WO2023088295A1

WO2023088295A1 - Multi-specific antibody and pharmaceutical use thereof

Info

Publication number: WO2023088295A1
Application number: PCT/CN2022/132228
Authority: WO
Inventors: 杨翠青; 邵小慧; 张韫; 付雅媛; 曹卓晓; 唐任宏; 任晋生
Original assignee: 江苏先声药业有限公司
Priority date: 2021-11-17
Filing date: 2022-11-16
Publication date: 2023-05-25
Also published as: TW202340240A

Abstract

A multi-specific antibody and a pharmaceutical use thereof. The multi-specific antibody comprises at least three parts: (A) a target antigen binding part, (B) a half-life extending part, and (C) a T cell engaging part. By binding to two or more targets at the same time, the multi-specific antibody can simultaneously exert multiple functions, so as to prevent and/or treat proliferative diseases.

Description

A kind of multispecific antibody and its pharmaceutical use

Cross References to Related Applications

This application claims the benefit and priority of the following 2 Chinese invention patent applications, the entire contents of which are hereby incorporated by reference in their entirety: Patent No. 202111361168.1 filed with the State Intellectual Property Office of China on November 17, 2021 application; and patent application No. 202211362184.7 filed with the State Intellectual Property Office of China on November 02, 2022.

technical field

The present invention relates to the field of biomedicine, in particular to a multispecific antibody and its medicinal use.

Background technique

The concept of multispecific antibodies can be traced back to the 1960s, when Nisonoff and his team first proposed that antibody-based molecules could simultaneously bind to different antigens. So far, more than 100 kinds of multispecific antibody structures have been developed, among which bispecific antibodies are the main research and development focus.

Due to the complexity of the pathological mechanisms of various diseases, monoclonal antibodies targeting one antigen alone have certain limitations and deficiencies in actual clinical treatment. Therefore, the research and development of multispecific antibodies tends to be hot in recent years. By binding to two or more targets at the same time, multispecific antibodies can perform multiple functions at the same time, so as to achieve the effect that monoclonal antibodies cannot achieve.

Contents of the invention

The disclosure of the present invention provides a multispecific antibody, a nucleic acid fragment encoding the multispecific antibody, a vector, a cell, a composition, a preparation method, a pharmaceutical use and a treatment method thereof.

In a first aspect, the present invention provides a multispecific antibody comprising at least three parts: (A) a target antigen binding part, (B) a half-life extending part, and (C) a T cell engaging part;

(A) Target antigen-binding portion: preferably a target antigen-binding antibody or a target antigen-binding ligand; the target antigen-binding antibody may be selected from any antigen-binding fragment;

(B) half-life extension moiety: preferably anti-HSA antibody;

(C) T cell engaging portion: wherein the T cell engaging portion is preferably an anti-CD3 antibody or an antigen-binding fragment;

The antigen-binding fragment is preferably Fd, Fv, scFv, diabody or single domain antibody (VHH);

The fragments of the three parts (A), (B) and (C) can be connected through a linker, or can be directly connected without a linker.

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

In some embodiments, the multispecific antibody comprises two target antigen-binding portions, A1 and A2, and A1 and A2 may be the same or different; in some embodiments, A1 and A2 bind different antigen targets; in some embodiments In the scheme, A1 and A2 bind different epitopes of the same antigen target;

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

In a specific embodiment, the structural order of the multispecific antibody from the N-terminal to the C-terminal 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, said multispecific antibody (C) T cell engaging portion 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:

The heavy chain CDR1 is shown in SEQ ID NO.34, 39, 42, 47;

The heavy chain CDR2 is shown in SEQ ID NO.35, 40, 43, 45, 48;

The 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, 64;

The light chain CDR2 is shown in SEQ ID NO.51, 56, 59, 62, 65;

The light chain CDR3 is shown in SEQ ID NO.52, 53, 54, 57, 60, 63, 66;

In some embodiments, the (C) T cell engaging portion comprises 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% of the above heavy chain CDRs and/or light chain CDRs % identity sequence. In a specific embodiment, the target antigen of the multispecific antibody (A) part 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 comprises two target antigen-binding portions, A1 and A2, and A1 and A2 respectively bind to different epitopes of MSLN; for example, MLSN-R1 epitope or MSLN-R3 epitope.

In some embodiments, the multispecific antibody comprises two target antigen-binding portions, A1 and A2, and A1 and A2 bind target antigens CD70 and CD33, respectively.

In some embodiments, the target antigen binding portion comprises complementarity determining regions (CDRs) selected from the following fragments: SEQ ID NO.18-28, 103-106;

In some embodiments, the (A) target antigen binding portion comprises heavy chain CDRs and/or light chain CDRs selected from:

The heavy chain CDR1 is shown in SEQ ID NO.76, 80, 89, 92, 110, 113, 116;

The heavy chain CDR2 is shown in SEQ ID NO.77, 79, 81, 90, 93, 111, 114, 117;

The 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, 108;

The light chain CDR3 is shown in SEQ ID NO.85, 88, 109;

In some embodiments, the (A) target antigen binding portion comprises 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% of the above heavy chain CDRs and/or light chain CDRs % identity sequence.

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

In some embodiments, said portion (B) comprises CDRs selected from the group consisting of:

CDR1 is shown in SEQ ID NO.67, 70, 73;

CDR2 as shown in SEQ ID NO. 68, 71, 74; and

CDR3 is shown in SEQ ID NO.69, 72, 75;

In some embodiments, said portion (B) comprises sequences that are 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% identical to the above-mentioned CDRs.

In a specific embodiment, the multispecific antibody uses a linker to connect the fragments of the three parts (A), (B), and (C); each of the linkers is 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 GGGGSGGGGSGGGGSGGGGS (Linker5).

