Multi-specific antigen-binding protein targeting BCMA, CD19 and CD3 and use thereof

Abstract

A multi-specific antigen-binding protein targeting BCMA, CD19 and CD3. Specifically, provided is a tri-specific T cell adapter formed by linking an anti-BCMA scFv, an anti-CD19 scFv and an anti-CD3 Fab. The multi-specific T cell adapter is capable of simultaneously targeting BCMA, CD19 and CD3 with high binding affinity, thereby linking target cells and T cells, and promoting the killing effect of T cells. The present invention has the prospect of being applied in the anti-tumor field.

Description

Multispecific antigen binding proteins targeting BCMA, CD19 and CD3 and their applications Technical Field

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to a multispecific antigen-binding protein targeting BCMA, CD19 and CD3 and its application. Background Art

[0002] Currently, the main treatment options for cancer patients are still surgery, traditional chemotherapy, radiotherapy, or small molecule targeted drugs. However, with the gradual application of immunotherapy, especially the publication of breakthrough results from various clinical trials, cancer immunotherapy has become a new treatment option, bringing hope to cancer patients.

[0003] Current cancer immunotherapy strategies mainly focus on enhancing the activation of effector T cells (exogenous or endogenous) in tumor patients, or releasing suppressed anti-cancer T cell responses, or breaking T cell tolerance to tumor cells, thereby killing and eliminating tumor cells to achieve the purpose of treating patients.

[0004] Currently, the immunotherapies for treating cancer patients mainly include: 1) immune checkpoint inhibitors (CPI), 2) chimeric antigen receptor T cells (CAR-T), 3) antibody-drug conjugates (ADC), and 4) T cell engagers (TCE).

[0005] Among them, the T cell engager is an artificially designed molecule that can specifically bind to two or more different antigens, one end of which binds to the CD3 antigen and the other end binds to one or more tumor-associated antigens (TAAs) and / or tumor-specific antigens (TSAs). This T cell engager can cross-link tumor cells and T cells, namely the T cell driver (T cell engager), and then form synapses at the contact site, activating cytotoxic signals and leading to tumor cell lysis. When T cells are activated, the CD25 and CD69 cell surface markers are upregulated and cytokines are released. The secreted cytokines, such as interleukin-2 (IL-2), can further activate T cells and promote their proliferation to enhance their ability to kill tumor cells.

[0006] CD19 is a transmembrane glycoprotein that is primarily expressed in the early stages of human B cell development and persists on mature B cells. As part of the B cell antigen receptor complex, it plays a key role in B cell activation, proliferation, and differentiation. High expression of CD19 is not only observed on normal B cells, but is also prevalent in a variety of B cell tumors, such as B cell acute lymphoblastic leukemia (B-ALL) and non-Hodgkin's lymphoma (NHL). Therefore, CD19 has become an ideal biomarker for the treatment of targeted B cell malignancies.

[0007] B-cell maturation antigen (BCMA) is a transmembrane glycoprotein that belongs to the tumor necrosis factor receptor superfamily (TNFRSF). BCMA is primarily expressed on mature B cells and plays a critical role in the B-cell life cycle. It regulates B-cell survival, proliferation, and antibody secretion by binding to its two major ligands, APRIL (proliferation-inducing ligand) and BAFF (B-cell activating factor).

[0008] In B-cell malignancies such as multiple myeloma, BCMA expression is significantly increased and is closely associated with tumor cell proliferation and survival. Tumor cells can activate BCMA through autocrine APRIL and BAFF, thereby promoting self-proliferation and evading immune system surveillance. High BCMA expression is also associated with disease progression and poor prognosis, making it a clinically significant biomarker.

[0009] BCMA's unique expression pattern and key role in the progression of B-cell malignancies make it an ideal therapeutic target. BCMA-targeted therapeutic strategies can achieve specific attacks on diseased B cells while having relatively minimal effects on normal tissues. Furthermore, BCMA-targeted therapies can work through multiple mechanisms, including directly inducing tumor cell apoptosis, blocking tumor growth signals, and activating the immune system's attack on tumor cells.

[0010] Although CD19 or BCMA targeted therapies have achieved remarkable clinical results, such as CarT therapy, their complex manufacturing process and high cost limit the widespread application of these therapies. In clinical applications, there are still some unresolved issues, such as the resistance of some patients to CD19 mono-targeted therapy or BCMA mono-targeted therapy, the improvement of long-term survival rate, and the optimization of efficacy in children and the elderly. With the in-depth understanding of the tumor microenvironment and tumor immunology, future treatments may include the combination of targeted therapy with other treatment methods, such as radiotherapy, chemotherapy, immunomodulators or other emerging targeted therapies. For example, simultaneous targeting of CD19 and BCMA can avoid antigen escape of a single target. This multimodal treatment strategy may improve the treatment effect and provide patients with a more comprehensive treatment plan.

[0011] Therefore, there is a need in the art to develop a T cell engager targeting CD19 and BCMA. Summary of the Invention

[0012] The purpose of the present invention is to provide a T cell engager targeting CD19 and BCMA.

[0013] In a first aspect of the present invention, an anti-BCMA antibody or an antigen-binding fragment thereof is provided, wherein the antibody comprises a heavy chain variable region and a light chain variable region,

[0014] Wherein, the heavy chain variable region has the following complementarity determining regions CDR:

[0015] VH-CDR1 shown in SEQ ID NO: 14,

[0016] VH-CDR2 shown in SEQ ID NO: 15, and

[0017] VH-CDR3 shown in SEQ ID NO: 16;

[0018] Furthermore, the light chain variable region has the following complementarity determining regions (CDRs):

[0019] VL-CDR1 shown in SEQ ID NO: 17,

[0020] VL-CDR2 shown in SEQ ID NO: 18, and

[0021] VL-CDR3 shown in SEQ ID NO:19.

[0022] In another preferred embodiment, the antigen-binding fragment is selected from the group consisting of Fab, (Fab')2, scFv, or a combination thereof.

[0023] In another preferred embodiment, the antibody is a monospecific antibody, a bispecific antibody or a multispecific antibody.

[0024] In another preferred embodiment, the antibody is a single-chain antibody having a structure shown in Formula Ia or Ib from N-terminus to C-terminus:

[0025] VL BCMA -L1-VH BCMA (Ia);

[0026] VH BCMA -L1-VL BCMA (Ib);

[0027] In the formula, “-” is each independently a bond;

[0028] VL BCMAThe light chain variable region of the anti-BCMA antibody;

[0029] VH BCMA The heavy chain variable region of an anti-BCMA antibody;

[0030] L1 is a connecting peptide.

[0031] In another preferred embodiment, the amino acid sequence of the antibody heavy chain variable region is as shown in SEQ ID No: 25 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto, and / or

[0032] The amino acid sequence of the antibody light chain variable region is shown in SEQ ID No: 26 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto.

[0033] In another preferred embodiment, L1 is a flexible connecting peptide.

[0034] In another preferred embodiment, L1 has an amino acid sequence as shown in (GGGGS)n or GG, wherein n is an integer of 1-6, preferably 2, 3 or 4.

[0035] In another preferred embodiment, L1 has the amino acid sequence shown in SEQ ID NO: 34.

[0036] In a second aspect of the present invention, an anti-CD3 antibody or an antigen-binding fragment thereof is provided, wherein the antibody comprises a heavy chain variable region and a light chain variable region.

[0037] Wherein, the heavy chain variable region has the following complementarity determining regions CDR:

[0038] VH-CDR1 shown in SEQ ID NO: 1,

[0039] VH-CDR2 shown in SEQ ID NO:7, and

[0040] VH-CDR3 shown in SEQ ID NO: 3;

[0041] Furthermore, the light chain variable region has the following complementarity determining regions (CDRs):

[0042] VL-CDR1 shown in SEQ ID NO:4,

[0043] VL-CDR2 shown in SEQ ID NO:5, and

[0044] VL-CDR3 shown in SEQ ID NO:6.

[0045] In another preferred embodiment, the antigen-binding fragment is selected from the group consisting of Fab, (Fab')2, scFv, or a combination thereof.

[0046] In another preferred embodiment, the antibody is a monospecific antibody, a bispecific antibody or a multispecific antibody.

[0047] In another preferred embodiment, the amino acid sequence of the antibody heavy chain variable region is as shown in SEQ ID No: 22 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto, and / or

[0048] The amino acid sequence of the antibody light chain variable region is shown in SEQ ID No: 21 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto.

[0049] In another preferred embodiment, the antibody further comprises a light chain constant region and / or a heavy chain constant region.

[0050] In another preferred embodiment, the antibody comprises a light chain constant region CL and a heavy chain constant region CH1.

[0051] In another preferred embodiment, the constant region is a human constant region or a mouse constant region.

[0052] In another preferred example, the light chain constant region CL has the sequence shown in SEQ ID NO: 36.

[0053] In another preferred example, the heavy chain constant region CH1 has the sequence shown in SEQ ID NO: 35.

[0054] In the third aspect of the present invention, a multispecific antibody is provided, wherein the multispecific antibody comprises the anti-BCMA antibody or antigen-binding fragment thereof according to the first aspect of the present invention, and / or the anti-CD3 antibody or antigen-binding fragment thereof according to the second aspect of the present invention.

[0055] In another preferred embodiment, the multispecific antibody is a trispecific antibody, and the trispecific antibody comprises:

[0056] 1) a CD19 targeting binding domain comprising one or more antigen binding domains that specifically bind to the CD19 protein;

[0057] 2) a BCMA targeting binding domain comprising one or more antigen binding domains that specifically bind to the BCMA protein; and

[0058] 3) CD3 targeting binding domain, which comprises one or more antigen binding domains that specifically bind to CD3 protein.

[0059] In another preferred embodiment, the antigen binding domain that specifically binds to the CD19 protein includes a heavy chain variable region and a light chain variable region.

[0060] Wherein, the heavy chain variable region has the following complementarity determining regions CDR:

[0061] VH-CDR1 shown in SEQ ID NO:8,

[0062] VH-CDR2 shown in SEQ ID NO:9, and

[0063] VH-CDR3 shown in SEQ ID NO: 10;

[0064] Furthermore, the light chain variable region has the following complementarity determining regions (CDRs):

[0065] VL-CDR1 shown in SEQ ID NO: 11,

[0066] VL-CDR2 shown in SEQ ID NO: 12, and

[0067] VL-CDR3 shown in SEQ ID NO:13.

[0068] In another preferred embodiment, the antigen binding domain that specifically binds to CD19 protein is selected from the following group: single-chain antibody, double-chain antibody, or a combination thereof.

[0069] In another preferred embodiment, the antigen binding domain that specifically binds to CD19 protein is an anti-CD19 single-chain antibody or Fab.

[0070] In another preferred embodiment, the anti-CD19 single-chain antibody has a structure shown in the following formula IIa or IIb from N-terminus to C-terminus:

[0071] VL CD19 -L2-VH CD19 (IIa);

[0072] VH CD19 -L2-VL CD19 (IIb);

[0073] In the formula, “-” is each independently a bond;

[0074] VL CD19 The light chain variable region of the anti-CD19 antibody;

[0075] VH CD19 is the heavy chain variable region of an anti-CD19 antibody;

[0076] L2 is a connecting peptide.

