, and apply the supernatant sample to the Ni Sepharose excel column at a certain flow rate. Rinse the column with PBS buffer until the A280 reading drops to the baseline, then rinse the column with PBS+10mM imidazole to remove non-specifically bound impurity proteins, and collect the effluent, and finally use 300mM imidazole to elute the target The eluted peaks were collected, and the solution was concentrated and exchanged. The obtained protein was identified as correct by electrophoresis, peptide map, and LC-MS.

Example 3. Construction and identification of cell lines expressing recombinant human BCMA and cynomolgus monkey BCMA

In order to screen for antibodies that can better bind to cell surface BCMA, the present disclosure constructed a K562-BCMA cell line expressing human BCMA. The full-length human BCMA gene was cloned into the mammalian cell expression vector pCDH, and HEK293T cells were co-transfected with the three plasmids pVSV-G, pCMV-dR8.91 and pCDH-humanBCMA (

CRL-11268) to package the virus, and 48 hours after transfection, the virus was collected to infect K562 cells (

CCL-243). K562 monoclonal cells with high expression of humanBCMA were obtained by flow sorting 72 hours after infection. The present disclosure also constructs a CHO-K1-cynoBCMA cell line expressing cynomolgus monkeys. The cynomolgus monkey BCMA full-length gene was cloned into mammalian cell expression vector pCDH, and HEK293T cells were co-transfected with three plasmids pVSV-G, pCMV-dR8.91 and pCDH-cynoBCMA (

CRL-11268) to package the virus, and 48 hours after transfection, the virus was collected to infect CHOK1 cells (

CCL-61). 72 hours after infection, CHOK1 monoclonal cells with high expression of cynoBCMA were obtained by flow sorting.

Example 4. Screening and identification of anti-human BCMA hybridoma antibodies

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

Using humanBCMA-ECD-his and cynoBCMA-ECD-his as cross-immunization reagents,

Gold Adjuvant (Sigma Cat No.T2684) with Thermo

Mice were cross-immunized with Alum (Thermo Cat No. 77161) as adjuvant. After primary immunization and 7 booster immunizations, mice with high antibody titer in serum 10-6# (titer 625K) were selected for splenocyte fusion. After fusion, according to the growth density of hybridoma cells, the culture supernatant of hybridoma is detected, and the antibody that specifically binds to cell surface BCMA is screened.

The active monoclonal hybridoma cell lines 4E3, 33H4, 3B2 and 78 were obtained by screening. Hybridoma cells in logarithmic growth phase were collected separately, RNA was extracted with NucleoZol (MN) (according to the kit instructions), and reverse transcribed (PrimeScript ^™ Reverse Transcriptase, Takara, cat#2680A). The cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326Rev.B 0503) and sequenced. The amino acid sequences of the CDRs and variable regions of 4E3, 33H4, 3B2 and 78 are as follows:

Table 5. BCMA antibody CDRs

>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 regions (SEQ ID NO: 35)

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

Note: The underlined area is the CDR area obtained according to the Kabat numbering rule.

Example 5. Humanized design of anti-human BCMA monoclonal antibody

Humanization of murine monoclonal antibodies is carried out according to methods disclosed in many literatures in the art. Briefly, on the basis of the obtained canonical structures of murine antibody VH/VL CDRs, homologous sequences of light chain variable region (VL) and heavy chain variable region (VH) were searched from human germline databases. , transplant the CDR region of the murine antibody to the human template, mutate some residues of VL and VH, and replace the constant region of the murine antibody with the human constant region to obtain the final humanized molecule.

1. Selection and backmutation of the human FR region of 4E3. Among them, the FR1-3 of the variable region of the heavy chain adopts IGHV1-46*01, and the FR4 adopts IGHJ6*01; the FR1-3 of the variable region of the light chain adopts IGKV1-39*01, and the FR4 adopts IGKJ2*01. The sources and back mutations of FR1-3 are shown in the table below.

Table 6. Human FR region selection and backmutation of 4E3

Note: Graft stands for murine antibody CDR implanted human germline FR region sequence. Exemplarily, R71A represents the mutation of the R at position 71 back to A according to the Kabat numbering system. The same below.

In addition, the first amino acid residue of the variable region of the antibody heavy chain was also mutated from Q to E, and the specific sequence of the antibody variable region obtained by humanization of the mouse antibody 4E3 was as follows (underline indicates CDR, the same below):

>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)

2. Selection and backmutation of the human FR region of 33H4. Among them, the FR1-3 of the heavy chain variable region adopts IGHV7-4-1*02, and the FR4 adopts IGHJ1*01; the FR1-3 of the light chain variable region adopts IGKV1-8*01 or IGKV1-27*01, and the FR4 adopts IGKJ4*01. The sources and back mutations of FR1-3 are shown in the table below.

Table 7. Human FR region selection and backmutation of 33H4

In addition, the first amino acid residue of the variable region of the antibody heavy chain was mutated from Q to E, and the specific sequence of the antibody variable region obtained by humanization of the mouse antibody 33H4 is 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)

3. Selection and backmutation of the human FR region of 3B2. Among them, the FR1-3 of the heavy chain variable region adopts IGHV3-11*01, and the FR4 adopts IGHJ1*01; the FR1-3 of the light chain variable region adopts IGKV1-39*01, and the FR4 adopts IGKJ2*01. The sources and back mutations of FR1-3 are shown in the table below.

Table 8. Human FR region selection and backmutation for 3B2

The specific sequence of the antibody variable region obtained by humanization of mouse 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)

Selection and backmutation of the human FR region of 4.78. Among them, IGHV2-70*01 was used for FR1-3 of the heavy chain variable region, and IGHJ6*01 was used for FR4; IGKV1-33*01 was used for FR1-3 of the light chain variable region, and IGKJ2*01 was used for FR4. The sources and back mutations of FR1-3 are shown in the table below.

Table 9. Human FR region selection and backmutation for 78

In addition, the first amino acid residue of the variable region of the antibody heavy chain was mutated from Q to E, and the specific sequence of the antibody variable region obtained by humanization of mouse antibody 78 is 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/CD3 bispecific antibodies

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

The CDR and variable region sequences of the anti-CD3 arms in the bispecific antibodies of the present disclosure are shown below:

Table 10. CDRs of the 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)

The humanized anti-BCMA antibody variable regions were combined with the variable regions of S107E and 6164 of anti-CD3 antibodies and the IgG ₁ mutant IgG ₁ (AA) (L234A/L235A), respectively. Several different forms of bispecific antibodies are each formed, Format-11, Format-11-6164, Format-14 and Format-15.

Format-11 is a symmetrical structure 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, the schematic diagram of which is shown in Figure 1A.