In some embodiments, the structural order of the multispecific antibody from the N-terminus to the C-terminus 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) a humanized antibody or fragment 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 has 99%, 98%, 97%, 96 %, 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 described in the first aspect of the present invention.

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

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

In a fourth aspect, the present invention provides a method for preparing the multispecific antibody of the present invention, characterized in that the method includes culturing the cell described in the fourth aspect, and isolating the multispecific antibody expressed by the cell.

In the fifth aspect, the present invention also provides a pharmaceutical composition, the 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 third aspect of the present invention The carrier described in the aspect; or the product prepared by the method described in the fourth aspect of the present invention; optionally, the pharmaceutical composition also includes a pharmaceutically acceptable carrier (carrier), diluent or adjuvant; optionally , the pharmaceutical composition further comprises an additional antineoplastic agent.

In a sixth aspect, the present invention provides a method for preventing and/or treating proliferative diseases, tumor diseases, inflammatory diseases, immune disorders, autoimmune diseases, infectious diseases, viral diseases, allergies, parasitic reactions, transplants A method for host-versus-graft disease or host-versus-graft disease, comprising administering an effective amount of 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 present invention to a patient in need thereof. The carrier described in the third aspect of the 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 tumor 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 tumor 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 neoplastic disease is epithelioid Malignant pleural mesothelioma, lung adenocarcinoma, triple-negative breast cancer, pancreatic cancer, ovarian cancer, cervical cancer, monocytic leukemia, histiocytic lymphoma, clear cell adenocarcinoma of the kidney, T lymphocytic leukemia, or acute myeloid leukemia.

In the seventh aspect, the present invention provides the multispecific antibody described in the first aspect, or the nucleic acid fragment described in the second aspect of the present invention, or the vector described in the third aspect of the present invention, or the vector described in the fourth aspect of the present invention The use of the product obtained by the method or the pharmaceutical composition described in the fifth aspect of the present invention in the preparation of drugs for the prevention and/or treatment of diseases; the diseases include proliferative diseases, tumor diseases, inflammatory diseases, immune disorders, autoimmune immune disease, infectious disease, viral disease, allergy, parasitic reaction, graft-versus-host disease or host-versus-graft disease;

Definitions and Explanations of Terms

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

Also, unless otherwise specified herein, terms in the singular shall include pluralities, and terms in the plural shall include the singular, unless otherwise specified herein. More specifically, as used in this specification and the appended claims, the singular forms "a" and "the" include plural referents unless expressly stated otherwise.

The terms "comprising", "including" and "having" are used interchangeably herein and are intended to indicate the inclusiveness of the scheme, meaning that the scheme may have other elements besides the listed elements. At the same time, it should be understood that the terms "comprising", "comprising" and "having" are used herein to also provide "consisting of". Exemplarily, "a composition including A and B" should be understood as the following technical scheme: a composition composed of A and B, and a composition containing other components in addition to A and B, all fall into Into the scope of the aforementioned "a composition".

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

The term "specifically binds" herein means that an antigen-binding molecule (eg, an antibody) specifically binds an antigen and substantially the same antigen with high affinity, typically, but does not bind an unrelated antigen with high affinity. Affinity is usually reflected in an equilibrium dissociation constant (KD), where a lower KD indicates a 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 on-rate. The equilibrium dissociation constant KD can be measured by methods 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 to refer to a molecule that specifically binds an antigen. Exemplary, antigen binding molecules include, but are not limited to, antibodies or antibody mimetics. "Antibody mimic" 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. Exemplary, antibody mimics include but are not limited to affibody, affitin, affilin, designed ankyrin repeat proteins (DARPins), aptamers or Kunitz-type domain peptides.

The term "antibody" is used herein in the broadest sense to refer to a polypeptide comprising sufficient sequence from the variable region of an immunoglobulin heavy chain and/or sufficient sequence from the variable region of an immunoglobulin light chain to be capable of specifically binding to an antigen or peptide combinations. "Antibody" herein encompasses various forms and various structures as long as they exhibit the desired antigen-binding activity. "Antibody" herein includes alternative protein scaffolds or artificial scaffolds with grafted complementarity determining regions (CDRs) or CDR derivatives. Such scaffolds include antibody-derived scaffolds comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds comprising, eg, biocompatible polymers. See, eg, 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.

"Antibody" herein includes a typical "four-chain antibody", which belongs to the immunoglobulins composed of two heavy chains (HC) and two light chains (LC); In the N-terminal to C-terminal direction, 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, a heavy chain constant region CH3 domain; and, When the full-length antibody is of the 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 in the N-terminal to C-terminal direction. The polypeptide chain composed of the region (CL); the heavy chain and the heavy chain, and the heavy chain and the light chain are connected by disulfide bonds to form a "Y"-shaped structure. Because the amino acid composition and sequence of the constant region of the immunoglobulin heavy chain are different, their antigenicity is also different. Accordingly, "immunoglobulins" herein can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are respectively the μ chain and the delta chain , γ chain, α chain and ε chain. The same class of Ig can be divided into different subclasses according to 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, IgG4, and IgA can be divided into IgA1 and IgA. IgA2. Light chains are classified as either kappa chains or lambda chains by difference in the constant region. Each of the five Ig classes can have either a kappa chain or a lambda chain.

"Antibody" herein also includes "antigen-binding fragment" or "antibody fragment", and "antigen-binding fragment" and "antibody fragment" are used interchangeably herein. Partial or partial variants that possess the ability to bind 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 yields two identical antigen-binding fragments, termed "Fab" fragments, each containing the variable domains of the heavy and light chains, as well as the constant domain of the light chain and the first constant domain of the heavy chain (CH1 ). Thus, the term "Fab fragment" herein refers to a light chain fragment comprising the VL domain and the 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 CH1 domain of the heavy chain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine residue of the constant domain bears a free thiol group. Pepsin treatment yields an F(ab')2 fragment with two antigen-combining sites (two Fab fragments) and part of the Fc region.

