Trispecific antibody targeting CD19, BCMA, and CD3 and use thereof

Abstract

The present invention relates to a trispecific antibody molecule targeting CD19, BCMA, and CD3. The present invention further provides a nucleic acid molecule encoding the antibody, an expression vector for expressing the antibody, a host cell, and a preparation method therefor. The present invention also provides diagnostic and therapeutic methods using the antibody of the present invention.

Description

Trispecific antibodies targeting CD19, BCMA, and CD3 and their applications

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to Chinese patent applications CN202510116675.0, filed on January 24, 2025, and CN202511131820.9, filed on August 13, 2025, the entire contents of which are incorporated herein by reference.

[0003] This invention relates to a trispecific antibody molecule targeting CD19, BCMA, and CD3. The invention further provides a nucleic acid molecule encoding the antibody, an expression vector for expressing the antibody, a host cell, and a method for preparing the same. The invention also provides diagnostic and therapeutic methods using the antibodies of this invention. Background technology:

[0004] CD3 (Cluster 3) is a protein complex that, together with T cell antigen receptor α, T cell antigen receptor β, and two ζ chains, constitutes the T cell receptor complex, participating in the activation of cytotoxic T cells (CD8+ T cells) and T helper cells (CD4+ T cells). The CD3 protein complex is a definitive marker of the T cell lineage; therefore, anti-CD3 antibodies can be effectively used as T cell markers.

[0005] CD3 antibodies recognize all T cells and react with 70%-80% of human peripheral blood lymphocytes and 65%-85% of thymocytes. Activated T cells by CD3 antibodies direct themselves to the vicinity of target cells, where they come into contact and form synapses. This triggers activation of the T cell receptor (TCR) signaling pathway, leading to the expression and release of granzymes, which in turn causes perforation of the tumor cell membrane, resulting in cytolysis and apoptosis. Activation of the TCR signaling pathway also induces the expression and release of a series of cytokines, such as IL-2, which in turn stimulates T cell proliferation, amplifying the immune killing effect. Preclinical studies have demonstrated that, in the presence of target cells, CD3 bispecific antibody molecules targeting tumor-associated antigens can effectively activate T cells and stimulate their proliferation, while simultaneously causing target cell death. Several CD3-binding antibody molecules are now known, particularly bispecific antibody molecules containing CD3 binding specificity.

[0006] Existing technologies have constructed a variety of bispecific antibody molecules based on CD3, such as bispecific antibodies that specifically bind to CD3 and CD19 (US11780920B2, US11365254B2) or bispecific antibodies that specifically bind to CD3 and BCMA (US10072088B2).

[0007] However, there remains a need to develop trispecific antibodies targeting CD19, BCMA, and CD3, especially trispecific antibodies with improved performance. Summary of the Invention

[0008] This invention develops a novel trispecific antibody with a specific structure targeting CD19, BCMA, and CD3. In some embodiments, the trispecific antibody has high affinity for cell surface CD19 and BCMA antigens and weak affinity for T cell surface CD3, which helps to obtain stronger T cell activation and / or cell killing function, and optionally can also reduce off-target T cell activation caused by cells that do not express CD19 or BCMA.

[0009] In some embodiments, the trispecific antibody of the present invention comprises or is composed of the following:

[0010] Heavy chain 1: From the N-terminus to the C-terminus, it contains or consists of the following: VH BCMA -First CH1-First Fc region, wherein the heavy chain constant region CH1 is connected at its C end to the N end of the first Fc region directly or via a connector (preferably directly);

[0011] Light chain 1: Composed of or consisting of the following from the N-terminus to the C-terminus: VL BCMA -First CL;

[0012] Heavy chain 2: From the N-terminus to the C-terminus, it contains or consists of the following: VH CD19 -Second CH1-scFv CD3 - Second Fc region, where the C-terminus of the second CH1 is connected to scFv CD3 The N-terminus is connected directly or via connector 1, and scFv CD3 The C end is directly or via connector 3 connected to the N end of the second Fc region;

[0013] Light chain 2: From the N-terminus to the C-terminus, it includes or consists of the following: VL CD19 -Second CL; or

[0014] Heavy chain 1: From the N-terminus to the C-terminus, it contains or consists of the following: VH CD19 -First CH1-First Fc region, wherein the heavy chain constant region CH1 is connected at its C end to the N end of the first Fc region directly or via a connector (preferably directly);

[0015] Light chain 1: Composed of or consisting of the following from the N-terminus to the C-terminus: VL CD19 -First CL;

[0016] Heavy chain 2: From the N-terminus to the C-terminus, it contains or consists of the following: VH BCMA-Second CH1-scFv CD3 - Second Fc region, where the C-terminus of the second CH1 is connected to scFv CD3 The N-terminus is connected directly or via connector 1, scFv C The C end is directly or via connector 3 connected to the N end of the second Fc region;

[0017] Light chain 2: From the N-terminus to the C-terminus, it includes or consists of the following: VL BCMA -Second CL;

[0018] Where “-” represents a connector or direct connection;

[0019] VH BCMA This refers to the heavy chain variable region VH that specifically binds to the antigen-binding region of BCMA, and VL BCMA It refers to the light chain variable region (VL) of the antigen-binding region that specifically binds to BCMA;

[0020] VH CD19 This refers to the heavy chain variable region VH that specifically binds to the antigen-binding region of CD19, and VL CD19 It refers to the light chain variable region (VL) of the antigen-binding region that specifically binds to CD19;

[0021] The first CH1 and the second CH1 refer to the CH1 domain of the heavy chain constant region, respectively.

[0022] The first CL and the second CL refer to the constant regions of the light chain, respectively.

[0023] VH BCMA -CH1 and VL BCMA -CL constitutes the Fab region, which is the antigen-binding region that specifically binds to BCMA. BCMA ;

[0024] VH CD19 -CH1 and VL CD19 -CL constitutes the Fab region, which serves as the antigen-binding region specifically binding to CD19. CD19 ;

[0025] scFv CD3 This refers to the scFv, which is the antigen-binding region that specifically binds to CD3, and consists of VH cells directly or via linker 2. CD3 and VL CD3 The polypeptide chain composition, preferably the scFv CD3 From the N-terminus to the C-terminus, it includes or consists of the following: VH CD3 -VL CD3 VH CD3 C-terminus and VL CD3 The N-terminus is connected directly or via connector 2;

[0026] Optionally, the first CH1 and the second CH1 are the same, and / or the first CL and the second CL are the same, and / or the first Fc region and the second Fc region are different, for example, by introducing different mutations in the first Fc region and the second Fc region based on the Innobody platform to prevent heavy chain mismatch.

[0027] In a preferred embodiment, the BCMA-targeting Fab of the trispecific antibody constructed in this invention is located on the first heavy chain and directly linked to the Fc region, while the CD19-targeting Fab is located on the second heavy chain and linked to the CD3-targeting scFv. This Fab placement maximizes the killing effect of BCMA and CD19-positive cells, respectively. Furthermore, due to the CD19 Fab masking effect, the CD3-binding scFv lacks the ability to bind to T cells when the antibody does not bind to BCAM or CD19. Only when the antibody binds to BCMA or CD19 can it bind to and activate T cells. Thus, the obtained trispecific antibody maintains high binding capacity to both CD19 and BCMA while reducing antibody waste caused by ineffective binding to T cells when not binding to BCMA or CD19, and also avoids the release of inflammatory factors caused by target-dependent T cell activation. In addition, the tandem arrangement of Fab and scFv in the trispecific antibody effectively prevents light and heavy chain mismatches. Combined with the Innobody platform that prevents heavy chain mismatches, the trispecific antibodies can be correctly paired at a high proportion when expressed in the same cell.

[0028] In some embodiments, the trispecific antibody targeting CD19, BCMA, and CD3 of the present invention has one or more of the following properties:

[0029] (1) Specific binding to CD19, such as human CD19, for example, the binding affinity K for specific binding to human CD19. D Values ​​less than or equal to approximately 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4.5 nM, 4 nM, or 3.5 nM, such as those determined by biolayer interferometry;

[0030] (2) Specific binding to BCMA, such as human BCMA, cynomolgus monkey BCMA, or mouse BCMA, for example, the binding affinity KD value of specific binding to human BCMA is less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3.5 nM, 3 nM, 2.5 nM, or 2 nM; the binding affinity KD value of specific binding to cynomolgus monkey BCMA is less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3.5 nM, 3 nM, or 2.5 nM; and / or the binding affinity KD value of specific binding to mouse BCMA is less than or equal to about 10 nM, 9 nM, 8 nM, 7 nM, 6.5 nM, 6 nM, 5.5 nM, 5 nM, or 4.5 nM, for example, as determined by biolayer interferometry;

[0031] (3) It specifically binds to CD19 positive cells, BCMA positive cells, or CD19 positive and BCMA positive cells, such as CD19 or BCMA on the surface of tumor cells, and the binding ability is comparable to or better than that of the control antibody. For example, the control antibody may be the TCB antibody CD19(2B11)CD3opt or Teclistamab disclosed in US20240132590A1.

[0032] (4) It can effectively block the binding of the antibody to the CD3 antigen on the surface of T cells, preventing the antibody from being ineffectively distributed on T cells;

[0033] (5) In the presence of CD19 and / or BCMA antigens, it effectively binds to CD3 on T cells and activates T cells, mediating T cell killing; in contrast, control antibodies such as the TCB antibody CD19(2B11)CD3opt disclosed in US20240132590A1 and Teclistamab have their CD3 binding domains fully exposed, and the antibody still binds to the CD3 antigen even in the absence of CD19 or BCMA antigen binding.

[0034] (6) Mediates T cells to effectively kill cells expressing CD19 antigen (CD19 positive cells), such as tumor cells, such as leukemia tumor cells, such as human acute lymphoblastic leukemia cells, for example, its killing ability is superior to control antibodies such as the TCB antibody CD19(2B11)CD3opt disclosed in US20240132590A1.

[0035] (7) Mediates T cells to effectively kill cells expressing BCMA antigen (BCMA positive cells), such as tumor cells, such as myeloma cells, such as multiple myeloma cells, such as tumor cells with different BCMA expression levels (low, medium or high), such as myeloma cells, such as multiple myeloma cells.

[0036] (8) In the presence of CD19 positive cells, the antibody mediates the activation of T cells and kills CD19 positive cells, for example, the CD19 positive cells are CD19 positive tumor cells, such as leukemia tumor cells, such as human acute lymphoblastic leukemia cells.

[0037] (9) In the presence of BCMA-positive cells, the antibody mediates the activation of T cells and kills BCMA-positive cells, such as BCMA-positive tumor cells, such as myeloma cells, such as multiple myeloma cells, such as tumor cells with different BCMA expression levels (low, medium or high).

[0038] (10) It has no killing effect on cells that do not express CD19 and BCMA and / or no activating effect on T cells. Its toxicity is lower than that of control antibodies such as the TCB antibody CD19(2B11)CD3opt and / or Teclistamab disclosed in US20240132590A1.

[0039] (11) It has a clearing effect on CD19 positive cells, such as B cells, and is superior to control antibodies such as W3438-T3U4.E17-1.uIgG4.SP disclosed in CN109535257B, for example, as detected in the KLH mouse model;

[0040] (12) Has the ability to clear CD19 positive cells such as B cells and / or BCMA positive cells such as antibody-secreting cells (e.g. plasma cells), for example, superior to control antibodies such as the TCB antibody CD19(2B11)CD3opt and / or Teclistamab disclosed in US20240132590A1.

[0041] (13) It has the effect of inhibiting the production of immunoglobulins (e.g., IgM, IgA, IgG) or inhibiting the production of anti-KLH immunoglobulins (e.g., anti-KLH IgM, anti-KLH IgA or anti-KLH IgG) produced by the KLH response, for example, it is superior to control antibodies such as W3438-T3U4.E17-1.uIgG4.SP disclosed in CN109535257B, for example, as detected in the KLH mouse model;

[0042] (13) Has a scavenging effect on BCMA-positive cells, such as antibody-secreting cells (e.g., plasma cells), as detected in the KLH mouse model; and / or

[0043] (14) It has the ability to clear both CD19-positive and BCMA-positive cells, such as clearing both B cells and antibody-secreting cells (e.g., plasma cells), and is more effective than the combination of anti-CD19 antibody and anti-BCMA antibody, or more effective than the combination of CD19(2B11)CD3opt and Teclistamab (CD3×BCMA) disclosed in US20240132590A1, or more effective than the combination of W3438-T3U4.E17-1.uIgG4.SP and Teclistamab (CD3×BCMA) disclosed in CN109535257B. Attached Figure Description

[0044] Figure 1 shows two structures of the trispecific antibody CD19×BCMA TCE of the present invention;

[0045] Figure 2 illustrates the two structures of the trispecific antibody of the present invention mediating T cell killing of CD19+ cells;

[0046] Figure 3 illustrates the two structures of the trispecific antibody of the present invention mediating T cell killing of BCMA+ cells;

[0047] Figure 4 shows the configuration of the trispecific antibody CD19×BCMA TCE of the present invention;

[0048] Figure 5 shows the binding of CD19×BCMA TCE to the CD19 antigen on the surface of NALM-6;

[0049] Figure 6 shows the binding of CD19×BCMA TCE to the BCMA antigen on the surface of NCI-H929;

[0050] Figure 7 shows the binding of CD19×BCMA TCE to BCMA antigen on the surface of MM.1S;

[0051] Figure 8 shows the binding of CD19×BCMA TCE to the BCMA antigen on the L363 surface;

[0052] Figure 9 shows the binding of CD19×BCMA TCE to CD3 antigen on the surface of T cells;

[0053] Figure 10 shows CD19×BCMA TCE-mediated T cell killing of NALM-6 cells;

[0054] Figure 11 shows CD19×BCMA TCE-mediated T cell killing of NCI-H929 cells;

[0055] Figure 12 shows CD19×BCMA TCE-mediated T cell killing of MM.1S cells;

[0056] Figure 13 shows CD19×BCMA TCE-mediated T cell killing of L363 cells;

[0057] Figure 14 shows T cell activation mediated by CD19×BCMA TCE-mediated NALM-6 cell killing;

[0058] Figure 15 shows T cell activation mediated by CD19×BCMA TCE-mediated killing of NCI-H929 cells;

[0059] Figure 16 shows T cell activation mediated by CD19×BCMA TCE-mediated MM.1S cell killing;

[0060] Figure 17 shows T cell activation mediated by CD19×BCMA TCE-mediated killing of L363 cells;

[0061] Figure 18 shows CD19×BCMA TCE-mediated T cell killing of 293T cells;

[0062] Figure 19 shows T cell activation induced by CD19×BCMA TCE-mediated killing of 293T cells;

[0063] Figure 20 shows the effect of CD19×BCMA TCE on the clearance of B cells in B-hCD3EDG / hCD19 transgenic mice.

[0064] Figure 21 shows the effect of CD19×BCMA TCE on inhibiting immunoglobulin production in B-hCD3EDG / hCD19 transgenic mice;

[0065] Figure 22 shows the effect of CD19×BCMA TCE on inhibiting the production of anti-KLH immunoglobulin in B-hCD3EDG / hCD19 transgenic mice;

[0066] Figure 23 shows the effect of CD19×BCMA TCE on the clearance of plasma cells in B-hCD3EDG / hBCMA transgenic mice;

[0067] Figure 24 shows the effect of CD19×BCMA TCE on inhibiting immunoglobulin production in B-hCD3EDG / hBCMA transgenic mice;

[0068] Figure 25 shows the effect of CD19×BCMA TCE on inhibiting the production of anti-KLH immunoglobulin in B-hCD3EDG / hBCMA transgenic mice;

[0069] Figure 26 shows the effect of CD19×BCMA TCE on B cell clearance in B-hCD3EDG / hBCMA / hCD19 transgenic mice;

[0070] Figure 27 shows the effect of CD19×BCMA TCE on the clearance of antibody-secreting cells in B-hCD3EDG / hBCMA / hCD19 transgenic mice;

[0071] Figure 28 shows the effect of CD19×BCMA TCE on inhibiting immunoglobulin production in B-hCD3EDG / hBCMA / hCD19 transgenic mice;

[0072] Figure 29 shows the effect of CD19×BCMA TCE on inhibiting the production of anti-KLH immunoglobulin in B-hCD3EDG / hBCMA / hCD19 transgenic mice.

[0073] Invention Details

[0074] It should be understood that the present invention is not limited to the specific methods, schemes, embodiments, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0075] I. Definition

[0076] 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 pertains.

[0077] To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0078] The term “about” when used in conjunction with a numeric value means a range of numeric values ​​that have a lower limit of 5% (e.g., 4%, 3%, 2%, or 1%) smaller than the specified numeric value and an upper limit of 5% (e.g., 4%, 3%, 2%, or 1%) larger than the specified numeric value.

[0079] As used herein, the term “and / or” means any one of the options or two or more or all of the options.

[0080] When “first” and “second” are mentioned in this article, it is only to distinguish between two structural domains or two chains, and does not indicate the location of the two structural domains in any way.

[0081] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, they also cover situations consisting of the stated elements, integers, or steps, unless otherwise specified. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0082] The term “BCMA” stands for B cell maturation antigen (BCMA, i.e., CD269, TNFRSF17), a member of the tumor necrosis factor receptor superfamily (TNFRSF), and refers to any naturally occurring BCMA from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term covers “full-length” unprocessed BCMA, as well as any form of BCMA produced by cellular processing. The term also covers naturally occurring variants of BCMA, such as splice variants or allelic variants. The term also covers variants, isotypes, species homologs, and analogs of BCMA that share at least one epitope with BCMA (e.g., human BCMA). In one respect, BCMA is human BCMA, such as human BCMA under UniProt (www.uniprot.org) accession number Q02223 or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_001183.2 accession number. The BCMA protein may also include fragments of BCMA, such as extracellular domains and fragments of extracellular domains, for example, fragments that maintain the ability to bind to any antibody of the present invention.

[0083] As used herein, the terms “BCMA antibody,” “antibody against BCMA,” “antibody that specifically binds to BCMA,” “antibody that specifically targets BCMA,” and “antibody that specifically recognizes BCMA” are used interchangeably to refer to antibodies that can specifically bind to B cell maturation antigen (BCMA) (e.g., human BCMA, cynomolgus monkey BCMA, or mouse BCMA). In some respects, the anti-BCMA antibodies described herein also encompass multispecific antibodies that simultaneously and specifically bind to BCMA and other target antigens.

[0084] The term "CD19" stands for cluster 19 of differentiation (also known as B lymphocyte antigen CD19 or B lymphocyte surface antigen B4) and refers to any naturally occurring CD19 from any vertebrate source, including mammals such as primates (e.g., humans), non-human primates (e.g., cynomolgus monkeys), and rodents (e.g., mice and rats). The term covers "full-length" unprocessed CD19, as well as any form of CD19 produced by cellular processing. The term also covers naturally occurring variants of CD19, such as splice variants or allelic variants. The term also covers variants, isotypes, species homologs, and analogs of CD19 that have at least one identical epitope to CD19 (e.g., human CD19). In one respect, CD19 is human CD19, such as Human Protein UniProt (www.uniprot.org) accession number P15391 (version 211) or NCBI (www.ncbi.nlm.nih.gov / ) RefSeq NP_001761.3. The CD19 protein may also include fragments of CD19, such as extracellular domains and fragments of the extracellular domains, for example, fragments that maintain the ability to bind to any antibody of the present invention.

[0085] As used herein, "antibody that binds to CD19," "anti-CD19 antibody," or "antibody that specifically binds to CD19" refers to an antibody capable of binding to CD19 (e.g., human CD19) or its domains with appropriate affinity. The antibodies and antigen-binding fragments of the present invention can bind to CD19 expressed on the cell surface. In some aspects, the anti-CD19 antibodies described herein also encompass multispecific antibodies that simultaneously and specifically bind to CD19 and other target antigens.

[0086] As used herein, the term "CD3" refers to the antigen expressed on T cells as a portion of the multimolecular T-cell receptor (TCR), specifically the T-cell surface glycoprotein CD3, which is a T-cell adaptor antigen composed of homodimers or heterodimers formed from two of the following four receptor chains: CD3-ε, CD3-δ, CD3-ζ, and CD3-γ. Human CD3-εn (hCD3ε) contains the amino acid sequence described in UniProtKB / Swiss-Prot: P07766. Human CD3-δ (hCD3δ) contains the amino acid sequence described in UniProtKB / Swiss-Prot: P04234. In some embodiments, the CD3 described herein refers to CD3 derived from humans or monkeys (e.g., cynomolgus monkeys).

[0087] As used herein, the terms "CD3-binding antibody" or "anti-CD3 antibody" encompass antibodies and their antigen-binding fragments that specifically recognize or bind to a single CD3 subunit (e.g., ε, δ, γ, or ζ), and antibodies and their antigen-binding fragments that specifically recognize or bind to a dimer complex (e.g., γ / ε, δ / ε, and ζ / ζ CD3 dimers) of two CD3 subunits. The antibodies and antigen-binding fragments of the present invention can bind to soluble CD3, bound CD3, and / or CD3 expressed on the cell surface. Soluble CD3 comprises native CD3 protein and recombinant CD3 protein variants, such as monomeric and dimer CD3 structures lacking a transmembrane region or otherwise not binding to the cell membrane. In one embodiment, the CD3-binding antigen-binding region of the multispecific antibody of the present invention may have low binding activity to CD3 or CD3-expressing cells (e.g., T cells). The binding affinity of the antibody to CD3 can be detected by flow cytometry or biomembrane layer optical interferometry. In some embodiments, the CD3-binding antigen-binding region of the anti-CD3 antibody of the present invention or its antigen-binding fragment or multispecific antibody binds to human and / or monkey (e.g., cynomolgus monkey) CD3 with low binding affinity, thereby enabling the activation of human and / or monkey (e.g., cynomolgus monkey) T cells. In some aspects, the anti-CD3 antibodies described herein also encompass multispecific antibodies that simultaneously and specifically bind to CD3 and other target antigens.

[0088] General information on the amino acid and nucleotide sequences of the light and heavy chains of human immunoglobulins is given in Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0089] The multispecific antibodies of the present invention may include linkers. As used herein, the term "linker" refers to any molecule that enables direct connection between different portions of a multispecific binding molecule. Examples of linkers that establish covalent links between different molecular portions include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyethylene, or copolymers of PEG and polypropylene glycol. In some embodiments, the linker is a peptide linker (also referred to as a "linking peptide"), which refers to a short amino acid sequence consisting of amino acids, such as glycine (G) and / or serine (S) and / or threonine residues (T) used alone or in combination, or a hinge region from an immunoglobulin, for linking the amino acid sequence of a first portion of the binding molecule to a second portion of the binding molecule. For example, a peptide linker can link a first target-binding region of the binding molecule to a second target-binding region. For example, a peptide linker can also link one portion of an antibody to another portion of the antibody, such as linking a light chain variable region to a heavy chain variable region. Preferably, the peptide linker has a length sufficient to link two entities in such a way that they maintain their conformation relative to each other without impeding the desired activity. In one embodiment, the linker peptide has a length of 5-50 amino acids, for example, 10, 15, 20, 25, or 30 amino acids. In one embodiment, the linker peptide comprises the amino acid sequences (GS)n, (GGS)n, (GSGGS)n (SEQ ID NO:45), (GGGGS)n (SEQ ID NO:47), (GGGS)n (SEQ ID NO:46), and (GGGGS)nG (SEQ ID NO:51), where n is an integer equal to or greater than 1, for example, n is an integer of 2, 3, 4, 5, 6, 7, 8, 9, or 10. Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers. In some embodiments, the peptide linker is (GGGGS)n, where n = any integer from 1 to 10, for example, 1, 2, 3, 4, or 5, such as the sequences shown in SEQ ID NO:47, 48, or 49. In some embodiments, the peptide linker is GSGGGGS, i.e., the sequence shown in SEQ ID NO:50.

[0090] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain containing at least a portion of its constant region. This term includes native sequence Fc regions and variant Fc regions. A native immunoglobulin "Fc domain" contains two or three constant domains: a CH2 domain, a CH3 domain, and optionally a CH4 domain. For example, in native antibodies, an immunoglobulin Fc domain contains the second and third constant domains (CH2 and CH3 domains) of two heavy chains derived from IgG, IgA, and IgD antibodies; or it contains the second, third, and fourth constant domains (CH2, CH3, and CH4 domains) of two heavy chains derived from IgM and IgE antibodies. Unless otherwise stated herein, amino acid residues in the Fc region or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD, 1991. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Two Fc regions can dimerize to form a dimer Fc, and two different Fc regions can heterodimerize to form a heterodimeric Fc. In this document, the terms “Fc region,” “Fc portion,” and “dimeric Fc (e.g., heterodimeric Fc)” do not include the heavy chain variable region VH and light chain variable region VL of immunoglobulins, nor the heavy chain constant region CH1 and light chain constant region CL, but in some cases may include the hinge region at the N-terminus of the heavy chain constant region. In one embodiment, the human IgG heavy chain Fc region extends from Asp221, Cys226, or Asp231 to the carboxyl terminus of the heavy chain. Unless otherwise specified herein, when referring to the Fc region, it means the Fc region containing a portion of the hinge region, corresponding to the Asp221 extension of the human IgG1 heavy chain Fc region to the carboxyl terminus of the heavy chain.