Format-11-6164 is a symmetrical molecule, containing 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;

in:

>IgG1( _CH1 ) (SEQ ID NO: 67)

>CL (SEQ ID NO: 68)

>IgG1 ₍ AA)Fc (SEQ ID NO:69)

Format-14 is an asymmetric structure molecule. The complete molecule consists of four chains, all of which are different. 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 Figure 1B shown (Ob stands for Obscurin).

in:

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

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

Format-15 is an asymmetric structure molecule. The complete molecule has four chains, and the four chains are different. The 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-Titin, the schematic diagram is shown in Figure 1C (Ob represents Obscurin).

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

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

Note: The single-underlined region is the CDR region of the CD3 binding domain obtained according to the Kabat numbering rule, the double-underlined region is the Titin or Obscurin sequence, and the italics is the constant region.

> _IgGi Fc (Knob) (SEQ ID NO: 153)

> _IgGi Fc (Hole) (SEQ ID NO: 154)

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

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

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

Table 11. Bispecific antibodies of the present disclosure

编号Numbering	链1chain 1	链2chain 2	链3chain 3	链4chain 4
4E3-11-14E3-11-1	hu4E3H1-11hu4E3H1-11	hu4E3L1-11hu4E3L1-11
4E3-11-24E3-11-2	hu4E3H2-11hu4E3H2-11	hu4E3L1-11hu4E3L1-11
4E3-11-34E3-11-3	hu4E3H3-11hu4E3H3-11	hu4E3L1-11hu4E3L1-11
4E3-11-44E3-11-4	hu4E3H1-11hu4E3H1-11	hu4E3L2-11hu4E3L2-11
4E3-11-54E3-11-5	hu4E3H1-11hu4E3H1-11	hu4E3L3-11hu4E3L3-11
4E3-11-64E3-11-6	hu4E3H1-11hu4E3H1-11	hu4E3L4-11hu4E3L4-11
4E3-11-6164-14E3-11-6164-1	hu4E3H2-11-6164hu4E3H2-11-6164	hu4E3L1-11hu4E3L1-11
4E3-11-6164-24E3-11-6164-2	hu4E3H3-11-6164hu4E3H3-11-6164	hu4E3L1-11hu4E3L1-11
4E3-11-6164-34E3-11-6164-3	hu4E3H1-11-6164hu4E3H1-11-6164	hu4E3L2-11hu4E3L2-11
4E3-11-6164-44E3-11-6164-4	hu4E3H1-11-6164hu4E3H1-11-6164	hu4E3L3-11hu4E3L3-11
4E3-11-6164-54E3-11-6164-5	hu4E3H1-11-6164hu4E3H1-11-6164	hu4E3L4-11hu4E3L4-11
33H4-11-133H4-11-1	hu33H4H1-11hu33H4H1-11	hu33H4L1-11hu33H4L1-11
33H4-11-233H4-11-2	hu33H4H2-11hu33H4H2-11	hu33H4L1-11hu33H4L1-11
33H4-11-333H4-11-3	hu33H4H3-11hu33H4H3-11	hu33H4L1-11hu33H4L1-11
33H4-11-433H4-11-4	hu33H4H1-11hu33H4H1-11	hu33H4L2-11hu33H4L2-11
33H4-11-533H4-11-5	hu33H4H2-11hu33H4H2-11	hu33H4L2-11hu33H4L2-11
33H4-11-633H4-11-6	hu33H4H3-11hu33H4H3-11	hu33H4L2-11hu33H4L2-11
33H4-15-133H4-15-1	hu33H4H2-15hu33H4H2-15	hu33H4L2-11hu33H4L2-11	Ob-HoleOb-Hole	VL-TitinVL-Titin
3B2-11-13B2-11-1	hu3B2H1-11hu3B2H1-11	hu3B2L1-11hu3B2L1-11
3B2-11-23B2-11-2	hu3B2H2-11hu3B2H2-11	hu3B2L1-11hu3B2L1-11
3B2-11-6164-13B2-11-6164-1	hu3B2H1-11-6164hu3B2H1-11-6164	hu3B2L1-11hu3B2L1-11
3B2-11-6164-23B2-11-6164-2	hu3B2H2-11-6164hu3B2H2-11-6164	hu3B2L1-11hu3B2L1-11
3B2-14-13B2-14-1	hu3B2H1-14hu3B2H1-14	hu3B2L1-11hu3B2L1-11	T-KnobT-Knob	VL-ObVL-Ob
3B2-14-23B2-14-2	hu3B2H2-14hu3B2H2-14	hu3B2L1-11hu3B2L1-11	T-KnobT-Knob	VL-ObVL-Ob
3B2-15-13B2-15-1	hu3B2H2-15hu3B2H2-15	hu3B2L1-11hu3B2L1-11	Ob-HoleOb-Hole	VL-TitinVL-Titin
78-11-178-11-1	hu78H1-11hu78H1-11	hu78L1-11hu78L1-11
78-11-6164-178-11-6164-1	hu78H1-11-6164hu78H1-11-6164	hu78L1-11hu78L1-11
78-11-6164-278-11-6164-2	hu78H2-11-6164hu78H2-11-6164	hu78L1-11hu78L1-11

In Table 11, hu4E3H1-11 in 4E3-11-1 indicates that hu4E3H1 is used as VH (anti-BCMA), and the structure is Format-11; hu4E3H2-11-6164 in 4E3-11-6164-1 indicates 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 used in this disclosure.

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

REGN5458 sequence source: patent WO2020018830

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

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

Chain 3/Chain 4: BCMA light/CD3 light chain (SEQ ID NO: 161)

Example 7. Antigen-binding molecules containing Titin-T chain/Obscurin-O chain

The Titin chain/Obscurin chain of the present disclosure may be derived from any suitable polypeptide, including those derived from PCT/CN2021/070832 and its publications (hereby incorporated by reference in their entirety) and CN202110527339.7 and its patents as priority documents (via The polypeptides are incorporated herein by reference in their entirety. Construct a bispecific antibody, wherein CL is the kappa light chain constant region in PCT/CN2021/070832, the amino acid sequences of Titin chain and Obscurin chain are shown in Table 3-1 and Table 3-2, and the linker sequence includes GGGGS (SEQ ID NO. : 157), ASTKG (SEQ ID NO: 173) or RTVAS (SEQ ID NO: 174), the amino acid sequences of Fc1, Fc2 and CH1 in this embodiment are respectively SEQ ID NO: 153, SEQ ID NO: 154 and SEQ ID NO: 157. ID NO: 67.