Herein the term "Fd" refers to an antibody consisting of VH and CH1 domains. The term "Fv" herein refers to an antibody fragment consisting of a single-armed VL and VH domain. The Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. It is generally believed that the six CDRs confer antigen-binding specificity to an antibody. However, even a variable region (such as the Fd fragment, which contains only three CDRs specific for an antigen) is capable of recognizing and binding antigen, although perhaps with a lower affinity than the full binding site.

The term "scFv" (single-chain variable fragment) herein refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker (see, e.g., Bird et al., Science 242:423- 426 (1988); Huston et al, Proc. , 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 the repeated GGGGS amino acid sequence 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 described 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, there may also be a disulfide bond between the VH and VL of the scFv, forming a disulfide-linked Fv (dsFv).

The term "diabody" herein, whose VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains of the same chain, thus forcing the domains to The complementary domains of the other chain pair 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" (single domain antibody, sdAb), "VHH" and "nanobody" have the same meaning and are used interchangeably herein, and refer to the variable region of the heavy chain of a cloned antibody, constructed by only one A single domain antibody composed of the heavy chain variable region, which is the smallest fully functional antigen-binding fragment. Usually, after obtaining the antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned 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 comprise light chains, for example, antibodies produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and alpaca ( The heavy-chain antibodies (HCAbs) produced by Vicugna pacos) and the new immunoglobulin antigen receptor (Ig new antigen receptor, IgNAR) found in cartilaginous fishes such as sharks.

An "antibody" herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as camelids, llamas , proto-ostrich, alpaca, sheep, rabbit, mouse, rat or cartilaginous fishes (eg sharks).

"Antibody" herein includes, but is not limited to, monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies), monovalent antibodies, multivalent antibodies, intact antibodies, fragments of intact antibodies, naked antibodies , conjugated antibody, chimeric antibody, humanized antibody or fully human antibody.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., except for possible variants (such as containing naturally occurring mutations or arising during the manufacture of a formulation, such variants typically appear as In addition to being 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 (epitopes), in monoclonal antibody preparations each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" herein should not be interpreted as requiring that the antibody or antigen-binding molecule be produced by any particular method. For example, monoclonal antibodies can be produced by a variety of techniques including, but not limited to, hybridoma technology, recombinant DNA methods, phage library display technology and the use of transgenic animals containing all or part of the human immunoglobulin loci 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" herein refers to a non-natural antibody obtained through genetic engineering, antibody engineering and other techniques. Exemplarily, "engineered antibody" includes humanized antibodies, small molecule antibodies (such as scFv, etc.), specific antibodies, etc.

The term "monospecific" herein refers to having one or more binding sites, wherein 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 is associated with a different epitope of the same antigen or with a different epitope of a different antigen. bit binding. Thus, terms such as "bispecific", "trispecific", "tetraspecific" and the like refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.

The term "valence" herein refers to the presence of a defined number of binding sites in an antibody/antigen binding molecule. Accordingly, the terms "monovalent", "bivalent", "tetravalent" and "hexavalent" denote one binding site, two binding sites, four binding sites and six binding sites in an antibody/antigen binding molecule, respectively. point of existence.

Herein "full-length antibody", "intact antibody" and "intact antibody" are used interchangeably herein to refer to having a structure substantially similar to that of a natural antibody.

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

The term "Chimeric antibody" herein refers to an antibody whose light chain and/or heavy chain are partly derived from an antibody (which may be derived from a specific species or belong to a specific class or subclass of antibodies). 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 to 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 antibody's heavy and light chain variable regions are derived from a primary antibody (e.g., a murine antibody), and the antibody's heavy and light chains are The light chain constant region is from a second antibody (eg, a human antibody).

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

The term "fully human antibody" herein refers to antibodies having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues 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 that is involved in making the antibody bind to an 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 (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures, and each domain contains four conserved framework regions (FR) and three hypervariable regions (HVR). See, eg, 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 herein, and generally refer to the hypervariable region (HVR) of the heavy chain variable region (VH) or the light chain variable region (VL). It can form a precise complementarity with the antigen epitope, so it is also called complementarity determining region. Among them, the CDR of the variable region of the heavy chain can be abbreviated as HCDR, and the CDR of the variable region of the light chain can be abbreviated as LCDR. The terms "framework region" or "FR region" are used interchangeably and refer to those amino acid residues in an antibody heavy chain variable region or light chain variable region other than the CDRs. 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 descriptions 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); People such as Chothia, J.Mol.Biol.196:901-917 (1987); People such as Al-Lazikani B., J.Mol.Biol., 273:927-948 (1997); People such as MacCallum, 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). The "CDR" herein can be marked and defined by methods known in the art, including but not limited to Kabat numbering system, Chothia numbering system or IMGT numbering system, and the tool websites used include but 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 classical rule for identifying the boundaries of CDR regions based on the position of structural loop regions (see, for example, 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 carboxy-terminal portion of the heavy chain of an antibody that is not directly involved in binding the antibody to an antigen, but exhibits effector functions, such as interaction with Fc receptors, which are relative to the antibody's available Variable domains have more conserved amino acid sequences. The "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 that of a natural antibody constant region, while the latter only includes "full-length heavy chain constant region" part". Exemplarily, a typical "full-length antibody heavy chain constant region" consists of a 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 In the case of an antibody, it does not include a CH1 domain. Exemplarily, a typical "heavy chain constant region segment" can be selected from CH1, Fc or CH3 domains.

The term "light chain constant region" herein refers to the carboxy-terminal part of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen, and 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 carboxy-terminal part of the antibody obtained by papain hydrolysis of the whole 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 carboxyl 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 heavy chain of the antibody, thus the Fc region can comprise or excluding Lys447.

