TW202325743A - Antigen-binding molecule that specifically binds to cd38, bcma and cd3 and medical uses thereof - Google Patents

Abstract

The present disclosure relates to antigen-binding molecule that specifically binds to CD38, BCMA, and CD3, and medicinal uses thereof.

Description

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

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

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

Multiple myeloma (MM) is the second most common blood cancer in the world. It is mainly characterized by the uncontrolled proliferation of plasma cells in the bone marrow. Cancerous plasma cells rapidly proliferate and spread, leading to the massive production of individual immunoglobulins. , thereby causing immunosuppression, osteolysis and end-organ damage. Globally, there are more than 138,500 newly diagnosed MM patients. In the past few decades, the emergence of new treatments such as proteasome inhibitors, immunomodulators, and CD38 antibodies has greatly improved the clinical treatment effects of MM patients, increasing the patient's life expectancy from 3 to 4 years to 7 years. to 8 years. However, most patients will still relapse due to drug resistance. Even patients with negative MRD (minimal residual disease) will still relapse. Therefore, there is an urgent clinical need. New, more effective treatments are needed. Immunotherapy that reshapes the anti-tumor activity of immune cells while targeting MM cells will be an excellent therapy for the treatment of MM.

CD38 is a type II transmembrane glycoprotein that not only interacts with the ligand CD31 to transmit downstream signals to regulate lymphocyte activation, B cell differentiation and other processes, but also functions through the action of exonucleases. CD38 is widely expressed in plasma cells and multiple myeloma cells, and the CD38 monoclonal antibodies Daratumumab and Isatuximab have good clinical response rates. As well as the depth of response, it fully confirms that CD38 is a better target for multiple myeloma.

BCMA (Tumor necrosis factor receptor superfamily member 17) is a type III transmembrane protein without a signal peptide and is a member of the TNFR protein superfamily. BCMA activates downstream signaling pathways such as ERK1/2 and NFkB to promote the growth and proliferation of B cells by binding to its ligands APRIL and BAFF. In normal tissue cells, BCMA is mainly expressed in the later stages of B cell differentiation, that is, more mature B cells (Plasmablast, PC), and is not expressed in early development B cells and other normal tissues. BCMA is highly selectively expressed on the surface of multiple myeloma cells, and the expression level is much higher than that in normal B cells. Moreover, the proximal membrane end of BCMA will be cleaved by γ-secretase to form soluble BCMA (sBCMA). The level of sBCMA in plasma is significantly higher in patients with multiple myeloma and relapsed myeloma than in healthy humans. It has been regarded as Important markers of disease progression in multiple myeloma.

Bispecific antibodies targeting CD3&TAA are a new type of immunotherapy that can combine with T cells and tumor cells at the same time, simulating the interaction between MHC and TCR, causing T cells to form lytic synapse and then release perforin and granzyme for specific killing. tumor cells. Activated T cells can release cytokines, activate other immune cells and expand the immune response against tumors, ultimately leading to the proliferation of T cells and a cascade reaction that kills tumor cells.

The present disclosure provides an antigen-binding molecule comprising at least one antigen-binding module that specifically binds BCMA, at least one antigen-binding module that specifically binds CD38, and at least one antigen-binding module that specifically binds CD3. These antigen-binding molecules can provide better therapeutic activity than BCMA/CD3 antibodies or CD38/CD3 antibodies, and cover a wider range of multiple myeloma patient groups.

In some embodiments, the antigen-binding molecule as described above, wherein the antigen-binding module refers to an antibody fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region and the light chain variable region Together they form a domain that binds the target antigen (BCMA, CD38 or CD3). In some embodiments, the antigen binding module is a Fab or scFv.

In one aspect, the disclosure provides an antigen-binding molecule comprising two antigen-binding modules that specifically bind BCMA, two antigen-binding modules that specifically bind CD38, and two antigen-binding modules that specifically bind CD3. and Fc area.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the antigen-binding module that specifically binds BCMA is BCMA-Fab, the antigen-binding module that specifically binds CD3 is CD3-scFv and the antigen-binding module that specifically binds CD38 The antigen-binding module is CD38-scFv. BCMA-Fab in this disclosure means that the antigen-binding module has a Fab structure, and CD3-scFv and CD38-scFv means that the antigen-binding module has a scFv structure.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the antigen-binding module that specifically binds CD38 is CD38-Fab, the antigen-binding module that specifically binds CD3 is CD3-scFv and the antigen-binding module that specifically binds BCMA The antigen-binding module is BCMA-scFv. CD38-Fab in this disclosure means that the antigen-binding module has a Fab structure, and CD3-scFv and BCMA-scFv mean that the antigen-binding module has a scFv structure.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the antigen-binding module that specifically binds BCMA is BCMA-Fab, the antigen-binding module that specifically binds CD3 is CD3-scFv and the antigen-binding module that specifically binds CD38 The antigen-binding module is CD38-scFv; and a heavy chain of BCMA-Fab, a CD3-scFv, a subunit of the Fc region and a CD38-scFv are connected directly or through a linker in order from N-terminus to C-terminus. . 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 expressed in N-terminal to C-terminal order, respectively, directly or via a linker. connection. In specific embodiments, the antigen-binding molecule has a structure as shown in Figure IB.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the antigen-binding module that specifically binds CD38 is CD38-Fab, the antigen-binding module that specifically binds CD3 is CD3-scFv and the antigen-binding module that specifically binds BCMA The antigen-binding module is BCMA-scFv; and a heavy chain of CD38-Fab, a CD3-scFv, a subunit of the Fc region and a BCMA-scFv are connected directly or through a linker in the order from 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 expressed in N-terminal to C-terminal order, respectively, directly or via a linker. connection. In specific embodiments, the antigen-binding molecule has a structure as shown in Figure ID.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the antigen-binding module that specifically binds to BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, and the antigen-binding module that specifically binds to CD3 The antigen-binding module includes the heavy chain variable region CD3-VH and the light chain variable region CD3-VL, and the antigen-binding module that specifically binds CD38 includes the heavy chain variable region CD38-VH and the light chain variable region CD38- VL; where,

The antigen-binding molecule includes 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]-[a subunit of the 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 the same 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 shown in SEQ ID NO: 99, and the amino acid sequence of linker 2 is as shown in SEQ ID NO: 96 shown.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the antigen-binding module that specifically binds to BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, and the antigen-binding module that specifically binds to CD3 The antigen-binding module includes the heavy chain variable region CD3-VH and the light chain variable region CD3-VL, and the antigen-binding module that specifically binds CD38 includes the heavy chain variable region CD38-VH and the light chain variable region CD38- VL; where,

The antigen-binding molecule includes 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]-[a subunit of the Fc region]-[linker 3 ]-[BCMA-VH]-[Connector 4]-[BCMA-VL],

(d)[CD38-VL]-[CL],

Linker 1, linker 2, linker 3 and linker 4 in formula (c) are the same 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 connection in formula (c) The amino acid sequences of linker 1, linker 3 and linker 4 are shown in SEQ ID NO: 99, and the amino acid sequence of linker 2 is shown in SEQ ID NO: 96.

In one aspect, the present disclosure provides an antigen-binding molecule comprising an antigen-binding module that specifically binds BCMA, two antigen-binding modules that specifically bind CD38, an antigen-binding module that specifically binds CD3, and Fc district.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the antigen-binding module that specifically binds BCMA is BCMA-Fab, the antigen-binding module that specifically binds CD3 is CD3-scFv and the antigen-binding module that specifically binds CD38 The antigen-binding module is CD38-scFv. In specific embodiments, the antigen-binding molecule has a structure as shown in Figure 1A.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the antigen-binding module that specifically binds CD38 is CD38-Fab, the antigen-binding module that specifically binds CD3 is CD3-scFv and the antigen-binding module that specifically binds BCMA The antigen-binding module is BCMA-scFv. In specific embodiments, the antigen-binding molecule has a structure as shown in Figure IE.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, 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 reduction Amino acid substitution for homodimerization of the Fc region; and, the antigen-binding module that specifically binds to BCMA is BCMA-Fab, the antigen-binding module that specifically binds to CD3 is CD3-scFv, and the antigen that specifically binds to CD38 The binding module is CD38-scFv; and the heavy chain of BCMA-Fab, Fc1 and a CD38-scFv are directly or connected to CD3-scFv, Fc2 and another CD38-scFv in order from N terminus to C terminus, respectively, directly or through a linker They are connected directly or through linkers in the order from N end to C end.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, 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 reduction Amino acid substitutions for homodimerization of the Fc region; and, the specific binding The antigen-binding module that specifically binds CD38 is CD38-Fab, the antigen-binding module that specifically binds CD3 is CD3-scFv, and the antigen-binding module that specifically binds BCMA is BCMA-scFv; and the heavy chain, Fc1, and Fc1 of a CD38-Fab CD3-scFv is connected directly or through a linker in the order from N-terminus to C-terminus, and BCMA-scFv, the heavy chain of another CD38-Fab and Fc2 are connected in order from N-terminus to C-terminus directly or through a linker. connection.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the antigen-binding module that specifically binds to BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, and the antigen-binding module that specifically binds to CD3 The antigen-binding module includes the heavy chain variable region CD3-VH and the light chain variable region CD3-VL, and the antigen-binding module that specifically binds CD38 includes the heavy chain variable region CD38-VH and the light chain variable region CD38- VL; where,

The antigen-binding molecule includes 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 of 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 amines in length acid residues. In some embodiments, the amino acid sequences of linker 1 and linker 2 in formula (e) are as shown in SEQ ID NO: 99, and the linker in formula (f) The amino acid sequences of linker 1, linker 3 and linker 4 are shown in SEQ ID NO: 99, and the amino acid sequence of linker 2 in formula (f) is shown in SEQ ID NO: 96.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the antigen-binding module that specifically binds to BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, and the antigen-binding module that specifically binds to CD3 The antigen-binding module includes the heavy chain variable region CD3-VH and the light chain variable region CD3-VL, and the antigen-binding module that specifically binds CD38 includes the heavy chain variable region CD38-VH and the light chain variable region CD38- VL; where,

The antigen-binding molecule includes 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 sequence of linker 1 and linker 2 in formula (g) is as shown in SEQ ID NO: 99, and the amino acid sequence of linker 1 in formula (h) is as shown in SEQ ID It is shown in NO: 97, and the amino acid sequence of linker 2 in formula (h) is shown in SEQ ID NO: 99.

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

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the antigen-binding module that specifically binds BCMA is BCMA-Fab, the antigen-binding module that specifically binds CD3 is CD3-scFv and the antigen-binding module that specifically binds CD38 The antigen-binding module is CD38-scFv.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, 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 reduction Amino acid substitution for homodimerization of the Fc region; and CD38-scFv, CD3-scFv and Fc1 are directly or connected to the heavy chain and Fc2 of BCMA-Fab in sequence from N-terminus to C-terminus, respectively. The sequences from N-terminus to C-terminus are connected directly or through linkers respectively.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the antigen-binding module that specifically binds to BCMA comprises a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, and the antigen-binding module that specifically binds to CD3 The antigen-binding module includes the heavy chain variable region CD3-VH and the light chain variable region CD3-VL, and the antigen-binding module that specifically binds CD38 includes the heavy chain variable region CD38-VH and the light chain variable region CD38- VL; where,

The antigen-binding molecule includes 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]-[Connexon 1]-[CD38-VL]-[Connexon 2]-[CD3-VH]-[Connexon 3]-[CD3-VL]-[Connexon 4] -[Fc1],

Among them, linker 1, linker 2, linker 3 and linker 4 in formula (j) 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, Linker 3 and Linker 4 in Formula (j) are 3-15 amino acid residues in length. In some embodiments, the amino acid sequence of linker 1 and linker 3 in formula (j) is as shown in SEQ ID NO: 99, and the amino acid sequence of linker 2 in formula (j) is as shown in SEQ ID It is shown in NO: 97, and the amino acid sequence of linker 4 in formula (j) is shown in SEQ ID NO: 96.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, 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 includes the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 23, and the BCMA-VL includes the BCMA-LCDR1, BCMA in SEQ ID NO: 24 -Amino acid sequences of LCDR2 and BCMA-LCDR3; or

(ii) The BCMA-VH includes the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 25, and the BCMA-VL includes the BCMA-LCDR1, BCMA in SEQ ID NO: 26 -Amino acid sequences of LCDR2 and BCMA-LCDR3; or

(iii) The BCMA-VH includes the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 27, and the BCMA-VL includes the BCMA-LCDR1, BCMA in SEQ ID NO: 28 -Amino acid sequences of LCDR2 and BCMA-LCDR3.

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

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

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

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

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

In some embodiments, the BCMA-HCDR1, BCMA-HCDR2, BCMA-HCDR3, BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 are defined according to the Kabat numbering rule as described above.