7.1 DI Bispecific Antibodies

With reference to Example 5 of PCT/CN2021/070832, DI bispecific antibodies against hNGF and hRANKL were constructed: DI-2 to DI-20, which comprise a first heavy chain, a second heavy chain, and a first light chain as described below and the second light chain:

The first heavy chain: from N-terminal to C-terminal: [VH1-I]-[Linker 1]-[Obscurin chain]-[Fc2],

The first light chain: from N-terminal to C-terminal: [VL1-I]-[Linker 2]-[Titin chain],

Second heavy chain: from N-terminus to C-terminus: [VH2-D]-[CH1]-[Fc1], and

The second light chain: from N-terminal to C-terminal: [VL2-D]-[CL];

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

Table i-1. Correspondence table of Obscurin-O chain/Titin-T chain and linker in DI bispecific antibody

Note: The numbers of Titin chain and Obscurin chain in the table are shown in Table 3-1 and Table 3-2.

The binding activity of the DI-2 to DI-20 bispecific antibodies to their antigens was detected by the method in Test Example 4 of PCT/CN2021/070832. Antibodies were subjected to thermal stability studies. Research methods: The concentration of the antibody was diluted to 5 mg/mL with PBS, and its thermal stability was measured by a high-throughput differential scanning fluorometer (UNCHAINED, specification model: Unit) (sample volume 9 μL; parameter setting: Start Temp 20°C; Incubation 0s; Rate 0.3℃/min; Plate Hold 5s; End Temp 95℃). The experimental results showed that the antigen-binding activity of the engineered bispecific antibody did not change significantly; and, compared with DI-2, the Tm1 of DI-4 to DI-8, DI-10 to DI-16, and DI-20 (°C) and Tonset (°C) were significantly improved, and the thermal stability of the bispecific antibody was better.

Table i-2. Detection of Binding Activity of DI Bispecific Antibodies

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

编号Numbering	Tm1(℃)Tm1(℃)	Tonset(℃)Tonset(℃)	编号Numbering	Tm1(℃)Tm1(℃)	Tonset(℃)Tonset(℃)
DI-2DI-2	55.655.6	48.348.3	DI-11DI-11	57.3557.35	--
DI-4DI-4	60.160.1	52.49352.493	DI-12DI-12	59.959.9	51.72651.726
DI-5DI-5	6161	51.96751.967	DI-13DI-13	6161	50.98850.988
DI-6DI-6	60.860.8	53.01253.012	DI-14DI-14	61.261.2	52.19152.191
DI-7DI-7	60.3460.34	52.00352.003	DI-15DI-15	60.4160.41	50.55850.558
DI-8DI-8	60.6160.61	50.42550.425	DI-16DI-16	61.561.5	50.69150.691
DI-10DI-10	60.260.2	52.76652.766	DI-20DI-20	60.760.7	51.85951.859

The DI bispecific antibody-containing solution was prepared in a buffer of 10 mM acetic acid, pH 5.5, and 9% sucrose, and the solution was incubated in a 40°C incubator for four weeks. After the end, the concentration of the antibody was concentrated to the concentration at the beginning of the incubation, and the solution was observed. Precipitation situation. The experimental results showed that the solution of DI-2 bispecific antibody group appeared precipitation, and DI-3 to DI-7 had better stability than DI-2.

Table i-8. Precipitation of DI Bispecific Antibodies

编号Numbering	开始浓度starting concentration	第4周浓缩到浓度Week 4 concentrated to concentration	溶液沉淀情况solution precipitation
DI-2DI-2	20mg/ml20mg/ml	20mg/ml20mg/ml	出现沉淀Precipitation occurs
DI-3DI-3	20mg/ml20mg/ml	20mg/ml20mg/ml	无沉淀No precipitation
DI-4DI-4	60mg/ml60mg/ml	60mg/ml60mg/ml	无沉淀No precipitation
DI-5DI-5	25mg/ml25mg/ml	25mg/ml25mg/ml	无沉淀No precipitation
DI-6DI-6	60mg/ml60mg/ml	60mg/ml60mg/ml	无沉淀No precipitation
DI-7DI-7	16mg/ml16mg/ml	16mg/ml16mg/ml	无沉淀No precipitation

7.2 PL Bispecific Antibodies

Construction of PL bispecific antibodies against hPDL1 and hCTLA4: 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:

The first heavy chain: from N-terminus to C-terminus: [VH1-P]-[Linker 1]-[Obscurin chain]-[Fc1],

The first light chain: from N-terminal to C-terminal: [VL1-P]-[Linker 2]-[Titin chain],

Second heavy chain: from N-terminus to C-terminus: [VH2-L]-[CH1]-[Fc2], and

The second light chain: from N-terminal to C-terminal: [VL2-L]-[CL];

Wherein, VH1-P and VL1-P are the heavy chain variable region and light chain variable region of the h1831K antibody in 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 Obscurin-O chain, Titin-T chain, linker 1 and 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

The binding activity of the PL bispecific antibody was detected with reference to the ELISA method in Test Example 4 in PCT/CN2021/070832, wherein the hPDL1 and hCTLA4 antigens were purchased from: Sino biology. Antibodies were subjected to thermal stability studies. Method: The concentration of the antibody was diluted to 1.4-3 mg/mL with PBS, and its thermal stability was determined by a high-throughput differential scanning fluorometer (UNCHAINED, specification model: Unit) (loading volume 9 μL; parameter setting: Start Temp 20°C ; Incubation 0s; Rate 0.3℃/min; Plate Hold 5s; End Temp95℃). The experimental results show that the PL bispecific antibody still has good binding activity to the antigen; and, compared with PL-1, the Tm1 (°C), Tagg 266 (°C), Tonset (°C) of PL-2 to PL-19 There is a significant improvement, and the thermal 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:

The first heavy chain: from N-terminus to C-terminus: [VH1-H]-[Linker 1]-[Titin chain]-[Fc1],

The first light chain: from N-terminal to C-terminal: [VL1-H]-[Linker 2]-[Obscurin chain],

Second heavy chain: from N-terminus to C-terminus: [VH2-J]-[CH1]-[Fc2], and

The second light chain: from N-terminal to C-terminal: [VL2-J]-[CL];

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

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

The antigen-binding activity of the HJ bispecific antibody was detected with reference to the method in Test Example 4 in PCT/CN2021/070832. The thermal stability of the antibody was studied. The method was as follows: HJ bispecific antibody dilution solution was prepared with a buffer of 10mM acetic acid pH5.5 and 9% sucrose, and then the bispecific antibody was concentrated by ultrafiltration to obtain different concentrations. HJ bispecific antibody solution (see Table 13-2 for the concentration of HJ bispecific antibody), then the concentrated solution was incubated in a 40°C incubator, on day 0 (that is, before the incubation at 40°C, D0), Day 7 (day 7 of incubation at 40°C, D7), day 14 (day 14 of incubation at 40°C, D14), day 21 (day 21 of incubation at 40°C, D21) and day 28 (day 21 of 40°C incubation, D21) 28 days, D28) to detect the SEC purity of the samples, after 28 days of incubation at 40°C, samples were taken immediately to detect the CE-SDS purity of the samples. The experimental results show that the HJ bispecific antibodies constructed in the present disclosure have no significant changes in the antigen-binding activity; and, compared with HJ-3, the HJ-5 to HJ-11 bispecific antibodies have better thermal stability.

