CN117642427A

CN117642427A - Antigen binding molecules that specifically bind CD38, BCMA and CD3 and medical uses thereof

Info

Publication number: CN117642427A
Application number: CN202280041322.3A
Authority: CN
Inventors: 叶鑫; 陈雨潇; 金薪盛; 应华; 陶维康
Original assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Jiangsu Hengrui Medicine Co Ltd; Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2021-07-14
Filing date: 2022-07-14
Publication date: 2024-03-01
Also published as: WO2023284806A1; WO2023284806A9; TW202325743A

Abstract

The present disclosure relates to antigen binding molecules that specifically bind CD38, BCMA and CD3 and their medical uses.

Description

Antigen binding molecules that specifically bind CD38, BCMA and CD3 and medical uses thereof

Technical Field

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

Background

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

Multiple Myeloma (MM) is the second most common hematological cancer worldwide, which is characterized by uncontrolled proliferation of plasma cells in the bone marrow, rapid proliferation and diffusion of cancerous plasma cells, which in turn leads to the massive production of monoclonal immunoglobulins, which in turn cause immunosuppression, osteolysis and end organ damage. Worldwide, there are over 13.85 tens of thousands of newly diagnosed MM patients. In the last decades, due to the emergence of new therapeutic means such as proteasome inhibitors, immunomodulators and CD38 antibodies, the clinical therapeutic effect of MM patients is greatly improved, and the life expectancy of patients is improved from 3 to 4 years to 7 to 8 years. However, most patients still relapse due to drug resistance, even MRD (minimal residual disease) negative patients still relapse, and thus, there is a great clinical need for more effective new treatments. Immunotherapy that targets MM cells while remodelling the antitumor activity of immune cells would be a very excellent therapy for treating MM.

CD38 is a type two transmembrane glycoprotein, which can not only regulate processes of lymphocyte activation, B cell differentiation and the like by interacting with ligand CD31 and transmitting downstream signals, but also can play a role by the action of exonuclease. CD38 is widely expressed in plasma cells and multiple myeloma cells, and the monoclonal antibodies of CD38 reach Lei Tuoyou monoclonal antibodies (Daratumumab) and Isatuximab both have better therapeutic response rate and response depth clinically, which fully proves that CD38 is a better target for multiple myeloma.

BCMA (Tumor necrosis factor receptor superfamily member 17) is a class of signal peptide-free three-type transmembrane proteins belonging to one of the TNFR protein superfamily members. BCMA activates downstream ERK1/2, NFkB, etc. signaling pathways by binding to ligands APRIL and BAFF, promoting B cell growth and proliferation. In normal tissue cells, BCMA is expressed mainly in B cells in the late differentiation phase, i.e. in more mature B cells (Plasmablast, PC), but not in early-developing B cells and other normal tissues. BCMA is highly selectively expressed on the surface of multiple myeloma cells in a much higher amount than in normal B cells. Moreover, the membrane proximal end of BCMA is cleaved by γ -secret to form soluble BCMA (sBCMA), which is significantly higher in plasma than healthy human in patients with multiple myeloma and relapsing myeloma, and has been used as an important marker for progression of multiple myeloma diseases.

Bispecific antibodies targeting CD3& TAA are a novel immunotherapy that can bind T cells and tumor cells simultaneously, mimic the interaction of MHC and TCR, and release perforin and granzyme-specific killing of tumor cells after T cells form lytic synapse. The activated T cells can release cytokines, initiate other immune cells and amplify immune responses against the tumor, ultimately leading to proliferation of T cells and a cascade of killing tumor cells.

Disclosure of Invention

The present disclosure provides an antigen binding molecule comprising at least one antigen binding moiety that specifically binds BCMA, at least one antigen binding moiety that specifically binds CD38, and at least one antigen binding moiety that specifically binds CD 3. These antigen binding molecules are capable of providing better therapeutic activity than BCMA/CD3 antibodies or CD38/CD3 antibodies, covering a broader population of patients with multiple myeloma.

In some embodiments, an antigen binding molecule as described previously, wherein the antigen binding moiety refers to an antibody fragment comprising a heavy chain variable region and a light chain variable region that together comprise a domain that binds an antigen of interest (BCMA, CD38 or CD 3). In some embodiments, the antigen binding moiety is a Fab or scFv.

In one aspect, the present disclosure provides an antigen binding molecule comprising two antigen binding moieties that specifically bind BCMA, two antigen binding moieties that specifically bind CD38, two antigen binding moieties that specifically bind CD3, and an Fc region.

In some embodiments, the antigen binding molecule of any one of the preceding claims, the antigen binding moiety that specifically binds BCMA is BCMA-Fab, the antigen binding moiety that specifically binds CD3 is CD3-scFv and the antigen binding moiety that specifically binds CD38 is CD38-scFv. BCMA-Fab of the present disclosure means that the antigen binding moiety has a Fab structure, and CD3-scFv and CD38-scFv mean that the antigen binding moiety has a scFv structure.

In some embodiments, the antigen binding molecule of any one of the preceding claims, the antigen binding moiety that specifically binds CD38 is a CD38-Fab, the antigen binding moiety that specifically binds CD3 is a CD3-scFv and the antigen binding moiety that specifically binds BCMA is a BCMA-scFv. The CD38-Fab of the present disclosure means that the antigen binding moiety has a Fab structure, and CD3-scFv and BCMA-scFv means that the antigen binding moiety has a scFv structure.

In some embodiments, the antigen binding molecule of any one of the preceding claims, the antigen binding moiety that specifically binds BCMA is BCMA-Fab, the antigen binding moiety that specifically binds CD3 is CD3-scFv and the antigen binding moiety that specifically binds CD38 is CD38-scFv; and a heavy chain of BCMA-Fab, a CD3-scFv, a subunit of Fc region and a CD38-scFv are linked directly or via a linker, respectively, in the order of N-terminal to C-terminal. In some embodiments, the heavy chain of another BCMA-Fab, another CD3-scFv, another subunit of the Fc region, and another CD38-scFv are also linked directly or via a linker, respectively, in the N-terminal to C-terminal order. In a specific embodiment, the antigen binding molecule has a structure as shown in fig. 1B.

In some embodiments, the antigen binding molecule of any one of the preceding claims, the antigen binding moiety that specifically binds CD38 is a CD38-Fab, the antigen binding moiety that specifically binds CD3 is a CD3-scFv and the antigen binding moiety that specifically binds BCMA is a BCMA-scFv; and a heavy chain of a CD38-Fab, a CD3-scFv, a subunit of the Fc region and a BCMA-scFv are linked directly or via a linker, respectively, in the order of N-terminus to C-terminus. In some embodiments, the heavy chain of another CD38-Fab, another CD3-scFv, another subunit of the Fc region, and another BCMA-scFv are also linked directly or via a linker, respectively, in the N-terminal to C-terminal order. In a specific embodiment, the antigen binding molecule has a structure as shown in fig. 1D.

In some embodiments, the antigen-binding molecule of any one of the preceding claims, the antigen-binding moiety that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the antigen-binding moiety that specifically binds CD38 comprises a heavy chain variable region CD38-VH and a light chain variable region CD38-VL; wherein:

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),

(a) [ BCMA-VH ] - [ CH1] - [ CD3-VH ] - [ linker 1] - [ CD3-VL ] - [ linker 2] - [ one subunit of Fc region ] - [ linker 3] - [ CD38-VH ] - [ linker 4] - [ CD38-VL ],

(b)[BCMA-VL]-[CL]，

linker 1, linker 2, linker 3 and linker 4 in formula (a) are identical or different peptide linkers. In some embodiments, linker 1, linker 2, linker 3, and linker 4 in formula (a) are 3-15 amino acid residues in length. In some embodiments, the amino acid sequences of linker 1, linker 3, and linker 4 in formula (a) are as set forth in SEQ ID NO:99, the amino acid sequence of the linker 2 is shown in SEQ ID NO: shown at 96.

The antigen binding molecule comprises two first chains having a structure represented by formula (c) and two second chains having a structure represented by formula (d),

(c) [ CD38-VH ] - [ CH1] - [ CD3-VH ] - [ linker 1] - [ CD3-VL ] - [ linker 2] - [ one subunit of the Fc region ] - [ linker 3] - [ BCMA-VH ] - [ linker 4] - [ BCMA-VL ],

(d)[CD38-VL]-[CL]，

linker 1, linker 2, linker 3 and linker 4 in formula (c) are identical or different peptide linkers. In some embodiments, linker 1, linker 2, linker 3, and linker 4 in formula (c) are 3-15 amino acid residues in length. In some embodiments, the amino acid sequences of linker 1, linker 3, and linker 4 in formula (c) are as set forth in SEQ ID NO:99, the amino acid sequence of the linker 2 is shown in SEQ ID NO: shown at 96.

In one aspect, the present disclosure provides an antigen binding molecule comprising one antigen binding moiety that specifically binds BCMA, two antigen binding moieties that specifically bind CD38, one antigen binding moiety that specifically binds CD3, and an Fc region.

In some embodiments, the antigen binding molecule of any one of the preceding claims, the antigen binding moiety that specifically binds BCMA is BCMA-Fab, the antigen binding moiety that specifically binds CD3 is CD3-scFv and the antigen binding moiety that specifically binds CD38 is CD38-scFv. In a specific embodiment, the antigen binding molecule has a structure as shown in fig. 1A.

In some embodiments, the antigen binding molecule of any one of the preceding claims, the antigen binding moiety that specifically binds CD38 is a CD38-Fab, the antigen binding moiety that specifically binds CD3 is a CD3-scFv and the antigen binding moiety that specifically binds BCMA is a BCMA-scFv. In a specific embodiment, the antigen binding molecule has a structure as shown in fig. 1E.

In some embodiments, the antigen binding molecule of any one of the preceding claims, the Fc region comprises a first subunit Fc1 and a second subunit Fc2 capable of associating, each of the Fc1 and Fc2 independently having one or more amino acid substitutions that reduce homodimerization of the Fc region; and, the antigen binding moiety that specifically binds BCMA is BCMA-Fab, the antigen binding moiety that specifically binds CD3 is CD3-scFv and the antigen binding moiety that specifically binds CD38 is CD38-scFv; and the heavy chain of the BCMA-Fab, fc1 and one CD38-scFv are linked directly or via a linker, respectively, in N-terminal to C-terminal order, and the CD3-scFv, fc2 and the other CD38-scFv are linked directly or via a linker, respectively, in N-terminal to C-terminal order.

In some embodiments, the antigen binding molecule of any one of the preceding claims, the Fc region comprises a first subunit Fc1 and a second subunit Fc2 capable of associating, each of the Fc1 and Fc2 independently having one or more amino acid substitutions that reduce homodimerization of the Fc region; and, the antigen binding moiety that specifically binds CD38 is a CD38-Fab, the antigen binding moiety that specifically binds CD3 is a CD3-scFv and the antigen binding moiety that specifically binds BCMA is a BCMA-scFv; and the heavy chain of one CD38-Fab, fc1 and CD3-scFv are linked directly or via a linker, respectively, in N-terminal to C-terminal order, and the heavy chain of the BCMA-scFv, the heavy chain of the other CD38-Fab and Fc2 are linked directly or via a linker, respectively, in N-terminal to C-terminal order.

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

(e) [ BCMA-VH ] - [ CH1] - [ Fc1] - [ linker 1] - [ CD38-VH ] - [ linker 2] - [ CD38-VL ],

(b)[BCMA-VL]-[CL]，

(f) [ CD3-VH ] - [ linker 1] - [ CD3-VL ] - [ linker 2] - [ Fc2] - [ linker 3] - [ CD38-VH ] - [ linker 4] - [ CD38-VL ] -,

wherein linker 1 and linker 2 in formula (e) are the same or different peptide linkers, and linker 1, linker 2, linker 3 and linker 4 in formula (f) are the same or different peptide linkers. In some embodiments, the peptide linker is a flexible peptide linker. In some embodiments, linker 1, linker 2 in formula (e), and linker 1, linker 2, linker 3, and linker 4 in formula (f) are 3-15 amino acid residues in length. In some embodiments, the amino acid sequences of linker 1 and linker 2 in formula (e) are as set forth in SEQ ID NO:99, the amino acid sequences of linker 1, linker 3 and linker 4 in formula (f) are as shown in SEQ ID NO:99, and linker 2 in formula (f) has the amino acid sequence shown in SEQ ID NO: shown at 96.

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

(g) [ BCMA-VL ] - [ linker 1] - [ BCMA-VH ] - [ linker 2] - [ CD38-VH ] - [ CH1] - [ Fc2],

(d)[CD38-VL]-[CL]，

(h) [ CD38-VH ] - [ CH1] - [ Fc1] - [ linker 1] - [ CD3-VL ] - [ linker 2] - [ CD3-VH ],

wherein linker 1 and linker 2 in formula (g) are the same or different peptide linkers, and linker 1 and linker 2 in formula (h) are the same or different peptide linkers. In some embodiments, the peptide linker is a flexible peptide linker. In some embodiments, linker 1, linker 2 in formula (g), and linker 1 and linker 2 in formula (h) are 3-15 amino acid residues in length. In some embodiments, the amino acid sequences of linker 1 and linker 2 in formula (g) are as set forth in SEQ ID NO:99, the amino acid sequence of linker 1 in formula (h) is shown in SEQ ID NO:97, and the amino acid sequence of linker 2 in formula (h) is as shown in SEQ ID NO: 99.

In one aspect, the present disclosure provides an antigen binding molecule comprising an antigen binding moiety that specifically binds BCMA, an antigen binding moiety that specifically binds CD38, an antigen binding moiety that specifically binds CD3, and an Fc region. In a specific embodiment, the antigen binding molecule has a structure as shown in fig. 1C.