In some embodiments, the antigen-binding molecule as described above, wherein,

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

In some embodiments, the antigen-binding molecule as described above, wherein,

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

In some embodiments, the antigen-binding molecule as described above, wherein,

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

In some embodiments, the antigen-binding molecule as described above, wherein,

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

In some embodiments, the antigen-binding molecule as described above, wherein,

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

In some embodiments, the antigen-binding molecule as described above, wherein,

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

In some embodiments, the antigen-binding molecule as described above, wherein,

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

In some embodiments, the antigen-binding molecule as described above, wherein,

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

In some embodiments, the antigen-binding molecule as described above, wherein,

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

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

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

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

In some embodiments, the antigen-binding molecule as described above, the CD3-HCDR1, CD3-HCDR2, CD3-HCDR3, CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3 are according to Kabat, IMGT, Chothia, AbM or Contact Numbering rules defined.

In some embodiments, the antigen-binding molecule as described above, wherein,

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

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

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

In some embodiments, the antigen-binding molecule as described above, wherein,

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

In some embodiments, the antigen-binding molecule as described above, wherein,

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

In some embodiments, the antigen-binding molecule as described above, the antigen-binding module that specifically binds to CD38 includes a heavy chain variable region CD38-VH and a light chain variable region CD38-VL, wherein in the CD38-VH CD38-HCDR1, CD38-HCDR2 and CD38-HCDR3 respectively comprise the amino acid sequences of CD38-HCDR1, CD38-HCDR2 and CD38-HCDR3 in SEQ ID NO: 53, and the CD38-LCDR1, CD38- in the CD38-VL LCDR2 and CD38-LCDR3 respectively comprise the amino acid sequences of CD38-LCDR1, CD38-LCDR2 and CD38-LCDR3 in SEQ ID NO: 54. 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 antigen-binding molecule as described above, the CD38-VH has: CD38-HCDR1 comprising the amino acid sequence of SEQ ID NO: 47, CD38 comprising the amino acid sequence of SEQ ID NO: 48 - HCDR2, and CD38-HCDR3 comprising the amino acid sequence of SEQ ID NO: 49; and the CD38-VL has: CD38-LCDR1 comprising the amino acid sequence of SEQ ID NO: 50, and CD38-LCDR3 comprising the amino acid sequence of SEQ ID NO: 51 CD38-LCDR2 of the amino acid sequence, and CD38-LCDR3 comprising the amino acid sequence of SEQ ID NO:52. In some embodiments, the CD38-HCDR1, CD38-HCDR2, CD38-HCDR3, CD38-LCDR1, CD38-LCDR2, and CD38-LCDR3 are defined according to the Kabat numbering rule.

In some embodiments, the antigen-binding molecule as described above, wherein,

The CD38-VH comprises an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 53, and the CD38-VL comprises an amino acid sequence identical to SEQ ID NO: 53 : 54 Amino acid sequences having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the antigen binding moiety that specifically binds CD38 is functionally equivalent to an antigen binding moiety comprising SEQ ID NO: 53 and SEQ ID NO: 54. In some embodiments, the CD38-VH comprises the amino acid sequence of SEQ ID NO:53, and the CD38-VL comprises the amino acid sequence of SEQ ID NO:54.

In some embodiments, the linker of the antigen-binding molecule of any one of the preceding items is a peptide linker. In some embodiments, the peptide linker is 3-15 amino acid residues in length. In some embodiments, the peptide linker each independently has an amino acid sequence represented by L ₁ -(GGGGS) _n -L ₂ , 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 GGGG (SEQ ID NO: 103), and the Connectors are not keys. In some embodiments, the peptide linker sequence is 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 items comprises an Fc region. In some embodiments, the Fc region is an IgG Fc region, preferably an _IgG1 Fc region. In some embodiments, the Fc region contains 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 Fcγ receptors. In some embodiments, the Fc region is a human _IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. In some embodiments, the Fcl has the amino acid sequence of SEQ ID NO: 68.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, comprising an Fc region, the 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 Fcl and Fc2 each independently have one or more amino acid substitutions according to the pestle and mortar technique. In some embodiments, the Fc1 has a convex structure according to the pestle and mortar technology, and the Fc2 has a pore structure according to the pestle and mortar technology. In some embodiments, the amino acid residue of the Fc1 at position 366 is W; and the amino acid residue of the Fc2 at position 366 is S, the amino acid residue at position 368 is A, and the amino acid residue at position 407 The amino acid residue at the position is V, and the numbering is based on the EU index. In some embodiments, the Fc1 has the amino acid sequence of SEQ ID NO:100, and the Fc2 has the amino acid sequence of SEQ ID NO:101.

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

The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 76 and two second chains comprising the amino acid sequence of SEQ ID NO: 74; or

The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 75 and two second chains comprising the amino acid sequence of SEQ ID NO: 74; or

The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 82 and two second chains comprising the amino acid sequence of SEQ ID NO: 81; or

The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 83 and two second chains comprising the amino acid sequence of SEQ ID NO: 81; or

The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 90 and two second chains comprising the amino acid sequence of SEQ ID NO: 89; or

The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 91 and two second chains comprising the amino acid sequence of SEQ ID NO: 89; or

The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 77, two second chains comprising the amino acid sequence of SEQ ID NO: 71; or

The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 78, and two second chains comprising the amino acid sequence of SEQ ID NO: 71; or

The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 85, and two second chains comprising the amino acid sequence of SEQ ID NO: 71; or

The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 86, and two second chains comprising the amino acid sequence of SEQ ID NO: 71; or

The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 93, and two second chains comprising the amino acid sequence of SEQ ID NO: 71; or

The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 94, and two second chains comprising the amino acid sequence of SEQ ID NO: 71; or

The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO:73, a second chain including the amino acid sequence of SEQ ID NO:74, and an amino group including SEQ ID NO:66 the third strand of the acid sequence; or

The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO:73, a second chain including the amino acid sequence of SEQ ID NO:74, and an amino group including SEQ ID NO:67 the third strand of the acid sequence; or

The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 80, a second chain including the amino acid sequence of SEQ ID NO: 81, and an amino group including SEQ ID NO: 66 the third strand of the acid sequence; or

The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 80, a second chain including the amino acid sequence of SEQ ID NO: 81, and an amino group including SEQ ID NO: 67 the third strand of the acid sequence; or

The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 88, a second chain including the amino acid sequence of SEQ ID NO: 89, and an amino group including SEQ ID NO: 66 the third strand of the acid sequence; or

The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 88, a second chain including the amino acid sequence of SEQ ID NO: 89, and an amino group including SEQ ID NO: 67 the third strand of the acid sequence; or

The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 84, a second chain including the amino acid sequence of SEQ ID NO: 81, and an amino group including SEQ ID NO: 69 the third strand of the acid sequence; or

The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 84, a second chain including the amino acid sequence of SEQ ID NO: 81, and an amino group including SEQ ID NO: 70 the third strand of the acid sequence; or

The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 92, a second chain including the amino acid sequence of SEQ ID NO: 89, and an amino group including SEQ ID NO: 69 the third strand of the acid sequence; or

The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 92, a second chain including the amino acid sequence of SEQ ID NO: 89, and an amino group including SEQ ID NO: 70 the third strand of the acid sequence; or

The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO:79, two second chains including the amino acid sequence of SEQ ID NO:71 and an amine including SEQ ID NO:72 The third strand of the amino acid sequence; or

The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 87, two second chains including the amino acid sequence of SEQ ID NO: 71, and an amine including SEQ ID NO: 72 The third strand of the amino acid sequence; or

The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 95, two second chains including the amino acid sequence of SEQ ID NO: 71, and an amine including SEQ ID NO: 72 The third strand of the amino acid sequence.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items has: two first chains comprising the amino acid sequence of SEQ ID NO: 76 and two first chains comprising the amino acid sequence of SEQ ID NO: 74 The second strand of the sequence.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items has: two first chains comprising the amino acid sequence of SEQ ID NO: 82 and two first chains comprising the amino acid sequence of SEQ ID NO: 81 The second strand of the sequence.

In another aspect, the present disclosure provides a pharmaceutical composition, which contains: a therapeutically effective amount of the antigen-binding molecule described in any of the preceding items, and one or more pharmaceutically acceptable carriers, diluents, and buffers or excipients. In some embodiments, the pharmaceutical composition further includes at least one second therapeutic agent.

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

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

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

In another aspect, the present disclosure also provides the use of any of the aforementioned antigen-binding molecules or compositions in the preparation of medicaments for treating or preventing diseases.

In another aspect, the present disclosure also provides the antigen-binding molecule or composition of any of the preceding for use as a medicament. In some embodiments, the drug is used to treat a disease.

In some embodiments, the disease is a hematopoietic tumor or autoimmune disease of the lymphoid or myeloid lineage. In some embodiments, the hematopoietic tumor of lymphoid lineage or myeloid lineage is selected from: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenstrom's macroglobulinemia, isolated Bone plasmacytoma, extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain disease, monoclonal gammapathies of undetermined significance, smoldering multiple myeloma, B-cell lymphoma, Burkitt 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's thyroiditis, ankylosing spondylitis, and graft versus host disease.

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

Figure 1A: Schematic structural diagram of Format5; Figure 1B: Schematic structural diagram of Format12; Figure 1C: Schematic structural diagram of Format20; Figure 1D: Schematic structural diagram of Format21; Figure 1E: Schematic structural diagram of Format24.

Figure 2: Ability of antibodies to bind to RPMI-8226 cells.

Figure 3: Cytotoxic activity of antibodies against CHO K1 cells.

Terminology

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

Unless the context clearly requires otherwise, throughout the specification and claims, the words "include," "have," "includes," and the like are to be understood in an inclusive sense and not in an exclusive or exhaustive sense; that is, " including but not limited to”. Unless otherwise stated, "comprising" includes "consisting of." For example, for BCMA-HCDR1 that includes the amino acid sequence of SEQ ID NO:5, it clearly includes BCMA-HCDR1 whose amino acid sequence is as shown in SEQ ID NO:5.

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

The term "and/or", such as "X and/or Y" should be understood to mean "X and Y" or "X or Y" and should be used to provide clear 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 mimetics 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 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 naturally occurring amino acids (i.e., alpha carbon bonded to hydrogen, carboxyl, amine and R groups), such as homoserine, norleucine, Methionine trisulfide, methionine methylthionine. Such analogs have modified R groups (eg, norleucine) or modified peptide backbones but retain the same basic chemical structure as the naturally occurring amino acid. Amino acid mimetics Refers to chemical compounds that have a structure that is different from the general chemical structure of amino acids, but act 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 can be made to achieve the final construct, as long as the final construct possesses the desired properties, such as reduction or binding to Fc receptors. Amino acid sequence deletions and insertions include deletions and insertions at the amino terminus and/or carboxyl terminus of the polypeptide chain. Specific amino acid mutations may be amino acid substitutions. In one embodiment, the amino acid mutation is a non-conservative amino acid substitution, ie, one amino acid is replaced with another amino acid with different structural and/or chemical properties. Amino acid substitutions include non-naturally occurring amino acids or derivatives of 20 natural amino acids (e.g., 4-hydroxyproline, 3-methylhistidine, ornithine, homoserine, 5 -Hydroxylysine) replacement. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is anticipated that methods other than genetic engineering to alter amino acid side chain groups, such as chemical modification, may also be available. Various names may be used herein to refer to the same amino acid mutation. Herein, the amino acid residue at a specific position can be represented by position + amino acid residue. For example, 366W means that the amino acid residue at position 366 is W. T366W means that the amino acid residue at position 366 has been mutated from the original T to W.

The term "antigen-binding molecule" is used in the broadest sense and covers various molecules that specifically bind to antigens, including but not limited to antibodies, other polypeptides with antigen-binding activity, and antibody fusion proteins formed by the fusion of the two. Exemplarily, the antigen-binding molecule herein is a trispecific antigen-binding molecule (eg, trispecific antibody). 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 encompasses 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), as long as they exhibit the desired antigen-binding activity. "sky "Native antibody" refers to a naturally occurring immunoglobulin molecule. For example, a natural IgG antibody is a heterotetrameric protein of approximately 150,000 daltons consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From From N to C-terminal, each heavy chain has a variable region (VH), also known as variable heavy domain, heavy chain variable region, followed by three constant domains (CH1, CH2 and CH3). Similarly, from From N to C-terminus, each light chain has a variable region (VL), also called a variable light domain, or a 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 domain of an antigen-binding molecule that binds the antigen. In this article, the heavy chain variable region in the antigen-binding module that specifically binds to BCMA is labeled BCMA-VH, and the light chain variable region is labeled BCMA-VL; the heavy chain variable region in the antigen-binding module that specifically binds to CD38 is labeled BCMA-VH. It is labeled as CD38-VH, and the light chain variable region is labeled as CD38-VL; the heavy chain variable region in the antigen-binding module that specifically binds to CD3 is labeled as CD3-VH, and the light chain variable region is labeled as CD3-VL. VH and VL each contain four conserved framework regions (FR) and three complementarity determining regions (CDR). Among them, the term "complementarity determining region" or "CDR" refers to the region within the variable domain that mainly contributes to binding to the antigen; "framework" or "FR" refers to the variable domain residues other than CDR residues. VH contains 3 CDR areas: HCDR1, HCDR2 and HCDR3; VL contains 3 CDR areas: LCDR1, LCDR2 and LCDR3. In this article, the three CDR regions in BCMA-VH are labeled BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 respectively; the three CDR regions in BCMA-VL are labeled BCMA-LCDR1, BCMA-LCDR2 and BCMA-LCDR3 respectively. ; The three CDR regions in CD38-VH are labeled CD38-HCDR1, CD38-HCDR2 and CD38-HCDR3 respectively; the three CDR regions in CD38-VL are labeled respectively CD38-LCDR1, CD38-LCDR2 and CD38-LCDR3; CD3 The three CDR regions in -VH are labeled CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 respectively; the three CDR regions in CD3-VL are labeled respectively CD3-LCDR1, CD3-LCDR2 and CD3-LCDR3. Each VH and VL consists of three elements arranged in the following order from the amine terminus to the carboxyl terminus. It consists of one CDR and four FRs: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A single VH or VL may be sufficient to confer antigen binding specificity.