The term "conserved amino acid" herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity). Exemplarily, the amino acids in each of the following groups belong to each other's conservative amino acid residues, and the substitution of amino acid residues in the group belongs to the conservative amino acid substitution:

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), Histidine (H);

(5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

(6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

The term "identity" as used herein may be calculated by aligning said sequences for optimal comparison purposes in order to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences (for example, may be optimal alignment to introduce 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 corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.

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

The comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm in the GAP program that has been integrated into the GCG software package (available at www.gcg.com), using the Blossum 62 matrix or The PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 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), using the NWSgapdna.CMP matrix with gap weights of 40, 50, 60, 70, or 80 and length weights of 1, 2, 3, 4, 5 or 6, determining the percent identity between two nucleotide sequences. Specifically in some embodiments the parameter set (and one that 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.

It is also possible to use a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4, using the E. Meyers and W. Miller algorithm that has been incorporated into the ALIGN program (version 2.0), ((1989) CABIOS, 4:11-17 ) to determine 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, eg 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. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 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 utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov.

The term "CD3" (cluster of differentiation 3) herein refers to a cluster of differentiation 3 protein derived from any vertebrate source, including mammals, such as primates (such as humans, monkeys) and rodents (such as mice and rats). mouse). In mammals, the CD3 molecule is a multiprotein complex of six chains, including: a CD3γ chain, a CD3δ chain, a homodimer of two CD3ε chains, and a CD3ζ chain, where the CD3ζ chain is the intracellular tail of the CD3 molecule, and The CD3 gamma, CD3 delta and CD3 epsilon chains all contain an extracellular domain (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) CD3ε protein (NCBI Ref Seq No. NP_001270544), cynomolgus monkey (Macaca fascicularis) (monkey) CD3δ protein (NCBI Ref SeqNo. NP_001274617), Crab monkey (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).

Herein the term "CD3ε" is intended to encompass any form of CD3ε subunit, for example, 1) a native unprocessed CD3ε molecule, a "full length" CD3ε chain or a naturally occurring CD3ε variant, including for example splice variants or alleles Variant; 2) any form of CD3ε produced by processing in the cell; or 3) full length, fragment (e.g. truncated form, ectodomain/transmembrane domain) or modification of the CD3ε subunit produced by recombinant means (e.g., mutated, glycosylated/pegylated, histidine-tagged/immunofluorescent fused forms).

The term "HSA" herein refers to human serum albumin, having a molecular weight of 67 kD. There are 17 disulfide bonds inside the HSA protein, which makes the whole protein have good stability. HSA has the advantages of prolonging the half-life and promoting the drug to pass through the blood-brain barrier. It is not easy to pass through the glomerulus, and the half-life in plasma is as long as 2 Zhou, which is widely distributed in the body and has no immunogenicity, is an ideal protein drug carrier.

The term "CD70" herein, also known as "TNFSF7" or "CD27L", is a member of the TNF ligand family and is a 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 (splice variants), orthologs (Orthologs) and the aforementioned Fragment of CD70. The "CD70" herein may be derived from humans, primates such as monkeys (eg rhesus monkeys, cynomolgus monkeys) and rodents such as mice and rats. Exemplarily, the amino acid sequence of human CD70 can be found in UniProt number: P32970, and the amino acid sequence of rhesus monkey CD70 can be found in UniProt number: F7GPA5.

The term "CD33" herein is the smallest member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family. A type I transmembrane receptor protein with a molecular weight of 67kDa and 364 amino acids. Its N-terminus is located extracellularly, and the terminal amino acid consists of a conserved V-set immunoglobulin-like domain and a variable C2-set domain, in which V-set specifically recognizes and binds to sialic acid; the cytoplasmic tail There is an immunoreceptor tyrosine-based inhibitory motif (immunoreceptor tyrosine-based inhibitory motif, ITIM) and an ITIM-like structure at the end, which transmits inhibitory signals to the cell by combining with tyrosine phosphatase, so as to regulate cell growth. Purpose. The ITIM sequence in the CD33 molecule is different from other Siglecs in that the hydrophobic amino acid in front of tyrosine is 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 normal pleura, pericardium and peritoneum. The expression in normal tissues is limited, but MSLN was found to be highly expressed in epithelioid malignant pleural mesothelioma, lung adenocarcinoma, breast cancer, esophageal cancer, pancreatic tumor 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.

Herein the term "nucleic acid" includes any compound and/or substance comprising a polymer of nucleotides. Each nucleotide consists of a base, especially a purine or pyrimidine base (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e. deoxyribose or ribose) and phosphate groups. Typically, nucleic acid molecules are 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 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), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and synthetic forms of DNA or RNA comprising both Mixed polymers of one 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- 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 with derivatized sugar or phosphate backbone linkages or chemically modified residues. Nucleic acid molecules also encompass DNA and RNA molecules suitable as vectors for direct expression of 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 expression of the encoded molecule, so that the mRNA can be injected into a subject to generate antibodies in vivo (see e.g. Stadler et al., Nature Medicine 2017, published online 12 June 2017, doi: 10.1038/nm.4356 or EP 2 101 823 B1). An "isolated" nucleic acid herein refers to a nucleic acid molecule that has been separated from components of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than its natural chromosomal location.

The term "vector" herein refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it has been 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 vectors are referred to herein as "expression vectors".

The term "host cell" herein refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of such a cell. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical to the parental cell in nucleic acid content, 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.

As used herein, the term "pharmaceutical composition" refers to a preparation that is present in a form that permits the biological activity of the active ingredients contained therein to be effective and that does not contain substances that are unacceptably toxic to the subject to which the pharmaceutical composition is administered. additional ingredients.

