TW202340240A - Multi-specific antibody and its pharmaceutical uses - Google Patents

Abstract

The invention discloses a multi-specific antibody and its pharmaceutical uses. Specifically, the multi-specific antibody of the invention comprises at least three domains: (A) target antigen binding domain, (B) half-life extension domain, and (C) T cell engaging domain. By binding to two or more targets at the same time, the multi-specific antibodies can perform multiple functions simultaneously to prevent and/or treat proliferative diseases.

Description

Multispecific antibodies and their pharmaceutical uses

The present invention relates to the field of biomedicine, specifically to multispecific antibodies and their pharmaceutical uses.

The concept of multispecific antibodies can be traced back to the 1960s, when Nisonoff and his team first proposed that antibody-based molecules can bind to different antigens at the same time. To date, more than a hundred types of multispecific antibody structures have been developed, with bispecific antibodies as the main research and development focus.

Since the pathological mechanisms of various diseases are very complex, monoclonal antibodies targeting one antigen alone have certain limitations and deficiencies in actual clinical treatment. Therefore, the research and development of multi-specific antibodies has become increasingly popular in recent years. By binding to two or more targets at the same time, multispecific antibodies can exert multiple functions at the same time to achieve effects that cannot be achieved by combining monoclonal antibodies.

The present invention discloses a multispecific antibody, nucleic acid fragments encoding the multispecific antibody, vectors, cells, compositions, preparation methods, pharmaceutical uses and treatment methods thereof.

In a first aspect, the present invention provides a multispecific antibody comprising at least three parts: (A) a targeting antigen-binding part, (B) a half-life extending part, and (C) a T cell engaging part ; (A) Targeting antigen-binding part: preferably a targeting antigen-binding antibody or a targeting antigen-binding ligand; the targeting antigen-binding antibody can be selected from any antigen-binding fragment; (B) Half-life extension part: better anti-HSA antibodies; (C) T cell engagement part: The T cell engagement part is preferably anti-CD3 antibody or antigen-binding fragment; The antigen-binding fragment is preferably Fd, Fv, scFv, diabody or single domain antibody (VHH); The segments of the three parts (A), (B), and (C) can be connected through connectors or directly connected without connectors.

In a specific embodiment, the multispecific antibody wherein (A) the targeting antigen-binding portion or (C) the T-cell engaging portion may be repeated or comprise multiple portions;

In some embodiments, the multispecific antibody contains two targeting antigen-binding portions, A1 and A2. A1 and A2 can be the same or different; in some embodiments, A1 and A2 bind different antigen targets; in In some embodiments, A1 and A2 bind to different epitopes of the same antigenic target.

In some embodiments, part (C) is a scFv that binds CD3; further in some embodiments, part (C) is a scFv that binds human CD3.

In a specific embodiment, the structural sequence from the N-terminus to the C-terminus of the multispecific antibody is: (1)A(VHH) - B(VHH) - C(VH) - C(VL) (2)A(VHH) - B(VHH) - C(VL) - C(VH) (3) A1(VL) - A1(VH) - A2(VHH) - B(VHH) - C(VH) - C(VL) (4) A1(VL) - A1(VH) - A2(VHH) - B(VHH) - C(VL) - C(VH) (5) A1(VHH) – A2(VL) – C(VH) – C(VL) – A2(VH) – B(VHH) (6) A1(VHH) – A2(VH) – C(VL) – C(VH) – A2(VL) – B(VHH) (7) B(VHH) – A2(VL) – C(VH) – C(VL) – A2(VH) – A1(VHH) (8) B(VHH) – A2(VH) – C(VL) – C(VH) – A2(VL) – A1(VHH).

In a specific embodiment, the T cell engaging portion of the multispecific antibody (C) comprises complementarity determining regions (CDRs) selected from the following antibodies: OKT3, TRX4, MGA031, Nuvion, SP34, X35, VIT3, BMA030, CLB -T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2 -8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, S004-2-03, S004-2-06, S004-2-08, S004-2 -10, S004-2-18, 6-35.22-hu, 1-22.6-1-hu, 7-35.6-hu, and 6-44.5-hu.

In some embodiments, the (C) T cell engaging moiety comprises heavy chain CDRs and/or light chain CDRs selected from: Heavy chain CDR1 is shown in SEQ ID NO. 34, 39, 42, and 47; Heavy chain CDR2 is shown in SEQ ID NO. 35, 40, 43, 45 and 48; Heavy chain CDR3 is shown in SEQ ID NO. 36, 37, 38, 41, 44, 46, 49; The light chain CDR1 is shown in SEQ ID NO. 50, 55, 58, 61, and 64; The light chain CDR2 is shown in SEQ ID NO. 51, 56, 59, 62, and 65; The light chain CDR3 is shown in SEQ ID NO. 52, 53, 54, 57, 60, 63, and 66.

In some embodiments, the (C) T cell engaging portion comprises 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% of the heavy chain CDRs and/or light chain CDRs described above. % identity sequence. In a specific embodiment, the targeting antigen in part (A) of the multispecific antibody can be selected from the following group: CD19, BCMA, HER2, EGFR, VEGF, MSLN, CD33, CD70, CD5, CD20, CD40, CD47, CD38, CD137, TNF-alpha, HER3, CD27, EphA2, EpCAM, MUC1, MUC17, CEA, Claudin18.2, folate receptor, Claudin6, WT1, NY-ESO-1, MAGE3, ASGPR1, CDH16, GPRC5D, DLL3, ROR1 or GUCY2C.

In some embodiments, the multispecific antibody contains two targeting antigen-binding portions, A1 and A2, respectively, and A1 and A2 respectively bind to different epitopes of MSLN; for example, the MLSN-R1 epitope or the MSLN-R3 epitope.

In some embodiments, the multispecific antibody simultaneously contains two targeting antigen-binding portions, A1 and A2, which bind to the targeting antigens CD70 and CD33, respectively.

In some embodiments, the targeting antigen binding portion comprises complementarity determining regions (CDRs) selected from the following fragments: SEQ ID NOs. 18-28, 103-106.

In some embodiments, the (A) targeting antigen binding moiety comprises heavy chain CDRs and/or light chain CDRs selected from: Heavy chain CDR1 is shown in SEQ ID NO. 76, 80, 89, 92, 110, 113, and 116; Heavy chain CDR2 is shown in SEQ ID NO. 77, 79, 81, 90, 93, 111, 114, and 117; Heavy chain CDR3 is shown in SEQ ID NO. 78, 82, 91, 94, 112, 115, 118; The light chain CDR1 is shown in SEQ ID NO. 83, 86, and 107; The light chain CDR2 is shown in SEQ ID NO. 84, 87, and 108; The light chain CDR3 is shown in SEQ ID NO. 85, 88, and 109.

In some embodiments, the (A) targeting antigen-binding portion comprises 99%, 98%, 97%, 96%, 95%, 90%, 85%, Sequences with 80% identity.

In a specific embodiment, part (B) of the multispecific antibody comprises complementarity determining regions (CDRs) selected from the following fragments: SEQ ID NO. 15-17.

In some embodiments, said part (B) comprises CDRs selected from: CDR1 is shown in SEQ ID NO. 67, 70, and 73; CDR2 is shown in SEQ ID NO. 68, 71, 74; and CDR3 is shown in SEQ ID NO. 69, 72, and 75;

In some embodiments, the part (B) includes sequences that are 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% identical to the CDRs described above.

In a specific embodiment, the multispecific antibody uses a linker to connect each fragment of the three parts (A), (B), and (C); the linker is each independently selected from: (GS)n, (GGS)n, (GGGS)n, (GGSG)n, (GGSGG)n, (GGGGS)n or (GGGGS)n (GGGS)n, where n is 1, 2, 3, 4, 5, 6, 7 ,8,9 or 10.

In some embodiments, the linker is GGGGS (Linker1), GGGGSGGGS (Linker2), GGGGSGGGGS (Linker3), GGGGSGGGGSGGGGS (Linker4), or GGGSGGGGSGGGGSGGGGS (Linker5).

In some embodiments, the structural sequence from the N-terminus to the C-terminus of the multispecific antibody is: (1)A(VHH)-linker2-B(VHH)-linker2-C(VH)-linker4-C(VL) (2)A(VHH)-linker2-B(VHH)-linker2-C(VL)-linker4-C(VH) (3) A1(VL)-linker4-A1(VH)-linker2-A2(VHH)-linker2-B(VHH)-linker2-C(VH)-linker4-C(VL) (4) A1(VL)-linker4-A1(VH)-linker2-A2(VHH)-linker2-B(VHH)-linker2-C(VL)-linker4-C(VH) (5) A1(VHH)-linker3-A2(VL)-linker1-C(VH)-linker5-C(VL)-linker1-A2(VH)-linker3-B(VHH) (6) A1(VHH)-linker3-A2(VH)-linker1-C(VL)-linker5-C(VH)-linker1-A2(VL)-linker3-B(VHH) (7) B(VHH)-linker3-A2(VL)-linker1-C(VH)-linker5-C(VL)-linker1-A2(VH)-linker3-A1(VHH) (8) B(VHH)-linker3-A2(VH)-linker1-C(VL)-linker5-C(VH)-linker1-A2(VL)-linker3-A1(VHH).

In a specific embodiment, the antibody or antigen-binding fragment used in the multispecific antibody is: (1) Chimeric antibodies or fragments thereof; (2) Humanized antibodies or fragments thereof; or, (3) Fully human antibodies or fragments thereof;

In some embodiments, the multispecific antibody comprises the sequence shown in SEQ ID NO. 97, 98, 99, 119, 120, 121, or 122, or is 99%, 98%, 97%, or 96% identical to the above sequence. %, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identical sequences.

In a second aspect, the present invention provides an isolated nucleic acid fragment encoding the multispecific antibody of the first aspect of the present invention.

In a third aspect, the present invention provides a vector, which contains the nucleic acid fragment described in the second aspect of the present invention.

In a fourth aspect, the present invention provides a host cell, the host cell comprising the vector according to the third aspect of the present invention; in some embodiments, the cell is a prokaryotic cell or a eukaryotic cell, such as a bacterium (E. coli ), fungi (yeast), insect cells or mammalian cells (CHO cell strain or 293T cell strain).

In a fourth aspect, the present invention provides a method for preparing the multispecific antibodies of the present invention, wherein the method includes culturing the cells of the fourth aspect, and isolating the multispecific antibodies expressed by the cells.

In a fifth aspect, the present invention also provides a pharmaceutical composition comprising the multispecific antibody described in the first aspect of the present invention, or the nucleic acid fragment described in the second aspect of the present invention, or the The carrier of the third aspect; or the product prepared by the method of the fourth aspect of the present invention; optionally, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, diluent or auxiliary agent ; Optionally, the pharmaceutical composition also contains additional anti-tumor agents.

In a sixth aspect, the present invention provides a method for preventing and/or treating proliferative diseases, tumor diseases, inflammatory diseases, immune diseases, autoimmune diseases, infectious diseases, viral diseases, allergies, parasitic reactions, transplants A method for versus host disease or host versus graft disease, comprising administering to a patient in need an effective amount of the multispecific antibody of the first aspect of the invention, or the nucleic acid fragment of the second aspect of the invention, Or the carrier described in the third aspect of the present invention, or the product prepared by the method described in the fourth aspect of the present invention, or the pharmaceutical composition described in the fifth aspect of the present invention;

In a specific embodiment, the neoplastic disease is a solid tumor expressing MSLN, CD70 and/or CD33 or a hematological tumor expressing MSLN, CD70 and/or CD33; in some embodiments, the neoplastic disease is mesothelioma , lung cancer, breast cancer, esophageal cancer, pancreatic cancer, ovarian cancer, pleural cancer, cholangiocarcinoma, cervical cancer, gastric cancer, leukemia, histiocytic lymphoma, or renal cancer; in some embodiments, the tumor disease is epithelial cancer malignant pleural mesothelioma, lung adenocarcinoma, triple-negative breast cancer, pancreatic cancer, ovarian cancer, cervical cancer, monocytic leukemia, histiocytic lymphoma, renal clear cell adenocarcinoma, T lymphocytic leukemia, or acute myeloid leukemia.

In a seventh aspect, the present invention provides a multispecific antibody as described in the first aspect, or a nucleic acid fragment as described in the second aspect of the present invention, or a vector as described in the third aspect of the present invention, or a fourth aspect of the present invention. The use of the product prepared by the method of the aspect or the pharmaceutical composition of the fifth aspect of the present invention in preparing drugs for preventing and/or treating diseases; the diseases include proliferative diseases, tumor diseases, inflammatory diseases, immune diseases Sexually transmitted diseases, autoimmune diseases, infectious diseases, viral diseases, allergies, parasitic reactions, graft-versus-host disease or host-versus-graft disease.

In a specific embodiment, the neoplastic disease is a solid tumor expressing MSLN, CD70 and/or CD33 or a hematological tumor expressing MSLN, CD70 and/or CD33; in some embodiments, the neoplastic disease is mesothelioma , lung cancer, breast cancer, esophageal cancer, pancreatic cancer, ovarian cancer, pleural cancer, cholangiocarcinoma, cervical cancer, gastric cancer, leukemia, histiocytic lymphoma, or renal cancer; in some embodiments, the tumor disease is epithelial cancer malignant pleural mesothelioma, lung adenocarcinoma, triple-negative breast cancer, pancreatic cancer, ovarian cancer, cervical cancer, monocytic leukemia, histiocytic lymphoma, renal clear cell adenocarcinoma, T lymphocytic leukemia, or acute myeloid leukemia.

Cross-references to related applications: This application claims the rights and priorities of the following two Chinese invention patent applications, and their entire contents are hereby incorporated into this article by reference: No. 202111361168.1 submitted to the State Intellectual Property Office of China on November 17, 2021 Patent application No. 202211362184.7 submitted to the State Intellectual Property Office of China on November 2, 2022.

Unless otherwise defined herein, all terms herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

Furthermore, unless otherwise indicated herein, singular terms herein shall include the plural form and plural terms shall include the singular form. More specifically, as used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

The terms "comprising," "comprising," and "having" are used interchangeably herein and are intended to indicate the inclusive nature of the scheme, meaning that there may be elements other than those listed. At the same time, it should be understood that the descriptions of "including", "including" and "having" are used in this article, and solutions of "consisting of" are also provided. For example, "a composition including A and B" should be understood as the following technical solution: a composition composed of A and B, as well as a composition containing other components in addition to A and B, all fall into Within the scope of the aforementioned "a composition".

The term "and/or" when used herein includes the meaning of "and", "or" and "all or any other combination of elements connected by the applicable term".

The term "specific binding" herein means that an antigen-binding molecule (eg, an antibody) specifically binds an antigen and substantially the same antigen, typically with high affinity, but does not bind with high affinity to an unrelated antigen. Affinity is usually reflected in terms of the equilibrium dissociation constant (KD), where a lower KD indicates higher affinity. Taking antibodies as an example, high affinity usually refers to having about 10 ^-7 M or lower, about 10 ^-8 M or lower, about 1×10 ^-9 M or lower, about 1×10 ^-10 M or lower, KD of 1×10 ^-11 M or lower or 1×10 ^-12 M or lower. KD is calculated as follows: KD = Kd/Ka, where Kd represents the dissociation rate and Ka represents the association rate. The equilibrium dissociation constant KD can be measured using methods commonly known in the art, such as surface plasmon resonance (eg Biacore) or equilibrium dialysis.

The term "antigen-binding molecule" is used herein in the broadest sense and refers to a molecule that specifically binds an antigen. Exemplary, antigen-binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimetic" refers to an organic compound or binding domain that can specifically bind to an antigen but has nothing to do with the structure of an antibody. For example, antibody mimetic includes but is not limited to affibody, affitin, affilin, and designed ankyrin repeat proteins. (DARPin), aptamer or Kunitz type domain peptide.

The term "antibody" is used herein in its broadest sense to refer to a polypeptide that contains sufficient sequence from the variable domain of an immunoglobulin heavy chain and/or sufficient sequence from the variable domain of an immunoglobulin light chain to be capable of specifically binding to an antigen. or peptide combinations. As used herein, "antibody" encompasses various forms and structures as long as they exhibit the desired antigen-binding activity. "Antibody" as used herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds, which contain mutations introduced to stabilize, for example, the three-dimensional structure of the antibody, as well as fully synthetic scaffolds, which contain, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Such scaffolds may also include non-antibody derived scaffolds, such as scaffold proteins known in the art to be useful for grafting CDRs, including but not limited to tenascin, fibronectin, peptide aptamers, and the like.