[0077] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the anti-CD19 antibody is as shown in SEQ ID No: 23 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto.

[0078] In another preferred embodiment, the amino acid sequence of the light chain variable region of the anti-CD19 antibody is as shown in SEQ ID No: 24 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto.

[0079] In another preferred embodiment, L2 is a flexible connecting peptide.

[0080] In another preferred embodiment, L2 has an amino acid sequence as shown in (GGGGS)n or GG, wherein n is an integer of 1-6, preferably 2, 3 or 4.

[0081] In another preferred embodiment, L2 has the amino acid sequence shown in SEQ ID NO: 34.

[0082] In another preferred embodiment, the antigen-binding domain that specifically binds to the BCMA protein is the anti-BCMA antibody or antigen-binding fragment thereof as described in the first aspect of the present invention.

[0083] In another preferred embodiment, the antigen-binding domain that specifically binds to the BCMA protein is a single-chain antibody or Fab, preferably a single-chain antibody represented by Formula Ia or Ib.

[0084] In another preferred embodiment, the CD3 targeting binding domain includes a heavy chain variable region and a light chain variable region.

[0085] Wherein, the heavy chain variable region has the following complementarity determining regions CDR:

[0086] VH-CDR1 shown in SEQ ID NO: 1,

[0087] VH-CDR2 shown in SEQ ID NO: 2 or 7, and

[0088] VH-CDR3 shown in SEQ ID NO: 3;

[0089] Furthermore, the light chain variable region has the following complementarity determining regions (CDRs):

[0090] VL-CDR1 shown in SEQ ID NO:4,

[0091] VL-CDR2 shown in SEQ ID NO:5, and

[0092] VL-CDR3 shown in SEQ ID NO:6.

[0093] In another preferred example, the CD3 targeting binding domain has a heavy chain variable region with an amino acid sequence as shown in SEQ ID No: 20 or 22, and / or a light chain variable region with an amino acid sequence as shown in SEQ ID No: 21.

[0094] In another preferred example, the CD3 targeting binding domain further includes a light chain constant region and / or a heavy chain constant region.

[0095] In another preferred embodiment, the CD3 targeting binding domain is an anti-CD3 single-chain antibody or Fab.

[0096] In another preferred embodiment, the CD3 targeting binding domain is Fab, which includes a light chain constant region CL and a heavy chain constant region CH1.

[0097] In another preferred embodiment, the constant region is a human constant region or a mouse constant region.

[0098] In another preferred example, the light chain constant region CL has the sequence shown in SEQ ID NO: 36.

[0099] In another preferred example, the heavy chain constant region CH1 has the sequence shown in SEQ ID NO: 35.

[0100] In another preferred embodiment, the CD3 targeting binding domain is the anti-CD3 antibody or antigen-binding fragment thereof as described in the second aspect of the present invention.

[0101] In another preferred example, the CD19 targeting binding domain is connected to the N-terminus of the heavy chain variable region of the CD3 targeting binding domain; and the BCMA targeting binding domain is connected to the N-terminus of the light chain variable region of the CD3 targeting binding domain.

[0102] In another preferred example, the CD19 targeting binding domain is connected to the N-terminus of the light chain variable region of the CD3 targeting binding domain; and the BCMA targeting binding domain is connected to the N-terminus of the heavy chain variable region of the CD3 targeting binding domain.

[0103] In another preferred embodiment, the light chain and / or heavy chain of the trispecific antibody further comprises an Fc domain.

[0104] In another preferred embodiment, the Fc domain is the Fc domain from IgG protein.

[0105] In another preferred embodiment, the trispecific antibody has a structure as shown in Formula IIIa, IIIb, IIIc, IIId or IIIe:

[0106] Wherein, “-” is each independently a bond or a peptide linker;

[0107] ScFv CD19 is the CD19 targeting binding domain;

[0108] ScFv BCMA is the BCMA targeting binding domain;

[0109] ScFv CD3 is the CD3 targeting binding domain;

[0110] VH CD3 The heavy chain variable region is the CD3 targeting binding domain;

[0111] VL CD3 The light chain variable region is the CD3 targeting binding domain;

[0112] VH BCMA The heavy chain variable region is the BCMA targeting binding domain;

[0113] VL BCMA The light chain variable region is the BCMA targeting binding domain;

[0114] VH CD19 The heavy chain variable region is the CD19 targeting binding domain;

[0115] VL CD19 The light chain variable region is the CD19 targeting binding domain;

[0116] CH1 is the heavy chain constant region 1;

[0117] CL is the light chain constant region;

[0118] Fc1 and Fc2 are each independently free or Fc domain;

[0119] L3 and L4 are each independently a connecting peptide;

[0120] It is a disulfide bond or a covalent bond.

[0121] In another preferred embodiment, the trispecific antibody has the structure shown in IIIa or IIIb, and Fc1 and Fc2 are absent.

[0122] In another preferred embodiment, L3 and L4 are flexible connecting peptides.

[0123] In another preferred embodiment, L3 and L4 each independently have an amino acid sequence as shown in (GGGGS)n or GG, wherein n is an integer of 1-6, preferably 1, 2, or 3.

[0124] In another preferred embodiment, L3 has the amino acid sequence shown in SEQ ID NO: 33.

[0125] In another preferred embodiment, L4 has the amino acid sequence shown in SEQ ID NO: 33.

[0126] In another preferred embodiment, Fc1 has the amino acid sequence shown in SEQ ID NO: 46.

[0127] In another preferred embodiment, Fc2 has the amino acid sequence shown in SEQ ID NO: 47.

[0128] In another preferred example, the CD19 targeting binding domain includes the heavy chain variable region shown in SEQ ID NO: 23 and the light chain variable region shown in SEQ ID NO: 24.

[0129] In another preferred embodiment, the BCMA targeting binding domain includes the heavy chain variable region shown in SEQ ID NO: 25 and the light chain variable region shown in SEQ ID NO: 26.

[0130] In another preferred embodiment, the CD3 targeting binding domain includes the heavy chain variable region shown in SEQ ID NO: 20 and the light chain variable region shown in SEQ ID NO: 21.

[0131] In another preferred embodiment, the CD3 targeting binding domain includes the heavy chain variable region shown in SEQ ID NO: 22 and the light chain variable region shown in SEQ ID NO: 21.

[0132] In another preferred embodiment, the heavy chain amino acid sequence of the trispecific antibody is as shown in SEQ ID NO: 27 or 31, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto; and

[0133] The light chain amino acid sequence of the trispecific antibody is as shown in SEQ ID NO: 28 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto.

[0134] In another preferred embodiment, the heavy chain amino acid sequence of the trispecific antibody is as shown in SEQ ID NO: 29 or 32, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto; and

[0135] The light chain amino acid sequence of the trispecific antibody is as shown in SEQ ID NO: 30 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto.

[0136] In another preferred embodiment, the heavy chain amino acid sequence of the trispecific antibody is as shown in SEQ ID NO: 39 or 45, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto; and

[0137] The light chain amino acid sequence of the trispecific antibody is as shown in SEQ ID NO: 40 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto.

[0138] In another preferred embodiment, the heavy chain amino acid sequence of the trispecific antibody is as shown in SEQ ID NO: 41 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto; and

[0139] The light chain amino acid sequence of the trispecific antibody is as shown in SEQ ID NO: 42 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto.

[0140] In another preferred embodiment, the heavy chain amino acid sequence of the trispecific antibody is as shown in SEQ ID NO: 43 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto; and

[0141] The light chain amino acid sequence of the trispecific antibody is as shown in SEQ ID NO: 44 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto.

[0142] In another preferred embodiment, the trispecific antibody has a heavy chain and a light chain selected from the group consisting of:

[0143] 1) a heavy chain as set forth in SEQ ID NO: 27; and a light chain as set forth in SEQ ID NO: 28;

[0144] 2) a heavy chain as shown in SEQ ID NO: 29; and a light chain as shown in SEQ ID NO: 30;

[0145] 3) a heavy chain as shown in SEQ ID NO: 31; and a light chain as shown in SEQ ID NO: 28;

[0146] 4) a heavy chain as shown in SEQ ID NO: 32; and a light chain as shown in SEQ ID NO: 30;

[0147] 5) a heavy chain as shown in SEQ ID NO: 39; and a light chain as shown in SEQ ID NO: 40;

[0148] 6) a heavy chain as shown in SEQ ID NO:41; and a light chain as shown in SEQ ID NO:42;

[0149] 7) a heavy chain as shown in SEQ ID NO: 43; and a light chain as shown in SEQ ID NO: 44;

[0150] 8) the heavy chain shown in SEQ ID NO:45; and the light chain shown in SEQ ID NO:40.

[0151] In another preferred example, the heavy chain amino acid sequence of the trispecific antibody is shown in SEQ ID NO: 31; and the light chain amino acid sequence of the trispecific antibody is shown in SEQ ID NO: 28.

[0152] In another preferred example, the heavy chain amino acid sequence of the trispecific antibody is shown in SEQ ID NO: 45; and the light chain amino acid sequence of the trispecific antibody is shown in SEQ ID NO: 40.

[0153] In another preferred embodiment, the multispecific antibody is a bispecific antibody.

[0154] In another preferred embodiment, the bispecific antibody comprises:

[0155] 1) a BCMA targeting binding domain comprising one or more antigen binding domains that specifically bind to a BCMA protein; and

[0156] 2) CD3 targeting binding domain, which comprises one or more antigen binding domains that specifically bind to CD3 protein.

[0157] In another preferred example, the N-terminus of the heavy chain variable region and / or light chain variable region of the CD3 targeting binding domain is connected to the BCMA targeting binding domain, preferably through a flexible connecting peptide.

[0158] In another preferred embodiment, the connecting peptide has an amino acid sequence as shown in (GGGGS)n or GG, wherein n is an integer of 1-6, preferably 1, 2, or 3.

[0159] In another preferred embodiment, the bispecific antibody comprises:

[0160] 1) a CD19 targeting binding domain comprising one or more antigen binding domains that specifically bind to the CD19 protein; and

[0161] 2) A CD3 targeting binding domain comprising one or more antigen binding domains that specifically bind to the CD3 protein. In another preferred embodiment, the bispecific antibody has a structure as shown in Formula IIIf, IIIg, or IIIh:

[0162] Wherein, “-” is each independently a bond or a peptide linker;

[0163] ScFv CD19 is the CD19 targeting binding domain;

[0164] ScFv BCMA is the BCMA targeting binding domain;

[0165] VH CD3 The heavy chain variable region is the CD3 targeting binding domain;

[0166] VL CD3 The light chain variable region is the CD3 targeting binding domain;

[0167] CH1 is the heavy chain constant region 1;

[0168] CL is the light chain constant region;

[0169] It is a disulfide bond or a covalent bond.