[0091] In one embodiment, the human IgG1 Fc region polypeptide (including a portion of the hinge region) comprises or is composed of the following amino acid sequence:

[0092] In one embodiment, the Fc region is a human-derived Fc region. In one embodiment, the Fc region comprises all or part of the human constant region. The antibody Fc region is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. In one embodiment, the Fc region is a human Fc region. In one embodiment, the Fc region belongs to the human IgG4 subclass. In one embodiment, the Fc region belongs to the human IgG1 subclass. In one embodiment, the Fc region is an Fc region derived from human IgG1, IgG2, IgG3, or IgG4.

[0093] In this article, "heterodimolecular Fc scaffold" refers to a scaffold containing two different Fc regions or formed by dimerization of two different Fc regions, which can be connected to an antigen-binding domain (e.g., the heavy chain and / or light chain variable region of an antibody that can bind to a target molecule, or the antigen-binding fragment of an antibody, or the soluble portion of a ligand or receptor that can bind to a target molecule) at its N-terminus or C-terminus to form a multispecific binding molecule, such as a multispecific antibody, for example a trispecific antibody.

[0094] The term "CH1 region" refers to the portion of the antibody heavy chain polypeptide extending from EU position 118 to EU position 215 or EU position 220 (including a portion of the hinge region) (EU numbering system). Unless otherwise specified herein, when referring to the CH1 region, it means the portion of the antibody heavy chain polypeptide extending from EU position 118 to EU position 220 (EU numbering system). In one embodiment, the CH1 domain includes...

[0095] The amino acid sequence of or composed of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC (SEQ ID NO:40).

[0096] In this document, specific amino acid residues in the constant region of antibody IgG are referred to according to the EU numbering system. For example, "S364" refers to serine at position 364 in the EU. Amino acid mutations at a specific position in the constant region are represented by (original amino acid, amino acid position, mutant amino acid). For example, "S364R" means that serine (S) at position 364 in the EU is replaced by arginine (R). The above amino acid mutations can also represent mutations in other constant regions corresponding to that position in a specific constant region, and the original amino acid may not be the amino acid shown. For example, "S364R" can also mean that an amino acid (which can be serine or other amino acids) at position 364 of IgG1 in the constant region of other IgG subclasses or IgA, etc., is mutated to R. When referring to combinations of mutations, the combined mutations are connected by a plus sign (+), a "-", or an "and". "S364R+D399K", S364R-D399K, or "S364R and D399K" means that the Fc region contains both the mutations S364R and D399K. When multiple mutation possibilities exist at a particular position, this is indicated by the symbol " / ". For example, the mutation "K370T / S" means that the K residue at position 370 can be replaced by either a T or an S residue.

[0097] For polypeptide sequences, "conservative alteration" includes substitutions, deletions, or additions to the polypeptide sequence that do not substantially change the desired functional activity of the polypeptide sequence. For example, a conserved substitution often results in a particular amino acid being replaced by a chemically similar amino acid. Tables providing conserved substitutions of functionally similar amino acids are well known in the art. The following lists eight groups of amino acids containing mutually conserved substitutions: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). In some embodiments, the term "conserved sequence alteration" is used to refer to amino acid modifications that do not significantly affect or alter the target antigen binding characteristics of the antibody molecule or binding protein molecule of the present invention containing the amino acid sequence. For example, conserved modified variants maintain at least 80%, 85%, 90%, 95%, 98%, 99%, or higher binding affinity to the target antigen relative to the parent antibody or binding protein, such as 100-110% or higher.

[0098] The term "vector," as used herein, refers to a nucleic acid molecule capable of replicating another nucleic acid linked to it. This term includes vectors that function as self-replicating nucleic acid structures as well as vectors that bind to the genome of a host cell that has already been introduced therein. Some vectors are capable of directing the expression of nucleic acids operatively linked to them. Such vectors are referred to herein as "expression vectors."

[0099] The term "binding molecule" refers to any molecule that can specifically bind to a target, such as an antibody or its antigen-binding fragment or fusion protein.

[0100] The term "target" refers to the substance to which a binding molecule is directed. A target can be an antigen, a ligand, or a receptor. The term "antigen" refers to a molecule that elicits an immune response. This immune response may involve antibody production or activation of specific immune cells, or both. Those skilled in the art will understand that any macromolecule, including virtually all proteins or peptides, can be used as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to a portion of an antigen that specifically interacts with an antibody molecule. When the binding molecule of the present invention relates to a target-binding region derived from an antibody, "target" and "antigen" may be used interchangeably.

[0101] As used herein, the term "target-binding region" refers to the portion of a binding molecule, such as a multispecific binding molecule or a trispecific binding molecule, that binds to a specific target or antigen. The target-binding region can be, for example, an antibody or immunoglobulin itself or an antibody fragment. Such a target-binding region may or may not have a tertiary structure independent of the remaining portion of the binding molecule and can bind to its target as a standalone entity or not. The target-binding region can also be a receptor or ligand, or a ligand-binding domain of a receptor. In the case of multispecific antibodies, such as trispecific antibodies, the "target-binding region" is also referred to as the "antigen-binding region."

[0102] As used herein, the term "antigen-binding region" refers to any portion of an antibody or its antigen-binding fragment, such as a multispecific antibody or a trispecific antibody, that binds to a specific target or antigen or epitope. An antigen-binding region can be, for example, the antibody or immunoglobulin itself or an antibody fragment. Such an antigen-binding region may or may not have a tertiary structure independent of the remaining portion of the multispecific or trispecific antibody, and may bind or not bind its antigen / epitaxy as a standalone entity. When the binding molecule of the present invention relates to a target-binding region derived from an antibody, the terms "target-binding region" and "antigen-binding region" may be used interchangeably.

[0103] The term "multispecific binding molecule" refers to a multispecific binding molecule that is at least bispecific, such as a bispecific binding molecule containing at least a first target binding region and a second target binding region, wherein the first target binding region binds to one target or antigen and the second target binding region binds to another antigen or target. The multispecific binding molecule according to the present invention also encompasses multispecific binding molecules containing multiple target binding regions / binding sites. In some embodiments, the multispecific binding molecule of the present invention is a multispecific antibody. In some embodiments, the trispecific binding molecule of the present invention is a trispecific antibody.

[0104] As used herein, a binding molecule, such as an antibody, refers to having at least two target-binding regions, each of which binds to a different site / structure of the same target or to a different target. A multispecific binding molecule is a binding molecule that has binding specificity to at least two different targets or sites / structures. In one embodiment, this document provides such a trispecific binding molecule having binding specificity against a first target, a second target, and a third target.

[0105] As used herein, the term "multispecific" antibody refers to an antibody having at least two antigen-binding regions, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. A multispecific antibody is an antibody that has binding specificity to at least two different antigens or epitopes. In one embodiment, this document provides a trispecific antibody having binding specificity against a first antigen, binding specificity against a second antigen, and binding specificity against a third antigen. In some embodiments, the present invention provides an antibody that specifically binds to BCMA, CD19, and CD3, also referred to as CD19×BCMA TCE or BCMA×CD19 TCE.

[0106] When "first antigen-binding region" is mentioned in the context of multispecific or trispecific antibodies, it refers to the binding region that binds to the first antigen, and is not intended to limit the number of such antigen-binding regions contained in the antibody. For example, a multispecific or bispecific antibody may contain one or more first antigen-binding regions. Similarly, a trispecific antibody may contain a first antigen-binding region, a second antigen-binding region, and a third antigen-binding region, but may contain one or more first antigen-binding regions, one or more second antigen-binding regions, and one or more third antigen-binding regions.

[0107] When it is mentioned that "the antigen-binding region is derived from the antibody", it means that the binding domain constituting the antigen-binding region is or is derived from the binding domain of the antibody that specifically binds to the antigen. For example, the Fab or scFv of the antigen-binding region is or is derived from the corresponding fragment of the antibody, such as Fab, or the heavy chain variable region and / or light chain variable region of the antigen-binding region is or is derived from the heavy chain variable region and / or light chain variable region of the antibody, or one, two, three, four, five or six CDRs of the antigen-binding region are CDRs of the antibody.

[0108] The term "derived from" means that the fragment in the antigen-binding region is substantially identical to the fragment from the antibody from which it originated, but has mutations at one or more sites, such as substitution, deletion, or addition. In one specific embodiment, the mutation is not in the antibody's CDR.

[0109] The terms "full-length antibody" or "complete antibody" are used interchangeably herein and refer to antibody molecules that have the molecular structure of natural immunoglobulins. In the case of a conventional four-chain IgG antibody, a full-length antibody consists of two heavy chains (H) and two light chains (L) linked together by disulfide bonds. In the case of a heavy chain antibody that has only heavy chains and lacks light chains, a full-length antibody consists of two heavy chains (H) linked together by disulfide bonds. For a conventional four-chain IgG antibody, the heavy chain of a full-length antibody typically consists of a heavy chain variable region (abbreviated as VH in this document) and a heavy chain constant region, wherein the heavy chain constant region contains at least three domains CH1, CH2, and CH3. The light chain of a full-length antibody consists of a light chain variable region (abbreviated as VL in this document) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody fragment" includes a portion of a complete antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0110] The term "antigen-binding fragment" in antibody refers to a molecule distinct from a full-length antibody. It contains a portion of the full-length antibody but can bind to the antigen of the full-length antibody or compete with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment originates) for antigen binding. Antigen-binding fragments can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), and nanobodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. Furthermore, digestion of a complete antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, a dimer of Fab' and a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by breaking the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into Fab' monomers. Fab' monomers are essentially Fab fragments with hinge regions. Fv fragments consist of the VL and VH domains of the antibody's single arm. The two domains VL and VH of the Fv fragment can be encoded by independent genes, but they can also be produced as a single protein chain by using a recombinant approach, connecting the two domains with a synthetic linker peptide, and pairing the VL and VH regions in the single protein chain to form a single-chain Fv (scFv).

[0111] The term "Fab fragment" or "Fab" is used interchangeably herein to refer to an immunoglobulin fragment consisting of two polypeptide chains containing the immunoglobulin heavy chain variable region VH, the heavy chain constant domain CH1, the light chain variable region VL, and the light chain constant region CL. One polypeptide chain contains VH and a constant region selected from CH1 and CL from the N-terminus to the C-terminus, and the other polypeptide chain contains VL and another constant region selected from CL and CH1 from the N-terminus to the C-terminus. The VH and VL domains pair to form an antigen-binding site. In this document, the Fab polypeptide chain containing the heavy chain constant region CH1 is also referred to as the "Fab heavy chain"; correspondingly, the Fab polypeptide chain containing the light chain constant region CL is also referred to as the "Fab light chain."

[0112] The complementarity-determining region (CDR) or CDR is a region in the antibody variable region that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable region are called HCDR1, HCDR2, and HCDR3, while those located within the antibody light chain variable region are called LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many known antibody CDR assignment schemes, including, for example: Chothia (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody three-dimensional structure and CDR loop topology; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., USDapartment of Health and Human Services, National Institutes of Health (1987)) based on antibody sequence variability; AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics. The database (IMGT) (www.imgt.cines.fr / ) and the North CDR definition based on affinity propagation clustering utilizing a large number of crystal structures. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" covers a CDR sequence determined in any of the foregoing methods or combinations thereof. A CDR may also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of this invention).

[0113] In some embodiments, the CDRs in the heavy chain variable region and the light chain variable region of the antibody of the present invention are determined according to Kabat, Chothia, AbM or IMGT or any combination thereof (e.g., a combination of Kabat and Chothia). For example, HCDR1 of the heavy chain variable region is determined according to the AbM scheme, and HCDR2, HCDR3 of the heavy chain variable region and LCDR1, LCDR2 and LCDR3 of the light chain variable region are determined according to the Kabat scheme.

[0114] An example CDR definition scheme is as follows:

[0115] The following is a calculation of sequence identity between sequences.

[0116] To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the reference sequence length. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at that position.

[0117] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody while exhibiting low immunogenicity when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen binding site and replacing the remaining portion of the antibody with its corresponding human portion (i.e., replacing the non-binding portion of the variable region with the corresponding portion of the human antibody).

[0118] As used herein, the terms “anti,” “binding,” or “specific binding” mean that the binding interaction is selective for the target or antigen and can be distinguished from unwanted or nonspecific interactions. The ability of a binding site to bind to a specific target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as radioimmunoassay (RIA), thin-layer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0119] "Affinity" or "binding affinity" refers to the inherent binding affinity that reflects the interaction between members of a binding pair. The affinity of molecule X for its partner Y can usually be determined by the dissociation constant (K).D This indicates that the dissociation constant is the dissociation rate constant and the association rate constant (Kdissociation and Kassociation, respectively). dis and K on The ratio of affinity to kinetic binding affinity. Affinity can be measured by common methods known in the art. One specific method used to measure affinity is the ForteBio kinetic binding assay described in this paper.

[0120] The term "host cell" refers to a cell into which exogenous polynucleotides have been introduced, including the progeny of such cells. Host cells include "transformers" and "transformed cells," which include primary transformed cells and their derived progeny.

[0121] The terms “individual” or “subject” are used interchangeably and refer to mammals. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In particular, an individual is a human.

[0122] The term "treatment" refers to slowing down, interrupting, blocking, relieving, stopping, reducing, or reversing the onset of symptoms, complications, or biochemical indicators of a disease, relieving symptoms, or preventing or inhibiting the further development of the disease, condition, or symptom.

[0123] The term "prevention" includes the suppression of the occurrence or development of a disease or condition or the symptoms of a particular disease or condition.

[0124] The term “therapeutic agent” as used herein encompasses any substance that is effective in the prevention or treatment of cancer or autoimmune diseases, including chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies (e.g., immune checkpoint molecules), small molecule drugs or immunomodulators (e.g., immunosuppressants or agonists), nonsteroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs), antirheumatic drugs, etc.

[0125] The term "combination therapy" refers to the administration of two or more therapeutic agents or modes of treatment to treat the disease described herein. Such administration includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule containing active ingredients in a fixed proportion. Alternatively, such administration includes the co-administration of individual active ingredients in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose prior to administration. Furthermore, such administration includes the sequential administration of each type of therapeutic agent at substantially the same time or at different times. In either case, the treatment regimen will provide the beneficial effect of the combination of drugs in treating the condition or symptom described herein.

[0126] The term "drug combination" or "drug combination product" refers to a non-fixed or fixed combination. In some embodiments, the drug combination may be in a pillbox. The term "non-fixed combination" means that the active ingredients (e.g., (i) the antibodies of the present invention, and (ii) other therapeutic agents) are administered to a patient simultaneously, without a specific time limit, or sequentially at the same or different time intervals, in separate entities, wherein such administration to the patient provides a preventive or therapeutically effective level. The term "fixed combination" means that two or more active ingredients are administered to a patient simultaneously in the form of a single entity. Preferably, the dosage and / or time interval of the two or more active ingredients are selected so that the combined use of the portions can produce an effect greater than that achieved by using any one ingredient alone in treating a disease or condition. The components may each be in a separate formulation, and their formulations may be the same or different.

[0127] The term "pharmaceutical composition" refers to a composition which is present in a form that allows the biological activity of the active ingredient contained therein to be effective, and which does not contain any additional ingredients that would have unacceptable toxicity to a subject administering the composition.

[0128] The term "pharmaceutical excipients" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, or stabilizers that are applied together with the active substance.

[0129] "Subject / Patient / Individual Sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of the tissue or cell sample can be solid tissue, such as fresh, frozen and / or preserved organ or tissue samples or biopsy samples or puncture samples; body fluid, such as tears, vitreous fluid, cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid.

[0130] II. Multispecific antibodies

[0131] In one aspect, the present invention relates to multispecific binding molecules, such as trispecific binding molecules, capable of specifically binding to CD19, BCMA, and CD3. In some embodiments, the multispecific binding molecule is a multispecific antibody, such as a trispecific antibody.

[0132] In some embodiments, the multispecific antibody of the present invention, such as a trispecific antibody, comprises an antigen-binding region that specifically binds to CD19, an antigen-binding region that specifically binds to BCMA, and an antigen-binding region that specifically binds to CD3. In some embodiments, the trispecific antibody of the present invention comprises an antigen-binding region that specifically binds to CD19, an antigen-binding region that specifically binds to BCMA, and an antigen-binding region that specifically binds to CD3.

[0133] In some embodiments, in the multispecific antibody, the antigen-binding region that specifically binds to CD19 is the Fab (Fab group) that specifically binds to CD19. CD19 The antigen-binding region that specifically binds to BCMA is the Fab (Fab group) that specifically binds to BCMA. BCMA ); and / or the antigen-binding region that specifically binds to CD3 is a CD3-specific scFv (scFv CD3 ).

[0134] In some embodiments, the multispecific antibody of the present invention is an IgG-like multispecific antibody. In some embodiments, the IgG-like multispecific antibody of the present invention refers to a multispecific antibody comprising an Fc dimer (e.g., a heterodimeric Fc scaffold comprising a first Fc region and a second Fc region). Therefore, in some embodiments, the multispecific antibody of the present invention comprises an Fc dimer, such as a heterodimeric Fc scaffold composed of different first Fc regions and second Fc regions.

[0135] In some specific implementation schemes, Fab BCMA The Fab heavy chain is connected directly or via a connector (preferably directly) to the N end of the first Fc region at the C end of its CH1 region. CD19 The Fab heavy chain has scFv at the C-terminus of its CH1. CD3 The N-terminus of VH is connected directly or via a connector (preferably via connector 1, such as the connector shown in SEQ ID NO:48), and scFv CD3 The C end of the VL is connected directly or via a connector (preferably via a connector (connector 3), such as the connector shown in SEQ ID NO:50) to the N end of the second Fc region.

[0136] In some embodiments, the configuration of the trispecific antibody of the present invention is shown in Figure 1 (left) or Figure 4.

[0137] In one embodiment, the multispecific antibody, such as a trispecific antibody, comprises or is composed of the following:

[0138] Heavy chain 1: From the N-terminus to the C-terminus, it contains or consists of the following: VH BCMA -First CH1-First Fc region, wherein the heavy chain constant region CH1 is connected at its C end to the N end of the first Fc region directly or via a connector (preferably directly);

[0139] Light chain 1: Composed of or consisting of the following from the N-terminus to the C-terminus: VL BCMA -First CL;

[0140] Heavy chain 2: From the N-terminus to the C-terminus, it contains or consists of the following: VH CD19 -Second CH1-scFvCD3 - Second Fc region, where the C-terminus of the second CH1 is connected to scFv CD3 The N-terminus is connected directly or via connector 1, and scFv CD3 The C end is directly or via connector 3 connected to the N end of the second Fc region;

[0141] Light chain 2: From the N-terminus to the C-terminus, it includes or consists of the following: VL CD19 -Second CL;

[0142] Where “-” represents a connector or direct connection;

[0143] VH BCMA This refers to the heavy chain variable region VH that specifically binds to the antigen-binding region of BCMA, and VL BCMA It refers to the light chain variable region (VL) of the antigen-binding region that specifically binds to BCMA;

[0144] VH CD19 This refers to the heavy chain variable region VH that specifically binds to the antigen-binding region of CD19, and VL CD19 It refers to the light chain variable region (VL) of the antigen-binding region that specifically binds to CD19;

[0145] The first CH1 and the second CH1 refer to the CH1 domain of the heavy chain constant region, respectively.

[0146] The first CL and the second CL refer to the constant regions of the light chain, respectively.

[0147] VH BCMA - First CH1 and VL BCMA - The first CL constitutes the Fab, which is the antigen-binding region that specifically binds to BCMA. BCMA ;

[0148] VH CD19 -Second CH1 and VL CD19 - The second CL constitutes the Fab, which serves as the antigen-binding region specifically binding to CD19. CD19 ;

[0149] scFv CD3 This refers to the scFv, which is the antigen-binding region that specifically binds to CD3, and consists of VH CD3 and VL CD3 Composed of polypeptide chains directly or via linker 2, preferably the scFv CD3 From the N-terminus to the C-terminus, it includes or consists of the following: VH CD3 -VL CD3 VH CD3 C-terminus and VL CD3 The N-terminus is connected directly or via connector 2;

[0150] Optionally, the first CH1 and the second CH1 are the same, and / or the first CL and the second CL are the same, and / or the first Fc region and the second Fc region are different.

[0151] In some implementation schemes, Fab CD19 Fab heavy chain and scFv CD3 The linker is connected via a linker 1, which may be a peptide linker composed of amino acid residues. Suitable peptide linkers are known to those skilled in the art. In one embodiment, the linker is 5-50 amino acid long, for example 5-30 amino acid long, for example 15 or 20 amino acid long. In one embodiment, the linker 1 comprises the amino acid sequence (G4S)n, where n = 1, 2, 3, 4 or 5, preferably n = 2. In one embodiment, the linker 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 47 or 48.

[0152] In some implementations, scFv CD3 The Fc region is connected via a linker 3, wherein the linker 3 may be a peptide linker composed of amino acid residues. Suitable peptide linkers are known to those skilled in the art. In one embodiment, the linker is 5-50 amino acid long, for example 5-30 amino acid long, such as 15 or 20 amino acid long. In one embodiment, the linker 3 comprises or consists of the amino acid sequence shown in SEQ ID NO:47 or 50.

[0153] In some specific implementation schemes, Fab CD19 The Fab heavy chain is connected directly or via a connector (preferably directly) to the N end of the first Fc region at the C end of its CH1 region. BCMA The Fab heavy chain has scFv at the C-terminus of its CH1. CD3 The N-terminus of VH is connected directly or via a connector (preferably via connector 1, such as the connector shown in SEQ ID NO:48), and scFv CD3 The C end of the VL is connected directly or via a connector (preferably via a connector (connector 3), such as the connector shown in SEQ ID NO:50) to the N end of the second Fc region.

[0154] In some embodiments, the configuration of the trispecific antibody of the present invention is shown in Figure 1 (right figure).

[0155] In one embodiment, the multispecific antibody, such as a trispecific antibody, comprises or is composed of the following:

[0156] Heavy chain 1: From the N-terminus to the C-terminus, it contains or consists of the following: VH CD19-First CH1-First Fc region, wherein the heavy chain constant region CH1 is connected at its C end to the N end of the first Fc region directly or via a connector (preferably directly);

[0157] Light chain 1: Composed of or consisting of the following from the N-terminus to the C-terminus: VL CD19 -First CL;

[0158] Heavy chain 2: From the N-terminus to the C-terminus, it contains or consists of the following: VH BCMA -Second CH1-scFv CD3 - Second Fc region, where the C-terminus of the second CH1 is connected to scFv CD3 The N-terminus is connected directly or via connector 1, and scFv CD3 The C end is directly or via connector 3 connected to the N end of the second Fc region;

[0159] Light chain 2: From the N-terminus to the C-terminus, it includes or consists of the following: VL BCMA -Second CL;

[0160] in,

[0161] "-" indicates a connector or direct connection;

[0162] VH CD19 This refers to the heavy chain variable region VH that specifically binds to the antigen-binding region of CD19, and VL CD19 It refers to the light chain variable region (VL) of the antigen-binding region that specifically binds to CD19;

[0163] VH BCMA This refers to the heavy chain variable region VH that specifically binds to the antigen-binding region of BCMA, and VL BCMA It refers to the light chain variable region (VL) of the antigen-binding region that specifically binds to BCMA;

[0164] The first CH1 and the second CH1 refer to the CH1 domain of the heavy chain constant region, respectively.

[0165] The first CL and the second CL refer to the constant regions of the light chain, respectively.

[0166] VH CD19 - First CH1 and VL CD19 - The first CL constitutes the Fab, which serves as the antigen-binding region specifically binding to CD19. CD19 ;

[0167] VH BCMA -Second CH1 and VL BCMA - The second CL constitutes the Fab, which is the antigen-binding region that specifically binds to BCMA. BCMA ;

[0168] scFv CD3This refers to the scFv, which is the antigen-binding region that specifically binds to CD3, and consists of VH CD3 and VL CD3 Composed of polypeptide chains directly or via linker 2, preferably the scFv CD3 From the N-terminus to the C-terminus, it includes or consists of the following: VH CD3 -VL CD3 VH CD3 C-terminus and VL CD3 The N-terminus is connected directly or via connector 2;

[0169] Optionally, the first CH1 and the second CH1 are the same, and / or the first CL and the second CL are the same, and / or the first Fc region and the second Fc region are different.

[0170] In some implementation schemes, Fab BCMA Fab heavy chain and scFv CD3 The linker is connected via a linker 1, which may be a peptide linker composed of amino acid residues. Suitable peptide linkers are known to those skilled in the art. In one embodiment, the linker is 5-50 amino acid long, for example 5-30 amino acid long, for example 15 or 20 amino acid long. In one embodiment, the linker 1 comprises the amino acid sequence (G4S)n, where n = 1, 2, 3, 4 or 5, preferably n = 2. In one embodiment, the linker 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO: 47 or 48.