"Antibodies" in this article include a typical "quadruplex antibody", which is an immunoglobulin composed of two heavy chains (HC) and two light chains (LC); the heavy chain refers to such a polypeptide chain, which is in N It consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the direction from end to C end; and, when When the full-length antibody is of IgE isotype, it optionally also includes a heavy chain constant region CH4 domain; the light chain is composed of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction. ); heavy chains and heavy chains, heavy chains and light chains are connected through disulfide bonds to form a "Y"-shaped structure. Since the amino acid composition and arrangement order of the immunoglobulin heavy chain constant region are different, their antigenicity is also different. Based on this, "immunoglobulins" in this article can be divided into five categories, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE. Their corresponding heavy chains are μ chain, δ chain, γ chain, α chain and ε chain. The same type of Ig can be divided into different subclasses based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. Light chains are divided into kappa or lambda chains through differences in constant regions. Each of the five types of Ig can have either a kappa chain or a lambda chain.

"Antibody" herein also includes "antigen-binding fragments" or "antibody fragments." "Antigen-binding fragments" and "antibody fragments" are used interchangeably in this article. They do not have the entire structure of a complete antibody, but only include parts of a complete antibody. or localized variants that have the ability to bind to the antigen. Including but not limited to Fab, Fab’, Fab’-SH, F(ab’)2, Fd, Fv, scFv, diabody and single domain antibody.

Papain digestion of intact antibodies generates two identical antigen-binding fragments, termed "Fab" fragments, each containing the heavy and light chain variable domains, as well as the light chain constant domain and the heavy chain first constant domain (CH1). ). Thus, the term "Fab fragment" herein refers to a light chain fragment comprising the VL domain and constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine residues of the constant domain carry free thiol groups. Pepsin treatment produces an F(ab')2 fragment with two antigen binding site sites (two Fab fragments) and part of the Fc region.

The term "Fd" herein refers to an antibody consisting of VH and CH1 domains. The term "Fv" herein refers to an antibody fragment consisting of a single arm of VL and VH domains. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind the antigen, although its affinity may be lower than that of the intact binding site.

The term "scFv" (single-chain variable fragment) herein refers to a single polypeptide chain containing VL and VH domains, wherein the VL and VH are connected by a linker (see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers useful in the present invention are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bond may also exist between the VH and VL of scFv, forming a disulfide-linked Fv (dsFv).

The term "diabody" is used herein, in which the VH and VL domains are expressed on a single polypeptide chain, but a linker that is too short is used to allow pairing between the two domains of the same chain, thus forcing the domains to The complementary domains of the other chain pair up and create two antigen-binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123(1994)).

The terms "single domain antibody" (sdAb), "VHH" and "nanobody" have the same meaning and can be used interchangeably in this article. They refer to the variable region of the heavy chain of a replicating antibody, constructed of only one A single-domain antibody composed of a heavy chain variable region, which is the smallest fully functional antigen-binding fragment. Usually, after obtaining an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is copied to construct a single domain antibody consisting of only one heavy chain variable region. Single domain antibodies can be derived from camelid heavy chain antibodies or cartilaginous fish IgNARs.

"Antibody" herein also includes antibodies that do not contain a light chain, for example, those produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and alpaca ( Heavy-chain antibodies (HCAbs) produced by Vicugna pacos, etc., and immunoglobulin neoantigen receptors (IgNAR) discovered in sharks and other cartilaginous fishes.

The "antibody" herein can be derived from any animal, including but not limited to humans and non-human animals. The non-human animals can be selected from primates, mammals, rodents and vertebrates, such as camelids and llamas. , ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fish (e.g. shark).

"Antibodies" herein include, but are not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (such as bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, and naked antibodies , conjugated antibodies, chimeric antibodies, humanized antibodies or fully human antibodies.

The term "monoclonal antibody" herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., except for possible variants (e.g., containing naturally occurring mutations or arising during the manufacture of the preparation), such variants typically present in small amounts. ), the individual antibodies comprising the population are identical and/or bind the same epitope. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (etopes), each monoclonal antibody in a monoclonal antibody preparation is directed against a single determinant on the antigen. The modifier "single strain" herein should not be construed as requiring production of the antibody or antigen-binding molecule by any specific method. For example, monoclonal antibodies can be produced through a variety of techniques, including (but not limited to) fusionoma technology, recombinant DNA methods, phage library display technology, and the use of transgenic animals containing all or part of the human immunoglobulin locus. methods and other methods known in the art.

The term "natural antibody" herein refers to antibodies produced and paired by the immune system of a multicellular organism. The term "engineered antibody" in this article refers to non-natural antibodies obtained through genetic engineering, antibody engineering and other technologies. For example, "engineered antibodies" include humanized antibodies, small molecule antibodies (such as scFv, etc.), bis(antibodies), etc. Specific antibodies, etc.

The term "monospecific" herein refers to having one or more binding sites, where each binding site binds the same epitope of the same antigen.

The term "multispecific antibody" herein refers to an antibody having at least two antigen-binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. bit combination. Thus, terms such as "bispecific," "trispecific," "tetraspecific," etc. refer to the number of different epitopes to which the antibody/antigen-binding molecule can bind.

The term "valency" herein refers to the presence of a defined number of binding sites in the antibody/antigen binding molecule. Therefore, the terms "monovalent", "bivalent", "tetravalent" and "hexavalent" respectively represent one binding site, two binding sites, four binding sites and six binding sites in the antibody/antigen binding molecule. bit existence.

"Full-length antibody", "intact antibody" and "intact antibody" are used interchangeably herein and refer to a structure that is substantially similar to the structure of a natural antibody.

The term "naked antibody" herein refers to an antibody that is not conjugated to a therapeutic agent or tracer; the term "conjugated antibody" refers to an antibody that is conjugated to a therapeutic agent or tracer.

The term "chimeric antibody" herein refers to an antibody in which part of the light chain or/and heavy chain is derived from an antibody (which may be derived from a specific species or belong to a specific antibody class or subclass). class), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but in any case, it still retains the Binding activity of the target antigen (U.S.P 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 6855 (1984)). For example, the term "chimeric antibody" may include antibodies (e.g., human-mouse chimeric antibodies) in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody) and the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine antibody). The light chain constant region is derived from a secondary antibody (eg, a human antibody).

The term "humanized antibody" herein refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase sequence homology with that of a human antibody. Generally speaking, all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) comes from a human source. Immunoglobulins (receptor antibodies). Humanized antibodies usually retain or partially retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, the ability to increase immune cell activity, the ability to enhance immune response, etc.

The term "fully human antibody" herein refers to an antibody having variable regions in which both FRs and CDRs are derived from human germline immunoglobulin sequences. In addition, if the antibody contains a constant region, the constant region is also derived from human germline immunoglobulin sequences. The fully human antibodies herein may include amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, "fully human antibodies" herein do not include antibodies in which CDR sequences derived from the germline of another mammalian species (eg, mouse) have been grafted onto human framework sequences.

The term "variable region" herein refers to the region in the heavy or light chain of an antibody involved in allowing the antibody to bind to the antigen. "Heavy chain variable region" is used interchangeably with "VH" and "HCVR", and "light chain variable region" Can be used interchangeably with "VL" and "LCVR". The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, with each domain containing four conserved framework regions (FR) and three highly variable regions (HVR). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007). A single VH or VL domain may be sufficient to confer antigen binding specificity. The terms "complementarity determining region" and "CDR" are used interchangeably in this article, and usually refer to the highly variable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). This region is spatially structurally variable. It forms precise complementarity with the epitope, so it is also called a complementarity determining region. Among them, the CDR of the heavy chain variable region can be abbreviated as HCDR, and the CDR of the light chain variable region can be abbreviated as LCDR. The term "framework region" or "FR region" is used interchangeably and refers to those amino acid residues other than CDRs in the variable region of the heavy chain or light chain of an antibody. Usually, a typical antibody variable region consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

For further description of CDRs, refer to Kabat et al., J. Biol. Chem., 252:6609-6616 (1977); Kabat et al., U.S. Department of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol .Biol. 262:732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc M.P. et al., Dev. Comp.Immunol., 27: 55-77 (2003) ; and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001). "CDR" in this article can be annotated and defined by methods commonly known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system. The tool websites used include but are not limited to AbRSA website (http://cao.labshare .cn/AbRSA/cdrs.php), abYsis website (www.abysis.org/abysis/sequence_input/key_annotation/key_annotation.cgi) and IMGT website (http://www.imgt.org/3Dstructure-DB/cgi/DomainGapAlign .cgi#results). CDRs herein include overlaps and subsets of amino acid residues defined in different ways.

The term "Kabat numbering system" herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991).

The term "Chothia numbering system" herein generally refers to the immunoglobulin numbering system proposed by Chothia et al., which is a classic rule for identifying CDR region boundaries based on the position of structural loop regions (see, e.g., Chothia & Lesk (1987) J. Mol. Biol .196:901-917; Chothia et al. (1989) Nature 342:878-883).

The term "IMGT numbering system" herein generally refers to the numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., see Lefranc et al., Dev.Comparat.Immunol. 27:55-77, 2003.

The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of the antibody heavy chain that is not directly involved in the binding of the antibody to the antigen, but exhibits effector functions, such as interaction with Fc receptors, that are relative to the antibody's Variable domains have more conserved amino acid sequences. "Heavy chain constant region" at least includes: CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof. "Heavy chain constant region" includes "full-length heavy chain constant region" and "heavy chain constant region fragment", the former has a structure substantially similar to the natural antibody constant region, while the latter only includes "a portion of the full-length heavy chain constant region" . Illustratively, a typical "full-length antibody heavy chain constant region" consists of CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody , then it does not include the CH1 domain. Illustratively, a typical "heavy chain constant region fragment" can be selected from CH1, Fc or CH3 domains.

The term "light chain constant region" herein refers to the carboxyl-terminal part of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen. The light chain constant region can be selected from a constant kappa domain or a constant lambda domain.

The term "Fc" herein refers to the carboxyl-terminal portion of the antibody resulting from papain hydrolysis of the intact antibody, which typically includes the CH3 and CH2 domains of the antibody. Fc regions include, for example, native sequence Fc regions, recombinant Fc regions, and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxy terminus. The C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during the production or purification of the antibody, or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Thus, the Fc region can include Or excluding Lys447.

The term "conserved amino acids" herein generally refers to amino acids that belong to the same class or have similar characteristics (such as charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity). For example, the amino acids in each of the following groups belong to each other's conserved amino acid residues, and the substitution of amino acid residues within the group belongs to the substitution of conservative amino acids: Illustratively, the following six groups are examples of amino acids that are considered conservative substitutions for each other: (1) Alanine (A), serine (S), threonine (T); (2) Aspartic acid (D), glutamic acid (E); (3) Asparagine (N), glutamine (Q); (4) Arginine (R), lysine (K), histamine (H); (5) Isoleucine (I), leucine (L), methionine (M), valine (V); and (6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

The term "identity" herein may be calculated by aligning two amino acid sequences or two nucleic acid sequences for optimal comparison purposes in order to determine the percent "identity" of the sequences (e.g., may be Optimal alignment introduces gaps in one or both of the first and second amino acid sequences or nucleic acid sequences or non-homologous sequences may be discarded for comparison purposes). The amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. When a position in a first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

The percent identity between two sequences varies as a function of the identical positions shared by the sequences, taking into account the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap.

Mathematical algorithms can be used to perform sequence comparison and calculation of percent identity between two sequences. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm that has been integrated into the GAP program of the GCG software package (available at www.gcg.com), the Blossum 62 matrix or PAM250 matrix with gap weights 16, 14, 12, 10, 8, 6 or 4 and length weights 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences. As another example, using the GAP program in the GCG software package (available at www.gcg.com), use the NWSgapdna.CMP matrix with gap weights 40, 50, 60, 70 or 80 and length weights 1, 2, 3, 4, 5 or 6, determine the percent identity between two nucleotide sequences. Specifically in some embodiments the parameter set (and the one parameter set should be used unless otherwise stated) is a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

You can also use the PAM120 weighted remainder table, gap length penalty 12, gap penalty 4, and use the E. Meyers and W. Miller algorithm that has been incorporated into the ALIGN program (version 2.0) ((1989) CABIOS, 4:11-17 ) determines the percent identity between two amino acid sequences or nucleotide sequences.

Additionally or alternatively, the nucleic acid and protein sequences described herein may further be used as "query sequences" to perform searches against public databases, for example to identify other family member sequences or related sequences. For example, such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10. A BLAST nucleotide search can be performed using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed using the XBLAST program, score = 50, word length = 3, to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When using BLAST and gapped BLAST programs, you can use the default parameters of the corresponding programs (for example, XBLAST and NBLAST). See www.ncbi.nlm.nih.gov.

The term "CD3" (cluster of differentiation 3) herein refers to cluster of differentiation 3 proteins derived from any vertebrate source, including mammals, such as primates (e.g., humans, monkeys) and rodents (e.g., mice and rats). mouse). In mammals, the CD3 molecule is a multi-protein complex with six chains, including: CD3γ chain, CD3δ chain, two CD3ε chains and a homodimer of CD3ζ chain, where the CD3ζ chain is the intracellular tail of the CD3 molecule. And the CD3γ chain, CD3δ chain and CD3ε chain all contain extracellular domains (ECD) expressed on the surface of T cells. Exemplary sequences of human CD3 include human CD3ε protein (NCBI Ref Seq No. NP_000724 or NCBI: AAH49847.1), human CD3δ protein (NCBI Ref Seq No. NP_000723), and human CD3γ protein (NCBI Ref Seq No. NP_000064). Exemplary sequences of non-human CD3 include Macaca fascicularis (monkey) CD3 epsilon protein (NCBI Ref Seq No. NP_001270544), Macaca fascicularis (monkey) CD3 delta protein (NCBI Ref Seq No. NP_001274617), Macaca fascicularis (monkey) CD3γ protein (NCBI Ref Seq No.NP_001270839); mouse CD3ε protein (NCBI Ref Seq No.NP_031674), mouse CD3δ protein (NCBI Ref SeqNo.NP_038515), mouse CD3γ protein ( NCBI Ref Seq No. AAA37400); Rattus norvegicus (rat) CD3ε protein (NCBI Ref Seq No.NP_001101610), Rattus norvegicus (rat) CD3δ protein (NCBI Ref Seq No.NP_037301), Rattus norvegicus (rat )CD3γ protein (NCBI Ref Seq No. NP_001071114).

The term "CD3ε" herein is intended to include any form of CD3ε subunit, e.g., 1) native unprocessed CD3ε molecule, "full-length" CD3ε chain or naturally occurring CD3ε variants, including, for example, splice variants or allelic variants 2) Any form of CD3ε produced by processing in the cell; or 3) Full-length, fragmented (e.g., truncated forms, extracellular/transmembrane domain) or modified forms of the CD3ε subunit produced by recombinant methods (eg mutated forms, glycosylated/PEGylated, histidine-tagged/immunofluorescent fusion forms).

The term "HSA" herein refers to human serum albumin, with a molecular weight of 67 kD. There are 17 disulfide bonds inside the HSA protein, which makes the entire protein very stable. HSA has the advantages of extending the half-life and promoting the passage of drugs through the blood-brain barrier. It is not easy to pass through the glomerulus, and its half-life in the plasma is as long as 2 weeks. It is widely distributed in the body and has no immunogenicity, making it an ideal protein drug carrier.

The term "CD70" herein is also known as "TNFSF7" or "CD27L", which is a member of the TNF ligand family and is the ligand of CD27 (also known as TNFRSF27). “CD70” herein includes mature or immature full-length wild-type CD70 protein or its mutants (such as point mutations, insertion mutations or deletion mutations), splice variants, orthologs (Orthologs) and the aforementioned CD70 fragment. "CD70" herein can be derived from humans, primates, such as monkeys (such as rhesus monkeys, cynomolgus monkeys), and rodents, such as mice and rats. For example, the human CD70 amino acid sequence can be found in UniProt No.: P32970, and the rhesus monkey CD70 amino acid sequence can be found in UniProt No.: F7GPA5.

The term "CD33" herein is the smallest member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family. It is a type I transmembrane receptor protein with a molecular weight of 67 kDa and consists of 364 amino acids. Its N-terminus is located outside the cell, and the terminal amino acids constitute a conserved V-set immunoglobulin-like domain and a variable C2-set domain, in which V-set specifically recognizes and binds to sialic acid; There is an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an ITIM-like structure at the end of the cytoplasm, which transmit inhibitory signals into cells by binding to tyrosine phosphatase, thereby regulating cells. purpose of growth. The ITIM sequence in the CD33 molecule is different from other Siglecs in that the hydrophobic amino acids in front of tyrosine are replaced by leucine and threonine. Analysis of its primary structure shows that CD33 molecules are highly conserved in various organisms. The term "CD33" includes CD33 proteins of any human and non-human animal species, and specifically includes human CD33 as well as CD33 of non-human mammals.

The term "MSLN" herein refers to mesothelin (MSLN), which is a differentiation antigen present on normal mesothelial cells and can be expressed in mesothelial cells of the normal pleura, pericardium and peritoneum. The expression in normal tissues is limited, but MSLN has been found to be highly expressed on cells such as epithelioid malignant pleural mesothelioma, lung adenocarcinoma, breast cancer, esophageal cancer, pancreatic tumors, and ovarian cancer. The term "MSLN" includes MSLN proteins of any human and non-human animal species, and specifically includes human MSLN as well as MSLN of non-human mammals.