In some embodiments, the antigen binding molecule of any one of the preceding claims, the antigen binding moiety that specifically binds BCMA is BCMA-Fab, the antigen binding moiety that specifically binds CD3 is CD3-scFv and the antigen binding moiety that specifically binds CD38 is CD38-scFv.

In some embodiments, the antigen binding molecule of any one of the preceding claims, the Fc region comprises a first subunit Fc1 and a second subunit Fc2 capable of associating, each of the Fc1 and Fc2 independently having one or more amino acid substitutions that reduce homodimerization of the Fc region; and the CD38-scFv, CD3-scFv and Fc1 are linked directly or via a linker, respectively, in N-terminal to C-terminal order, and the heavy chain of the BCMA-Fab and Fc2 are linked directly or via a linker, respectively, in N-terminal to C-terminal order.

The antigen binding molecule comprises a first chain having a structure represented by formula (i), a second chain having a structure represented by formula (b), and a third chain having a structure represented by formula (j),

(i)[BCMA-VH]-[CH1]-[Fc2]，

(b)[BCMA-VL]-[CL]，

(j) [ CD38-VH ] - [ linker 1] - [ CD38-VL ] - [ linker 2] - [ CD3-VH ] - [ linker 3] - [ CD3-VL ] - [ linker 4] - [ Fc1] - [ linker 3-VL ] -.

Wherein linker 1, linker 2, linker 3 and linker 4 in formula (j) are identical or different peptide linkers. In some embodiments, the peptide linker is a flexible peptide linker. In some embodiments, linker 1, linker 2, linker 3, and linker 4 in formula (j) are 3-15 amino acid residues in length. In some embodiments, the amino acid sequences of linker 1 and linker 3 in formula (j) are as set forth in SEQ ID NO:99, the amino acid sequence of linker 2 in formula (j) is shown in SEQ ID NO:97, and linker 4 in formula (j) has the amino acid sequence shown in SEQ ID NO: shown at 96.

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

(i) The BCMA-VH comprises SEQ ID NO:23, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3, and said BCMA-VL comprises the amino acid sequence of SEQ ID NO:24, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(ii) The BCMA-VH comprises SEQ ID NO:25, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3, and said BCMA-VL comprises the amino acid sequence of SEQ ID NO:26, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(iii) The BCMA-VH comprises SEQ ID NO:27, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3, and said BCMA-VL comprises the amino acid sequence of SEQ ID NO:28, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3.

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

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

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

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

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

In some embodiments, the antigen binding molecules as described previously, the BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR3 are defined according to the Kabat numbering convention.

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

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

(i) The BCMA-VH comprises a sequence identical to SEQ ID NO:23, and the BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:24 has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the BCMA-VH comprises SEQ ID NO:23, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:24, and an amino acid sequence of seq id no.

(i) The BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 29. SEQ ID NO:30 and SEQ ID NO:31, and the BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 32. SEQ ID NO: 33. SEQ ID NO:34 and SEQ ID NO:35, the amino acid sequences of the group consisting of at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity, respectively. In some embodiments, the BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 29. SEQ ID NO:30 and SEQ ID NO:31, and the BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 32. SEQ ID NO: 33. SEQ ID NO:34 and SEQ ID NO:35, and a sequence of amino acids of the group consisting of seq id no. In some embodiments, the antigen binding moiety that specifically binds BCMA binds to a polypeptide comprising SEQ ID NO:29 and SEQ ID NO:32 is functionally equivalent. In some embodiments, the BCMA-VH comprises SEQ ID NO:29, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:32, and a sequence of amino acids.

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

(ii) The BCMA-VH comprises a sequence identical to SEQ ID NO:25, and the BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:26 has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the BCMA-VH comprises SEQ ID NO:25, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:26, and a sequence of amino acids.

(ii) The BCMA-VH comprises and is selected from the group consisting of SEQ ID NO: 36. SEQ ID NO:37 and SEQ ID NO:38, and the BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:39 or SEQ ID NO:40, having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity, respectively. In some embodiments, the BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 36. SEQ ID NO:37 and SEQ ID NO:38, and the BCMA-VL comprises the amino acid sequence of the group consisting of SEQ ID NO:39 or SEQ ID NO: 40. In some embodiments, the antigen binding moiety that specifically binds BCMA binds to a polypeptide comprising SEQ ID NO:36 and SEQ ID NO:40 is functionally equivalent. In some embodiments, the BCMA-VH comprises SEQ ID NO:36, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO: 40.

(iii) The BCMA-VH has: comprising SEQ ID NO:17, BCMA-HCDR1 comprising the amino acid sequence of SEQ ID NO:18 and a BCMA-HCDR2 comprising the amino acid sequence of SEQ ID NO:19, BCMA-HCDR3 of the amino acid sequence of 19; and the BCMA-VL has: comprising SEQ ID NO:20, BCMA-LCDR1 comprising the amino acid sequence of SEQ ID NO:21 and a BCMA-LCDR2 comprising the amino acid sequence of SEQ ID NO:22, and a BCMA-LCDR3 of the amino acid sequence of seq id no. In some embodiments, the BCMA-VH and/or the BCMA-VL is murine or humanized. In some embodiments, the BCMA-VH and/or the BCMA-VL is humanized. In some embodiments, the BCMA-VH has a heavy chain framework region derived from IGHV1-46 x 01 and is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 38K, 48I, 66T, 67A, 71S, 73K, and 78A; and/or the BCMA-VL has a light chain framework region derived from IGKV2-28 x 01 and which is unsubstituted or has one or more amino acid substitutions selected from the group consisting of 2V, 45K and 87F. In some embodiments, the variable regions and CDRs described above are defined according to the Kabat numbering convention.

(iii) The BCMA-VH comprises a sequence identical to SEQ ID NO:27, and the BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:28 has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the BCMA-VH comprises SEQ ID NO:27, and the BCMA-VL comprises the amino acid sequence of SEQ ID NO:28, and a sequence of amino acids.

(iii) The BCMA-VH comprises and is selected from the group consisting of SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO:43 and SEQ ID NO:44, and the BCMA-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 45 or SEQ ID NO:46, the amino acid sequences of the group consisting of at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity, respectively. In some embodiments, the BCMA-VH comprises a sequence selected from the group consisting of SEQ ID NOs: 41. SEQ ID NO: 42. SEQ ID NO:43 and SEQ ID NO:44, and the BCMA-VL comprises an amino acid sequence selected from the group consisting of SEQ ID NO:45 or SEQ ID NO:46, and a sequence of amino acids of the group consisting of seq id no. In some embodiments, the antigen binding moiety that specifically binds BCMA binds to a polypeptide comprising SEQ ID NO:43 and SEQ ID NO:45 is functionally equivalent. In some embodiments, the BCMA-VH comprises SEQ ID NO:43, and said BCMA-VL comprises the amino acid sequence of SEQ ID NO: 45.

In some embodiments, the antigen binding molecule that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, as described previously, wherein:

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

(ii) The CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 comprise the amino acid sequences of SEQ ID NO:65, CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3; and the CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3 comprise the amino acid sequences of SEQ ID NO:64, CD3-LCDR1, CD3-LCDR2 and CD3-LCDR 3.

In some embodiments, the antigen binding molecules, as described above, are defined according to Kabat, IMGT, chothia, abM or the Contact numbering convention for CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2 and CD3-LCDR 3.

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

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

In some embodiments, the antigen binding molecules, as described previously, are defined according to the Kabat numbering convention for CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2, and CD3-LCDR3.

(i) The CD3-VH comprises a sequence identical to SEQ ID NO:63, and said CD3-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:64 has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the antigen binding moiety that specifically binds CD3 binds to a polypeptide comprising SEQ ID NO:63 and SEQ ID NO:64 is functionally equivalent. In some embodiments, the CD3-VH comprises SEQ ID NO:63, and said CD3-VL comprises the amino acid sequence of SEQ ID NO:64, and a sequence of amino acids.

(ii) The CD3-VH comprises a sequence identical to SEQ ID NO:65, and said CD3-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:64 has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the antigen binding moiety that specifically binds CD3 binds to a polypeptide comprising SEQ ID NO:65 and SEQ ID NO:64 is functionally equivalent. In some embodiments, the CD3-VH comprises SEQ ID NO:65, and said CD3-VL comprises the amino acid sequence of SEQ ID NO:64, and a sequence of amino acids.

In some embodiments, the antigen binding molecule that specifically binds to CD38 comprises a heavy chain variable region CD38-VH and a light chain variable region CD38-VL, as described previously, wherein the CD38-HCDR1, CD38-HCDR2, and CD38-HCDR3 in the CD38-VH comprise the amino acid sequence of SEQ ID NO:53, and the CD38-LCDR1, CD38-LCDR2, and CD38-LCDR3 of the CD38-VL comprises the amino acid sequences of SEQ ID NOs: 54, CD38-LCDR1, CD38-LCDR2 and CD38-LCDR 3. In some embodiments, the CD38-HCDR1, CD38-HCDR2, CD38-HCDR3, CD38-LCDR1, CD38-LCDR2, and CD38-LCDR3 are defined according to Kabat, IMGT, chothia, abM or Contact numbering rules.

In some embodiments, the CD38-VH has: comprising SEQ ID NO:47, a CD38-HCDR1 comprising the amino acid sequence of SEQ ID NO:48, and a CD38-HCDR2 comprising the amino acid sequence of SEQ ID NO:49, CD38-HCDR3 of the amino acid sequence of; and the CD38-VL has: comprising SEQ ID NO:50, a CD38-LCDR1 comprising the amino acid sequence of SEQ ID NO:51, and a CD38-LCDR2 comprising the amino acid sequence of SEQ ID NO:52, and a CD38-LCDR3 of the amino acid sequence of seq id no. In some embodiments, the CD38-HCDR1, CD38-HCDR2, CD38-HCDR3, CD38-LCDR1, CD38-LCDR2, and CD38-LCDR3 are defined according to the Kabat numbering convention.

the CD38-VH comprises a sequence identical to SEQ ID NO:53, and said CD38-VL comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:54 has an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the antigen binding moiety that specifically binds CD38 binds to a polypeptide comprising SEQ ID NO:53 and SEQ ID NO:54 are functionally equivalent. In some embodiments, the CD38-VH comprises SEQ ID NO:53, and said CD38-VL comprises the amino acid sequence of SEQ ID NO:54, an amino acid sequence of seq id no.

In some embodiments, the antigen binding molecule of any one of the preceding claims, the linker thereof is a peptide linker. In some embodiments, the peptide linker is 3-15 amino acid residues in length. In some embodiments, the peptide linkers each independently have a peptide as L ₁ -(GGGGS) _n -L ₂ The amino acid sequence is shown, wherein L ₁ Is a bond, A, GS, GGS or GGGS (SEQ ID NO: 102), n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, L ₂ Is a bond, G, GG, GGG or GGGGGG (SEQ ID NO: 103), and the linker is not a bond. In some embodiments, the peptide linker sequence is as set forth in SEQ ID NO:96 to SEQ ID NO: 99.

GGG(SEQ ID NO：96)

GGGGS(SEQ ID NO：97)

GGGGSGGGGS(SEQ ID NO：98)

GGGGSGGGGSGGGGS(SEQ ID NO：99)

In some embodiments, the antigen binding molecule of any one of the preceding claims, comprising an Fc region. In some embodiments, the Fc region is an IgG Fc region, preferably IgG ₁ An Fc region. In some embodiments, the Fc region comprises one or more amino acid substitutions that reduce binding of the Fc region to an Fc receptor. In some embodiments, the amino acid substitution reduces binding to an fcγ receptor. In some embodiments, the Fc region is a human IgG ₁ The Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. In some embodiments, the Fc1 has the amino acid sequence of SEQ ID NO:68, and a sequence of amino acids.

In some embodiments, the antigen binding molecule of any one of the preceding claims, comprising an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating with each other, each of the Fc1 and Fc2 independently having one or more amino acid substitutions that reduce homodimerization of the Fc region. In some embodiments, the Fc1 and Fc2 each independently have one or more amino acid substitutions according to the mortar and pestle technique. In some embodiments, the Fc1 has a convex structure according to the pestle and socket technique, and the Fc2 has a pore structure according to the pestle and socket technique. In some embodiments, the amino acid residue of Fc1 at position 366 is W; and the amino acid residue at position 366, the amino acid residue at position 368, and the amino acid residue at position 407 of said Fc2 are S, A, and V, respectively, according to the EU index. In some embodiments, the Fc1 has the amino acid sequence of SEQ ID NO:100, and said Fc2 has the amino acid sequence of SEQ ID NO: 101.

the antigen binding molecule has: two comprise SEQ ID NOs: 76 and two strands comprising the amino acid sequence of SEQ ID NO:74, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 75 and two strands comprising the amino acid sequence of SEQ ID NO:74, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 82 and two strands comprising the amino acid sequence of SEQ ID NO:81, and a second strand of the amino acid sequence of 81; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 83 and two strands comprising the amino acid sequence of SEQ ID NO:81, and a second strand of the amino acid sequence of 81; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 90 and two strands comprising the amino acid sequence of SEQ ID NO:89, a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 91 and two strands comprising the amino acid sequence of SEQ ID NO:89, a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 77, two strands comprising the amino acid sequence of SEQ ID NO:71, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 78, two strands comprising the amino acid sequence of SEQ ID NO:71, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 85, two strands comprising the amino acid sequence of SEQ ID NO:71, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 86, two strands comprising the amino acid sequence of SEQ ID NO:71, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 93, two strands comprising the amino acid sequence of SEQ ID NO:71, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 94, two strands comprising the amino acid sequence of SEQ ID NO:71, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:73, a first strand comprising the amino acid sequence of SEQ ID NO:74 and a second strand comprising the amino acid sequence of SEQ ID NO:66, a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:73, a first strand comprising the amino acid sequence of SEQ ID NO:74 and a second strand comprising the amino acid sequence of SEQ ID NO:67, and a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:80, 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:66, a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:80, 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:67, and a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:88, 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:66, a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:88, 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:67, and a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:84, 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:69, a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:84, 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:70, and a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:92, 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:69, a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:92, 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:70, and a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:79, two strands comprising the amino acid sequence of SEQ ID NO:71 and a second strand comprising the amino acid sequence of SEQ ID NO:72, and a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:87, two strands comprising the amino acid sequence of SEQ ID NO:71 and a second strand comprising the amino acid sequence of SEQ ID NO:72, and a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:95, two strands comprising the amino acid sequence of SEQ ID NO:71 and a second strand comprising the amino acid sequence of SEQ ID NO:72, and a third strand of an amino acid sequence of seq id no.