[0171] In some implementations, scFv CD3 The Fc region is connected via a linker 3, wherein the linker 3 may be a peptide linker composed of amino acid residues. Suitable peptide linkers are known to those skilled in the art. In one embodiment, the linker is 5-50 amino acid long, for example 5-30 amino acid long, such as 15 or 20 amino acid long. In one embodiment, the linker 3 comprises or consists of the amino acid sequence shown in SEQ ID NO:47 or 50.

[0172] The present invention describes various components / domains for multispecific binding molecules, such as multispecific antibodies. Those skilled in the art will understand that, unless the context explicitly indicates otherwise, any combination of any technical features of any component for multispecific binding molecules, such as multispecific antibodies, described herein is within the scope of this invention. Furthermore, those skilled in the art will understand that, unless the context explicitly indicates otherwise, the antibodies of this invention (including antibodies of any form) may comprise any such combination.

[0173] II-1 specifically binds to the antigen-binding region of CD3.

[0174] In some embodiments, the CD3-specific antigen-binding region comprises three complementarity-determining regions (HCDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the CD3-specific antigen-binding region comprises three complementarity-determining regions (LCDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3. In some embodiments, the CD3-specific antigen-binding region comprises three HCDRs from the heavy chain variable region and three LCDRs from the light chain variable region.

[0175] In some respects, the antigen-binding region that specifically binds to CD3 includes the heavy chain variable region (VH, also referred to herein as "VH"). CD3 In some respects, the antigen-binding region that specifically binds to CD3 contains a light chain variable region (VL, also referred to herein as "VL"). CD3 In some aspects, the antigen-binding region that specifically binds to CD3 includes a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region includes three complementarity-determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region includes three complementarity-determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the HCDRs and LCDRs can be determined according to any scheme for determining CDRs, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, and HCDR2 and HCDR3 are determined according to the Kabat scheme; and LCDR1, LCDR2, and LCDR3 are determined according to the Kabat scheme.

[0176] In some embodiments, the antigen-binding region is derived from an antibody that specifically binds to CD3, such as the CD3 antibody disclosed in WO2022068809A1 (incorporated herein in its entirety), for example, sp34.24 therein. In some embodiments, the antigen-binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to CD3 (e.g., the CD3 antibody disclosed in WO2022068809A1, for example, sp34.24 therein). In some embodiments, the antigen-binding region comprises 1, 2, or 3 heavy chain variable region CDRs of a known antibody that specifically binds to CD3 (e.g., the CD3 antibody disclosed in WO2022068809A1, for example, sp34.24 therein), namely HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding region comprises one, two, or three light chain variable regions (CDRs) of a known CD3-specific antibody (e.g., the CD3 antibody disclosed in WO2022068809A1, such as sp34.24 therein), namely LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen-binding region comprises three heavy chain variable regions and three light chain variable regions of a known CD3-specific antibody (e.g., the CD3 antibody disclosed in WO2022068809A1, such as sp34.24 therein). In some embodiments, the antigen-binding region comprises a heavy chain variable region and a light chain variable region of a known CD3-specific antibody (e.g., the CD3 antibody disclosed in WO2022068809A1, such as sp34.24 therein), particularly a combination of the heavy chain variable regions and light chain variable regions.

[0177] In some embodiments, the heavy chain variable region VH included in the antigen-binding region that specifically binds to CD3

[0178] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:25; or

[0179] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:25; or

[0180] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:25, preferably, the amino acid changes do not occur in the CDR region.

[0181] In some embodiments, the light chain variable region VL included in the antigen-binding region that specifically binds to CD3

[0182] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:26; or

[0183] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:26; or

[0184] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:26, preferably, the amino acid changes do not occur in the CDR region.

[0185] In some embodiments, the three complementary determinant regions (HCDRs) from the heavy chain variable region of the antigen-binding region that specifically binds to CD3, HCDR1, HCDR2, and HCDR3, are the three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO:25. Preferably, the HCDRs can be determined according to any scheme for determining CDRs, such as Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, and HCDR2 and HCDR3 are determined according to Kabat.

[0186] In some embodiments, the three complementary determinant regions (LCDRs) from the light chain variable region of the antigen-binding region that specifically binds to CD3, LCDR1, LCDR2, and LCDR3, are the three complementary determinant regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO:26. Preferably, the LCDRs can be determined according to any scheme for determining CDRs, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, LCDR1, LCDR2, and LCDR3 are determined by the Kabat scheme.

[0187] In some embodiments, in the specific CD3-binding antigen-binding region of the present invention, HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:27; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:28; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:29; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:30; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:31; and / or LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:32.

[0188] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD3 includes VH and VL, wherein

[0189] The VH contains the amino acid sequence shown in SEQ ID NO:25 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:26 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0190] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD3 includes three complementary determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:25, and three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:26. Preferably, the HCDR and LCDR can be determined according to any scheme for determining CDR, such as Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, and HCDR2 and HCDR3 are determined according to Kabat; and LCDR1, LCDR2, and LCDR3 are determined according to the Kabat scheme.

[0191] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD3 includes: HCDR1 as shown in SEQ ID NO:27, HCDR2 as shown in SEQ ID NO:28, HCDR3 as shown in SEQ ID NO:29; LCDR1 as shown in SEQ ID NO:30, LCDR2 as shown in SEQ ID NO:31, and LCDR3 as shown in SEQ ID NO:32.

[0192] In some specific embodiments of the present invention, the antigen-binding region specifically binding to CD3 comprises VH and VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO:25, and the VL contains the amino acid sequence shown in SEQ ID NO:26. In some specific embodiments of the present invention, the antigen-binding region specifically binding to CD3 comprises VH and VL, wherein the VH consists of the amino acid sequence shown in SEQ ID NO:25, and the VL consists of the amino acid sequence shown in SEQ ID NO:26.

[0193] In one embodiment, the antigen-binding region that specifically binds to CD3 is an antigen-binding fragment of an anti-CD3 antibody, such as an antigen-binding fragment selected from: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), or linear antibodies. Preferably, the antigen-binding region that specifically binds to CD3 is scFv (scFv CD3 ).

[0194] In some embodiments, the scFv fragment consists of a polypeptide chain comprising VH and VL domains, wherein the VH and VL are linked (e.g., via a linker) to pair and form an antigen-binding site. In some embodiments, the scFv is in a trans configuration, comprising or consisting of VH, a linker, and VL (VH-linker-VL) from the N-terminus to the C-terminus. In other embodiments, the scFv is in a cis configuration, comprising or consisting of VL, a linker, and VH (VL-linker-VH) from the N-terminus to the C-terminus. In some embodiments, the linker is a peptide linker consisting of amino acid residues. Suitable peptide linkers are known to those skilled in the art. In one embodiment, the linker is 5-50 amino acid long, for example 5-30 amino acid long, for example 15 or 20 amino acid long. In one embodiment, the linker comprises an amino acid sequence (G4S). nWhere n = 1, 2, 3, 4, or 5, preferably n = 3 or 4, more preferably n = 4. In one embodiment, the linker comprises or consists of the amino acid sequence shown in SEQ ID NO: 47 or 49. In some preferred embodiments, the scFv is a disulfide-stabilized scFv.

[0195] In some embodiments, the specific CD3-binding antigen-binding region of the present invention is scFv, which comprises or consists of the following from the N-terminus to the C-terminus: VH CD3 -VL CD3 Where "-" represents a connector or direct connection, and VH CD3 C-terminus and VL CD3 The N-terminus is connected directly or via a connector (e.g., connector 2).

[0196] Therefore, in some embodiments, the CD3-specific scFv of the present invention comprises the amino acid sequence shown in SEQ ID NO:52, or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0197] II-2 specifically binds to the antigen-binding region of BCMA.

[0198] In some embodiments, the antigen-binding region that specifically binds to BCMA includes three complementarity-determining regions (HCDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding region that specifically binds to BCMA includes three complementarity-determining regions (LCDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3. In some embodiments, the antigen-binding region that specifically binds to BCMA includes both three HCDRs from the heavy chain variable region and three LCDRs from the light chain variable region.

[0199] In some respects, the antigen-binding region that specifically binds to BCMA includes a heavy chain variable region (VH, also referred to herein as "VH"). BCMA In some respects, the antigen-binding region that specifically binds to BCMA contains a light chain variable region (VL, also referred to herein as "VL"). BCMAIn some aspects, the antigen-binding region that specifically binds to BCMA includes a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region includes three complementarity-determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region includes three complementarity-determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the HCDRs and LCDRs can be determined according to any scheme for determining CDRs, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, HCDR2 and HCDR3 are determined according to the Kabat scheme, and LCDR1, LCDR2, and LCDR3 are determined according to the Kabat scheme.

[0200] In some embodiments, the antigen-binding region is derived from an antibody that specifically binds to BCMA, such as the BCMA antibody disclosed in WO2023104100A1 (integrated herein in its entirety), for example, ADI38497 therein. In some embodiments, the antigen-binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to BCMA (e.g., the BCMA antibody disclosed in WO2023104100A1, for example, ADI38497 therein). In some embodiments, the antigen-binding region comprises 1, 2, or 3 heavy chain variable region CDRs of a known antibody that specifically binds to BCMA (e.g., the BCMA antibody disclosed in WO2023104100A1, for example, ADI38497 therein), namely HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding region comprises one, two, or three light chain variable region CDRs, namely LCDR1, LCDR2, and LCDR3, of a known BCMA-specific antibody (e.g., the BCMA antibody disclosed in WO2023104100A1, such as ADI38497). In some embodiments, the antigen-binding region comprises three heavy chain variable region CDRs and three light chain variable region CDRs of a known BCMA-specific antibody (e.g., the BCMA antibody disclosed in WO2023104100A1, such as ADI38497). In some embodiments, the antigen-binding region comprises both heavy chain and light chain variable regions of a known BCMA-specific antibody (e.g., the BCMA antibody disclosed in WO2023104100A1, such as ADI38497), particularly a combination of the heavy chain and light chain variable regions.

[0201] In some embodiments, the heavy chain variable region VH included in the antigen-binding region that specifically binds to BCMA

[0202] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 17; or

[0203] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:17; or

[0204] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:17, preferably, the amino acid changes do not occur in the CDR region.

[0205] In some embodiments, the light chain variable region (VL) included in the antigen-binding region that specifically binds to BCMA

[0206] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 18; or

[0207] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:18; or

[0208] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:18, preferably, the amino acid changes do not occur in the CDR region.

[0209] In some embodiments, the three complementary determinant regions (HCDRs) from the heavy chain variable region of the antigen-binding region that specifically binds to BCMA, HCDR1, HCDR2, and HCDR3, are the three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO:17. Preferably, the HCDRs can be determined according to any scheme for determining CDRs, such as Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, and HCDR2 and HCDR3 are determined according to Kabat.

[0210] In some embodiments, the three complementary determinant regions (LCDRs) from the light chain variable region of the antigen-binding region that specifically binds to BCMA, LCDR1, LCDR2, and LCDR3, are the three complementary determinant regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO:18. Preferably, the LCDRs can be determined according to any scheme for determining CDRs, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, LCDR1, LCDR2, and LCDR3 are determined by the Kabat scheme.

[0211] In some embodiments, in the antigen-binding region of the present invention that specifically binds to BCMA, HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:19; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:20; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:21; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:22; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:23; and / or LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:24.

[0212] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to BCMA includes VH and VL, wherein

[0213] The VH contains the amino acid sequence shown in SEQ ID NO:17 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:18 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0214] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to BCMA includes three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:17, and three complementary determinant regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:18. Preferably, the HCDR and LCDR can be determined according to any scheme for determining CDR, such as Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, HCDR2 and HCDR3 are determined according to Kabat, and LCDR1, LCDR2, and LCDR3 are determined according to the Kabat scheme.

[0215] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to BCMA includes: HCDR1 as shown in SEQ ID NO:19, HCDR2 as shown in SEQ ID NO:20, HCDR3 as shown in SEQ ID NO:21; LCDR1 as shown in SEQ ID NO:22, LCDR2 as shown in SEQ ID NO:23, and LCDR3 as shown in SEQ ID NO:24.

[0216] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to BCMA comprises VH and VL, wherein VH contains the amino acid sequence shown in SEQ ID NO:17, and VL contains the amino acid sequence shown in SEQ ID NO:18. In some specific embodiments of the present invention, the antigen-binding region that specifically binds to BCMA comprises VH and VL, wherein VH consists of the amino acid sequence shown in SEQ ID NO:17, and VL consists of the amino acid sequence shown in SEQ ID NO:18.

[0217] In one embodiment, the antigen-binding region that specifically binds to BCMA is an antigen-binding fragment of an anti-BCMA antibody, such as an antigen-binding fragment selected from: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), or linear antibodies. Preferably, the antigen-binding region that specifically binds to BCMA is Fab (Fab')2. BCMA ).

[0218] In some implementations, the Fab fragment consists of the VH(VH) BCMA CH1 of the heavy chain constant region, and the VL (VL) BCMA The Fab consists of two polypeptide chains, VH and CH1, forming a light chain constant region CL, wherein VH pairs with VL and CH1 pairs with CL to form a Fab, serving as an antigen-binding region. In some embodiments, in the Fab, one chain from the N-terminus to the C-terminus contains VH and CH1 (i.e., VH-CH1) or consists of VH and CH1 (where the C-terminus of VH is connected to the N-terminus of CH1), and the other chain from the N-terminus to the C-terminus contains VL and CL (i.e., VL-CL) or consists of VL and CL (where the C-terminus of VL is connected to the N-terminus of CL). In this document, the Fab chain containing VH-CH1 or consisting of VH-CH1 is also referred to as the Fab heavy chain, and the Fab chain containing VL-CL or consisting of VL-CL is also referred to as the Fab light chain.

[0219] Therefore, in some embodiments, the antigen-binding region of the present invention that specifically binds to BCMA is a BCMA-specific Fab, wherein the Fab comprises or is composed of the following:

[0220] Fab heavy chain: from N end to C end: VH BCMA -CH1, where VH BCMA The C-terminus of CH1 is directly connected to the N-terminus of CH1;

[0221] Fab Light Chain: From N-end to C-end: VL BCMA -CL, where VL BCMA The C-terminus of CL is directly connected to the N-terminus of CL;

[0222] Where "-" represents a direct connection, VH BCMA and VL BCMA These are VH and VL, respectively, the antigen-binding regions that specifically bind to BCMA as defined in this article.

[0223] In some embodiments, CH1 is derived from IgG, such as CH1 of (human) IgG1, IgG2, IgG3, or IgG4, preferably CH1 of human IgG1. In some embodiments, CH1

[0224] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:40;

[0225] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:40; or

[0226] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:40.

[0227] In some embodiments, the CL is a light chain constant region derived from the Kappa light chain constant region or the Lambda light chain constant region (e.g., the human Kappa light chain constant region or the human Lambda light chain constant region). In some embodiments, the CL is a Kappa light chain constant region or a Lambda light chain constant region, such as the human Kappa light chain constant region or the human Lambda light chain constant region. In some embodiments, the CL is a human Kappa light chain constant region. In some embodiments, the CL...

[0228] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:41;

[0229] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:41; or

[0230] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:41.

[0231] II-3 specifically binds to the CD19 antigen-binding region.

[0232] In some embodiments, the CD19-specific antigen-binding region includes three complementarity-determining regions (HCDRs) from the heavy chain variable region: HCDR1, HCDR2, and HCDR3. In some embodiments, the CD19-specific antigen-binding region includes three complementarity-determining regions (LCDRs) from the light chain variable region: LCDR1, LCDR2, and LCDR3. In some embodiments, the CD19-specific antigen-binding region includes three HCDRs from the heavy chain variable region and three LCDRs from the light chain variable region.

[0233] In some respects, the antigen-binding region that specifically binds to CD19 includes a heavy chain variable region (VH, also referred to herein as "VH"). CD19 In some respects, the antigen-binding region that specifically binds to CD19 includes a light chain variable region (VL, also referred to herein as "VL"). CD19 In some aspects, the antigen-binding region that specifically binds to CD19 comprises a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementarity-determining regions (CDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementarity-determining regions (CDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3. In some embodiments, the HCDRs and LCDRs can be determined according to any scheme for determining CDRs, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, HCDR2 and HCDR3 are determined according to the Kabat scheme, and LCDR1, LCDR2, and LCDR3 are determined according to the Kabat scheme.

[0234] In some embodiments, the antigen-binding region is derived from an antibody that specifically binds to CD19, such as the CD19 antibody disclosed in US20120082664 or WO2007002223A2 (both incorporated herein by reference in their entirety). In some embodiments, the antigen-binding region comprises 1, 2, 3, 4, 5, or 6 CDRs of a known antibody that specifically binds to CD19 (e.g., the CD19 antibody disclosed in US20120082664 or WO2007002223A2). In some embodiments, the antigen-binding region comprises 1, 2, or 3 heavy chain variable region CDRs of a known antibody that specifically binds to CD19 (e.g., the CD19 antibody disclosed in US20120082664 or WO2007002223A2), namely HCDR1, HCDR2, and HCDR3. In some embodiments, the antigen-binding region comprises one, two, or three light chain variable regions (CDRs), namely LCDR1, LCDR2, and LCDR3, of a known antibody that specifically binds to CD19 (e.g., the CD19 antibody disclosed in US20120082664 or WO2007002223A2). In some embodiments, the antigen-binding region comprises three heavy chain variable regions and three light chain variable regions of a known antibody that specifically binds to CD19 (e.g., the CD19 antibody disclosed in US20120082664 or WO2007002223A2). In some embodiments, the antigen-binding region comprises both heavy chain and light chain variable regions of a known antibody that specifically binds to CD19 (e.g., the CD19 antibody disclosed in US20120082664 or WO2007002223A2), particularly a combination of the heavy chain and light chain variable regions.

[0235] In some embodiments, the heavy chain variable region VH included in the antigen-binding region that specifically binds to CD19

[0236] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1; or

[0237] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:1; or

[0238] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:1, preferably, the amino acid changes do not occur in the CDR region.

[0239] In some embodiments, the light chain variable region VL included in the antigen-binding region that specifically binds to CD19

[0240] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:2 or 70; or

[0241] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:2 or 70; or

[0242] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:2 or 70, preferably, the amino acid changes do not occur in the CDR region.

[0243] In some embodiments, the three complementary determinant regions (HCDRs) from the heavy chain variable region of the antigen-binding region that specifically binds to CD19, HCDR1, HCDR2, and HCDR3, are the three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO:1. Preferably, the HCDRs can be determined according to any scheme for determining CDRs, such as Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, and HCDR2 and HCDR3 are determined according to Kabat.

[0244] In some embodiments, the three complementary determinant regions (LCDRs) from the light chain variable region of the antigen-binding region that specifically binds to CD19, LCDR1, LCDR2, and LCDR3, are the three complementary determinant regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO:2 or 70. Preferably, the LCDRs can be determined according to any scheme for determining CDRs, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, LCDR1, LCDR2, and LCDR3 are determined by the Kabat scheme.

[0245] In some embodiments, the antigen-binding region of the present invention specifically binds to CD19,

[0246] HCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:3; HCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:4; HCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:5; LCDR1 contains or is composed of the amino acid sequence shown in SEQ ID NO:6 or 71; LCDR2 contains or is composed of the amino acid sequence shown in SEQ ID NO:7; and / or LCDR3 contains or is composed of the amino acid sequence shown in SEQ ID NO:8.

[0247] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD19 includes VH and VL, wherein

[0248] The VH contains the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:2 or 70 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0249] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD19 includes three complementary determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:1, and three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:2 or 70. Preferably, the HCDR and LCDR can be determined according to any scheme for determining CDR, such as Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, HCDR2 and HCDR3 are determined according to Kabat; and LCDR1, LCDR2, and LCDR3 are determined according to the Kabat scheme.

[0250] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD19 includes: HCDR1 as shown in SEQ ID NO:3, HCDR2 as shown in SEQ ID NO:4, HCDR3 as shown in SEQ ID NO:5; LCDR1 as shown in SEQ ID NO:6 or 71, LCDR2 as shown in SEQ ID NO:7, and LCDR3 as shown in SEQ ID NO:8.

[0251] In some specific embodiments of the present invention, the antigen-binding region specifically binding to CD19 comprises VH and VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO:1, and the VL contains the amino acid sequence shown in SEQ ID NO:2 or 70. In some specific embodiments of the present invention, the antigen-binding region specifically binding to CD19 comprises VH and VL, wherein the VH consists of the amino acid sequence shown in SEQ ID NO:1, and the VL consists of the amino acid sequence shown in SEQ ID NO:2 or 70.

[0252] In some embodiments, the heavy chain variable region VH included in the antigen-binding region that specifically binds to CD19

[0253] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:9; or

[0254] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:9; or

[0255] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:9, preferably, the amino acid changes do not occur in the CDR region.

[0256] In some embodiments, the light chain variable region VL included in the antigen-binding region that specifically binds to CD19

[0257] (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO: 10; or

[0258] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:10; or

[0259] (iii) An amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:10, preferably, the amino acid changes do not occur in the CDR region.

[0260] In some embodiments, the three complementary determinant regions (HCDRs) from the heavy chain variable region of the antigen-binding region that specifically binds to CD19, HCDR1, HCDR2, and HCDR3, are the three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO:9. Preferably, the HCDRs can be determined according to any scheme for determining CDRs, such as Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, and HCDR2 and HCDR3 are determined according to Kabat.

[0261] In some embodiments, the three complementary determinant regions (LCDRs) from the light chain variable region of the antigen-binding region that specifically binds to CD19, LCDR1, LCDR2, and LCDR3, are the three complementary determinant regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO:10. Preferably, the LCDRs can be determined according to any scheme for determining CDRs, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, LCDR1, LCDR2, and LCDR3 are determined by the Kabat scheme.

[0262] In some embodiments, in the specific CD19-binding antigen-binding region of the present invention, HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:11; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:12; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:13; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:14; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:15; and / or LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:16.

[0263] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD19 includes VH and VL, wherein

[0264] The VH contains the amino acid sequence shown in SEQ ID NO:9 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0265] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD19 includes three complementary determining regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:9, and three complementary determining regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:10. Preferably, the HCDR and LCDR can be determined according to any scheme for determining CDR, such as Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, HCDR2 and HCDR3 are determined according to Kabat, and LCDR1, LCDR2, and LCDR3 are determined according to the Kabat scheme.

[0266] In some specific embodiments of the present invention, the antigen-binding region that specifically binds to CD19 includes: HCDR1 as shown in SEQ ID NO:11, HCDR2 as shown in SEQ ID NO:12, HCDR3 as shown in SEQ ID NO:13; LCDR1 as shown in SEQ ID NO:14, LCDR2 as shown in SEQ ID NO:15, and LCDR3 as shown in SEQ ID NO:16.

[0267] In some specific embodiments of the present invention, the antigen-binding region specifically binding to CD19 comprises VH and VL, wherein VH contains the amino acid sequence shown in SEQ ID NO:9, and VL contains the amino acid sequence shown in SEQ ID NO:10. In some specific embodiments of the present invention, the antigen-binding region specifically binding to CD19 comprises VH and VL, wherein VH consists of the amino acid sequence shown in SEQ ID NO:9, and VL consists of the amino acid sequence shown in SEQ ID NO:10.

[0268] In one embodiment, the antigen-binding region that specifically binds to CD19 is an antigen-binding fragment of an anti-CD19 antibody, such as an antigen-binding fragment selected from: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies such as VHH, dAb (domain antibody), or linear antibodies. Preferably, the antigen-binding region that specifically binds to CD19 is Fab (Fab'-SH). CD19 ).

[0269] In some implementations, the Fab fragment consists of the VH(VH) CD19 CH1 of the heavy chain constant region, and the VL (VL) CD19 The Fab consists of two polypeptide chains, VH and CH1, forming a light chain constant region CL, wherein VH pairs with VL and CH1 pairs with CL to form a Fab, serving as an antigen-binding region. In some embodiments, in the Fab, one chain from the N-terminus to the C-terminus contains VH and CH1 (i.e., VH-CH1) or consists of VH and CH1 (where the C-terminus of VH is connected to the N-terminus of CH1), and the other chain from the N-terminus to the C-terminus contains VL and CL (i.e., VL-CL) or consists of VL and CL (where the C-terminus of VL is connected to the N-terminus of CL). In this document, the Fab chain containing VH-CH1 or consisting of VH-CH1 is also referred to as the Fab heavy chain, and the Fab chain containing VL-CL or consisting of VL-CL is also referred to as the Fab light chain.