The amino acid sequence boundaries of CDRs can be determined by various well-known schemes, such as: "Kabat" numbering rule (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th 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 rules (Lefranc, M.P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); Front Immunol. 2018 Oct 16; 9: 2278), etc.; the correspondence between various numbering systems is a technical field Well known to those with ordinary knowledge. The numbering convention for this disclosure is as shown in Table 1 below.

Unless otherwise stated, the variable region and CDR sequences in the embodiments of the present disclosure are all subject to the "Kabat" numbering rule.

The term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the 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 composed of immunoglobulin VH and CH1 (Fab heavy chain) and VL and CL (Fab light chain). In this disclosure, BCMA-Fab represents an antigen-binding module with a Fab structure that specifically binds to BCMA, BCMA-scFv represents an antigen-binding module with a scFv structure that specifically binds to BCMA, and so on.

The term "scFv" refers to a single chain antibody fragment (scFv), which consists of an antibody heavy chain variable region (VH) and a light chain variable region (VL) through a short peptide of 15 to 20 amino acids. Antibodies linked by linkers.

The term "Fc region" or "fragment crystallizable region" is used to define the C-terminal region of an antibody heavy chain, including native Fc regions and engineered Fc regions. In some embodiments, the Fc region contains two subunits that are the same or different. In some embodiments, the Fc region of a human IgG heavy chain is defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. Suitable native sequence Fc regions for use in the antibodies described herein include human IgGl, IgG2 (IgG2A, IgG2B), IgG3 and IgG4. Unless otherwise stated, the numbering convention for the Fc region is the EU index.

The term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a specific source or species and the remaining portion of the heavy and/or light chain is 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. This can be accomplished, for example, by retaining the non-human CDR regions and replacing the remainder of the antibody with their human counterparts (ie, the constant regions and the framework portion of the variable regions).

The term "affinity" refers to the overall strength of non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the internal binding affinity that reflects a 1:1 interaction between the members of a binding pair (eg, antibody and 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 "kassoc" or "ka" refers to the association rate of a particular antibody-antigen interaction, while the term "kdis" or "kd" as used herein is intended to refer to the dissociation rate 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 value of the antibody can be determined using methods known in the art. For example, methods for determining the KD of the antibody include using a biosensing system such as a system to measure surface plasmon resonance, or measuring in solution by solution equilibrium titration (SET). affinity.

The terms "capable of specifically binding", "specifically binding" or "binding" refer to the ability of an antibody to bind with higher affinity to an antigen or an epitope within the antigen than to other antigens or epitopes. Typically, an antibody binds an antigen or an epitope within an antigen with an equilibrium dissociation constant (KD) of about 1×10 ⁻⁶ M or less (eg, about 1×10 ⁻⁷ M, about 1×10 ⁻⁸ M or less) . In some embodiments, the KD of the antibody binding to the antigen is 10% or less (eg, 1%) of the KD of the antibody binding to a non-specific antigen (eg, BSA, casein). KD can be measured using known methods, such as by FACS or ^BIACORE® surface plasmon resonance assay. However, antibodies that specifically bind to an antigen or an epitope within the antigen may be cross-reactive to other related antigens, e.g., to antibodies from other species (homologous) such as humans or monkeys, e.g., Macaca fascicularis Corresponding antigens from cynomolgus (cynomolgus, cyno), chimpanzees (Pan troglodytes) (chimpanzee, chimp)) or marmosets (Callithrix jacchus) (commonmarmoset, marmoset) are cross-reactive.

The term "antigen-binding module" refers to a polypeptide molecule that specifically binds a target antigen. Antigen binding modules include antibodies as defined herein and fragments thereof. Specific antigen-binding moieties include the antigen-binding domain of an antibody, which includes an antibody heavy chain variable region and an antibody light chain variable region. The term "antigen-binding module that specifically binds BCMA" refers to a module that is capable of binding BCMA with sufficient affinity. In certain embodiments, the antigen-binding module that specifically binds BCMA has the following equilibrium dissociation constant (KD): < about 1 μM, < about 100 nM, or < about 10 nM, as measured by FACS, surface plasmon resonance, etc. . In certain embodiments, an antigen-binding moiety that specifically binds BCMA binds to a conserved epitope in BCMA from different species. The term "antigen-binding module that specifically binds CD38" refers to a module that is capable of binding CD38 with sufficient affinity. In certain embodiments, the antigen-binding module that specifically binds CD38 has the following equilibrium dissociation constant (KD): < about 1 μM, < about 100 nM, or < about 10 nM, as measured by FACS, surface plasmon resonance, etc. . In certain embodiments, an antigen-binding module that specifically binds CD38 binds to a conserved epitope in CD38 from different species. The term "antigen-binding module that specifically binds CD3" refers to a module 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 constant (KD): < about 1 μM, < about 200 nM, < about 100 nM, and/or > 50 nM, as determined by FACS, surface Measured by plasma resonance and other methods. In certain embodiments, anti-CD3 antibodies bind to conserved epitopes in CD3 from different species. Antigen binding moieties include antibody fragments as defined herein, such as Fab or scFv.

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

The term "monoclonal antibody" refers to a population of antibodies that are substantially homogeneous, ie, the antibody molecules contained in the population are identical in amino acid sequence, except for natural mutations that may be present in minor amounts. In contrast, polyclonal antibody preparations typically contain multiple different antibodies with different amino acid sequences in their variable domains, often specific for different epitopes. "Single strain" refers to the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced 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 (eg, an antibody) and that is otherwise capable of being used in an animal to generate antibodies capable of binding the antigen. An antigen may have one or more epitopes capable of interacting with different antigen-binding proteins (eg, antibodies).

The term "linker" refers to a linking unit that joins two polypeptide fragments. In this article, linkers appearing in the same structural formula may be the same or different. The linker can be a peptide linker, which contains 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 structural formula, it means that the units on both sides are directly connected by covalent bonds. When the term "bond" appears in a structural unit, it means that the unit has no amino acids and the units on either side of the unit are directly connected.

The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages that are synthetic, naturally occurring and non-naturally occurring, have similar binding properties to the reference nucleic acid, and are produced in a manner similar to Metabolism in the manner of reference nucleotides. Examples of such analogs include, but are not limited to, phosphorothioates, aminophosphates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acids ( PNA). An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that typically contain The nucleic acid molecule exists extrachromosomally or at a chromosomal location different from its native chromosomal location. Isolated nucleic acid encoding the antigen-binding molecule refers to one or more nucleic acid molecules encoding antibody heavy and light chains (or fragments thereof), including such one or more nucleic acid molecules in a single vector or separate vectors , and such one or more nucleic acid molecules present at one or more locations in the host cell. Unless otherwise stated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences thereof as well as sequences explicitly indicated. Specifically, as detailed below, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected (or all) codons is mixed bases and/or deoxygenated. Inosine residue substitution.

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

The term sequence "identity" means that when two sequences are optimally aligned, gaps are introduced when necessary to achieve maximum percent sequence identity, and any conservative substitutions are not considered part of the sequence identity of the two sequences. The degree to which amino acids/nucleic acids of a sequence are identical at equivalent positions (percentage). To determine percent sequence identity, alignment can be performed by techniques known to those skilled in the art, for example using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. One of ordinary skill in the art can determine parameters suitable for measuring alignment, including any algorithms required to achieve maximal alignment over the entire length of the sequences being compared.

The term "fusion" or "linking" means that the components (eg, the antigen-binding module and the Fc domain) are covalently linked, either 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 circle into which additional DNA segments can be ligated. Another type of vector is a viral vector, such as an adeno-associated viral vector (AAV or AAV2), in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (eg, bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be introduced into the host cell and integrated into the host cell's genome, thereby replicating with the host genome. The term "expression vector" or "expression construct" refers to a vector suitable for transformation of a host cell and containing the direction and/or control of the expression of one or more heterologous coding regions (together with the host cell) operably linked thereto. Nucleic acid sequence vectors. Expression constructs may include, but are not limited to, sequences that affect or control transcription, translation, and, in the presence of introns, RNA splicing of the coding region operably linked thereto.

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 the primary transformed cell and progeny derived therefrom, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parent cells, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the original transformed cell are included herein. 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, porcine, 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 (eg, Hep G2), A549 cells, 3T3 cells, and HEK-293 cells. Fungal cells include yeast and filamentous fungal cells including, for example, Pichia pastoris (Pichia pastoris), Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta )(Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pupil Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia pastoris, Saccharomycescerevisiae, Saccharomyces cerevisiae, Hansenula polymorpha , Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens and Neurospora crassa. Pichia pastoris, any Saccharomyces spp., Hansenula polymorpha, any Kluyveromyces spp., Candida albicans, any Aspergillus spp., Trichoderma reesei, Luck Chrysosporium lucknowense, any species of Fusarium, Yarrowia lipolytica and Neurospora crassa.

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

"As appropriate" 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 does or does not occur.

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

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

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

"Administration" or "administration", when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, means the administration of an exogenous drug, therapeutic, diagnostic or composition to an animal, human , contact with subjects, cells, tissues, organs or biological fluids.

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 a subject. Exemplary samples are biological fluids such as blood, serum and serosal fluids, plasma, lymph fluid, urine, saliva, cyst fluid, tears, excreta, sputum, mucosal secretions of secretory tissues and organs, vaginal secretions, ascites, Fluid from the pleura, pericardium, peritoneum, peritoneal cavity and other body cavities, fluid collected from bronchial lavage fluid, Synovial fluid, liquid solutions in contact with subjects or biological sources, such as cell and organ culture media (including cell or organ conditioned media), lavage fluid, etc., tissue biopsy samples, fine needle aspiration, surgically resected tissue, organ culture or cell culture.

"Treatment" and "treatment" (and their grammatical variations) refer to clinical intervention that attempts to alter the natural course of the individual being treated, and may be performed for prevention or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, alleviating/reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and regressing or ameliorating the disease. Prognosis. In some embodiments, the antibodies of the present disclosure are used to delay the development of disease or slow the progression of disease.

An "effective amount" is generally sufficient to reduce the severity and/or frequency of symptoms, eliminate these symptoms and/or underlying causes, prevent the occurrence of symptoms and/or their underlying causes, and/or ameliorate or ameliorate impairments caused by or associated with a disease state. (e.g. lung disease). In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount. A "therapeutically effective amount" is one sufficient to treat a disease state or symptom, particularly a condition or symptom associated with that disease state, or to otherwise prevent, hinder, delay or reverse the disease state or any other adverse effect in any way related to the disease. The ideal amount of symptomatic progression. 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 associated symptoms. . 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. "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, as well as the ability of the therapeutic agent or combination of therapeutic agents to elicit the desired response in the individual. Exemplary indicators of an effective therapeutic agent or combination of therapeutic agents include, for example, improved health status of the patient.

Antigen binding molecules of the present disclosure

The present disclosure provides antigen-binding molecules that have many 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 druggability (such as yield, purity and stability, etc.).