The term "nucleic acid" herein includes any compound and/or substance that contains a polymer of nucleotides. Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), Composed of sugar (i.e. deoxyribose or ribose) and phosphate groups. Typically, a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule. The sequence of bases is usually expressed as 5' to 3'. In this context, the term nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA), synthetic forms of DNA or RNA, as well as A polymer that is a mixture of two or more of these molecules. Nucleic acid molecules can be linear or circular. Furthermore, the term nucleic acid molecule includes both sense and antisense strands, as well as single-stranded and double-stranded forms. Furthermore, the nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides. Examples of non-naturally occurring nucleotides include modified nucleotide bases having derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for the direct expression of the antibodies of the invention in vitro and/or in vivo, for example in a host or patient. Such DNA (eg cDNA) or RNA (eg mRNA) vectors may be unmodified or modified. For example, mRNA can be chemically modified to enhance the stability of the RNA vector and/or the performance of the encoded molecule, so that the mRNA can be injected into a subject to produce antibodies in vivo (see, e.g., Stadler et al., Nature Medicine 2017, published online June 12, 2017, doi: 10.1038/nm.4356 or EP 2 101 823 B1). "Isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from components of its natural environment. Isolated nucleic acids include nucleic acid molecules contained in cells that normally contain the nucleic acid molecule but which are present extrachromosomally or at a chromosomal location that is different from its native chromosomal location.

The term "vector" herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vehicles are referred to herein as "expression vehicles".

The term "host cell" herein refers to a cell into which exogenous nucleic acid is introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include the original transformed cells and their progeny, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

The term "pharmaceutical composition" herein refers to a preparation that is in a form effective to permit the biological activity of the active ingredients contained therein and does not contain unacceptable toxicity to the subject administered the pharmaceutical composition of additional ingredients.

The term "treatment" as used herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) undesirable physiological changes or pathologies in the subject, such as cancer, autoimmune diseases and viral infections. progress. Beneficial or desirable clinical outcomes include, but are not limited to, alleviation of symptoms, less severe disease, stable disease status (i.e., no worsening), delay or slowing of disease progression, improvement or remission of disease status, and remission (whether partial response or complete response), whether detectable or undetectable. Those in need of treatment include those already suffering from the condition or disease as well as those susceptible to the condition or disease or those in whom the condition or disease is intended to be prevented. When referring to terms such as slow down, alleviation, weakening, alleviation, alleviation, their meanings also include elimination, disappearance, non-occurrence, etc.

The term "subject" herein refers to an organism undergoing treatment for a particular disease or condition as described herein. Examples of subjects and patients include mammals such as humans, primates (eg, monkeys), or non-primate mammals undergoing treatment for a disease or condition.

The term "effective amount" herein refers to an amount of a therapeutic agent that is effective to prevent or alleviate the symptoms of a disease or the progression of a disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject. "Effective amount" also refers to an amount of a compound sufficient to alleviate symptoms, such as to treat, cure, prevent, or alleviate a related medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such conditions. When the active ingredient is administered to an individual alone, the therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amount of active ingredients that produces a therapeutic effect, whether administered in combination, sequentially, or simultaneously.

The term "autoimmune disease" herein refers to a condition in which a subject develops an immune response to its own cells, tissues and/or organs and causes damage to cells, tissues and/or organs.

The term "cancer" herein refers to or describes a physiological condition in mammals that is typically characterized by unregulated cell growth. This definition includes both benign and malignant cancers. The term "tumor" or "tumor" herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" when used herein are not mutually exclusive.

The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. If the specific conditions are not specified in the examples, the conditions should be carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.

The embodiments of the present invention are only exemplary and do not limit the scope of the present invention in any way. Those skilled in the art should understand that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention.

Example 1 Construction and Preparation of Multispecific Antibodies 1.1 Structural Design of Multispecific Antibodies The multispecific antibodies of the present invention can be tri-specific (tri-specific) or tetra-specific (tetra-specific) antibodies. Specificity can refer to binding to different antigenic targets, or it can refer to binding to different epitopes of the same antigenic target. The schematic structure of a multispecific antibody is shown in Figures 1A-1B and Table 1. The multispecific antibody contains at least three parts: (A) targeting antigen-binding part, (B) half-life extending part, and (C) T cell engagement part. The (A) targeting antigen-binding part can select a targeting antigen-binding antibody or a targeting antigen-binding ligand. The targeting antigen-binding antibody can be selected from any antigen-binding fragment, preferably Fd, Fv, scFv, or diabody. (diabody) or single domain antibody (VHH). The half-life extending portion of (B) is a preferred anti-HSA antibody. Said (C) T cell engaging part, wherein the T cell engaging part is preferably an anti-CD3 antibody or antibody fragment. The three parts (A), (B) and (C) of the multispecific antibody can be arranged or connected in various ways, as shown in Figure 1A, where (A) targets the antigen-binding part or (C) T cells The conjugating part can appear repeatedly or contain multiple parts (for example, a multispecific antibody contains two targeting antigen-binding parts, A1 and A2, and A1 and A2 can be the same or different). The connection between the various parts of the multispecific antibody can be through a linker or directly without a linker. Table 1. Schematic structure of multispecific antibodies Format No. Multispecific antibody structure (from N-terminus to C-terminus, the connection between each part can be connected through or not through a linker) 1 A(VHH) - B(VHH) - C(VH) - C(VL) 2 A(VHH) - B(VHH) - C(VL) - C(VH) 3 A1(VL) - A1(VH) - A2(VHH) - B(VHH) - C(VH) - C(VL) 4 A1(VL) - A1(VH) - A2(VHH) - B(VHH) - C(VL) - C(VH) 5 A1(VHH) – A2(VL) – C(VH) – C(VL) – A2(VH) – B(VHH) 6 A1(VHH) – A2(VH) – C(VL) – C(VH) – A2(VL) – B(VHH) 7 B(VHH) – A2(VL) – C(VH) – C(VL) – A2(VH) – A1(VHH) 8 B(VHH) – A2(VH) – C(VL) – C(VH) – A2(VL) – A1(VHH)

1.2 Preparation of multispecific antibodies Construct multispecific antibodies according to the multispecific antibody structure described in 1.1, which uses (A) targeting antigen binding part, (B) half-life extending part, (C) T cell engaging part, and/or linker, which can be selected from the sequences shown in Table 2 below. The CDR regions of the antibody sequences shown in Table 2 can be divided by the numbering rules or definition methods common in the field such as IMGT, Kabat, Chothia, AbM, and Contact. Table 3 only The CDRs defined by Kabat method are shown as an example, but not as a limitation: Table 2. Antigen-binding fragment sequence information sequence name Serial number amino acid sequence CD3-VH1 SEQ ID NO.1 EVQLVESGGGLVQPGGSLKLSCAASGFTFN TYAMN WVRQAPGKGLEWVA RIRSKYNSYATYYADSVKD RFTISRDDSKNTAYLQMNNLKTEDTAVYYCVR HGNFGNAYISYWAY WGQGTLVTVSS CD3-VH2 SEQ ID NO.2 EVQLVESGGGLVQPGGSLKLSCAASGFTFN TYAMN WVRQAPGKGLEWVA RIRSKYNSYATYYADSVKD RFTISRDDDSKNTAYLQMNNLKTEDTAVYYCVR HSNFGNSYISYWAY WGQGTLVTVSS CD3-VH3 SEQ ID NO.3 EVQLVESGGGLVQPGGSLKLSCAASGFTFN TYAMN WVRQAPGKGLEWVA RIRSKYNSYATYYADSVKD RFTISRDDDSKNTAYLQMNNLKTEDTAVYYCVR HGNFGNSYISYWAH WGQGTLVTVSS CD3-VH4 SEQ ID NO.4 EVQLVESGGGVVQPGRSLRLSCAASGFTFT DYYMS WVRQAPGKGLEWVA MSRNKAKGHTIEYSSSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR DTYESYDGYFDV WGQGTTVTVSS CD3-VH5 SEQ ID NO.5 EVQLVQSGSELKKPGASVKVSCKTSGYTFT SYGMS WVRQAPGQGLEWMG WINTYSGVPTYAQDFKG RFVFSLDTSVSTAYLQISSLKAEDTAVYYCAR LKNNPFV WGQGTTVTVSS CD3-VH6 SEQ ID NO.6 EVQLVESGGGLVQPGGSLKLSCAASGFTFN TYAMN WVRQASGKGLEWVG RIRSKINNYATYYGDSVKD RFTISRDDSKNTFYLQMNSLKTEDTAVYYCVI HENYGSISWFAY WGQGTLVTVSS CD3-VH7 SEQ ID NO.7 EVQLVQSGAEVKKPGSSVKVSCKASGHTFS SHIMH WVRQAPGQGLEWMG YINPNNDRTEYSEKFKG RVTITADKSTSTAYMELSSLRSEDTAVYYCAG EAYYSNSLYAMDY WGQGTTVTVSS CD3-VL1 SEQ ID NO.8 QTVVTQEPSLTVSPGGTVTLTC SSSTGAVTSGNYPN WVQQKPGQAPRGLIG GTKFLSP GTPARFSGSLLGGKAALTLSGVQPEDEAEYYC VLWFSNRWV FGGGTKLTVL CD3-VL2 SEQ ID NO.9 QTVVTQEPSLTVSPGGTVTLTC SSSTGAVTSGNYPN WVQQKPGQAPRGLIG GTKFLSP GTPARFSGSLLGGKAALTLSGVQPEDEAEYYC SLWYSNRWV FGGGTKLTVL CD3-VL3 SEQ ID NO.10 QTVVTQEPSLTVSPGGTVTLTC SSSTGAVTSGNYPN WVQQKPGQAPRGLIG GTKFLSP GTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWTFGGGTKLTVL CD3-VL4 SEQ ID NO.11 DIVMTQSPDSLAVSLGERATINC KSSQSLFNSRSRKNYLA WYQQKPGQTPKLLIY WASIRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC KQSYYLYT FGQGTKLEIK CD3-VL5 SEQ ID NO.12 DIVMTQSPDSLAVSLGERATINC RSSQLILHSSGNTYLE WYQQKPGQSPKLLIY KVSNRFS GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC FQGSHFPWT FGGGTKVEIK CD3-VL6 SEQ ID NO.13 ETVVTQEPSLTVSPGGTVTLTC RSSTGAVTTSNYAN WVQQKPGQAFTGLIG GTNNRAP WTPARFSGSLLGGKAALTLSGVQPEDEAEYYC ALWYSNHWV FGGGTKLTVL CD3-VL7 SEQ ID NO.14 DIVMTQSPDSLAVSLGERATINC KSSQSLFNSRTRKNYLA WYQQKPGQSPKLLIY WASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCKQSYYLRTFGGGTKVEIK HSA-VHH1 SEQ ID NO.15 ELQLVESGGGLVQPGGSLRLSCAASGFAFS KFGMA WVRQAPGKGLEWVS RINSDGDTTMYADSAKG RFTISRDNAKNTLYLQMNSLRAEDTAVYYCSI GGGSP SGQGTMVTVSS HSA-VHH2 SEQ ID NO.16 EVQLVESGGGLVQPGGSLRLSCAASGFTFN YYAMS WFRQAPGKGLEWVS SVSMLGGGTTYADSVKG RFTVSRDNSKNTLYLEMNSLRAEDTAVYYCRQ GGRDALGSRP RGRGTLVTVSS HSA-VHH3 SEQ ID NO.17 EVQLVESGGGLVQPGGSLRLSCAASGVDFR LTTMS WVRQAPGKGLEWVS SISMLGGDTRYADSVKG RFTISRDNSKNTLYLQMNSLKFEDMAVYYCVQ GFGASNPRIKSP PGQGTMVTVSS MSLN-VH1 SEQ ID NO.18 EVQLVQSGAEVKKPGASVKVSCKASGYTFT DYNMD WVRQAPGQGLEWMG DINPSTGGTIYNQKFQG RVTMTEDKSTSTVYMELSSLRSEDTAVYYCAR RRIGTGYFDV WGQGTTVTVSS MSLN-VH2 SEQ ID NO.19 EVQLVQSGAEVKKPGASVKVSCKASGYTFT DYNMD WVRQAPGQGLEWMG DINPSTGGTIYNQKFNG RVTMTEDKSASTVYMELSSLRSEDTAVYYCAR RRIGTGYFD VWGQGTTVTVSS MSLN-VH3 SEQ ID NO.20 EVQLVQSGAEVKKPGASVKVSCKASGYTFT NYWMH WVRQAPGQGLEWMG NINPSSGDSYNERFMS RVTMTVDKSTSTVYMELSSLRSEDTAVYYCAR SGGLWLAF WGQGTLVTVSS MSLN-VH4 SEQ ID NO.21 EVQLVQSGAEVKKPGASVKVSCKASGYTFT NYWMH WVRQAPGQGLEWMG NINPSSGDSYYNERFMS RVTMTVDKSTSTAYMELSSLRSEDTAVYYCAR SGGLWLA FWGQGTLVTVSS MSLN-VL1 SEQ ID NO.22 EIVLTQSPDFQSVTPKEKVTITC SASSSVISSYLS WYQQKPDQSPKLLIY RTSNLAS GVPSRFSGSGSGTDFTLTINSLEAEDAATYYC HQWSSFPYT FGGGTKVEIK MSLN-VL2 SEQ ID NO.23 DIVMTQSPDSLAVSLGERATINC KSSQTLLNSVSQNNYLA WYQQKPGQSPTLLIY FASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQHYRTPYT FGGGTKVEIK MSLN-VL3 SEQ ID NO.24 DIVMTQSPDSLAVSLGERATINC KSSQTLLNSVSQNNYLA WYQQKPGQSPKLLIY FASTRES GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQHYRTPYT FGGGTKVEIK MSLN-VHH1 SEQ ID NO.25 EVQLVESGGGLVKPGGSLRLSCAASESGFS ANYMG WYRQAPGKERELVA TINRFGSTNYADSVKG RFTISRDNAKNSVYLQMNSLRAEDTAVYYCRI MRPGNWY WGQGTMVTVSS MSLN-VHH2 SEQ ID NO.26 EVQLVESGGGLVKPGGSLRLSCAASESGFS ANYMG WYRQAPGKERELVA TINRFGSTNYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCRI MRPGNWY WGQGTMVTVSS MSLN-VHH3 SEQ ID NO.27 QVQLVESGGGLVKPGGSLRLSCAASESGFS ANYMG WYRQAPGKERELVA TINRFGSTNYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTAVYYCRI MRPGNWY WGQGTMVTVSS MSLN-VHH4 SEQ ID NO.28 EVQLVESGGGVVQPGRSLRLSCAASGSIFS GNAVA WYRQAPGKEREAVA VITRDGDTYYADSVKG RFTISRDNSKNTAYLQMNSLRAEDTAVYYCNT ARGAAVDS WGQGTMVTVSS CD70-VL SEQ ID NO.103 DIVMTQTPLSLSVTPGQPASISC HASQGINSNIG WLLQKPGQSFQGLIY HGTNLED GVPDRFSGSGSGTDYTLKISRVEAEDVGVYYC VQYAQFPYT FGGGTKVEIK CD70-VH SEQ ID NO.104 EVQLVQSGAEVKKPGASVKVSCKASGYTFT DYNMN WVRQAPGQRLEWMG IINPYNAGTIYNQKFKG RVTITVDKSASTAYMELSSLRSEDTAVYYCAR QHYYAMDY WGQGTTVTVSS CD33-VHH1 SEQ ID NO.105 EVQLVESGGGLVQPGGSLRLSCAASGSIFT SQDMDYAMG WYRQAPGKQREWVA TITSDEDTNYVDSVKG RFTISRDNAKNSAYLQMNSLRAEDTAVYYCFA NVRSDRSSLYRNY WGQGTMVTVSS CD33-VHH2 SEQ ID NO.106 EVQLVESGGGLVQPGGSLRLSCAASGGTFV INAA WYRQAPGKQRELVA SISYGGSTNYADRVAG RFTISRDNAKNSVYLQMNSLRAEDTAVYYCRL DPGLALNNRPLYY WGQGTMVTVSS Linker1 SEQ ID NO.29 GGGGS Linker2 SEQ ID NO.30 GGGGSGGGS Linker3 SEQ ID NO.31 GGGGSGGGGS Linker4 SEQ ID NO.32 GGGGSGGGGSGGGGS Linker5 SEQ ID NO.33 GGGGSGGGGSGGGGSGGGGS Pairing of antibody heavy chain variable regions and light chain variable regions Antibody name sequence pairing Antibody name sequence pairing S004-2-03 CD3-VH1+ CD3-VL1 F7.44.20-H5 MSLN-VH1 + MSLN-VL1 S004-2-06 CD3-VH2+ CD3-VL2 F7.44.20-H9 MSLN-VH2 + MSLN-VL1 S004-2-08 CD3-VH2 + CD3-VL1 F2.39.3-H2 MSLN-VH3 + MSLN-VL2 S004-2-10 CD3-VH2 + CD3-VL3 F2.39.3-H5 MSLN-VH4 + MSLN-VL3 S004-2-18 CD3-VH3 + CD3-VL1 6-35.22-hu CD3-VH4 + CD3-VL4 1-22.6-1-hu CD3-VH5 + CD3-VL5 7-35.6-hu CD3-VH6 + CD3-VL6 6-44.5-hu CD3-VH7 + CD3-VL7 Table 3. CDR sequence information sequence name CDR1 CDR2 CDR3 CD3-VH1 TYAMN (SEQ ID NO.34) RIRSKYNSYATYYADSVKD (SEQ ID NO.35) HGNFGNAYISYWAY (SEQ ID NO.36) CD3-VH2 TYAMN (SEQ ID NO.34) RIRSKYNSYATYYADSVKD (SEQ ID NO. 35) HSNFGNSYISYWAY (SEQ ID NO.37) CD3-VH3 TYAMN (SEQ ID NO.34) RIRSKYNSYATYYADSVKD (SEQ ID NO. 35) HGNFGNSYISYWAH (SEQ ID NO.38) CD3-VH4 DYYMS (SEQ ID NO.39) MSRNKAKGHTIEYSSSVKG (SEQ ID NO.40) DTYESYDGYFDV (SEQ ID NO.41) CD3-VH5 SYGMS (SEQ ID NO.42) WINTYSGVPTYAQDFKG (SEQ ID NO.43) LKNNPFV (SEQ ID NO.44) CD3-VH6 TYAMN (SEQ ID NO.34) RIRSKINNYATYYGDSVKD (SEQ ID NO.45) HENYGSISWFAY (SEQ ID NO.46) CD3-VH7 SHIMH (SEQ ID NO.47) YINPNNDRTEYSEKFKG (SEQ ID NO.48) EAYYSNSLYAMDY (SEQ ID NO.49) CD3-VL1 SSSTGAVTSGNYPN (SEQ ID NO.50) GTKFLSP (SEQ ID NO.51) VLWFSNRWV (SEQ ID NO.52) CD3-VL2 SSSTGAVTSGNYPN (SEQ ID NO.50) GTKFLSP (SEQ ID NO.51) SLWYSNRWV (SEQ ID NO.53) CD3-VL3 SSSTGAVTSGNYPN (SEQ ID NO.50) GTKFLSP (SEQ ID NO.51) VLWYSNRWT (SEQ ID NO.54) CD3-VL4 KSSQSLFNSRSRKNYLA (SEQ ID NO.55) WASIRES (SEQ ID NO.56) KQSYYLYT (SEQ ID NO.57) CD3-VL5 RSSQLILHSSGNTYLE (SEQ ID NO.58) KVSNRFS (SEQ ID NO.59) FQGSHFPWT (SEQ ID NO.60) CD3-VL6 RSSTGAVTTSNYAN (SEQ ID NO.61) GTNNRAP (SEQ ID NO.62) ALWYSNHWV (SEQ ID NO.63) CD3-VL7 KSSQSLFNSRTKNYLA (SEQ ID NO.64) WASTRES (SEQ ID NO.65) KQSYYLRT (SEQ ID NO.66) HSA-VHH1 KFGMA (SEQ ID NO.67) RINSDGDTMYADSAKG (SEQ ID NO.68) GGGSP (SEQ ID NO.69) HSA-VHH2 YYAMS (SEQ ID NO.70) SVSMLGGGTTYADSVKG (SEQ ID NO.71) GGRDALGSRP (SEQ ID NO.72) HSA-VHH3 LTTMS (SEQ ID NO.73) SISMLGGDTRYADSVKG (SEQ ID NO.74) GFGASNPRIKSP (SEQ ID NO.75) MSLN-VH1 DYNMD (SEQ ID NO.76) DINPSTGGTIYNQKFQG (SEQ ID NO.77) RRIGTGYFDV (SEQ ID NO.78) MSLN-VH2 DYNMD (SEQ ID NO.76) DINPSTGGTIYNQKFNG (SEQ ID NO.79) RRIGTGYFDV (SEQ ID NO.78) MSLN-VH3 NYWMH (SEQ ID NO.80) NINPSSGDSYYNERFMS (SEQ ID NO.81) SGGLWLAF (SEQ ID NO.82) MSLN-VH4 NYWMH (SEQ ID NO.80) NINPSSGDSYYNERFMS (SEQ ID NO.81) SGGLWLAF (SEQ ID NO.82) MSLN-VL1 SASSSVISSYLS (SEQ ID NO.83) RTSNLAS (SEQ ID NO.84) HQWSSFPYT (SEQ ID NO.85) MSLN-VL2 KSSQTLLNSVSQNNYLA (SEQ ID NO.86) FASTRES (SEQ ID NO.87) QQHYRTPYT (SEQ ID NO.88) MSLN-VL3 KSSQTLLNSVSQNNYLA (SEQ ID NO.86) FASTRES (SEQ ID NO.87) QQHYRTPYT (SEQ ID NO.88) MSLN-VHH1 ANYMG (SEQ ID NO.89) TINRFGSTNYADSVKG (SEQ ID NO.90) MRPGNWY (SEQ ID NO.91) MSLN-VHH2 ANYMG (SEQ ID NO.89) TINRFGSTNYADSVKG (SEQ ID NO.90) MRPGNWY (SEQ ID NO.91) MSLN-VHH3 ANYMG (SEQ ID NO.89) TINRFGSTNYADSVKG (SEQ ID NO.90) MRPGNWY (SEQ ID NO.91) MSLN-VHH4 GNAVA (SEQ ID NO.92) VITRDGDTYYADSVKG (SEQ ID NO.93) ARGAAVDS (SEQ ID NO.94) CD70-VL HASQGINSNIG (SEQ ID NO.107) HGTNLED (SEQ ID NO.108) VQYAQFPYT (SEQ ID NO.109) CD70-VH DYNMN (SEQ ID NO.110) IINPYNAGTIYNQKFKG (SEQ ID NO.111) QHYYAMDY (SEQ ID NO.112) CD33-VHH1 SQDMDYAMG (SEQ ID NO.113) TITSDEDTNYVDSVKG (SEQ ID NO.114) NVRSDRSSLYRNY (SEQ ID NO.115) CD33-VHH2 INAA (SEQ ID NO.116) SISYGGSTNYADRVAG (SEQ ID NO.117) DPGLALNNRPLYY (SEQ ID NO.118) Recombinant plasmids were constructed using nucleotide sequences encoding multispecific antibody amino acid sequences (see Table 4). A schematic representation of the multispecific antibody structure is shown in Figure 1B. The plasmid construction and antibody expression and purification work were completed by Taizhou Baiying Biotechnology Co., Ltd. In the large-scale screening stage of multi-specific antibodies, in order to facilitate the expression and purification of small-volume antibodies and rapid detection, His tag (HHHHHH) was added to the C-terminus of multi-specific antibodies; after preliminary screening of candidate antibodies, the The antibody sequence of His tag optimizes the antibody expression and purification method. The multispecific antibody sequences used in animal experiments are shown in Table 4, and there is no His tag at the C terminus. Add the plasmid and transfection reagent (Thermofisher, Cat. No.: A29133) to OptiPRO SFM (Thermofisher, Cat. No.: 12309019), mix well, let stand for 5 minutes, and add to ExpiCHO-S™ cells (Manufacturer: Thermofisher, Cat. No.: A29127). Put in 5% CO ₂ , 120rpm, 37℃ shaker culture. On the second day of transfection, feed was added, and the shaker temperature was adjusted to 32°C to continue culturing. On the 9th day of transfection, the cell supernatant was collected. Centrifuge the cell expression supernatant sample at high speed to remove impurities, replace the buffer with PBS, and add imidazole to a final concentration of 5mM. Equilibrate the nickel column with PBS solution containing 5mM imidazole and rinse 2-5 times the column volume. Place the replaced supernatant sample onto the column for binding, and flush the column with a PBS solution containing 5mM imidazole until the A280 reading drops to the baseline. Then rinse the chromatography column with PBS+10mM imidazole to remove non-specifically bound impurity proteins, and collect the flow-through. Then use PBS solution containing 300mM imidazole to elute the target protein, and collect the elution peaks. The collected eluted products can be further purified by gel chromatography Superdex200 (GE) after concentration. The mobile phase is PBS to remove aggregates and impurity protein peaks, and collect the eluted peaks of the target product. The obtained protein was analyzed by electrophoresis, SEC-HPLC and then separated for use.