[0170] In another preferred embodiment, the heavy chain amino acid sequence of the bispecific antibody is as shown in SEQ ID NO: 29 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto; and

[0171] The light chain amino acid sequence of the bispecific antibody is as shown in SEQ ID NO: 28 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto.

[0172] In another preferred embodiment, the heavy chain amino acid sequence of the bispecific antibody is as shown in SEQ ID NO: 29; and

[0173] The light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 28.

[0174] In another preferred embodiment, the heavy chain amino acid sequence of the bispecific antibody is as shown in SEQ ID NO: 31; and

[0175] The light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 37.

[0176] In another preferred embodiment, the heavy chain amino acid sequence of the bispecific antibody is as shown in SEQ ID NO: 38; and

[0177] The light chain amino acid sequence of the bispecific antibody is shown in SEQ ID NO: 28.

[0178] In another preferred embodiment, the antibody has an EC50 binding affinity to target cells expressing CD19 and BCMA ≤ 1×10 -10 M, preferably ≤6×10 -11 M, more preferably ≤2×10 -11 M.

[0179] In a fourth aspect of the present invention, a recombinant protein is provided, comprising:

[0180] (i) the anti-BCMA antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof according to the second aspect of the present invention, or the multispecific antibody according to the third aspect of the present invention; and

[0181] (ii) Tag sequences that facilitate expression and / or purification.

[0182] In another preferred embodiment, the tag sequence includes a 6His tag, a GGGS sequence, and a FLAG tag.

[0183] In another preferred embodiment, the recombinant protein is a fusion protein.

[0184] In another preferred embodiment, the recombinant protein is a monomer, a dimer, or a multimer.

[0185] In the fifth aspect of the present invention, a polynucleotide is provided, which encodes the anti-BCMA antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof as described in the second aspect of the present invention, or the multispecific antibody as described in the third aspect of the present invention.

[0186] In the sixth aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide according to the fifth aspect of the present invention.

[0187] In another preferred embodiment, the expression vector is selected from the following group: DNA, RNA, viral vector, plasmid, transposon, other gene transfer systems, or a combination thereof.

[0188] In another preferred embodiment, the expression vector is a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

[0189] In the seventh aspect of the present invention, a genetically engineered host cell is provided, wherein the host cell contains the vector as described in the sixth aspect of the present invention, or an exogenous polynucleotide as described in the fifth aspect of the present invention integrated into its genome.

[0190] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0191] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, and mammalian cells.

[0192] In an eighth aspect of the present invention, an antibody conjugate is provided, wherein the antibody conjugate comprises:

[0193] (a) an antibody portion, wherein the antibody portion is selected from the group consisting of the anti-BCMA antibody or antigen-binding fragment thereof according to the first aspect of the invention, the anti-CD3 antibody or antigen-binding fragment thereof according to the second aspect of the invention, or the multispecific antibody according to the third aspect of the invention; and

[0194] (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, or a combination thereof.

[0195] In another preferred embodiment, the detectable marker comprises a radionuclide.

[0196] In another preferred embodiment, the drugs include toxins, cytokines, and enzymes.

[0197] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, nanomagnetic particles, prodrug-activating enzymes (for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (for example, cisplatin) or any form of nanoparticles, etc.

[0198] In another preferred embodiment, the antibody portion and the coupling portion are coupled via a chemical bond or a linker.

[0199] In the ninth aspect of the present invention, there is provided a use of an active ingredient in the preparation of a medicament for preventing and / or treating diseases associated with high expression of CD19 and / or BCMA, wherein the active ingredient is selected from the group consisting of the anti-BCMA antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof as described in the second aspect of the present invention, the multispecific antibody as described in the third aspect of the present invention, the recombinant protein as described in the fourth aspect of the present invention, the antibody conjugate as described in the eighth aspect of the present invention, or a combination thereof.

[0200] In another preferred embodiment, the disease is cancer or tumor.

[0201] In another preferred embodiment, the cancer or tumor is a solid tumor or a hematological tumor.

[0202] In another preferred embodiment, the cancer or tumor is selected from the group consisting of multiple myeloma, refractory or relapsed B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin's lymphoma (NHL), or a combination thereof.

[0203] In a tenth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0204] (i) an active ingredient selected from the group consisting of the anti-BCMA antibody or antigen-binding fragment thereof according to the first aspect of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof according to the second aspect of the present invention, the multispecific antibody according to the third aspect of the present invention, the recombinant protein according to the fourth aspect of the present invention, the antibody conjugate according to the eighth aspect of the present invention, or a combination thereof; and

[0205] (ii) a pharmaceutically acceptable carrier.

[0206] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0207] In another preferred embodiment, the pharmaceutical composition is an injection.

[0208] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of active ingredient and 0.01 to 99.99% of pharmaceutical carrier, and the percentages are the mass percentages of the pharmaceutical composition.

[0209] In another preferred embodiment, the pharmaceutical composition is used to prevent and / or treat diseases associated with high expression of CD19 and / or BCMA.

[0210] In the eleventh aspect of the present invention, a method for treating a disease associated with high expression of CD19 and / or BCMA is provided, comprising administering to a subject in need thereof an effective amount of the anti-BCMA antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, the anti-CD3 antibody or antigen-binding fragment thereof as described in the second aspect of the present invention, the multispecific antibody as described in the third aspect of the present invention, the recombinant protein as described in the fourth aspect of the present invention, the antibody conjugate as described in the eighth aspect of the present invention, or the pharmaceutical composition as described in the tenth aspect of the present invention, or a combination thereof.

[0211] In another preferred embodiment, the disease is cancer or tumor.

[0212] In another preferred embodiment, the cancer or tumor is a solid tumor or a hematological tumor.

[0213] In another preferred embodiment, the cancer or tumor is selected from the group consisting of multiple myeloma, refractory or relapsed B-cell acute lymphoblastic leukemia (B-ALL), non-Hodgkin's lymphoma (NHL), or a combination thereof.

[0214] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0215] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention defined by the claims.

[0216] Figure 1 shows the structures of trispecific antibody molecules (IMP-2023-031, IMP-2023-032, IMP-2023-033, IMP-2023-034), bispecific molecules (IMP-2023-035), control molecules (IMC-035 and IMC-036), and long-acting molecules (IMP-2023-036, IMP-2023-037, IMP-2023-038, IMP-2023-039).

[0217] FIG2 shows a heavy chain transient expression vector and a light chain transient expression vector.

[0218] Figure 3A-D shows cell binding curves. A is a T cell binding curve; B is a NCI-H929, K562-CD19, and Daudi cell binding curves, respectively.

[0219] Figure 4 shows CD19 and BCMA expression.

[0220] Figures 5A-J show target cell killing curves for IMP-2023-033. In Figure D, the effector cells were hPBMCs, the target cells were Raji, and the E:T ratio was 10:1; in Figure F, the effector cells were hPBMCs T cells, the target cells were hPBMCs B cells, and the incubation time was 24 hours. In the remaining experiments, the effector cells were Pri-T cells, the E:T ratio was 4:1, and the incubation time was 24 hours.

[0221] Figures 6A-B show target cell killing curves for IMP-2023-033 and control molecules. A) Effector cells are T cells, target cells are K562-BCMA, E:T ratio is 4:1, and incubation time is 24 hours. B) Effector cells are T cells, target cells are K562-CD19, E:T ratio is 4:1, and incubation time is 24 hours.

[0222] FIG7 shows the results of IMP-2023-033 stimulating T cell activation.

[0223] FIG8 shows the results of IMP-2023-033 stimulating T cell proliferation.

[0224] Figures 9A-E show that dexamethasone inhibits cytokine release.

[0225] FIG10 shows the tumor growth curves of the in vivo pharmacodynamic study.

[0226] Figure 11 shows a graph of cell binding curves.

[0227] Figure 12 shows the results of IMP-2023-induced target cell killing. The effector cells are T cells. The target cells in Figures A, B, and C are NCI-H929 expressing BCMA, K562-CD19 expressing CD19, and Daudi expressing both BCMA and CD19, respectively. The E:T ratio is 4:1, and the incubation time is 24 hours.

[0228] FIG13 shows the T cell proliferation results.

[0229] FIG14 shows the results of dexamethasone inhibition of cytokine release. DETAILED DESCRIPTION

[0230] After extensive and in-depth research, the inventors unexpectedly discovered for the first time a multispecific antigen-binding protein targeting BCMA, CD19, and CD3. This multispecific antigen-binding protein is composed of an anti-BCMA scFv, an anti-CD19 scFv, and an anti-CD3 Fab linked together. The multispecific antigen-binding protein binds BCMA, CD19, and CD3 simultaneously with high affinity and can function as a T cell engager, connecting target cells and T cells, inducing T cell activation and proliferation, and promoting T cell cytotoxicity. This led to the completion of the present invention.

[0231] the term

[0232] In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings generally understood by those of ordinary skill in the art to which the present invention belongs. Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions can change. It should also be understood that the terms used herein are intended only to describe specific embodiments and are not intended to be restrictive, and the scope of the present invention will be limited only by the appended claims.

[0233] 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 invention belongs. As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0234] The three-letter and one-letter codes for amino acids used in the present invention are as described in J. biol. chem, 243, p3558 (1968).

[0235] As used herein, the term "treatment" refers to administering an internal or external therapeutic agent, including antibodies against respiratory syncytial virus fusion protein (preferably pre-fusion F protein) and compositions thereof, to a patient experiencing one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered to the patient in an amount effective to alleviate one or more symptoms of the disease (a therapeutically effective amount).

[0236] As used herein, the term "optionally" or "optionally" means that the event or situation described subsequently may occur but need not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable regions of a specific sequence may have but need not have, and may have 1, 2, or 3.

[0237] As used herein, "sequence identity" refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between a sequence described herein and a sequence to which it is identical may be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0238] Antibody

[0239] As used herein, the terms "antibody" or "immunoglobulin" are heterotetrameric glycoproteins of approximately 150,000 daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite the first constant region of the heavy chain, and the variable region of the light chain is opposite the variable region of the heavy chain. Specific amino acid residues form an interface between the variable regions of the light and heavy chains.

[0240] As used herein, the term "variable" refers to certain parts of the variable region in an antibody that are different in sequence, which form the binding and specificity of various specific antibodies to their specific antigens. However, variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementarity determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved parts of the variable region are called framework regions (FRs). The variable regions of natural heavy and light chains each contain four FR regions, which are generally in a β-pleated configuration and are connected by three CDRs that form a connecting loop, and in some cases can form a partial β-pleated structure. The CDRs in each chain are closely together through the FR region and form the antigen-binding site of the antibody together with the CDRs of the other chain (see Kabat et al., NIH Publ. No. 91-3242, Volume 1, pages 647-669 (1991)). The constant regions do not directly participate in the binding of the antibody to the antigen, but they exhibit different effector functions, such as participating in the antibody-dependent cytotoxicity of the antibody.