[0270] Therefore, in some embodiments, the antigen-binding region of the present invention that specifically binds to CD19 is a Fab that specifically binds to BCMA, wherein the Fab comprises or is composed of the following:

[0271] Fab heavy chain: from N end to C end: VH CD19 -CH1, where VH CD19 The C-terminus of CH1 is directly connected to the N-terminus of CH1;

[0272] Fab Light Chain: From N-end to C-end: VL CD19 -CL, where VL CD19 The C-terminus of CL is directly connected to the N-terminus of CL;

[0273] Where "-" represents a direct connection, VH CD19 and VL CD19 These are VH and VL, which are the antigen-binding regions that specifically bind to CD19, as defined in this article.

[0274] In some embodiments, CH1 is derived from IgG, such as CH1 of (human) IgG1, IgG2, IgG3, or IgG4, preferably CH1 of human IgG1. In some embodiments, CH1

[0275] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:40;

[0276] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:40; or

[0277] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:40.

[0278] In some embodiments, the CL is a light chain constant region derived from the Kappa light chain constant region or the Lambda light chain constant region (e.g., the human Kappa light chain constant region or the human Lambda light chain constant region). In some embodiments, the CL is a Kappa light chain constant region or a Lambda light chain constant region, such as the human Kappa light chain constant region or the human Lambda light chain constant region. In some embodiments, the CL is a human Kappa light chain constant region. In some embodiments, the CL...

[0279] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:41;

[0280] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:41; or

[0281] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10 or 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid alterations (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:41.

[0282] II-4.Fc area

[0283] In some embodiments, the multispecific binding molecules of the present invention, such as multispecific antibodies (e.g., trispecific antibodies), further include an Fc region, wherein the included Fc regions may be the same or different.

[0284] In some implementations, the first Fc region and the second Fc region are different, and they are capable of dimerizing to form a heterodimeric Fc scaffold.

[0285] In this document, the Fc region refers to the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region, and may include native sequence Fc regions and variant Fc regions. Native sequence Fc regions encompass various naturally occurring immunoglobulin Fc sequences, such as the Fc regions of various Ig subtypes and their allotypes (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi:10.3389 / fimmu.2014.00520). In some embodiments, the Fc region of the present invention comprises antibodies CH2 and CH3. In some embodiments, the antibody Fc region may also have an IgG hinge region or a portion of an IgG hinge region at its N-terminus, for example, an IgG1 hinge region or a portion of an IgG1 hinge region, such as sequences D221 to P230 according to EU numbers. Mutations may be contained in said hinge region. Unless otherwise specified, when referring to the Fc region in this article, it means the Fc region with a portion of the IgG hinge region (corresponding to the EU numbers D221 to P230 of IgG1) at the N-terminus.

[0286] Unless otherwise noted herein, the amino acid residues in the Fc region are numbered according to the EU numbering system, also known as the EU index, as described in Kabat, EA, et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242.

[0287] In some embodiments, the immunoglobulin Fc region is preferably derived from IgG1, IgG2, IgG3, or IgG4, or their subtypes, such as human IgG1, IgG2, IgG3, or IgG4, or their subtypes. Preferably, the immunoglobulin Fc region comprises an Fc region sequence derived from a human. In some embodiments, the Fc region is a human IgG Fc region, for example, human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, or human IgG4 Fc. In one embodiment, the Fc region comprises or consists of the amino acid sequence shown in SEQ ID NO:42 or an amino acid sequence having at least 90% identity with it, such as 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher.

[0288] The Fc region of the binding molecule of the present invention, such as an antibody, can be mutated to obtain the desired properties. Mutations of the Fc region are known in the art.

[0289] As those skilled in the art will understand, in order to promote the formation of heterodimers in the multispecific antibodies of the present invention, the Fc regions comprising the multispecific antibodies of the present invention may contain mutations that facilitate heterodimerization of the first Fc region and the second Fc region. In one embodiment, mutations are introduced in the CH3 regions of both Fc regions.

[0290] Methods for promoting heterodimerization of Fc regions are known in the art. For example, the CH3 regions of the first and second Fc regions are engineered in a complementary manner such that each CH3 region (or the heavy chain containing it) can no longer homodimerize with itself but is forced to heterodimerize with other complementary engineered CH3 regions (so that the CH3 regions of the first and second Fc regions heterodimerize and no homodimer is formed between the two first CH3 regions or the two second CH3 regions). For example, based on the Knob-into-Hole technique, corresponding Knob mutations and Hole mutations are introduced into the first and second Fc regions. This technique is described in, for example, US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9,617-621 (1996) and Carter, J Immunol Meth 248,7-15 (2001).

[0291] Alternatively, based on Innobody technology, corresponding mutations can be introduced into the first and second Fc regions. This technology is illustrated, for example, by PCT / CN2021 / 143141 (the entire contents of which are incorporated herein by reference).

[0292] In a particular implementation scheme

[0293] The first CH3 region contains the S364R / K mutation (preferably S364R), and optionally one or more other mutations. In some embodiments, the second CH3 region contains the K370S / T / A / V mutation (preferably K370S), and optionally one or more other mutations. In some embodiments, the first CH3 region contains the S364R mutation, and the second CH3 region contains the K370S mutation. In some embodiments, the first CH3 region contains the S364R mutation, and the second CH3 region contains the K370S mutation.

[0294] In some embodiments, the first CH3 region contains the S364R / K (preferably S364R) and D399K / R (preferably D399K) mutations. In some embodiments, the second CH3 region contains the K370S / T / A / V (preferably K370S) mutation and the K409D / E (preferably K409D) mutation. In some embodiments, the first CH3 region contains S364R / K+D399K / R, and the second CH3 region contains K370S / T / A / V+Y349T / S / A / V. In some embodiments, the first CH3 region contains S364R+D399K, and the second CH3 region contains K370S+Y349T. In some embodiments, the first CH3 region further contains E375N / Q (preferably E375N) and / or T350V / A (preferably T350V). In some embodiments, the second CH3 region further includes K409D / E (preferably K409D), Q347D / E (preferably Q347D) and / or T350V / A (preferably T350V).

[0295] In some embodiments, the first CH3 region includes S364R+D399K, and the second CH3 region includes K370S+Y349T+K409D. In some embodiments, the first CH3 region further includes E357N. In some embodiments, the second CH3 region further includes Q347D. In some embodiments, the first CH3 region further includes E357N, and the second CH3 region further includes Q347D. In some embodiments, the first CH3 region and the second CH3 region further each include T350V, or both include T350V.

[0296] Therefore, in some embodiments, the first CH3 region includes S364R+D399K, and the second CH3 region includes K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region includes S364R+D399K+E357N, and the second CH3 region includes K370S+Y349T+K409D+Q347D. In some embodiments, the first CH3 region includes S364R+D399K+E357N+T350V, and the second CH3 region includes K370S+Y349T+K409D+Q347D+T350V.

[0297] In some embodiments, the first CH3 region comprises K409E / D (preferably K409E). In some embodiments, the second CH3 region comprises D399K / R (preferably D399K) or K370T / S / A / V (preferably K370T). In some embodiments, the first CH3 region comprises K409E / D (preferably K409E), and the second CH3 region comprises D399K / R (preferably D399K). In some embodiments, the first CH3 region further comprises T411R / K (preferably T411R). In some embodiments, the second CH3 region further comprises K370T / S / A / V (preferably K370T). In some embodiments, the CH3 region comprises K409E / D+T411R / K, and the second CH3 region comprises D399K / R+K370T / S / A / V. In some implementations, the CH3 region includes K409E+T411R, and the second CH3 region includes D399K+K370T.

[0298] In some specific implementations, the first and second CH3 regions have a combination of five mutations selected from the following:

[0299] In one implementation, CH3 in one Fc region contains S364R and D399K mutations, and CH3 in another Fc region contains Y349T, K370S, and K409D mutations.

[0300] The CH3 mutation location described herein refers to the human IgG1 Fc region, for example, the amino acid sequence shown in SEQ ID NO:42.

[0301] In some implementations, the Fc region also contains other mutations that facilitate heterodimer purification.

[0302] As those skilled in the art will understand, according to the present invention, in conjunction with the intended use of a molecule such as an antibody molecule, the Fc region can be modified to possess characteristics of effector functions (e.g., complement activation functions of the Fc region, such as ADCC, ADCP, or CDC effector functions). In one embodiment, the effector function has been reduced or eliminated relative to the wild-type isotype Fc region. In one embodiment, the effector function is reduced or eliminated by using a naturally occurring Fc isotype with reduced or eliminated effector function and / or Fc region modification. In a preferred embodiment, the Fc region has reduced Fc region-mediated effector functions, such as reduced or eliminated ADCC, ADCP, or CDC effector functions, for example, by including mutations that achieve the aforementioned functions.

[0303] In some embodiments, the binding molecules of the present invention, such as antibody molecules, may also include modifications in the Fc region that alter the binding affinity to one or more Fc receptors. In one embodiment, the Fc receptor is an Fcγ receptor, particularly a human Fcγ receptor. In some embodiments, the Fc region contains mutations that reduce binding to the Fcγ receptor. For example, in some embodiments, the Fc region used in the present invention has mutations that reduce binding to the Fcγ receptor, such as the L234A / L235A mutation. In yet another preferred embodiment, the Fc fragment may have mutations that result in an increased serum half-life, such as mutations that improve the binding of the Fc fragment to FcRn.

[0304] Therefore, in a specific embodiment, the multispecific antibody of the present invention, such as a trispecific antibody, comprises a first Fc region and a second Fc region, wherein the two Fc regions are heterodimerized, wherein

[0305] One Fc-region polypeptide contains the mutants S364R and D399K, while the other Fc-region polypeptide contains the mutants Y349T, K370S, and K409D;

[0306] Optionally, the Fc region may also contain L234A / L235A mutations.

[0307] Therefore, in a specific embodiment, the multispecific antibody of the present invention, such as a trispecific antibody, comprises a first Fc region and a second Fc region, wherein the two Fc regions are heterodimerized, wherein

[0308] a) One Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:43, while the other Fc region polypeptide contains or is composed of the amino acid sequence shown in SEQ ID NO:44.

[0309] In one specific embodiment, the multispecific antibody of the present invention, such as a trispecific antibody, comprises two Fc regions (a first Fc region and a second Fc region), wherein the two Fc regions are heterodimerized, wherein

[0310] a) One Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:43, while the other Fc region polypeptide contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:44.

[0311] Therefore, in one specific embodiment, the multispecific antibody of the present invention, such as a trispecific antibody, comprises two Fc regions (a first Fc region and a second Fc region), wherein the two Fc regions are heterodimerized, wherein

[0312] a) One Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:43 and contains the mutations S364R and D399K, while the other Fc region polypeptide contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:44 and contains the mutations Y349T, K370S, and K409D;

[0313] b) One Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:43 and contains the mutations S364R and D399K and L234A / L235A mutations, while the other Fc region polypeptide contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:44 and contains the mutations Y349T, K370S, and K409D and L234A / L235A mutations.

[0314] II-5 Exemplary Multispecific Antibody

[0315] In some specific embodiments of the present invention, the trispecific antibody of the present invention specifically binds to CD3, CD19, and BCMA, wherein the trispecific antibody comprises heavy chain 1, heavy chain 2, light chain 1, and light chain 2, or is composed of said chains.

[0316] Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:33, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:34, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:35, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:35. The amino acid sequence shown in NO:36 or 73, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or consisting of said amino acid sequence; or

[0317] Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:33, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:34, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:37, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:37. The amino acid sequence shown in NO:38, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or consisting of said amino acid sequence; or

[0318] Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:72, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:73, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:74, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:74. The amino acid sequence shown in NO:34, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence.

[0319] In some specific embodiments of the present invention, the trispecific antibody of the present invention specifically binds to CD3, CD19, and BCMA, wherein the trispecific antibody comprises heavy chain 1, heavy chain 2, light chain 1, and light chain 2, or is composed of said chains.

[0320] Heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:33; light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:34; heavy chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:35; and light chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:36 or 73; or

[0321] Heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:33; light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:34; heavy chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:37; and light chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:38; or

[0322] Heavy chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:72; light chain 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:73; heavy chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:74; and light chain 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:34.

[0323] III. Anti-CD3 antibody or its antigen-binding fragment

[0324] In some embodiments, the present invention also relates to an anti-CD3 antibody or an antigen-binding fragment thereof, which includes an antigen-binding region specifically binding to CD3 as defined above.

[0325] In some specific embodiments of the present invention, the anti-CD3 antibody or its antigen-binding fragment comprises VH and VL, wherein the VH contains or is composed of the amino acid sequence shown in SEQ ID NO:25, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:26.

[0326] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention is an antibody in the form of IgG1, IgG2, IgG3, or IgG4, or its antigen-binding fragment, for example, an antibody in the form of IgG1 or its antigen-binding fragment.

[0327] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention further comprises an antibody heavy chain constant region (HC). In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention further comprises an antibody light chain constant region (LC). In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention further comprises both a heavy chain constant region (HC) and a light chain constant region (LC). In some embodiments, the heavy chain constant region of the present invention comprises an Fc region, such as an Fc region as defined herein.

[0328] In some embodiments, the heavy chain constant region comprising the anti-CD3 antibody or its antigen-binding fragment of the present invention is a heavy chain constant region derived from IgG1, IgG2, IgG3, or IgG4 (e.g., human IgG1, IgG2, IgG3, or IgG4). In some embodiments, the heavy chain constant region is a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, for example, the heavy chain constant region of human IgG1.

[0329] In some implementations, the heavy chain constant region

[0330] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:39;

[0331] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:39; or

[0332] (iii) An amino acid sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO:39 having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions).

[0333] In some embodiments, the light chain constant region of the anti-CD3 antibody of the present invention is a light chain constant region as defined herein. In some embodiments, the light chain constant region is a light chain constant region derived from the Kappa light chain constant region or the Lambda light chain constant region (e.g., the human Kappa light chain constant region or the human Lambda light chain constant region). In some embodiments, the light chain constant region is the Kappa light chain constant region or the Lambda light chain constant region, such as the human Kappa light chain constant region or the human Lambda light chain constant region. In some embodiments, the light chain constant region is the human Kappa light chain constant region.

[0334] In some implementations, the light chain constant region

[0335] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:41;

[0336] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:41; or

[0337] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:41.

[0338] In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises an antibody heavy chain. In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises an antibody light chain. In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises a heavy chain and a light chain. In some embodiments, the anti-CD3 antibody or its antigen-binding fragment of the present invention comprises two heavy chains and two light chains. In some embodiments, the heavy chain comprises a heavy chain variable region and a heavy chain constant region, or is composed of a heavy chain variable region and a heavy chain constant region. In some embodiments, the light chain comprises a light chain variable region and a light chain constant region, or is composed of a light chain variable region and a light chain constant region.

[0339] In some implementations, the anti-CD3 antibody is a monoclonal antibody.

[0340] In some implementations, the anti-CD3 antibody is humanized.

[0341] In one embodiment, the anti-CD3 antibody of the present invention is a full-length antibody. In another embodiment, the anti-CD3 antibody of the present invention further encompasses its antibody fragments (e.g., antigen-binding fragments), preferably selected from the following antigen-binding fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), dAb (domain antibody), heavy-chain antibodies, or linear antibodies.

[0342] In one embodiment, the anti-CD3 antibody fragment of the present invention is a single-chain antibody comprising an anti-CD3 antibody fragment scFv and a heavy chain constant region Fc (optionally the Fc region includes a hinge region or a partial hinge region), or is composed of scFv and a heavy chain constant region Fc (optionally the Fc region includes a hinge region or a partial hinge region).

[0343] In one embodiment, the anti-CD3 antibody of the present invention may also be a trispecific antibody or a multispecific antibody that specifically binds to CD3, as well as one or more other antigens.

[0344] IV. Anti-CD19 antibody or its antigen-binding fragment

[0345] In some embodiments, the present invention also relates to an anti-CD19 antibody or an antigen-binding fragment thereof, which includes an antigen-binding region specifically binding to CD19 as defined above.

[0346] In some specific embodiments, the antibody that specifically binds to CD19 or its antigen-binding fragment contains three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, which are the three complementarity-determining regions HCDR1, HCDR2, and HCDR3 contained in the VH as shown in SEQ ID NO:1. Preferably, the HCDRs can be determined according to any scheme for determining CDRs, such as Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, and HCDR2 and HCDR3 are determined according to Kabat.

[0347] In some embodiments, the antibody that specifically binds to CD19 or its antigen-binding fragment includes three complementary determinant regions (LCDRs) from the variable region of the light chain, LCDR1, LCDR2, and LCDR3, which are the three complementary determinant regions LCDR1, LCDR2, and LCDR3 contained in the VL as shown in SEQ ID NO:2. Preferably, the LCDRs can be determined according to any scheme for determining CDRs, such as the Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, LCDR1, LCDR2, and LCDR3 are determined by the Kabat scheme.

[0348] In some specific embodiments of the present invention, the antibody that specifically binds to CD19 or its antigen-binding fragment includes three complementary determinant regions HCDR1, HCDR2, and HCDR3 contained in VH as shown in SEQ ID NO:1, and three complementary determinant regions LCDR1, LCDR2, and LCDR3 contained in VL as shown in SEQ ID NO:2. Preferably, the HCDR and LCDR can be determined according to any scheme for determining CDR, such as Kabat, AbM, Chothia, Contact, or IMGT schemes or combinations thereof; for example, HCDR1 is determined according to the AbM rule, and HCDR2 and HCDR3 are determined according to Kabat; and LCDR1, LCDR2, and LCDR3 are determined according to the Kabat scheme.

[0349] In some embodiments, in the CD19-specific antibody or antigen-binding fragment of the present invention, HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:3; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:4; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:5; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:6; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:7; and / or LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:8.

[0350] In some specific embodiments of the present invention, the antibody or its antigen-binding fragment that specifically binds to CD19 includes: HCDR1 as shown in SEQ ID NO:3, HCDR2 as shown in SEQ ID NO:4, HCDR3 as shown in SEQ ID NO:5; LCDR1 as shown in SEQ ID NO:6, LCDR2 as shown in SEQ ID NO:7, and LCDR3 as shown in SEQ ID NO:8.

[0351] In some specific embodiments of the present invention, the antibody or its antigen-binding fragment that specifically binds to CD19 comprises VH and VL, wherein

[0352] The VH contains the amino acid sequence shown in SEQ ID NO:1 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence, and the VL contains the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0353] In some specific embodiments of the present invention, the antibody or antigen-binding fragment that specifically binds to CD19 comprises VH and VL, wherein the VH contains the amino acid sequence shown in SEQ ID NO:1, and the VL contains the amino acid sequence shown in SEQ ID NO:2.

[0354] In some embodiments, the anti-CD19 antibody or its antigen-binding fragment of the present invention is an antibody in the form of IgG1, IgG2, IgG3, or IgG4, or its antigen-binding fragment, for example, an antibody in the form of IgG1 or its antigen-binding fragment.

[0355] In some embodiments, the anti-CD19 antibody or its antigen-binding fragment of the present invention further comprises an antibody heavy chain constant region (HC). In some embodiments, the anti-CD19 antibody or its antigen-binding fragment of the present invention further comprises an antibody light chain constant region (LC). In some embodiments, the anti-CD19 antibody or its antigen-binding fragment of the present invention further comprises both a heavy chain constant region (HC) and a light chain constant region (LC). In some embodiments, the heavy chain constant region of the present invention comprises an Fc region, such as an Fc region as defined herein.

[0356] In some embodiments, the heavy chain constant region comprising the anti-CD19 antibody or its antigen-binding fragment is a heavy chain constant region derived from IgG1, IgG2, IgG3, or IgG4 (e.g., human IgG1, IgG2, IgG3, or IgG4). In some embodiments, the heavy chain constant region is a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4, for example, the heavy chain constant region of human IgG1.

[0357] In some implementations, the heavy chain constant region

[0358] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:39;

[0359] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:39; or

[0360] (iii) An amino acid sequence comprising or consisting of the amino acid sequence shown in SEQ ID NO:39 having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions).

[0361] In some embodiments, the light chain constant region comprised of the anti-CD19 antibody or its antigen-binding fragment is a light chain constant region as defined herein. In some embodiments, the light chain constant region is a light chain constant region derived from the Kappa light chain constant region or the Lambda light chain constant region (e.g., the human Kappa light chain constant region or the human Lambda light chain constant region). In some embodiments, the light chain constant region is the Kappa light chain constant region or the Lambda light chain constant region, such as the human Kappa light chain constant region or the human Lambda light chain constant region. In some embodiments, the light chain constant region is the human Kappa light chain constant region.

[0362] In some implementations, the light chain constant region

[0363] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:41;

[0364] (ii) Contains or is composed of the amino acid sequence shown in SEQ ID NO:41; or

[0365] (iii) An amino acid sequence comprising or consisting of one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence shown in SEQ ID NO:41.

[0366] In some embodiments, the anti-CD19 antibody or its antigen-binding fragment of the present invention comprises an antibody heavy chain. In some embodiments, the anti-CD19 antibody or its antigen-binding fragment of the present invention comprises an antibody light chain. In some embodiments, the anti-CD19 antibody or its antigen-binding fragment of the present invention comprises both the heavy chain and the light chain. In some embodiments, the anti-CD19 antibody or its antigen-binding fragment of the present invention comprises two of the heavy chains and two of the light chains. In some embodiments, the heavy chain comprises, or is composed of, the heavy chain variable region and the heavy chain constant region. In some embodiments, the light chain comprises, or is composed of, the light chain variable region and the light chain constant region.

[0367] In some implementations, the anti-CD19 antibody is a monoclonal antibody.

[0368] In some implementations, the anti-CD19 antibody is humanized.

[0369] In one embodiment, the anti-CD19 antibody of the present invention is a full-length antibody. In another embodiment, the anti-CD19 antibody of the present invention also encompasses its antibody fragments (e.g., antigen-binding fragments), preferably selected from the following antigen-binding fragments: Fab, Fab', Fab'-SH, Fv, single-chain antibodies (e.g., scFv), (Fab')2, single-domain antibodies (e.g., VHH), dAb (domain antibody), heavy-chain antibodies, or linear antibodies.

[0370] In one embodiment, the anti-CD19 antibody of the present invention may also be a trispecific antibody or a multispecific antibody that specifically binds to CD19, as well as one or more other antigens.

[0371] V. Polynucleotides, carriers, and host cells

[0372] This invention provides nucleic acid molecules encoding any antibody molecule of the invention (e.g., the multispecific antibody of the invention, such as a trispecific antibody, or an anti-CD3 antibody or its antigen-binding fragment, or an anti-CD19 antibody or its antigen-binding fragment).

[0373] In one aspect, the present invention provides a nucleic acid molecule comprising or composed of a polynucleotide encoding any antibody molecule of the present invention (e.g., a multispecific antibody of the present invention such as a trispecific antibody, or an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD19 antibody or an antigen-binding fragment thereof).

[0374] To facilitate production and purification, any antibody molecule of the present invention (e.g., the multispecific antibody of the present invention, such as a trispecific antibody, or an anti-CD3 antibody or its antigen-binding fragment, or an anti-CD19 antibody or its antigen-binding fragment) may be fused to a secretory signal peptide at the N-terminus or C-terminus (e.g., C-terminus), and / or a tagged peptide that facilitates purification, such as a hexahistine tag or biotin label.

[0375] As will be apparent to those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences.

[0376] In some embodiments, the nucleic acid of the present invention comprises a polynucleotide encoding an amino acid sequence selected from any one of SEQ ID NO: 1, 2, 25, 26 and 33-38, or a polynucleotide encoding an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 1, 2, 25, 26 and 33-38, or is composed of said polynucleotide.

[0377] The nucleic acid molecules encoding the molecules of the present invention can be produced using methods well known in the art, such as de novo solid-phase DNA synthesis or PCR amplification.

[0378] In one aspect, the present invention also provides a vector comprising the nucleic acid molecules of the present invention. In one embodiment, the vector is an expression vector, such as a prokaryotic expression vector or a eukaryotic expression vector. The vector includes, but is not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YAC). In a preferred embodiment, the expression vector is pCDNA, such as pCDNA3.4.

[0379] In one aspect, the invention also provides a host cell comprising the nucleic acid molecule or the vector. In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells and mammalian cells (e.g., CHO cells or 293 cells, such as ExpiCHO cells, HEK 293, 293F, 293FT, or Expi293 cells). In yet another embodiment, the host cell is prokaryotic.

[0380] Suitable host cells include prokaryotic microorganisms such as *Escherichia coli*, eukaryotic microorganisms such as filamentous fungi or yeast, or various eukaryotic cells such as Chinese hamster ovary cells (CHO), insect cells, etc. Mammalian cell lines suitable for suspension culture can be used. Examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney lines (HEK 293 or 293F cells or 293FT cells or Expi293 cells), young hamster kidney cells (BHK), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), Buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), CHO cells, NSO cells, myeloma cell lines such as YO, NSO, P3X63, or Sp2 / 0, etc. Mammalian host cell lines suitable for antibody production are known in the art. In a preferred embodiment, the host cell is a CHO, such as an ExpiCHO cell, or a 293 cell, such as a HEK293 cell, a 293FT cell, or an Expi293 cell.