Table i-8. Detection of Binding Activity of PL Bispecific Antibodies

Table i-9. HJ Bispecific Antibody Accelerated Stability Test Results

test case

Test Example 1. Determination of the affinity of BCMA/CD3 bispecific antibody to BCMA by FACS

In order to test the binding ability of the BCMA/CD3 bispecific antibody of the present disclosure to BCMA, flow cytometry was used to detect the BCMA/CD3 bispecific antibody and the stably transfected cell line K562-humanBCMA-High overexpressing human BCMA. (K562) and the stably transfected cell line CHOK1-cynoBCMA-High (CHOK1) overexpressing cynomolgus BCMA.

The details are as follows: the above cells were cultured in 10% FBS IMDM medium, placed in a 37°C, 5% CO ₂ incubator for 2 days, and the cells were added to the cell plate according to the number of cells per well 1×10 ⁵ medium, centrifuged at 300 g for 5 min, and washed once with 1% BSA. The antibody was serially diluted to 8 concentrations, 100 μL per well was added to the cell plate, incubated at 4°C for 1 hour, washed once with 1% BSA, and 100 μL of APC-Goat Anti-human IgG Fc fluorescent secondary antibody dilution (1:400) was added to each well. , and incubated at 4°C for 1 hour. The plate was washed three times with 1% BSA, and 100 μL of PBS was added to each well to read the plate. The EC50 (nM) of each antibody binding to the corresponding cells is shown in the table below.

Table 12. Determination of the ability of antibodies to bind BCMA

The results show that different BCMA-CD3 double antibodies constructed using the BCMA antibodies screened in the present disclosure have good binding ability to BCMA on the membrane surface, and have good cross-binding activity with CynoBCMA.

Test Example 2. The extent to which the antibody-binding membrane BCMA of the present disclosure is affected by soluble BCMA

High concentrations of soluble BCMA (average 30 ng/mL) present in the blood of patients with multiple myeloma interfere with the specific binding of BCMA-CD3 bispecific antibodies to BCMA on the membrane surface. This test case tested whether the presence of high concentrations of soluble BCMA affects the binding of bispecific antibodies of the present disclosure to BCMA antigens on membrane surfaces.

The endogenous BCMA-expressing myeloma cell line NCIH929 with a viability of ≥90% was added to the 3795 cell plate, 1×10 ⁵ cells per well, centrifuged at 300 g for 5 min, washed once with 1% BSA, and added 50 μL A solution containing 1% BSA and 200ng/mL soluble human BCMA was then added with serially diluted bispecific antibodies and incubated at 4°C for 1 hour. After washing the plate once with 1% BSA, add 100 μL of APC Goat Anti-Mouse Ig diluent (1:400) to each well and incubate at 4°C for 1 hour. After washing the plate three times with 1% BSA, add 100 μL of PBS to each well and read the plate. The results are shown in the table below. In the presence of 200 ng/mL soluble BCMA solution, the affinity of the antibody to BCMA on the cell surface did not change significantly, and the antibody had a very good binding ability to BCMA on the membrane surface, and was not affected by soluble BCMA. However, the control antibody AMGEN701 was significantly affected by soluble BCMA.

Table 13. Determination of the ability of antibodies to bind to membrane surface BCMA

Test Example 3. Activity of Bispecific Antibodies of the Disclosure in Activating T Cells in Vitro

This test case studies the activation ability of the antibodies described in this disclosure on T cells, using the BCMA-expressing cell line U266B and the BCMA-non-expressing CHOK1 as target cells to detect the activating ability of the bispecific antibody on Jurkat-Lucia ^™ NFAT cells .

Jurkat-Lucia ^™ NFAT cells were collected and centrifuged, resuspended and counted in 1640+10% FBS medium, the number of cells was adjusted to 1×10 ⁶ cells/mL, and 50 μL (5×10 ⁴ /well) was added to each well. After the target cells U266B or CHOK1 were collected and centrifuged, they were resuspended and counted in medium 1640+10% FBS, and the number of cells was adjusted to 8×10 ⁵ cells/mL. The bispecific antibody was diluted with 1640+10% FBS medium, the initial concentration was 80 nM, and after 5-fold dilution to generate 9 gradients, 25 μL of the solution was added to each well. Then put the treated cells in a 37°C, 5% CO ₂ incubator for 5 hours, take out the culture plate, add 100 μL QUANTI-Luc ^TM Gold at a ratio of 1:1, incubate at room temperature for 5 minutes, and use a microplate reader to detect luminescence The data were processed and analyzed using Graphpad Prism 5.

The results show that in the presence of target cell U266B, the bispecific antibody of the present disclosure can specifically activate Jurkat-Lucia ^™ NFAT cells, and the activation capacity is significantly improved compared with AMGEN701; while on CHOK1 cells that do not express BCMA, the disclosed bispecific antibodies Bispecific antibodies did not activate Jurkat-Lucia ^™ NFAT cells, demonstrating that the ability of bispecific antibodies of the present disclosure to activate T cells is dependent on their target, BCMA.

Table 14. Activity of antibodies to activate T cells in vitro

Note: "--" means negative.

Test Example 4. In Vitro Cytotoxic Activity of Bispecific Antibodies of the Disclosure

This test case studies the killing activity of the bispecific antibodies of the present disclosure as T cell adaptors on tumor cells. The BCMA-expressing cell lines RPMI8226, H929, and CHOK1, which does not express BCMA, were used as target cells to test the target-specific cytotoxic activity of the bispecific antibodies of the present disclosure.

Fresh PBMC (purchased from Xuanfeng Biotechnology Co., Ltd.) were centrifuged at 300g for 10 min, the supernatant was discarded, resuspended with phenol red-free 1640+4% FBS, resuspended and counted after centrifugation again, and the number of cells was adjusted to 1.5×10 ⁶ cells/mL , add 50 μL to each well. The target cells were collected, centrifuged at 1000 rpm for 3 min, resuspended and counted, the number of cells was adjusted to 3×10 ⁵ cells/mL, 25 μL was added to each well, and E:T was 10:1. Antibodies were diluted with phenol red-free complete medium at an initial concentration of 400 nM (4× final concentration), 10-fold diluted in 9 gradients, and 25 μL per well. The cells were then placed in a 37°C, 5% _CO2 incubator for 48h. Before detection, aspirate 10 μL of medium from two wells with only target cells, then add 10 μL of Lysis Solution (10×), lyse for 45 min, take out the culture plate, centrifuge at 1000 rpm for 3 min, and aspirate 50 μL of supernatant into a new 96-well plate ( #3590), add dissolved 50μL CytoTox at a ratio of 1:1

Reagent was incubated at room temperature for 0.5 h, and then 50 μL of stop solution was added, and the absorption light (490 nm) was detected by FlexStation 3 (Molecular Devices).