1.3 Preparation of control group antibodies The sequences of the positive control group antibody and negative control group antibody of Anti-MSLN come from SEQ ID NO: 101 and SEQ ID NO: 99 of patent US20180327508A1, and are named MB001 and MB060 respectively (see Table 4 for the sequences). All are trispecific antibodies, and the structural diagram is shown in Figure 1B. The antibodies in the positive control group and the antibodies in the negative control group were completed by Taizhou Baiying Biotechnology Co., Ltd. to complete the plasmid construction and antibody production and purification. For specific methods, see Example 1.2. Table 4. Multispecific antibody sequence information Multispecific antibody number Serial number amino acid sequence MB001 SEQ ID NO.95 Question VYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLHHHHHH MB060 SEQ ID NO.96 Question VYYCTIGGSLSVSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAINWVRQAPGKGLEWVARIRSKYNNYATYYADQVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHANFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCASSTGAVTSGNYPNWVQQKPG QAPRGLIGGTKFLVPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCTLWYSNRWVFGGGTKLTVLHHHHHH MB048 SEQ ID NO.97 EVQLVESGGGLVKPGGSLRLSCAASESGFSANYMGWYRQAPGKERELVATINRFGSTNYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCRIMRPGNWYWGQGTMVTVSSGGGGSGGGSELQLVESGGGLVQPGGSLRLSCAASGFAFSKFGMAWVRQAPGKGLEWVSRINSDGDTTMYADSAKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSIGGGSPSGQGTMVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNSYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNAYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCSSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLSPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWFSNRWVFGGGTKLTVL (MSLN-VHH1)-Linker2-(HSA-VHH1)-Linker2-(CD3-VH1)-Linker4-(CD3-VL1) MB065 SEQ ID NO.98 EIVLTQSPDFQSVTPKEKVTITCSASSSVISSYLSWYQQKPDQSPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHQWSSFPYTFGGGTKVEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWMGDINPSTGGTIYNQKFQGRVTMTEDKSTSTVYMELSSLRSEDTAVYYCARRRIGTGYFDVWGQGTTVTVSSGGGGSGGGSEVQLVESGGGLVKPGGSLRLSCAASESGFSANYMGWYRQAPGKERELVATINRFGSTNYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCRIMRPGNWYWGQGTMVTVSSGGGGSGGGSELQLVESGGGLVQPGGSLRLSCAASGFAFSKFGMAWVRQAPGKGLEWVSRINSDGDTTMYADSAKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSIGGGSPSGQGTMVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNSYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNAYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCSSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLSPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWFSNRWVFGGGTKLTVL (MSLN-VL1)-Linker4-(MSLN-VH1)-Linker2-(MSLN-VHH1)-Linker2-(HSA-VHH1)- Linker2-(CD3-VH1)- Linker4-(CD3-VL1) MB069 SEQ ID NO.99 EVQLVESGGGLVKPGGSLRLSCAASESGFSANYMGWYRQAPGKERELVATINRFGSTNYADSVKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCRIMRPGNWYWGQGTMVTVSSGGGGSGGGGSEIVLTQSPDFQSVTPKEKVTITCSASSSVISSYLSWYQQKPDQSPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHQWSSFPYTFGGGTKVEIKGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNSYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNAYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCSSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLSPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWFSNRWVFGGGTKLTVLGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMDWVRQAPGQGLEWMGDINPSTGGTIYNQKFQGRVTMTEDKSTSTVYMELSSLRSEDTAVYYCARRRIGTGYFDVWGQGTTVTVSSGGGGSGGGGSELQLVESGGGLVQPGGSLRLSCAASGFAFSKFGMAWVRQAPGKGLEWVSRINSDGDTTMYADSAKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSIGGGSPSGQGTMVTVSS (MSLN-VHH1)-Linker3-(MSLN-VL1)-Linker1-(CD3-VH1)-Linker5-(CD3-VL1) -Linker1-(MSLN-VH1) -Linker3-(HSA-VHH1) 1# (Same as BDD20-09-1#) SEQ ID NO.119 DIVMTQTPLSLSVTPGQPASISCHASQGINSNIGWLLQKPGQSFQGLIYHGTNLEDGVPDRFSGSGSGTDYTLKISRVEAEDVGVYYCVQYAQFPYTFGGGTKVEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMNWVRQAPGQRLEWMGIINPYNAGTIYNQKFKGRVTITVDKSASTAYMELSSLRSEDTAVYYCARQHYYAMDYWGQGTTVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSIFTSQDMDYAMGWYRQAPGKQREWVATITSDEDTNYVDSVKGRFTISRDNAKNSAYLQMNSLRAEDTAVYYCFANVRSDRSSLYRNYWGQGTMVTVSSGGGGSGGGSELQLVESGGGLVQPGGSLRLSCAASGFAFSKFGMAWVRQAPGKGLEWVSRINSDGDTTMYADSAKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSIGGGSPSGQGTMVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNSYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHSNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCSSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLSPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWFSNRWVFGGGTKLTVL (CD70-VL)-Linker4-(CD70-VH)-Linker2-(CD33-VHH1)-Linker2-(HSA-VHH1)- Linker2-(CD3-VH2)- Linker4-(CD3-VL1) 2# (Same as BDD20-09-2#) SEQ ID NO.120 DIVMTQTPLSLSVTPGQPASISCHASQGINSNIGWLLQKPGQSFQGLIYHGTNLEDGVPDRFSGSGSGTDYTLKISRVEAEDVGVYYCVQYAQFPYTFGGGTKVEIKGGGGSGGGGSGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMNWVRQAPGQRLEWMGIINPYNAGTIYNQKFKGRVTITVDKSASTAYMELSSLRSEDTAVYYCARQHYYAMDYWGQGTTVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGGTFVINAAWYRQAPGKQRELVASISYGGSTNYADRVAGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCRLDPGLALNNRPLYYWGQGTMVTVSSGGGGSGGGSELQLVESGGGLVQPGGSLRLSCAASGFAFSKFGMAWVRQAPGKGLEWVSRINSDGDTTMYADSAKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSIGGGSPSGQGTMVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNSYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHSNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCSSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLSPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWFSNRWVFGGGTKLTVL (CD70-VL)-Linker4-(CD70-VH)-Linker2-(CD33-VHH2)-Linker2-(HSA-VHH1)- Linker2-(CD3-VH2)- Linker4-(CD3-VL1) 9# (Same as BDD20-09-9#) SEQ ID NO.121 EVQLVESGGGLVQPGGSLRLSCAASGGTFVINAAWYRQAPGKQRELVASISYGGSTNYADRVAGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCRLDPGLALNNRPLYYWGQGTMVTVSSGGGGSGGGSELQLVESGGGLVQPGGSLRLSCAASGFAFSKFGMAWVRQAPGKGLEWVSRINSDGDTTMYADSAKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSIGGGSPSGQGTMVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNSYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHSNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCSSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLSPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWFSNRWVFGGGTKLTVL (CD33-VHH2)-Linker2-(HSA-VHH1)-Linker2-(CD3-VH2)-Linker4-(CD3-VL1) 15# (Same as BDD20-09-15#) SEQ ID NO.122 ELQLVESGGGLVQPGGSLRLSCAASGFAFSKFGMAWVRQAPGKGLEWVSRINSDGDTTMYADSAKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCSIGGGSPSGQGTMVTVSSGGGGSGGGGSDIVMTQTPLSLSVTPGQPASISCHASQGINSNIGWLLQKPGQSFQGLIYHGTNLEDGVPDRFSGSGSGTDYTLKISRVEAEDVGVYYCVQYAQFPYTFGGGTKVEIKGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNSYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHSNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCSSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLSPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWFSNRWVFGGGTKLTVLGGGGSEVQLVQSGAEVKKPGASVKVSCKASGYTFTDYNMNWVRQAPGQRLEWMGIINPYNAGTIYNQKFKGRVTITVDKSASTAYMELSSLRSEDTAVYYCARQHYYAMDYWGQGTTVTVSSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGGTFVINAAWYRQAPGKQRELVASISYGGSTNYADRVAGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCRLDPGLALNNRPLYYWGQGTMVTVSS (HSA-VHH1)-Linker3-(CD70-VL)-Linker1-(CD3-VH2)-Linker5-(CD3-VL1) -Linker1-(CD70-VH) -Linker3-(CD33-VHH2)