[0381] VI. Production and purification of the molecules of the present invention

[0382] In another aspect, the present invention provides a method for producing the antibody molecules of the present invention (e.g., multispecific antibodies such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments), the method comprising: culturing a nucleic acid molecule encoding the polypeptide chain or a host cell containing the polypeptide chain under conditions suitable for expressing a polypeptide chain of the antibody molecule; optionally further comprising assembling the polypeptide chain to produce the antibody molecule under conditions suitable for the polypeptide chain to assemble into the antibody molecule.

[0383] For recombinant production, polynucleotides encoding the polypeptide chain of the antibody molecule of the present invention can be inserted into one or more vectors for further cloning and / or expression in host cells. Expression vectors can be constructed using methods well known to those skilled in the art. Expression vectors include, but are not limited to, viruses, plasmids, granules, λ phages, or yeast artificial chromosomes (YACs). Once an expression vector containing one or more polynucleotides of the present invention has been prepared for expression, the expression vector can be transfected or introduced into suitable host cells. Various techniques can be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection, or other conventional techniques.

[0384] The antibody molecules prepared as described herein can be purified using known existing techniques such as high-performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, affinity chromatography (e.g., Protein A affinity chromatography), size exclusion chromatography, etc. The actual conditions used to purify a specific protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which are obvious to those skilled in the art.

[0385] The purity of the antibody molecules of the present invention can be determined by any of a variety of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, etc.

[0386] VI. Determination Method

[0387] The antibody molecules of the present invention (e.g., multispecific antibodies such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments) can be identified, screened, or characterized for their physical / chemical properties and / or biological activity using a variety of assays known in the art. Examples illustrate methods for determining the properties of the trispecific antibodies of the present invention, such as biolayer interferometry, physicochemical property analysis, binding or killing effects on BCMA, CD19, or CD3 positive cells, determination of T cell activation induced by BCMA or CD19 positive cells, and determination of their effect on the clearance of B cells and / or antibody-secreting cells (e.g., plasma cells) and inhibition of antibody production in a KLH mouse model.

[0388] VII. Immunoconjugates

[0389] In one aspect, the present invention provides an immunoconjugate comprising the antibody molecule of the present invention (e.g., a multispecific antibody such as a trispecific antibody, or an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD19 antibody or an antigen-binding fragment thereof) and one or more other active ingredients (e.g., active ingredients derived from a medicament or therapeutic agent for treating the disease of the present invention, such as small molecules that enhance the therapeutic effect of the molecule of the present invention).

[0390] In some implementations, the immune conjugate is an antibody-drug conjugate (ADC).

[0391] VIII. Pharmaceutical Compositions

[0392] This invention relates, in one aspect, to compositions (e.g., pharmaceutical compositions) comprising the molecules of this invention (e.g., multispecific antibodies such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments, or their immunoconjugates). In some embodiments, the pharmaceutical composition may be a formulation.

[0393] In one embodiment, the composition further comprises pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. In one embodiment, the composition, such as a pharmaceutical composition, comprises molecules of the present invention (e.g., multispecific antibodies such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments, or their immunoconjugates), and a combination of one or more other therapeutic agents.

[0394] The compositions of the present invention may also contain suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers.

[0395] As used herein, “pharmaceutical carrier” or “pharmaceutical excipient” encompasses any physiologically compatible solvent, dispersion medium, isotonic agent, or absorption delay agent.

[0396] For information on the use and applications of pharmaceutical excipients, see "Handbook of Pharmaceutical Excipients", 8th edition, R.C. Rowe, P.J. Seskey and S.C. Swen, Pharmaceutical Press, London, Chicago.

[0397] In some embodiments, the composition may be in the form of a formulation; for example, the compositions of the present invention may be formulations in a variety of forms. These forms include, for example, liquid, semi-solid, or solid dosage forms, such as liquid solutions (e.g., injectable and infusionable solutions), powders, suspensions, liposomes, or suppositories. Preferred forms depend on the intended administration method and therapeutic use.

[0398] A pharmaceutical product comprising the molecules described herein can be prepared, for example in the form of a lyophilized formulation or an aqueous solution, by mixing molecules of the present invention having the desired purity (e.g., multispecific antibodies such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments, or their immunoconjugates) with one or more optional pharmaceutical excipients.

[0399] IX. Drug Combinations

[0400] In one aspect, the present invention provides pharmaceutical combinations or pharmaceutical combination products comprising the molecules of the present invention (e.g., multispecific antibodies such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments, or their immunoconjugates), and one or more other therapeutic agents.

[0401] Another object of the present invention is to provide a complete pillbox containing the drug combination of the present invention, preferably said pillbox in the form of drug dosage units. This allows dosage units to be provided according to a dosing regimen or drug administration interval.

[0402] In one embodiment, the complete medicine box of the present invention comprises, within the same package:

[0403] - A first container containing a pharmaceutical composition comprising molecules of the present invention (e.g., multispecific antibodies of the present invention, such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments, or their immunoconjugates).

[0404] - A second container containing a pharmaceutical composition comprising other therapeutic agents.

[0405] In some embodiments, when the molecules of the present invention (e.g., the multispecific antibodies of the present invention, such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments, or their immunoconjugates) are used to treat tumors, the other therapeutic agents are, for example, various therapeutic agents used to treat tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0406] In some embodiments, when the molecules of the present invention (e.g., the multispecific antibodies of the present invention, such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments, or their immunoconjugates) are used to treat autoimmune diseases, the other therapeutic agents are, for example, therapeutic agents for treating autoimmune diseases, such as nonsteroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs), antirheumatic drugs, or immunomodulators such as immunosuppressants.

[0407] X. Methods and Applications

[0408] This invention provides, in one aspect, a method for preventing or treating a disease in a subject, comprising administering to the subject an effective amount of a molecule of the invention (e.g., a multispecific antibody of the invention, such as a trispecific antibody, or an anti-CD3 antibody or its antigen-binding fragment, or an anti-CD19 antibody or its antigen-binding fragment, or an immunoconjugate thereof), a pharmaceutical composition, a combination of drugs, or a kit. In some embodiments, the disease is selected from tumors such as cancer or autoimmune diseases.

[0409] In some embodiments, the present invention relates to molecules of the invention (e.g., multispecific antibodies of the invention, such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments, or their immunoconjugates) or pharmaceutical compositions or combinations or kits of the invention for use in therapies, such as for treating tumors such as cancer or autoimmune diseases.

[0410] In some embodiments, the present invention relates to methods of treating diseases, such as tumors such as cancer or autoimmune diseases, using molecules of the present invention (e.g., multispecific antibodies of the present invention such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments, or their immunoconjugates) or pharmaceutical compositions or combinations or kits of the present invention.

[0411] In some embodiments, the present invention relates to the use of molecules of the present invention (e.g., multispecific antibodies of the present invention, such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments, or their immunoconjugates) or pharmaceutical compositions or combinations or kits of the present invention for therapeutic purposes, such as for treating tumors such as cancer or autoimmune diseases.

[0412] In some embodiments, the present invention relates to the use of the molecules of the present invention (e.g., the multispecific antibodies of the present invention, such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments, or their immunoconjugates) or pharmaceutical compositions or pods of the present invention in the preparation of a medicine, such as for treating tumors such as cancer or autoimmune diseases.

[0413] In some embodiments, the tumor is a solid tumor, a hematologic malignancy, or a metastatic lesion. The cancer can be in its early, intermediate, or late stages, or it can be metastatic. In some embodiments, the tumor exhibits tumor immune evasion. In some embodiments, the tumor is a hematologic malignancy, such as leukemia like acute lymphoblastic leukemia or myeloma like multiple myeloma.

[0414] In some embodiments, the tumor is a BCMA-positive tumor or cancer. In some embodiments, the tumor is associated with aberrant expression or activity of BCMA. In some embodiments, the tumor is a CD19-positive tumor or cancer. In some embodiments, the tumor is associated with aberrant expression or activity of CD19. In some embodiments, the tumor is a BCMA and CD19-positive tumor or cancer. In some embodiments, the tumor is associated with aberrant expression or activity of both BCMA and CD19.

[0415] In some embodiments, a BCMA-positive tumor or cancer refers to a subject suffering from said tumor, such as cancer, having tumor cells that express BCMA. In some embodiments, the subject's tumor cells express BCMA, for example, low, moderate, or high levels of BCMA. In some embodiments, the subject has BCMA, for example, with increased BCMA nucleic acid or protein levels or activity compared to healthy subjects. In some embodiments, the subject's biological sample (e.g., tumor cells or tumor tissue or blood or blood cells) has BCMA, for example, with increased BCMA nucleic acid or protein levels or activity compared to a biological sample of a healthy subject (e.g., corresponding tissue or cells in a healthy subject, such as blood or blood cells), or compared to adjacent healthy tissue or cells of the subject. In some embodiments, a CD19-positive tumor or cancer refers to a subject suffering from said tumor, such as cancer, having tumor cells that express CD19. In some embodiments, the subject's tumor cells express CD19, for example, low, moderate, or high levels of CD19. In some embodiments, the subject has CD19, for example, with increased CD19 nucleic acid or protein levels or activity compared to healthy subjects. In some embodiments, the subject's biological sample (e.g., tumor cells or tumor tissue or blood or blood cells) contains CD19, for example, with increased CD19 nucleic acid or protein levels or activity compared to a biological sample of a healthy subject (e.g., corresponding tissue or cells in a healthy subject, such as blood or blood cells), or compared to adjacent healthy tissue or cells of the subject.

[0416] In some embodiments, a BCMA and CD19 positive tumor or cancer refers to tumor cells in a subject suffering from said tumor, such as cancer, that express both BCMA and CD19. In some embodiments, the subject's tumor cells express both BCMA and CD19, for example, at low, moderate, or high levels. In some embodiments, the subject possesses both BCMA and CD19, for example, with increased nucleic acid or protein levels or activity of both BCMA and CD19 compared to a healthy subject. In some embodiments, the subject's biological sample (e.g., tumor cells or tumor tissue or blood or blood cells) contains both BCMA and CD19, for example, with increased nucleic acid or protein levels or activity of both BCMA and CD19 compared to a biological sample from a healthy subject (e.g., corresponding tissue or cells in a healthy subject, such as blood or blood cells), or compared to adjacent healthy tissue or cells of the subject.

[0417] In some embodiments, the tumor is a myeloma such as multiple myeloma, for example, a BCMA-positive myeloma. In some embodiments, the tumor is, for example, leukemia such as acute lymphoblastic leukemia, for example, a CD19-positive leukemia such as acute lymphoblastic leukemia.

[0418] In some implementations, the tumor is a BCMA-positive and CD19-positive tumor, such as multiple myeloma or acute lymphoblastic leukemia.

[0419] In some embodiments, the molecules of the present invention (e.g., the multispecific antibodies of the present invention, such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments, or their immunoconjugates) are used to treat autoimmune diseases.

[0420] In some implementations, autoimmune diseases are B cell-mediated and / or antibody-secreting cell (e.g., plasma cells)-mediated and / or autoantibody-mediated autoimmune diseases.

[0421] In some embodiments, the autoimmune disease benefits from the elimination of B cells and / or antibody-secreting cells (e.g., plasma cells). In some embodiments, the autoimmune disease benefits from the inhibition of autoantibody production. In some embodiments, the autoimmune disease benefits from the elimination of B cells and antibody-secreting cells (e.g., plasma cells) and the inhibition of autoantibody production.

[0422] In some embodiments, the multispecific antibodies of the present invention, such as trispecific antibodies, treat autoimmune diseases by purging B cells and / or antibody-secreting cells (e.g., plasma cells) while inhibiting the production of autoantibodies.

[0423] In some implementations, the autoimmune disease is selected from systemic lupus erythematosus, lupus nephritis, or rheumatoid arthritis.

[0424] Depending on their therapeutic use, the molecules of the present invention (e.g., the multispecific antibodies of the present invention, such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments, or their immunoconjugates) or the pharmaceutical compositions of the present invention may also be administered in combination with one or more other therapies, such as modes of treatment and / or other therapeutic agents, for the methods and / or uses described herein, such as for the prevention and / or treatment of the relevant diseases or conditions mentioned herein.

[0425] In some embodiments, when the molecules of the present invention (e.g., the multispecific antibodies of the present invention, such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments, or their immunoconjugates) or the pharmaceutical compositions of the present invention are used to treat tumors, the other therapeutic agents are, for example, various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists); and / or the treatment may include surgery; radiotherapy, such as local irradiation or focused irradiation, etc.

[0426] In some embodiments, when the molecules of the present invention (e.g., the multispecific antibodies of the present invention, such as trispecific antibodies, or anti-CD3 antibodies or their antigen-binding fragments, or anti-CD19 antibodies or their antigen-binding fragments, or their immunoconjugates) or the pharmaceutical compositions of the present invention are used to treat autoimmune diseases, the other therapeutic agents are, for example, therapeutic agents for treating autoimmune diseases, such as nonsteroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs), antirheumatic drugs, or immunomodulators such as immunosuppressants.

[0427] XI. Specific Implementation Plan

[0428] 1. A trispecific antibody that specifically binds to CD3, BCMA, and CD19, comprising heavy chain 1, light chain 1, heavy chain 2, and light chain 2, or composed of heavy chain 1, light chain 1, heavy chain 2, and light chain 2, wherein heavy chain 1, light chain 1, heavy chain 2, and light chain 2 are shown below:

[0429] Heavy chain 1: From the N-terminus to the C-terminus, it contains or consists of the following: VH BCMA -First CH1-First Fc region, wherein the heavy chain constant region CH1 is connected at its C end to the N end of the first Fc region directly or via a connector (preferably directly);

[0430] Light chain 1: Composed of or consisting of the following from the N-terminus to the C-terminus: VL BCMA -First CL;

[0431] Heavy chain 2: From the N-terminus to the C-terminus, it contains or consists of the following: VH CD19 -Second CH1-scFv CD3 - Second Fc region, where the C-terminus of the second CH1 is connected to scFv CD3 The N-terminus is connected directly or via connector 1, and scFv CD3 The C end is directly or via connector 3 connected to the N end of the second Fc region;

[0432] Light chain 2: From the N-terminus to the C-terminus, it includes or consists of the following: VL CD19 -Second CL;

[0433] or

[0434] Heavy chain 1: From the N-terminus to the C-terminus, it contains or consists of the following: VH CD19 -First CH1-First Fc region, wherein the heavy chain constant region CH1 is connected at its C end to the N end of the first Fc region directly or via a connector (preferably directly);

[0435] Light chain 1: Composed of or consisting of the following from the N-terminus to the C-terminus: VL CD19 -First CL;

[0436] Heavy chain 2: From the N-terminus to the C-terminus, it contains or consists of the following: VH BCMA -Second CH1-scFv CD3 - Second Fc region, where the C-terminus of the second CH1 is connected to scFv CD3 The N-terminus is connected directly or via connector 1, scFv CD3 The C end is directly or via connector 3 connected to the N end of the second Fc region;

[0437] Light chain 2: From the N-terminus to the C-terminus, it includes or consists of the following: VL BCMA -Second CL;

[0438] in

[0439] "-" indicates a connector or direct connection;

[0440] VH BCMA This refers to the heavy chain variable region VH that specifically binds to the antigen-binding region of BCMA, and VL BCMA It refers to the light chain variable region (VL) of the antigen-binding region that specifically binds to BCMA;

[0441] VH CD19 This refers to the heavy chain variable region VH that specifically binds to the antigen-binding region of CD19, and VL CD19 It refers to the light chain variable region (VL) of the antigen-binding region that specifically binds to CD19;

[0442] The first CH1 and the second CH1 refer to the CH1 domain of the heavy chain constant region, respectively.

[0443] The first CL and the second CL refer to the constant regions of the light chain, respectively.

[0444] VH BCMA -CH1 and VL BCMA -CL constitutes the Fab region, which is the antigen-binding region that specifically binds to BCMA. BCMA ;

[0445] VH CD19 -CH1 and VL CD19-CL constitutes the Fab region, which serves as the antigen-binding region specifically binding to CD19. CD19 ;

[0446] scFv CD3 This refers to the scFv, which is the antigen-binding region that specifically binds to CD3, and consists of VH CD3 VL CD3 VH connected directly or via connector 2 CD3 and VL CD3 The polypeptide chain composition, preferably the scFv CD3 From the N-terminus to the C-terminus, it includes or consists of the following: VH CD3 -VL CD3 , of which VH CD3 C-terminus and VL CD3 The N-terminus is connected directly or via connector 2;

[0447] Optionally, the first CH1 and the second CH1 are the same, and / or the first CL and the second CL are the same, and / or the first Fc region and the second Fc region are different.

[0448] 2. The trispecific antibody of implementation plan 1, wherein...

[0449] The first CH1 is directly connected to the first Fc region;

[0450] Second CH1 and scFv CD3 The N-terminal is connected via connector 1;

[0451] scFv CD3 VH CD3 C-terminus and VL CD3 The N-terminus is connected via connector 2; and / or

[0452] scFv CD3 The C end is connected to the second Fc region via connector 3;

[0453] Optionally, connector 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:47 or 48; connector 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:47 or 49; and / or connector 3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:47 or 50.

[0454] 3. The trispecific antibody according to embodiment 1 or 2, wherein the first Fc region and the second Fc region are respectively Fc regions derived from human IgG1, IgG2, IgG3 or IgG4, optionally, the Fc region is the human IgG1 Fc region or the human IgG4 Fc region, for example, the Fc region

[0455] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:42; or

[0456] (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:42.

[0457] 4. The trispecific antibody according to any one of embodiments 1-3, wherein the first Fc region and the second Fc region are different, and based on Innobody technology, mutations are introduced into the first Fc region and the second Fc region to promote heterodimerization of the first Fc region and the second Fc region. Preferably, the CH3 of one Fc region contains S364R and D399K mutations, and the CH3 of the other Fc region contains Y349T, K370S and K409D mutations. Optionally, the first and / or second Fc regions contain mutations that reduce binding to the Fcγ receptor, such as L234A / L235A mutations.

[0458] Optional, of which

[0459] a) One Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:43, and the other Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:44;

[0460] b) One Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:43 and contains the mutants S364R and D399K, while the other Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:44 and contains the mutants Y349T, K370S, and K409D; or

[0461] c) One Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:43 and contains the mutations S364R and D399K and L234A / L235A mutations, while the other Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:44 and contains the mutations Y349T, K370S, and K409D and L234A / L235A mutations.

[0462] 5. The trispecific antibody according to any one of embodiments 1-4, wherein the first CH1 and the second CH1 are respectively CH1 derived from human IgG1, IgG2, IgG3 or IgG4, preferably the first CH1 and the second CH1 are CH1 of human IgG1 or CH1 of human IgG4, preferably, the CH1

[0463] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:40; or

[0464] (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:40.

[0465] 6. The trispecific antibody according to any one of embodiments 1-5, wherein the first CL and the second CL are light chain constant regions derived from the Kappa light chain constant region or the Lambda light chain constant region, preferably, the CL is the human Kappa light chain constant region or the human Lambda light chain constant region, preferably, the CL

[0466] (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:41; or

[0467] (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:41.

[0468] 7. The trispecific antibody described in any one of embodiments 1-6, wherein the VH BCMAIncludes complementary determinant regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and wherein the VL BCMA The device comprises complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:19; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:20; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:21; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:22; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:23; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:24.

[0469] 8. The trispecific antibody according to any one of embodiments 1-7, wherein the VH BCMA Containing the amino acid sequence shown in SEQ ID NO:17 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence, and said VL BCMA It contains the amino acid sequence shown in SEQ ID NO:18 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or is composed of said amino acid sequence.

[0470] 9. The trispecific antibody according to any one of implementation schemes 1-8, wherein

[0471] The VH BCMA Contains the amino acid sequence shown in SEQ ID NO:17, and the VL BCMA Contains the amino acid sequence shown in SEQ ID NO:18; or

[0472] The VH BCMA Composed of the amino acid sequence shown in SEQ ID NO:17, and the VL BCMA It consists of the amino acid sequence shown in SEQ ID NO:18.

[0473] 10. The trispecific antibody described in any one of embodiments 1-9, wherein the VH CD3 Includes complementary determinant regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and wherein the VLCD3 The device comprises complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:27; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:28; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:29; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:30; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:31; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:32.

[0474] 11. The trispecific antibody according to any one of embodiments 1-10, wherein the VH CD3 Containing or consisting of the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the VL CD3 It contains the amino acid sequence shown in SEQ ID NO:26 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or is composed of said amino acid sequence.

[0475] 12. The trispecific antibody according to any one of implementation schemes 1-11, wherein

[0476] The VH CD3 Contains the amino acid sequence shown in SEQ ID NO:25, and the VL CD3 Contains the amino acid sequence shown in SEQ ID NO:26; or

[0477] The VH CD3 Composed of the amino acid sequence shown in SEQ ID NO:25, and the VL CD3 It consists of the amino acid sequence shown in SEQ ID NO:26.

[0478] 13. The trispecific antibody described in any one of embodiments 1-12, wherein the VH CD19 The complementary determinant region (HCDR) containing the heavy chain variable region, HCDR1, HCDR2 and HCDR3, and the VL CD19The device comprises a complementarity-determining region (LCDR) of a light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:3; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:4; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:5; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:6 or 71; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:7; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:8.

[0479] 14. The trispecific antibody according to any one of embodiments 1-13, wherein the VH CD19 Containing the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence, and said VL CD19 It contains the amino acid sequence shown in SEQ ID NO:2 or 70, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

[0480] 15. The trispecific antibody according to any one of implementation schemes 1-14, wherein

[0481] The VH CD19 Contains the amino acid sequence shown in SEQ ID NO:1, and the VL CD19 Contains the amino acid sequence shown in SEQ ID NO:2 or 70; or

[0482] The VH CD19 Composed of the amino acid sequence shown in SEQ ID NO:1, and the VL CD19 It consists of the amino acid sequence shown in SEQ ID NO:2 or 70.

[0483] 16. The trispecific antibody described in any one of embodiments 1-12, wherein the VH CD19 It includes complementary determinant regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and the VL CD19The device comprises complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:11; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:12; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:13; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:14; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:15; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:16.

[0484] 17. The trispecific antibody according to any one of embodiments 1-12 and 16, wherein the VH CD19 Containing the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence, and said VL CD19 It contains the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or is composed of said amino acid sequence.

[0485] 18. The trispecific antibody according to any one of embodiments 1-12 and 16-17, wherein

[0486] The VH CD19 Contains the amino acid sequence shown in SEQ ID NO:9, and the VL CD19 Contains the amino acid sequence shown in SEQ ID NO:10; or

[0487] The VH CD19 Composed of the amino acid sequence shown in SEQ ID NO:9, and the VL CD19 It consists of the amino acid sequence shown in SEQ ID NO:10.

[0488] 19. The trispecific antibody according to any one of implementation schemes 1-18, wherein

[0489] Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:33, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:34, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:35, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:35. The amino acid sequence shown in NO:36 or 73, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or consisting of said amino acid sequence; or

[0490] Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:33, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:34, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:37, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:37. The amino acid sequence shown in NO:38, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or consisting of said amino acid sequence; or

[0491] Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:72, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:73, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:74, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:74. The amino acid sequence shown in NO:34, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence.

[0492] 20. The trispecific antibody according to any one of implementation schemes 1-19, wherein

[0493] Heavy chain 1 contains the amino acid sequence shown in SEQ ID NO:33; light chain 1 contains the amino acid sequence shown in SEQ ID NO:34; heavy chain 2 contains the amino acid sequence shown in SEQ ID NO:35; and light chain 2 contains the amino acid sequence shown in SEQ ID NO:36 or 73.

[0494] Heavy chain 1 consists of the amino acid sequence shown in SEQ ID NO:33; light chain 1 consists of the amino acid sequence shown in SEQ ID NO:34; heavy chain 2 consists of the amino acid sequence shown in SEQ ID NO:35; and light chain 2 consists of the amino acid sequence shown in SEQ ID NO:36 or 73;

[0495] Heavy chain 1 contains the amino acid sequence shown in SEQ ID NO:33; light chain 1 contains the amino acid sequence shown in SEQ ID NO:34; heavy chain 2 contains the amino acid sequence shown in SEQ ID NO:37; and light chain 2 contains the amino acid sequence shown in SEQ ID NO:38.

[0496] Heavy chain 1 consists of the amino acid sequence shown in SEQ ID NO:33; light chain 1 consists of the amino acid sequence shown in SEQ ID NO:34; heavy chain 2 consists of the amino acid sequence shown in SEQ ID NO:37; and light chain 2 consists of the amino acid sequence shown in SEQ ID NO:38.