[0241] Generally, an antibody's antigen-binding properties are described by three specific regions located in the variable regions of the heavy and light chains, known as the variable regions (CDRs). These regions are divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and do not directly participate in the binding reaction. These CDRs form a ring structure, spatially close to each other through the β-sheet formed by the FRs between them. The CDRs on the heavy chain and the corresponding CDRs on the light chain constitute the antibody's antigen-binding site. The amino acid sequences of antibodies of the same type can be compared to determine which amino acids constitute the FR or CDR regions.

[0242] The term "antigen-binding fragment" (or simply "antibody fragment") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used to perform the antigen-binding function of an antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment of an antibody" include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; and (iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody. Fv antibodies are the smallest antibody fragments that contain the variable regions of the heavy and light chains of an antibody, but lack the constant region, and possess all the antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains, and are capable of forming the structure required for antigen binding.

[0243] The present invention includes not only complete monoclonal antibodies, but also antibody fragments with immunological activity, such as Fab or (Fab')2 fragments; antibody heavy chains; and antibody light chains.

[0244] The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds. An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. An epitope can be a discontinuous three-dimensional site on an antigen that is recognized by the antibodies or antigen-binding fragments of the present invention.

[0245] The terms "specific binding", "selective binding", "selectively binds" and "specifically binds" refer to the binding of an antibody to a predetermined epitope on an antigen. -7 M, for example, less than approximately 10 -8 M, 10 -9 M or l0 -10 Binds with an affinity (KD) of M or less.

[0246] The present invention includes not only complete antibodies, but also fragments of antibodies with immunological activity or fusion proteins formed by antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives and analogs of the antibodies.

[0247] In the present invention, antibodies include murine, chimeric, humanized or fully human antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies, such as chimeric and humanized monoclonal antibodies, including human and non-human parts, can be obtained by standard DNA recombinant techniques, and they are all useful antibodies. A chimeric antibody is a molecule in which different parts are derived from different animal species, such as a chimeric antibody having a variable region from a mouse monoclonal antibody and a constant region from a human immunoglobulin (see, for example, U.S. Patent No. 4,816,567 and U.S. Patent No. 4,816,397, which are hereby incorporated by reference in their entirety). A humanized antibody refers to an antibody molecule derived from a non-human species, having one or more complementary determining regions (CDRs) derived from a non-human species and a framework region derived from a human immunoglobulin molecule (see U.S. Patent No. 5,585,089, which is hereby incorporated by reference in its entirety). These chimeric and humanized monoclonal antibodies can be prepared using DNA recombinant techniques well known in the art.

[0248] In the present invention, the antibodies can be monospecific, bispecific, trispecific, or more multispecific.

[0249] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat EA et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242).

[0250] As used herein, the term "heavy chain constant region (CH)" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region includes at least one of the following: a CH1 domain, a hinge region (e.g., an upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.

[0251] As used herein, the term "light chain constant region" includes an amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.

[0252] The term "scFv" or "single-chain antibody" refers to a small molecule antibody consisting of VH and VL domains in a VL-VH or VH-VL configuration with a linker region between the VH and VL domains.

[0253] The term "Fab" refers to a monovalent fragment consisting of the VL, VH, CL and CH1 domains, which form a dimer via a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0254] The terms "multispecific antibody" and "multispecific antigen-binding protein" are used interchangeably to refer to molecules that are able to simultaneously bind to multiple different types of epitopes, which can be located on the same target cell or on different target cells.

[0255] In one embodiment, the present invention provides a bispecific antibody (also known as a bispecific T cell engager) that simultaneously targets BCMA and CD3. In another embodiment, the present invention provides a trispecific antibody (also known as a trispecific T cell engager) that simultaneously targets CD19, BCMA, and CD3. Its advantage is that it can redirect specific polyclonal immune cells (such as T cells and NK cells) to tumor cells to enhance tumor killing. The antibodies of the present invention comprise antigen-binding fragments from two or more (e.g., three) monoclonal antibodies and rely on their antigen-binding ability to exert their effects. In one embodiment, the antibodies of the present invention comprise Fab fragments from anti-CD3 antibodies. In one embodiment, the antibodies of the present invention comprise scFv from anti-BCMA antibodies. In one embodiment, the antibodies of the present invention comprise scFv from anti-CD19 antibodies.

[0256] Bispecific or trispecific antibodies can be produced by chemical crosslinking or hybridoma technology. Alternatively, bispecific or trispecific antibody molecules can be produced by recombinant technology, for example, by linking one or more scFv molecules to Fab molecules via a connecting peptide. The length of the connecting peptide is between 2 and 15 amino acids. The connecting peptide can be composed of multiple amino acids, such as repeating units of GGGGS.

[0257] The trispecific antibodies of the present invention may further comprise an Fc domain, thereby extending the antibody half-life and forming a long-acting molecule. In one embodiment, the Fc domain is derived from an IgG protein. In one embodiment, the Fc domain is linked to the C-terminus of the CH1 domain and the CL domain of the Fab fragment.

[0258] In one embodiment, the multispecific antibody has a structure as shown in any one of the following formulae IIIa-IIIh:

[0259] Wherein, “-” is each independently a bond or a peptide linker;

[0260] ScFv CD19 is the CD19 targeting binding domain;

[0261] ScFv BCMA is the BCMA targeting binding domain;

[0262] ScFv CD3 is the CD3 targeting binding domain;

[0263] VH CD3 The heavy chain variable region is the CD3 targeting binding domain;

[0264] VL CD3 The light chain variable region is the CD3 targeting binding domain;

[0265] VH BCMA The heavy chain variable region is the BCMA targeting binding domain;

[0266] VL BCMA The light chain variable region is the BCMA targeting binding domain;

[0267] VH CD19 The heavy chain variable region is the CD19 targeting binding domain;

[0268] VL CD19 The light chain variable region is the CD19 targeting binding domain;

[0269] CH1 is the heavy chain constant region 1;

[0270] CL is the light chain constant region;

[0271] Fc1 and Fc2 are each independently free or Fc domain;

[0272] L3 and L4 are each independently a connecting peptide;

[0273] It is a disulfide bond or a covalent bond.

[0274] The present invention also provides a nucleic acid encoding the above-mentioned antibody or recombinant protein of the present invention or a chimeric antigen receptor (CAR) construct of the antibody of the present invention.

[0275] The method for preparing the nucleic acid is a conventional method in the art, and preferably comprises the following steps: obtaining a nucleic acid molecule encoding the above protein by gene cloning technology, or obtaining a nucleic acid molecule encoding the above protein by artificial full sequence synthesis.

[0276] Those skilled in the art will appreciate that the base sequence encoding the amino acid sequence of the aforementioned protein can be appropriately substituted, deleted, altered, inserted, or added to provide a polynucleotide homolog. The polynucleotide homologs of the present invention can be prepared by substituting, deleting, or adding one or more bases in the gene encoding the protein sequence while maintaining antibody activity.

[0277] Polynucleotides, vectors and host cells

[0278] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence of the antibody of the present invention or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence encoding an amino acid sequence identical to that of the polypeptide of the present invention, but having a different coding region sequence.

[0279] Polynucleotides encoding mature polypeptides of the present invention include: coding sequences encoding only the mature polypeptide; coding sequences for the mature polypeptide and various additional coding sequences; coding sequences for the mature polypeptide (and optional additional coding sequences) and non-coding sequences. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide or a polynucleotide further including additional coding and / or non-coding sequences.

[0280] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide constituting the antibody of the present invention.

[0281] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0282] The sequence of the DNA molecule of the antibody or fragment thereof of the present invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening.

[0283] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, then transferring it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods.

[0284] In addition, the sequences can also be synthesized by artificial synthesis, especially when the fragment length is shorter. Usually, a long fragment can be obtained by synthesizing multiple small fragments and then connecting them.

[0285] Currently, DNA sequences encoding the antibodies (or fragments thereof, or derivatives thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the present invention through chemical synthesis.

[0286] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0287] The host cell can be a prokaryotic cell, such as a bacterial cell, a lower eukaryotic cell, such as a yeast cell, or a higher eukaryotic cell, such as a mammalian cell. Preferred animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0288] Typically, the transformed host cells are cultured under conditions suitable for expression of the antibodies of the present invention. The antibodies of the present invention are then purified using conventional immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, among other conventional separation and purification methods well known to those skilled in the art.

[0289] The resulting monoclonal or polyclonal antibodies can be characterized using conventional methods. For example, the binding specificity of the antibody can be determined by immunoprecipitation or in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0290] The antibodies of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be separated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock, ultrasonic treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0291] Antibody-drug conjugates (ADCs)

[0292] The present invention also provides an antibody-drug conjugate (ADC) based on the antibody of the present invention.

[0293] Typically, the antibody-drug conjugate comprises the antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a therapeutically active drug. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic drug, a small molecule drug, or a radionuclide.

[0294] The antibody of the present invention and the effector molecule can be coupled via a coupling agent. Examples of the coupling agent may include any one or more of a non-selective coupling agent, a coupling agent utilizing a carboxyl group, a peptide chain, and a coupling agent utilizing a disulfide bond. The non-selective coupling agent refers to a compound that forms a covalent bond between the effector molecule and the antibody, such as glutaraldehyde. The coupling agent utilizing a carboxyl group may include any one or more of a cis-aconitic anhydride coupling agent (such as cis-aconitic anhydride) and an acylhydrazone coupling agent (where the coupling site is an acylhydrazone).

[0295] Certain residues on antibodies (such as Cys or Lys, etc.) are used to connect to a variety of functional groups, including imaging agents (such as chromophores and fluorescent groups), diagnostic agents (such as MRI contrast agents and radioisotopes), stabilizers (such as ethylene glycol polymers) and therapeutic agents. Antibodies can be coupled to functional agents to form antibody-functional agent conjugates. Functional agents (such as drugs, detection reagents, stabilizers) are coupled (covalently linked) to antibodies. Functional agents can be directly or indirectly connected to antibodies through linkers.

[0296] Antibodies can be conjugated to drugs to form antibody-drug conjugates (ADCs). Typically, ADCs contain a linker positioned between the drug and the antibody. The linker can be degradable or non-degradable. Degradable linkers typically readily degrade in the intracellular environment, for example, at the target site, thereby releasing the drug from the antibody. Suitable degradable linkers include, for example, enzymatically degradable linkers, including linkers containing peptidyl groups that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronide-containing linkers that can be degraded by glucuronidases. Peptide linkers can include, for example, dipeptides such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at a pH below 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug when the antibody is hydrolyzed by proteases.

[0297] Prior to attachment to the antibody, the linker has an active reactive group capable of reacting with certain amino acid residues, and attachment is achieved via the active reactive group. Thiol-specific active reactive groups are preferred and include, for example, maleimides, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethylketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones, pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, where the counter ion is acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. Linkers may include, for example, maleimides attached to the antibody via thiosuccinimide.

[0298] The drug can be any cytotoxic, cytostatic, or immunosuppressive drug. In embodiments, a linker connects the antibody and the drug, and the drug has a functional group capable of forming a bond with the linker. For example, the drug can have an amino, carboxyl, sulfhydryl, hydroxyl, or keto group capable of forming a bond with the linker. In cases where the drug is directly attached to the linker, the drug has a reactive group prior to attachment to the antibody.