Example 2 Identification of the binding ability of Anti-MSLN multispecific antibodies to proteins or cells 2.1 Enzyme binding immunosorbent assay (ELISA) to detect the binding of antibodies to human serum albumin To detect the binding of Anti-MSLN multispecific antibodies to human serum albumin The binding activity of human serum albumin (HSA, purchased from Chengdu Rongsheng, product number: S10940024), monkey serum albumin (CSA, purchased from: Abcam, product number: ab184894), mouse serum albumin (MSA) , purchased from: Alpha Diagnist, Catalog No.: ALB14-N-10) was diluted with PBS to a final concentration of 4µg/mL, then added to a 96-well ELISA plate at 50µl/well, sealed with plastic film and incubated at 4°C overnight, the next day Wash the plate twice with PBST, add blocking solution [PBS + 2% (w/w) BSA] and block at room temperature for 2 hours. Pour off the blocking solution, wash the plate twice with PBST, and add 50 µl/well of the antibody to be tested or the control group antibody at a starting concentration of 100 nM or 200 nM and a 1:3 gradient dilution. After incubation at 37°C for 1 hour, the plate was washed three times with PBST. Add HRP (horseradish peroxidase)-labeled Anti-his secondary antibody (purchased from Genscript, Cat. No.: A00612), incubate at 37°C for half an hour, and wash the plate 5 times with PBST. Add 50µl/well of TMB substrate, incubate at room temperature for 10 to 15 minutes, then add 50µl/well of stop solution (1.0N HCl). Use an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer) to read the OD450nm value. The experimental results are shown in Figures 2A~2C and Table 5-7. Among them, MB060 is the negative control group; MB001 is the positive control group. The data in the table are OD450nm values. The results showed that all Anti-MSLN multispecific antibodies had binding activity to human serum albumin, and had cross-binding activity to monkey serum albumin and mouse serum albumin. Table 5 Binding of Anti-MSLN multispecific antibody to HSA Antibody concentration (nM) Antibody name MB001 MB048 MB065 MB069 MB060 2.00E+02 3.20 3.03 3.07 3.06 2.99 2.22E+01 3.02 2.86 2.89 2.99 2.82 7.41E+00 2.78 2.79 2.70 2.85 2.61 2.47E+00 2.21 2.20 2.01 2.33 1.76 8.23E-01 0.92 1.10 1.06 1.31 0.72 2.74E-01 0.27 0.30 0.33 0.51 0.21 9.14E-02 0.10 0.10 0.10 0.18 0.10 3.05E-02 0.07 0.06 0.06 0.09 0.07 1.02E-02 0.06 0.06 0.06 0.07 0.06 3.39E-03 0.06 0.06 0.06 0.07 0.07 0 0.07 0.06 0.06 0.06 0.06 Table 6 Binding of Anti-MSLN multispecific antibody to CSA Antibody concentration (nM) Antibody name MB001 MB048 MB065 MB069 MB060 2.00E+02 3.10 2.86 2.82 2.90 2.75 6.67E+01 2.95 2.88 2.76 2.86 2.79 2.22E+01 2.97 2.77 2.74 2.80 2.68 7.41E+00 2.67 2.68 2.55 2.75 2.35 2.47E+00 1.99 2.11 1.93 2.27 1.58 8.23E-01 0.87 1.08 0.96 1.30 0.61 2.74E-01 0.26 0.30 0.30 0.48 0.19 9.14E-02 0.10 0.11 0.11 0.17 0.09 3.05E-02 0.07 0.07 0.08 0.09 0.07 1.02E-02 0.07 0.07 0.07 0.07 0.07 3.39E-03 0.07 0.07 0.07 0.07 0.07 0 0.07 0.07 0.07 0.06 0.06 Table 7 Binding of Anti-MSLN multispecific antibody to MSA Antibody concentration (nM) Antibody name MB001 MB048 MB065 MB069 MB060 2.00E+02 2.90 2.81 2.70 2.85 2.73 6.67E+01 2.46 2.59 2.51 2.72 2.58 2.22E+01 1.80 2.46 2.16 2.57 1.68 7.41E+00 0.97 2.18 1.46 2.23 0.77 2.47E+00 0.29 1.35 0.63 1.51 0.25 8.23E-01 0.10 0.52 0.19 0.69 0.11 2.74E-01 0.07 0.14 0.08 0.21 0.08 9.14E-02 0.06 0.07 0.06 0.08 0.07 3.05E-02 0.06 0.05 0.05 0.06 0.06 1.02E-02 0.06 0.06 0.05 0.05 0.06 3.39E-03 0.06 0.06 0.05 0.05 0.06 0 0.07 0.06 0.06 0.06 0.06

2.2 Enzyme binding immunosorbent assay (ELISA) to detect the ability of multispecific antibodies to simultaneously bind MSLN protein and CD3 protein. Human MSLN outer membrane segment protein (296-580, GenBank: AAH09272.1, His-tag, self-expression) was washed with PBS. Dilute to a final concentration of 2µg/mL, then add 50µl/well to a 96-well ELISA plate, seal with plastic film and incubate at 4°C overnight. Wash the plate twice with PBST the next day, and add blocking solution [PBS +2% (w /w) BSA] blocked at room temperature for 2 hours. Pour off the blocking solution, wash the plate twice with PBST, and add 50 µl/well of the antibody to be tested or the control group antibody at a starting concentration of 100 nM or 200 nM and a 1:3 gradient dilution. After incubation at 37°C for 1 hour, the plate was washed three times with PBST. Dilute human CD3 protein (purchased from Sino Biological, Catalog No.: 10977-H03H) and monkey CD3 protein (purchased from: Acro, Catalog No.: CDE-C5254) to 2µg/mL, and add 50µl/well to the 96-well ELISA plate. After incubation at 37°C for 1 hour, the plate was washed three times with PBST. Add HRP (horseradish peroxidase)-labeled Anti-hFc secondary antibody (purchased from Merck, product number: AP113P), incubate at 37°C for half an hour, and wash the plate 5 times with PBST. Add 50µl/well of TMB substrate, incubate at room temperature for 10 to 15 minutes, then add 50µl/well of stop solution (1.0N HCl). Use an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer) to read the OD450nm value. The experimental results are shown in Figures 3A~3B and Table 8-9. Among them, MB060 is the negative control group; MB001 is the positive control group. The data in the table are OD450nm values. The results show that all Anti-MSLN multispecific antibodies have the ability to bind to MSLN protein and CD3 protein at the same time; and have cross-binding activity with monkey CD3 protein. Table 8 Binding of Anti-MSLN multispecific antibody to human CD3 protein Antibody concentration (nM) Antibody name MB001 MB048 MB065 MB069 MB060 2.00E+02 2.62 1.84 1.88 2.03 0.09 6.67E+01 2.30 1.69 1.74 1.87 0.08 2.22E+01 2.20 1.61 1.65 2.01 0.07 7.41E+00 2.29 1.47 1.56 1.77 0.07 2.47E+00 2.28 1.17 1.17 1.37 0.07 8.23E-01 1.60 0.82 0.76 0.82 0.07 2.74E-01 0.96 0.29 0.18 0.30 0.07 9.14E-02 0.45 0.11 0.09 0.11 0.07 3.05E-02 0.15 0.08 0.07 0.08 0.07 1.02E-02 0.09 0.07 0.08 0.08 0.07 3.39E-03 0.08 0.07 0.07 0.07 0.07 0 0.07 0.07 0.08 0.07 0.08 Table 9 Binding of Anti-MSLN multispecific antibody to monkey CD3 protein Antibody concentration (nM) Antibody name MB001 MB048 MB065 MB069 MB060 2.00E+02 2.85 1.94 1.96 2.11 0.07 6.67E+01 2.48 1.99 1.97 2.16 0.06 2.22E+01 2.25 1.80 2.02 2.21 0.06 7.41E+00 2.17 1.92 1.90 2.09 0.07 2.47E+00 2.26 1.64 1.72 1.90 0.06 8.23E-01 1.87 0.94 1.10 1.33 0.06 2.74E-01 1.18 0.37 0.31 0.43 0.06 9.14E-02 1.03 0.21 0.10 0.12 0.06 3.05E-02 0.14 0.07 0.07 0.07 0.07 1.02E-02 0.08 0.06 0.06 0.06 0.06 3.39E-03 0.07 0.07 0.06 0.06 0.06 0 0.06 0.06 0.06 0.06 0.06

2.3 In the flow cytometry experiment (FACS) to detect the binding of antibodies to tumor cells, the human ovarian cancer cell line OVCAR3 (purchased from ATCC, product number: HTB-161) with high expression in MSLN and the human cervical cancer cell line with high expression in MSLN were used. Hela and human pancreatic cancer cell line Hs766T (purchased from the Cell Resource Center of the Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences), human lung cancer cell line NCI-H292 with low MSLN expression (purchased from ATCC, Cat. No.: CRL-1848), and human MSLN that does not express Proteins of human skin cancer cell line A431. Expand the required cells in a T175 cell culture flask to the logarithmic growth phase, aspirate the medium, wash twice with PBS buffer, digest the cells with trypsin, then use complete medium to terminate digestion, and pipet the cells to a single cell suspension. . After counting the cells, centrifuge, wash the cell pellet twice with PBS, resuspend the cell pellet in FACS buffer (PBS + 2% fetal calf serum) to 2×10 ⁶ cells/mL, and add 50 µl per well to 96 wells. In the FACS reaction plate, add 50 µl/well of the antibody to be tested or the control group antibody (200 nM as the starting concentration, 3-fold or 5-fold gradient dilution), mix with the cell suspension, and incubate at 4°C for 1 hour. Centrifuge and wash 3 times with PBS buffer, add 50 µl iFluor 647-labeled Anti-His secondary antibody (purchased from Genscript, Cat. No.: A01802-100) per well, and incubate at 4°C for 1 hour. Centrifuge and wash three times with PBS buffer, resuspend in 100 µl PBS, and use FACS (FACS Canto ^TM , purchased from BD Company) to detect and analyze the results. Data analysis was performed through software (CellQuest) to obtain the mean fluorescence intensity (MFI) of the cells. Then use software (GraphPad Prism8) to analyze, perform data fitting, and calculate EC50. The analysis results are shown in Figures 4A to 4E and Table 10, in which MB060 is the negative control group; MB001 is the positive control group. The results show that the Anti-MSLN multispecific antibody has binding activity to tumor cells with different expression levels, and has no binding activity to A431 cells that do not express human MSLN protein, indicating that the Anti-MSLN multispecific antibody can specifically bind to express human MSLN protein. MSLN protein in tumor cells. Table 10 Binding reaction of Anti-MSLN multispecific antibody and tumor cells Antibody name MSLN performance Detection value MB001 MB048 MB065 MB069 MB060 OVCAR3 +++ Max_MFI 2322 1715 2109 1503 97 EC50 (nM) 4.76 2.67 1.26 1.39 Not combined Hela ++ Max_MFI 1224 929 1131 1069 109 EC50 (nM) 0.14 0.09 0.16 0.14 Not combined Hs766T ++ Max_MFI 771 534 749 638 97 EC50 (nM) 0.03 0.03 0.12 0.11 Not combined NCI-H292 + Max_MFI 211 189 201 196 112 EC50 (nM) 1 0.27 0.22 0.21 Not combined A431 - Max_MFI 86 83 86 86 87 EC50 (nM) Not combined Not combined Not combined Not combined Not combined

2.4 Flow cytometry experiment (FACS) to detect the binding of antibodies to HEK293T-hMSLN-R3. For the preparation method of the recombinant HEK293T cell line HEK293T-hMSLN-R3 that expresses human MSLN-R3 protein, please refer to PCT/CN2021/136419 (encoding human MSLN-R3 The nucleotide sequence of the amino acid sequence (NCBI: Met487-Ser606 of AAH09272.1) was copied into the pcDNA3.1 vector and plastids were prepared. HEK293T cell line (purchased from ATCC) was subjected to plastid transfection (Lipofectamine® 3000 Transfection Kit (purchased from Invitrogen, Cat. No.: L3000-015), use puromycin and selective culture for 2 weeks, sort positive individual cells into a 96-well plate and select some individual wells for amplification. After amplification The replicas were detected and analyzed by FACS flow cytometry using specific antibodies. Cell lines with better growth trends and higher fluorescence intensity were selected to continue to be expanded and cultured and frozen in liquid nitrogen. The test showed that after puromycin pressure screening HEK293T-hMSLN-R3 has a relatively single positive peak). For the preparation of detection cells and antibodies to be tested and the detection method, please refer to Example 2.3. The analysis results are shown in Figure 5 and Table 11. The results show that MB065 and MB069 have binding activity to recombinant cells expressing human MSLN-R3 protein, indicating that Anti-MSLN monoclonal antibodies retain the characteristics of monoclonal antibodies after being combined into multispecific antibodies; while MB001 does not bind to recombinant cells expressing human MSLN-R3. Recombinant cell binding of proteins. Table 11 Binding reaction of Anti-MSLN multispecific antibody and HEK293T-hMSLN-R3 Antibody name HEK293T-hMSLN-R3 Max_MFI EC50 (nM) MB065 9674 3.67 MB069 14800 4.22 MB001 519 Not combined

2.5 Flow cytometry experiment (FACS) to detect the cross-binding of antibodies and HEK293T-monkey MSLN. Recombinant HEK293T cell line HEK293T-monkey MSLN expressing monkey MSLN protein. For the preparation method, please refer to PCT/CN2021/136419 (encoding the full-length amino acid sequence of monkey MSLN The nucleotide sequence of (NCBI: : L3000-015), use puromycin and selective culture for 2 weeks, sort the positive individual cells into a 96-well plate and select some of the individual wells for amplification. The amplified replicas are treated with specific antibodies. FACS flow cytometry detection and analysis, select cell lines with better growth trends and higher fluorescence intensity to continue to expand culture and freeze in liquid nitrogen. The test shows that HEK293T-monkey MSLN after puromycin pressure screening has a relatively single positive peak). For the preparation of detection cells and antibodies to be tested and the detection method, please refer to Example 2.3. The analysis results are shown in Figures 6A~6B and Table 12, in which MB060 is the negative control group. The results showed that all Anti-MSLN multispecific antibodies had specific binding activity to recombinant cells expressing monkey MSLN protein. Table 12 Binding reaction of Anti-MSLN multispecific antibody and HEK293T-monkey MSLN Antibody name HEK293T-Monkey MSLN HEK293T Max_MFI EC50 (nM) Max_MFI EC50 (nM) MB001 4803 1.16 650 Not combined MB048 4735 1.74 557 Not combined MB065 6322 1.66 516 Not combined MB069 3760 1.17 489 Not combined MB060 168 Not combined 545 Not combined

2.6 Flow cytometry experiment (FACS) to detect the binding of antibodies to human T cells expressing CD3 protein. Human PBMC cells (purchased from Aucells Biotechnology (Shanghai) Co., Ltd.) were tested according to T Cell Activation/Expansion Kit, human (purchased from Complete T cell isolation and in vitro activation and expansion experiments as described in the instructions of Miltenyi (Cat. No. 130-091-441). When the cells were cultured for 14 days, the culture supernatant was discarded by centrifugation, and the cell pellet was washed twice with PBS. SP34 antibody was used as the primary antibody and Alexa647-labeled secondary antibody (purchased from Jackson Immuno, Cat. No.: 109-605-098) was used for FACS (FACS CantoTM, purchased from BD Company) detection. The results showed that human T cells had a relatively high amplification rate. High CD3 protein expression. For the preparation of the above detection cells and antibodies to be tested and the detection method, please refer to Example 2.3. The analysis results are shown in Figures 7A~7B and Table 13. The results showed that all Anti-MSLN multispecific antibodies had binding activity to human T cells expressing human CD3 protein, and had cross-binding activity to monkey T cells expressing monkey CD3 protein. Table 13 Binding reaction of Anti-MSLN multispecific antibody to cells expressing CD3 protein Antibody name MB001 MB048 MB065 MB069 MB060 human T cells Max_MFI 10273 3002 1442 1639 9415 EC50 (nM) 8.54 31.85 97.52 88.89 17.94 monkey T cells Max_MFI 8867 2079 747 650 8578 EC50 (nM) 19.96 93.74 265 64.69 20.04