[0497] Heavy chain 1 contains the amino acid sequence shown in SEQ ID NO:72; light chain 1 contains the amino acid sequence shown in SEQ ID NO:73; heavy chain 2 contains the amino acid sequence shown in SEQ ID NO:74; and light chain 2 contains the amino acid sequence shown in SEQ ID NO:34; or

[0498] Heavy chain 1 consists of the amino acid sequence shown in SEQ ID NO:72; light chain 1 consists of the amino acid sequence shown in SEQ ID NO:73; heavy chain 2 consists of the amino acid sequence shown in SEQ ID NO:74; and light chain 2 consists of the amino acid sequence shown in SEQ ID NO:34.

[0499] 21. Anti-CD3 antibody or its antigen-binding fragment, comprising VH and VL, wherein

[0500] The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:25, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:26.

[0501] 22. The anti-CD3 antibody or its antigen-binding fragment as described in embodiment 21 further comprises a heavy chain constant region and / or a light chain constant region, preferably,

[0502] The heavy chain constant region is a constant region of IgG1, IgG2, IgG3 or IgG4, such as the heavy chain constant region of human IgG1 or human IgG4; and / or the light chain constant region is a Kappa light chain constant region or a Lambda light chain constant region, such as the human Kappa light chain constant region or the human Lambd light chain constant region.

[0503] 23. An anti-CD19 antibody or its antigen-binding fragment, comprising three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:3; HCDR2 comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:4; HCDR3 comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:5; LCDR1 comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:6; LCDR2 comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:7; and LCDR3 comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:8.

[0504] 24. The antibody or antigen-binding fragment thereof described in embodiment 23, comprising VH and VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:1, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:2.

[0505] 25. The anti-CD19 antibody or its antigen-binding fragment as described in embodiment 23 or 24, further comprising a heavy chain constant region and / or a light chain constant region, preferably,

[0506] The heavy chain constant region is a constant region of IgG1, IgG2, IgG3 or IgG4, such as the heavy chain constant region of human IgG1 or human IgG4; and / or the light chain constant region is a Kappa light chain constant region or a Lambda light chain constant region, such as the human Kappa light chain constant region or the human Lambd light chain constant region.

[0507] 26. A nucleic acid molecule comprising, or consisting of, a nucleic acid sequence encoding a trispecific antibody as described in any one of embodiments 1-20, an anti-CD3 antibody as described in embodiments 21 or 22, or an antigen-binding fragment thereof, or an anti-CD19 antibody as described in any one of embodiments 23-25, or an antigen-binding fragment thereof.

[0508] 27. An expression vector comprising the nucleic acid molecule described in embodiment 26, preferably, the expression vector being pCDNA, such as pCDNA3.4.

[0509] 28. A host cell comprising the nucleic acid molecule of embodiment 26 or the expression vector of embodiment 27, preferably, the host cell being prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as ExpiCHO cells.

[0510] 29. A method for preparing a trispecific antibody according to any one of embodiments 1-20, or an anti-CD3 antibody or antigen-binding fragment thereof according to embodiments 21 or 22, or an anti-CD19 antibody or antigen-binding fragment thereof according to any one of embodiments 23-25, wherein the method comprises culturing a host cell containing a nucleic acid molecule according to embodiment 26 or an expression vector according to embodiment 27 under conditions suitable for chain expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

[0511] 30. An immunoconjugate comprising the trispecific antibody of any one of embodiments 1-20, or the anti-CD3 antibody of embodiment 21 or 22, or the antigen-binding fragment thereof, or the anti-CD19 antibody of any one of embodiments 23-25, or the antigen-binding fragment thereof.

[0512] 31. A pharmaceutical composition comprising the trispecific antibody of any one of embodiments 1-20, or the anti-CD3 antibody or its antigen-binding fragment as described in embodiments 21 or 22, or the anti-CD19 antibody or its antigen-binding fragment as described in any one of embodiments 23-25, or the immunoconjugate as described in embodiment 30, and optionally pharmaceutical excipients.

[0513] 32. A drug combination comprising the trispecific antibody of any one of embodiments 1-20, or the anti-CD3 antibody or its antigen-binding fragment as described in embodiments 21 or 22, or the anti-CD19 antibody or its antigen-binding fragment as described in any one of embodiments 23-25, and one or more other therapeutic agents.

[0514] Preferably, the other therapeutic agents are various therapeutic agents used to treat tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists); or

[0515] The other therapeutic agents are those used to treat autoimmune diseases, such as nonsteroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs), antirheumatic drugs, or immunomodulators such as immunosuppressants.

[0516] 33. A method for preventing or treating tumors or autoimmune diseases in a subject, comprising administering to the subject an effective amount of the trispecific antibody of any one of embodiments 1-20, or the anti-CD3 antibody or its antigen-binding fragment of embodiments 21 or 22, or the anti-CD19 antibody or its antigen-binding fragment of any one of embodiments 23-25, or the immune conjugate of embodiment 30, or the pharmaceutical composition of embodiment 31, or the pharmaceutical combination of embodiment 32.

[0517] 34. Use of the trispecific antibody of any one of embodiments 1-20, or the anti-CD3 antibody or its antigen-binding fragment of embodiments 21 or 22, or the anti-CD19 antibody or its antigen-binding fragment of any one of embodiments 23-25, or the immune conjugate of embodiment 30, or the pharmaceutical composition of embodiment 31, or the pharmaceutical combination of embodiment 32 in the preparation of a medicament for the prevention or treatment of tumors or autoimmune diseases.

[0518] 35. The trispecific antibody of any one of embodiments 1-20, or the anti-CD3 antibody or its antigen-binding fragment of embodiments 21 or 22, or the anti-CD19 antibody or its antigen-binding fragment of any one of embodiments 23-25, or the immunoconjugate of embodiment 30, or the pharmaceutical composition of embodiment 31, or the pharmaceutical composition of embodiment 32, used for therapy.

[0519] 36. The trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immune conjugate, pharmaceutical composition or combination of drugs described in Implementation Scheme 35, used for the prevention or treatment of tumors or autoimmune diseases.

[0520] 37. The method described in embodiment 33, the use described in embodiment 34, or the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immune conjugate, pharmaceutical composition or combination of drugs described in embodiment 36, wherein the tumor is a solid tumor or a hematologic malignancy.

[0521] 38. The method described in embodiment 33 or 37, the use described in embodiment 34 or 37, or the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immune conjugate, pharmaceutical composition or combination of drugs described in embodiment 36 or 37, wherein the tumor is a BCMA-positive and / or CD19-positive tumor or cancer.

[0522] 39. The method of any one of embodiments 33 and 37-38, the use of any one of embodiments 34 and 37-38, or the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immunoconjugate, pharmaceutical composition or combination of drugs of any one of embodiments 36 and 37-38, wherein the tumor is a hematologic malignancy, such as leukemia such as acute lymphoblastic leukemia or myeloma such as multiple myeloma.

[0523] 40. The method of any one of embodiments 33, 37-39, the use of any one of embodiments 34, 37-39, or the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immunoconjugate, pharmaceutical composition, or combination of drugs of any one of embodiments 36, 37-39, wherein the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immunoconjugate, pharmaceutical composition, or combination of drugs, or the drugs are also administered in combination with other therapies such as treatment modalities (e.g., surgery or radiotherapy) and / or other therapeutic agents, preferably, the therapeutic agents are various therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

[0524] 41. The method described in embodiment 33, the use described in embodiment 34, or the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immune conjugate, pharmaceutical composition, or pharmaceutical combination described in embodiment 36, wherein the autoimmune disease is an autoimmune disease mediated by B cells and / or antibody-secreting cells (e.g., plasma cells) and / or autoantibodies.

[0525] 42. The method described in embodiment 33 or 41, the use described in embodiment 34 or 41, or the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immune conjugate, pharmaceutical composition or combination of drugs described in embodiment 36 or 41, wherein the autoimmune disease is systemic lupus erythematosus, lupus nephritis or rheumatoid arthritis.

[0526] 43. The method of any one of embodiments 33, 41, and 42; the use of any one of embodiments 34, 41, and 42; or the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immunoconjugate, pharmaceutical composition, or pharmaceutical combination of any one of embodiments 36, 41, and 42, wherein the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immunoconjugate, pharmaceutical composition, or pharmaceutical combination, or the pharmaceutical combination, is further administered in combination with other therapeutic agents, preferably, the other therapeutic agents being therapeutic agents for treating autoimmune diseases, such as nonsteroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs), antirheumatic drugs, or immunomodulators such as immunosuppressants. Example

[0527] Example 1. Structural design of a trispecific antibody molecule targeting CD19, BCMA, and CD3

[0528] 1.1 Acquisition and Optimization of Anti-CD19 Sequences

[0529] The sequences of the antigen-binding regions of the three specific antibodies against CD19 are derived from the following parental CD19 antibodies:

[0530] Tafa(NA): The sequence is derived from patent US20120082664, specifically 4G7 H1.52 Hybrid S239D / I332E (SEQ ID NO:87) and 4G7L1.155 (SEQ ID NO:106). Analysis of patent US20120082664 SEQ ID NO:106 revealed an NG deamidation site in the light chain CDR1. During sequence optimization, glycine (G) at position 34 of the light chain was selectively mutated to alanine (A), and the light chain sequence was labeled 4G7L1.155(NA). The resulting antibody is also represented as Tafa(NA) (and in bispecific antibodies, it is also represented as Tafa(NA)).

[0531] MDX: The sequence is derived from WO2007002223A2, specifically 21D4-VL (SEQ ID NO:8) and 21D4-VH (VH:SEQ ID NO:1), and is also referred to as MDX in this paper (and in bispecific antibodies). The specific sequences and numbers of Tafa(NA) and MDX are shown in Sequence Information Table A.

[0532] 1.2 Obtaining Anti-BCMA Sequences

[0533] The sequence of the antigen-binding region of the trispecific antibody against BCMA is derived from the fully human BCMA antibody ADI38497, whose sequence is derived from patent WO2023104100A1. The specific sequence and serial number of ADI38497 are shown in Sequence Information Table A in this paper.

[0534] 1.3 Obtaining anti-CD3 sequences

[0535] The sequence of the antigen-binding region of the anti-CD3 antibody in the trispecific antibody is derived from the fully human CD3 antibody sp34.24, whose sequence is derived from WO2022068809A1, and the framework region has been optimized. The specific sequence and number of sp34.24 are shown in Sequence Information Table A in this paper.

[0536] 1.4. Structural design and screening of trispecific antibody molecules targeting CD19, BCMA, and CD3

[0537] Based on humanized antibodies targeting CD19, BCMA, and CD3, a trispecific antibody molecule (CD19×BCMA TCE) was designed. The antibody structure is shown in Figure 1.

[0538] The antibody has the following composition:

[0539] Heavy chain 1: Contains the heavy chain variable region VH, the heavy chain constant region CH1, and the first Fc region.

[0540] Light chain 1: Contains the variable region VL and the constant region CL of the light chain.

[0541] Heavy chain 2: includes the heavy chain variable region VH, the heavy chain constant region CH1, linker 1, the single-chain antibody scFv formed by linker 2, linker 3, and the second Fc region, as well as

[0542] Light chain 2: includes the light chain variable region VL and the light chain constant region CL;

[0543] The antibodies targeting CD19 and BCMA are in Fab form, the antibody targeting CD3 is in scFv form, and Fc is the heavy chain constant region of IgG1.

[0544] The VH and CH1 regions of heavy chain 1 and the VL and CL regions of light chain 1 constitute the Fab targeting BCMA. The VH and CH1 regions of heavy chain 2 and the VL and CL regions of light chain 2 constitute the Fab targeting CD19. The scFv in heavy chain 2 is the scFv targeting CD3. (Structure 1: Antibodies targeting CD19 and BCMA are in Fab form, antibodies targeting CD3 are in scFv form. The Fab targeting BCMA is directly linked to the Fc region, while the Fab targeting CD19 is linked to the scFv targeting CD3 via a linker. The antibody is named ADI38497 x Tafa-sp34.24;) or

[0545] The VH and CH1 regions of heavy chain 1 and the VL and CL regions of light chain 1 constitute the Fab targeting CD19. The VH and CH1 regions of heavy chain 2 and the VL and CL regions of light chain 2 constitute the Fab targeting BCMA. The scFv in heavy chain 2 is the scFv targeting CD3 (Structure 2: Antibodies targeting CD19 and BCMA are in Fab form, and antibodies targeting CD3 are in scFv form. The Fab targeting CD19 is directly linked to the Fc region, while the Fab targeting BCMA is linked to the scFv targeting CD3 through a linker. The antibody is named Tafa x ADI38497-sp34.24).

[0546] Specifically, S364R and D399K mutations (SEQ ID NO:45, Innobody A Fc) were introduced in the first Fc region, and Y349T, K370S, and K409D mutations (SEQ ID NO:46, Innobody B Fc) were introduced in the second Fc region (Innobody platform, WO2022143912A1, EU number). Through charge complementarity and spatial complementarity, the pairing of heterologous heavy chains was increased. At the same time, amino acid mutations L234A and L235A (EU number) that weaken the effector function were introduced in the Fc region.

[0547] The full-length amino acid sequences of ADI38497 x Tafa-sp34.24 and Tafa x ADI38497-sp34.24 constructed in this invention are shown in Sequence Information Table B.

[0548] Detection of the in vitro killing effect of T cells on CD19-positive cells mediated by different molecular structures of trispecific antibodies:

[0549] Cell resuscitation: 1) Remove peripheral blood mononuclear cells (PBMCs) (Shanghai Miaoshun TPCS) from the liquid nitrogen tank and gently shake them in a 37℃ water bath until thawed. Keep the sealing ring and cap above the water surface to prevent contamination; 2) After thawing, immediately disinfect with 75% alcohol and transfer to a clean bench. Strictly follow aseptic techniques thereafter; 3) Prepare 1640 complete culture medium in advance: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010), 1% Sodium 3) Pyruvate (Gibco, 1136-070) and 0.1% β-ME (Gibco, 21985-023); 4) Transfer the cell suspension from the cryopreservation tube to preheated 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to precipitate the cells, and discard the supernatant; 5) Resuspend the cell pellet in 15mL of complete medium, take a portion and count the viable cells using trypan blue (Gibco, 15250-061).

[0550] Killing assay: 1) Tumor cell labeling: Collect NALM-6 (human acute lymphoblastic leukemia cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.) in logarithmic growth phase, centrifuge at 300g for 5 minutes, discard the supernatant, wash once with 5mL PBS, centrifuge at 300g for 5 minutes, discard the supernatant, resuspend the tumor cells in 1mL CFSE working solution (Invitrogen, C34554), incubate at room temperature in the dark for 15 minutes, stop staining with 1mL complete culture medium, centrifuge at 300g for 5 minutes, discard the supernatant, wash once with PBS. 2) Resuspend the tumor cells in RPMI 1640 complete culture medium containing 10% serum, and adjust the cell concentration to 4×10⁶ cells / year. 5 3) Add PBMC cells at a rate of 2 x 10⁶ cells / mL to each well of a 96-well round-bottom plate. 6 3) Antibody preparation: Prepare antibodies using complete culture medium. Dilute the antibodies in a sterile 96-well V-plate (Beyotime, FPT019) at 400 nM, 10-fold dilutions, for a total of 11 gradients. Add 50 μL of each antibody to the cell culture plate and incubate at 37°C with 5% CO2 for 24 hours. 4) Flow cytometry staining: Prepare cell viability and inactivation dyes in advance. After 24 hours of culture, centrifuge at 300g for 5 minutes, transfer cells to a sterile 96-well V-plate (Beyotime, FPT019), stain with viability and inactivation dyes for 15 minutes, wash away unbound and non-specifically bound dyes, and detect by flow cytometry.

[0551] The experimental results are shown in Figure 2. ADI38497 x Tafa-sp34.24 exhibits a stronger killing effect on CD19-positive tumor cells. In contrast, the Tafa x ADI38497-sp34.24 trispecific antibody has a weaker killing effect on CD19-positive tumor cells.

[0552] Detection of the in vitro killing effect of T cells on BCMA-positive cells mediated by different trispecific antibody molecular structures:

[0553] Cell resuscitation: 1) Remove peripheral blood mononuclear cells (PBMCs) (Shanghai Miaoshun TPCS) from the liquid nitrogen tank and gently shake them in a 37℃ water bath until thawed. Keep the sealing ring and cap above the water surface to prevent contamination; 2) After thawing, immediately disinfect with 75% alcohol and transfer to a clean bench. Strictly follow aseptic techniques thereafter; 3) Prepare 1640 complete culture medium in advance: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010), 1% Sodium 3) Pyruvate (Gibco, 1136-070) and 0.1% β-ME (Gibco, 21985-023); 4) Transfer the cell suspension from the cryopreservation tube to preheated 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to precipitate the cells, and discard the supernatant; 5) Resuspend the cell pellet in 15mL of complete medium, take a portion and count the viable cells using trypan blue (Gibco, 15250-061).

[0554] Killing assay: 1) Tumor cell labeling: Collect L-363 (human myeloma cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.) tumor cells expressing BCMA in logarithmic growth phase, centrifuge at 300g for 5 minutes, discard the supernatant, wash once with PBS, centrifuge at 300g for 5 minutes, discard the supernatant, resuspend the tumor cells in 1mL CFSE working solution (Invitrogen, C34554), incubate at room temperature in the dark for 15 minutes, stop staining with 1mL complete culture medium, centrifuge at 300g for 5 minutes, discard the supernatant, wash once with PBS. 2) Resuspend the tumor cells in RPMI 1640 complete culture medium containing 10% serum, and adjust the cell concentration to 4x10⁻⁶ cells / mL. 5 3) Add PBMC cells at a rate of 2 x 10⁶ cells / mL to each well of a 96-well round-bottom plate. 6 3) Antibody preparation: Prepare antibodies using complete culture medium. Dilute the antibodies in a sterile 96-well V plate (Beyotime, FPT019) at 400 nM, 10-fold dilutions, for a total of 8 gradients. Add 50 μL of each antibody to the cell culture plate and incubate at 37°C with 5% CO2 for 24 hours. 4) Flow cytometry staining: Prepare cell viability and inactivation dyes in advance. After 24 hours of culture, centrifuge at 300g for 5 minutes, transfer cells to a sterile 96-well V plate (Beyotime, FPT019), stain with viability and inactivation dyes for 15 minutes, wash away unbound and non-specifically bound dyes, and detect by flow cytometry.

[0555] The experimental results are shown in Figure 3. Different structural trispecific antibodies have similar killing effects on BCMA-positive tumor cells.

[0556] 1.5. Molecular structure of a trispecific antibody targeting CD19, BCMA, and CD3

[0557] Based on humanized antibodies targeting CD19, BCMA, and CD3, a trispecific antibody molecule (CD19×BCMA TCE) was designed. The antibody structure is shown in Figure 4.

[0558] The antibody has the following composition:

[0559] Heavy chain 1: Contains the heavy chain variable region VH, the heavy chain constant region CH1, and the first Fc region.

[0560] Light chain 1: Contains the variable region VL and the constant region CL of the light chain.

[0561] Heavy chain 2: includes the heavy chain variable region VH, the heavy chain constant region CH1, linker 1, the single-chain antibody scFv formed by linker 2, linker 3, and the second Fc region, as well as

[0562] Light chain 2: includes the light chain variable region VL and the light chain constant region CL;

[0563] The antibodies targeting CD19 and BCMA are in Fab form, the antibody targeting CD3 is in scFv form, and Fc is the heavy chain constant region of IgG1.

[0564] The VH and CH1 of heavy chain 1 and the VL and CL of light chain 1 constitute a Fab targeting BCMA, while the VH and CH1 of heavy chain 2 and the VL and CL of light chain 2 constitute a Fab targeting CD19, and the scFv in heavy chain 2 is the scFv targeting CD3.

[0565] Specifically, S364R and D399K mutations (SEQ ID NO:45, Innobody A Fc) were introduced in the first Fc region, and Y349T, K370S, and K409D mutations (SEQ ID NO:46, Innobody B Fc) were introduced in the second Fc region (Innobody platform, WO2022143912A1, EU number). Through charge complementarity and spatial complementarity, the pairing of heterologous heavy chains was increased. At the same time, amino acid mutations L234A and L235A (EU number) that weaken the effector function were introduced in the Fc region.

[0566] The tandem arrangement of Fab and scFv effectively prevents light and heavy chain mismatches. Combined with the Innobody platform, which prevents heavy chain mismatches, bispecific antibodies can be correctly paired at a high rate when expressed in the same cell. Furthermore, combining Innobody in vitro recombinant technology can achieve a high rate of correct pairing of trispecific antibodies. Furthermore, the BCMA-targeting Fab of the trispecific antibody constructed in this invention is located on the first heavy chain and directly linked to the Fc region, while the CD19-targeting Fab is located on the second heavy chain and linked to the CD3-targeting scFv. This Fab placement maximizes the killing effect of BCMA and CD19-positive cells. In addition, due to the CD19 Fab blocking effect, the CD3-binding scFv does not have the ability to bind to T cells when the antibody does not bind to BCAM or CD19. Only when the antibody binds to BCMA or CD19 can the antibody bind and activate T cells. Thus, the obtained trispecific antibody maintains high binding capacity to CD19 and BCMA, reduces antibody waste caused by ineffective binding on T cells when the antibody does not bind to BCMA or CD19, and avoids the release of inflammatory factors caused by non-target-dependent T cell activation.

[0567] The full-length amino acid sequences of CD19×BCMA TCE:ADI38497×Tafa(NA)-sp34.24 and ADI38497×MDX-sp34.24 constructed in this invention are shown in Sequence Information Table B.

[0568] Example 2. Preparation of TCE antibody protein

[0569] 1): Plasmid construction: Plasmids were synthesized by Baiying, and the DNA encoding each strand was constructed into pcDNA3.4 respectively.

[0570] 2) Protein expression: Protein was produced using the ExpiCHO™ Expression system (Gibco, A29133). Specifically, ExpiCHO-STM cells (Gibco) were passaged according to the required transfection volume, and the cell density was adjusted to 3.5 × 10⁶ cells / day before transfection. 6 Cells / mL. On the day of transfection, the cell density was adjusted to 6 × 10⁶ cells / mL. 6Cells / mL. Take a 50mL centrifuge tube and add OptiPRO™ SFM (Gibco, 12309019) transfection buffer at 8% of the cell volume. Calculate the total required plasmid amount based on 0.8μg / mL of transfection cells. Parental antibodies are premixed at a light chain:heavy chain plasmid mass ratio of 1:1. Filter the transfection buffer containing DNA plasmids through a 0.22μm filter membrane into another new 50mL centrifuge tube. Add DNA:Reagent to the filtered mixture at a ratio of 1:4. (Polyplus, 101000019) Mix thoroughly and incubate at room temperature for 10 minutes. Then, immediately and slowly add the transfection reagent and plasmid DNA mixture to the cells while gently shaking the flask. Incubate the transfected cells at 37°C in a shaker with 8% CO2. After 18-22 hours, add 6 μL / mL Enhancer (Gibco, 100033019) and 300 μL / mL Feed (Gibco, A29101-01) to the cells. Continue culturing at 37°C, 120 rpm, and 8% CO2. Collect the cell culture medium on day 7 or when cell viability is ≤70%. Mix the cell culture medium with diatomaceous earth (Sartorius, Cat 1000037025) (40 g diatomaceous earth per L of cell culture medium) and filter using a 0.22 μm disposable vacuum filter. Use the supernatant for subsequent affinity purification.

[0571] 3) Affinity chromatography purification of the target protein: A HiTrap MabSelect PrismA (GE Healthcare, Cat#17549853) affinity chromatography column was used for affinity capture. Before purification, 10-20 column volumes of 0.1M NaOH were passed through the tubing and the affinity chromatography column, followed by 10-20 column volumes of distilled water to wash the tubing and the column. The packing material was equilibrated with 5 column volumes of 1×PBS (Gibco). The filtered cell material was passed through the column, and the packing material was washed with 10 column volumes of 1×PBS to remove non-specific binding proteins. The packing material was washed with 5 column volumes of elution buffer (100mM sodium citrate, pH 3.5), and the eluent was collected. The pH was adjusted to 6.0 with 2M Tris, and the solution was filtered for sterilization. The two parental antibodies obtained were named Innobody A antibody (Innobody A in the Fc region) and Innobody B antibody (Innobody B in the Fc region).

[0572] 4) In vitro reduction and oxidation: The purified innobody A and innobody B were mixed in a 1:1 molar ratio, and an appropriate amount of GSH was added. The pH of the reaction was adjusted to 8.0 with 1M arginine. The mixture was incubated overnight at room temperature. The reaction mixture was then transferred to PBS and stored at 4°C for later use.

[0573] 5) Ion exchange chromatography purification of bispecific antibodies: A Mono S 5 / 50GL ion exchange chromatography column (from GE Healthcare) was used and placed in an AKTApure system (from GE Healthcare). Endotoxins were removed from the AKTApure system equipped with the Mono S 5 / 50GL ion exchange chromatography column using 0.5M NaOH for 2 hours. The system and column were then washed with distilled water. The column was equilibrated with 5-10 column volumes of loading buffer (20mM NaPO4, pH 6.6) until conductivity and pH stabilized. The protein obtained from affinity chromatography was diluted 10-fold with loading buffer and then loaded. The column was reequilibrated with 5 column volumes of loading buffer. Linear elution was performed using a gradient of 0-40% elution buffer (20mM NaPO4, 1M NaCl, pH 6.6) for a total of 30 column volumes. Samples were collected based on UV absorbance.