Exemplary antigen binding molecules

In one aspect, the present disclosure provides an antigen-binding molecule comprising at least one 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 CD3. 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&CD3e. 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 10 nM and binds 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. For specific test methods, see Test Example 2.

c. In vitro specific killing activity against BCMA-expressing cells. 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 CD38. For specific test methods, see Test Example 4.

d. In vitro specific killing activity was not affected by soluble BCMA/CD38/APRIL. For specific test methods, see Test Example 5.

e. Induces low-level cytokine (IL-6 and IL-2) release. For specific test methods, see Test Example 6.

f. Greater therapeutic activity in vivo. For specific test methods, see 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 CD3. The antigen-binding module that specifically binds BCMA includes a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein,

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

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

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

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

In some embodiments, the antigen-binding molecule as described above, wherein,

The amino acid sequence of the heavy chain variable region BCMA-VH is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain variable region BCMA-VL is shown in SEQ ID NO: 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: 45,

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding module 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: CD3-HCDR1 with an amino acid sequence as shown in SEQ ID NO: 55, CD3-HCDR2 with an amino acid sequence as shown in SEQ ID NO: 56, and CD3-HCDR2 with an amino acid sequence as shown in SEQ ID NO. CD3-HCDR3 shown in NO: 57; and the CD3-VL has: CD3-LCDR1 whose amino acid sequence is shown in SEQ ID NO: 58, and CD3-LCDR2 whose amino acid sequence is shown in SEQ ID NO: 59 and CD3-LCDR3 with an amino acid sequence as shown in SEQ ID NO: 60; or

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

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

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, the amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO: 63, and the light chain variable region CD3-VL The amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO: 64; or the amino acid sequence of the heavy chain variable region CD3-VH is shown in SEQ ID NO: 65, and the amine of the light chain variable region CD3-VL The amino acid sequence is shown in SEQ ID NO: 64.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding module 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: CD38-HCDR1 with an amino acid sequence as shown in SEQ ID NO: 47, CD38-HCDR2 with an amino acid sequence as shown in SEQ ID NO: 48, and CD38-HCDR2 with an amino acid sequence as shown in SEQ ID NO: 49. CD38-HCDR3 as shown; and the CD38-VL has: CD38-LCDR1 with an amino acid sequence as shown in SEQ ID NO: 50, CD38-LCDR2 with an amino acid sequence as shown in SEQ ID NO: 51 and an amino group The acid sequence is CD38-LCDR3 shown in SEQ ID NO: 52;

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

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, 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 The amino acid sequence is shown in SEQ ID NO: 54.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein

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

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

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

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

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

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

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 Kabat numbering rules.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, 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 The amino acid sequence 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, and the amino acid sequence of the light chain variable region CD3-VL As shown in SEQ ID NO: 64, the amino acid sequence of the heavy chain variable region CD38-VH is as shown in SEQ ID NO: 53, and the amino acid sequence of the light chain variable region CD38-VL is as shown in SEQ ID NO:54 shown.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, 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 The amino acid sequence is shown in SEQ ID NO: 40, 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 As shown in SEQ ID NO: 64, the amino acid sequence of the heavy chain variable region CD38-VH is as shown in SEQ ID NO: 53, and the amino acid sequence of the light chain variable region CD38-VL is as shown in SEQ ID NO:54 shown.

In some embodiments, the antigen-binding molecule as described in any one of the preceding items, wherein the antigen-binding molecule further comprises an Fc region, the Fc region comprising two subunits capable of associating. In some embodiments, the amino acid sequence of the two subunits is shown in SEQ ID NO: 68. In some embodiments, the amino acid sequences of the two subunits are as shown in SEQ ID NO: 100 and SEQ ID NO: 101, respectively.

The structure of an antigen-binding molecule

The present disclosure provides a trispecific antigen-binding molecule, which may be trivalent, tetravalent, pentavalent, hexavalent, and more. The antigen-binding molecule includes an antigen-binding module, and the antigen-binding module can be any polypeptide molecule capable of binding to the target antigen, such as an antibody fragment. In some embodiments, the antigen-binding module refers to an antibody fragment comprising a heavy chain variable region and a light chain variable region that together constitute a domain that binds the target antigen. In some embodiments, the antigen binding module is a Fab or scFv. The antigen-binding modules in the antigen-binding molecules of the present disclosure can be connected in any manner that does not affect the binding activity. Exemplarily, the structural schematic diagram of the antigen-binding molecule is shown in Figure 1A, Figure 1B, Figure 1C, Figure 1D or Figure 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 the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions, and/or substitutions of residues within the amino acid sequence of the antigen-binding molecule. Any combination of deletions, insertions, and substitutions can be made to obtain the final construct, so long as the final construct possesses the desired characteristics, such as antigen-binding properties.

Substitute, insert, and delete variants

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

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

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

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

(3) Acidic: Asp, Glu;

(4) Basic: His, Lys, Arg;

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

(6) Aromatic: Trp, Tyr, Phe.

Non-conservative substitutions would require the substitution of a member of one of these classes for a member of another class.

One type of substitution variant involves the substitution of one or more CDR residues of a parent antibody (eg, a humanized or human antibody). Generally, the resulting variants selected for further study will have alterations (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent antibody, and/or will be substantially Retains certain biological properties of the parent antibody. One exemplary substitution variant is an affinity matured antibody, which may 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 antibodies are displayed on phage and screened for specific biological activity (e.g., binding affinity). Changes (eg substitutions) can be made to the CDRs, for example to improve antibody affinity. Such changes can be made to CDR "hotspots," residues encoded by codons that undergo mutations at high frequency during the somatic maturation process, and/or residues that contact the antigen, while simultaneously modifying the resulting variant VH or VL tests binding affinity. In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods, such as error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis. . Then, create secondary libraries. The library is then screened to identify any antibody variants with the desired affinity. Another way to introduce diversity involves methods of CDR orientation, in which several CDR residues (eg 4-6 residues at a time) are randomized. One can e.g. use alanine scanning mutagenesis or construction Models are used to specifically identify CDR residues involved in antigen binding. In particular, HCDR3 and LCDR3 are often targeted.

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

Amino acid sequence insertions include amino and/or carboxyl 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 the antibody with enzymes or polypeptides that extend the serum half-life of the antibody.

Renovation of Fc area

In one aspect, the Fc region of an antigen-binding molecule of the present disclosure contains one or more amino acid substitutions that reduce its binding to an Fc receptor, such as its binding to an Fcγ receptor, and reducing or eliminating effector functions. The native IgG Fc region, specifically the _IgG1 Fc region or the _IgG4 Fc region, may cause the antigen-binding molecules of the present disclosure to target cells expressing Fc receptors rather than cells expressing the antigen. The modified Fc region of the present disclosure exhibits reduced binding affinity for Fc receptors and/or reduced effector function. In some embodiments, the engineered Fc region has a binding affinity for the Fc receptor that is reduced by more than 50%, 80%, 90%, or 95% compared to the native Fc region. In some embodiments, the Fc receptor is an Fcγ receptor. In some embodiments, the Fc receptor is a human Fcγ receptor, such as FcγRI, FcγRIIa, FcγRIIB, FcγRIIIa. In some embodiments, the engineered Fc region also has reduced binding affinity for complement, such as Clq, compared to the native Fc region. In some embodiments, the engineered Fc region has no reduced binding affinity for the 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 immune complex-mediated antigen uptake by antigen-presenting cells, reduced interaction with NK cells Binding, reduced binding to macrophages, reduced binding to monocytes, reduced binding to polymorphonuclear cells, reduced direct signaling-induced apoptosis, reduced dendritic cell maturation, or reduced T cell priming. For the _IgG1 Fc region, substitution of amino acid residues at positions 238, 265, 269, 270, 297, 327, and 329 may reduce effector function. In some embodiments, the Fc region is a human _IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. For the IgG ₄ Fc region, substitution of amino acid residues at positions such as 228 may reduce effector function.

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

Antigen-binding molecules may contain different antigen-binding modules fused to the two subunits of the Fc region, thus potentially leading to undesirable homodimerization. In order to increase yield and purity, it would therefore be advantageous to introduce modifications that promote heterodimerization into the Fc region of the antigen-binding molecules of the invention. In some embodiments, the Fc region of the present disclosure includes modifications according to the knob-into-hole (KIH) technique, which involves introducing a knob at the interface of the first subunit and a knob at the interface of the second subunit. A hole structure (hole) is introduced at the interface of the base. This allows the protruding structure to be positioned in the pore structure, promoting the formation of heterodimers and inhibiting the production of homodimers. The raised structure is built by replacing small amino acid side chains from the interface of the first subunit with larger side chains, such as tyrosine or tryptophan. The pore structure is created in the interface of the second subunit by replacing large amino acid side chains with smaller amino acid side chains (such as alanine or threonine). The bulge and pore structures are prepared by altering the nucleic acid encoding the polypeptide. Optional amino acid substitutions are shown in the table below:

In some embodiments, the specific amino acid sequence of the 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 pestle and mortar technology, other techniques for modifying the CH3 domain of the heavy chain of multispecific antibodies to achieve heterodimerization are also known in the art, such as WO96/27011, WO98/050431, EP1870459, WO2007/ 110205, WO 007/147901, WO2009/089004, WO2010/129304, WO2011/90754, WO2011/143545, WO2012/058768, WO2013/157954 and WO 013/096291.

The C-terminus of the Fc region can be a complete C-terminus ending with the amino acid residue PGK; it can also be a shortened C-terminus, for example, one or two C-terminal amino acid residues have been removed from the shortened C-terminus. . In a preferred aspect, the C-terminus of the heavy chain is a shortened C-terminus ending in PG. Thus, in some embodiments, a composition of intact antibodies may include a population of antibodies with all K447 residues and/or G446+K447 residues removed. In some embodiments, the composition of intact antibodies can include a population of antibodies without removal of the K447 residue and/or G446+K447 residues. In some embodiments, the composition of intact antibodies has a population of antibodies with and without a K447 residue and/or an antibody mixture of G446+K447 residues.

Recombination method

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

In the case of natural antibodies, natural antibody fragments or bispecific antigen-binding molecules with homodimeric heavy chains, two nucleic acids are required, one for the light chain or fragments thereof and one for the heavy chain or fragments thereof. Such nucleic acids encode amino acid sequences comprising antibody VL and/or amino acid sequences comprising antibody VH (eg, light and/or heavy chains of an antibody). These nucleic acids can be on the same expression vector or on different expression vectors.

In the case of a bispecific antigen-binding molecule with a heterodimeric heavy chain, four nucleic acids are required, one for the first light chain and one for the first heavy chain comprising the first heterologous monomeric Fc region polypeptide, One for the second light chain and one for the second heavy chain comprising a second heterologous monomeric Fc region polypeptide. These four nucleic acids can be contained in one or more nucleic acid molecules or expression vectors, usually these nucleic acids are located on two or three expression vectors, that is, one vector can contain more than one of these nucleic acids.

In one embodiment, the present disclosure provides an isolated nucleic acid encoding an antigen-binding molecule as described above. Such nucleic acids may independently encode any of the aforementioned polypeptide chains. In another aspect, the present disclosure provides one or more vectors (eg, expression vectors) comprising such nucleic acids. In another aspect, the present disclosure provides host cells comprising such nucleic acids. In one embodiment, 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 under conditions suitable for expression of the antigen-binding molecule, as provided above, and viewing Desirably, the antigen-binding molecule is recovered from the host cell (or host cell culture medium).

To recombinantly produce an antigen-binding molecule, the nucleic acid encoding the protein is isolated and inserted into one or more vectors for further selection and/or expression in host cells. such nucleic acids Can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of antigen-binding molecules), or produced by recombinant methods or by chemical synthesis obtain.

Suitable host cells for colonization or expression of vectors encoding antigen-binding molecules include prokaryotic or eukaryotic cells described herein. For example, antigen-binding molecules can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required for the antigen-binding molecules. After expression, the antigen-binding molecules can be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable strains or expression hosts for vectors encoding antigen-binding molecules, including fungal and yeast strains whose glycosylation pathways have been "humanized", resulting in Antigen-binding molecules with partially or fully human glycosylation patterns are generated. Suitable host cells suitable for expressing (glycosylated) antigen-binding molecules 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 that can be used in combination with insect cells, especially for transfection of Spodoptera frugiperda cells; plant cell cultures can also be used as hosts, such as US5959177, US6040498, US6420548 , US7125978 and US6417429; vertebrate cells can also be used as hosts, such as mammalian cell lines adapted to growth in suspension. Other examples of suitable mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7); the human embryonic kidney line (293 or 293T cells); baby hamster kidney cells (BHK); mouse Sertoli ( sertoli) cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical cancer cells (HELA); canine kidney cells (MDCK); buffalo rat liver cells ( BRL3A); human lung cells (W138); human liver cells (Hep G2); mouse breast tumors (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells; and myeloma cell lines, such as Y0, NSO, and Sp2/0. For a review of certain mammalian host cell lines suitable for the production of antigen-binding molecules see, for example, Yazaki, P. and Wu, AM, Methods in Molecular Biology , Vol. 248, Lo, BKC (Eds.), Humana Press, Totowa, NJ ( 2004), pp. 255-268.

Immunoconjugate

The present disclosure also provides immunoconjugates comprising an antigen-binding molecule conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., bacteria, fungi) , proteinaceous toxins, enzymatically active toxins, or fragments thereof of plant or animal origin), or radioactive isotopes.

Diagnostic and therapeutic compositions

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

In one embodiment, antigen binding molecules for use in diagnostic or detection methods are provided. In yet another aspect, a method of detecting the presence of a target antigen in a biological sample is provided. In certain embodiments, the method includes contacting a biological sample with an antigen-binding molecule under appropriate conditions and detecting whether a complex is formed between the detection reagent and the antigen. Such methods may be in vitro or in vivo methods. In one embodiment, antigen binding molecules are used to select subjects suitable for treatment, for example BCMA, CD38 or CD3 are biomarkers used to select patients.

Exemplary conditions that can be diagnosed using the antigen-binding molecules of the present disclosure are, for example, B-cell disorders or autoimmune diseases, the B-cell disorders being selected from the group consisting of: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia , amyloidosis, Waldenstrom's macroglobulinemia, solitary plasmacytoma of bone, extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain disease, unknown significance monoclonal gammopathy and smoldering 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 detectable labels or moieties (such as fluorescent, chromogenic, electron dense, chemiluminescent, and radioactive labels), and indirect detection moieties (e.g., indirect detection via enzymatic reactions or molecular interactions). modules, such as enzymes or ligands).