Example 3 Anti-MSLN multispecific antibody reporter gene experiment In order to detect whether the Anti-MSLN multispecific antibody can activate the NFAT signaling pathway in T cells, an experimental method for the reporter gene was specially established. Multispecific antibodies can simultaneously bind to Jurkat-NFAT-Luciferase-CD16a cells (purchased from: Promega, CD3 positive) and tumor cells expressing MSLN (OVCAR3, Hela, Hs766T, NCI-H292), thereby causing Jurkat-NFAT-Luciferase- The NFAT-related signaling pathway is activated in CD16a cells, and the expression level of luciferase increases. The fluorescent signal can be detected after adding substrate. The intensity of the fluorescent signal can indicate the intensity of activation of the signaling pathway. For the preparation process of tumor cells, refer to Example 2.3. Adjust the cell density to 4×10 ⁵ cells/ml. Add 100 µl per well into a 96-well cell culture plate and place it in a cell culture incubator for overnight culture. Discard the cell culture medium and wash it twice with PBS. Add 50 µl/well of the antibody to be tested or the control group antibody (60 nM as the starting concentration, 5-fold gradient dilution, 10~12 dilution points), and then add 50 µl/well of Jurkat-NFAT-Luciferase-CD16a cells (density: 1×10 ⁶ cells/ml) were mixed evenly and placed in a cell culture incubator for 6 hours. After taking it out and letting it stand at room temperature, add 50µl/well of Bright-Glo™ Luciferase Assay System (purchased from Promega, product number: E2620), shake and react in the dark for 5 minutes, then transfer the suspension to the detection plate (purchased from Costar, product number: 3610), detected with EnSight (purchased from: Perkin Elmer), and then analyzed through software (GraphPad Prism8), performed data fitting, and calculated EC50. The analysis results are shown in Figures 8A~8D and Tables 14-17, in which MB060 is the negative control group and MB001 is the positive control group. The results show that all Anti-MSLN multispecific antibodies can activate the NFAT-related signaling pathway in Jurkat-NFAT-Luciferase-CD16a cells. Table 14 Anti-MSLN multispecific antibody reporter gene experiment (OVCAR3) Antibody concentration (nM) Antibody name MB001 MB048 MB065 MB069 MB060 3.00E+01 2669780 2978410 3031250 2928470 467360 6.00E+00 2859040 3030520 3088000 2903260 449640 1.20E+00 2817120 3040160 3188030 2880810 219530 2.40E-01 2846900 2830030 3100560 2857610 36710 4.80E-02 2507910 2017310 2399640 2202880 26660 9.60E-03 1827120 1077920 979840 1310840 25220 1.92E-03 1099460 531840 220610 582660 24690 3.84E-04 662990 435600 74030 299550 24540 7.68E-05 363140 255920 62160 145620 24660 1.54E-05 208200 178430 39010 119200 25160 Table 15 Anti-MSLN multispecific antibody reporter gene experiment (Hela) Antibody concentration (nM) Antibody name MB001 MB048 MB065 MB069 MB060 3.00E+01 1624850 1371380 1457630 1445790 412000 6.00E+00 1560890 1513810 1474420 1462710 437920 1.20E+00 1563880 1477800 1533790 1563470 325430 2.40E-01 1603960 1454290 1539750 1531740 93810 4.80E-02 1566340 1347460 1410610 1434700 30510 9.60E-03 1452180 1042220 892750 1173970 22740 1.92E-03 1285340 755810 375140 789530 22200 3.84E-04 1133150 585890 275830 637690 21940 7.68E-05 848650 389790 97380 387040 23190 1.54E-05 643670 318190 50600 292920 20890 3.07E-06 528710 236070 38100 205550 22480 6.14E-07 461280 227070 35280 190000 21700 Table 16 Anti-MSLN multispecific antibody reporter gene experiment (Hs766T) Antibody concentration (nM) Antibody name MB001 MB048 MB065 MB069 MB060 3.00E+01 1136130 683640 736940 723940 386320 6.00E+00 1114530 785650 745140 752060 407940 1.20E+00 1127870 819140 775390 803680 277220 2.40E-01 1127160 806510 793190 813700 81930 4.80E-02 1151880 724150 729440 784540 27860 9.60E-03 1132410 567440 536400 698180 21360 1.92E-03 1040140 406470 247870 476560 21720 3.84E-04 922670 329930 182260 387210 21730 7.68E-05 665330 205550 68120 203440 20930 1.54E-05 515340 168390 42830 148290 20920 3.07E-06 423660 138360 35050 121850 19580 6.14E-07 357560 115560 27850 102790 22120 Table 17 Anti-MSLN multispecific antibody reporter gene experiment (NCI-H292) Antibody concentration (nM) Antibody name MB001 MB048 MB065 MB069 MB060 3.00E+01 650420 391250 430310 397540 241360 6.00E+00 761410 499590 495150 484630 239490 1.20E+00 825780 518760 499220 518500 124810 2.40E-01 834800 482590 515440 537080 28000 4.80E-02 775860 375410 421330 516290 19830 9.60E-03 570130 200470 186370 308240 19240 1.92E-03 340460 95230 46000 151120 19270 3.84E-04 225070 75680 26770 90840 17810 7.68E-05 131330 46280 24740 47240 19710 1.54E-05 78220 34700 21220 35820 18030

Example 4 Anti-MSLN multispecific antibody blocks the binding of CA125. CA125 has been confirmed to bind to MSLN, and the binding position is in the R1 region at the far membrane end. To confirm that antibodies targeting the R1 epitope can block the binding of CA125 to MSLN protein, we evaluated it at both the protein and cellular levels. The main experimental procedures of ELISA and FACS can be referred to Examples 2.1 and 2.3 respectively. Preparation instructions for primary antibodies for ELISA and FACS: Add 50µl/well of the test antibody or control group antibody (400nM as the starting concentration, 3-fold gradient dilution, 11 dilution points), incubate at room temperature for 30 minutes, then add 50µl/well 0.4µg/ml CA125 solution (purchased from Baiying, Cat. No.: B475601), mix evenly and continue to incubate at room temperature for 1 hour. The ELISA secondary antibody is wasabi peroxidase-coupled Streptavidin (purchased from Sigma, Catalog No.: S2438), and the FACS secondary antibody is APC-coupled Streptavidin (purchased from Biolegend, Catalog No.: 405243). The analysis results are shown in Figures 9 to 10 and Tables 18 to 19, in which MB060 is the negative control group. The results showed that Anti-MSLN multispecific antibody could block the binding of CA125 to human MSLN protein at both the protein and OVCAR3 cellular levels. Table 18 ELISA detection of anti-MSLN multispecific antibody blocking CA125 binding Antibody concentration (nM) Antibody name MB001 MB048 MB065 MB069 MB060 2.00E+02 0.05 0.05 0.05 0.05 1.81 6.67E+01 0.96 0.98 0.96 1.54 1.73 2.22E+01 1.82 1.74 1.78 1.86 1.76 7.41E+00 1.82 1.77 1.80 1.89 1.81 2.47E+00 1.79 1.79 1.79 1.94 1.81 8.23E-01 1.79 1.80 1.80 1.90 1.81 2.74E-01 1.76 1.79 1.79 1.92 1.84 9.14E-02 1.72 1.78 1.78 1.88 1.83 3.05E-02 1.76 1.73 1.77 1.83 1.77 1.02E-02 1.72 1.68 1.72 1.81 1.70 3.39E-03 1.73 1.65 1.63 1.70 1.63 0 1.77 1.70 1.67 1.75 1.69 Table 19 FACS detection of anti-MSLN multispecific antibody blocking CA125 binding Antibody concentration (nM) Antibody name MB001 MB048 MB065 MB069 MB060 2.00E+02 96 107 121 98 467 6.67E+01 106 116 99 109 494 2.22E+01 104 113 99 115 477 7.41E+00 139 110 125 110 431 2.47E+00 186 151 159 141 462 8.23E-01 275 238 264 262 465 2.74E-01 372 383 431 340 465 9.14E-02 449 457 501 428 464 3.05E-02 457 439 457 432 489 1.02E-02 502 458 540 465 462 3.39E-03 506 461 449 457 436 1.13E-03 476 452 485 465 472

Example 5 Binding status of Anti-MSLN bi-topic multi-specific antibody In order to verify whether the two MSLN-binding fragments of the bi-topic multi-specific antibody simultaneously bind to OVCAR3 tumor cells expressing human MSLN protein, the multi-specific antibody and cells were A high concentration of a monoclonal antibody against the R1 epitope (NB149-95) or a monoclonal antibody against the R3 epitope (F7.44.20) is added to the combined system in order to detect blocking or weakening one of the multispecific antibodies. After the antibody at the epitope binds, whether the antibody at the other epitope has binding activity. NB149-95, SEQ ID NO.100 QVQLVESGGGLVQAGGSLRLSCTASESGFSANYMGWYRQEAPGKERELVATINRFGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKSEDTGVYYCRIMRPGNWYWGQGTQVTVSS F7.44.20-VH, SEQ ID NO.101 EVQLQQSGPELVKPGASVKIPCKASG YTFTDYNMDWVKQSHGKSLEWIGDINPSTGGTIYNQKFNGKATLTEDKSASTVYMEFRSLTSDDTAVYYCARRRIGTGYFDVWGTGTTVTVSS F7.44.20-VL, SEQ ID NO.102 QIVLTQSPALMSASPGEKVTLTCSASSSVISSYLSWYQQKPGSSPKLLIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFC HQWSSFPYTFGGGTKLEIK The main experimental process can be referred to Example 2.3. Primary antibody preparation instructions: Add 50 µl/well of the antibody to be tested or the control group antibody (200 nM as the starting concentration, 5-fold gradient dilution, 8 dilution points), then add 50 µl/well of the 500 nM monoclonal antibody solution, and mix evenly . The analysis results are shown in Figures 11A to 11D and Table 20. The results show that the binding activity of MB001 and MB048 decreases in the presence of high concentrations of NB149-95 monoclonal antibodies, while the binding activity of MB065 (MSLN R1/R3) and MB069 (MSLN R1/R3) has no binding activity in the presence of monoclonal antibodies with different epitopes. Significantly reduced, demonstrating that antibodies targeting two determinants (MSLN R1/R3) can bind to MSLN membrane proteins simultaneously. Table 20 FACS detection of binding status of Anti-MSLN dual epitope multispecific antibody Antibody concentration (nM) Competitive antibody concentration (250nM) 100 20 4 0.8 0.16 0.032 0.0064 0.00128 MB001 / 3292 2764 2086 1061 517 339 281 220 NB149-95 1397 927 501 277 182 183 160 163 F7.44.20 3464 3119 2280 1039 483 289 227 211 MB048 / 2325 2158 1651 862 447 307 337 272 NB149-95 1849 1471 901 455 284 214 191 181 F7.44.20 2358 2327 1855 955 520 319 296 240 MB065 / 2850 2728 2646 1305 511 256 203 177 NB149-95 2765 2679 2232 1217 474 260 179 191 F7.44.20 2683 2623 2245 1055 423 256 184 155 MB069 / 2047 1867 1587 1028 488 318 240 228 NB149-95 1892 1628 1384 813 376 231 186 202 F7.44.20 2225 2063 1812 1060 508 301 230 232

Example 6 Endophytic activity of Anti-MSLN multispecific antibody OVCAR3 cell preparation and detection methods refer to Example 2.3. Instructions for primary antibody preparation and endophagy conditions: Add 50µl/well of the antibody to be tested or the control group antibody (200nM as the starting concentration, 10-fold gradient dilution, 3 dilution points), mix evenly and incubate at 4°C for 1 hour in PBS After washing 3 times, discard the supernatant. Add 50 µl/well of FACS buffer, resuspend the cells, place one part at 4°C and incubate for 1 hour, and the other part at 37°C for 1 hour. Centrifuge and discard the supernatant, then add diluted secondary antibody. The analysis results are shown in Figures 12A~12C and Tables 21-22. The results showed that both MB065 and MB069 had endophagy activity, and both were weaker than the endophagy activity of the positive control group antibody MB001, among which MB065 had the weakest endophagy activity. If CD3 multispecific antibodies bind to tumor cells and most of the antibodies are rapidly internalized, the probability of binding to T cells will be reduced, resulting in greatly weakened T cell activation and tumor killing activity. Table 21 FACS detection of endophagy of Anti-MSLN multispecific antibody Antibody name Incubation temperature Antibody concentration (nM) MB001 MB048 MB065 MB069 MB060 4℃ 100 3836 2900 3483 2501 174 10 3183 2655 3663 2294 204 1 1683 1318 2005 1517 195 37℃ 100 2061 1713 3141 1507 217 10 1419 1287 2951 1257 163 1 666 591 1458 836 160 Table 22 FACS detection of endophagy rate of Anti-MSLN multispecific antibody The endophagy rate of antibodies placed at 37°C for 1 hour Antibody concentration (nM) MB001 MB048 MB065 MB069 100 46% 41% 10% 40% 10 55% 52% 19% 45% 1 60% 55% 27% 45% Endophage rate = (4℃_MFI-37℃_MFI)/ 4℃_MFI*100%

Example 7 Detection of T lymphocyte activation status in vitro mediated by multispecific antibodies CD69 molecule is an early marker of T cell activation. T cells cultured in a resting state rarely express CD69 molecules. Once T lymphocytes are activated, CD69 The expression is significantly up-regulated, which is a marker for detecting whether T lymphocytes are effectively induced and activated. Use a T cell separation kit (purchased from STEMCELL technologies, product number 17951, please refer to the kit instructions for usage) to isolate and enrich primary T cells from PBMC of healthy donors, and culture them overnight. Digest NCI-H292 cells with trypsin to prepare single cell suspension. Use complete culture medium to adjust the tumor cell density to 2×10 ⁵ cells/ml and the T cell density to 1×10 ⁶ cells/ml. Take 50 μl of adjusted NCI-H292 cells and 50 μl of primary T cells in equal volumes, mix them evenly, and add them to each well of a flat-bottomed 96-well plate using a micropipette. The volume of the cell mixture is 100 μl/well (tumor per well). The numbers of cells and T cells were 1×10 ⁴ and 5×10 ⁴ respectively). The antibodies were serially diluted in complete culture medium (the initial concentration of the antibody was 2 nM, 6-fold dilution, 8 concentration gradients), and 100 μl of antibody dilutions of different concentrations were added to each well to make the final volume of each well 200 μl. Incubate in vitro for 24 hours. Centrifuge to remove the supernatant, resuspend in flow buffer, add APC-anti-human CD3 (purchased from Biolegend, Catalog No. 300312), FITC-anti-human CD69 (purchased from Biolegend, Catalog No. 317408), and react in the dark for 30 minutes. , using flow cytometry to measure the changes in the proportion of CD69+CD3+T cells after treatment with different concentrations of antibodies. The results were calculated and graphed using GraphPad Prism 9.0 software, see Figure 13. Because MB001 uses high-affinity CD3 antibodies, it has the strongest T cell activation activity, while MB048, MB065 and MB069, which use low-affinity CD3, have weaker T cell activation activities; the MB060 negative control group has no T cells because it cannot bind MSLN. activation activity.

Example 8 Detection of the in vitro killing activity and cytokine release of T cells mediated by multispecific antibodies. In this experiment, three tumor cell lines were selected as target cells for the T cell killing experiment in vitro, among which OVCAR3 was a high-expressing MSLN Human ovarian cancer cells, NCI-H292 are human lung cancer cells with low expression of MSLN, and A431 are human cutaneous squamous cell carcinoma cells that do not express MSLN, serving as the negative control group. Use a T cell isolation kit (purchased from STEMCELL technologies, product number 17951, please refer to the kit instructions for usage) to isolate primary T cells from PBMC of healthy donors and culture them overnight. On the second day, OVCAR3, NCI-H292 or A431 cells were digested with trypsin to prepare single cell suspension. Use complete culture medium to adjust the tumor cell density to 2×10 ⁵ cells/ml and the T cell density to 1×10 ⁶ cells/ml. Take 50 μl of adjusted OVCAR3, NCI-H292 or A431 cells and 50 μl of primary T cells in equal volumes and mix evenly (the number of tumor cells and T cells in each well is 1×10 ⁴ and 5×10 ⁴ respectively), and use Add a micropipette to each well of a flat-bottomed 96-well plate, and the cell mixture volume is 100 μl/well. The antibodies were serially diluted in complete culture medium (the starting concentration of the antibody was 25 nM, 6-fold dilution, 8 gradients), and 100 μl of antibody diluent of different concentrations was added to each well to make the final volume of each well 200 μl. After culturing for 24 hours, take the supernatant and use ELISA method to detect the production of IFNγ, TNFα and IL-6 cytokines by T cells (the kit was purchased from BD, the product numbers are 555142, 555212 and 555220 respectively, please refer to the kit instructions for usage). CellTiter-Glo kit (purchased from Promega, product number G9243, please refer to the product instructions for usage) to detect cell viability. Define the wells with only T cells as a positive control group with 100% killing, and define the wells with an antibody concentration of 0 as a blank negative control group. Cell killing rate = (blank well reading - sample well reading) / (blank well reading) -Positive control group well reading value) × 100%. The results were calculated and graphed using GraphPad Prism 9.0 software, see Figure 14. Combining the results of cell killing experiments and the results of cytokine release, it can be seen that whether it is OVCAR3 cells with high MSLN performance or NCI-H292 cells with low MSLN performance, MB069 shows the strongest T cell activation and in vitro tumor cell killing. activity, while the T cell activation and in vitro tumor cell killing activities of MB048 and MB065 are comparable to MB001. MB001, MB048, MB065 and MB069 have no killing effect on MSLN-negative A431 cells, indicating that the killing effect of the CD3 multispecific antibody has good target antigen specificity and will not produce abnormality in normal tissues that do not express the target antigen. Specific killing. The degree of cytokine release reflects the activity of multispecific antibodies and is generally positively correlated with the degree of T cell activation, in vitro killing activity, and in vivo tumor suppressive activity. At the same time, the stronger the T cell activation activity of multispecific antibodies, the higher the risk of developing cytokine storm (CRS) after entering the human body.