Table 15-1. Cytotoxic activity of antibodies against RPMI8266

Table 15-2. Cytotoxic activity of antibodies against H929

抗体Antibody	EC50(pM)EC50(pM)	裂解率％Cracking rate %
3B2-15-13B2-15-1	46.3846.38	85.0185.01
33H4-15-133H4-15-1	58.2758.27	80.3880.38
AMGEN701AMGEN701	88.6088.60	87.7387.73

Table 15-3. Cytotoxic activity of antibodies against CHOK1

The results show that the cytotoxic activity of the bispecific antibody of the present disclosure is stronger than that of AMGEN701; and, the cytotoxic activity of the bispecific antibody of the present disclosure is BCMA target-specific, showing no expression of BCMA on the CHOK1 cell line The weaker cytotoxic activity suggests that the bispecific antibody of the present disclosure has better safety.

Test Example 5. Effect of soluble BCMA on the in vitro cytotoxic activity of the antibodies of the present disclosure

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

The BCMA-expressing cell line U266B was used as target cells, and soluble BCMA was additionally added in the cytotoxic activity assay. Take fresh PBMC (purchased from Xuanfeng Biological Company), centrifuge at 300g for 10min, discard the supernatant, resuspend with phenol red-free 1640+4% FBS, resuspend and count after centrifugation again, adjust the number of cells to 1.5×10 ⁶ cells /mL, add 50 μL of cell suspension to each well. The target cells U266B were collected, centrifuged at 1000 rpm for 3 min, resuspended, counted, adjusted to 3×10 ⁵ cells/mL, and 25 μL of cell suspension was added to each well to ensure E:T ratio of 10:1. Antibodies were diluted with phenol red-free complete medium containing soluble BCMA, starting at 400 nM (4× final concentration), 10-fold dilution in 9 gradients, adding 25 μL to each well, and the final concentration of soluble BCMA was 30 ng/mL. The cells were then placed in a 37°C, 5% _CO2 incubator for 48h. Before detection, aspirate 10 μL of medium from two wells containing only target cells, then add 10 μL of Lysis Solution (10×), lyse for 45 min, take out the culture plate, centrifuge at 1000 rpm for 3 min, and aspirate 50 μL of supernatant into a new 96-well plate ( #3590), add dissolved 50μL CytoTox at a ratio of 1:1

Reagent, incubated at room temperature for 0.5h, added 50μL of Stop solution, and detected the absorption light (490nm) with FlexStation 3 (Molecular Devices).

Table 16. Effect of Soluble BCMA on Antibody Cell Killing Activity

The results showed that the cytotoxic activity of the bispecific antibodies of the present disclosure was less affected by soluble BCMA, and 30 ng/mL of soluble BCMA only increased the cytotoxicity EC50 to 4-9 fold in the absence of soluble BCMA. , while AMGEN701 increased to 20.5-fold; REGN5458 was affected by soluble BCMA comparable to the bispecific antibody of the present disclosure, but the cell killing activity was significantly lower than that of the bispecific antibody of the present disclosure.

Test Example 6. Cytokine Release Level of BCMA/CD3 Bispecific Antibody

CD3 T cell adaptor molecules cause cytokine storm. Therefore, when developing CD3 T cell-engaging molecules, it is necessary to keep cytokines (especially IL6, which is irrelevant to drug efficacy but can cause side effects) at a low level. This test case investigated the cytokine IFNy and IL6 release levels of the bispecific antibodies of the present disclosure.

Fresh PBMC (purchased from Xuanfeng Biotechnology Co., Ltd.) were centrifuged at 300g for 10 min, the supernatant was discarded, resuspended with phenol red-free 1640+4% FBS, resuspended and counted after centrifugation again, and the number of cells was adjusted to 1.5×10 ⁶ cells/mL , add 50 μL to each well. The target cells U266B or H929 were collected, centrifuged at 1000 rpm for 3 min, resuspended, counted, adjusted to 3×10 ⁵ cells/mL, 25 μL was added to each well, and E:T was 10:1. Antibodies were diluted with phenol red-free complete medium, starting at a concentration of 400 nM (4× final concentration), and serially diluted, adding 25 μL to each well. The cells were then placed in a 37°C, 5% _CO2 incubator for 48h for later use.

IL6 was detected by ELISA method. Centrifuge the above cell samples at 1000 rpm for 3 min, collect 50 μL of the cell supernatant and dilute it 6 times with the universal sample dilution solution, and also dilute the IL6 standard by 6 times. Add the diluted samples or different concentrations of standards (100 μL/well) to the detection plate. , seal the reaction wells with sealing tape, incubate at 37°C for 90 min, wash the plate 5 times, add biotinylated antibody working solution (100 μL/well), seal the reaction wells with new sealing tape, and incubate at 37°C for 60 minutes After washing the plate 5 times, adding the enzyme conjugate working solution (100 μL/well), sealing the reaction well with new sealing tape, incubating at 37°C for 30 minutes, then washing the plate 5 times, adding 100 μL of chromogenic substrate (TMB) Incubate at 37°C for 8 minutes in the dark, add 100 μL/well of reaction stop solution, and measure the OD450 value immediately after mixing (within 3 minutes).

The detection of IFNγ adopts the HTRF method. The above cell samples were centrifuged at 1000 rpm for 3 min, 50 μL of cell supernatant was collected, the detection kit was equilibrated to room temperature, and the two detection antibodies in the kit were diluted with detection buffer (20-fold dilution). Take 16 μL of 20-fold diluted samples and IFNγ standard in a 384-well plate, add 4 μL of the diluted corresponding detection antibody; paste the sealing material, shake and mix, centrifuge at 1000 rpm for 1 minute, incubate at room temperature overnight, and centrifuge at 1000 rpm for 1 After 10 minutes, the sealing material was removed, the absorption values at 665 nm and 620 nm were read using a PHERAstar multi-function microplate reader, and the data were processed and analyzed using Graphpad Prism 5.

Table 17-1. IL-6 release in U266B cytotoxic activity assay

Table 17-2. Release of IFNγ in U266B cytotoxic activity assay

Table 17-3. Release of IFNγ in H929 cytotoxic activity assay

The results show that the bispecific antibodies of the present disclosure release levels of IL6 and IFNγ lower than AMGEN701, suggesting that the bispecific antibodies of the present disclosure have better safety.