In a further aspect, pharmaceutical compositions comprising the antigen-binding molecule are provided, for example, for use in any of the following methods of treatment. In one aspect, a pharmaceutical composition includes any of the antigen-binding molecules provided herein and a pharmaceutically acceptable carrier. In another aspect, a pharmaceutical composition includes any of the antigen-binding molecules provided herein and at least one additional therapeutic agent.

The pharmaceutical composition of the antigen-binding molecule disclosed in the present disclosure is prepared by mixing such antigen-binding molecule with the required purity and one or more pharmaceutically acceptable carriers as needed, and the pharmaceutical composition is lyophilized. in the form of compositions or aqueous solutions. Formulations for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile membrane.

Treatment methods and routes of administration

Any of the antigen-binding molecules provided herein can be used in therapeutic methods.

In yet another aspect, the present disclosure provides use of an antigen-binding molecule in the manufacture or preparation of a medicament. In one embodiment, the medicament is used to treat a B cell disorder or an autoimmune disease selected from: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenstrom's macroglobulinemia, solitary plasmacytoma of bone, extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain disease, monoclonal gammopathy of undetermined significance, and smoldering multiple myeloma; the self The immune disease is systemic lupus erythematosus. And the drug is present in an effective amount 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 includes An effective amount of at least one additional therapeutic agent (eg, one, two, three, four, five, or six additional therapeutic agents) is administered to the subject. A "subject" according to any of the above embodiments may be a human.

In yet another aspect, there is provided a pharmaceutical composition comprising the antigen-binding molecule, for example, for use in any of the above pharmaceutical uses or therapeutic methods. In another embodiment, the pharmaceutical composition further includes at least one additional therapeutic agent.

The antigen-binding molecules of the present disclosure can be used alone or in combination with other agents for treatment. For example, the antigen-binding molecules of the present disclosure can 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 parenterally, intrapulmonary, and intranasal, and, if local 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 the administration is short-term or long-term. Various dosing schedules are contemplated herein, including, but not limited to, single or multiple administrations at multiple time points, bolus administration, and pulse infusion.

The antigen-binding molecules 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 specific condition being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the agent, the method of administration, the timing of administration, and others known to the medical practitioner factor. The antigen-binding 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 condition 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 empirically/clinically determined to be appropriate.

To prevent or treat disease, appropriate dosages 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 administration is for prophylactic or therapeutic purposes, previous treatments, the patient's clinical history and response to the therapeutic molecule, and the judgment of the attending physician. The therapeutic molecules are appropriately administered to the patient in one session or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg of the antigen-binding molecule may be an initial candidate dose for administration to the patient, whether, for example, by one or more divided administrations or by continuous infusion. A typical daily dose may range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. Correspondingly, taking a body weight of 50kg as an example, the exemplary unit daily dose is 50μg-5g.

Products

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

Examples and test cases

The disclosure is further described below in conjunction with embodiments and test examples, but these embodiments and test examples do not limit the scope of the disclosure. Experimental methods without specifying specific conditions in the disclosed embodiments and test examples usually follow conventional conditions, such as Cold Spring Harbor's Antibody Technology Experiment Manual and Molecular Cloning Manual; or follow the conditions recommended by the raw material or product manufacturer. Reagents whose specific sources are not indicated are conventional reagents purchased in the market.

Example 1. Expression of human BCMA and cynomolgus monkey BCMA

The extracellular region sequences encoding human BCMA and cynomolgus monkey BCMA with human IgG1-Fc tag were inserted into the phr vector to construct expression plasmids, and then transfected into HEK293. The sequence encoding the extracellular region of human BCMA with His tag was inserted into the phr vector to construct an expression plasmid, and then transfected into HEK293. The specific transfection steps are: the day before, HEK293E cells were inoculated into freestyle expression medium (containing 1% FBS, Gibco, 12338-026) at 1×10 ⁶ /mL, placed in a 37-degree constant-temperature shaker (120 rpm) and continued to culture for 24 hours. . After 24 hours, sterilize the transfection plasmid and transfection reagent PEI with a 0.22 μm filter, and then adjust the transfection plasmid to 100 μg/100 mL cells. The mass ratio of PEI (1 mg/mL) and plasmid is 3:1. Take Mix 10 mL of Opti-MEM and 200 μg of plasmid and let stand for 5 minutes; mix another 10 mL of Opti-MEM and 400 μg of PEI and let stand for 5 minutes. Mix plasmid and PEI and let stand for 15 minutes. Slowly add the plasmid and PEI mixture to 200 mL of HEK293E cells, and culture in a shaker with 8% CO ₂ , 120 rpm, and 37°C. On the 3rd day of transfection, add 10% volume of feed medium (20mM glucose + 2mM L-glutamic acid). On the 6th day after transfection, take a sample and centrifuge at 4500 rpm for 10 minutes to collect the cell supernatant, and purify according to the method in Test Example 2. The purified protein can be used in the following examples or test examples.

The amino acid sequences of human BCMA-ECD-Fc, human BCMA-ECD-his, cynoBCMA-ECD-his and cynoBCMA-ECD-Fc are as follows. cynoBCMA-ECD-his was purchased from ACRO.

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

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

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

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

(3) ECD of cynomolgus monkey BCMA with His tag: cynoBCMA-ECD-His (SEQ ID NO: 3)

MLQMARQCSQNEYFDSLLHDCKPCQLRCSSTPPLTCQRYCNASMTNSVKGMNA GGGSGGGSHHHHHHHHHH

Note: The italicized part is the linker-10His-tag

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

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

Example 2. Protein A affinity chromatography purification of recombinant proteins with Fc tags and nickel column purification of recombinant proteins with His tags

Purification of proteins with human Fc tags: The BCMA supernatant sample expressed in cells was centrifuged at high speed to remove impurities and purified through a Protein A column. Flush the column with PBS until the A280 reading drops to baseline. Rinse the target protein with 100mM acetic acid, pH3.5, and neutralize with 1M Tris-HCl, pH8.0. The eluted sample is appropriately concentrated and then the liquid is changed into PBS. The obtained protein is identified as correct by electrophoresis, peptide map, and LC-MS before being aliquoted and ready for use.

Purification of human His-tagged proteins: Centrifuge the cell-expressed BCMA supernatant sample at high speed to remove impurities. Equilibrate the nickel column with PBS buffer, flush the column volume 2-5 times, and load the supernatant sample onto the Ni Sepharose excel column at a certain flow rate. Flush the column with PBS buffer until the A280 reading drops to the baseline, then flush the chromatography column with PBS+10mM imidazole and remove Non-specifically bound impurities were collected, and the flow-through was collected. Finally, the target protein was washed with PBS solution containing 300mM imidazole, and the washed peak was collected, concentrated and replaced, and the obtained protein was identified by electrophoresis, peptide map, and LC-MS. After correct allocation, use it.

Example 3. Construction and identification of cell lines expressing recombinant human CD38, human BCMA and cynomolgus monkey BCMA

This disclosure constructs a cell line expressing human CHO-K1/hCD38. The full-length human CD38 gene was cloned into the mammalian cell expression vector pCDH, and HEK293T cells (ATCC ^® CRL-11268) were co-transfected with three plasmids: pVSV-G, pCMV-dR8.91 and pCDH-hCD38 to package the virus and transfect After 48 hours, virus-infected CHO-K1 cells (ATCC ^® CCL-61) were collected. CHOK1 cells that highly express hCD38 were obtained by flow sorting 72 hours after infection.

In order to screen for antibodies that can bind well to BCMA on the cell surface, the present disclosure constructed a K562-BCMA cell line expressing human BCMA. The full-length human BCMA gene was cloned into the mammalian cell expression vector pCDH, and HEK293T cells (ATCC ^® CRL-11268) were co-transfected with three plasmids: pVSV-G, pCMV-dR8.91 and pCDH-human BCMA to package the virus. After 48 hours of infection, virus-infected K562 cells (ATCC ^® CCL-243) were collected. K562 single cell lines that highly express human BCMA were obtained by flow sorting 72 hours after infection.

This disclosure also constructs a CHO-K1-cynoBCMA cell line expressing cynomolgus monkey, selects the full-length cynomolgus BCMA gene into the mammalian cell expression vector pCDH, and uses pVSV-G, pCMV-dR8.91 and pCDH-cynoBCMA The three plasmids were co-transfected into HEK293T cells (ATCC ^® CRL-11268) to package the virus. 48 hours after transfection, the virus was collected to infect CHOK1 cells (ATCC ^® CCL-61). 72 hours after infection, a single CHOK1 cell line with high expression of cynoBCMA was obtained by flow sorting.

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

This disclosure uses fusion tumor technology to prepare monoclonal antibodies against human BCMA. The obtained antibody specifically binds to human BCMA with high affinity and can cross-react with cynomolgus BCMA; the obtained antibody has good binding activity to human BCMA and cynomolgus BCMA on the cell surface, and the binding activity is affected by soluble BCMA interference is minimal.

Human BCMA-ECD-his and cynoBCMA-ECD-his were used as cross-immunization reagents, and TiterMax® Gold Adjuvant (Sigma Cat No. T2684) and Thermo Imject® Alum (Thermo Cat No. 77161) were used as adjuvants to cross-immunize mice. After the initial immunization and 7 boosted immunizations, mouse 10-6# (titer 625K) with high antibody titer in serum was selected for spleen cell fusion. After fusion, the fusion tumor culture supernatant was detected according to the growth density of the fusion tumor cells, and antibodies that specifically bind to BCMA on the cell surface were screened.

Single fusion tumor cell lines 4E3, 33H4 and 27 with good activity were screened. Fusion tumor cells in the logarithmic growth phase were collected respectively, RNA was extracted with NucleoZol (MN) (according to the kit instructions), and reverse transcription was performed (PrimeScript ^TM Reverse Transcriptase, Takara, cat # 2680A). The cDNA obtained by reverse transcription was PCR amplified and sequenced using mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503). The amino acid sequences of the CDRs and variable regions of 4E3, 33H4 and 27 are as follows:

>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 regions (SEQ ID NO: 27)

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

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

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

Humanization of mouse monoclonal antibodies is carried out according to methods published in many documents in this field. In short, on the basis of the obtained typical structure of mouse antibody VH/VL CDR, from human Search the homologous sequences of the light chain variable region (VL) and heavy chain variable region (VH) in the germline database, transplant the CDR region of the mouse antibody to the human template, and perform some analysis on some residues of VL and VH. Mutation, replacing the constant region of the murine antibody with the human constant region, results in the final humanized molecule.

1.Selection and backmutation of the human FR region of 4E3

The specific sequence of the antibody variable region obtained by humanizing mouse antibody 4E3 is as follows (underlined indicates CDR, the same below):

>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)

2.Selection and backmutation of the human FR region of 33H4

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

The specific sequence of the antibody variable region obtained by humanizing mouse 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 present disclosure

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

The CDR and variable region sequences of the anti-CD38 arm in the antibody of the present disclosure are as follows:

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 can be derived from any suitable antibody. Particularly suitable antibodies are described, for example, in International Application No. WO2020114478 (incorporated herein in its entirety).

The CDR and variable region sequences of the anti-CD3 arm in the bispecific antibody of the present disclosure are as follows:

Table 9. CDRs 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-CD38 antibody variable region, anti-BCMA antibody variable region, anti-CD3 antibody variable region and IgG ₁ mutant IgG ₁ (AA) (L234A/L235A) were combined. Each forms different forms of antigen-binding molecules, namely Format5, Format5-6164, Format12, Format12-6164, Format20, Format20-6164, Format21, Format21-6164, and Format24.

Format5 is an asymmetric structure molecule. The complete molecule has three chains, all of which are different. Its 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: The single underlined region is the CDR region of CD38 and CD3 binding domains obtained according to Kabat numbering rules, and italics are the constant regions.

Format5-6164 is an asymmetric structure molecule. The complete molecule has three chains, all of which are different. Its 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-5-6164): 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)

Note: The single underlined region is the CDR region of CD38 and CD3 binding domains obtained according to Kabat numbering rules, and italics are the constant regions.

Format12 is a symmetric structure molecule, including two identical heavy chains (chain 1) and two identical light chains (chain 2). Its structure is shown in Figure 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 symmetric structure molecule, including two identical heavy chains (chain 1) and two identical light chains (chain 2). Its structure is shown in Figure 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. The complete molecule has three chains, all of which are different. Its structure is shown in Figure 1C.

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

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

Chain 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)

Note: The single underlined region is the CDR region of CD38 and CD3 binding domains obtained according to Kabat numbering rules, and italics are the constant regions.

Format20-6164 is an asymmetric structure molecule. The complete molecule has three chains, all of which are different. Its structure is shown in Figure 1C.

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

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

Chain 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)

Note: The single underlined region is the CDR region of CD38 and CD3 binding domains obtained according to Kabat numbering rules, and italics are the constant regions.