The term "treatment" herein refers to surgical or therapeutic treatment, the purpose of which is to prevent, slow down (reduce) unwanted physiological changes or pathological changes in the subject of treatment, such as cancer, autoimmune diseases and viral infections. progress. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms, diminished extent of disease, stable disease state (i.e., not worsening), delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial response or complete response), whether detectable or undetectable. Those in need of treatment include those already with the condition or disease as well as those prone to have the condition or disease or those in which the condition or disease is to be prevented. When referring to the terms slow down, lessen, weaken, moderate, alleviate, etc., the meaning of eliminate, disappear, not occur, etc. is also included.

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

The term "effective amount" herein refers to an amount of a therapeutic agent effective to prevent or alleviate a disease condition or the progression of the 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, eg, treat, cure, prevent or alleviate the associated medical condition, or to increase the rate of treatment, cure, prevent or alleviate such condition. When the active ingredient is administered to a subject alone, a therapeutically effective dose refers to that ingredient alone. When a combination is used, a therapeutically effective dose refers to the combined amounts of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.

The term "autoimmune disease" herein refers to a condition in which cells, tissues and/or organs are damaged by a subject's immune response to its own cells, tissues and/or organs.

The term "cancer" herein refers to or describes the physiological condition in mammals typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition. The term "tumor" or "neoplastic" herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues. The terms "cancer" and "tumor" are not mutually exclusive when referred to herein.

Description of drawings

1A-1B. Schematic structure of multispecific antibody.

2A-2C. ELISA detection of the binding reaction between MSLN multispecific antibody and albumin: 2A. HAS; 2B. CSA; 2C. MSA.

3A-3B. ELISA detection of the binding reaction of MSLN multispecific antibody to MSLN protein and CD3 protein simultaneously: 3A. Human CD3; 3B. Monkey CD3.

4A-4E. FACS detection of the binding reaction of MSLN multispecific antibodies to tumor cells: 4A.OVCAR3; 4B.Hela; 4C.Hs766T; 4D.NCI-H292; 4E.A431.

Figure 5. FACS detection of the binding reaction of MSLN multispecific antibody to HEK293T-hMSLN-R3 cells.

Figure 6A. FACS detection of the binding reaction of MSLN multispecific antibody to HEK293T-monkey MSLN cells;

Fig. 6B. FACS detection of the binding reaction of MSLN multispecific antibody to HEK293T cells.

Figure 7A. FACS detection of the binding reaction of MSLN multispecific antibody to human T cells;

Fig. 7B. FACS detection of the binding reaction of MSLN multispecific antibody to 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 antibodies.

Figure 10. FACS detection of the effect of CA125 on the binding activity of MSLN multispecific antibodies.

Figures 11A-11D. Binding of bi-epitope MSLN multispecific antibodies.

Figure 12A. Endocytic activity of 100 nM MSLN multispecific antibody;

Figure 12B. Endocytic activity of 10 nM MSLN multispecific antibody;

Figure 12C. Endocytic activity of 1 nM MSLN multispecific antibody.

Figure 13. Detection of T lymphocyte activation state mediated by MSLN multispecific antibody in vitro.

Figure 14. MSLN multispecific antibody mediates T cell killing activity of tumor cells in vitro and cytokine detection.

Figure 15. Multispecific antibody inhibits humanized ovarian cancer tumor growth curve

Figure 16. The tumor growth curve of the mixed subcutaneous model of ovarian cancer cell OVCAR-3 and PBMC inhibited by multispecific antibodies.

Figure 17. Multispecific antibody inhibition of humanized lung cancer tumor growth curve.

Figure 18. Comparison of pharmacokinetics of multispecific antibodies in cynomolgus monkeys.

Figure 19A. Stability testing of multispecific antibodies in human serum.

Figure 19B. Stability testing of multispecific antibodies in cynomolgus monkey serum.

Fig. 20A. FACS detection of the binding reaction of CD33×CD70 multispecific antibody to THP-1 cells.

Fig. 20B. FACS detection of the binding reaction of CD33×CD70 multispecific antibody to U937 cells.

Fig. 20C. FACS detection of the binding reaction of CD33×CD70 multispecific antibody to 786-O cells.

Fig. 20D. FACS detection of the binding reaction of CD33×CD70 multispecific antibody to Jurkat cells.

Figure 21. FACS detection of the binding reaction of CD33×CD70 multispecific antibody to CHO-K1-human CD33 cells.

Figure 22. FACS detection of the binding reaction of CD33×CD70 multispecific antibody to CHO-K1-human CD70 cells.

Figure 23. Antibody killing activity experiment on tumor cells in different formats.

Figure 24. Antibody killing activity experiment on monocytes in different formats.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

The embodiments of the present invention are merely exemplary, and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

Example 1 Construction and preparation of multispecific antibodies

1.1 Structure design of multispecific antibody

The multispecific antibody of the present invention can be a tri-specific (tri-specific) or tetra-specific (tetra-specific) antibody, and the specificity can refer to binding to different antigenic targets, or can refer to binding to the same antigenic target. different epitopes. Please refer to Figures 1A-1B and Table 1 for the schematic structure of the multispecific antibody. The multispecific antibody contains at least three parts: (A) target antigen binding part, (B) half-life extension part, and (C) T cell joint part.

The (A) target antigen-binding portion can be selected from a target antigen-binding antibody or a target antigen-binding ligand, and the target antigen-binding antibody can be selected from any antigen-binding fragment, preferably Fd, Fv, scFv, diabody or single Domain antibody (VHH). The (B) half-life extending moiety is preferably an anti-HSA antibody. The (C) T cell engaging moiety, wherein the T cell engaging moiety is preferably an anti-CD3 antibody or an 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, wherein (A) target antigen binding part or (C) T The cell-engaging part may appear repeatedly or contain multiple parts (for example, a multispecific antibody contains two target antigen-binding parts, A1 and A2, and A1 and A2 may be the same or different). The connection between the various parts of the multispecific antibody may be directly connected through a linker or not through a linker.