Biological evaluation of in vivo activity

Test Example 7. The efficacy of BCMA/CD3 bispecific antibody in NCI-H929 subcutaneous xenograft model

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

NOG mice, female, 8-10 weeks old, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.

The human multiple myeloma cell line NCI-H929 was purchased from Shanghai Mingjin Biotechnology Co., Ltd.

NCI-H929 cells were added to RPMI-1640 medium containing 10% FBS and 0.05 mM 2-mercaptoethanol and cultured in a saturated humidity incubator at 5% CO ₂ 37°C. Logarithmic growth phase NCI-H929 cells were collected, resuspended in RPMI-1640 medium containing 50% Matrigel, and the cell concentration was adjusted to 2×10 ⁷ /mL. Under sterile conditions, inoculate 0.1 mL of the cell suspension subcutaneously on the right back of the mouse at a concentration of 2×10 ⁶ /0.1 mL/mouse. One day after tumor cell inoculation, hPBMCs frozen in liquid nitrogen were recovered, cultured in PRMI-1640 medium containing 10% HIFBS (FBS, 56 °C for 30 min), and incubated for 6 h in a 37 °C incubator containing 5% _CO2 . After incubation, hPBMCs were collected, resuspended in PBS buffer, and the cell concentration was adjusted to 2.5×10 ⁷ /mL. Under sterile conditions, 0.2 mL of cell suspension was injected intraperitoneally into mice at an injection concentration of 5×10 ⁶ /0.2 mL/mouse. When the average tumor volume reached about 100 mm ³ , animals were randomly divided into groups according to tumor volume, so that the difference in tumor volume between groups was less than 10% of the mean. The day of grouping was D0, and the animals were dosed according to their body weight. Administered immediately after grouping, intraperitoneal administration twice a week, 4 consecutive administrations (IP, BIW×2). The animals were sacrificed on the 17th day (D17), and the tumor was weighed and recorded by photographing.

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

Table 18. Antibody efficacy in NCI-H929 subcutaneous xenograft model

给药组G	给药剂量(mg/kg)Dosage (mg/kg)	抑瘤率(体积)％Tumor inhibition rate (volume)%	抑瘤率(重量)％Tumor inhibition rate (weight) %
空白(PBS)Blank (PBS)
4E3-11-14E3-11-1	0.50.5	95.1495.14	98.0798.07
4E3-11-14E3-11-1	0.050.05	63.5563.55	63.7463.74
AMGEN701AMGEN701	0.50.5	47.3947.39	42.7142.71
AMGEN701AMGEN701	0.10.1	13.0913.09	8.128.12
REGN5458REGN5458	0.750.75	49.6949.69	43.2343.23

The experimental results show that the 4E3-11-1 of the present disclosure has higher tumor growth inhibitory activity in vivo than the corresponding doses of AMGEN701 and REGN5458 under the condition of lower dosage. During the experiment, no drug-related animal death and other obvious drug-related side effects were observed.

Test Example 8. The efficacy of BCMA/CD3 bispecific antibody in RPMI-8226 orthotopic tumor model

In this experiment, NDG mice were inoculated with human myeloma RPMI-8226-lucG cells through the tail vein to establish an orthotopic tumor model, and BCMA-CD3 bispecific antibodies were administered respectively to measure the bioluminescence signal value of tumor-bearing mice after administration (Total Flux) and body weight (BW) to evaluate the efficacy of BCMA-CD3 bispecific antibody on human myeloma RPMI-8226-lucG mouse orthotopic xenograft tumors.

RPMI-8226-lucG cells were inoculated into NDG mice (purchased from Biositu Experimental Animal Co., Ltd.) at 5×10 ⁶ cells/200 μL/mouse tail vein. Fourteen days after tumor cell inoculation, freshly isolated PBMCs from two donors were mixed at a ratio of 1:1 and injected into the abdominal cavity of mice at 4×10 ⁶ cells/100 μL/mouse. Eighteen days after tumor cell inoculation, each mouse was intraperitoneally injected with a bioluminescent substrate (15 mg/mL) in a volume of 10 mL/kg, anesthetized with isoflurane, and photographed by a small animal imaging system 10 minutes after injection. Excessive and too small values of body weight and bioluminescence signal were removed, and the mice were randomly divided into groups according to the bioluminescence signal, with 6 mice in each group, and the day of grouping was defined as the beginning of the experiment on D0 and D1 by intraperitoneal injection of each antibody, twice a week, A total of 6 doses were administered (Table 19). Photographs were imaged twice a week, body weights were weighed, and data were recorded. The following tumor inhibition rates were calculated based on the data of D21.

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

Table 19. Antibody efficacy in the RPMI-8226 orthotopic tumor model

给药组G	给药剂量(mg/kg)Dosage (mg/kg)	抑瘤率％Tumor inhibition rate %
空白(PBS)Blank (PBS)	//	//
4E3-11-14E3-11-1	0.050.05	>100％>100%
3B2-11-6164-23B2-11-6164-2	0.20.2	69.22％69.22%
4E3-11-6164-54E3-11-6164-5	0.20.2	>100％>100%
REGN5458REGN5458	0.150.15	-5.35％-5.35%

The experimental results showed that the tumor inhibition rate of 4E3-11-1 and 4E3-11-6164-5 was more than 100%, and 3B2-11-6164-2 also showed a strong tumor inhibition effect; while REGN5458 showed no efficacy. .

RPMI-8226-lucG cells were inoculated into NDG mice at 5×10 ⁶ cells/200 μL/mouse tail vein. Fourteen days after tumor cell inoculation, each mouse was intraperitoneally injected with a bioluminescent substrate (15 mg/mL) in a volume of 10 mL/kg, anesthetized with isoflurane, and photographed by a small animal imaging system 10 minutes after the injection. Excessive body weight, too large and too small bioluminescence signal values were excluded, and the mice were randomly divided into two groups, Vehicle (PBS) and 33H4-15-1 (0.2mpk), according to bioluminescence signal, with 9 mice in each group. On the 2nd day after grouping, freshly isolated PBMCs from one donor were injected into the abdominal cavity of mice at 4.5×10 ⁶ /mouse. Antibodies were injected intraperitoneally on the 5th day after grouping, and this day was defined as the D0 of the experiment, once a week for a total of 2 doses (Table 20). Photographs were imaged twice a week, body weights were weighed, and data were recorded. The following tumor inhibition rates were calculated based on the data of D14.