Example 9 In vivo anti-tumor activity of multispecific antibodies in human ovarian cancer OVCAR-3 (MSLN high expression) xenograft model 9.1 Multispecific antibodies in human ovarian cancer OVCAR-3 (MSLN high expression) xenograft reconstituted with human PBMC In vivo anti-tumor activity in transplantation model In this example, human PBMC cells were injected into immunodeficient NPG mice (5-6 w, female, Beijing Vitong Lever Experimental Animal Technology Co., Ltd.) through tail vein injection, thereby obtaining human PBMC reconstruction. model, and then established the human ovarian cancer OVCAR-3 xenograft model (xenograft model) with high MSLN performance and the human lung cancer NCI-H292 xenograft model (xenograft model) with low MSLN performance respectively in this animal model, and performed experiments on these two models respectively. Validate the in vivo anti-tumor activity of multispecific antibodies in this model. Expand and culture OVCAR-3 cells (American Type Culture Collection, ATCC) in T-300 cell culture flasks to the required number, and in their logarithmic growth phase (confluence is about 80%; cells need to be replaced one day before inoculation) fresh culture medium) to start collection. First, remove the culture medium from the cell culture flask, wash it twice with phosphate buffered saline (PBS, Thermo Fisher Scientific Co., Ltd., Hyclone, 29588138), and then add an appropriate amount of 0.25% trypsin digestion solution (Thermo Fisher Scientific Co., Ltd. Company, Gibco, #25200-072/219085), shake the bottom of the bottle to make the trypsin digestion solution evenly cover the cell surface, digest at 37°C for 5 minutes, and add 20% fetal bovine serum (FBS, Thermo Fisher) Terminate the digestion reaction with the complete culture medium of Er Technology Co., Ltd., Gibco, #10099-141C/2261480CP), and gently blow the cells adhering to the bottom of the bottle away from the culture bottle. The digested cell suspension is collected into a 50 mL centrifuge tube, 350 g. , centrifuge for 5 minutes, absorb an appropriate amount of serum-free RPMI-1640 medium (Thermo Fisher Scientific Co., Ltd., Gibco, #61870-036/2192781) to resuspend the cells, and filter through a 70 μm mesh. Take 500 μL of cell suspension in Count with a cell counter. Finally, according to the cell count results, use serum-free medium to adjust the cell density to 1×10 ⁷ cells/mL, place on ice, and transfer to the SPF animal room through the transfer window for inoculation modeling. Before inoculation, mix the above cell suspension with Matrigel (Corning Biotechnology Co., Ltd., #356237) in equal proportions, and inoculate 200 μL of the above cell mixture subcutaneously into the right axilla of each mouse. The day after the above tumor cells were inoculated, resuscitate the corresponding number of human peripheral blood mononuclear cells (PBMC, Shanghai Saili Biotechnology Co., Ltd., #200256), filter it with a 70 μm mesh, take 100 μL cell suspension, and add 400 μL RPMI -1640 serum-free medium for counting, and based on the counting results, use RPMI-1640 serum-free medium to adjust the PBMC cell concentration to 2.5×10 ⁷ cells/mL, place it on ice, and transfer it to the SPF animal room through the transfer window for inoculation modeling. Each mouse was inoculated into the tail vein with 200 μL of the above cell suspension for immune reconstitution. After inoculation, monitor tumor growth. When the overall tumor volume is around 50-100 ^mm3 , blood is collected from the eye sockets for human immune system reconstitution testing. Mice with appropriate tumor volume and immune reconstitution results are selected and grouped (n=5) and given For multi-specific antibody drug treatment, the dosage regimen is shown in Table 23, in which MB060 is the negative control group, MB001 is the positive control group, and MB048, MB065 and MB069 are the antibodies to be tested. The dosage is based on the molar amount of positive molecule MB001 administered per unit body weight. The dosage per kilogram of mouse mass is converted into the molar amount per unit body weight according to the formula m=n*M, and finally the unit is obtained. The weight of the mouse is the mass of drug administration, where m is the mass of drug administration, n is the molar amount of drug administration, and M is the molecular weight of the drug. The dosages in this article are calculated based on this principle. Table 23 In vivo drug efficacy dosing regimen in humanized ovarian cancer tumor-bearing mice Group multispecific antibodies Dosage (mg/kg) Dosage (nmol/kg) Dosing frequency Group 1 MB060 3 56.60 Once a day, 15 times in total Group 2 MB001 1 18.87 Group 3 MB048 2.94 56.60 Group 4 MB065 1.47 18.87 Group 5 MB069 1.47 18.87 The results are shown in Figure 15. MB065 (tumor inhibition rate = 69.98%) showed better antitumor activity than MB048 (tumor inhibition rate = 42.56%) and the positive molecule MB001 (tumor inhibition rate = 47.05%), but all three Significantly weaker than MB069 (tumor inhibition rate = 106.85%). The calculation method of tumor inhibition rate is as follows, tumor inhibition rate (%) = (1-(Vn(experimental group)-V0(experimental group))/(Vn(same type control group)-V0(same type control group)))*100% , where Vn is the tumor volume on the day of measurement, V0 is the tumor volume on the day of grouping, and the tumor inhibition rate in this article is calculated using this formula.

9.2 In vivo anti-tumor activity of multispecific antibodies in a xenograft model in which human PBMC and human ovarian cancer cell OVCAR-3 (MSLN high expression) were mixed subcutaneously and inoculated into tumors. The processing method of OVCAR-3 cells was the same as 9.1, using serum-free Adjust the cell density of the culture medium to 12×10 ⁷ cells/mL and place it on ice for later use. At the same time, the PBMC treatment method is the same as 9.1. Use RPMI-1640 serum-free medium to adjust the PBMC cell concentration to 3 × 10 ⁷ cells/mL, place it on ice, and transfer the above cell suspensions and Matrigel (Corning) through the transfer window. Biotechnology Co., Ltd., #356237) was transferred to the SPF animal room for inoculation modeling, mixed in the ultra-clean table of the SPF animal room according to Table 24, and then 200 μL of the above cell mixture was subcutaneously inoculated into the right axilla of each mouse. After inoculation, tumor growth was monitored. When the overall tumor volume was around 50-100 ^mm3 , the tumors were divided into groups (n=5) and given multi-specific antibody drug treatment. The dosage regimen is shown in Table 23, in which MB060 is the negative control group. MB001 is the positive control group, and MB048, MB065 and MB069 are the antibodies to be tested. Table 24 Cell mixing scheme for subcutaneous inoculation of OVCAR-3 and PBMC OVCAR-3 stock solution OVCAR-3 dosage PBMC stock solution PBMC dosage Matrigel 12 × 10 ⁷ pieces/mL 5mL 3 × 10 ⁷ pieces/mL 1mL 6mL The results are shown in Figure 16. MB048 (tumor inhibition rate = 101.37%), MB065 (tumor inhibition rate = 106.60%) and MB069 (tumor inhibition rate = 106.97%) all showed better performance than the positive molecule MB001 (tumor inhibition rate = 89.09 %) anti-tumor activity.

Example 10 In vivo anti-tumor activity of multispecific antibodies in the human lung cancer NCI-H292 (MSLN low expression) xenograft model reconstituted with human PBMC. This example selects the human lung cancer cell NCI-H292 with low MSLN expression for mouse ( NPG, 5-6 w, female, Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) in vivo model was established, and the in vivo anti-tumor activity of the multispecific antibody was verified on this model. Human lung cancer cells NCI-H292 (American Type Culture Collection, ATCC) were processed according to the method described in Example 9.1 (the complete culture medium containing 10% fetal calf serum to stop digestion), and finally based on the cell counting results, using The serum culture medium was adjusted to a cell density of 5 × 10 ⁷ cells/mL, placed on ice, and transferred to the SPF animal room through the transfer window for inoculation modeling. 200 μL of the above cell suspension was subcutaneously inoculated into the right axilla of each mouse. On the second day after the above tumor cells were inoculated, the PBMC treatment method was the same as 9.1. Use RPMI-1640 serum-free medium to adjust the PBMC cell concentration to 2.5×10 ⁷ cells/mL, place it on ice, and transfer it to the SPF animal room through the transfer window. For inoculation modeling, 200 μL of the above cell suspension was inoculated into the tail vein of each mouse for immune reconstitution. After inoculation, the tumor growth was monitored. When the overall tumor volume was ^about 100 mm, blood was collected from the eye sockets for human immune system reconstitution testing. Mice with appropriate tumor volume and immune reconstitution results were selected and grouped (n=8) and given multispecific drugs. Sexual antibody drug treatment, the dosage regimen is shown in Table 25, in which MB060 is the negative control group, MB001 is the positive control group, and MB048, MB065 and MB069 are the antibodies to be tested. Table 25 In vivo drug efficacy dosing regimen in humanized lung cancer tumor-bearing mice Group candidate antibodies Dosage (mg/kg) Dosage (nmol/kg) Dosing frequency Group 1 MB060 9 169.79 Once a day, 22 times in total Group 2 MB001 0.3 5.66 Group 3 MB001 1.00 18.87 Group 4 MB001 3.00 56.60 Group 5 MB048 0.98 18.87 Group 6 MB048 2.94 56.60 Group 7 MB048 8.83 169.79 Group 8 MB065 0.44 5.66 Group 9 MB065 1.47 18.87 Group 10 MB065 4.42 56.60 Group 11 MB069 0.44 5.66 Group 12 MB069 1.47 18.87 Group 13 MB069 4.42 56.60 The results are shown in Figure 17. The low-dose group: MB048 (tumor inhibition rate = 48.18%) and MB065 (tumor inhibition rate = 62.57%) showed better antitumor activity than the positive molecule MB001 (tumor inhibition rate = 3.69%). But all three were significantly weaker than MB069 (tumor inhibition rate = 80.25%); medium-dose group: MB001 (tumor inhibition rate = 57.94%), MB048 (tumor inhibition rate = 54.74%) and MB065 (tumor inhibition rate = 49.39%) The tumor inhibition rates are equivalent and significantly weaker than MB069 (tumor inhibition rate = 80.21%); high-dose group: MB001 (tumor inhibition rate = 40.48%), MB048 (tumor inhibition rate = 37.35%) and MB065 (tumor inhibition rate = The tumor inhibition rates are basically the same (54.48%), both are significantly weaker than MB069 (tumor inhibition rate = 84.05%), and MB048 is significantly weaker than MB065.

Example 11 Pharmacokinetic experiment of multispecific antibodies in cynomolgus monkeys. Seven cynomolgus monkeys aged 3-5 weeks, weighing 3.7-5.7kg, were grouped according to different test products and dosages. The drugs were diluted with preparation solvent. Different individuals were given a single intravenous injection of MB001, MB069 and MB065 to compare their pharmacokinetic differences. Blood samples were collected from the veins of the hind limbs. The blood collection time points were before administration and 0.25 hours, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours after administration. 504 hours, 672 hours and 744 hours. After blood samples are collected, they are stored on ice for blood sample separation. Then, centrifuge at 1500 × g, 4°C for 10 minutes to separate the serum into a low-adsorption centrifuge tube, indicate the compound code and time point, and freeze it at -80°C before analysis. Blood samples should be collected, centrifuged and frozen within 2 hours. The hMSLN-FL-his protein is coated, and the concentration of the compounds in the control group and the test group of the present invention in the serum is measured using the indirect enzyme-binding immunoadsorption method. Pharmacokinetic parameters were calculated based on blood drug concentrations at different time points for each animal. It can be seen from the results that the half-lives of MB001, MB069 and MB065 can support clinical administration once a week. Table 26 Pharmacokinetics test in cynomolgus monkeys

Example 12 Stability experiments of multispecific antibodies in human serum and cynomolgus monkey serum 12.1 Stability experiment of multispecific antibodies in human serum The culture and treatment of PBMC cells are the same as in Example 7; the culture and treatment of Hela cells are the same as the treatment of NCI-H292 cells in Example 7. There are two types of antibody treatment: 1. The antibody is not pre-treated; 2. The antibody is pre-incubated in human serum at 37°C for two days. The antibodies of different treatments were serially diluted in complete culture medium (the starting concentration of the antibody was 25 nM, 6-fold dilution, 10 concentration gradients). Add 100 μl of antibody dilution solutions of different concentrations to each well, so that the final volume of each well is 200 μl. The in vitro cytotoxic activity experimental treatments are divided into two types: 1. The in vitro killing experiment is conducted in 10% FBS complete medium; 2. An additional 20% human serum is added to the in vitro killing experimental system. After 24 hours of culture, the cell viability was measured using the CellTiter-Glo kit (purchased from Promega, please refer to the product instructions for usage) to detect the effect of human serum on the in vitro cytotoxic activity of T cells mediated by multispecific antibodies. The results are shown in Figure 19A . After two days of incubation in human serum at 37°C, the activities of MB001 and MB048 decreased more, while the activities of MB065 and MB069 hardly decreased. However, comparing the killing activity in the unmanned serum system and the 20% human serum system, it can be seen that the addition of 20% human serum will significantly weaken the killing activity of multi-specific antibodies, especially MB065 and MB069, the EC50 increased by dozens of times. It may be that after the anti-HSA in the multispecific antibody binds to the HSA protein in human serum, the spatial conformation changes, which leads to changes in the binding activity of the multispecific antibody to tumor cells and T cells, leading to T cell activation. Reduced activity. Based on the experimental results, it is inferred that after MB065 and MB069 enter the human body, the T cell activation activity will be lower than the current in vitro test results. Therefore, the risk of CRS may be reduced after MB065 and MB069 enter clinical trials in the future.

12.2 Stability experiment of multispecific antibodies in cynomolgus monkey serum The experimental method is the same as 12.1, except that the human serum part is replaced with cynomolgus monkey serum. After culturing for 24 hours, cell viability was measured using the CellTiter-Glo kit (purchased from Promega, please refer to the product instructions for usage). The results are shown in Figure 19B. After two days of incubation in cynomolgus monkey serum at 37°C, the activities of MB001 and MB048 decreased slightly, while the activities of MB065 and MB069 showed almost no decrease. However, comparing the killing activity in the system without cynomolgus monkey serum and the system with 20% cynomolgus monkey serum, it can be seen that the addition of 20% cynomolgus monkey serum has little effect on MB001, but will significantly weaken MB048, MB065 and MB069. The EC50 of the killing activity of multispecific antibodies increased more than tenfold. It may be that after the anti-HSA in the multispecific antibody binds to the CSA protein in monkey serum, the spatial conformation changes, which leads to changes in the binding activity of the multispecific antibody to tumor cells and T cells, leading to T cell activation. Reduced activity. Based on the experimental results, it is inferred that after MB065 and MB069 enter monkeys, the T cell activation activity will be lower than the current in vitro test results.

Example 13 Identification of the binding ability of Anti-CD33×CD70 multispecific antibodies to proteins or cells 13.1 Flow cytometry experiment (FACS) to detect the binding of multispecific antibodies to tumor cells. Tumor cells THP-1 (purchased from the Chinese Academy of Sciences) were used in the experiment. Cell bank, catalog number: TCHu 57), U937 (purchased from ATCC, catalog number: CRL-1593.2), 786-O (purchased from ATCC, catalog number: CRL-1932), Jurkat (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number: TCHU123). Expand the required cells in a T-175 cell culture flask to the logarithmic growth phase, aspirate the medium, wash twice with PBS buffer, digest the cells with trypsin, then use complete medium to terminate the digestion, and pipet the cells until they are single cells suspension. After counting the cells, centrifuge, wash the cell pellet twice with PBS, resuspend the cell pellet in FACS buffer (PBS + 2% fetal calf serum) to 2×10 ⁶ cells/mL, and add 50 µl per well to 96 wells. In the FACS reaction plate, add 50 µl/well of the antibody to be tested (400 nM or 100 nM as the starting concentration, 3-fold or 5-fold gradient dilution), mix with the cell suspension, and incubate at 4°C for 1 hour. Centrifuge and wash 3 times with PBS buffer, add 50 µl iFluor 647-labeled Anti-His secondary antibody (purchased from Genscript, Cat. No.: A01802-100) per well, and incubate at 4°C for 1 hour. Centrifuge and wash 3 times with PBS buffer, resuspend in 100 µl PBS and use FACS (FACS CantoTM, purchased from BD Company) to detect and analyze the results. Data analysis was performed through software (CellQuest) to obtain the mean fluorescence intensity (MFI) of the cells. Then use software (GraphPad Prism8) to analyze, perform data fitting, and calculate EC50. The analysis results are shown in Figures 20A to 20D and Table 27, in which MB060 is the negative control group; the results show that Anti-CD33×CD70 multispecific antibodies have binding activity to tumor cells with different manifestations, indicating that Anti-CD33×CD70 multi-specific Specific antibodies can specifically bind to tumor cells expressing human CD33 protein. Table 27 Binding reaction between CD33×CD70 multispecific antibody and tumor cells Antibody name 1# 2# 9# 15# Mab060 THP-1 Max_MFI 1571 1096 1281 982 130 EC50 (nM) 2.59 1.12 25.11 1.04 Not combined U937 Max_MFI 393 330 357 307 112 EC50 (nM) 12.55 20.14 3.40 18.10 Not combined 786-O Max_MFI 1224 1181 74 1212 71 EC50 (nM) 24.57 20.61 Not combined 83.73 Not combined Jurkat Max_MFI 1242 1442 1121 1507 805 EC50 (nM) 4.37 4.88 3.78 5.02 3.28

13.2 Flow cytometry experiment (FACS) to detect the binding of antibodies to CHO-K1-human CD33 For the preparation method of the recombinant cell line CHO-K1-human CD33 expressing human CD33 protein, please refer to PCT/CN2022/075621. The nucleotide sequence encoding the full-length amino acid sequence of human CD33 (NCBI: XP_011525834.1) is copied to pcDNA3. 1 Vector and prepare plastids (completed by General Biosystems (Anhui) Co., Ltd.). After plastid transfection (Lipofectamine® 3000 Transfection Kit, purchased from Invitrogen, Cat. No.: L3000-015) on the CHO-K1 cell line (purchased from Shanghai Institutes for Life Sciences, Chinese Academy of Sciences), puromycin was used for pressure screening. Select the single cell line B8 with better growth trend, higher fluorescence intensity, continue to expand the culture and freeze it in liquid nitrogen. For the preparation of detection cells and antibodies to be tested and the detection method, please refer to Example 13.1. The analysis results are shown in Figure 21, in which MB060 is the negative control group. The results show that Anti-CD33×CD70 multispecific antibodies have binding activity to recombinant cells expressing human CD33 protein, indicating that Anti-CD33 monoclonal antibodies retain the characteristics of monoclonal antibodies after being combined into multispecific antibodies.