[0299] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folate antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, and the like. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines, and oxazolidinobenzodiazepines), and vinca alkaloids.

[0300] In the present invention, drug-linkers can be used to form ADCs in a single step. In other embodiments, bifunctional linker compounds can be used to form ADCs in a two-step or multi-step process. For example, a cysteine ​​residue is reacted with a reactive moiety of a linker in a first step, and in a subsequent step, the functional group on the linker reacts with the drug to form an ADC.

[0301] Typically, the functional group on the linker is selected to facilitate specific reaction with an appropriate reactive group on the drug moiety. As a non-limiting example, an azide-based moiety can be used to specifically react with a reactive alkynyl group on the drug moiety. The drug is covalently attached to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphines (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Technology, 2nd Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will appreciate that, when a complementary pair of reactive functional groups is selected for selective reaction between the drug moiety and the linker, each member of the complementary pair can be used for both the linker and the drug.

[0302] The present invention also provides a method for preparing an ADC, which may further comprise: combining an antibody and a drug-linker compound under conditions sufficient to form an antibody conjugate (ADC).

[0303] In certain embodiments, the methods of the present invention comprise conjugating an antibody to a bifunctional linker compound under conditions sufficient to form an antibody-linker conjugate. In these embodiments, the methods of the present invention further comprise conjugating the antibody-linker conjugate to a drug moiety under conditions sufficient to covalently attach the drug moiety to the antibody via the linker.

[0304] Pharmaceutical composition

[0305] The present invention also provides a composition. In preferred embodiments, the composition is a pharmaceutical composition comprising the above-mentioned antibody, active fragment thereof, fusion protein thereof, or ADC, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the nature of the substance being formulated and the condition to be treated.

[0306] The prepared pharmaceutical composition can be administered by conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the route of administration of the pharmaceutical composition of the present invention is preferably injection or oral administration. The injection preferably includes intravenous injection, arterial injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in the form of solid, semi-solid or liquid, and can be an aqueous solution, non-aqueous solution or suspension, more preferably tablets, capsules, granules, injections or infusions, etc.

[0307] The pharmaceutical composition of the present invention is used to prevent and / or treat diseases associated with high expression of CD19 and / or BCMA. Preferably, the pharmaceutical composition of the present invention is used to prevent and / or treat cancers or tumors with high expression of CD19 and / or BCMA.

[0308] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 5 mg / kg body weight per day. In addition, the polypeptide of the present invention can also be used in conjunction with other therapeutic agents.

[0309] In one embodiment of the present invention, the multispecific antibodies of the present invention can be used together with a glucocorticoid to inhibit cytokine release without affecting target cell killing activity. Therefore, the pharmaceutical composition of the present invention can comprise the multispecific antibodies of the present invention and a glucocorticoid (e.g., dexamethasone).

[0310] In the present invention, preferably, the pharmaceutical composition of the present invention further comprises one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and the pharmaceutical carrier can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, and preferably includes a pharmaceutically acceptable excipient, filler or diluent, etc. More preferably, the pharmaceutical composition comprises 0.01 to 99.99% of the above-mentioned protein and 0.01 to 99.99% of a pharmaceutical carrier, and the percentages are the mass percentages of the pharmaceutical composition.

[0311] In the present invention, the pharmaceutical composition is preferably administered in an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative, or damaging condition. The effective amount can be determined on an individual basis and will be based in part on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by utilizing the above factors on an individual basis and using no more than routine experimentation.

[0312] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 50 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 20 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0313] The main advantages of the present invention include:

[0314] 1) The multispecific T cell engager of the present invention can simultaneously target BCMA, CD19, and CD3 with high binding force, thereby connecting target cells and T cells and promoting T cell killing.

[0315] 2) The multispecific T cell engager of the present invention can induce T cell activation and proliferation in human PBMCs.

[0316] 3) The multispecific T cell engager of the present invention, when used in combination with dexamethasone, can inhibit cytokine release without affecting the killing activity induced by the T cell engager, and has good safety.

[0317] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0318] Example 1 TriTE-Ab molecule expression and purification

[0319] TriTE-Ab molecular cloning

[0320] The trispecific antibody molecule contains three antibody sequences that bind to different antigen targets (CD19, BCMA, and CD3). The three different target antibody sequences were amplified by overlap extension PCR (overlap PCR) to obtain the first peptide chain gene (heavy chain) and the second peptide chain gene (light chain), and both peptide chains carried their own Kozak sequence and signal peptide sequence. The DNA fragments encoding the heavy and light chains of the trispecific antigen-binding protein were double-digested with restriction endonucleases (EcoR I and Not I) and cloned into a transient expression vector to obtain a transient expression vector expressing the heavy and light chains (Figure 2). At the same time, the transient expression vector contains an ampicillin resistance gene and a strong promoter CMV gene, which can promote high-level expression of the target gene in eukaryotic cells. The colonies were picked and sent for sequencing, and after sequence alignment, they were consistent with the theoretical sequence.

[0321] The positive clone plasmid was transformed into competent cells DH5a, and the selected clone was inoculated into 50 mL of E. coli culture medium and cultured overnight. The next day, the cells were collected and the transfection-grade plasmid was extracted. The transfection-grade plasmid was extracted according to the instructions of Plamid Plus Midi Kit (100) (QIAGEN, Cat No. 12945).

[0322] TriTE-Ab protein expression

[0323] Transient expression was performed using CHO-S cells. CHO-S cells were cultured and expanded using ExpiCHO Expression Medium (Gibco, Cat No. A29100). Transient transfection of CHO-S cells was performed according to the instructions of the ExpiFectamine CHO Transfection Kit (Gibco, Cat No. A29129). The specific steps are as follows:

[0324] On the first day, prepare 50 mL of culture medium with a cell density of 6 × 10 6 cells / mL of cell solution; add 20 μg of heavy chain plasmid and 20 μg of light chain plasmid to 2.0 mL of OptiPRO TM SFM, and 160 μL ExpiFectamine TM Add 1.84 mL of OptiPRO to CHO Reagent TM SFM; mix the plasmid solution with ExpiFectamine TM Mix the CHO Reagent solution and let it sit at room temperature. Within 5 minutes, add 4 mL of the mixed solution to the cell suspension. Transfer the cells to a 37°C, 5% CO2 incubator with a shaker at 110 rpm and continue incubation.

[0325] The next day, add 300 μL Enhancer and 8 mL ExpiCHOTM Feed the cells to the shaker flask. Transfer the cells to a 37°C, 5% CO2 incubator with a shaker speed of 110 rpm and continue culturing.

[0326] On the fifth day, the supernatant was collected by centrifugation and filtered through a 0.22 μM filter.

[0327] TriTE-Ab molecule purification

[0328] The target protein was purified using an IgG-CH1 affinity chromatography column (Thermo Fisher Scientific). The IgG-CH1 affinity column was equilibrated with phosphate buffer (10 mM sodium phosphate buffer + 50 mM sodium chloride, pH 7.2), loaded with the cell culture supernatant, and then washed with phosphate buffer (10 mM sodium phosphate buffer + 50 mM sodium chloride, pH 7.2). The target protein was eluted with 50 mM NaAc buffer (pH 3.5). The pH of the eluate was adjusted to 4.5-5.5 using Tris buffer.

[0329] Example 2 TriTE-Ab target binding experiment

[0330] TriTE-Ab antigen protein binding assay

[0331] The three-target antigen affinity of IMP-2023-031 and IMP-2023-033 was detected using a Gator instrument using an anti-human IgG Fc GenII (HFCII) probe (Gator; Cat: 160024). The three antigen proteins are fusion proteins of the extracellular region of the protein and human Fc (CD19 / Fc was purchased from Acro Biosystems, BCMA / Fc and CD3 / Fc were purchased from Sino Biological). The antigen concentration was diluted to 2.5-5.0 ug / mL with buffer PBST and solidified onto the anti-human IgG Fc probe. The drug molecules IMP-2023-031 or IMP-2023-033 to be tested were gradiently diluted into 96-well black microplate wells and reacted with the antigen solidified on the probe. The test results were analyzed using Gator data analysis software.

[0332] As shown in Table 1, the KD value of IMP-2023-031 binding to CD3 antigen protein was 1.83×10 -9 M, and the KD value of IMP-2023-033 binding to CD3 antigen protein was 1.04×10 -8 The KD value of IMP-2023-031 binding to CD19 antigen protein is 5.49×10 -10The KD value of IMP-2023-033 binding to CD19 antigen protein was 5.36×10 -10 M, indicating that both have similar binding abilities to CD19 antigen protein. At the same time, the KD values ​​for binding to BCMA antigen protein are both 10 -9 Within the order of M.

[0333] Table 1. Affinity of drug molecules binding to three antigenic proteins

[0334] TriTE-Ab cell binding assay

[0335] T cell preparation: Human peripheral blood mononuclear cells (hPBMCs) (purchased from OriBiotech) were activated for 3 days under the conditions of 5 μg / mL CD3 antibody (eBioscience, 16-0037-85) and 1 μg / mL CD28 antibody (eBioscience, 16-0289-85), and then co-cultured with 10 ng / mL IL-2 (PeproTech, 200-02) for 3 days, and the cells were collected and frozen. When necessary, the cells were revived and cultured under the conditions of 10 ng / mL IL-2 for 1-3 days, and the cells were collected for use. The culture medium used in the experiment was RPMI-1640 (Life technologies) containing 10% fetal bovine serum (Life technologies), hereinafter referred to as complete medium.

[0336] NCI-H929, K562-CD19, and Daudi are all suspension cells. NCI-H929 expresses BCMA; K562-CD19 expresses CD19; and Daudi expresses both BCMA and CD19. The culture medium is complete medium (10% fetal bovine serum + RPMI-1640).

[0337] First, dilute the cells to be used in PBS (flow cytometry buffer) containing 1% fetal bovine serum, and then add 45 μL per tube, 1×10 5 Cells were seeded into EP tubes and 5 μL of serially diluted antibody to be tested was added. Mix well and incubate at room temperature. After 60 minutes, cells were washed and resuspended in 50 μL of flow cytometry buffer. PE-anti-human lambda light chain antibody (BD Pharmingen, 555797) was added and incubated at room temperature in the dark. After 45-60 minutes, cells were analyzed using flow cytometry.

[0338] Since the antibody molecule to be tested contains human λ light chain, λ(+) positive cells represent cells that bind to the antibody molecule to be tested. GraphPad Prism software was used to analyze the data, with T cell binding rate as the ordinate and drug concentration as the abscissa. A 4-parameter curve was fitted to plot and calculate EC 50 The results are shown in Table 2 and Figure 3.

[0339] The data showed that IMP-2023-031 binds to T cells and EC 50 The value is 2.8×10 -9 M, while IMP-2023-033 binds to T cells EC 50 The value is 3.0×10 -8 ECs of IMP-2023-031 or IMP-2023-033 binding to T cells (CD3+), NCI-H929 cells (BCMA+), K562-CD19 cells (CD19+), and Daudi cells (BCMA+CD19+) 50 The values ​​are 10 -9 M, 10 -10 M and 10 -11 Within the M range, the binding abilities of the two molecules are close.