[0574] The purity of samples collected from each fraction was determined using size exclusion chromatography (SEC). Samples from fractions with a purity greater than 95% were pooled based on the SEC results. Antibody concentration was then determined. Further qualitative and quantitative analysis of antibody purity and impurities was performed using a combination of capillary electrophoresis (CE-SDS) and liquid chromatography-mass spectrometry (LC-MS).

[0575] Similarly, control antibodies gp120×gp120-sp34.24, gp120-hIgG1-LALA (also referred to as Isotype in this article), Teclistamab, CD19(2B11)CD3opt (US20240132590A1), and W3438-T3U4.E17-1.uIgG4.SP (CN109535257B) were prepared. The sequences of the control antibodies are shown in Table D.

[0576] Example 3. ForteBio determined the binding kinetics of CD19×BCMA TCE antibody and control antibody to the antigen.

[0577] The equilibrium dissociation constant (KD) of the antibodies of this invention binding to human CD19 and human, cynomolgus monkey and mouse BCMA was determined using the ForteBio assay. ForteBio affinity assays were performed according to existing methods (Estep, P et al., High throughput solution Based measurement of antibody antigen affinity and epitope binning. MAbs, 2013.5(2)).

[0578] Half an hour before the experiment, according to the number of samples, take an appropriate number of AHC (18-5060, Sartorius) sensors and immerse them in SD buffer (PBS 1×, BSA 0.1%, Tween 20 0.05%).

[0579] 100 μL of SD buffer, antibody, and antigen [including human CD19 (CD9-H52H2, Acro biosystems), human BCMA (10620-H41H-B, Sino Biological), cynomolgus monkey BCMA (BCA-C5253, Acro biosystems), and mouse BCMA (BCA-M52H3, Acro biosystems)] were added to 96-well black polystyrene semi-mass microplates (Greiner, 675076). The plate was arranged according to the sample location, and the sensor position was selected. The instrument settings were as follows: Run steps: Baseline, Loading ~1 nm, Baseline, Association, and Dissociation; the run time for each step depended on the sample binding and dissociation rates, the rotation speed was 1000 rpm, and the temperature was 30℃. KD values ​​were analyzed using ForteBio analysis software. The results are shown in Table 1.

[0580] Based on the results in Table 1, the CD19×BCMA TCE of this invention and the control antibody CD19(2B11)CD3opt (US20240132590A1) exhibit comparable affinity for human CD19. Based on the results in Tables 2, 3, and 4, the CD19×BCMA TCE of this invention and the control antibody Teclistamab exhibit comparable affinity for human BCMA, and the CD19×BCMA TCE of this invention exhibits higher affinity for BCMA in cynomolgus monkeys and mice than Teclistamab.

[0581] Table 1. Affinity constants (M) for antigen-antibody binding detection by ForteBio

[0582] Table 2. Affinity constants (M) for antigen-antibody binding detection by ForteBio

[0583] Table 3. Affinity constants (M) for antigen-antibody binding detection by ForteBio

[0584] Table 4. Affinity constants (M) for antigen-antibody binding detection by ForteBio

[0585] Example 4. Detection of antibody binding to CD19 and BCMA-positive tumor cells

[0586] Cell resuscitation: 1) Remove tumor cells NALM-6 (human acute lymphoblastic leukemia cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.), NCI-H929 (human myeloma cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.), MM.1S (human myeloma cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.), and L-363 (human multiple myeloma cells, purchased from Nanjing Kebai Biotechnology Co., Ltd.) from the liquid nitrogen tank and place them in a 37℃ water bath. Gently shake until thawed, keeping the sealing ring and cap above the water surface to prevent contamination; 2) After thawing, immediately disinfect with 75% alcohol and transfer to a clean bench. Strictly follow aseptic techniques thereafter; 3) Prepare complete culture medium in advance: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010), 1% Sodium Pyruvate (Gibco, 1136-070); 3) Transfer the cell suspension from the cryopreservation tube to preheated 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to precipitate the cells, and discard the supernatant; 4) Resuspend the cell pellet in 15mL of complete medium, take out a portion and count the viable cells using trypan blue (Gibco, 15250-061), and culture the remaining cells.

[0587] Activity assay: 1) Once cell growth is stable, centrifuge an appropriate amount of cells at 300g for 5 minutes, resuspend in FACS buffer (PBS + 5% FBS + 2nM EDTA), adjust the cell concentration to 20k-40k cells / well, mix well, and seed the cells into a 96-well plate. 2) Centrifuge at 300g for 5 minutes, resuspend in FACS buffer containing Fc blocker (1:200), and incubate at 4°C in the dark for 10 minutes. 3) Centrifuge at 300g for 5 minutes, resuspend in 200μL FACS buffer, and wash twice. 4) Prepare the triple antibody and control antibody of this invention (the full-length sequence of the control antibody is shown in Sequence Information Table D, and the preparation method is described in Example 2) (300nM, 5-fold dilution). After incubation, centrifuge at 300g for 5 minutes, discard the unbound antibody in the plate, add 200μL of FACS washing buffer to each well, and wash twice. 5) Add 50 μL of anti-human Fc flow cytometry antibody (1:5000) to each well and incubate at 4°C for 30 minutes. 6) After incubation, centrifuge at 300g for 5 minutes, discard the supernatant, and add 200 μL of FACS washing buffer to each well, washing three times. 7) After washing, resuspend the cells in 100 μL of FACS buffer to each well and analyze using flow cytometry.

[0588] The experimental results are shown in Figure 5. CD19×BCMA TCE can bind to the CD19 antigen on the surface of NALM-6 tumor cells, and the binding ability is comparable to that of the control antibody CD19(2B11)CD3opt (US20240132590A1).

[0589] The experimental results are shown in Figures 6, 7, and 8. CD19×BCMA TCE can bind to BCMA antigens on the surface of NCI-H929, MM.1S, and L363 tumor cells, and its binding ability is superior to that of Teclistamab.

[0590] Example 5. Detection of antibody binding to CD3-positive cells (Jurkat)

[0591] Cell resuscitation: 1) Remove Jurkat tumor cells (Nanjing Kebai, catalog number CBP60942) from the liquid nitrogen tank and gently agitate them in a 37℃ water bath until thawed, keeping the sealing ring and cap above the water surface to prevent contamination; 2) After thawing, immediately disinfect with 75% alcohol and transfer to a clean bench, then strictly follow aseptic techniques thereafter; 3) Prepare complete culture medium in advance: 1640 (Gibco, 22400-071), 10%... FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010); 3) Transfer the cell suspension from the cryopreservation tube to preheated 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to precipitate the cells, and discard the supernatant; 4) Resuspend the cell pellet in 15mL of complete medium, take a portion and count the viable cells using trypan blue (Gibco, 15250-061).

[0592] Activity assay: 1) Digest cultured Jurkat cells, centrifuge at 1000 rpm for 5 minutes, resuspend in FACS buffer (PBS + 5% FBS + 2 nM EDTA), adjust cell concentration to (50k-100k cells / well), mix well, and seed cells into 96-well plates. 2) Centrifuge at 300g for 5 minutes, resuspend in FACS buffer containing Fc blocker (1:200), and incubate at 4°C in the dark for 10 minutes. 3) Centrifuge at 300g for 5 minutes, resuspend in 200 μL FACS buffer, and wash twice. 4) Prepare the antibody and control antibody of this invention (the full-length sequence of the control antibody is shown in Sequence Information Table D, and the preparation method is described in Example 2) (300 nM, 5-fold dilution). After incubation, centrifuge at 300g for 5 minutes, discard unbound antibody in the plate, add 200 μL of FACS washing buffer to each well, and wash twice. 5) Add secondary antibody (1:5000), 50 μL per well, and incubate at 4°C for 30 minutes. 6) After incubation, centrifuge at 300g for 5 minutes, discard the supernatant, and add 200 μL of FACS washing buffer to each well, washing three times. 7) After washing, resuspend the cells in 100 μL of FACS buffer to each well and analyze using flow cytometry.

[0593] The experimental results are shown in Figure 9. To reduce cytokine release syndrome (CRS) caused by T cell overactivation and decrease the binding of TCE to CD3 in the absence of target binding, thereby prolonging the half-life of TCE molecules in animals, the CD19×BCMA TCE was formulated in a 2+1 form. This antibody form effectively blocks the CD3 binding domain and the CD3 binding epitope in the absence of CD19 or BCMA antigen binding, thus preventing the antibody from being ineffectively distributed on T cells. In the presence of CD19 or BCMA antigen binding, the antibody can effectively bind to CD3 on T cells and activate T cells, mediating T cell killing. In contrast, the control antibodies CD19(2B11)CD3opt (US20240132590A1) and Teclistamab do not possess this characteristic; the CD3 antibody is fully exposed, and even in the absence of CD19 or BCMA antigen binding, the antibody still binds to the CD3 antigen.

[0594] Example 6. Detection of the in vitro killing effect of antibody-mediated T cells on CD19-positive cells.

[0595] Cell resuscitation: 1) Remove peripheral blood mononuclear cells (PBMCs) (Shanghai Miaoshun TPCS) from the liquid nitrogen tank and gently shake them in a 37℃ water bath until thawed. Keep the sealing ring and cap above the water surface to prevent contamination; 2) After thawing, immediately disinfect with 75% alcohol and transfer to a clean bench. Strictly follow aseptic techniques thereafter; 3) Prepare 1640 complete culture medium in advance: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010), 1% Sodium 3) Pyruvate (Gibco, 1136-070) and 0.1% β-ME (Gibco, 21985-023); 4) Transfer the cell suspension from the cryopreservation tube to preheated 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to precipitate the cells, and discard the supernatant; 5) Resuspend the cell pellet in 15mL of complete medium, take a portion and count the viable cells using trypan blue (Gibco, 15250-061).

[0596] Killing assay: 1) CD8 T cell sorting: After PBMC cell counting and stabilization overnight at 37°C with 5% CO2, CD8 T cells were isolated using a CD8 T cell isolation kit (Stem Cell, catalog number 19053) for subsequent in vitro experiments. 2) Tumor cell labeling: NALM-6 cells in logarithmic growth phase were collected, centrifuged at 300g for 5 minutes, the supernatant was discarded, and the cells were washed once with 5mL PBS. After centrifugation at 300g for 5 minutes, the supernatant was discarded, and the tumor cells were resuspended in 1mL CFSE working solution (Invitrogen, C34554). The cells were incubated at room temperature in the dark for 15 minutes, staining was stopped with 1mL complete culture medium, centrifuged at 300g for 5 minutes, the supernatant was discarded, and the cells were washed once with PBS. 3) The tumor cells were resuspended in RPMI 1640 complete culture medium containing 10% serum, and the cell concentration was adjusted to 4×10⁶ cells / mL. 5 4) Add 50 μL of CD8 T cells per well to a 96-well round-bottom plate at a rate of 4 x 10⁻⁶ cells / mL. 5 5) Antibody preparation: Prepare antibodies using complete culture medium. Dilute the antibodies in a sterile 96-well V-plate (Beyotime, FPT019) at 400 nM, 10-fold dilutions, for a total of 10 gradients. Add 50 μL of the antibody to each well of the cell culture plate and incubate at 37°C with 5% CO2 for 24 hours. 6) Flow cytometry staining: Prepare cell viability and inactivation stains in advance. After 24 hours of culture, centrifuge at 300g for 5 minutes to collect the supernatant for subsequent T cell activation cytokine release detection. Transfer cells to a sterile 96-well V-plate (Beyotime, FPT019), stain with viability and inactivation stains for 15 minutes, wash away unbound and non-specifically bound stains, and analyze using a flow cytometer.

[0597] The experimental results are shown in Figure 10. The CD19×BCMA TCE antibody can kill NALM-6 expressing CD19 antigen in vitro, and its killing ability is better than that of the control antibody CD19(2B11)CD3opt (US20240132590A1).

[0598] Example 7. Detection of the in vitro killing effect of antibody-mediated T cells on BCMA-positive cells.

[0599] Cell resuscitation: 1) Remove peripheral blood mononuclear cells (PBMCs) (Shanghai Miaoshun TPCS) from the liquid nitrogen tank and gently shake them in a 37℃ water bath until thawed. Keep the sealing ring and cap above the water surface to prevent contamination; 2) After thawing, immediately disinfect with 75% alcohol and transfer to a clean bench. Strictly follow aseptic techniques thereafter; 3) Prepare 1640 complete culture medium in advance: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010), 1% Sodium 3) Pyruvate (Gibco, 1136-070) and 0.1% β-ME (Gibco, 21985-023); 4) Transfer the cell suspension from the cryopreservation tube to preheated 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to precipitate the cells, and discard the supernatant; 5) Resuspend the cell pellet in 15mL of complete medium, take a portion and count the viable cells using trypan blue (Gibco, 15250-061).

[0600] Killing assay: 1) CD8 T cell sorting: After counting PBMC cells and stabilizing overnight at 37°C with 5% CO2, CD8 T cells were isolated using a CD8 T cell isolation kit (Stem Cell, catalog number 19053) for subsequent in vitro experiments. 2) Tumor cell labeling: BCMA-expressing tumor cells (NCI-H929, MM.1S, L363) in logarithmic growth phase were collected, centrifuged at 300g for 5 minutes, the supernatant was discarded, washed once with PBS, centrifuged at 300g for 5 minutes, the supernatant was discarded, and the tumor cells were resuspended in 1 mL of CFSE working solution (Invitrogen, C34554). The cells were incubated at room temperature in the dark for 15 minutes, staining was stopped with 1 mL of complete culture medium, centrifuged at 300g for 5 minutes, the supernatant was discarded, and the cells were washed once with PBS. 3) The tumor cells were resuspended in RPMI 1640 complete culture medium containing 10% serum, and the cell concentration was adjusted to 4 x 10⁻⁶ cells / mL. 5 4) Add 50 μL of CD8 T cells per well to a 96-well round-bottom plate at a rate of 4 x 10⁻⁶ cells / mL. 55) Antibody preparation: Prepare antibodies using complete culture medium. Dilute the antibodies in a sterile 96-well V-plate (Beyotime, FPT019) at 400 nM, 10-fold dilutions, for a total of 10 gradients. Add 50 μL of the antibody to each well of the cell culture plate and incubate at 37°C with 5% CO2 for 24 hours. 6) Flow cytometry staining: Prepare cell viability and inactivation stains in advance. After 24 hours of culture, centrifuge at 300g for 5 minutes to collect the supernatant for subsequent T cell activation cytokine release detection. Transfer cells to a sterile 96-well V-plate (Beyotime, FPT019), stain with viability and inactivation stains for 15 minutes, wash away unbound and non-specifically bound stains, and analyze using a flow cytometer.

[0601] The experimental results are shown in Figures 11, 12, and 13. The CD19×BCMA TCE antibody can kill NCI-H929, MM.1S, and L363 tumor cells with different BCMA expression levels in vitro, and its killing ability is comparable to that of the control antibody Teclistamab.

[0602] Example 8. Detection of the effect of antibody-mediated T cell activation induced by CD19-positive and BCMA-positive cells.

[0603] Cell resuscitation: 1) Remove peripheral blood mononuclear cells (PBMCs) (Shanghai Miaoshun TPCS) from the liquid nitrogen tank and gently shake them in a 37℃ water bath until thawed. Keep the sealing ring and cap above the water surface to prevent contamination; 2) After thawing, immediately disinfect with 75% alcohol and transfer to a clean bench. Strictly follow aseptic techniques thereafter; 3) Prepare 1640 complete culture medium in advance: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010), 1% Sodium 3) Pyruvate (Gibco, 1136-070) and 0.1% β-ME (Gibco, 21985-023); 4) Transfer the cell suspension from the cryopreservation tube to preheated 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to precipitate the cells, and discard the supernatant; 5) Resuspend the cell pellet in 15mL of complete medium, take a portion and count the viable cells using trypan blue (Gibco, 15250-061).

[0604] Killing assay: 1) CD8 T cell sorting: After PBMC cell counting and overnight stabilization at 37°C with 5% CO2, CD8 T cells were isolated using a CD8 T cell isolation kit (Stem Cell, catalog number 19053) for subsequent in vitro experiments. 2) Tumor cell labeling: CD19-positive NALM-6, NCI-H929, MM.1S, and L363 tumor cells in logarithmic growth phase were collected, centrifuged at 300g for 5 minutes, the supernatant was discarded, and the cells were washed once with 5mL PBS. The cells were then centrifuged again at 300g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 1mL CFSE working solution (Invitrogen, C34554). The cells were incubated at room temperature in the dark for 15 minutes, staining was stopped with 1mL complete culture medium, centrifuged at 300g for 5 minutes, the supernatant was discarded, and the cells were washed once with PBS. 3) The tumor cells were resuspended in RPMI 1640 complete culture medium containing 10% serum, and the cell concentration was adjusted to 4×10⁶ cells / mL. 5 4) Add 50 μL of CD8 T cells per well to a 96-well round-bottom plate at a rate of 4 × 10⁻⁶ cells / mL. 5 5) Antibody preparation: Prepare antibodies using complete culture medium. Dilute the antibodies in a sterile 96-well V plate (Beyotime, FPT019) with 400 nM, 10-fold dilutions for a total of 10 gradients. Add 50 μL of the antibody to each well of the cell culture plate and incubate at 37°C with 5% CO2 for 24 hours. 6) Flow cytometry staining: Prepare T cell activation-related marker flow cytometry dyes in advance, including Live Dead (Invitrogen, L34968A), CD8 (Biolegend, 344724), CD25 (Biolegend, 356140), CD69 (Biolegend, 310906), and PD-1 (Biolegend 329918). After 24 hours of culture, the supernatant was collected by centrifugation at 300g for 5 minutes for subsequent detection of cytokine release after T cell activation. Cells were transferred to sterile 96-well V plates (Beyotime, FPT019), stained for 1 hour, and unbound and non-specifically bound antibodies were washed away. Flow cytometry was then used for detection.

[0605] The experimental results are shown in Figure 14. When T cells were incubated with CD19-positive NALM-6 cells, the antibody could mediate T cell activation and cause NALM-6 tumor cell killing. The ability to induce T cell activation was comparable to that of the control antibody CD19(2B11)CD3opt(US20240132590A1).

[0606] The experimental results are shown in Figures 15, 16, and 17. When T cells were incubated with BCMA-positive NCI-H929, MM.1S, and L363 cells, the antibody could mediate T cell activation and cause the killing of BCMA-positive NCI-H929, MM.1S, and L363 tumor cells. Moreover, the ability to induce T cell activation was comparable to that of the control antibody Teclistamab.

[0607] Example 9. Detection of antibody-mediated T cell activation and killing activity in the absence of BCMA and CD19.

[0608] Cell resuscitation: 1) Remove peripheral blood mononuclear cells (PBMCs) (Shanghai Miaoshun TPCS) from the liquid nitrogen tank and gently shake them in a 37℃ water bath until thawed. Keep the sealing ring and cap above the water surface to prevent contamination; 2) After thawing, immediately disinfect with 75% alcohol and transfer to a clean bench. Strictly follow aseptic techniques thereafter; 3) Prepare 1640 complete culture medium in advance: 1640 (Gibco, 22400-071), 10% FBS (Gibco, 10099-141C), 1% pen / strep (Hyclone, SV30010), 1% Sodium 3) Pyruvate (Gibco, 1136-070) and 0.1% β-ME (Gibco, 21985-023); 4) Transfer the cell suspension from the cryopreservation tube to preheated 1640 complete medium containing DNase1 (SIGMA, D5025-375KU), mix gently, centrifuge at 300g for 6 minutes to precipitate the cells, and discard the supernatant; 5) Resuspend the cell pellet in 15mL of complete medium, take a portion and count the viable cells using trypan blue (Gibco, 15250-061).

[0609] Killing assay: 1) CD8 T cell sorting: After PBMC cell counting and stabilization overnight at 37°C with 5% CO2, CD8 T cells were isolated using a CD8 T cell isolation kit (Stem Cell, catalog number 19053) for subsequent in vitro experiments. 2) Tumor cell labeling: 293T cells in logarithmic growth phase that do not express CD19 and BCMA were collected, centrifuged at 300g for 5 minutes, the supernatant was discarded, washed once with PBS, centrifuged at 300g for 5 minutes, the supernatant was discarded, and the tumor cells were resuspended in 1 mL of CFSE working solution (Invitrogen, C34554). The cells were incubated at room temperature in the dark for 15 minutes, staining was stopped with 1 mL of complete culture medium, centrifuged at 300g for 5 minutes, the supernatant was discarded, and the cells were washed once with PBS. 3) The tumor cells were resuspended in RPMI 1640 complete culture medium containing 10% serum, and the cell concentration was adjusted to 4 x 10⁻⁶ cells / mL. 5 4) Add 50 μL of CD8 T cells per well to a 96-well round-bottom plate at a rate of 4 x 10⁻⁶ cells / mL. 55) Antibody preparation: Prepare antibodies using complete culture medium. Dilute the antibodies in a sterile 96-well V-plate (Beyotime, FPT019) at 400 nM, 10-fold dilutions, for a total of 10 gradients. Add 50 μL of each antibody to the cell culture plate and incubate at 37°C with 5% CO2 for 24 hours. 6) Flow cytometry staining: Prepare T cell activation-related marker flow cytometry dyes in advance, including Live Dead, CD8, CD25, CD69, and PD-1. After 24 hours of culture, centrifuge at 300g for 5 minutes to collect the supernatant for subsequent detection of cytokine release after T cell activation. Transfer cells to a sterile 96-well V-plate (Beyotime, FPT019), stain for 1 hour, wash away unbound and non-specifically bound antibodies, and perform flow cytometry analysis.

[0610] The experimental results are shown in Figures 18 and 19. The CD19×BCMA and control antibodies had no killing effect on 293T cells that did not express CD19 and BCMA antigens. However, the control antibodies CD19(2B11)CD3opt (US20240132590A1) and Teclistamab could induce stronger T cell activation, including upregulation of CD25 and CD69 expression. In contrast, the CD19×BCMA TCE of this invention does not cause T cell activation in the absence of CD19 and BCMA, which helps to reduce toxicity issues.

[0611] Example 10. Detection of the effect of antibody on B cell clearance and inhibition of antibody production in a B-hCD3EDG / hCD19 mouse KLH model.

[0612] To demonstrate that the CD19×BCMA TCE molecule of this invention possesses the in vivo efficacy of clearing CD19-positive B cells, we used a hemocyanin (KLH) model to verify the in vivo efficacy of CD19×BCMA TCE in clearing B cells and thereby inhibiting antibody production. The 6-8 week old B-hCD3EDG / hCD19 transgenic mice used in the experiment were purchased from Biocytogen Pharmaceuticals Co., Ltd. After arrival, they were domesticated and raised for 3 days before the experiment began.

[0613] In this experiment, mice were intraperitoneally injected with 250 μg KLH (Solarbio, K8160) on Day 0 and Day 12. On Day 4 and Day 11, mice were administered CD19×BCMA TCE 5.833 mg / kg, positive control antibody W3438-T3U4.E17-1.uIgG4.SP (CN109535257B) 5 mg / kg, and negative control antibody Isotype (gp120-hIgG1-LALA) 5 mg / kg, respectively. Subsequently, on Day 20, samples were collected, including lymph nodes, spleen, and blood. Cells were extracted from lymph nodes and spleen by grinding, and cells were extracted from blood samples by erythropoiesis. Cells were stained by B220 flow cytometry to detect the drug's effect on B cell clearance in different tissue samples. Serum was extracted from blood samples, and the anti-KLH antibody content in serum was detected by ELISA to detect the inhibitory effect of the drug on anti-KLH antibodies produced by KLH immune response.

[0614] The results are shown in Figure 20. At the same molar dose, the CD19×BCMA TCE antibody of this invention has a better B cell clearance effect than the positive control antibody W3438-T3U4.E17-1.uIgG4.SP (CN109535257B) in lymph nodes, spleen, and blood.

[0615] As shown in Figures 21 and 22, at the same molar dose, the CD19×BCMA TCE antibody of the present invention has a better effect on inhibiting the production of IgM, IgA, and IgG than the positive control antibody W3438-T3U4.E17-1.uIgG4.SP (CN109535257B) (Figure 21, IgG content is the detection data after serum sample is diluted 20 times). At the same time, the efficacy of inhibiting the production of anti-KLH IgM, IgA, and IgG antibodies in the KLH immune response is also better than that of the positive control W3438-T3U4.E17-1.uIgG4.SP (CN109535257B) (Figure 22).

[0616] Example 11. Detection of the efficacy of antibody in plasma cell clearance and inhibition of antibody production in B-hCD3EDG / hBCMA mouse KLH model.