Format21 is a symmetric structure molecule, containing two identical heavy chains (chain 1) and two identical light chains (chain 2). Its structure is shown in Figure 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 symmetric structure molecule, including two identical heavy chains (chain 1) and two identical light chains (chain 2). Its structure is shown in Figure 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, its structure is shown in Figure 1D.

>VL(1149)-CL(SEQ ID NO:71)

Note: The single underlined region is the CDR region of the CD38 binding domain obtained according to Kabat numbering rules, and the italicized region is the constant region.

Format24 is an asymmetric structure molecule. The complete molecule has four chains in total. Its structure is shown in Figure 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;

Chain 3 (Knob-24): VH(1149)-CH1- _IgG1 Fc(Knob,AA)-linker 1-VL(107E)-linker 2-VH(107E).

>Knob-24(SEQ ID NO:72)

Note: The single underlined region is the CDR region of CD38 and CD3 binding domains obtained according to Kabat numbering rules, and italics are the constant regions.

Construct an antigen-binding molecule according to Table 10. Among the numbers, T5 indicates that hu4E3L1 and hu4E3H1 are used as variable regions that bind BCMA, T3 indicates that hu33H4L2 and hu33H4H1 are used as variable regions that bind BCMA, and T1 indicates that hu27L1 and hu27H3 are used as BCMA-binding variable regions. Change area.

>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

Note: The single underlined area is the CDR area obtained according to the Kabat numbering rule, and the italicized area is the constant area.

>CH1(SEQ ID NO:104)

>CL(SEQ ID NO:105)

The positive control molecules used in this disclosure are GBR-1342 and AMGEN701. The amino acid sequence of GBR-1342 includes SEQ ID NO: 178, SEQ ID NO: 179 and SEQ ID NO: 128 in WO2016071355. The amino acid sequence of AMGEN701 includes SEQ ID NO: 74 in WO2017134134.

test case

Test Example 1. FACS determination of hBCMA and hCD38 expression on MM cell surface

In order to test the expression levels of BCMA and CD38 on the surface of MM cells, the expression levels of MM-related cell lines were detected by FACS. The specific determination of CD38 expression is as follows: cells with a viability rate >90% are added to a 96-well plate (3590#, Corning) at a cell number of 1× ¹⁰⁵ per well, centrifuged at 300g for 5 minutes, and washed once with 1% BSA . Add 100 μL of 10 μg/mL CD38 antibody prepared in 1% BSA and incubate at 4°C for 1 hour. Wash twice with 1% BSA. Add 50 μL of PE-human F(ab') ₂ (1:400) and incubate at 4°C for 0.5 hours. Wash twice with 1% BSA. Add 100 μL 1% BSA to resuspend, and read the value on the machine. The specific determination of BCMA expression is as follows: cells with a viability rate >90% are added to a 96-well plate (3590#, Corning) at a cell number of 1× ¹⁰⁵ per well, centrifuged at 300g for 5 minutes, and washed once with 1% BSA . Add the BCMA antibody (BL2-A: BCMA-APC-A) diluted according to the instructions and incubate at 4°C for 1 hour. Wash twice with 1% BSA. Add 100 μL 1% BSA to resuspend, and then use a FACS instrument to detect the fluorescence values of PE and APC on the cell surface, and use the geometric mean for parallel comparison.

Test Example 2. Affinity of the disclosed antibodies to CD38/BCMA/CD3d&CD3e proteins

In order to test the affinity of the disclosed antibody to CD38/BCMA/CD3D&E, this test example uses the instrument Biacore T200 to detect the antibody and hCD3d&CD3e (31.2Kda, Cat.# CT038-H2508H, S.B), hCD38 (30.7Kda, HR) and hBCMA (7.8Kda, Cat.# BCA-H52y, Acro) binding ability. The specific method is as follows: Use a CM5 biosensor chip coupled with an anti-human antibody to affinity capture the antibody, and then flow a solution containing 100nM BCMA-His over the surface of the chip for 180 seconds to saturate the BCMA binding site of the antibody, and then add hCD3d&CD3e or hCD38, detected in real time with a Biacore T200 instrument. The data fitting model adopts 1:1 Model. The affinity data of the antibodies of the present disclosure binding to the corresponding antigens are shown in the table below.

The results show that the disclosed antibody has better CD38/BCMA binding ability and can bind to CD3d&CD3e.

Test Example 3. FACS determination of the affinity of antibodies to MM cell surface antigens

In order to test the binding ability of the disclosed antibodies to hCD38 and hBCMA on the surface of MM cells, this test example used flow cytometry to detect the CD38/BCMA-CD3 trispecific antibody and MM tumor cells RPMI-8226 co-expressing CD38 and BCMA. combination ability. The details are as follows: The above cells were cultured in 1640 culture medium containing 10% FBS, placed in a 37°C, 5% _CO2 incubator, and cultured for 2 days. The cells were added to the cell plate at a cell number of 1× ¹⁰⁵ per well. medium, centrifuge at 300g for 1 minute, and wash once with 1% BSA. Use Mix-n-Stain CF 647 Antibody Labeling Kits (MX647S100-1KT#, Sigma) to directly label the antibody, dilute the fluorescently labeled antibody in a gradient, add 100 μL per well to the cell plate, incubate at 4°C for 1 hour, 1 Wash the plate three times with % BSA, add 100 μL PBS to each well and read the plate. The results are shown in Figure 2. The disclosed antibody can bind very well to the surface of tumor cells co-expressing BCMA and CD38, and its effect is better than that of the positive control.

Test Example 4. In vitro cytotoxic activity of antibodies

To test the killing activity of the disclosed antibodies as T cell adapter molecules against tumor cells. K562/hBCMA, a stably transduced cell line overexpressing human BCMA, CHO K1/hCD38, a stably transduced cell line overexpressing human CD38, and MM tumor cell RPMI-8226 co-expressing CD38 and BCMA were used as target cells to detect the dual specificity of the present disclosure. The target-specific cytotoxic activity of the bispecific antibody was determined, and CHOK1, which does not express CD38 and BCMA, was used as a target cell to detect the non-specific cytotoxic activity of the bispecific antibody of the present disclosure. Fresh PBMC (purchased from Xuanfeng Biological Company) were centrifuged at 300g for 10 minutes, resuspended in 1640+10% FBS (A solution) containing 200U IL2, and cultured overnight in a T75 culture bottle (density is 2×10 ⁶ /mL) ; After centrifugation, the cells were resuspended and counted in IMDM+10% FBS, DMEM/F-12 1:1+10% FBS and 1640+10% FBS culture media. Adjust the cell number to 1.5×10 ⁶ cells/mL. Add 50 μL to the well. Collect the target cells, centrifuge at 1000 rpm for 3 minutes, resuspend and count, adjust the cell number to 3×10 ⁵ cells/mL, add 25 μL to each well, and the E:T Ratio is 10:1. The antibody was diluted with the culture medium corresponding to the cells. The starting concentration was 400nM (4×final concentration), diluted 10 times in 9 gradients, and 25 μL was added to each well. The cells were then cultured in a 37°C, 5% CO ₂ incubator for 48 h. Fluorescence signal detection: Take out the culture plate, centrifuge at 1000 rpm for 3 minutes, pipet 50 μL of supernatant into a new 96-well plate, freeze it in a -20°C refrigerator, and add 50 μL one- Glo, incubate at room temperature for 5 minutes, detect the luminescence value (define the luminescence value of cells in the unantibody group as 0 kill), and calculate the killing percentage of different antibodies at different concentrations.

The results are shown in Tables 13-1 to 13-3 and Figure 3. The cytotoxic activity of the disclosed antibody is specific to CD38 and BCMA targets, and its effect is better than that of the positive control; at the same time, Figure 3 shows that in the absence of CD38 and BCMA expression On the CHOK1 cell line, the disclosed antibody exhibits weaker cytotoxic activity and has better safety.

Test Example 5. Effect of soluble BCMA/CD38/APRIL on the in vitro cytotoxic activity of the disclosed antibody

The high concentration of soluble BCMA (average 30ng/mL) present in the blood of multiple myeloma patients will interfere with the specific binding of the BCMA-CD3 bispecific antibody to BCMA on the membrane surface. Secondly, the content of APRIL in the serum of MM patients is also that of healthy people. 5.9 times, and soluble CD38 also increased to a certain extent in MM patients. In order to test whether the in vitro tumor cell killing activity of the disclosed antibody is affected by soluble BCMA/CD38/APRIL, the cell line RPMI-8226, which co-expresses CD38 and BCMA, was used as the target cell. During the cytotoxic activity test, they were added separately or together. Added soluble BCMA, CD38 and APRIL. Fresh PBMC (purchased from Xuanfeng Biological Company) were centrifuged at 300g for 10 minutes, resuspended in 1640+10% FBS (liquid A) containing 200U IL2, placed in a T75 culture bottle and cultured overnight (density is 2E6/mL); collected after centrifugation Resuspend and count the cells in 1640+10% FBS culture medium, adjust the number of cells to 1.5×10 ⁶ cells/mL, and add 50 μL to each well. Collect the target cells, centrifuge at 1000 rpm for 3 minutes, resuspend and count, adjust the cell number to 3×10 ⁵ cells/mL, add 25 μL to each well, and the E:T Ratio is 10:1. The antibody was diluted with the culture medium corresponding to the cells. The starting concentration was 400nM (4×final concentration), diluted 10 times in 9 gradients, and 25 μL was added to each well. The cells were then cultured in a 37°C, 5% CO ₂ incubator for 48 h. Fluorescence signal detection: Take out the culture plate, centrifuge at 1000 rpm for 3 minutes, pipet 50 μL of supernatant into a new 96-well plate, freeze it in a -20°C refrigerator, and add 50 μL one- Glo, incubate at room temperature for 5 minutes, detect the luminescence value (define the luminescence value of cells in the unantibody group as 0 kill), and calculate the killing percentage of different antibodies at different concentrations.

The results show that the cytotoxic activity of the disclosed antibodies is minimally affected by soluble BCMA/CD38/APRIL. Compared with the absence of soluble protein, the presence of 30 ng/mL soluble BCMA, 5 ng/mL soluble CD38 and 100 ng/mL soluble APRIL, respectively or together, only changed the cytotoxicity IC ₅₀ of the disclosed antibody to However, in the presence of soluble protein, the IC ₅₀ of AMGEN701 increased to 10 times the original value.

Test Example 6. Detection of antibody-induced cytokine release levels

The CD3 T cell adapter molecule causes a cytokine storm. Therefore, when developing CD3 T cell adapter molecules, it is necessary to keep cytokines, especially IL-6, a factor that has nothing to do with drug efficacy but can cause side effects, at a low level.

Centrifuge CHO-K1 cells at 1000 rpm for 1 minute, collect 50 μL of cell supernatant and dilute it 6 times with universal sample diluent. IL-6 or IL-2 standard is also diluted 6 times, and placed in the detection plate. Add diluted samples or standards of different concentrations (100 μL/well), seal the reaction wells with sealing tape, incubate at 37°C for 90 minutes, wash the plate 5 times, and add biotinylated antibody working solution (100 μL/well). Seal the reaction well with new sealing tape, incubate at 37°C for 60 minutes, wash the plate 5 times, add enzyme conjugate working solution (100 μL/well), seal the reaction well with new sealing tape, and incubate at 37°C for 30 minutes. Then wash the plate 5 times, add 100 μL/well of chromogenic substrate (TMB), incubate at 37°C for 8 minutes in the dark, add 100 μL/well of reaction stop solution, and measure the OD450 value immediately after mixing (within 3 minutes).

The results show that the release levels of IL6 and IL-2 brought about by the antibody of the present disclosure are lower than those of GBR-1342, suggesting that the antibody of the present disclosure has better safety.

Biological evaluation of in vivo activity

Test Example 7. The efficacy of antibodies in Molp-8 orthotopic tumor model

Molp-8, as a typical MM cell line, has higher CD38 expression and lower BCMA expression.

In order to test the efficacy of the disclosed antibodies in the Molp-8 orthotopic tumor model, 1.5×10 ⁶ cells/200 μL/mouse of Molp-8/lucG cells were inoculated into 79 NDG mice through the tail vein. One day after tumor cell inoculation, freshly isolated PBMCs from the two donors were mixed at a 1:1 ratio and injected into the abdominal cavity of mice at 5 × 10 ⁶ /mouse. Four days after tumor cell inoculation, each mouse was intraperitoneally injected with a bioluminescent substrate (15 mg/mL) at a volume of 10 ml/kg, anesthetized with isoflurane, and photographed using a small animal imaging system 10 minutes after injection. The mice with too small body weight, too large and too small bioluminescence signal values were removed, and the mice were randomly divided into groups according to the bioluminescence signal, with 7 mice in each group. On the day of grouping, each antibody was injected intraperitoneally, and this day was defined as Day 0 of the experiment, twice a week. Take pictures and images twice a week, weigh and record the data. All data were graphed and statistically analyzed using Excel and GraphPad Prism 8 software.