In some embodiments, the antigen binding molecule of any one of the preceding claims, having: two comprise SEQ ID NOs: 76 and two strands comprising the amino acid sequence of SEQ ID NO:74, and a second strand of an amino acid sequence of seq id no.

In some embodiments, the antigen binding molecule of any one of the preceding claims, having: two comprise SEQ ID NOs: 82 and two strands comprising the amino acid sequence of SEQ ID NO:81, and a second strand of the amino acid sequence of 81.

In another aspect, the present disclosure provides a pharmaceutical composition comprising: a therapeutically effective amount of an antigen binding molecule of any one of the preceding claims, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients. In some embodiments, the pharmaceutical composition further comprises at least one second therapeutic agent.

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

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

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

In another aspect, the present disclosure also provides the use of an antigen binding molecule or composition of any one of the preceding claims in the manufacture of a medicament for the treatment or prophylaxis of a disease.

In another aspect, the disclosure also provides an antigen binding molecule or composition of any one of the preceding claims for use as a medicament. In some embodiments, the medicament is for treating a disease.

In some embodiments, the disease is a hematopoietic tumor or autoimmune disease of lymphoid or myeloid lineage. In some embodiments, the hematopoietic tumor of lymphoid or myeloid lineage is selected from: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, fahrenheit macroglobulinemia, isolated bone plasmacytoma, extramedullary plasmacytoma, bone sclerosis myeloma, heavy chain disease, meaningless monoclonal gammaglobinopathy, and stasis type multiple myeloma, B-cell lymphoma, burkitt's lymphoma, hodgkin's lymphoma, and hairy cell lymphoma; the autoimmune disease is selected from: rheumatoid arthritis, systemic lupus erythematosus, asthma, inflammatory bowel disease, multiple sclerosis, crohn's disease, gastritis, hashimoto thyroiditis, ankylosing spondylitis, and graft versus host disease.

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

Drawings

Fig. 1A: a structural schematic diagram of Format 5; fig. 1B: a structural schematic diagram of the Format 12; fig. 1C: a structural schematic diagram of the Format 20; fig. 1D: a structural schematic diagram of the Format 21; fig. 1E: the Format24 is schematically shown.

Fig. 2: ability of antibodies to bind to RPMI-8226 cells.

Fig. 3: cytotoxic activity of antibodies against CHO K1 cells.

Detailed Description

Terminology

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

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

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

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

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

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

The term "antigen binding molecule" is used in its broadest sense to encompass a variety of molecules that specifically bind to an antigen, including but not limited to antibodies, other polypeptides having antigen binding activity, and antibody fusion proteins fused to both. Illustratively, the antigen binding molecules herein are trispecific antigen binding molecules (e.g., trispecific antibodies). The term "trispecific antigen-binding molecule" refers to an antigen-binding molecule capable of specifically binding to three different antigens. The term "antibody" is used in the broadest sense and covers a variety of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies; monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), full length antibodies and antibody fragments (or antigen-binding fragments, or antigen-binding portions) so long as they exhibit the desired antigen-binding activity. "Natural antibody" refers to a naturally occurring immunoglobulin molecule. For example, a natural IgG antibody is an iso-tetrasaccharide protein of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains that are disulfide bonded. From N to C-terminal, each heavy chain has a variable region (VH), also known as a variable heavy domain, a heavy chain variable region, followed by three constant domains (CH 1, CH2 and CH 3). Similarly, from N-to C-terminus, each light chain has a variable region (VL), also known as a variable light domain, or light chain variable domain, followed by a constant light domain (light chain constant region, CL).

The term "variable region" or "variable domain" refers to the antigen-binding domain of an antigen-binding molecule. Herein, the heavy chain variable region in the antigen binding module that specifically binds BCMA is denoted BCMA-VH, and the light chain variable region is denoted BCMA-VL; the heavy chain variable region in the antigen binding module that specifically binds CD38 is designated CD38-VH and the light chain variable region is designated CD38-VL; the heavy chain variable region in the antigen binding module that specifically binds CD3 is designated CD3-VH and the light chain variable region is designated CD3-VL. VH and VL each comprise four conserved Framework Regions (FR) and three Complementarity Determining Regions (CDRs). Wherein the term "complementarity determining region" or "CDR" refers to a region within the variable domain that primarily contributes to binding to an antigen; "framework" or "FR" refers to variable domain residues other than CDR residues. VH comprises 3 CDR regions: HCDR1, HCDR2 and HCDR3; VL comprises 3 CDR regions: LCDR1, LCDR2, and LCDR3. Herein, 3 CDR regions in BCMA-VH are denoted as BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3, respectively; the 3 CDRs in BCMA-VL are labeled BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3, respectively; the 3 CDRs in the CD38-VH are designated CD38-HCDR1, CD38-HCDR2 and CD38-HCDR3, respectively; the 3 CDRs in CD38-VL are designated as CD38-LCDR1, CD38-LCDR2 and CD38-LCDR3, respectively; the 3 CDRs in the CD3-VH are designated CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3, respectively; the 3 CDRs in CD3-VL are designated as CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3, respectively. Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen binding specificity.

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

TABLE 1 relationship between CDR numbering systems

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

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

The term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of the intact antibody that binds to an antigen to which the intact antibody binds. 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. Fab refers to a protein consisting of VH and CH1 (Fab heavy chain) and VL and CL (Fab light chain) of an immunoglobulin. In the present disclosure, BCMA-Fab means an antigen binding moiety having a Fab structure that specifically binds BCMA, BCMA-scFv means an antigen binding moiety having a scFv structure that specifically binds BCMA, and so on.

The term "scFv" refers to a single chain antibody (single chain antibody fragment, scFv) which is an antibody in which the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody are linked by a short peptide linker of 15 to 20 amino acids.

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

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

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

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

The term "capable of specifically binding", "specifically binding" or "binding" refers to an antibody that is capable of binding with higher affinity to an antigen or epitope within the antigen than other antigens or epitopes. Typically, the antibodies are present in an amount of about 1X 10 ^-6 M or less (e.g., about 1X 10) ^-7 M, about 1X 10 ^-8 M or less) binds to an antigen or an epitope within an antigen. In some embodiments, the antibody binds to an antigen with a KD of 10% or less (e.g., 1%) of the KD of the antibody to a non-specific antigen (e.g., BSA, casein). KD can be measured using known methods, for example by FACS orAs measured by surface plasmon resonance. However, antibodies that specifically bind to an antigen or an epitope within an antigen may be cross-reactive to other related antigens, for example, to corresponding antigens from other species (homologous), such as humans or monkeys, e.g., cynomolgus macaque (Macaca fascicularis) (cynomolgus, cyno), chimpanzee (Pan troglodes) (chimpanzee, chimp)) or marmoset (Callithrix jacchus) (common marmoset).

The term "antigen binding moiety" refers to a polypeptide molecule that specifically binds to an antigen of interest. Antigen binding moieties include antibodies and fragments thereof as defined herein. Specific antigen binding moieties include antigen binding domains of antibodies, which comprise an antibody heavy chain variable region and an antibody light chain variable region. The term "antigen binding moiety that specifically binds BCMA" refers to a moiety that is capable of binding BCMA with sufficient affinity. In certain embodiments, the antigen binding moiety that specifically binds BCMA has the following equilibrium dissociation constant (KD): < about 1 μΜ, < about 100nM, or < about 10nM, as measured by FACS, surface plasmon resonance, or the like. In certain embodiments, the antigen binding moiety that specifically binds BCMA binds to a conserved epitope in BCMA from a different species. The term "antigen binding moiety that specifically binds CD 38" refers to a moiety that is capable of binding CD38 with sufficient affinity. In certain embodiments, the antigen binding moiety that specifically binds CD38 has the following equilibrium dissociation constants (KD): < about 1 μΜ, < about 100nM, or < about 10nM, as measured by FACS, surface plasmon resonance, or the like. In certain embodiments, the antigen binding moiety that specifically binds CD38 binds a conserved epitope in CD38 from a different species. The term "antigen binding moiety that specifically binds CD 3" refers to a moiety that is capable of binding CD3 with sufficient affinity. In certain embodiments, the antigen binding moiety that specifically binds CD3 has the following equilibrium dissociation constants (KD): < about 1 μΜ, < about 200nM, < about 100nM, and/or >50nM, as measured by FACS, surface plasmon resonance, and the like. In certain embodiments, the anti-CD 3 antibody binds to a conserved epitope in CD3 from a different species. Antigen binding moieties include antibody fragments as defined herein, such as Fab or scFv.

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

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

The term "antigen" refers to a molecule or portion of a molecule that is capable of being bound by a selective binding agent, such as an antigen binding protein (e.g., an antibody), and that is otherwise capable of being used in an animal to produce an antibody capable of binding the antigen. An antigen may have one or more epitopes that are capable of interacting with different antigen binding proteins (e.g., antibodies).

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

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

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

The term "sequence identity" refers to the degree to which the amino acids/nucleic acids of two sequences are identical at equivalent positions (percent) when optimally aligned to the two sequences, with gaps introduced as necessary to obtain the maximum percent sequence identity, and without any conservative substitutions being considered part of the sequence identity. To determine percent sequence identity, the alignment may be accomplished by techniques known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN-2, or Megalign (DNASTAR) software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences compared.

The term "fusion" or "linkage" refers to covalent attachment of components (e.g., antigen binding moiety and Fc domain) directly or via a linker.

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

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

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

"optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not.

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

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

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

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

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

"treatment" and "treatment" (and grammatical variations thereof) refer to clinical interventions that attempt to alter the natural course of the treated individual, and may be performed for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and regression or improved prognosis. In some embodiments, the antibodies of the disclosure are used to delay the formation of a disease or to slow the progression of a disease.

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

Antigen binding molecules of the disclosure

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

Exemplary antigen binding molecules

In one aspect, the present disclosure provides an antigen binding molecule comprising at least one antigen binding moiety that specifically binds BCMA, at least one antigen binding moiety that specifically binds CD38, and at least one antigen binding moiety that specifically binds CD 3. In particular, the antigen binding molecules of the present disclosure have at least one of the following functional activities:

a. high affinity for CD38/BCMA/CD3d & CD3 e. In some embodiments, the antigen binding molecule binds to CD38/BCMA/CD3d & CD3e with a KD of less than 200 nM. In some embodiments, the antigen binding molecule binds to CD38 or BCMA with a KD of less than 10nM and to CD3d & CD3e with a KD of less than 200 nM. The KD is determined by surface plasmon resonance.

b. High affinity for cell surface antigens. Specific test methods are shown in test example 2.

c. Specific killing activity against BCMA expressing cells in vitro. In some embodiments, the antigen binding molecule can specifically kill cells that express BCMA and/or CD38, but cannot kill cells that do not express BCMA and CD 38. Specific test methods are shown in test example 4.

d. The in vitro specific killing activity is not affected by the soluble BCMA/CD 38/APRIL. Specific test methods are shown in test example 5.

e. Inducing low levels of cytokine (IL-6 and IL-2) release. Specific test methods are shown in test example 6.

f. Stronger therapeutic activity in vivo. Specific test methods are shown in test examples 7 and 8.

In one aspect, the present disclosure provides an antigen binding molecule comprising at least one antigen binding moiety that specifically binds BCMA, at least one antigen binding moiety that specifically binds CD38, and at least one antigen binding moiety that specifically binds CD 3. The antigen binding module that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein:

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

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

(iii) The BCMA-VH has: the amino acid sequence is shown in SEQ ID NO:17, and the amino acid sequence of the BCMA-HCDR1 is shown as SEQ ID NO:18 and the amino acid sequence of the BCMA-HCDR2 is shown in SEQ ID NO:19 a BCMA-HCDR3; and the BCMA-VL has: the amino acid sequence is shown in SEQ ID NO:20, and the amino acid sequence of the BCMA-LCDR1 is shown as SEQ ID NO:21 and the amino acid sequence of BCMA-LCDR2 is shown in SEQ ID NO: BCMA-LCDR3 shown at 22;

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

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:32 or

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO:36 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:43 and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: as indicated by the numeral 45,

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

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

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

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

In some embodiments, the antigen binding molecule of any one of the preceding claims, the heavy chain variable region CD3-VH has an amino acid sequence set forth in SEQ ID NO:63 and the light chain variable region CD3-VL has the amino acid sequence set forth in SEQ ID NO: indicated at 64; or the amino acid sequence of the heavy chain variable region CD3-VH is shown as SEQ ID NO:65 and the light chain variable region CD3-VL having the amino acid sequence set forth in SEQ ID NO: shown at 64.

In some embodiments, the antigen binding molecule of any one of the preceding claims, wherein the antigen binding moiety that specifically binds CD38 comprises a heavy chain variable region CD38-VH and a light chain variable region CD38-VL, wherein:

the CD38-VH has: the amino acid sequence is shown in SEQ ID NO:47, the amino acid sequence of the CD38-HCDR1 is shown in SEQ ID NO:48 and the CD38-HCDR2 and the amino acid sequence shown in SEQ ID NO: CD38-HCDR3 shown in FIG. 49; and the CD38-VL has: the amino acid sequence is shown in SEQ ID NO:50, the amino acid sequence of the CD38-LCDR1 is shown as SEQ ID NO:51 and the CD38-LCDR2 and amino acid sequence shown in SEQ ID NO:52, CD38-LCDR3;

The CD38-HCDR1, CD38-HCDR2, CD38-HCDR3, CD38-LCDR1, CD38-LCDR2 and CD38-LCDR3 are defined according to the Kabat numbering convention.