[0340] Table 2. Cell-bound EC 50 (M) value

[0341] Example 3 Detection of TriTE-Ab Target Cell Killing Ability

[0342] 1.1 Detection of cell target protein expression

[0343] 1.1.1 Experimental methods

[0344] The cells used in this experiment included Raji, Su-DHL-4, Daudi, K562, K562-CD19, K562-CD20, K562-BCMA, hPBMCs, NCI-H929, and RPMI-8226. All cells were suspension culture medium, using RPMI-1640 (Life Technologies) supplemented with 10% fetal bovine serum (Life Technologies), a complete medium.

[0345] The three cell lines K562-CD19, K562-CD20 and K562-BCMA are artificially constructed cells obtained by stably transfecting CD19, CD20 or BCMA into K562 cells, respectively.

[0346] Prepare the cells to be tested to the same concentration, about 0.5-1×10 6Cells were plated at 400 μL / mL and 50-100 μL of culture medium was added with APC anti-CD19 antibody (BD Pharmingen, 555415) or FITC anti-CD20 antibody (Biolegend, 357504). Incubate at room temperature in the dark. After 30-60 minutes, analyze the cells using flow cytometry and calculate the mean fluorescence intensity (MFI) for each cell line.

[0347] 1.1.2 Experimental Results

[0348] The results are shown in Figure 4 and Table 3. Raji, Su-DHL-4, and Daudi expressed both CD19 and BCMA, but Raji expressed very low BCMA levels, near background detection. K562-CD19 and hPBMCs expressed only CD19; NCI-H929, RPMI-8226, and K562-BCMA expressed only BCMA; and K562-CD20 expressed neither CD19 nor BCMA.

[0349] 1.2 Cell killing assay

[0350] 1.2.1 Experimental methods

[0351] The cells tested for protein expression were used as target cells, and the specific preparation was as described above.

[0352] Before the experiment, tumor target cells were washed and centrifuged, then stained with CFSE (carboxyfluorescein succinimidyl amino ester) (eBioscience, 65-0850-85) at room temperature for 10 minutes. The cells were then stained, washed, and resuspended in complete culture medium. If the effector cells were T cells, the cells were seeded at a 4:1 effector cell:target cell ratio. If the effector cells were hPBMCs, the cells were seeded at a 10:1 effector cell:target cell ratio. For hPBMC autologous B cell cytotoxicity assays, hPBMCs were directly plated. The molecule to be tested was added and incubated.

[0353] After 24 hours, 7-AAD (BD, 51-68981E or Biolegend, 420404) was added to the corresponding wells of the 96-well plate. After staining for 15 minutes, the cells were analyzed by flow cytometry.

[0354] CFSE + Represents target cells, 7-AAD - Represents viable cells, CFSE + 7-AAD - Represents surviving target cells. Cytotoxicity can be calculated using the following formula:

[0355] hPBMCs autologous B cell killing assay: Before adding 7-AAD, add PE-anti-CD20 (Biolegend, 375503) and incubate at room temperature for 30-60 minutes. Finally, analyze the cells by flow cytometry.

[0356] CD20 + Represents target cells, 7-AAD - Represents viable cells, CD20 + 7-AAD - Represents surviving target cells. Cytotoxicity can be calculated using the following formula:

[0357] GraphPad Prism software was used to analyze the data. The cell killing rate was used as the vertical axis and the drug concentration was used as the horizontal axis. A four-parameter curve was fitted to plot the data and calculate the EC. 50 value.

[0358] 1.2.2 Results of IMP-2023-033 Killing Target Cells Experiment

[0359] The results are shown in Figure 5. The target cells in Figures 5A-D are CD19 and BCMA dual-expressing cells, and IMP-2023-033 kills them. 50 The target cells in Figure 5C-D are all Raji, and the effector cells are hPBMCs and T cells, respectively. The results show that the killing effect of hPBMCs and T cells is basically the same. The target cells in Figure 5E-F are cells that express CD19 but do not express BCMA. IMP-2023-033 kills ECs on them. 50 The range is 3.3-13 pM, indicating that IMP-2023-033 can act through anti-CD19. The target cells in Figure 5G-I are cells that express BCMA but do not express CD19. IMP-2023-033 kills ECs on them. 50 The concentration range is between 16 and 34 pM, indicating that IMP-2023-033 can exert its effect through anti-BCMA. The target cells in Figure 5J are K562-CD20 cells, which express neither CD19 nor BCMA. IMP-2023-033 has no cytotoxic activity against them, while the positive molecule IMP-2023-026 (anti-CD3 x anti-CD20) has cytotoxic activity against them. This indicates that IMP-2023-033 mediates specific target cell killing and has no activity against cells that do not express the target protein, indicating a relatively good safety profile.

[0360] Table 3 Target cell target protein expression and IMP-2023-033 molecule lethality

[0361] *: MFI, mean fluorescence intensity.

[0362] IMP-2023-033 kills ECs on target cells that express only CD19 50 Killing ECs on target cells expressing only BCMA at 3.3-13pM 50 Between 16-34 pM, and killing EC on target cells expressing both CD19 and BCMA 50 It is between 0.045-0.844 pM, indicating that dual-expressing target cells are more easily killed by IMP-2023-033.

[0363] 1.2.3 Target Cell Killing Experimental Results of IMP-2023-033 and Control Molecules

[0364] The cytotoxicity of two bispecific antibodies, IMC-035 (anti-CD3 x anti-CD19) and IMC-036 (anti-CD3 x anti-BCMA), was compared with that of IMP-2023-033 (anti-CD3 x anti-CD19 x anti-BCMA). The structures of IMC-035 and IMC-036 are shown in Figure 1.

[0365] Using K562-BCMA and K562-CD19, which highly express CD19 and BCMA, respectively, as target cells, the results are shown in Table 4 and Figures 6A-B. IMP-2023-033 demonstrated high cytotoxicity against target cells expressing both antigens and reduced off-target activity compared to the two control molecules, IMC-035 and IMC-036.

[0366] Table 4 Comparison of the killing effect of IMP-2023-033 and control molecules on target cells

[0367] 1.3 T cell activation

[0368] 1.3.1 Experimental methods

[0369] hPBMCs were resuspended in complete culture medium and seeded into 96-well cell culture plates. The detection antibody was diluted to different concentrations with complete culture medium, and the test antibody IMP-2023-033 or diluent was added to the corresponding wells. Finally, the 96-well cell culture plate containing cells was placed in a CO2 incubator and incubated at 37°C. After 24 hours, APC-anti-CD4 antibody (BD Pharmingen, 551980), PE-anti-CD8 antibody (Biolegend, 301008) and FITC-anti-CD69 antibody (BD Pharmingen, 555530) were added and incubated at room temperature for 30-60 minutes. The cells were analyzed using a flow cytometer to calculate CD4 + T or CD8 + The proportion of CD69-positive cells in T subsets.

[0370] 1.3.2 Experimental Results

[0371] CD69 is a marker of T cell activation. This study examined CD69 expression on T cells to confirm whether IMP-2023-033 mediates T cell activation.

[0372] Graphpad Prism software was used to fit the data obtained by flow cytometry to a four-parameter equation to obtain the dose-response curve and the half-effective concentration (EC) 50 , as shown in Figure 7. The experimental results showed that IMP-2023-033 could induce CD4 + T cells or CD8 + CD69 expression on T cells, and its EC 50 26 and 29 pM respectively.

[0373] Conclusion: IMP-2023-033 can induce T cell activation in human PBMCs in a concentration-dependent manner.

[0374] 1.4 T cell proliferation

[0375] 1.4.1 Experimental methods

[0376] Resuspend hPBMCs in complete medium and seed into 96-well cell culture plates. Dilute the test antibody to various concentrations in complete medium and add the test antibody IMP-2023-033 or its dilution to the corresponding wells. Finally, incubate the 96-well cell culture plates containing cells in a CO2 incubator at 37°C.

[0377] After 72 hours, T cells were collected and processed according to the Fixation / Permeabilization Solution Kit (Invitrogen, 00-5523-00), and APC-anti-CD4 antibody (Biolegend, 344614), PE-anti-CD8 antibody (Biolegend, 301008), and FITC-anti-Ki67 antibody (BD Pharmingen, 556026) were added. Then, the cells were incubated in the dark at room temperature for 45-60 minutes. Finally, flow cytometry was used to analyze the CD4 T cell population and count the number of T cells. + Ki67 + or CD8 + Ki67 + Proportion.

[0378] 1.4.2 Experimental Results

[0379] Ki67 is a marker protein for cell proliferation. In this study, we detected the expression of Ki67 in T cells to investigate the activity of IMP-2023-033 in inducing T cell proliferation.

[0380] The results showed that under the same conditions, CD4 + T cell proliferation rate is lower than CD8 + T cell proliferation rate, but EC 50 The difference is not big, 59 and 80 pM respectively, the results are shown in Figure 8.

[0381] Conclusion: The results showed that IMP-2023-033 could induce T cell proliferation in a concentration-dependent manner.

[0382] 1.5 Dexamethasone inhibits cytokine release

[0383] 1.5.1 Experimental methods

[0384] hPBMCs and Raji cells were resuspended in complete culture medium and seeded into 96-well plates at a 10:1 effector cell:target cell ratio. Serial dilutions of IMP-2023-033 or IMP-2023-033 plus dexamethasone (Dex) (Sigma, 50-02-2) were added and incubated.

[0385] After 24 hours, save the cell supernatant or cell lysate for the following experiments.

[0386] The LDH kit (Promega, batch number: G1782) was used to determine the killing curve of IMP-2023-033-mediated T cells against Raji cells in the presence or absence of Dex according to the method provided in the instructions. The specific method is: take the cell supernatant or cell lysate, add the LDH substrate, stop the reaction after 30 minutes, and measure the absorbance value at 490nm (OD490). LDH is released by dead cells, so the LDH content in the cell supernatant is related to the degree of cell death. In this experiment, the cell death rate was calculated according to the following formula:

[0387] in,

[0388] [OD490] 样品 represents the absorbance value of the supernatant of wells containing Raji cells, PBMCs, and IMP-2023-033;

[0389] [OD490] 最小 represents the absorbance value of the supernatant of the wells containing Raji cells and PBMCs;

[0390] [OD490] 最大 Represents the absorbance value of the mixed liquid in the wells containing Raji cells, supernatant, and cell lysate added during detection;

[0391] [OD490] 本底 Represents the absorbance value of the mixed liquid in the wells of cell culture medium and cell lysis solution added during detection.

[0392] The same sample was used to detect relevant cytokines according to the requirements of IL-2 ELISA kit (R&D, DY202), IL-6 ELISA kit (R&D, DY206), TNF-α ELISA kit (R&D, DY210), and IFN-γ ELISA kit (R&D, DY285B).