13.3 Flow cytometry experiment (FACS) to detect the cross-binding of antibodies and CHO-K1-human CD70 The recombinant cell line CHO-K1-human CD70 expressing human CD70 protein was purchased from Kangyuan Bochuang (KC-1267) for detection cells, and the preparation of the antibody to be tested and the detection method were as described in Example 13.1. The analysis results are shown in Figure 22, in which MB060 is the negative control group, and BDD20-09-9# only has CD33 monoclonal antibodies but no CD70 monoclonal antibodies. The results show that Anti-CD33×CD70 multispecific antibodies have binding activity to recombinant cells expressing human CD70 protein, indicating that Anti-CD70 monoclonal antibodies retain the characteristics of monoclonal antibodies after being combined into multispecific antibodies.

Example 14 Different formats of polyclonal antibodies mediate differences in killing activity against tumor cells and normal cells expressing CD33. In this experiment, Molm13, an AML3 cell line with high expression of CD33, was added to healthy donor PBMC to simulate the in vivo environment of AML patients and verify different formats. Polyclonal antibodies mediate differences in the killing activity of tumor cells and normal cells expressing CD33 (mainly CD14+ monocytes in peripheral blood). Resuscitate frozen healthy donor PBMC cells, label the recovered PBMC cells with CellTrace™ Violet dye, adjust Molm13 tumor cells to 2.5 × ¹⁰ cells/ml with complete culture medium, and adjust the PBMC cell density to 1 × 10 ⁶ pieces/ml. Take 50 μl of adjusted monocytes or Molm13 cells and 50 μl of primary T cells and mix them evenly (the number of Molm13 cells and PBMC cells in each well is 1.25 × 10 ⁵ and 5 × 10 ⁴ respectively), and use a micropipette to The cell mixture was added to each well of a flat-bottomed 96-well plate, and the volume of cell mixture was 100 μl/well. The antibodies were serially diluted in complete culture medium (the starting concentration of the antibody was 5 μg/ml, 20-fold dilution, 8 gradients), and 100 μl of antibody diluent of different concentrations was added to each well so that the final volume of each well was 200 μl. . After culturing for 48 hours, centrifuge to remove the supernatant, resuspend in flow buffer, add Fc Receptor Blocking Solution (purchased from Biolegend, Cat. No. 422302), block at room temperature for 15 minutes, and perform CD14-PerCPCy5.5 (purchased from Biolegend, Cat. No. 367110) staining, protect from light, react for 30 minutes, centrifuge to remove the supernatant, wash twice with flow buffer, add PI (purchased from Thermo Fisher, Cat. No. P3566) to the cells, incubate for 5 minutes, and proceed Flow cytometry (FACS Canto Plus, purchased from BD Company), determines the proportion of PI+ cells in CellTrace™ Violet+CD14+ double-positive cells and the proportion of PI+ cells in CellTrace™ Violet-negative cells, respectively. Different formats of polyclonal antibodies against normal cells expressing CD33 and tumor cell killing activity. The results were calculated and graphed using GraphPad Prism 9.0 software. The results are shown in Figure 23-24. Different format antibodies have killing activity against tumor cells and monocytes, but BDD20-09-15# has better killing activity against tumor cells than BDD20-09-09#.

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Figure 1A~1B. Schematic representation of the structure of multispecific antibodies. Figure 2A~2C. ELISA detection of binding reaction between MSLN multispecific antibody and albumin: 2A.HAS; 2B.CSA; 2C.MSA. Figure 3A~3B. ELISA detection of the binding reaction of MSLN multispecific antibodies that simultaneously bind to MSLN protein and CD3 protein: 3A. Human CD3; 3B. Monkey CD3. Figure 4A~4E. FACS detection of the binding reaction between MSLN multispecific antibodies and tumor cells: 4A.OVCAR3; 4B.Hela; 4C.Hs766T; 4D.NCI-H292; 4E.A431. Figure 5. FACS detection of binding reaction between MSLN multispecific antibody and HEK293T-hMSLN-R3 cells. Figure 6A. FACS detection of the binding reaction between MSLN multispecific antibodies and HEK293T-monkey MSLN cells; Figure 6B. FACS detection of binding reaction between MSLN multispecific antibody and HEK293T cells. Figure 7A. FACS detection of binding reaction between MSLN multispecific antibody and human T cells; Figure 7B. FACS detection of binding reaction between MSLN multispecific antibody and monkey T cells. Figure 8A~8D. MSLN multispecific antibody reporter assay: 8A.OVCAR3; 8B.Hela; 8C.Hs766T; 8D.NCI-H292. Figure 9. ELISA detection of the effect of CA125 on the binding activity of MSLN multispecific antibody. Figure 10. FACS detection of the effect of CA125 on the binding activity of MSLN multispecific antibody. Figure 11A ~ 11D. Binding status of dual-topic MSLN multispecific antibodies. Figure 12A. Endophytic activity of 100nM MSLN multispecific antibody; Figure 12B. Endophytic activity of 10nM MSLN multispecific antibody; Figure 12C. Endophytic activity of 1 nM MSLN multispecific antibody. Figure 13. In vitro activation status detection of T lymphocytes mediated by MSLN multispecific antibodies. Figure 14. MSLN multispecific antibody mediates T cell tumor cell killing activity and cytokine detection in vitro. Figure 15. Multispecific antibody inhibits humanized ovarian cancer tumor growth curve. Figure 16. Multispecific antibodies inhibit tumor growth curve of ovarian cancer cell OVCAR-3 and PBMC mixed subcutaneous model. Figure 17. Multispecific antibody inhibits humanized lung cancer tumor growth curve. Figure 18. Comparison of pharmacokinetics of multispecific antibodies in cynomolgus monkeys. Figure 19A. Stability test of multispecific antibodies in human serum. Figure 19B. Stability testing of multispecific antibodies in cynomolgus monkey serum. Figure 20A. FACS detection of binding reaction between CD33×CD70 multispecific antibody and THP-1 cells. Figure 20B. FACS detection of binding reaction between CD33×CD70 multispecific antibody and U937 cells. Figure 20C. FACS detection of binding reaction between CD33×CD70 multispecific antibody and 786-O cells. Figure 20D. FACS detection of binding reaction between CD33×CD70 multispecific antibody and Jurkat cells. Figure 21. FACS detection of binding reaction between CD33×CD70 multispecific antibody and CHO-K1-human CD33 cells. Figure 22. FACS detection of binding reaction between CD33×CD70 multispecific antibody and CHO-K1-human CD70 cells. Figure 23. Experiment on the killing activity of different format antibodies against tumor cells. Figure 24. Experiment on the killing activity of different format antibodies against monocytes.

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TW202340240A_111143652_SEQL.xml

Claims (14)

A multispecific antibody, characterized in that the multispecific antibody contains at least three parts: (A) a targeting antigen-binding part, (B) a half-life extending part, and (C) a T cell engaging part; (A) Targeting antigen-binding part: preferably a targeting antigen-binding antibody or a targeting antigen-binding ligand; the targeting antigen-binding antibody can be selected from any antigen-binding fragment; (B) Half-life extension part: better anti-HSA antibodies; (C) T cell engagement part: The T cell engagement part is preferably anti-CD3 antibody or antigen-binding fragment; The antigen-binding fragment is preferably Fd, Fv, scFv, diabody or single domain antibody (VHH); The segments of the three parts (A), (B), and (C) can be connected through connectors or directly connected without connectors. The multispecific antibody as described in claim 1, wherein (A) the targeting antigen-binding portion or (C) the T-cell engaging portion can appear repeatedly or include multiple portions; Preferably, the multispecific antibody contains two target antigen-binding parts, A1 and A2. A1 and A2 can be the same or different; preferably, A1 and A2 bind different antigen targets; preferably, A1 Binds to different epitopes of the same antigen target as A2; Preferably, part (C) is a scFv that binds CD3; More preferably, part (C) is a scFv that binds human CD3. The multispecific antibody according to claim 1, wherein the structural sequence of the multispecific antibody from the N-terminus to the C-terminus is: (1)A(VHH) - B(VHH) - C(VH) - C(VL) (2)A(VHH) - B(VHH) - C(VL) - C(VH) (3) A1(VL) - A1(VH) - A2(VHH) - B(VHH) - C(VH) - C(VL) (4) A1(VL) - A1(VH) - A2(VHH) - B(VHH) - C(VL) - C(VH) (5) A1(VHH) – A2(VL) – C(VH) – C(VL) – A2(VH) – B(VHH) (6) A1(VHH) – A2(VH) – C(VL) – C(VH) – A2(VL) – B(VHH) (7) B(VHH) – A2(VL) – C(VH) – C(VL) – A2(VH) – A1(VHH) (8) B(VHH) – A2(VH) – C(VL) – C(VH) – A2(VL) – A1(VHH). The multispecific antibody according to any one of claims 1-3, wherein (C) the T cell engaging portion includes complementarity determining regions (CDRs) selected from the following antibodies: OKT3, TRX4, MGA031, Nuvion, SP34, X35, VIT3, BMA030, CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII- 87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, S004-2-03, S004-2-06, S004- 2-08, S004-2-10, S004-2-18, 6-35.22-hu, 1-22.6-1-hu, 7-35.6-hu, and 6-44.5-hu; Preferably, the (C) T cell engaging portion includes heavy chain CDRs and/or light chain CDRs selected from the following: Heavy chain CDR1 is shown in SEQ ID NO. 34, 39, 42, and 47; Heavy chain CDR2 is shown in SEQ ID NO. 35, 40, 43, 45 and 48; Heavy chain CDR3 is shown in SEQ ID NO. 36, 37, 38, 41, 44, 46, 49; The light chain CDR1 is shown in SEQ ID NO. 50, 55, 58, 61, and 64; The light chain CDR2 is shown in SEQ ID NO. 51, 56, 59, 62, and 65; The light chain CDR3 is shown in SEQ ID NO. 52, 53, 54, 57, 60, 63, and 66; Preferably, the (C) T cell engaging portion contains 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% identity with the above heavy chain CDRs and/or light chain CDRs. sexual sequence. The multispecific antibody according to any one of claims 1-3, wherein the targeting antigen described in part (A) can be selected from the following group: CD19, BCMA, HER2, EGFR, VEGF, MSLN, CD33 , CD70, CD5, CD20, CD40, CD47, CD38, CD137, TNF-alpha, HER3, CD27, EphA2, EpCAM, MUC1, MUC17, CEA, Claudin18.2, folate receptor, Claudin6, WT1, NY-ESO-1 , MAGE3, ASGPR1, CDH16, GPRC5D, DLL3, ROR1 or GUCY2C; Preferably, the targeting antigen-binding portion includes complementarity determining regions (CDRs) selected from the following fragments: SEQ ID NO. 18-28, 103-106; Preferably, the (A) targeting antigen-binding portion includes heavy chain CDRs and/or light chain CDRs selected from the following: Heavy chain CDR1 is shown in SEQ ID NO. 76, 80, 89, 92, 110, 113, and 116; Heavy chain CDR2 is shown in SEQ ID NO. 77, 79, 81, 90, 93, 111, 114, and 117; Heavy chain CDR3 is shown in SEQ ID NO. 78, 82, 91, 94, 112, 115, 118; The light chain CDR1 is shown in SEQ ID NO. 83, 86, and 107; The light chain CDR2 is shown in SEQ ID NO. 84, 87, and 108; The light chain CDR3 is shown in SEQ ID NO. 85, 88, and 109; Preferably, the (A) targeting antigen-binding portion contains 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% of the above heavy chain CDRs and/or light chain CDRs. Identity sequence. The multispecific antibody according to any one of claims 1-3, wherein part (B) includes complementarity determining regions (CDRs) selected from the following fragments: SEQ ID NO. 15-17; Preferably, said part (B) contains CDRs selected from the following: CDR1 is shown in SEQ ID NO. 67, 70, and 73; CDR2 is shown in SEQ ID NO. 68, 71, 74; and CDR3 is shown in SEQ ID NO. 69, 72, and 75; Preferably, part (B) includes sequences having 99%, 98%, 97%, 96%, 95%, 90%, 85%, and 80% identity with the above-mentioned CDRs. The multispecific antibody according to any one of claims 1 to 3, wherein a linker is used to connect the fragments of the three parts (A), (B), and (C); the linkers are each independently selected. From: (GS)n, (GGS)n, (GGGS)n, (GGSG)n, (GGSGG)n, (GGGGS)n or (GGGGS)n (GGGS)n, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Preferably, the linker is GGGGS, GGGGSGGGS, GGGGSGGGGS, GGGGSGGGGSGGGGS, or GGGSGGGGSGGGGSGGGGS. The multispecific antibody according to any one of claims 1-7, wherein the antibody or antigen-binding fragment is: (1) Chimeric antibodies or fragments thereof; (2) Humanized antibodies or fragments thereof; or, (3) Fully human antibodies or fragments thereof; Preferably, the multispecific antibody includes the sequence shown in SEQ ID NO. 97, 98, 99, 119, 120, 121, or 122, or has 99%, 98%, 97%, 96%, or Sequences with 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% identity. An isolated nucleic acid fragment, characterized in that the nucleic acid fragment encodes a multispecific antibody as described in any one of claims 1-8. A vector, characterized in that the vector contains the nucleic acid fragment described in claim 9. A host cell, characterized in that the host cell contains the vector according to claim 10; preferably, the cell is a prokaryotic cell or a eukaryotic cell, such as bacteria (E. coli), fungi (yeast), insects cells or mammalian cells (CHO cell line or 293T cell line). A method for preparing the multispecific antibody according to any one of claims 1 to 8, wherein the method includes culturing the cell according to claim 11, and isolating the multispecific antibody expressed by the cell. A pharmaceutical composition, wherein the pharmaceutical composition comprises the multispecific antibody as described in any one of claims 1-8, or the nucleic acid fragment as described in claim 9, or as described in claim 10 Carrier; or a product prepared by the method described in claim 12; optionally, the pharmaceutical composition also includes a pharmaceutically acceptable carrier, diluent or auxiliary agent; optionally, the pharmaceutical composition The pharmaceutical compositions also contain additional antineoplastic agents. A method for the prevention and/or treatment of proliferative diseases, neoplastic diseases, inflammatory diseases, immune disorders, autoimmune diseases, infectious diseases, viral diseases, allergies, parasitic reactions, graft-versus-host disease or host-versus-graft A method for treating the disease, comprising administering to a patient in need an effective amount of the multispecific antibody described in any one of claims 1-8, or the nucleic acid fragment described in claim 9, or the vector described in claim 10, or The product prepared by the method described in claim 12 or the pharmaceutical composition described in claim 13; The tumor disease is preferably a solid tumor expressing MSLN, CD70 and/or CD33 or a hematological tumor expressing MSLN, CD70 and/or CD33; Better mesothelioma, lung cancer, breast cancer, esophageal cancer, pancreatic cancer, ovarian cancer, pleural cancer, cholangiocarcinoma, cervical cancer, gastric cancer, leukemia, histiocytic lymphoma, or kidney cancer; Better epithelioid malignant pleural mesothelioma, lung adenocarcinoma, triple-negative breast cancer, pancreatic cancer, ovarian cancer, cervical cancer, monocytic leukemia, histiocytic lymphoma, renal clear cell adenocarcinoma, T lymphocytic leukemia or Acute myelogenous leukemia.

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