In some embodiments, the antigen binding molecule of any one of the preceding claims, the heavy chain variable region CD38-VH has an amino acid sequence set forth in SEQ ID NO:53 and the light chain variable region CD38-VL has the amino acid sequence set forth in SEQ ID NO: indicated at 54.

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

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

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

The CD38-VH has: the amino acid sequence is shown in SEQ ID NO:47, the amino acid sequence of the CD38-HCDR1 is shown in SEQ ID NO:48 and the CD38-HCDR2 and the amino acid sequence shown in SEQ ID NO: CD38-HCDR3 shown in FIG. 49; and the CD38-VL has: the amino acid sequence is shown in SEQ ID NO:50, the amino acid sequence of the CD38-LCDR1 is shown as SEQ ID NO:51 and the CD38-LCDR2 and amino acid sequence shown in SEQ ID NO:52, CD38-LCDR3; or (b)

(ii) The BCMA-VH has: the amino acid sequence is shown in SEQ ID NO:11, and the amino acid sequence of the BCMA-HCDR1 is shown as SEQ ID NO:12 and the amino acid sequence of BCMA-HCDR2 is shown in SEQ ID NO:13, BCMA-HCDR3; and the BCMA-VL has: the amino acid sequence is shown in SEQ ID NO:14, and the amino acid sequence of the BCMA-LCDR1 is shown in SEQ ID NO:15 and the amino acid sequence of BCMA-LCDR2 is shown in SEQ ID NO:16, and

the CD3-VH has: the amino acid sequence is shown in SEQ ID NO:55, and the amino acid sequence of the CD3-HCDR1 is shown as SEQ ID NO:56 and the amino acid sequence of the CD3-HCDR2 is shown in SEQ ID NO: 57-CD 3-HCDR3; and the CD3-VL has: the amino acid sequence is shown in SEQ ID NO:58, the amino acid sequence of the CD3-LCDR1 is shown as SEQ ID NO:59 and the CD3-LCDR2 and amino acid sequence shown in SEQ ID NO:60, and CD3-LCDR3, and

the BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2, BCMA-LCDR3, CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2, CD3-LCDR3, CD38-HCDR1, CD38-HCDR2, CD38-HCDR3, CD38-LCDR1, CD38-LCDR2 and CD38-LCDR3 are defined according to the Kabat numbering convention.

In some embodiments, the antigen binding molecule of any one of the preceding claims, the heavy chain variable region BCMA-VH has the amino acid sequence set forth in SEQ ID NO:29, wherein the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:32, the amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO:65, the amino acid sequence of the light chain variable region CD3-VL is shown in SEQ ID NO:64, the amino acid sequence of the heavy chain variable region CD38-VH is shown in SEQ ID NO:53 and the light chain variable region CD38-VL has the amino acid sequence set forth in SEQ ID NO: indicated at 54.

In some embodiments, the antigen binding molecule of any one of the preceding claims, the heavy chain variable region BCMA-VH has the amino acid sequence set forth in SEQ ID NO:36, wherein the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO:40, wherein 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 amino acid sequence of the heavy chain variable region CD38-VH is shown in SEQ ID NO:53 and the light chain variable region CD38-VL has the amino acid sequence set forth in SEQ ID NO: indicated at 54.

In some embodiments, the antigen binding molecule of any one of the preceding claims, wherein the antigen binding molecule further comprises an Fc region comprising two subunits capable of associating. In some embodiments, the amino acid sequences of the two subunits are as set forth in SEQ ID NO: shown at 68. In some embodiments, the amino acid sequences of the two subunits are set forth in SEQ ID NOs: 100 and SEQ ID NO: shown at 101.

Structure of antigen binding molecule

The present disclosure provides a trispecific antigen-binding molecule that may be trivalent, tetravalent, pentavalent, hexavalent and more multivalent. The antigen binding molecule comprises an antigen binding moiety, which may be any polypeptide molecule, such as an antibody fragment, capable of binding to an antigen of interest. In some embodiments, the antigen binding moiety refers to an antibody fragment comprising a heavy chain variable region and a light chain variable region that together comprise a domain that binds an antigen of interest. In some embodiments, the antigen binding moiety is a Fab or scFv. The antigen binding moieties in the antigen binding molecules of the present disclosure may be linked in any manner that does not affect binding activity. Exemplary schematic structural diagrams of antigen binding molecules are shown in fig. 1A, 1B, 1C, 1D, or 1E.

Variants of antigen binding molecules

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

Substitution, insertion, and deletion variants

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

TABLE 2 substitution of amino acids

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

Trp(W)	Tyr；Phe	Tyr
Tyr(Y)	Trp；Phe；Thr；Ser	Phe
Val(V)	Ile; leu; met; phe; ala; norleucine (N-leucine)	Leu

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

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

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

(3) Acidic: asp, glu;

(4) Alkaline: his, lys, arg;

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

(6) Aromatic: trp, tyr, phe.

Non-conservative substitutions may require replacement of a member of one of these classes with a member of another class.

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

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

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

Amino acid sequence insertions include amino and/or carboxy terminal fusions ranging in length from 1 residue to polypeptides containing 100 or more residues, and intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertional variants of antibody molecules include fusions of the N-or C-terminus of an antibody with an enzyme or polypeptide that extends the serum half-life of the antibody.

Engineering of the Fc region

In one aspect, the Fc region of an antigen binding molecule of the present disclosure comprises one or more amino acid substitutions that reduce its binding to an Fc receptor, e.g., its binding to an fcγ receptor, and reduce or eliminate effector function. Native IgG Fc region, in particular IgG ₁ Fc region or IgG ₄ The Fc region, may result in the antigen binding molecules of the present disclosure targeting cells expressing Fc receptors, rather than cells expressing antigens. The engineered Fc regions of the present disclosure exhibit reduced binding affinity to Fc receptors and/or reduced effector function. In some embodiments, the engineered Fc region has a 50%, 80%, 90%, or more than 95% decrease in binding affinity to Fc receptors as compared to the native Fc region. In some embodiments, the Fc receptor is an fcγ receptor. In some embodiments, the Fc receptor is a human fcγ receptor, e.g., fcγri, fcγriia, fcγriib, fcγriiia. In some embodiments, the engineered Fc region also has reduced binding affinity for complement, such as C1q, compared to the native Fc region. In some embodiments, the engineered Fc region has no reduced binding affinity for neonatal Fc receptor (FcRn) compared to the native Fc region. In some embodiments, the engineered Fc region has reduced effector functions, which may include, but are not limited to, one or more of the following: reduced Complement Dependent Cytotoxicity (CDC), reduced antibody dependent cell mediated cytotoxicity (ADCC), reduced Antibody Dependent Cellular Phagocytosis (ADCP), reduced cytokine secretion, reduced antigen uptake by immune complex mediated antigen presenting cells, reduced binding to NK cells, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling Number transduction induces apoptosis, reduced dendritic cell maturation or reduced T cell priming. For IgG ₁ Amino acid residue substitutions at positions 238, 265, 269, 270, 297, 327 and 329 etc. of the Fc region may reduce effector function. In some embodiments, the Fc region is a human IgG ₁ The Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. For IgG ₄ The Fc region, amino acid residue substitution at position 228, etc., can reduce effector function.

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

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

TABLE 3 KIH mutant combinations

In some embodiments, the amino acid sequence of a particular Fc region is as follows:

>IgG ₁ Fc(Knob)(SEQ ID NO：100)

>IgG ₁ Fc(Hole)(SEQ ID NO：101)

in addition to the knob and socket technique, other techniques for modifying the CH3 domain of the heavy chain of a multispecific antibody to achieve heterodimerization are also known in the art, such as WO96/27011, WO98/050431, EP1870459, WO2007/110205, WO 007/147901, WO2009/089004, WO2010/129304, WO2011/90754, WO2011/143545, WO2012/058768, WO2013/157954 and WO013/096291.

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

Recombination method

Antigen binding molecules can be produced using recombinant methods. For these methods, one or more isolated nucleic acids encoding an antigen binding molecule are provided.

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

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

In one embodiment, the disclosure provides an isolated nucleic acid encoding an antigen binding molecule as described previously. Such nucleic acids may be given from the independent encoding of any of the polypeptide chains described previously. In another aspect, the disclosure provides one or more vectors (e.g., expression vectors) comprising such nucleic acids. In another aspect, the disclosure provides host cells comprising such nucleic acids. In one embodiment, a method of preparing an antigen binding molecule is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antigen binding molecule, as provided above, under conditions suitable for expression of the antigen binding molecule, and optionally recovering the antigen binding molecule from the host cell (or host cell culture medium).

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

Suitable host cells for cloning or expressing a vector encoding an antigen binding molecule include prokaryotic or eukaryotic cells as described herein. For example, antigen binding molecules may be produced in bacteria, particularly when glycosylation and Fc effector function are not required for the antigen binding molecule. After expression, the antigen binding molecules may be isolated from the bacterial cell paste in a soluble fraction and may be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding antigen binding molecules, including fungal and yeast strains, whose glycosylation pathways have been "humanized" resulting in the production of antigen binding molecules with a partially or fully human glycosylation pattern. Suitable host cells suitable for expression (glycosylation) of the antigen-binding molecule may also be derived from multicellular organisms (invertebrates and vertebrates); examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified which can be used in combination with insect cells, in particular for transfection of Spodoptera frugiperda (Spodoptera frugiperda) cells; plant cell cultures may also be used as hosts, for example US5959177, US6040498, US6420548, US7125978 and US6417429; vertebrate cells can also be used as hosts, for example mammalian cell lines adapted to grow in suspension. Other examples of suitable mammalian host cell lines are the SV40 transformed monkey kidney CVl line (COS-7); human embryonic kidney lines (293 or 293T cells); baby hamster kidney cells (BHK); mouse sertoli (sertoli) cells (TM 4 cells); monkey kidney cells (CV 1); african green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat (buffalo rate) hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines, such as Y0, NS0, and Sp2/0. For a review of certain mammalian host cell lines suitable for producing antigen binding molecules see, e.g., yazaki, p. And Wu, a.m., methods in Molecular Biology, vol.248, lo, b.k.c. (ed.), humana Press, totowa, NJ (2004), pages 255-268.

Immunoconjugates

The present disclosure also provides immunoconjugates comprising an antigen binding molecule conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent or drug, a growth inhibitor, a toxin (e.g., a bacterial, fungal, plant, or animal-derived protein toxin, an enzymatically active toxin, or fragments thereof), or a radioisotope.

Diagnostic and therapeutic compositions

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

In one embodiment, antigen binding molecules are provided for use in diagnostic or detection methods. In yet another aspect, a method of detecting the presence of an antigen of interest in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with the antigen binding molecule under suitable conditions and detecting whether a complex is formed between the detection reagent and the antigen. Such methods may be in vitro or in vivo. In one embodiment, the use of an antigen binding molecule to select a subject suitable for treatment, e.g., BCMA, CD38, or CD3, is a biomarker for selecting patients.

Exemplary disorders that can be diagnosed using the antigen binding molecules of the present disclosure, such as B cell disorders selected from the group consisting of: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, fahrenheit macroglobulinemia, isolated bone plasmacytoma, extramedullary plasmacytoma, bone sclerosis myeloma, heavy chain disease, meaningless monoclonal gammaglobulosis, and stasis type multiple myeloma; the autoimmune disease is systemic lupus erythematosus.

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

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

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

Methods of treatment and routes of administration

Any of the antigen binding molecules provided herein may be used in a method of treatment.

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

In a further aspect, a pharmaceutical composition comprising the antigen binding molecule is provided, e.g., for use in any of the above pharmaceutical uses or methods of treatment. In another embodiment, the pharmaceutical composition further comprises at least one additional therapeutic agent.

The antigen binding molecules of the present disclosure may be used alone or in combination with other agents for therapy. For example, the antigen binding molecules of the present disclosure may be co-administered with at least one additional therapeutic agent.

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

The antigen binding molecules of the present disclosure will be formulated, administered, and administered in a manner consistent with good medical practice. Factors considered in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the timing of administration, and other factors known to the medical practitioner. The antigen binding molecules need not be, but are 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 present in the pharmaceutical composition, the type of disorder or treatment, and other factors discussed above. These are generally used at the same dosages and routes of administration as described herein, or at about 1 to 99% of the dosages described herein, or at any dosage, and by any route of empirical/clinical determination as appropriate.

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

Article of manufacture

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

Examples and test examples

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

Example 1 expression of human BCMA and cynomolgus monkey BCMA

The sequences encoding human BCMA with the human IgG1-Fc tag and the cynomolgus BCMA extracellular domain were inserted into phr vector, constructed into expression plasmids, and then transfected with HEK293. The sequence encoding the His-tagged human BCMA extracellular domain was inserted into the phr vector, constructed as an expression plasmid, and then HEK293 was transfected. The specific transfection steps are as follows: HEK293E cells were grown at 1X 10 on the previous day ⁶ the/mL was inoculated into fresh expression medium (containing 1% FBS, gibco, 12338-026) and placed on a 37℃constant temperature shaker (120 rpm) for further culture for 24 hours. After 24 hours, the transfected plasmid and the transfection reagent PEI were sterilized with a 0.22 μm filter, and then the transfected plasmid was adjusted to 100. Mu.g/100 mL cells, the mass ratio of PEI (1 mg/mL) to plasmid was 3:1, 10mL of Opti-MEM and 200. Mu.g of plasmid were mixed evenly and allowed to stand for 5min; another 10mL of Opti-MEM and 40 were taken Mix 0 μg PEI and stand for 5min. Mixing the plasmid and PEI, and standing for 15min. The plasmid and PEI mixture was slowly added to 200mL HEK293E cells and 8% CO was added ₂ Culturing in a shaker at 120rpm and 37 ℃. Day 3 of transfection, 10% volume of feed medium (20 mM glucose+2 mM L-glutamic acid) was supplemented. On day 6 of transfection, the cell supernatant was collected by centrifugation at 4500rpm for 10min and purified according to the method of test example 2. The purified proteins can be used in the various examples or test case experiments described below.