[0393] 1.5.2 Experimental Results

[0394] T cell activation and proliferation can cause cytokine release syndrome (CRS), and fulminant CRS can be life-threatening. Glucocorticoids, such as dexamethasone (Dex), can effectively alleviate CRS caused by T cell activation and are therefore a commonly used clinical approach to manage CRS.

[0395] The results showed that 1.5×10 -7M Dex had no significant effect on IMP-2023-033-mediated T cell killing activity against Raji cells ( Figure 9A ), almost completely blocked the release of IL-6 ( Figure 9C ), and inhibited the secretion of IL-2, TNF-α, and IFNγ to varying degrees ( Figures 9B, D, and E ).

[0396] Table 5 Dexamethasone inhibits cytokine release

[0397] Conclusion: The results showed that dexamethasone Dex could inhibit the release of cytokines without affecting the killing activity of IMP-2023-033.

[0398] 1.6 In vivo antitumor pharmacodynamics studies

[0399] 1.6.1 Experimental methods

[0400] Human PBMCs were resuspended in PBS and the cell density was adjusted to 2.5 × 10 7 / mL, 5 days before tumor cell inoculation (Day-5), 5×10 6 The cells were injected intraperitoneally for immune reconstitution, with 0.2 mL injected into each mouse.

[0401] After 5 days (Day 0), NCI-H929 cells in the logarithmic growth phase of in vitro culture were collected, resuspended in PBS and the cell density was adjusted to 3×10 7 / mL, 3×10 per mouse 6 The cells were injected subcutaneously, with 0.1 mL injected into each mouse.

[0402] On Day 7 after inoculation, when the tumor grew to an average volume of approximately 80 mm 3 Mice with normally distributed tumor volumes were randomly divided into five groups: a vehicle control (PBS) group, a subcutaneous low-dose IMP-2023-033 group (300 μg / kg, SC), a subcutaneous medium-dose IMP-2023-033 group (1000 μg / kg, SC), a subcutaneous high-dose IMP-2023-033 group (3000 μg / kg, SC), and an intraperitoneal administration group (1000 μg / kg, IP). Each group was administered subcutaneously or intraperitoneally every other day. The high-dose IMP-2023-033 group (3000 μg / kg, SC) and the intraperitoneal administration group (1000 μg / kg, IP) were administered for two weeks, while the other groups were administered for three weeks, after which the drugs were discontinued for observation.

[0403] The long diameter (L) and short diameter (W) of the tumor of the tumor-bearing mice were measured with a vernier caliper twice a week, and the tumor volume (TV) and tumor growth inhibition rate (TGI%) were calculated according to the following formula. i The average tumor volumes of mice in the treatment group at grouping (Day 7) and Day 7+i, avC0 and avC i The mean tumor volumes of mice in the vehicle control group at grouping (Day 7) and Day 7+1, respectively.

[0404] Tumor volume (TV) = (L × W 2 ) / 2

[0405] Experimental results

[0406] Subcutaneous injection of IMP-2023-033 exhibited dose-dependent antitumor efficacy in NCI-H929 tumor-bearing mice reconstituted with human PBMC immunity. The TGI% on Day 27 of the subcutaneous low-dose IMP-2023-033 group (IMP-2023-033, 300 μg / kg, SC), subcutaneous medium-dose group (IMP-2023-033, 1000 μg / kg, SC), and subcutaneous high-dose group (IMP-2023-033, 3000 μg / kg, SC) were 43.1%, 65.9%, and 94.2%, respectively. Compared with the solvent control group during the same period, the P values ​​were 0.0303, 0.0009, and 0.0002, respectively, and the differences were statistically significant.

[0407] After subcutaneous administration of 3000 μg / kg or intraperitoneal administration of 1000 μg / kg IMP-2023-033 to NCI-H929 tumor-bearing mice reconstituted with human PBMCs, 60% (3 / 5) and 80% (4 / 5) of the mice showed tumor regression, respectively.

[0408] 1.6.2 Conclusion

[0409] The results are shown in Figure 10. Subcutaneous administration of IMP-2023-033 can dose-dependently inhibit the growth of BCMA-positive NCI-H929 mouse xenograft tumors, and high-dose IMP-2023-033 can cause tumor regression.

[0410] Example 4 Detection of the long-acting molecular effect of TriTE-Ab

[0411] 1.1 Cell Binding Assay of TriTE-Ab Long-Acting Molecules

[0412] The experimental method was the same as that of the TriTE-Ab cell binding experiment. The results are shown in Table 6 and Figure 11.

[0413] The data showed that IMP-2023-036 and IMP-2023-037 bind to T cells in ECs 50 The values ​​are 2.2×10 -8 M, 1.4×10 -8 M, while T cell binding of IMP-2023-038 and IMP-2023-039 to EC 50 The values ​​are 1.3×10 -9 M, 2.6×10 -9 M. In binding to NCI-H929 cells (BCMA+), IMP-2023-037 binds to EC 50 Value is 10 -8 M level, other EC 50 The values ​​are all 10 -9 M level. IMP-2023-037 binds weakly to K562-CD19 cells (CD19+), EC 50 The value is 1.0×10 -8 M; while the other three molecules bind to EC 50 The values ​​are all above 10 -10 M level. IMP-2023-037 has the weakest binding to Daudi cells (BCMA+CD19+), EC 50 The value is 1.7×10 -9 M; IMP-2023-039 combined with EC 50 The value is 1.6×10 -10 M; while the other two molecules bind to EC 50 The values ​​are all above 10 -11 M level.

[0414] Table 6. Cell-bound EC 50 (M) value

[0415] 1.2 Cell killing assay

[0416] 1.2.1 Experimental methods

[0417] The cells tested for protein expression were used as target cells, and the specific preparation was as described above.

[0418] Before the experiment, target cells were washed and centrifuged, then stained with CFSE (eBioscience, 65-0850-85) for 10 minutes at room temperature. The cells were then washed and resuspended in complete culture medium. The cells were then seeded at a 4:1 ratio of effector (T) cells to target cells and incubated with the test molecule. After 24 hours, 7-AAD (Biolegend, 420404) was added to the corresponding wells of the 96-well plate. After staining for 10-15 minutes, the cells were analyzed by flow cytometry.

[0419] CFSE + Represents target cells, 7-AAD - Represents viable cells, CFSE + 7-AAD - Represents surviving target cells. Cytotoxicity can be calculated using the following formula:

[0420] GraphPad Prism software was used to analyze the data. The cell killing rate was used as the vertical axis and the drug concentration was used as the horizontal axis. A four-parameter curve was fitted to plot the data and calculate the EC. 50 value.

[0421] 1.2.2 Killing test results

[0422] The results are shown in Figure 12 and Table 7. The target cells in Figure 12A are NCI-H929 cells that express BCMA but do not express CD19. The data show that IMP-2023-037 and IMP-2023-038 have anti-tumor activity against EC. 50 were greater than 1000 pM, while IMP-2023-036 and IMP-2023-039 had the same killing activity EC 50 The target cells in Figure 12B are K562-CD19 cells that express CD19 but not BCMA. The data show that IMP-2023-038 has the best killing activity against them. 50 was 1.3 pM, while IMP-2023-037 had the weakest killing activity, EC 50 The killing activity EC of IMP-2023-036 and IMP-2023-039 is in the middle. 50 The target cells in Figure 12C are Daudi cells that express both CD19 and BCMA. The data show that IMP-2023-036, IMP-2023-038, and IMP-2023-039 have similar cytotoxicity against them. 50 The killing activity of IMP-2023-037 was weak, and the EC 50 It is 204pM.

[0423] Table 7 Summary of IMP-2023 Killing Activity

[0424] IMP-2023-037 and IMP-2023-038 had weak anti-cytotoxic activity against NCI-H929 cells that only expressed BCMA. 50Greater than 1000pM; but they have killing activity against K562-CD19 and Daudi expressing CD19, indicating that IMP-2023-037 and IMP-2023-038 may act through CD19 rather than BCMA and are not suitable triple-target antibodies. IMP-2023-036 and IMP-2023-039 have killing activity against all three target cell lines and are triple-target antibodies. Considering that the killing activity of IMP-2023-039 against target cells expressing only BCMA or CD19 is relatively close, EC 50 They were 80 pM and 14 pM respectively, while those of IMP-2023-039 were 242 pM and 7.2 pM respectively. Therefore, IMP-2023-039 should be better than IMP-2023-036, so IMP-2023-039 was selected for further experiments.

[0425] 1.3 T cell proliferation

[0426] 1.3.1 Experimental methods

[0427] hPBMCs and Daudi were seeded into 96-well cell culture plates at a ratio of 10:1, and then the test antibody IMP-2023-039 was added. Finally, the 96-well cell culture plates containing cells were placed in a CO2 incubator and incubated at 37°C.

[0428] After 72 hours, T cells were collected and processed according to the Fixation / Permeabilization Solution Kit (Invitrogen, 00-5523-00), and APC-anti-CD4 antibody (Biolegend, 344614), PE-anti-CD8 antibody (Biolegend, 301008), and FITC-anti-Ki67 antibody (BD Pharmingen, 556026) were added. Then, the cells were incubated in the dark for 30-60 minutes. Finally, flow cytometry was used to analyze the CD4 T cell population and count the number of T cells. + Ki67 + or CD8 + Ki67 + Proportion.

[0429] 1.3.2 Experimental Results

[0430] Ki67 is a marker protein for cell proliferation. In this study, we detected the expression of Ki67 in T cells to investigate the activity of IMP-2023-039 in inducing T cell proliferation.

[0431] The results showed that under the same conditions, CD4 + T cell proliferation rate is lower than that of CD8 + T cell proliferation rate, but EC 50The difference is not big, both are 1.6pM, the results are shown in Figure 13.

[0432] Conclusion: The results showed that IMP-2023-039 could induce T cell proliferation in a concentration-dependent manner.

[0433] 1.4 Dexamethasone inhibits cytokine release

[0434] 1.4.1 Experimental methods

[0435] Cell Killing Assay: hPBMCs and CFSE-prestained Daudi were resuspended in complete culture medium at a ratio of 10:1 and seeded into 96-well plates. Serial dilutions of IMP-2023-039 or IMP-2023-039 plus dexamethasone (Dex) (Sigma, 50-02-2) were added and incubated for 24 hours for cell killing experiments. Specific methods are as described above.

[0436] Cytokine detection experiment: hPBMCs and Daudi were resuspended in complete medium at a ratio of 10:1 and seeded into 48-well plates. Serial dilutions of IMP-2023-039 or IMP-2023-039 plus dexamethasone (Dex) (Sigma, 50-02-2) were added and incubated. After 24 hours, the 48-well plates containing cells and supernatant were frozen at -80°C to prepare cell lysates. During the experiment, the prepared cell lysates were thawed and the relevant cytokines were detected according to the IL-2 ELISA kit (R&D, DY202), IL-6 ELISA kit (R&D, DY206), TNF-α ELISA kit (R&D, DY210), and IFN-γ ELISA kit (R&D, DY285B).