Table 1. Schematic structure of multispecific antibody

1.2 Preparation of multispecific antibodies

Construct a multispecific antibody according to the multispecific antibody structure described in 1.1, where the (A) target antigen binding part, (B) half-life extension part, (C) T cell engaging part, and/or linker used can be selected from The sequence shown in Table 2 below, the CDR region division of the antibody sequence shown in Table 2 can use the numbering rules or definition methods commonly used in the field such as IMGT, Kabat, Chothia, AbM, Contact, etc. Table 3 only shows the Kabat method to define the division CDRs , but not as limited:

Table 2. Sequence information of antigen-binding fragments

Table 3. CDR sequence information

The nucleotide sequences containing the amino acid sequences encoding the multispecific antibodies (see Table 4) were respectively constructed as recombinant plasmids. The schematic structure of the multispecific antibody is shown in Fig. 1B. Plasmid construction and antibody expression and purification were completed by Taizhou Baiying Biotechnology Co., Ltd. In the stage of large-scale screening of multispecific antibodies, in order to facilitate small-volume antibody expression and purification, as well as rapid detection, a tag His tag (HHHHHH) was added to the C-terminus of multispecific antibodies; The antibody sequence of His tag optimizes the method of antibody expression and purification. The sequences of multispecific antibodies 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, product number: A29133) to OptiPRO SFM (Thermofisher, product number: 12309019) and mix well, then let it stand for 5 minutes, add it to ExpiCHO-S ^TM cells (manufacturer: Thermofisher, product number: A29127), put Into 5% CO ₂ , 120 rpm, 37°C shaker culture. On the second day after transfection, feed was added, and the shaker temperature was adjusted to 32°C to continue culturing. On day 9 of transfection, the cell supernatant was collected. The cell expression supernatant sample was centrifuged at high speed to remove impurities, and the buffer was replaced with PBS, and imidazole was added to a final concentration of 5 mM. Equilibrate the nickel column with PBS solution containing 5mM imidazole, wash 2-5 times the column volume. The replaced supernatant sample was loaded onto the column for binding, and the column was washed with PBS solution containing 5 mM imidazole until the A280 reading dropped to the baseline. Afterwards, the chromatographic column was washed with PBS+10mM imidazole to remove non-specifically bound impurity proteins, and the effluent was collected. The target protein was then eluted with PBS solution containing 300 mM imidazole, and the elution peaks were collected. After concentration, the collected eluted product can be further purified by gel chromatography Superdex200 (GE), the mobile phase is PBS, the polymer and foreign protein peaks are removed, and the eluted peak of the target product is collected. The obtained proteins were electrophoresed and identified by SEC-HPLC and sorted for use.

1.3 Preparation of control antibody

The sequences of the positive control antibody and negative control antibody of Anti-MSLN come from the SEQ ID NO: 101 and SEQ ID NO: 99 of the patent US20180327508A1, named MB001 and MB060 respectively (see Table 4 for the sequence), all of which are trispecific antibodies with a structure A schematic is shown in Figure 1B. Both the positive control antibody and the negative control antibody were constructed by Taizhou Baiying Biotechnology Co., Ltd. and the production and purification of antibodies were completed. For specific methods, see Example 1.2.

Table 4. Multispecific antibody sequence information

Example 2 Identification of the binding ability of Anti-MSLN multispecific antibody to protein or cell

2.1 Enzyme-linked immunosorbent assay (ELISA) to detect the binding of antibody to human serum albumin

In order to detect the binding activity of Anti-MSLN multispecific antibody to human serum albumin, human serum albumin (HSA, purchased from Chengdu Rongsheng, article number: S10940024), monkey serum albumin (CSA, purchased from: Abcam, product number: ab184894), mouse serum albumin (MSA, purchased from: Alpha Diagnist, product number: ALB14-N-10) was diluted with PBS to a final concentration of 4 μg/mL, and then added to a 96-well ELISA plate at 50 μl/well , sealed with a plastic film and incubated at 4°C overnight, washed the plate twice with PBST the next day, and added blocking solution [PBS+2% (w/w) BSA] to block at room temperature for 2 hours. Pour off the blocking solution, wash the plate twice with PBST, and add the test antibody or control antibody with an initial concentration of 100nM or 200nM and a 1:3 gradient dilution at 50μl/well. After incubation at 37°C for 1 hour, the plate was washed 3 times with PBST. Add HRP (horseradish peroxidase) labeled Anti-his secondary antibody (purchased from Genscript, product number: A00612), incubate at 37°C for half an hour, and wash the plate 5 times with PBST. Add TMB substrate at 50 μl/well, incubate at room temperature for 10-15 minutes, then add 50 μl/well stop solution (1.0N HCl). Read the OD450nm value with an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The experimental results are shown in Figures 2A-2C and Table 5-7. MB060 is a negative control; MB001 is a positive control. 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

Table 6 The binding of Anti-MSLN multispecific antibody to CSA

Table 7 The binding of Anti-MSLN multispecific antibody to MSA

2.2 Enzyme-linked immunosorbent assay (ELISA) to detect the ability of multispecific antibodies to simultaneously bind to MSLN protein and CD3 protein