Wherein the amino acid sequences of human BCMA-ECD-Fc, human BCMA-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: human BCMA-ECD-Fc (SEQ ID NO: 1)

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

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

Note that: underlined is the signal peptide sequence; the italic part is His 6-linker-tag

(3) ECD of His-tagged cynomolgus BCMA: cynoBCMA-ECD-His (SEQ ID NO: 3)

Note that: the italic part is linker-10 His-tag

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

And (3) injection: underlined is the signal peptide sequence; the italic part is human Fc-tag

EXAMPLE 2 purification of Fc-tagged recombinant protein by protein A affinity chromatography and purification of His-tagged recombinant protein by Nickel column

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

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

EXAMPLE 3 construction and identification of cell lines expressing recombinant human CD38, human BCMA and cynomolgus monkey BCMA

The present disclosure constructs cell lines expressing human CHO-K1/hCD 38. Cloning human CD38 full-length gene onto mammal cell expression vector pCDH, co-transfecting HEK293T cell with three plasmids of pVSV-G, pCMV-dR8.91 and pCDH-hCD38CRL-11268) to package virus, and collecting virus-infected CHO-K1 cells after 48 hours of transfectionCCL-61). CHOK1 monoclonal cells highly expressing hCD38 were obtained by flow sorting 72 hours after infection.

To screen for antibodies that bind well to cell surface BCMA, the present disclosure constructs K562-BCMA cell lines expressing human BCMA. Cloning human BCMA full-length gene onto mammal cell expression vector pCDH, co-transfecting HEK293T cell with three plasmids of pVSV-G, pCMV-dR8.91 and pCDH-human BCMACRL-11268) to package virus, and collecting virus-infected K562 cells after 48 hours of transfectionCCL-243). K562 monoclonal cells highly expressing human BCMA were obtained 72 hours after infection by flow sorting.

The present disclosure also constructs a CHO-K1-cynoBCMA cell strain expressing cynomolgus monkey, clones the full-length gene of cynomolgus monkey BCMA onto a mammalian cell expression vector pCDH, and co-transfects HEK293T cells with three plasmids of pVSV-G, pCMV-dR8.91 and pCDH-cynoBCMA CRL-11268) to package virus, and collecting virus-infected CHOK1 cells after 48 hr transfectionCCL-61). CHOK1 monoclonal cells highly expressing cynoBCMA were obtained by flow sorting 72 hours after infection.

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

The present disclosure prepares monoclonal antibodies against human BCMA by hybridoma technology. The obtained antibody specifically binds with high affinity to human BCMA and can cross react with cynomolgus monkey BCMA; the obtained antibody has better binding activity with human BCMA and cynomolgus monkey BCMA on the cell surface, and the binding activity is little interfered by soluble BCMA.

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

The monoclonal hybridoma cell strains 4E3, 33H4 and 27 with good activity are obtained by screening. Collecting hybridoma cells in logarithmic growth phase, extracting RNA with NucleoZol (MN) (according to the procedure of the kit instructions), and performing reverse transcription (PrimeScript) ^TM Reverse Transcriptase, takara, cat# 2680A). The cDNA obtained by reverse transcription was subjected to PCR amplification using mouse Ig-Primer Set (Novagen, TB326Rev. B0503) and then sequenced. The amino acid sequences of the CDRs and variable regions of 4E3, 33H4 and 27 are as follows:

TABLE 4 BCMA antibody CDR

4E3 murine heavy chain variable region (SEQ ID NO: 23)

4E3 murine light chain variable region (SEQ ID NO: 24)

33H4 murine heavy chain variable region (SEQ ID NO: 25)

33H4 murine light chain variable region (SEQ ID NO: 26)

27 murine heavy chain variable region (SEQ ID NO: 27)

27 murine light chain variable region (SEQ ID NO: 28)

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

Example 5 humanized design of anti-human BCMA monoclonal antibodies

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

Selection and back-mutation of the human FR region of 1.4E3

Human FR region selection and back mutation of table 5.4E3

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

The specific sequences of the antibody variable regions obtained by humanization of murine antibody 4E3 are as follows (the CDRs are underlined, the following are the same):

>hu4E3H1(SEQ ID NO：29)

>hu4E3H2(SEQ ID NO：30)

>hu4E3H3(SEQ ID NO：31)

>hu4E3L1(SEQ ID NO：32)

>hu4E3L2(SEQ ID NO：33)

>hu4E3L3(SEQ ID NO：34)

>hu4E3L4(SEQ ID NO：35)

selection and back-mutation of the human FR region of 2.33H4

Human FR region selection and back mutation of table 6.33H4

The specific sequence of the antibody variable region obtained by humanizing the murine antibody 33H4 is as follows:

>hu33H4H1(SEQ ID NO：36)

>hu33H4H2(SEQ ID NO：37)

>hu33H4H3(SEQ ID NO：38)

>hu33H4L1(SEQ ID NO：39)

>hu33H4L2(SEQ ID NO：40)

human FR region selection and back mutation of Table 7.27

The specific sequence of the antibody variable region obtained by humanizing the murine antibody 27 is as follows:

>hu27H1(SEQ ID NO：41)

>hu27H2(SEQ ID NO：42)

>hu27H3(SEQ ID NO：43)

>hu27H4(SEQ ID NO：44)

>hu27L1(SEQ ID NO：45)

>hu27L2(SEQ ID NO：46)

example 6 preparation and identification of antibodies of the disclosure

The CD38 binding molecules of the present disclosure may be derived from any suitable antibody. Particularly suitable antibodies are described, for example, in patent publication No. WO2020052546 (incorporated herein by reference in its entirety).

The CDR and variable region sequences of the anti-CD 38 arm in the antibodies of the present disclosure are shown below:

TABLE 8 CDR of CD38 arm

The specific sequence of the variable region is as follows:

>1149-VH(SEQ ID NO：53)

>1149-VL(SEQ ID NO：54)

the CD3 binding molecules of the present disclosure may be derived from any suitable antibody. Particularly suitable antibodies are described, for example, in International application No. WO2020114478 (incorporated herein by reference in its entirety).

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

TABLE 9 CDR of CD3 arm

The specific sequence of the variable region is as follows:

>S107E-VH(SEQ ID NO：63)

>S107E-VL(SEQ ID NO：64)

>6164-VH(SEQ ID NO：65)

>6164-VL(SEQ ID NO：64)

anti-CD 38 antibody variable region, anti-BCMA antibody variable region, anti-CD 3 antibody variable region and IgG ₁ Mutant IgG ₁ (AA) (L234A/L235A) combinations. The antigen binding molecules are respectively formed into different forms, namely Format5, format5-6164, format12-6164, format20-6164, format21-6164 and Format24.

Format5 is an asymmetric structure molecule, and the whole molecule has three chains, wherein the three chains are different, and the structure is shown in figure 1A.

Chain 1: VH (anti-BCMA) -CH1-IgG ₁ Fc (Knob, AA) -linker 1-VH (1149) -linker 2-VL (1149);

chain 2: VL (anti-BCMA) -CL;

chain 3 (Hole-5): VH (S107E) -linker 1-VL (S107E) -linker 2-IgG ₁ Fc (Hole, AA) -linker 3-VH (1149) -linker 4-VL (1149).

>Hole-5(SEQ ID NO：66)

Note that: the single underlined tag region is the CDR region of the CD38, CD3 binding domain obtained according to the Kabat numbering convention, italics is the constant region.

Format5-6164 is an asymmetric structure molecule, and the whole molecule has three chains which are different, and the structure is shown in figure 1A.

chain 2: VL (anti-BCMA) -CL;

chain 3: (Hole-5-6164): VH (6164) -linker 1-VL (6164) -linker 2-IgG ₁ Fc (Hole, AA) -linker 3-VH (1149) -linker 4-VL (1149)。

>Hole-5-6164(SEQ ID NO：67)

Format12 is a symmetrical structural molecule comprising two identical heavy chains (chain 1) and two identical light chains (chain 2), the structure of which is shown in FIG. 1B.

Chain 1: VH (anti-BCMA) -CH1-VH (S107E) -linker 1-VL (S107E) -linker 2-IgG ₁ Fc (AA) -linker 3-VH (1149) -linker 4-VL (1149);

chain 2: VL (anti-BCMA) -CL.

Format12-6164 is a symmetrical structural molecule comprising two identical heavy chains (chain 1) and two identical light chains (chain 2), the structure of which is shown in FIG. 1B.

Chain 1: VH (anti-BCMA) -CH1-VH (6164) -linker 1-VL (6164) -linker 2-IgG ₁ Fc (AA) -linker 3-VH (1149) -linker 4-VL (1149);

chain 2: VL (anti-BCMA) -CL.

>IgG ₁ Fc(AA)(SEQ ID NO：68)

Format20 is an asymmetric structure molecule, and the whole molecule has three chains, wherein the three chains are different, and the structure is shown in figure 1C.

Chain 1: VH (anti-BCMA) -CH1-IgG ₁ Fc(Hole,AA)；

Chain 2: VL (anti-BCMA) -CL;

Strand 3 (Knob-20): VH (1149) -linker 1-VL (1149) -linker 2-VH (S107E) -linker 3-VL (S107E) -linker 4-IgG ₁ Fc(Knob,AA)。

>Knob-20(SEQ ID NO：69)

Format20-6164 is an asymmetric structure molecule, and the whole molecule has three chains which are different, and the structure is shown in figure 1C.

Chain 1: VH (anti-BCMA) -CH1-IgG ₁ Fc(Hole,AA)；

Chain 2: VL (anti-BCMA) -CL;

strand 3 (Knob-20-6164): VH (1149) -linker 1-VL (1149) -linker 2-VH (6164) -linker 3-VL (6164) -linker 4-IgG ₁ Fc(Knob,AA)。

>Knob-20-6164(SEQ ID NO：70)

Format21 is a symmetrical structural molecule comprising two identical heavy chains (chain 1) and two identical light chains (chain 2), the structure of which is shown in FIG. 1D.

Chain 1: VH (1149) -CH1-VH (S107E) -linker 1-VL (S107E) -linker 2-IgG ₁ Fc (AA) -linker 3-VL (anti-BCMA) -linker 4-VH (anti-BCMA);

chain 2: VL (1149) -CL.

Format21-6164 is a symmetrical structural molecule comprising two identical heavy chains (chain 1) and two identical light chains (chain 2), the structure of which is shown in FIG. 1D.

Chain 1: VH (1149) -CH1-VH (6164) -linker 1-VL (6164)Linker 2-IgG ₁ Fc (AA) -linker 3-VL (anti-BCMA) -linker 4-VH (anti-BCMA);

chain 2: VL (1149) -CL, the structure of which is shown in FIG. 1D.

>VL(1149)-CL(SEQ ID NO：71)

Note that: the single underlined tag region is the CDR region of the CD38 binding domain obtained according to the Kabat numbering convention, italicized as the constant region.

Format24 is an asymmetric structural molecule, and the complete molecule has four chains, and the structure is shown in FIG. 1E.

Chain 1: VL (anti-BCMA) -linker 1-VH (anti-BCMA) -linker 2-VH (1149) -CH1-IgG ₁ Fc(Hole,AA)；

Chain 2 (two): VL (1149) -CL;

strand 3 (Knob-24): VH (1149) -CH1-IgG ₁ Fc (Knob, AA) -linker 1-VL (107E) -linker 2-VH (107E).

>Knob-24(SEQ ID NO：72)

Antigen binding molecules were constructed according to table 10, numbering where T5 represents the use of hu4E3L1, hu4E3H1 as BCMA-binding variable regions, T3 represents the use of hu33H4L2, hu33H4H1 as BCMA-binding variable regions, and T1 represents the use of hu27L1, hu27H3 as BCMA-binding variable regions.

TABLE 10 trispecific antibodies of the disclosure

Numbering device	Chain 1	Chain 2	Chain 3
T5-5	SEQ ID NO：73	SEQ ID NO：74	SEQ ID NO：66
T5-5-6164	SEQ ID NO：73	SEQ ID NO：74	SEQ ID NO：67
T5-12	SEQ ID NO：75	SEQ ID NO：74
T5-12-6164	SEQ ID NO：76	SEQ ID NO：74
T5-21	SEQ ID NO：77	SEQ ID NO：71
T5-21-6164	SEQ ID NO：78	SEQ ID NO：71
T5-24	SEQ ID NO：79	SEQ ID NO：71	SEQ ID NO：72
T3-5	SEQ ID NO：80	SEQ ID NO：81	SEQ ID NO：66
T3-5-6164	SEQ ID NO：80	SEQ ID NO：81	SEQ ID NO：67

T3-12	SEQ ID NO：82	SEQ ID NO：81
T3-12-6164	SEQ ID NO：83	SEQ ID NO：81
T3-20	SEQ ID NO：84	SEQ ID NO：81	SEQ ID NO：69
T3-20-6164	SEQ ID NO：84	SEQ ID NO：81	SEQ ID NO：70
T3-21	SEQ ID NO：85	SEQ ID NO：71
T3-21-6164	SEQ ID NO：86	SEQ ID NO：71
T3-24	SEQ ID NO：87	SEQ ID NO：71	SEQ ID NO：72
T1-5	SEQ ID NO：88	SEQ ID NO：89	SEQ ID NO：66
T1-5-6164	SEQ ID NO：88	SEQ ID NO：89	SEQ ID NO：67
T1-12	SEQ ID NO：90	SEQ ID NO：89
T1-12-6164	SEQ ID NO：91	SEQ ID NO：89
T1-20	SEQ ID NO：92	SEQ ID NO：89	SEQ ID NO：69
T1-20-6164	SEQ ID NO：92	SEQ ID NO：89	SEQ ID NO：70
T1-21	SEQ ID NO：93	SEQ ID NO：71
T1-21-6164	SEQ ID NO：94	SEQ ID NO：71
T1-24	SEQ ID NO：95	SEQ ID NO：71	SEQ ID NO：72

Note that: -5 in T5-5 represents a structure using Format 5; -5-6164 in T5-5-6164 represents a structure using Format 5-6164; and-12 in T5-12 represents a structure adopting Format12, and so on.