Table 20. Antibody efficacy in the RPMI-8226 orthotopic tumor model

给药组G	给药剂量(mg/kg)Dosage (mg/kg)	抑瘤率％Tumor inhibition rate %
空白(PBS)Blank (PBS)	//	//
33H4-15-133H4-15-1	0.20.2	>100％>100%

Although the foregoing invention has been described in detail with the aid of the drawings and examples for a clear understanding, the description and examples should not be construed as limiting the scope of the present 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 and at least one second antigen-binding domain that specifically binds CD3, the first antigen-binding domain that specifically binds BCMA comprising a heavy chain variable region BCMA-VH and light chain variable region BCMA-VL, wherein:

(i) BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in the BCMA-VH comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 29, respectively; and the BCMA- BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in VL comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 30, respectively; or

(ii) the BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in the BCMA-VH respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 31; and the BCMA- BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in VL comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 32, respectively; or

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

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

Preferably:

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

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

(iii) the BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and BCMA comprising the amino acid sequence of SEQ ID NO: 19 -HCDR3; and the BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:20, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:21, and BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:22 BCMA-LCDR3; or

(iv) the BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:23, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:24, and BCMA comprising the amino acid sequence of SEQ ID NO:25 -HCDR3; and the BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:26, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:27, and BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:28 BCMA-LCDR3.
The antigen-binding molecule of claim 1, wherein:

(i) the BCMA-VH comprises the amino acid sequence of SEQ ID NO:29, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:30; or

The BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: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:39 : the amino acid sequence of the group consisting of 41, SEQ ID NO: 42 and SEQ ID NO: 43;

Preferably,

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

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

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

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

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

The BCMA-VH comprises the amino acid sequence of SEQ ID NO:37, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:43; or

(ii) the BCMA-VH comprises the amino acid sequence of SEQ ID NO:31, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:32; or

The BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: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:46 : the amino acid sequence of the group consisting of 48;

Preferably,

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

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

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

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

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

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

(iii) the BCMA-VH comprises the amino acid sequence of SEQ ID NO:33, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:34; or

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

Preferably,

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

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

(iv) the BCMA-VH comprises the amino acid sequence of SEQ ID NO:35, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:36; or

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

Preferably,

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

The BCMA-VH comprises the amino acid sequence of SEQ ID NO:53, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:54.
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) CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in said CD3-VH comprise the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 63, respectively; and said CD3-HCDR1 CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in VL comprise the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in SEQ ID NO: 64, respectively; or

(ii) CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in said CD3-VH comprise the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 65, respectively; and said CD3-HCDR1 CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in VL comprise the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 in SEQ ID NO: 66, respectively;

Preferably,

(i) the CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:56, and CD3 comprising the amino acid sequence of SEQ ID NO:57 -HCDR3; and said CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:58, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59, and CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:60 CD3-LCDR3; or

(ii) the CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:61, and CD3 comprising the amino acid sequence of SEQ ID NO:62 -HCDR3; and said CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:58, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59, and CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:60 CD3-LCDR3;

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

The CD3-VH comprises the amino acid sequence of SEQ ID NO:65, and the 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 to CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein:

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

The CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:56, and CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO:57; And the CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:58, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO:60 ;or

(ii) the BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:5, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and BCMA comprising the amino acid sequence of SEQ ID NO:7 -HCDR3; and the BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:8, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:9, and BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:10 BCMA-LCDR3, and

The CD3-VH has: CD3-HCDR1 comprising the amino acid sequence of SEQ ID NO:55, CD3-HCDR2 comprising the amino acid sequence of SEQ ID NO:61, and CD3-HCDR3 comprising the amino acid sequence of SEQ ID NO:62; And the CD3-VL has: CD3-LCDR1 comprising the amino acid sequence of SEQ ID NO:58, CD3-LCDR2 comprising the amino acid sequence of SEQ ID NO:59, and CD3-LCDR3 comprising the amino acid sequence of SEQ ID NO:60 ;

Preferably,

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

The CD3-VH comprises the amino acid sequence of SEQ ID NO:63, and the CD3-VL comprises the amino acid sequence of SEQ ID NO:64; or

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

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

Preferably, the Fc region comprises one or more amino acid substitutions that can reduce its binding to Fc receptors, more preferably, said amino acid substitutions can reduce its binding to Fcγ receptors;

More preferably, the Fc region is a human IgGi 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 Fc region;

Preferably, wherein 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.
The antigen-binding molecule according to any one of claims 1 to 6, wherein the antigen-binding molecule comprises two first chains having a structure represented by formula (a) and two first chains having a structure represented by formula (b) two chains;

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

(b) [BCMA-VL]-[CL];

Preferably, the antigen binding molecule has:

a first strand comprising the amino acid sequence of SEQ ID NO:76, and a second strand comprising the amino acid sequence of SEQ ID NO:72; or

a first strand comprising the amino acid sequence of SEQ ID NO:77, and a second strand comprising the amino acid sequence of SEQ ID NO:72; or

a first strand comprising the amino acid sequence of SEQ ID NO:78, and a second strand comprising the amino acid sequence of SEQ ID NO:72; or

a first strand comprising the amino acid sequence of SEQ ID NO:76, and a second strand comprising the amino acid sequence of SEQ ID NO:73; or

a first strand comprising the amino acid sequence of SEQ ID NO:76, and a second strand comprising the amino acid sequence of SEQ ID NO:74; or

a first strand comprising the amino acid sequence of SEQ ID NO:76, and a second strand comprising the amino acid sequence of SEQ ID NO:75; or

a first strand comprising the amino acid sequence of SEQ ID NO:80, and a second strand comprising the amino acid sequence of SEQ ID NO:72; or

a first strand comprising the amino acid sequence of SEQ ID NO:81, and a second strand comprising the amino acid sequence of SEQ ID NO:72; or

a first strand comprising the amino acid sequence of SEQ ID NO:79, and a second strand comprising the amino acid sequence of SEQ ID NO:73; or

a first strand comprising the amino acid sequence of SEQ ID NO:79, and a second strand comprising the amino acid sequence of SEQ ID NO:74; or

a first strand comprising the amino acid sequence of SEQ ID NO:79, and a second strand comprising the amino acid sequence of SEQ ID NO:75; or

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

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

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

a first strand comprising the amino acid sequence of SEQ ID NO:84, and a second strand comprising the amino acid sequence of SEQ ID NO:83; or

a first strand comprising the amino acid sequence of SEQ ID NO:85, and a second strand comprising the amino acid sequence of SEQ ID NO:83; or

a first strand comprising the amino acid sequence of SEQ ID NO:86, and a second strand comprising the amino acid sequence of SEQ ID NO:83; or

a first strand comprising the amino acid sequence of SEQ ID NO:88, and a second strand comprising the amino acid sequence of SEQ ID NO:87; or

a first strand comprising the amino acid sequence of SEQ ID NO:89, and a second strand comprising the amino acid sequence of SEQ ID NO:87; or

a first strand comprising the amino acid sequence of SEQ ID NO:90, and a second strand comprising the amino acid sequence of SEQ ID NO:87; or

a first strand comprising the amino acid sequence of SEQ ID NO:91, and a second strand comprising the amino acid sequence of SEQ ID NO:87; or