[0617] To demonstrate the in vivo efficacy of the CD19×BCMA TCE molecule of this invention in clearing plasma cells, we used a KLH model to verify the in vivo efficacy of CD19×BCMA TCE in clearing plasma cells and thus inhibiting antibody production. The 6-8 week old B-hCD3EDG / hBCMA transgenic mice used in the experiment were purchased from Biocytogen Pharmaceuticals Co., Ltd. After arrival, they were domesticated and raised for 3 days before the experiment began.

[0618] In this experiment, mice were intraperitoneally injected with 250 μg of KLH on Day 0 and Day 12. On Day 4 and Day 11, they were given CD19×BCMA TCE 5.833 mg / kg, positive control antibody Teclistamab 5 mg / kg, and negative control antibody Isotype (gp120-hIgG1-LALA) 5 mg / kg, respectively. Subsequently, on Day 20, samples were collected from lymph nodes, spleen, bone marrow, and blood to detect the drug's effect on plasma cell clearance and its effect on inhibiting the production of anti-KLH antibodies in different tissue samples (methods are the same as in Example 10).

[0619] The results are shown in Figure 23. At the same molar dose, the CD19×BCMA TCE antibody of this invention and the positive control antibody Teclistamab both showed comparable plasma cell clearance effects in lymph nodes, spleen, and bone marrow (bone marrow of the two hind limbs of mice).

[0620] As shown in Figures 24 and 25, at the same molar dose, the CD19×BCMA TCE antibody of the present invention has a comparable effect on inhibiting the production of IgM, IgA, and IgG with the positive control antibody Teclistamab (Figure 24). At the same time, its efficacy in inhibiting the production of anti-KLH IgM, IgA, and IgG antibodies in the KLH immune response is also comparable to that of the positive control Teclistamab (Figure 25).

[0621] Example 12. Detection of the antibody's effect on the clearance of B cells and plasma cells in B-hCD3EDG / hCD19 / hBCMA mice.

[0622] To demonstrate the efficacy of the CD19×BCMA TCE molecule of this invention in clearing B cells and plasma cells in vivo, we conducted this experiment. The 6-8 week old B-hCD3EDG / hCD19 / hBCMA transgenic mice used in the experiment were purchased from Biocytogen Pharmaceutical Technology Co., Ltd. After arrival, they were domesticated and raised for 3 days before the experiment began.

[0623] On Day 0, mice in each group were administered CD19×BCMA TCE 3.5 mg / kg, positive control antibody CD19(2B11)CD3opt (US20240132590A1) 4 mg / kg, positive control antibody Teclistamab 3 mg / kg, and negative control antibody Isotype (gp120-hIgG1-LALA) 3 mg / kg. On Day 6, samples were collected from lymph nodes, spleen, bone marrow, and blood to detect the drug's clearance effect on B cells and antibody-secreting cells in different tissue samples.

[0624] The results are shown in Figures 26 and 27. At the same molar dose, the CD19×BCMA TCE antibody of the present invention was found to have a better clearance effect on B cells and antibody-secreting cells than the positive control antibody in different tissue samples (bone marrow from a mouse hind limb).

[0625] Example 13. Detection of the efficacy of antibody in inhibiting antibody production in a B-hCD3EDG / hCD19 / hBCMA mouse KLH model.

[0626] To demonstrate the efficacy of the CD19×BCMA TCE molecule of this invention in inhibiting immunoglobulin production in vivo, we used the KLH model for verification. The 6-8 week old B-hCD3EDG / hCD19 / hBCMA transgenic mice used in this experiment were purchased from Biocytogen Pharmaceutical Technology Co., Ltd. After arrival, they were domesticated and raised for 3 days before the experiment began.

[0627] In this experiment, mice were intraperitoneally injected with 250 μg of KLH on Day 0 and Day 12. On Day 4 and Day 11, mice were administered the following antibodies: CD19×BCMA TCE 5.0 mg / kg, positive control antibody CD3opt (US20240132590A1) 5.7 mg / kg, positive control antibody Teclistamab 4.3 mg / kg, and negative control antibody Isotype (gp120-hIgG1-LALA) 4.3 mg / kg, respectively. Samples were collected on Day 20, and blood was collected to detect the effect of inhibiting the production of anti-KLH antibodies in the KLH immune response (method as described above).

[0628] Example 10).

[0629] At the same molar dose, the CD19×BCMA TCE antibody of the present invention is superior to the positive control in inhibiting the production of IgM, IgA and IgG (Figure 28), and its efficacy in inhibiting the production of anti-KLH IgM and IgG antibodies in the KLH immune response is also superior to the positive control antibody (Figure 29).

Claims

1. A trispecific antibody that specifically binds to CD3, BCMA, and CD19, comprising heavy chain 1, light chain 1, heavy chain 2, and light chain 2, or composed of heavy chain 1, light chain 1, heavy chain 2, and light chain 2, wherein heavy chain 1, light chain 1, heavy chain 2, and light chain 2 are shown below: Heavy chain 1: From the N-terminus to the C-terminus, it contains or consists of the following: VH BCMA -First CH1-First Fc region, wherein the heavy chain constant region CH1 is connected at its C end to the N end of the first Fc region directly or via a connector (preferably directly); Light chain 1: Composed of or consisting of the following from the N-terminus to the C-terminus: VL BCMA -First CL; Heavy chain 2: From the N-terminus to the C-terminus, it contains or consists of the following: VH CD19 -Second CH1-scFv CD3 - Second Fc region, where the C-terminus of the second CH1 is connected to scFv CD3 The N-terminus is connected directly or via connector 1, and scFv CD3 The C end is directly or via connector 3 connected to the N end of the second Fc region; Light chain 2: From the N-terminus to the C-terminus, it includes or consists of the following: VL CD19 -Second CL; or Heavy chain 1: From the N-terminus to the C-terminus, it contains or consists of the following: VH CD19 -First CH1-First Fc region, wherein the heavy chain constant region CH1 is connected at its C end to the N end of the first Fc region directly or via a connector (preferably directly); Light chain 1: Composed of or consisting of the following from the N-terminus to the C-terminus: VL CD19 -First CL; Heavy chain 2: From the N-terminus to the C-terminus, it contains or consists of the following: VH BCMA -Second CH1-scFv CD3 - Second Fc region, where the C-terminus of the second CH1 is connected to scFv CD3 The N-terminus is connected directly or via connector 1, scFv CD3 The C end is directly or via connector 3 connected to the N end of the second Fc region; Light chain 2: From the N-terminus to the C-terminus, it includes or consists of the following: VL BCMA -Second CL; in "-" indicates a connector or direct connection; VH BCMA This refers to the heavy chain variable region VH that specifically binds to the antigen-binding region of BCMA, and VL BCMA It refers to the light chain variable region (VL) of the antigen-binding region that specifically binds to BCMA; VH CD19 This refers to the heavy chain variable region VH that specifically binds to the antigen-binding region of CD19, and VL CD19 It refers to the light chain variable region (VL) of the antigen-binding region that specifically binds to CD19; The first CH1 and the second CH1 refer to the CH1 domain of the heavy chain constant region, respectively. The first CL and the second CL refer to the constant regions of the light chain, respectively. VH BCMA -CH1 and VL BCMA -CL constitutes the Fab region, which is the antigen-binding region that specifically binds to BCMA. BCMA ; VH CD19 -CH1 and VL CD19 -CL constitutes the Fab region, which serves as the antigen-binding region specifically binding to CD19. CD19 ; scFv CD3 This refers to the scFv, which is the antigen-binding region that specifically binds to CD3, and consists of VH CD3 VL CD3 VH connected directly or via connector 2 CD3 and VL CD3 The polypeptide chain composition, preferably the scFv CD3 From the N-terminus to the C-terminus, it includes or consists of the following: VH CD3 -VL CD3 , of which VH CD3 C-terminus and VL CD3 The N-terminus is connected directly or via connector 2; Optionally, the first CH1 and the second CH1 are the same, and / or the first CL and the second CL are the same, and / or the first Fc region and the second Fc region are different.

2. The trispecific antibody of claim 1, wherein The first CH1 is directly connected to the first Fc region; Second CH1 and scFv CD3 The N-terminal is connected via connector 1; scFv CD3 VH CD3 C-terminus and VL CD3 The N-terminus is connected via connector 2; and / or scFv CD3 The C end is connected to the second Fc region via connector 3; Optionally, connector 1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:47 or 48; connector 2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:47 or 49; and / or connector 3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:47 or 50.

3. The trispecific antibody of claim 1 or 2, wherein the first Fc region and the second Fc region are respectively Fc regions derived from human IgG1, IgG2, IgG3 or IgG4, optionally, the Fc region is a human IgG1 Fc region or a human IgG4 Fc region, for example, the Fc region (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:42; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:

42.

4. The trispecific antibody according to any one of claims 1-3, wherein the first Fc region and the second Fc region are different, and based on Innobody technology, mutations are introduced into the first Fc region and the second Fc region to promote heterodimerization of the first Fc region and the second Fc region, preferably one Fc region CH3 contains S364R and D399K mutations, and the other Fc region CH3 contains Y349T, K370S and K409D mutations, and optionally the first and / or the second Fc region contains mutations that reduce binding to the Fcγ receptor, such as L234A / L235A mutations; Optional, of which a) One Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:43, and the other Fc region contains or is composed of the amino acid sequence shown in SEQ ID NO:44; b) One Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:43 and contains the mutants S364R and D399K, while the other Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:44 and contains the mutants Y349T, K370S, and K409D; or c) One Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:43 and contains the mutations S364R and D399K and L234A / L235A mutations, while the other Fc region contains an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:44 and contains the mutations Y349T, K370S, and K409D and L234A / L235A mutations.

5. The trispecific antibody according to any one of claims 1-4, wherein the first CH1 and the second CH1 are respectively CH1 derived from human IgG1, IgG2, IgG3 or IgG4, preferably the first CH1 and the second CH1 are respectively CH1 of human IgG1 or CH1 of human IgG4, preferably, the CH1 (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:40; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:

40.

6. The trispecific antibody according to any one of claims 1-5, wherein the first CL and the second CL are light chain constant regions respectively derived from the Kappa light chain constant region or the Lambda light chain constant region, preferably, the CL is the human Kappa light chain constant region or the human Lambda light chain constant region, preferably, the CL (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:41; or (ii) Contains or consists of the amino acid sequence shown in SEQ ID NO:

41.

7. The trispecific antibody according to any one of claims 1-6, wherein the VH BCMA Includes complementary determinant regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and wherein the VL BCMA The device comprises complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:19; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:20; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:21; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:22; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:23; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

24.

8. The trispecific antibody according to any one of claims 1-7, wherein the VH BCMA Containing the amino acid sequence shown in SEQ ID NO:17 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence, and said VL BCMA It contains the amino acid sequence shown in SEQ ID NO:18 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or is composed of said amino acid sequence.

9. The trispecific antibody according to any one of claims 1-8, wherein The VH BCMA Contains the amino acid sequence shown in SEQ ID NO:17, and the VL BCMA Contains the amino acid sequence shown in SEQ ID NO:18; or The VH BCMA Composed of the amino acid sequence shown in SEQ ID NO:17, and the VL BCMA It consists of the amino acid sequence shown in SEQ ID NO:

18.

10. The trispecific antibody according to any one of claims 1-9, wherein the VH CD3 Includes complementary determinant regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and wherein the VL CD3 The device comprises complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:27; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:28; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:29; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:30; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:31; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

32.

11. The trispecific antibody according to any one of claims 1-10, wherein the VH CD3 Containing or consisting of the amino acid sequence shown in SEQ ID NO:25, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, and the VL CD3 It contains the amino acid sequence shown in SEQ ID NO:26 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or is composed of said amino acid sequence.

12. The trispecific antibody according to any one of claims 1-11, wherein The VH CD3 Contains the amino acid sequence shown in SEQ ID NO:25, and the VL CD3 Contains the amino acid sequence shown in SEQ ID NO:26; or The VH CD3 Composed of the amino acid sequence shown in SEQ ID NO:25, and the VL CD3 It consists of the amino acid sequence shown in SEQ ID NO:

26.

13. The trispecific antibody according to any one of claims 1-12, wherein the VH CD19 The complementary determinant region (HCDR) containing the heavy chain variable region, HCDR1, HCDR2 and HCDR3, and the VL CD19 The device comprises a complementarity-determining region (LCDR) of a light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:3; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:4; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:5; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:6 or 71; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:7; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

8.

14. The trispecific antibody according to any one of claims 1-13, wherein the VH CD19 Containing the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence, and said VL CD19 It contains the amino acid sequence shown in SEQ ID NO:2 or 70, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence.

15. The trispecific antibody according to any one of claims 1-14, wherein The VH CD19 Contains the amino acid sequence shown in SEQ ID NO:1, and the VL CD19 Contains the amino acid sequence shown in SEQ ID NO:2 or 70; or The VH CD19 Composed of the amino acid sequence shown in SEQ ID NO:1, and the VL CD19 It consists of the amino acid sequence shown in SEQ ID NO:2 or 70.

16. The trispecific antibody according to any one of claims 1-12, wherein the VH CD19 It includes complementary determinant regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and the VL CD19 The device comprises complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:11; HCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:12; HCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:13; LCDR1 comprises or is composed of the amino acid sequence shown in SEQ ID NO:14; LCDR2 comprises or is composed of the amino acid sequence shown in SEQ ID NO:15; and LCDR3 comprises or is composed of the amino acid sequence shown in SEQ ID NO:

16.

17. The trispecific antibody according to any one of claims 1-12 and 16, wherein the VH CD19 Containing the amino acid sequence shown in SEQ ID NO:9, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence, and said VL CD19 It contains the amino acid sequence shown in SEQ ID NO:10 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with it, or is composed of said amino acid sequence.

18. The trispecific antibody according to any one of claims 1-12 and 16-17, wherein The VH CD19 Contains the amino acid sequence shown in SEQ ID NO:9, and the VL CD19 Contains the amino acid sequence shown in SEQ ID NO:10; or The VH CD19 Composed of the amino acid sequence shown in SEQ ID NO:9, and the VL CD19 It consists of the amino acid sequence shown in SEQ ID NO:

10.

19. The trispecific antibody according to any one of claims 1-18, wherein Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:33, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:34, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:35, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:

35. The amino acid sequence shown in NO:36 or 73, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or consisting of said amino acid sequence; or Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:33, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:34, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:37, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:

37. The amino acid sequence shown in NO:38, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or consisting of said amino acid sequence; or Heavy chain 1 comprises the amino acid sequence shown in SEQ ID NO:72, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; light chain 1 comprises the amino acid sequence shown in SEQ ID NO:73, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; heavy chain 2 comprises the amino acid sequence shown in SEQ ID NO:74, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or is composed of said amino acid sequence; and light chain 2 comprises SEQ ID NO:

74. The amino acid sequence shown in NO:34, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with it, or composed of said amino acid sequence.

20. The trispecific antibody according to any one of claims 1-19, wherein Heavy chain 1 contains the amino acid sequence shown in SEQ ID NO:33; light chain 1 contains the amino acid sequence shown in SEQ ID NO:34; heavy chain 2 contains the amino acid sequence shown in SEQ ID NO:35; and light chain 2 contains the amino acid sequence shown in SEQ ID NO:36 or 73. Heavy chain 1 consists of the amino acid sequence shown in SEQ ID NO:33; light chain 1 consists of the amino acid sequence shown in SEQ ID NO:34; heavy chain 2 consists of the amino acid sequence shown in SEQ ID NO:35; and light chain 2 consists of the amino acid sequence shown in SEQ ID NO:36 or 73; Heavy chain 1 contains the amino acid sequence shown in SEQ ID NO:33; light chain 1 contains the amino acid sequence shown in SEQ ID NO:34; heavy chain 2 contains the amino acid sequence shown in SEQ ID NO:37; and light chain 2 contains the amino acid sequence shown in SEQ ID NO:

38. Heavy chain 1 consists of the amino acid sequence shown in SEQ ID NO:33; light chain 1 consists of the amino acid sequence shown in SEQ ID NO:34; heavy chain 2 consists of the amino acid sequence shown in SEQ ID NO:37; and light chain 2 consists of the amino acid sequence shown in SEQ ID NO:

38. Heavy chain 1 contains the amino acid sequence shown in SEQ ID NO:72; light chain 1 contains the amino acid sequence shown in SEQ ID NO:73; heavy chain 2 contains the amino acid sequence shown in SEQ ID NO:74; and light chain 2 contains the amino acid sequence shown in SEQ ID NO:34; or Heavy chain 1 consists of the amino acid sequence shown in SEQ ID NO:72; light chain 1 consists of the amino acid sequence shown in SEQ ID NO:73; heavy chain 2 consists of the amino acid sequence shown in SEQ ID NO:74; and light chain 2 consists of the amino acid sequence shown in SEQ ID NO:

34.

21. An anti-CD3 antibody or its antigen-binding fragment, comprising VH and VL, wherein The VH contains or is composed of the amino acid sequence shown in SEQ ID NO:25, and the VL contains or is composed of the amino acid sequence shown in SEQ ID NO:

26.

22. The anti-CD3 antibody or its antigen-binding fragment according to claim 21, further comprising a heavy chain constant region and / or a light chain constant region, preferably, The heavy chain constant region is a constant region of IgG1, IgG2, IgG3 or IgG4, such as the heavy chain constant region of human IgG1 or human IgG4; and / or the light chain constant region is a Kappa light chain constant region or a Lambda light chain constant region, such as the human Kappa light chain constant region or the human Lambd light chain constant region.

23. An anti-CD19 antibody or an antigen-binding fragment thereof, comprising three complementarity-determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, and three complementarity-determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, wherein HCDR1 comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:3; HCDR2 comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:4; HCDR3 comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:5; LCDR1 comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:6; LCDR2 comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:7; and LCDR3 comprises, or is composed of, the amino acid sequence shown in SEQ ID NO:

8.

24. The antibody or antigen-binding fragment thereof of claim 23, comprising VH and VL, wherein the VH comprises or is composed of the amino acid sequence shown in SEQ ID NO:1, and the VL comprises or is composed of the amino acid sequence shown in SEQ ID NO:

2.

25. The anti-CD19 antibody or its antigen-binding fragment according to claim 23 or 24, further comprising a heavy chain constant region and / or a light chain constant region, preferably, The heavy chain constant region is a constant region of IgG1, IgG2, IgG3 or IgG4, such as the heavy chain constant region of human IgG1 or human IgG4; and / or the light chain constant region is a Kappa light chain constant region or a Lambda light chain constant region, such as the human Kappa light chain constant region or the human Lambd light chain constant region.

26. A nucleic acid molecule comprising, or consisting of, a nucleic acid sequence encoding, any one of the trispecific antibodies of claims 1-20, or the anti-CD3 antibody of claims 21 or 22, or an antigen-binding fragment thereof, or any one of the anti-CD19 antibody of claims 23-25, or an antigen-binding fragment thereof.

27. An expression vector comprising the nucleic acid molecule of claim 26, preferably, the expression vector being pCDNA, such as pCDNA3.

4.

28. A host cell comprising the nucleic acid molecule of claim 26 or the expression vector of claim 27, preferably, the host cell being prokaryotic or eukaryotic, such as 293 cells or CHO cells, such as ExpiCHO cells.

29. A method for preparing a trispecific antibody according to any one of claims 1-20, or an anti-CD3 antibody or an antigen-binding fragment thereof according to claims 21 or 22, or an anti-CD19 antibody or an antigen-binding fragment thereof according to any one of claims 23-25, wherein the method comprises culturing a host cell containing a nucleic acid molecule according to claim 26 or an expression vector according to claim 27 under conditions suitable for chain expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium).

30. An immunoconjugate comprising the trispecific antibody of any one of claims 1-20, or the anti-CD3 antibody of any one of claims 21 or 22, or the antigen-binding fragment thereof, or the anti-CD19 antibody of any one of claims 23-25, or the antigen-binding fragment thereof.

31. A pharmaceutical composition comprising the trispecific antibody of any one of claims 1-20, or the anti-CD3 antibody of any one of claims 21 or 22, or the antigen-binding fragment thereof, or the anti-CD19 antibody of any one of claims 23-25, or the immunoconjugate of claim 30, and optionally pharmaceutical excipients.

32. A pharmaceutical combination comprising the trispecific antibody of any one of claims 1-20, or the anti-CD3 antibody of any one of claims 21 or 22, or the antigen-binding fragment thereof, or the anti-CD19 antibody of any one of claims 23-25, and one or more other therapeutic agents. Preferably, the other therapeutic agents are various therapeutic agents used to treat tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists); or The other therapeutic agents are those used to treat autoimmune diseases, such as nonsteroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs), antirheumatic drugs, or immunomodulators such as immunosuppressants.

33. A method for preventing or treating tumors or autoimmune diseases in a subject, comprising administering to the subject an effective amount of the trispecific antibody of any one of claims 1-20, or the anti-CD3 antibody of any one of claims 21 or 22 or its antigen-binding fragment, or the anti-CD19 antibody of any one of claims 23-25 ​​or its antigen-binding fragment, or the immunoconjugate of claim 30, or the pharmaceutical composition of claim 31, or the pharmaceutical combination of claim 32.

34. Use of the trispecific antibody of any one of claims 1-20, or the anti-CD3 antibody or its antigen-binding fragment of any one of claims 21 or 22, or the anti-CD19 antibody or its antigen-binding fragment of any one of claims 23-25, or the immunoconjugate of claim 30, or the pharmaceutical composition of claim 31, or the pharmaceutical combination of claim 32, in the preparation of a medicament for the prevention or treatment of tumors or autoimmune diseases.

35. The trispecific antibody of any one of claims 1-20, or the anti-CD3 antibody or its antigen-binding fragment of any one of claims 21 or 22, or the anti-CD19 antibody or its antigen-binding fragment of any one of claims 23-25, or the immunoconjugate of claim 30, or the pharmaceutical composition of claim 31, or the pharmaceutical composition of claim 32, for use in a therapy.

36. The trispecific antibody, anti-CD3 antibody or its antigen-binding fragment thereof, anti-CD19 antibody or its antigen-binding fragment thereof, immune conjugate, pharmaceutical composition or combination thereof as described in claim 35, for the prevention or treatment of tumors or autoimmune diseases.

37. The method of claim 33, the use of claim 34, or the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immunoconjugate, pharmaceutical composition or combination of drugs of claim 36, wherein the tumor is a solid tumor or a hematologic malignancy.

38. The method of claim 33 or 37, the use of claim 34 or 37, or the trispecific antibody, anti-CD3 antibody or antigen-binding fragment thereof, anti-CD19 antibody or antigen-binding fragment thereof, immune conjugate, pharmaceutical composition or combination of drugs of claim 36 or 37, wherein the tumor is a BCMA-positive and / or CD19-positive tumor or cancer.

39. The method of any one of claims 33 and 37-38, the use of any one of claims 34 and 37-38, or the trispecific antibody, anti-CD3 antibody or antigen-binding fragment thereof, anti-CD19 antibody or antigen-binding fragment thereof, immunoconjugate, pharmaceutical composition or combination of drugs of any one of claims 36 and 37-38, wherein the tumor is a hematologic malignancy, such as leukemia such as acute lymphoblastic leukemia or myeloma such as multiple myeloma.

40. The method of any one of claims 33, 37-39, the use of any one of claims 34, 37-39, or the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immunoconjugate, pharmaceutical composition, or pharmaceutical combination of any one of claims 36, 37-39, wherein the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immunoconjugate, pharmaceutical composition, or pharmaceutical combination, or the pharmaceutical combination, is further administered in combination with other therapies such as treatment modalities (e.g., surgery or radiotherapy) and / or other therapeutic agents, preferably, the therapeutic agent is a variety of therapeutic agents for treating tumors, such as chemotherapeutic agents, angiogenesis inhibitors, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immune checkpoint inhibitors or agonists).

41. The method of claim 33, the use of claim 34, or the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immunoconjugate, pharmaceutical composition, or combination of drugs of claim 36, wherein the autoimmune disease is an autoimmune disease mediated by B cells and / or antibody-secreting cells (e.g., plasma cells) and / or autoantibodies.

42. The method of claim 33 or 41, the use of claim 34 or 41, or the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immune conjugate, pharmaceutical composition or combination of drugs of claim 36 or 41, wherein the autoimmune disease is systemic lupus erythematosus, lupus nephritis or rheumatoid arthritis.

43. The method of any one of claims 33, 41, and 42, the use of any one of claims 34, 41, and 42, or the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immunoconjugate, pharmaceutical composition, or pharmaceutical combination of any one of claims 36, 41, and 42, wherein the trispecific antibody, anti-CD3 antibody or its antigen-binding fragment, anti-CD19 antibody or its antigen-binding fragment, immunoconjugate, pharmaceutical composition, or pharmaceutical combination, or the pharmaceutical combination, is further administered in combination with other therapeutic agents, preferably, the other therapeutic agents being therapeutic agents for treating autoimmune diseases, such as nonsteroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs (SAIDs), antirheumatic drugs, or immunomodulators such as immunosuppressants.

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