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

Tumor inhibition rate (%) = 1-T/C (%). T/C(%)=(T-T0)/(C-C0)×100%, where T and C are the bioluminescence signal values of the treatment group and control group at the end of the experiment; T ₀ and C ₀ are at the beginning of the experiment bioluminescence signal value. Experimental results showed that both T3-12 and T5-12-6164 showed significant efficacy.

Test Example 8. The efficacy of antibodies in the RPMI-8226 orthotopic tumor model

RPMI-8226, as a typical MM cell line, has moderate CD38 expression and BCMA expression.

RPMI-8226-lucG cells 5×10 ⁶ cells/200 μL/mouse were inoculated into NDG mice through the tail vein. Fifteen days after the tumor cells were inoculated, each mouse was intraperitoneally injected with bioluminescent substrate (15 mg/mL) at a volume of 10 mL/kg, anesthetized with isoflurane, and photographed using a small animal imaging system 10 minutes after injection. The mice with too small body weight, too large and too small bioluminescence signal values were removed, and the mice were randomly divided into groups according to the bioluminescence signal, with 7 mice in each group. One day after grouping, freshly isolated PBMCs from the two donors were mixed in a 1:1 ratio and injected into the abdominal cavity of the mice at 5 × 10 ⁶ /mouse. Intraperitoneal injection of each antibody was started 4 days after grouping, and this day was defined as Day 0 of the experiment, administered twice a week, for a total of 4 times. Take pictures and images twice a week, weigh and record the data. All data were graphed and statistically analyzed using Excel and GraphPad Prism 8 software.

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

Tumor inhibition rate (%) = 1-T/C (%). T/C(%)=(T-T0)/(C-C0)×100%, where T and C are the bioluminescence signal values of the treatment group and control group at the end of the experiment; T ₀ and C ₀ are at the beginning of the experiment bioluminescence signal value.

The results showed that both T3-12 and T5-12-6164 showed obvious efficacy, and both showed better efficacy than GBR-1342 of equal molar amount. During the administration process, the tumor-bearing mice tolerated each antibody well, and their body weight did not fluctuate significantly.

Although the above invention has been described in detail with the aid of drawings and examples for clear understanding, the description and examples should not be construed as limiting the scope of the present disclosure. The disclosures of all patent and scientific documents cited herein are expressly incorporated by reference in their entirety.

TW202325743A_111126501_SEQL.xml

Claims (19)