Human MSLN extramembrane protein (296-580, GenBank: AAH09272.1, His-tag, self-expression) was diluted with PBS to a final concentration of 2 μg/mL, then added to 96-well ELISA plate at 50 μl/well, covered with plastic film Seal and incubate overnight at 4°C, wash the plate twice with PBST the next day, add blocking solution [PBS+2% (w/w) BSA] to block at room temperature for 2 hours. Pour off the blocking solution, wash the plate twice with PBST, and add the test antibody or control antibody with an initial concentration of 100nM or 200nM and a 1:3 gradient dilution at 50μl/well. After incubation at 37°C for 1 hour, the plate was washed 3 times with PBST. Dilute human CD3 protein (purchased from Sino Biological, catalog number: 10977-H03H) and monkey CD3 protein (purchased from: Acro, catalog number: CDE-C5254) to 2 μg/mL, and add 50 μl/well to a 96-well ELISA plate. After incubation at 37°C for 1 hour, the plate was washed 3 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 TMB substrate at 50 μl/well, incubate at room temperature for 10-15 minutes, then add 50 μl/well stop solution (1.0N HCl). Read the OD450nm value with an ELISA plate reader (Multimode Plate Reader, EnSight, purchased from Perkin Elmer). The experimental results are shown in Figures 3A-3B and Tables 8-9. MB060 is a negative control; MB001 is a positive control. The data in the table are OD450nm values. The results showed that all Anti-MSLN multispecific antibodies had the ability to simultaneously bind MSLN protein and CD3 protein; and had cross-binding activity with monkey CD3 protein.

Table 8 The binding of Anti-MSLN multispecific antibody to human CD3 protein

Table 9 The binding of Anti-MSLN multispecific antibody to monkey CD3 protein

2.3 Flow cytometry (FACS) detection of antibody binding to tumor cells

In the experiment, the human ovarian cancer cell line OVCAR3 (purchased from ATCC, product number: HTB-161) with high expression of MSLN, the human cervical cancer cell line Hela and the human pancreatic cancer cell line Hs766T (purchased from the Chinese Academy of Medical Sciences Foundation) were used in the experiment. Institute of Medicine Cell Resource Center), human lung cancer cell line NCI-H292 with low MSLN expression (purchased from ATCC, catalog number: CRL-1848), and human skin cancer cell line A431 that does not express human MSLN protein.

Expand the required cells to the logarithmic growth phase in a T175 cell culture flask, remove the medium, wash twice with PBS buffer, digest the cells with trypsin, then stop the digestion with complete medium, and pipette the cells to single cell suspension. After counting the cells, centrifuge, wash the cell pellet twice with PBS, resuspend the cell pellet with FACS buffer (PBS+2% fetal calf serum) to 2×10 ⁶ cells/mL, add 50 μl per well to 96-well FACS In the reaction plate, add the test antibody or control antibody (200nM as the initial concentration, 3-fold or 5-fold gradient dilution) at 50 μl/well, mix with the cell suspension, and incubate at 4°C for 1 hour. After centrifugation and washing with PBS buffer for 3 times, 50 μl of iFluor 647-labeled Anti-His secondary antibody (purchased from Genscript, catalog number: A01802-100) was added to each well, and incubated at 4°C for 1 hour. Centrifuge and wash 3 times with PBS buffer, resuspend in 100 μl PBS, detect and analyze the results with FACS (FACS Canto ^™ , purchased from BD Company). Data analysis was performed by software (CellQuest) to obtain the mean fluorescence intensity (MFI) of the cells. Then analyze by software (GraphPad Prism8), perform data fitting, and calculate EC50. The analysis results are shown in Figures 4A-4E and Table 10, wherein MB060 is a negative control; MB001 is a positive control. 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 the expression human Tumor cells with MSLN protein.

Table 10 The binding reaction of Anti-MSLN multispecific antibody to tumor cells

2.4 Flow cytometry (FACS) detection of antibody binding to HEK293T-hMSLN-R3

For the preparation method of recombinant HEK293T cell line HEK293T-hMSLN-R3 expressing human MSLN-R3 protein, please refer to the nucleotide sequence of PCT/CN2021/136419 (coding the amino acid sequence of human MSLN-R3 (NCBI: Met487-Ser606 of AAH09272.1) Be cloned to pcDNA3.1 carrier and prepare plasmid.HEK293T cell line (purchased from ATCC) is carried out plasmid transfection (

3000 Transfection Kit, purchased from Invitrogen, product number: L3000-015), using puromycin to selectively culture for 2 weeks, sorting positive monoclonal cells into 96-well plates and selecting some monoclonal wells for amplification. The amplified clones were detected and analyzed by FACS flow cytometer with specific antibodies, and the cell lines with better growth and higher fluorescence intensity were selected to continue to be expanded and cultured and frozen in liquid nitrogen. The detection shows that HEK293T-hMSLN-R3 after puromycin pressure selection has a relatively single positive peak). The preparation of the detection cells and the antibody to be tested and the detection method refer to Example 2.3. The analysis results are shown in Figure 5 and Table 11. The results showed that MB065 and MB069 had binding activity to recombinant cells expressing human MSLN-R3 protein, indicating that Anti-MSLN monoclonal antibodies combined into multispecific antibodies retained the characteristics of monoclonal antibodies; while MB001 did not bind to recombinant cells expressing human MSLN-R3 protein Recombinant cell binding.

Table 11 Binding reaction of Anti-MSLN multispecific antibody to HEK293T-hMSLN-R3

2.5 Flow cytometry (FACS) detection of antibody cross-binding with HEK293T-monkey MSLN

For the preparation method of recombinant HEK293T cell line HEK293T-monkey MSLN expressing monkey MSLN protein, please refer to PCT/CN2021/136419 (the nucleotide sequence encoding the full-length amino acid sequence of monkey MSLN (NCBI: XP_028696439.1) was cloned into the pcDNA3.1 vector And prepare plasmid. HEK293T cell line (purchased from ATCC) is carried out plasmid transfection (

3000 Transfection Kit, purchased from Invitrogen, product number: L3000-015), using puromycin to selectively culture for 2 weeks, sorting positive monoclonal cells into 96-well plates and selecting some monoclonal wells for amplification. The amplified clones were detected and analyzed by FACS flow cytometer with specific antibodies, and the cell lines with better growth and higher fluorescence intensity were selected to continue to be expanded and cultured and frozen in liquid nitrogen. The detection showed that the HEK293T-monkey MSLN after puromycin pressure selection had a relatively single positive peak). The preparation of the detection cells and the antibody to be tested and the detection method refer to Example 2.3. The analysis results are shown in Figures 6A-6B and Table 12, wherein MB060 is a negative control. The results showed that all the Anti-MSLN multispecific antibodies had specific binding activity to the recombinant cells expressing monkey MSLN protein.