>SEQ ID NO：73

>SEQ ID NO：74

>SEQ ID NO：75

>SEQ ID NO：76

>SEQ ID NO：77

>SEQ ID NO：78

>SEQ ID NO：79

>SEQ ID NO：80

>SEQ ID NO：81

>SEQ ID NO：82

>SEQ ID NO：83

>SEQ ID NO：84

>SEQ ID NO：85

>SEQ ID NO：86

>SEQ ID NO：87

>SEQ ID NO：88

>SEQ ID NO：89

>SEQ ID NO：90

>SEQ ID NO：91

>SEQ ID NO：92

>SEQ ID NO：93

>SEQ ID NO：94

>SEQ ID NO：95

And (3) injection: the single underlined tag region is the CDR region obtained according to the Kabat numbering convention, italics is the constant region.

>CH1(SEQ ID NO：104)

>CL(SEQ ID NO：105)

The positive control molecules GBR-1342 and AMGEN701 used in the present disclosure. The amino acid sequence of GBR-1342 comprises the amino acid sequence of SEQ ID NO in WO 2016071355: 178. SEQ ID NO:179 and SEQ ID NO:128. the amino acid sequence of amben 701 comprises SEQ ID NO:74.

test case

Test example 1 FACS determination of the expression level of hBCMA and hCD38 on the surface of MM cells

To test the expression levels of BCMA and CD38 on the surface of MM cells, the expression levels of MM-related cell lines were examined by FACS. The CD38 expression level was determined as follows: rate of activity>90% of the cells were 1X 10 per well ⁵ Cells were added to 96-well plates (3590 #, corning), centrifuged at 300g for 5 min, and washed once with 1% bsa. mu.L of 10. Mu.g/mL CD38 antibody in 1% BSA was added and incubated at 4℃for 1 hour. Washing with 1% BSA was performed twice. Addition of PE-human F (ab') ₂ (1:400) 50. Mu.L, incubated at 4℃for 0.5 h. Washing with 1% BSA was performed twice. 100. Mu.L of 1% BSA was added for resuspension and machine-readable values were set up. The BCMA expression level was determined as follows: rate of activity>90% of the cells were 1X 10 per well ⁵ Cells were added to 96-well plates (3590 #, corning), centrifuged at 300g for 5 min, and washed once with 1% bsa. The BCMA antibody diluted according to the instructions (BL 2-A: BCMA-aPC-A) was added and incubated at 4℃for 1 hour. Washing with 1% BSA was performed twice. 100. Mu.L of 1% BSA was added for resuspension, and the cell surface was then detected with a FACS instrument Fluorescence values of PE and APC were compared in parallel using geometric mean.

Identification of CD38 and BCMA expression levels of MM cell lines

Test example 2 affinity of antibodies of the disclosure for CD38/BCMA/CD3d & CD3e protein

To test the affinity of the antibodies of the present disclosure for CD38/BCMA/CD3D & E, the present test example tested the binding capacity of the antibodies to hCD3D & CD3E (31.2 kda, cat.#ct038-H2508H, S.B), hCD38 (30.7 kda, hr) and hbma (7.8 kda, cat.#bca-H52y, acro) using the instrument Biacore T200. The specific method comprises the following steps: the anti-human antibody-conjugated CM5 biological sensing chip is used for affinity capture of the antibody, then 100nM BCMA-His solution is flowed on the surface of the chip for 180 seconds, the BCMA binding site of the antibody is saturated, then hCD3d & CD3e or hCD38 is added, and the real-time detection is carried out by using a Biacore T200 instrument. The data fitting model uses a 1:1model. Affinity data for binding of antibodies of the present disclosure to the corresponding antigen are shown in the following table.

TABLE 12 affinity assay for antibodies

Note that: for convenient purification, the C-terminal of the Knob-24 chain of Format24 contains 6His.

The results show that the antibodies of the present disclosure have better CD38/BCMA binding and are able to bind CD3d & CD3e.

Test example 3 FACS determination of affinity of antibodies to MM cell surface antigens

To test the binding capacity of the antibodies of the present disclosure to hCD38 and hbma on the surface of MM cells, the present test example examined the binding capacity of CD38/BCMA-CD3 trispecific antibodies to RPMI-8226 on MM tumor cells co-expressed with CD38 and BCMA by flow cytometry. The method comprises the following steps: cell culture as described aboveCulturing in 1640 medium containing 10% FBS, standing at 37deg.C, and 5% CO ₂ Culturing in incubator for 2 days, wherein the number of cells per well is 1×10 ⁵ Cells were added to the cell plates, centrifuged at 300g for min and washed once with 1% BSA. The antibodies were directly labeled with Mix-n-Stain CF 647Antibody Labeling Kits (MX 647S100-1KT#, sigma) and the fluorescently labeled antibodies were serially diluted and added to the cell plates at 100. Mu.L per well, incubated for 1 hour at 4℃and washed three times with 1% BSA and 100. Mu.L PBS read plate per well. As shown in fig. 2, the antibodies of the present disclosure bind very well to the surface of tumor cells co-expressed with BCMA and CD38, which is better than the positive control.

Test example 4 in vitro cytotoxic Activity of antibodies

To test the killing activity of antibodies of the present disclosure as T cell adapter molecules against tumor cells. The target-specific cytotoxic activity of the bispecific antibodies of the present disclosure was tested using the stably transgenic cell line K562/hbma that overexpresses human BCMA, the stably transgenic cell line CHO K1/hCD38 that overexpresses human CD38, and MM tumor cell RPMI-8226 that coexpresses CD38 and BCMA as target cells, and the non-specific cytotoxic activity of the bispecific antibodies of the present disclosure was tested using CHOK1 that does not express CD38 and BCMA as target cells. 300g of fresh PBMC (from Xuanfeng Biometrics) was centrifuged for 10 min, resuspended in 1640+10% FBS (solution A) containing 200U IL2 and incubated overnight (density 2X 10) in T75 flasks ⁶ /mL); after centrifugation, the cells were resuspended and counted in medium containing IMDM+10% FBS, DMEM/F-12:1+10% FBS and 1640+10% FBS, respectively, and the cell number was adjusted to 1.5X10 ⁶ mu.L/mL was added to each well. Collecting target cells, centrifuging at 1000rpm for 3min, re-suspending, counting, and adjusting cell number to 3×10 ⁵ cells/mL, 25. Mu.L per well, E: T Ratio of 10:1. The antibodies were diluted with the corresponding medium of the cells at an initial concentration of 400nM (4 Xfinal concentration), 9 gradients at 10-fold dilution, and 25. Mu.L was added to each well. The cells were then placed at 37℃with 5% CO ₂ Is cultured for 48 hours. Fluorescent signal detection: taking out the culture plate, centrifuging at 1000rpm for 3min, sucking 50 μl of supernatant into a new 96-well plate, freezing in a refrigerator at-20deg.C, adding 50 μ into the original plate containing cells at a ratio of 1:1L one-Glo, incubated at room temperature for 5min, the luminescence values (luminescence values of cells of the non-antibody group were defined as 0 killing) were measured, and the percentage of killing of different antibodies at different concentrations was calculated.

TABLE 13-1 cytotoxic Activity of antibodies against K562/hBCMA

Antibodies to	IC50(pM)	Cleavage Rate%
T1-12	0.034	98.6
T3-12	0.110	98.7
T5-12	0.012	99.5
T5-12-6164	0.148	99.5
T1-21	0.085	98.8
T3-21	0.024	99.4
AMGEN701	0.343	99.5

TABLE 13-2 cytotoxic Activity of antibodies against CHO K1/hCD38

TABLE 13-3 cytotoxic Activity of antibodies against RPMI8266

Note that: the C-terminal of the Knob chain of Format5-6164 and Format24 contains 6His.

The results are shown in tables 13-1 to 13-3 and fig. 3, the cytotoxic activity of the antibodies of the present disclosure is specific for CD38 and BCMA targets, which is superior to the positive control; meanwhile, fig. 3 shows that the antibodies of the present disclosure exhibit weaker cytotoxic activity and better safety on CHOK1 cell lines without CD38 and BCMA expression.

Test example 5. Effect of soluble BCMA/CD38/APRIL on in vitro cytotoxic Activity of antibodies of the disclosure

The presence of high concentrations of soluble BCMA (30 ng/mL on average) in the blood of patients with multiple myeloma interferes with specific binding of BCMA-CD3 bispecific antibodies to membrane surface BCMA, and secondly, APRIL is also present in the serum of MM patients in 5.9 times that of healthy persons, with a proportional increase in soluble CD38 in MM patients. To test whether the in vitro tumor cell killing activity of the antibodies of the present disclosure is subject to solubilityEffect of BCMA/CD38/APRIL, the cell line RPMI-8226 co-expressing CD38 and BCMA was used as target cells, and soluble BCMA, CD38 and APRIL were added separately or together at the time of cytotoxic activity test. 300g of fresh PBMC (from Xuanfeng Biolabs) was centrifuged for 10min, resuspended in 1640+10% FBS (solution A) containing 200U IL2 and incubated overnight (density 2E 6/mL) in a T75 flask; after centrifugation, the cells were resuspended and counted in 1640+10% FBS medium to adjust the number of cells to 1.5X10 ⁶ cells/mL, 50. Mu.L per well. Target cells were collected, centrifuged at 1000rpm for 3 minutes, resuspended, counted and the cell number was adjusted to 3X 10 ⁵ cells/mL, 25. Mu.L per well, E: T Ratio of 10:1. The antibodies were diluted with the corresponding medium of the cells at an initial concentration of 400nM (4 Xfinal concentration), 9 gradients at 10-fold dilution, and 25. Mu.L was added to each well. The cells were then placed at 37℃with 5% CO ₂ Is cultured for 48 hours. Fluorescent signal detection: taking out the culture plate, centrifuging at 1000rpm for 3min, sucking 50 mu L of supernatant into a new 96-well plate, freezing in a refrigerator at-20 ℃, adding 50 mu L of one-Glo into the original plate containing cells according to the ratio of 1:1, incubating at room temperature for 5 min, detecting the value of luminescence (the value of luminescence of cells without antibody group is defined as 0 killer), and calculating the killing percentage of different antibodies at different concentrations.

TABLE 14 Effect of soluble proteins on antibody cell killing Activity

Note that: "relative IC ₅₀ "means the ratio of the IC50 with soluble protein added to the IC50 without soluble protein added.

The results show that the cytotoxic activity of the antibodies of the present disclosure is minimally affected by soluble BCMA/CD 38/APRIL. And have no solubilityComparison in the presence of protein, 30ng/mL of soluble BCMA, 5ng/mL of soluble CD38, and 100ng/mL of soluble APRIL, respectively or together, resulted in only cytotoxic IC of antibodies of the disclosure ₅₀ To 0.8-2.5 times the original, while AMGEN701 is IC in the presence of soluble protein ₅₀ But is increased by 10 times.

Test example 6 antibody-induced cytokine Release level detection

CD3T cell adaptor molecules can cause cytokine storms. Thus, there is a need to keep cytokines, particularly IL-6, which is not related to potency but causes side effects, at low levels in the development of CD3T cell adaptor molecules.

The CHO-K1 cells are centrifuged at 1000rpm for minutes, 50 mu L of cell supernatant is collected, the sample is diluted by a general dilution liquid for specimens, IL-6 or IL-2 standard substances are diluted by 6 times, diluted samples or standard substances with different concentrations (100 mu L/hole) are added into a detection plate, the reaction hole is sealed by using sealing plate gummed paper, the plates are washed 5 times after incubation for 90 minutes at 37 ℃, biotinylated antibody working liquid (100 mu L/hole) is added, the reaction hole is sealed by using new sealing plate gummed paper, the plates are washed 5 times after incubation for 60 minutes at 37 ℃, enzyme conjugate working liquid (100 mu L/hole) is added, the reaction hole is sealed by using new sealing plate gummed paper, the plates are washed 5 times after incubation for 30 minutes at 37 ℃, chromogenic substrate (TMB) is added, the plates are incubated for 8 minutes at 37 ℃ away from light, the reaction termination liquid is added for 100 mu L/hole, and OD450 value (within 3 minutes) is measured immediately after mixing.

TABLE 15 Release of IL-6 and IL-2 (CHO K1)

The results show that the antibodies of the present disclosure bring lower levels of release of both IL6 and IL-2 than GBR-1342, suggesting that the antibodies of the present disclosure have better safety.

Biological evaluation of in vivo Activity

Test example 7 efficacy of antibodies in Molp-8 in situ tumor model

Molp-8 is a typical MM cell line with higher CD38 expression and lower BCMA expression.

To test the efficacy of the antibodies of the present disclosure in a Molp-8 in situ tumor model, molp-8/lucG cells were used 1.5X10 ⁶ Cells/200. Mu.L/mouse were inoculated into 79 NDG mice via tail vein. 1 day after tumor cell inoculation, two donor freshly isolated PBMCs were mixed at a 1:1 ratio at 5X 10 ⁶ Mice were injected into the abdominal cavity of the mice. 4 days after tumor cell inoculation, each mouse was intraperitoneally injected with a bioluminescent substrate (15 mg/mL), injected at a volume of 10mL/kg, anesthetized by isoflurane, and imaged by a small animal imaging system after 10 minutes of injection. The weight was removed, the bioluminescence signal values were too large and too small, the mice were randomly grouped by bioluminescence signal, 7 per group. The Day of grouping was started with intraperitoneal injections of each antibody and the Day was defined as the experiment Day0, 2 times per week. Imaging was performed 2 times per week, body weight was weighed, and data was recorded. All data were plotted and statistically analyzed using Excel and GraphPad Prism 8 software.