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

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

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

More preferably, the antigen binding molecule has:

a first strand comprising the amino acid sequence of SEQ ID NO:76, and a second strand comprising the amino acid sequence of SEQ ID NO:72; or

A first strand comprising the amino acid sequence of SEQ ID NO:79, and a second strand comprising the amino acid sequence of SEQ ID NO:75.
The antigen-binding molecule of any one of claims 1 to 5, wherein the first antigen-binding domain that specifically binds BCMA is a Fab; the second antigen-binding domain that specifically binds CD3 is a substituted Fab, The substituted Fab comprises a Titin chain and an Obscurin chain connected directly or through a linker to the two polypeptide chains of the variable region, 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; the first antigen-binding domain that specifically binds BCMA is a substituted Fab, the substituted Fab comprising two polypeptide chains of the variable region, respectively Titin chain and Obscurin chain connected directly or through a linker, excluding light chain constant region and heavy chain constant region CH1;

Preferably, the Titin chain comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 98 to SEQ ID NO: 116, and the Obscurin chain comprises an amino acid sequence selected from SEQ ID NO: 117 to SEQ ID NO: 152 and SEQ ID NO : the amino acid sequence of the group consisting of 162 to SEQ ID NO: 166;

More preferably, the Titin chain comprises the amino acid sequence of SEQ ID NO: 114 and the Obscurin chain comprises the amino acid sequence of SEQ ID NO: 152.
The antigen-binding molecule according to claim 8, wherein the antigen-binding molecule further comprises an Fc region, the Fc region comprises a first subunit and a second subunit capable of association, and the C-terminus of the Titin chain is directly or It is fused to the N-terminus of the first subunit 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 a or more amino acid substitutions that reduce homodimerization of the Fc region; or

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

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 (e) the fourth strand of the structure shown,

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

(b) [BCMA-VL]-[CL];

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

(e) [CD3-VL]-[Linker]-[Obscurin chain];

wherein the first subunit of the Fc region has a raised structure according to the knob and hole technology, and the second subunit of the Fc region has a pore structure according to the knob and hole technology; linkers in formulas (d) and (e) preferably the same or different peptide linkers; or

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 having a structure represented by formula (h) The fourth strand of the structure shown,

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

(b) [BCMA-VL]-[CL];

(g) [CD3-VH]-[Linker]-[Obscurin chain]-[Second subunit of Fc region];

(h) [CD3-VL]-[Linker]-[Titin chain];

wherein the first subunit of the Fc region has a raised structure according to the knob and hole technology, and the second subunit of the Fc region has a pore structure according to the knob and hole technology; linkers in formulas (g) and (h) preferably the same or different peptide linkers;

More preferably, the antigen binding molecule has:

The first strand comprising the amino acid sequence of SEQ ID NO:92, the second strand comprising the amino acid sequence of SEQ ID NO:87, the third strand comprising the amino acid sequence of SEQ ID NO:70, and the third strand comprising the amino acid sequence of SEQ ID NO:71 the fourth strand of the amino acid sequence; or

The first strand comprising the amino acid sequence of SEQ ID NO:93, the second strand comprising the amino acid sequence of SEQ ID NO:87, the third strand comprising the amino acid sequence of SEQ ID NO:70, and the third strand comprising the amino acid sequence of SEQ ID NO:71 the fourth strand of the amino acid sequence; or

The first strand comprising the amino acid sequence of SEQ ID NO: 171, the second strand comprising the amino acid sequence of SEQ ID NO: 83, the third strand comprising the amino acid sequence of SEQ ID NO: 167, and the third strand comprising the amino acid sequence of SEQ ID NO: 168 the fourth strand of the amino acid sequence; or

The first strand comprising the amino acid sequence of SEQ ID NO: 172, the second strand comprising the amino acid sequence of SEQ ID NO: 87, the third strand comprising the amino acid sequence of SEQ ID NO: 167, and the third strand comprising the amino acid sequence of SEQ ID NO: 168 The fourth strand of the amino acid sequence.
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 the BCMA-VH comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 29, respectively; and the BCMA- BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in VL comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 30, respectively; or

(ii) the BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in the BCMA-VH respectively comprise the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 31; and the BCMA- BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in VL comprise the amino acid sequences of BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 in SEQ ID NO: 32, respectively; or

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

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

Preferably:

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

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

(iii) the BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO: 17, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO: 18, and BCMA comprising the amino acid sequence of SEQ ID NO: 19 -HCDR3; and the BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:20, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:21, and BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:22 BCMA-LCDR3; or

(iv) the BCMA-VH has: BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:23, BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:24, and BCMA comprising the amino acid sequence of SEQ ID NO:25 -HCDR3; and the BCMA-VL has: BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:26, BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:27, and BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:28 BCMA-LCDR3.
The anti-BCMA antibody of claim 11, wherein:

(i) the BCMA-VH comprises the amino acid sequence of SEQ ID NO:29, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:30; or

The BCMA-VH comprises an amino acid sequence selected from the group consisting of SEQ ID NO: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:39 : the amino acid sequence of the group consisting of 41, SEQ ID NO: 42 and SEQ ID NO: 43;

Preferably,

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

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

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

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

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

The BCMA-VH comprises the amino acid sequence of SEQ ID NO:37, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:43; or

(ii) the BCMA-VH comprises the amino acid sequence of SEQ ID NO:31, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:32; or

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

Preferably,

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

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

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

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

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

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

(iii) the BCMA-VH comprises the amino acid sequence of SEQ ID NO:33, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:34; or

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

Preferably,

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

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

(iv) the BCMA-VH comprises the amino acid sequence of SEQ ID NO:35, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:36; or

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

Preferably,

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

The BCMA-VH comprises the amino acid sequence of SEQ ID NO:53, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:54.
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 autoimmune disease associated with BCMA expression, the method comprising administering to a subject a therapeutically effective amount of the antigen binding molecule or claim 1 according to any one of claims 1 to 10 The step of claiming the anti-BCMA antibody described in 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 the group consisting of: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenstrom's macroglobulinemia, solitary plasmacytoma of bone, myeloid Extraplasmacytoma, sclerosing myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering 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 the group consisting of: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenstrom's macroglobulinemia, solitary plasmacytoma of bone, myeloid Extraplasmacytoma, sclerosing myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma; the autoimmune disease is systemic lupus erythematosus.