[0437] 1.4.2 Experimental Results

[0438] The results are shown in Figure 14 and Table 8. -7 M Dex had no significant effect on IMP-2023-039-mediated T cell killing activity against Daudi cells ( FIG. 14A ), but almost completely blocked the secretion of IL-2, IL-6, TNF-α, and IFNγ ( FIG. 14B-E ).

[0439] Table 8 Dexamethasone inhibits cytokine release

[0440] Conclusion: The results showed that dexamethasone Dex could inhibit the release of cytokines without affecting the killing activity of IMP-2023-039.

[0441] Table 9 Antibody CDR sequences

[0442] Table 10 Antibody variable region sequences

[0443] Table 11 Antibody light and heavy chain sequences

[0444] Connector peptide:

[0445] Connector peptide:

[0446] Connector peptide:

[0447] Heavy chain constant region 1 (CH1):

[0448] Light chain constant region (CL):

[0449] IgG Fc1:

[0450] IgG Fc2:

[0451] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An anti-BCMA antibody or an antigen-binding fragment thereof, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region, Wherein, the heavy chain variable region has the following complementarity determining regions CDR: VH-CDR1 shown in SEQ ID NO: 14, VH-CDR2 shown in SEQ ID NO: 15, and VH-CDR3 shown in SEQ ID NO: 16; Furthermore, the light chain variable region has the following complementarity determining regions (CDRs): VL-CDR1 shown in SEQ ID NO: 17, VL-CDR2 shown in SEQ ID NO: 18, and VL-CDR3 shown in SEQ ID NO:

19.

2. The anti-BCMA antibody or antigen-binding fragment thereof according to claim 1, wherein The amino acid sequence of the antibody heavy chain variable region is as shown in SEQ ID No: 25 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto, and / or The amino acid sequence of the antibody light chain variable region is shown in SEQ ID No: 26 or has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence homology thereto.

3. An anti-CD3 antibody or antigen-binding fragment thereof, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region, Wherein, the heavy chain variable region has the following complementarity determining regions CDR: VH-CDR1 shown in SEQ ID NO: 1, VH-CDR2 shown in SEQ ID NO:7, and VH-CDR3 shown in SEQ ID NO: 3; Furthermore, the light chain variable region has the following complementarity determining regions (CDRs): VL-CDR1 shown in SEQ ID NO:4, VL-CDR2 shown in SEQ ID NO:5, and VL-CDR3 shown in SEQ ID NO:

6.

4. A multispecific antibody, characterized in that The multispecific antibody comprises the anti-BCMA antibody or antigen-binding fragment thereof according to claim 1, and / or the anti-CD3 antibody or antigen-binding fragment thereof according to claim 3.

5. The multispecific antibody according to claim 4, wherein The multispecific antibody is a trispecific antibody, and the trispecific antibody comprises: 1) a CD19 targeting binding domain comprising one or more antigen binding domains that specifically bind to the CD19 protein; 2) a BCMA targeting binding domain, comprising one or more antigen-binding domains that specifically bind to a BCMA protein, wherein the antigen-binding domain that specifically binds to a BCMA protein is the anti-BCMA antibody or antigen-binding fragment thereof according to claim 1; and 3) CD3 targeting binding domain, which comprises one or more antigen binding domains that specifically bind to CD3 protein.

6. The multispecific antibody according to claim 4, wherein The multispecific antibody is a bispecific antibody, and the bispecific antibody comprises: 1) a BCMA targeting binding domain comprising one or more antigen-binding domains that specifically bind to a BCMA protein, wherein the antigen-binding domain that specifically binds to a BCMA protein is the anti-BCMA antibody or antigen-binding fragment thereof according to claim 1; and 2) a CD3 targeting binding domain comprising one or more antigen binding domains that specifically bind to the CD3 protein; or, The bispecific antibody comprises: 1) a CD19 targeting binding domain comprising one or more antigen binding domains that specifically bind to the CD19 protein; and 2) CD3 targeting binding domain, which comprises one or more antigen binding domains that specifically bind to CD3 protein.

7. The multispecific antibody according to claim 5 or 6, wherein The antigen binding domain that specifically binds to the CD19 protein includes a heavy chain variable region and a light chain variable region, Wherein, the heavy chain variable region has the following complementarity determining regions CDR: VH-CDR1 shown in SEQ ID NO:8, VH-CDR2 shown in SEQ ID NO:9, and VH-CDR3 shown in SEQ ID NO: 10; Furthermore, the light chain variable region has the following complementarity determining regions (CDRs): VL-CDR1 shown in SEQ ID NO: 11, VL-CDR2 shown in SEQ ID NO: 12, and VL-CDR3 shown in SEQ ID NO:

13.

8. The multispecific antibody according to any one of claims 5 to 7, wherein The CD3 targeting binding domain includes a heavy chain variable region and a light chain variable region, Wherein, the heavy chain variable region has the following complementarity determining regions CDR: VH-CDR1 shown in SEQ ID NO: 1, VH-CDR2 shown in SEQ ID NO: 2 or 7, and VH-CDR3 shown in SEQ ID NO: 3; Furthermore, the light chain variable region has the following complementarity determining regions (CDRs): VL-CDR1 shown in SEQ ID NO:4, VL-CDR2 shown in SEQ ID NO:5, and VL-CDR3 shown in SEQ ID NO:

6.

9. The multispecific antibody according to claim 5 or 6, wherein The trispecific antibody has a structure as shown in any one of the following formulae IIIa-IIIh: Wherein, "-" is each independently a bond or a peptide linker; ScFv CD19 is the CD19 targeting binding domain; ScFv BCMA is the BCMA targeting binding domain; ScFv CD3 is the CD3 targeting binding domain; VH CD3 The heavy chain variable region is the CD3 targeting binding domain; VL CD3 The light chain variable region is the CD3 targeting binding domain; VH BCMA The heavy chain variable region is the BCMA targeting binding domain; VL BCMA The light chain variable region is the BCMA targeting binding domain; VH CD19 The heavy chain variable region is the CD19 targeting binding domain; VL CD19 The light chain variable region is the CD19 targeting binding domain; CH1 is the heavy chain constant region 1; CL is the light chain constant region; Fc1 and Fc2 are each independently free or Fc domain; L3 and L4 are each independently a connecting peptide; It is a disulfide bond or a covalent bond.

10. The multispecific antibody according to claim 9, wherein The CD19 targeting binding domain includes a heavy chain variable region as shown in SEQ ID NO: 23 and a light chain variable region as shown in SEQ ID NO:

24.

11. The multispecific antibody according to claim 9, wherein The BCMA targeting binding domain includes a heavy chain variable region as shown in SEQ ID NO: 25 and a light chain variable region as shown in SEQ ID NO:

26.

12. The multispecific antibody according to claim 9, wherein The CD3 targeting binding domain includes the heavy chain variable region shown in SEQ ID NO: 20 and the light chain variable region shown in SEQ ID NO: 21; or The CD3 targeting binding domain includes a heavy chain variable region as shown in SEQ ID NO: 22 and a light chain variable region as shown in SEQ ID NO:

21.

13. The multispecific antibody according to claim 9, wherein The multispecific antibody has a heavy chain and a light chain selected from the group consisting of: 1) a heavy chain as set forth in SEQ ID NO: 27; and a light chain as set forth in SEQ ID NO: 28; 2) a heavy chain as shown in SEQ ID NO: 29; and a light chain as shown in SEQ ID NO: 30; 3) a heavy chain as shown in SEQ ID NO: 31; and a light chain as shown in SEQ ID NO: 28; 4) a heavy chain as shown in SEQ ID NO: 32; and a light chain as shown in SEQ ID NO: 30; 5) a heavy chain as shown in SEQ ID NO: 39; and a light chain as shown in SEQ ID NO: 40; 6) a heavy chain as shown in SEQ ID NO:41; and a light chain as shown in SEQ ID NO:42; 7) a heavy chain as shown in SEQ ID NO: 43; and a light chain as shown in SEQ ID NO: 44; 8) a heavy chain as shown in SEQ ID NO: 45; and a light chain as shown in SEQ ID NO: 40; 9) the heavy chain shown in SEQ ID NO: 29; and the light chain shown in SEQ ID NO: 28; 10) a heavy chain as shown in SEQ ID NO: 31; and a light chain as shown in SEQ ID NO: 37; 11) the heavy chain shown in SEQ ID NO: 38; and the light chain shown in SEQ ID NO:

28.

14. A recombinant protein, characterized in that The recombinant protein comprises: (i) the anti-BCMA antibody or antigen-binding fragment thereof according to claim 1, the anti-CD3 antibody or antigen-binding fragment thereof according to claim 3, or the multispecific antibody according to claim 4; and (ii) Tag sequences that facilitate expression and / or purification.

15. A polynucleotide, characterized in that The polynucleotide encodes the anti-BCMA antibody or antigen-binding fragment thereof according to claim 1, the anti-CD3 antibody or antigen-binding fragment thereof according to claim 3, or the multispecific antibody according to claim 4.

16. A carrier, characterized in that The vector contains the polynucleotide according to claim 15.

17. A genetically engineered host cell, characterized in that The host cell contains the vector according to claim 16, or the exogenous polynucleotide according to claim 15 is integrated into its genome.

18. An antibody conjugate, characterized in that The antibody conjugate contains: (a) an antibody portion selected from the group consisting of: The anti-BCMA antibody or antigen-binding fragment thereof according to claim 1, the anti-CD3 antibody or antigen-binding fragment thereof according to claim 3, or the multispecific antibody according to claim 4; and (b) a conjugated moiety conjugated to the antibody portion, wherein the conjugated moiety is selected from the group consisting of a detectable label, a drug, or a combination thereof.

19. Use of an active ingredient in the preparation of a medicament for preventing and / or treating a disease associated with high expression of CD19 and / or BCMA, characterized in that: The active ingredient is selected from the following group: The anti-BCMA antibody or antigen-binding fragment thereof according to claim 1, the anti-CD3 antibody or antigen-binding fragment thereof according to claim 3, the multispecific antibody according to claim 4, the recombinant protein according to claim 14, the antibody conjugate according to claim 18, or a combination thereof.

20. A pharmaceutical composition, characterized in that The pharmaceutical composition contains: (i) an active ingredient selected from the group consisting of: The anti-BCMA antibody or antigen-binding fragment thereof according to claim 1, the anti-CD3 antibody or antigen-binding fragment thereof according to claim 3, the multispecific antibody according to claim 4, the recombinant protein according to claim 14, the antibody conjugate according to claim 18, or a combination thereof; and (ii) a pharmaceutically acceptable carrier.

21. A method for treating a disease associated with high expression of CD19 and / or BCMA, characterized in that: administering to a subject in need thereof an effective amount of the anti-BCMA antibody or antigen-binding fragment thereof according to claim 1, the anti-CD3 antibody or antigen-binding fragment thereof according to claim 3, the multispecific antibody according to claim 4, the recombinant protein according to claim 14, the antibody conjugate according to claim 18, or the pharmaceutical composition according to claim 20, or a combination thereof.

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