The bioluminescence signal value is Total Flux (units, p/s) and the average is calculated as avg;

tumor inhibition rate (%) =1-T/C (%). T/C (%) = (T-T0)/(C-C0) ×100%, where T, C is the bioluminescence signal value of the treatment group and control group at the end of the experiment; t (T) ₀ 、C ₀ Is the bioluminescence signal value at the start of the experiment. The experimental results show that both T3-12 and T5-12-6164 show obvious drug effects.

TABLE 16 drug efficacy of antibodies in Molp-8 in situ tumor model

Test example 8 efficacy of antibodies in RPMI-8226 in situ tumor model

RPMI-8226 is a typical MM cell line with moderate CD38 expression and BCMA expression.

RPMI-8226-lucG cells were 5X 10 ⁶ Cell/200. Mu.L-Mice were inoculated into NDG mice via the tail vein. Tumor cells were inoculated for 15 days, and each mouse was intraperitoneally injected with a bioluminescent substrate (15 mg/mL), injected at a volume of 10mL/kg, anesthetized by isoflurane, and imaged by a small animal imaging system after 10 minutes of injection. The weight was removed, the bioluminescence signal values were too large and too small, the mice were randomly grouped by bioluminescence signal, 7 per group. 1 day after grouping, two donors were freshly isolated PBMCs mixed at a 1:1 ratio at 5X 10 ⁶ Mice were injected into the abdominal cavity of the mice. Intraperitoneal injections of each antibody were started 4 days after the grouping, and this Day was defined as the experiment Day0, 2 times per week, for a total of 4 administrations. Imaging was performed 2 times per week, body weight was weighed, and data was recorded. All data were plotted and statistically analyzed using Excel and GraphPad Prism 8 software.

tumor inhibition rate (%) =1-T/C (%). T/C (%) = (T-T0)/(C-C0) ×100%, where T, C is the bioluminescence signal value of the treatment group and control group at the end of the experiment; t (T) ₀ 、C ₀ Is the bioluminescence signal value at the start of the experiment.

TABLE 17 drug efficacy of antibodies in RPMI-8226 in situ tumor model

Administration group	Dosage (mg/kg)	Tumor inhibition rate%
Blank (PBS)	/	/
T3-12	0.01	14.4
T3-12	0.03	96.9
T3-12	0.1	>100
T5-12-6164	0.03	50.2

GBR-1342	0.015	-263.0
GBR-1342	0.05	-17.8

The results show that both T3-12 and T5-12-6164 show obvious drug effects, and better drug effects than the equivalent molar GBR-1342. In the process of administration, tumor-bearing mice can better tolerate each antibody, and the body weight does not obviously fluctuate.

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

Claims

An antigen binding molecule that specifically binds BCMA, CD38, and CD3 comprising at least one antigen binding moiety that specifically binds BCMA, at least one antigen binding moiety that specifically binds CD38, and at least one antigen binding moiety that specifically binds CD 3.
The antigen binding molecule of claim 1, comprising two antigen binding moieties that specifically bind BCMA, two antigen binding moieties that specifically bind CD38, two antigen binding moieties that specifically bind CD3, and an Fc region;

preferably, the method comprises the steps of,

the antigen binding moiety that specifically binds BCMA is BCMA-Fab, the antigen binding moiety that specifically binds CD3 is CD3-scFv and the antigen binding moiety that specifically binds CD38 is CD38-scFv; or (b)

The antigen binding moiety that specifically binds CD38 is a CD38-Fab, the antigen binding moiety that specifically binds CD3 is a CD3-scFv and the antigen binding moiety that specifically binds BCMA is a BCMA-scFv;

more preferably, the process is carried out,

the antigen binding moiety that specifically binds BCMA is BCMA-Fab, the antigen binding moiety that specifically binds CD3 is CD3-scFv and the antigen binding moiety that specifically binds CD38 is CD38-scFv; and the heavy chain of the BCMA-Fab, the CD3-scFv, one subunit of the Fc region and the CD38-scFv are linked directly or via a linker, respectively, in the order N-terminal to C-terminal; or (b)

The antigen binding moiety that specifically binds CD38 is a CD38-Fab, the antigen binding moiety that specifically binds CD3 is a CD3-scFv and the antigen binding moiety that specifically binds BCMA is a BCMA-scFv; and the heavy chain of the CD38-Fab, the CD3-scFv, one subunit of the Fc region and the BCMA-scFv are linked directly or via a linker, respectively, in the order of N-terminus to C-terminus.
The antigen-binding molecule of claim 1 or 2, wherein the antigen-binding moiety that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and wherein the antigen-binding moiety that specifically binds CD38 comprises a heavy chain variable region CD38-VH and a light chain variable region CD38-VL; wherein:

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):

(a) [ BCMA-VH ] - [ CH1] - [ CD3-VH ] - [ linker 1] - [ CD3-VL ] - [ linker 2] - [ one subunit of Fc region ] - [ linker 3] - [ CD38-VH ] - [ linker 4] - [ CD38-VL ],

(b)[BCMA-VL]-[CL]，

linker 1, linker 2, linker 3 and linker 4 in formula (a) are identical or different peptide linkers; or (b)

The antigen binding molecule comprises two first chains having a structure represented by formula (c) and two second chains having a structure represented by formula (d):

(c) [ CD38-VH ] - [ CH1] - [ CD3-VH ] - [ linker 1] - [ CD3-VL ] - [ linker 2] - [ one subunit of the Fc region ] - [ linker 3] - [ BCMA-VH ] - [ linker 4] - [ BCMA-VL ],

(d)[CD38-VL]-[CL]，

linker 1, linker 2, linker 3 and linker 4 in formula (c) are identical or different peptide linkers;

Preferably, the method comprises the steps of,

linker 1, linker 2, linker 3 and linker 4 in formula (a) are 3-15 amino acid residues in length; or (b)

Linker 1, linker 2, linker 3 and linker 4 in formula (c) are 3-15 amino acid residues in length;

more preferably, the process is carried out,

the amino acid sequences of the linker 1, the linker 3 and the linker 4 in the formula (a) are shown in SEQ ID NO:99, the amino acid sequence of the linker 2 is shown in SEQ ID NO: 96; or (b)

The amino acid sequences of the linker 1, the linker 3 and the linker 4 in the formula (c) are shown in SEQ ID NO:99, the amino acid sequence of the linker 2 is shown in SEQ ID NO: shown at 96.
The antigen binding molecule of claim 1, comprising one antigen binding moiety that specifically binds BCMA, two antigen binding moieties that specifically bind CD38, one antigen binding moiety that specifically binds CD3, and an Fc region;

preferably, the method comprises the steps of,

the antigen binding moiety that specifically binds BCMA is BCMA-Fab, the antigen binding moiety that specifically binds CD3 is CD3-scFv and the antigen binding moiety that specifically binds CD38 is CD38-scFv; or (b)

The antigen binding moiety that specifically binds CD38 is a CD38-Fab, the antigen binding moiety that specifically binds CD3 is a CD3-scFv and the antigen binding moiety that specifically binds BCMA is a BCMA-scFv;

More preferably, the Fc region comprises a first subunit Fc1 and a second subunit Fc2 capable of associating, each of the Fc1 and Fc2 independently having one or more amino acid substitutions that reduce homodimerization of the Fc region; and, in addition, the processing unit,

the antigen binding moiety that specifically binds BCMA is BCMA-Fab, the antigen binding moiety that specifically binds CD3 is CD3-scFv and the antigen binding moiety that specifically binds CD38 is CD38-scFv; and the heavy chain of the BCMA-Fab, fc1 and one CD38-scFv are linked directly or via a linker, respectively, in the order of N-terminus to C-terminus, and the CD3-scFv, fc2 and the other CD38-scFv are linked directly or via a linker, respectively, in the order of N-terminus to C-terminus; or (b)

The antigen binding moiety that specifically binds CD38 is a CD38-Fab, the antigen binding moiety that specifically binds CD3 is a CD3-scFv and the antigen binding moiety that specifically binds BCMA is a BCMA-scFv; and the heavy chain of one CD38-Fab, fc1 and CD3-scFv are linked directly or via a linker, respectively, in N-terminal to C-terminal order, and the heavy chain of the BCMA-scFv, the heavy chain of the other CD38-Fab and Fc2 are linked directly or via a linker, respectively, in N-terminal to C-terminal order.
The antigen-binding molecule of claim 1 or 4, wherein the antigen-binding moiety that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and wherein the antigen-binding moiety that specifically binds CD38 comprises a heavy chain variable region CD38-VH and a light chain variable region CD38-VL; wherein:

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

(e) [ BCMA-VH ] - [ CH1] - [ Fc1] - [ linker 1] - [ CD38-VH ] - [ linker 2] - [ CD38-VL ],

(b)[BCMA-VL]-[CL]，

(f) [ CD3-VH ] - [ linker 1] - [ CD3-VL ] - [ linker 2] - [ Fc2] - [ linker 3] - [ CD38-VH ] - [ linker 4] - [ CD38-VL ] -,

wherein linker 1 and linker 2 in formula (e) are the same or different peptide linkers, and linker 1, linker 2, linker 3 and linker 4 in formula (f) are the same or different peptide linkers; or (b)

The antigen binding molecule comprises a first chain having a structure represented by formula (g), two second chains having a structure represented by formula (d), and a third chain having a structure represented by formula (h):

(g) [ BCMA-VL ] - [ linker 1] - [ BCMA-VH ] - [ linker 2] - [ CD38-VH ] - [ CH1] - [ Fc2],

(d)[CD38-VL]-[CL]，

(h) [ CD38-VH ] - [ CH1] - [ Fc1] - [ linker 1] - [ CD3-VL ] - [ linker 2] - [ CD3-VH ],

wherein linker 1 and linker 2 in formula (g) are the same or different peptide linkers, and linker 1 and linker 2 in formula (h) are the same or different peptide linkers;

preferably, the method comprises the steps of,

linker 1, linker 2 in formula (e), and linker 1, linker 2, linker 3 and linker 4 in formula (f) are 3-15 amino acid residues in length; or (b)

Linker 1, linker 2 in formula (g), and linker 1 and linker 2 in formula (h) are 3-15 amino acid residues in length;

more preferably, the process is carried out,

the amino acid sequences of the linker 1 and the linker 2 in the formula (e) are shown in SEQ ID NO:99, the amino acid sequences of linker 1, linker 3 and linker 4 in formula (f) are as shown in SEQ ID NO:99, and linker 2 in formula (f) has the amino acid sequence shown in SEQ ID NO: 96; or (b)

The amino acid sequences of the linker 1 and the linker 2 in the formula (g) are shown in SEQ ID NO:99, the amino acid sequence of linker 1 in formula (h) is shown in SEQ ID NO:97, and the amino acid sequence of linker 2 in formula (h) is as shown in SEQ ID NO: 99.
The antigen binding molecule of claim 1, comprising one antigen binding moiety that specifically binds BCMA, one antigen binding moiety that specifically binds CD38, one antigen binding moiety that specifically binds CD3, and an Fc region;

preferably, the method comprises the steps of,

the antigen binding moiety that specifically binds BCMA is BCMA-Fab, the antigen binding moiety that specifically binds CD3 is CD3-scFv and the antigen binding moiety that specifically binds CD38 is CD38-scFv;

more preferably, the process is carried out,

The Fc region comprises a first subunit Fc1 and a second subunit Fc2 capable of associating, each of the Fc1 and Fc2 independently having one or more amino acid substitutions that reduce homodimerization of the Fc region; and the CD38-scFv, CD3-scFv and Fc1 are linked directly or via a linker, respectively, in N-terminal to C-terminal order, and the heavy chain of the BCMA-Fab and Fc2 are linked directly or via a linker, respectively, in N-terminal to C-terminal order.
The antigen-binding molecule of claim 1 or 6, wherein the antigen-binding moiety that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and wherein the antigen-binding moiety that specifically binds CD38 comprises a heavy chain variable region CD38-VH and a light chain variable region CD38-VL; wherein:

the antigen binding molecule comprises a first chain having a structure represented by formula (i), a second chain having a structure represented by formula (b), and a third chain having a structure represented by formula (j):

(i)[BCMA-VH]-[CH1]-[Fc2]，

(b)[BCMA-VL]-[CL]，

(j) [ CD38-VH ] - [ linker 1] - [ CD38-VL ] - [ linker 2] - [ CD3-VH ] - [ linker 3] - [ CD3-VL ] - [ linker 4] - [ Fc1] - [ linker 3-VL ] -.