An antigen-binding molecule that specifically binds BCMA, CD38 and CD3, comprising at least one antigen-binding module that specifically binds BCMA, at least one antigen-binding module that specifically binds CD38, and at least one antigen-binding module that specifically binds CD3. The antigen-binding molecule as described in claim 1, wherein the antigen-binding molecule includes two antigen-binding modules that specifically bind BCMA, two antigen-binding modules that specifically bind CD38, and two antigen-binding modules that specifically bind CD3. and Fc region; Preferably, The antigen-binding module that specifically binds BCMA is BCMA-Fab, the antigen-binding module that specifically binds CD3 is CD3-scFv, and the antigen-binding module that specifically binds CD38 is CD38-scFv; or The antigen-binding module that specifically binds to CD38 is CD38-Fab, the antigen-binding module that specifically binds to CD3 is CD3-scFv, and the antigen-binding module that specifically binds to BCMA is BCMA-scFv; Better yet, The antigen-binding module that specifically binds to BCMA is BCMA-Fab, the antigen-binding module that specifically binds to CD3 is CD3-scFv, and the antigen-binding module that specifically binds to CD38 is CD38-scFv; and the heavy chain of BCMA-Fab, CD3-scFv, a subunit of the Fc region, and CD38-scFv are connected in N-terminal to C-terminal order, either directly or through a linker; or The antigen-binding module that specifically binds to CD38 is CD38-Fab, the antigen-binding module that specifically binds to CD3 is CD3-scFv, and the antigen-binding module that specifically binds to BCMA is BCMA-scFv; and the heavy chain of CD38-Fab, CD3-scFv, a subunit of the Fc region and BCMA-scFv are connected directly or through a linker in order from N-terminus to C-terminus. The antigen-binding molecule according to claim 1 or 2, wherein the antigen-binding module that specifically binds to BCMA includes a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, and the antigen that specifically binds to CD3 The binding module includes a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the antigen-binding module that specifically binds CD38 includes a heavy chain variable region CD38-VH and a light chain variable region CD38-VL; in, The antigen-binding molecule includes 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]-[a subunit of the 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 the same or different peptide linkers; or The antigen-binding molecule includes 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]-[a subunit of the Fc region]-[linker 3 ]-[BCMA-VH]-[Connector 4]-[BCMA-VL], (d)[CD38-VL]-[CL], Linker 1, linker 2, linker 3 and linker 4 in formula (c) are the same or different peptide linkers; Preferably, The length of linker 1, linker 2, linker 3 and linker 4 in formula (a) is 3-15 amino acid residues; or The length of linker 1, linker 2, linker 3 and linker 4 in formula (c) is 3-15 amino acid residues; Better yet, The amino acid sequences of linker 1, linker 3 and linker 4 in formula (a) are shown in SEQ ID NO: 99, and the amino acid sequence of linker 2 is shown in SEQ ID NO: 96; or The amino acid sequences of linker 1, linker 3 and linker 4 in formula (c) are shown in SEQ ID NO: 99, and the amino acid sequence of linker 2 is shown in SEQ ID NO: 96. The antigen-binding molecule as described in claim 1, wherein the antigen-binding molecule includes one antigen-binding module that specifically binds BCMA, two antigen-binding modules that specifically bind CD38, one antigen-binding module that specifically binds CD3, and Fc district; Preferably, The antigen-binding module that specifically binds BCMA is BCMA-Fab, the antigen-binding module that specifically binds CD3 is CD3-scFv, and the antigen-binding module that specifically binds CD38 is CD38-scFv; or The antigen-binding module that specifically binds to CD38 is CD38-Fab, the antigen-binding module that specifically binds to CD3 is CD3-scFv, and the antigen-binding module that specifically binds to BCMA is BCMA-scFv; More preferably, the Fc region includes a first subunit Fc1 and a second subunit Fc2 capable of associating, and each of Fc1 and Fc2 independently has one or more amino acid substitutions that reduce homodimerization of the Fc region; and, The antigen-binding module that specifically binds to BCMA is BCMA-Fab, the antigen-binding module that specifically binds to CD3 is CD3-scFv, and the antigen-binding module that specifically binds to CD38 is CD38-scFv; and the heavy chain of BCMA-Fab, Fc1 and a CD38-scFv are connected directly or through a linker in the order from N-terminus to C-terminus, and CD3-scFv, Fc2 and another CD38-scFv are connected directly or through a linker in the order from N-terminus to C-terminus. Connect; or The antigen-binding module that specifically binds CD38 is CD38-Fab, the antigen-binding module that specifically binds CD3 is CD3-scFv and the antigen-binding module that specifically binds BCMA is BCMA-scFv; and a heavy chain of CD38-Fab , Fc1 and CD3-scFv are connected directly or through linkers in the order from N-terminus to C-terminus, and BCMA-scFv, the heavy chain of another CD38-Fab and Fc2 are directly or through the order from N-terminus to C-terminus. Connected by connectors. The antigen-binding molecule as described in claim 1 or 4, wherein the antigen-binding module that specifically binds to BCMA includes a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, and the antigen that specifically binds to CD3 The binding module includes a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the antigen-binding module that specifically binds CD38 includes a heavy chain variable region CD38-VH and a light chain variable region CD38-VL; in, The antigen-binding molecule includes 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 a peptide linker; or The antigen-binding molecule includes 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 length of linker 1, linker 2 in formula (e), and linker 1, linker 2, linker 3 and linker 4 in formula (f) is 3-15 amino acid residues; or The lengths of linker 1 and linker 2 in formula (g), and linker 1 and linker 2 in formula (h) are 3-15 amino acid residues; Better yet, The amino acid sequences of linker 1 and linker 2 in formula (e) are as shown in SEQ ID NO: 99, and the amino acid sequences of linker 1, linker 3 and linker 4 in formula (f) are as follows SEQ ID NO: 99, and the amino acid sequence of linker 2 in formula (f) is SEQ ID NO: 96; or The amino acid sequence of linker 1 and linker 2 in formula (g) is shown in SEQ ID NO: 99, and the amino acid sequence of linker 1 in formula (h) is shown in SEQ ID NO: 97. The amino acid sequence of linker 2 in formula (h) is shown in SEQ ID NO: 99. The antigen-binding molecule of claim 1, wherein the antigen-binding molecule includes an antigen-binding module that specifically binds BCMA, an antigen-binding module that specifically binds CD38, an antigen-binding module that specifically binds CD3, and an Fc region. ; Preferably, The antigen-binding module that specifically binds to BCMA is BCMA-Fab, the antigen-binding module that specifically binds to CD3 is CD3-scFv, and the antigen-binding module that specifically binds to CD38 is CD38-scFv; Better yet, The Fc region includes a first subunit Fc1 and a second subunit Fc2 capable of associating, each of Fc1 and Fc2 independently having one or more amino acid substitutions that reduce homodimerization of the Fc region; and CD38-scFv , CD3-scFv and Fc1 are connected directly or through a linker in the order from N-terminus to C-terminus, and the heavy chain and Fc2 of BCMA-Fab are connected directly or through a linker in the order from N-terminus to C-terminus. The antigen-binding molecule as described in claim 1 or 6, wherein the antigen-binding module that specifically binds to BCMA includes a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, and the antigen that specifically binds to CD3 The binding module includes a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the antigen-binding module that specifically binds CD38 includes a heavy chain variable region CD38-VH and a light chain variable region CD38-VL; in, The antigen-binding molecule includes 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]-[Connexon 1]-[CD38-VL]-[Connexon 2]-[CD3-VH]-[Connexon 3]-[CD3-VL]-[Connexon 4] -[Fc1], Wherein, linker 1, linker 2, linker 3 and linker 4 in formula (j) are the same or different peptide linkers; Preferably, The length of linker 1, linker 2, linker 3 and linker 4 in formula (j) is 3-15 amino acid residues; Better yet, The amino acid sequence of linker 1 and linker 3 in formula (j) is shown in SEQ ID NO: 99, and the amino acid sequence of linker 2 in formula (j) is shown in SEQ ID NO: 97. The amino acid sequence of linker 4 in formula (j) is shown in SEQ ID NO: 96. The antigen-binding molecule according to any one of claims 1 to 7, wherein the antigen-binding module that specifically binds BCMA includes a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, wherein, (i) The BCMA-VH includes the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 23, and the BCMA-VL includes the BCMA-LCDR1, BCMA in SEQ ID NO: 24 -Amino acid sequences of LCDR2 and BCMA-LCDR3; or (ii) The BCMA-VH includes the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 25, and the BCMA-VL includes the BCMA-LCDR1, BCMA in SEQ ID NO: 26 -Amino acid sequences of LCDR2 and BCMA-LCDR3; or (iii) The BCMA-VH includes the amino acid sequences of BCMA-HCDR1, BCMA-HCDR2 and BCMA-HCDR3 in SEQ ID NO: 27, and the BCMA-VL includes the BCMA-LCDR1, BCMA in SEQ ID NO: 28 -Amino acid sequences of LCDR2 and BCMA-LCDR3; Preferably, (i) The BCMA-VH has: BCMA-HCDR1 of SEQ ID NO: 5, BCMA-HCDR2 of SEQ ID NO: 6, and BCMA-HCDR3 of SEQ ID NO: 7; and the BCMA-VL has: SEQ ID NO: BCMA-LCDR1 of SEQ ID NO: 9, BCMA-LCDR2 of SEQ ID NO: 9, and BCMA-LCDR3 of SEQ ID NO: 10, or (ii) The BCMA-VH has BCMA-HCDR1 of SEQ ID NO: 11, BCMA-HCDR2 of SEQ ID NO: 12 and BCMA-HCDR3 of SEQ ID NO: 13; and the BCMA-VL has SEQ ID NO: 14 BCMA-LCDR1, BCMA-LCDR2 of SEQ ID NO: 15, and BCMA-LCDR3 of SEQ ID NO: 16, or (iii) The BCMA-VH has BCMA-HCDR1 of SEQ ID NO: 17, BCMA-HCDR2 of SEQ ID NO: 18 and BCMA-HCDR3 of SEQ ID NO: 19; and the BCMA-VL has SEQ ID NO: 20 BCMA-LCDR1, BCMA-LCDR2 of SEQ ID NO:21 and BCMA-LCDR3 of SEQ ID NO:22. The antigen-binding molecule as described in claim 8, wherein, (i) The BCMA-VH is as shown in SEQ ID NO: 23, and the BCMA-VL is as shown in SEQ ID NO: 24, or The BCMA-VH is as shown in any one of SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, and the BCMA-VL is as shown in SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34 and any one of SEQ ID NO: 35, Preferably, The BCMA-VH is set forth in SEQ ID NO: 29, and the BCMA-VL is set forth in SEQ ID NO: 32; or (ii) the BCMA-VH is as shown in SEQ ID NO: 25, and the BCMA-VL is as shown in SEQ ID NO: 26, or the BCMA-VH is as set forth in any one of SEQ ID NO:36, SEQ ID NO:37 and SEQ ID NO:38, and the BCMA-VL is as set forth in one of SEQ ID NO:39 or SEQ ID NO:40, Preferably, The BCMA-VH is set forth in SEQ ID NO: 36, and the BCMA-VL is set forth in SEQ ID NO: 40; or (iii) the BCMA-VH is as shown in SEQ ID NO: 27, and the BCMA-VL is as shown in SEQ ID NO: 28, or The BCMA-VH is as set forth in any one of 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 shown in one of 46, Preferably, The BCMA-VH is set forth in SEQ ID NO:43, and the BCMA-VL is set forth in SEQ ID NO:45. The antigen-binding molecule according to any one of claims 1 to 9, wherein the antigen-binding module that specifically binds to CD3 includes a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, wherein, (i) The CD3-VH comprises the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 63; and the CD3-VL comprises the CD3-LCDR1, CD3 in SEQ ID NO: 64 -Amino acid sequences of LCDR2 and CD3-LCDR3, or (ii) the CD3-VH comprises the amino acid sequences of CD3-HCDR1, CD3-HCDR2 and CD3-HCDR3 in SEQ ID NO: 65; and the CD3-VL comprises the CD3-LCDR1, CD3 in SEQ ID NO: 64 -Amino acid sequences of LCDR2 and CD3-LCDR3; Preferably, (i) the CD3-VH has CD3-HCDR1 as shown in SEQ ID NO: 55, CD3-HCDR2 as shown in SEQ ID NO: 56, and CD3-HCDR3 as shown in SEQ ID NO: 57; and the CD3-VL has CD3-LCDR1 as shown in SEQ ID NO:58 as SEQ ID CD3-LCDR2 as shown in NO:59, and CD3-LCDR3 as shown in SEQ ID NO:60, or (ii) the CD3-VH has CD3-HCDR1 as shown in SEQ ID NO: 55, CD3-HCDR2 as shown in SEQ ID NO: 61, and CD3-HCDR3 as shown in SEQ ID NO: 62; and the CD3-VL has CD3-LCDR1 as shown in SEQ ID NO: 58, CD3-LCDR2 as shown in SEQ ID NO: 59, and CD3-LCDR3 as shown in SEQ ID NO: 60; Better yet, The CD3-VH is set forth in SEQ ID NO: 63, and the CD3-VL is set forth in SEQ ID NO: 64; or The CD3-VH is set forth in SEQ ID NO:65, and the CD3-VL is set forth in SEQ ID NO:64. The antigen-binding molecule according to any one of claims 1 to 10, wherein the antigen-binding module that specifically binds to CD38 includes a heavy chain variable region CD38-VH and a light chain variable region CD38-VL, wherein the CD38 -VH comprises the amino acid sequences of CD38-HCDR1, CD38-HCDR2 and CD38-HCDR3 in SEQ ID NO: 53, and the CD38-VL comprises CD38-LCDR1, CD38-LCDR2 and CD38- in SEQ ID NO: 54 Amino acid sequence of LCDR3; Preferably, The CD38-VH has CD38-HCDR1 as shown in SEQ ID NO: 47, CD38-HCDR2 as shown in SEQ ID NO: 48, and CD38-HCDR3 as shown in SEQ ID NO: 49; and the CD38-VL Having CD38-LCDR1 as shown in SEQ ID NO: 50, CD38-LCDR2 as shown in SEQ ID NO: 51, and CD38-LCDR3 as shown in SEQ ID NO: 52; Better yet, The CD38-VH is set forth in SEQ ID NO:53, and the CD38-VL is set forth in SEQ ID NO:54. The antigen-binding molecule according to any one of claims 1 to 11, wherein the antigen-binding module that specifically binds BCMA includes a heavy chain variable region BCMA-VH and a light chain variable region BCMA-VL, and the specificity The antigen-binding module that binds CD3 includes a heavy chain variable region CD3-VH and a light chain variable region CD3-VL, and the antigen-binding module that specifically binds CD38 includes a heavy chain variable region CD38-VH and a light chain variable region CD38. -VL, where, (i) The BCMA-VH has: BCMA-HCDR1 as shown in SEQ ID NO: 5, BCMA-HCDR2 as shown in SEQ ID NO: 6 and BCMA-HCDR3 as shown in SEQ ID NO: 7; and the BCMA-VL has: BCMA-LCDR1 as shown in SEQ ID NO: 8, BCMA-LCDR2 as shown in SEQ ID NO: 9 and BCMA-LCDR3 as shown in SEQ ID NO: 10, and The CD3-VH has: CD3-HCDR1 as shown in SEQ ID NO: 55, CD3-HCDR2 as shown in SEQ ID NO: 61, and CD3-HCDR3 as shown in SEQ ID NO: 62; and the CD3- VL has: CD3-LCDR1 as shown in SEQ ID NO:58, CD3-LCDR2 as shown in SEQ ID NO:59, and CD3-LCDR3 as shown in SEQ ID NO:60, and The CD38-VH has: CD38-HCDR1 as shown in SEQ ID NO: 47, CD38-HCDR2 as shown in SEQ ID NO: 48, and CD38-HCDR3 as shown in SEQ ID NO: 49; and the CD38- VL has: CD38-LCDR1 as set forth in SEQ ID NO: 50, CD38-LCDR2 as set forth in SEQ ID NO: 51, and CD38-LCDR3 as set forth in SEQ ID NO: 52; or (ii) The BCMA-VH has: BCMA-HCDR1 as shown in SEQ ID NO: 11, BCMA-HCDR2 as shown in SEQ ID NO: 12 and SEQ ID NO: 13 BCMA-HCDR3; and the BCMA-VL has: BCMA-LCDR1 as shown in SEQ ID NO: 14, BCMA-LCDR2 as shown in SEQ ID NO: 15 and BCMA-LCDR3 as shown in SEQ ID NO: 16, and The CD3-VH has: CD3-HCDR1 as shown in SEQ ID NO: 55, CD3-HCDR2 as shown in SEQ ID NO: 56, and CD3-HCDR3 as shown in SEQ ID NO: 57; and the CD3- VL has: CD3-LCDR1 as shown in SEQ ID NO:58, CD3-LCDR2 as shown in SEQ ID NO:59, and CD3-LCDR3 as shown in SEQ ID NO:60, and The CD38-VH has: CD38-HCDR1 as shown in SEQ ID NO: 47, CD38-HCDR2 as shown in SEQ ID NO: 48, and CD38-HCDR3 as shown in SEQ ID NO: 49; and the CD38- VL has: CD38-LCDR1 as shown in SEQ ID NO: 50, CD38-LCDR2 as shown in SEQ ID NO: 51, and CD38-LCDR3 as shown in SEQ ID NO: 52; Preferably, (i) The BCMA-VH is as shown in SEQ ID NO: 29, the BCMA-VL is as shown in SEQ ID NO: 32, the CD3-VH is as shown in SEQ ID NO: 65, the CD3-VL is as shown in SEQ ID NO :64, the CD38-VH is represented by SEQ ID NO:53, and the CD38-VL is represented by SEQ ID NO:54; or (ii) The BCMA-VH is shown in SEQ ID NO: 36, the BCMA-VL is shown in SEQ ID NO: 40, the CD3-VH is shown in SEQ ID NO: 63, and the CD3-VL is shown in SEQ ID NO :64, the CD38-VH is represented by SEQ ID NO:53, and the CD38-VL is represented by SEQ ID NO:54. The antigen-binding molecule according to any one of claims 1 to 12, wherein the antigen-binding molecule includes an Fc region, and the Fc region is preferably an IgG Fc region, more preferably an IgG ₁ Fc region; Preferably, the Fc region contains one or more amino acid substitutions, which amino acid substitutions can reduce the binding of the Fc region to Fc receptors; More preferably, the amino acid substitution can reduce its binding to Fcγ receptor; In particular, the Fc region is a human _IgG1 Fc region, and the amino acid residues at positions 234 and 235 are A, numbered according to the EU index. The antigen-binding molecule according to any one of claims 1 to 13, wherein the antigen-binding molecule includes an Fc region, the Fc region includes a first subunit Fc1 and a second subunit Fc2 capable of associating, and the Fc1 and Each Fc2 independently has one or more amino acid substitutions that reduce homodimerization of the Fc region; Preferably, The Fc1 has a convex structure according to the pestle and mortar technology, and the Fc2 has a hole structure according to the pestle and mortar technology; Better yet, The amino acid residue of Fc1 at position 366 is W; and the amino acid residue of Fc2 at position 366 is S, the amino acid residue at position 368 is A, and the amino acid residue at position 407 The base is V and the numbering basis is the EU index. The antigen-binding molecule as described in claim 1, wherein, The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 76 and two second chains comprising the amino acid sequence of SEQ ID NO: 74; or The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 75 and two second chains comprising the amino acid sequence of SEQ ID NO: 74; or The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 82 and two second chains comprising the amino acid sequence of SEQ ID NO: 81; or The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 83 and two second chains comprising the amino acid sequence of SEQ ID NO: 81; or The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 90 and two second chains comprising the amino acid sequence of SEQ ID NO: 89; or The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 91 and two second chains comprising the amino acid sequence of SEQ ID NO: 89; or The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 77, two second chains comprising the amino acid sequence of SEQ ID NO: 71; or The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 78, and two second chains comprising the amino acid sequence of SEQ ID NO: 71; or The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 85, and two second chains comprising the amino acid sequence of SEQ ID NO: 71; or The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 86, and two second chains comprising the amino acid sequence of SEQ ID NO: 71; or The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 93, and two second chains comprising the amino acid sequence of SEQ ID NO: 71; or The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 94, and two second chains comprising the amino acid sequence of SEQ ID NO: 71; or The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO:73, a second chain including the amino acid sequence of SEQ ID NO:74, and an amino group including SEQ ID NO:66 the third strand of the acid sequence; or The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO:73, a second chain including the amino acid sequence of SEQ ID NO:74, and an amino group including SEQ ID NO:67 the third strand of the acid sequence; or The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 80, a second chain including the amino acid sequence of SEQ ID NO: 81, and an amino group including SEQ ID NO: 66 the third strand of the acid sequence; or The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 80, a second chain including the amino acid sequence of SEQ ID NO: 81, and an amino group including SEQ ID NO: 67 the third strand of the acid sequence; or The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 88, a second chain including the amino acid sequence of SEQ ID NO: 89, and an amino group including SEQ ID NO: 66 the third strand of the acid sequence; or The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 88, a second chain including the amino acid sequence of SEQ ID NO: 89, and an amino group including SEQ ID NO: 67 the third strand of the acid sequence; or The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 84, a second chain including the amino acid sequence of SEQ ID NO: 81, and an amino group including SEQ ID NO: 69 the third strand of the acid sequence; or The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 84, a second chain including the amino acid sequence of SEQ ID NO: 81, and an amino group including SEQ ID NO: 70 the third strand of the acid sequence; or The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 92, a second chain including the amino acid sequence of SEQ ID NO: 89, and an amino group including SEQ ID NO: 69 the third strand of the acid sequence; or The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 92, a second chain including the amino acid sequence of SEQ ID NO: 89, and an amino group including SEQ ID NO: 70 the third strand of the acid sequence; or The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO:79, two second chains including the amino acid sequence of SEQ ID NO:71 and an amine including SEQ ID NO:72 The third strand of the amino acid sequence; or The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 87, two second chains including the amino acid sequence of SEQ ID NO: 71, and an amine including SEQ ID NO: 72 The third strand of the amino acid sequence; or The antigen-binding molecule has: a first chain including the amino acid sequence of SEQ ID NO: 95, two second chains including the amino acid sequence of SEQ ID NO: 71, and an amine including SEQ ID NO: 72 The third strand of the amino acid sequence; Preferably, The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO: 76 and two second chains comprising the amino acid sequence of SEQ ID NO: 74; or The antigen-binding molecule has: two first chains comprising the amino acid sequence of SEQ ID NO:82 and two second chains comprising the amino acid sequence of SEQ ID NO:81. A pharmaceutical composition containing: A therapeutically effective amount of the antigen-binding molecule according to any one of claims 1 to 15, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients; Preferably, the pharmaceutical composition also contains 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 as described in any one of claims 1 to 15 or the pharmaceutical composition as described in claim 16; Preferably, the disease is a hematopoietic system tumor of lymphatic lineage or myeloid lineage or an autoimmune disease; More preferably, the hematopoietic system tumor of lymphoid lineage or myeloid lineage is selected from: multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia, amyloidosis, Waldenstrom's macroglobulinemia, solitary bone disease Plasmacytoma, extramedullary plasmacytoma, osteosclerotic myeloma, heavy chain disease, monoclonal gammapathies of undetermined significance and smoldering multiple myeloma, B-cell lymphoma, Burkitt lymphoma, Hodgkin's lymphoma and hairy cell lymphoma; this autoimmune disease is selected from: rheumatoid arthritis, systemic lupus erythematosus, asthma, inflammatory bowel disease, multiple sclerosis, Crohn's disease, gastritis , Hashimoto's thyroiditis, ankylosing spondylitis, and graft versus host disease.

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