Wherein linker 1, linker 2, linker 3 and linker 4 in formula (j) are identical or different peptide linkers;

Preferably, the method comprises the steps of,

linker 1, linker 2, linker 3 and linker 4 in formula (j) are 3-15 amino acid residues in length;

more preferably, the process is carried out,

the amino acid sequences of the linker 1 and the linker 3 in the formula (j) are shown in SEQ ID NO:99, the amino acid sequence of linker 2 in formula (j) is shown in SEQ ID NO:97, and linker 4 in formula (j) has the amino acid sequence shown in SEQ ID NO: shown at 96.
The antigen binding molecule of any one of claims 1 to 7, wherein the antigen binding moiety that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein:

(i) The BCMA-VH comprises SEQ ID NO:23, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3, and said BCMA-VL comprises the amino acid sequence of SEQ ID NO:24, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(ii) The BCMA-VH comprises SEQ ID NO:25, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3, and said BCMA-VL comprises the amino acid sequence of SEQ ID NO:26, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3; or (b)

(iii) The BCMA-VH comprises SEQ ID NO:27, BCMA-HCDR1, BCMA-HCDR2, and BCMA-HCDR3, and said BCMA-VL comprises the amino acid sequence of SEQ ID NO:28, BCMA-LCDR1, BCMA-LCDR2, and BCMA-LCDR 3;

Preferably, the method comprises the steps of,

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

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

(iii) The BCMA-VH has the amino acid sequence of SEQ ID NO:17, BCMA-HCDR1, SEQ ID NO:18 and BCMA-HCDR2 of SEQ ID NO:19 BCMA-HCDR3; and the BCMA-VL has the amino acid sequence of SEQ ID NO:20, BCMA-LCDR1, SEQ ID NO:21 and BCMA-LCDR2 of SEQ ID NO: BCMA-LCDR3 of 22.
The antigen binding molecule of claim 8, wherein:

(i) The BCMA-VH is shown in SEQ ID NO:23 and said BCMA-VL is as shown in SEQ ID NO:24, or

The BCMA-VH is shown in SEQ ID NO: 29. SEQ ID NO:30 and SEQ ID NO:31 and said BCMA-VL is as set forth in any one of SEQ ID NOs: 32. SEQ ID NO: 33. SEQ ID NO:34 and SEQ ID NO: as indicated in any one of the above-mentioned items 35,

Preferably, the method comprises the steps of,

the BCMA-VH is shown in SEQ ID NO:29 and said BCMA-VL is as set forth in SEQ ID NO: shown at 32; or (b)

(ii) The BCMA-VH is shown in SEQ ID NO:25 and said BCMA-VL is as set forth in SEQ ID NO: 26 or

The BCMA-VH is shown in SEQ ID NO: 36. SEQ ID NO:37 and SEQ ID NO:38, and the BCMA-VL is as set forth in SEQ ID NO:39 or SEQ ID NO: one of which is shown at 40,

preferably, the method comprises the steps of,

the BCMA-VH is shown in SEQ ID NO:36 and said BCMA-VL is as set forth in SEQ ID NO: shown at 40; or (b)

(iii) The BCMA-VH is shown in SEQ ID NO:27 and said BCMA-VL is as set forth in SEQ ID NO:28, or

The BCMA-VH is shown in SEQ ID NO: 41. SEQ ID NO: 42. SEQ ID NO:43 and SEQ ID NO:44, and the BCMA-VL is as set forth in SEQ ID NO:45 or SEQ ID NO: as indicated by one of the reference numerals 46,

preferably, the method comprises the steps of,

the BCMA-VH is shown in SEQ ID NO:43 and said BCMA-VL is as set forth in SEQ ID NO: 45.
The antigen-binding molecule of any one of claims 1 to 9, wherein the antigen-binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein:

(i) The CD3-VH comprises SEQ ID NO:63, CD3-HCDR1, CD3-HCDR2 and CD3-HCDR 3; and said CD3-VL comprises SEQ ID NO:64, the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3, or

(ii) The CD3-VH comprises SEQ ID NO:65, CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3; and said CD3-VL comprises SEQ ID NO:64, the amino acid sequences of CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3;

preferably, the method comprises the steps of,

(i) The CD3-VH has the sequence shown in SEQ ID NO:55, as shown in SEQ ID NO:56, and a CD3-HCDR2 as set forth in SEQ ID NO: 57-CD 3-HCDR3; and said CD3-VL has the amino acid sequence as set forth in SEQ ID NO:58, as set forth in SEQ ID NO:59, and a CD3-LCDR2 as set forth in SEQ ID NO:60, or CD3-LCDR3, or

(ii) The CD3-VH has the sequence shown in SEQ ID NO:55, as shown in SEQ ID NO:61, and a CD3-HCDR2 as set forth in SEQ ID NO:62 CD3-HCDR3; and said CD3-VL has the amino acid sequence as set forth in SEQ ID NO:58, as set forth in SEQ ID NO:59, and a CD3-LCDR2 as set forth in SEQ ID NO:60, CD3-LCDR3;

more preferably, the process is carried out,

the CD3-VH is shown in SEQ ID NO:63, and said CD3-VL is set forth in SEQ ID NO: indicated at 64; or (b)

The CD3-VH is shown in SEQ ID NO:65, and said CD3-VL is set forth in SEQ ID NO: 64. as shown.
The antigen-binding molecule of any one of claims 1 to 10, wherein the antigen-binding moiety that specifically binds CD38 comprises a heavy chain variable region CD38-VH and a light chain variable region CD38-VL, wherein the CD38-VH comprises the amino acid sequence of SEQ ID NO:53, CD38-HCDR1, CD38-HCDR2 and CD38-HCDR3, and said CD38-VL comprises the amino acid sequence of SEQ ID NO:54, CD38-LCDR1, CD38-LCDR2 and CD38-LCDR 3;

Preferably, the method comprises the steps of,

the CD38-VH has the sequence shown in SEQ ID NO:47, and the CD38-HCDR1, as shown in SEQ ID NO:48, and a CD38-HCDR2 as set forth in SEQ ID NO: CD38-HCDR3 shown in FIG. 49; and said CD38-VL has the amino acid sequence as set forth in SEQ ID NO:50, as set forth in SEQ ID NO:51, and a CD38-LCDR2 as set forth in SEQ ID NO:52, CD38-LCDR3;

more preferably, the process is carried out,

the CD38-VH is shown in SEQ ID NO:53 and said CD38-VL is set forth in SEQ ID NO: indicated at 54.
The antigen binding molecule of any one of claims 1 to 11, wherein the antigen binding moiety that specifically binds BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, the antigen binding moiety that specifically binds CD3 comprises a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the antigen binding moiety that specifically binds CD38 comprises a heavy chain variable region CD38-VH and a light chain variable region CD38-VL, wherein:

(i) The BCMA-VH has: as set forth in SEQ ID NO:5, BCMA-HCDR1 as shown in SEQ ID NO:6 and BCMA-HCDR2 as shown in SEQ ID NO: BCMA-HCDR3 shown in fig. 7; and the BCMA-VL has: as set forth in SEQ ID NO:8, BCMA-LCDR1 as shown in SEQ ID NO:9 and BCMA-LCDR2 as shown in SEQ ID NO:10, and

The CD3-VH has: as set forth in SEQ ID NO:55, as shown in SEQ ID NO:61, and a CD3-HCDR2 as set forth in SEQ ID NO:62 CD3-HCDR3; and the CD3-VL has: as set forth in SEQ ID NO:58, as set forth in SEQ ID NO:59, and a CD3-LCDR2 as set forth in SEQ ID NO:60, and CD3-LCDR3, and

the CD38-VH has: as set forth in SEQ ID NO:47, and the CD38-HCDR1, as shown in SEQ ID NO:48, and a CD38-HCDR2 as set forth in SEQ ID NO: CD38-HCDR3 shown in FIG. 49; and the CD38-VL has: as set forth in SEQ ID NO:5, and the CD38-LCDR1 is shown as SEQ ID NO:51, and a CD38-LCDR2 as set forth in SEQ ID NO:52, CD38-LCDR3; or (b)

(ii) The BCMA-VH has: as set forth in SEQ ID NO:11, BCMA-HCDR1 as shown in SEQ ID NO:12 and BCMA-HCDR2 as shown in SEQ ID NO:13, BCMA-HCDR3; and the BCMA-VL has: as set forth in SEQ ID NO:14, BCMA-LCDR1 as shown in SEQ ID NO:15 and BCMA-LCDR2 as shown in SEQ ID NO:16, and

the CD3-VH has: as set forth in SEQ ID NO:55, as shown in SEQ ID NO:56, and a CD3-HCDR2 as set forth in SEQ ID NO: 57-CD 3-HCDR3; and the CD3-VL has: as set forth in SEQ ID NO:58, as set forth in SEQ ID NO:59, and a CD3-LCDR2 as set forth in SEQ ID NO:60, and CD3-LCDR3, and

The CD38-VH has: as set forth in SEQ ID NO:47, and the CD38-HCDR1, as shown in SEQ ID NO:48, and a CD38-HCDR2 as set forth in SEQ ID NO: CD38-HCDR3 shown in FIG. 49; and the CD38-VL has: as set forth in SEQ ID NO:50, as set forth in SEQ ID NO:51, and a CD38-LCDR2 as set forth in SEQ ID NO:52, CD38-LCDR3;

preferably, the method comprises the steps of,

(i) The BCMA-VH is shown in SEQ ID NO:29, said BCMA-VL is as shown in SEQ ID NO:32, said CD3-VH is set forth in SEQ ID NO:65, said CD3-VL is set forth as SEQ ID NO:64, said CD38-VH is set forth in SEQ ID NO:53 and said CD38-VL is set forth in SEQ ID NO: indicated at 54; or (b)

(ii) The BCMA-VH is shown in SEQ ID NO:36, wherein the BCMA-VL is as shown in SEQ ID NO:40, said CD3-VH is set forth in SEQ ID NO:63, said CD3-VL is set forth in SEQ ID NO:64, said CD38-VH is set forth in SEQ ID NO:53 and said CD38-VL is set forth in SEQ ID NO: indicated at 54.
The antigen binding molecule of any one of claims 1 to 12, comprising an Fc region, preferably an IgG Fc region, more preferably an IgG ₁ An Fc region;

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

More preferably, the amino acid substitution reduces binding to fcγ receptor;

in particular, the Fc region is a human IgG ₁ The Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index.
The antigen binding molecule of any one of claims 4 to 13, comprising an Fc region comprising a first subunit Fc1 and a second subunit Fc2 capable of associating, each of the Fc1 and Fc2 independently having one or more amino acid substitutions that reduce homodimerization of the Fc region;

preferably, the method comprises the steps of,

the Fc1 has a convex structure according to a pestle-and-mortar technique, and the Fc2 has a hole structure according to a pestle-and-mortar technique;

more preferably, the process is carried out,

the amino acid residue at position 366 of said Fc1 is W; and the amino acid residue at position 366, the amino acid residue at position 368, and the amino acid residue at position 407 of said Fc2 are S, a, and V, respectively, according to the EU index.
The antigen binding molecule of claim 1,

the antigen binding molecule has: two comprise SEQ ID NOs: 76 and two strands comprising the amino acid sequence of SEQ ID NO:74, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 75 and two strands comprising the amino acid sequence of SEQ ID NO:74, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 82 and two strands comprising the amino acid sequence of SEQ ID NO:81, and a second strand of the amino acid sequence of 81; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 83 and two strands comprising the amino acid sequence of SEQ ID NO:81, and a second strand of the amino acid sequence of 81; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 90 and two strands comprising the amino acid sequence of SEQ ID NO:89, a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 91 and two strands comprising the amino acid sequence of SEQ ID NO:89, a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 77, two strands comprising the amino acid sequence of SEQ ID NO:71, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 78, two strands comprising the amino acid sequence of SEQ ID NO:71, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 85, two strands comprising the amino acid sequence of SEQ ID NO:71, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 86, two strands comprising the amino acid sequence of SEQ ID NO:71, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 93, two strands comprising the amino acid sequence of SEQ ID NO:71, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 94, two strands comprising the amino acid sequence of SEQ ID NO:71, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:73, a first strand comprising the amino acid sequence of SEQ ID NO:74 and a second strand comprising the amino acid sequence of SEQ ID NO:66, a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:73, a first strand comprising the amino acid sequence of SEQ ID NO:74 and a second strand comprising the amino acid sequence of SEQ ID NO:67, and a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:80, 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:66, a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:80, 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:67, and a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:88, 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:66, a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:88, 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:67, and a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:84, 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:69, a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:84, 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:70, and a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:92, 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:69, a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:92, 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:70, and a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:79, two strands comprising the amino acid sequence of SEQ ID NO:71 and a second strand comprising the amino acid sequence of SEQ ID NO:72, and a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:87, two strands comprising the amino acid sequence of SEQ ID NO:71 and a second strand comprising the amino acid sequence of SEQ ID NO:72, and a third strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: a polypeptide comprising SEQ ID NO:95, two strands comprising the amino acid sequence of SEQ ID NO:71 and a second strand comprising the amino acid sequence of SEQ ID NO:72, and a third strand of an amino acid sequence of seq id no;

Preferably, the method comprises the steps of,

the antigen binding molecule has: two comprise SEQ ID NOs: 76 and two strands comprising the amino acid sequence of SEQ ID NO:74, and a second strand of an amino acid sequence of seq id no; or (b)

The antigen binding molecule has: two comprise SEQ ID NOs: 82 and two strands comprising the amino acid sequence of SEQ ID NO:81, and a second strand of the amino acid sequence of 81.
A pharmaceutical composition comprising:

a therapeutically effective amount of the antigen binding molecule of any one of claims 1 to 15, 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 15.
A host cell comprising the isolated nucleic acid of claim 17.
A method of treating a disease, the method comprising the step of administering to a subject the antigen binding molecule of any one of claims 1 to 15 or the pharmaceutical composition of claim 16;

preferably, the disease is a hematopoietic tumor of lymphoid or myeloid lineage or an autoimmune disease;

More preferably, the hematopoietic tumor of lymphoid or myeloid lineage is selected from: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, fahrenheit macroglobulinemia, isolated bone plasmacytoma, extramedullary plasmacytoma, bone sclerosis myeloma, heavy chain disease, meaningless monoclonal gammaglobinopathy, and stasis type multiple myeloma, B-cell lymphoma, burkitt's lymphoma, hodgkin's lymphoma, and hairy cell lymphoma; the autoimmune disease is selected from: rheumatoid arthritis, systemic lupus erythematosus, asthma, inflammatory bowel disease, multiple sclerosis, crohn's disease, gastritis, hashimoto thyroiditis, ankylosing spondylitis, and graft versus host disease.