Table 12 Binding reaction of Anti-MSLN multispecific antibody to HEK293T-monkey MSLN

2.6 Flow cytometry (FACS) detection of antibody binding to human T cells expressing CD3 protein

Human PBMC cells (purchased from Aussells Biotechnology (Shanghai) Co., Ltd.) were prepared according to the instructions of T Cell Activation/Expansion Kit, human (purchased from Miltenyi, product number: 130-091-441) to complete the T cell activation. Isolation and in vitro activation amplification experiments. When the cells were cultured for 14 days, the medium 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, catalog number: 109-605-098) was used for FACS (FACS CantoTM, purchased from BD Company) detection. The results showed that human T cells had a higher CD3 protein expression.

For the preparation and detection methods of the above detection cells and antibodies to be tested, refer to Example 2.3. The analysis results are shown in FIGS. 7A to 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 The binding reaction of Anti-MSLN multispecific antibody to cells expressing CD3 protein

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, the experimental method of the reporter gene is specially established. Multispecific antibodies can simultaneously bind 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 in CD16a cells was activated, the expression level of luciferase increased, and the fluorescent signal could be detected after adding the substrate. The intensity of the fluorescent signal can characterize the intensity of the activation of the signaling pathway.

Refer to Example 2.3 for the preparation process of tumor cells, adjust the cell density to 4×10 ⁵ cells/ml, add 100 μl per well into a 96-well cell culture plate, and culture overnight in a cell incubator. Discard the cell culture medium, wash twice with PBS, add the antibody to be tested or control antibody (60nM as the initial concentration, 5-fold serial dilution, 10-12 dilution points) at 50 μl/well, and then add 50 μl/well Jurkat-NFAT-Luciferase-CD16a cells (at a density of 1×10 ⁶ cells/ml) were mixed evenly and placed in a cell incubator for 6 hours. Take it out and let it stand at room temperature, add 50 μl/well Bright-Glo ^TM Luciferase Assay System (purchased from Promega, product number: E2620), shake and react in the dark for 5 minutes, transfer the suspension to the detection plate (purchased from Costar , Cat. No.: 3610), detected by EnSight (purchased from: Perkin Elmer), and then analyzed by software (GraphPad Prism8) to perform data fitting and calculate EC50. The analysis results are shown in Figures 8A-8D and Tables 14-17, wherein MB060 is a negative control and MB001 is a positive control. The results showed that all Anti-MSLN multispecific antibodies could activate NFAT-related signaling pathways in Jurkat-NFAT-Luciferase-CD16a cells.

Table 14 Anti-MSLN multispecific antibody reporter gene experiment (OVCAR3)

Table 15 Anti-MSLN multispecific antibody reporter gene experiment (Hela)

Table 16 Anti-MSLN multispecific antibody reporter gene experiment (Hs766T)

Table 17 Anti-MSLN multispecific antibody reporter gene experiment (NCI-H292)

Example 4 Blocking of Anti-MSLN multispecific antibody binding to CA125

CA125 has been confirmed to bind to MSLN in the R1 region of the distal end of the membrane. To confirm that antibodies against the R1 epitope can block the binding of CA125 to MSLN protein, both protein and cellular levels were evaluated. The main experimental procedures of ELISA and FACS can refer to Examples 2.1 and 2.3, respectively. ELISA and FACS primary antibody preparation instructions: Add the antibody to be tested or the control antibody (400nM as the initial concentration, 3-fold serial dilution, 11 dilution points) at 50μl/well, incubate at room temperature for 30 minutes, and then add 0.4μg at 50μl/well /ml CA125 solution (purchased from Baiying, product number: B475601), mix well and continue to incubate at room temperature for 1 hour. The ELISA secondary antibody was horseradish peroxidase-coupled Streptavidin (purchased from Sigma, product number: S2438), and the FACS secondary antibody was APC-coupled Streptavidin (purchased from Biolegend, product number: 405243).

The analysis results are shown in Figures 9-10 and Tables 18-19, wherein MB060 is a negative control. The results showed that the Anti-MSLN multispecific antibody could block the binding of CA125 and human MSLN protein at the protein and OVCAR3 cell levels.

Table 18 ELISA detection of Anti-MSLN multispecific antibody binding to CA125 blocking situation

Table 19 FACS detection of Anti-MSLN multispecific antibody binding to CA125 blocking situation

Example 5 Binding of Anti-MSLN bi-epitope multispecific antibody

In order to verify whether the two MSLN-binding fragments of the bi-epitope multispecific antibody simultaneously bind to OVCAR3 tumor cells expressing human MSLN protein, a high concentration of monoclonal antibody against the R1 epitope was added to the system in which the multispecific antibody was bound to the cells (NB149-95) or monoclonal antibody against the R3 epitope (F7.44.20), the purpose is to detect whether the antibody binding to another epitope in the multispecific antibody blocks or weakens the binding of the antibody to another epitope active.

NB149-95, SEQ ID NO.100

F7.44.20-VH, SEQ ID NO.101

F7.44.20-VL, SEQ ID NO.102

The main experimental process can refer to Example 2.3. Primary antibody preparation instructions: Add the antibody to be tested or control antibody (200nM as the initial concentration, 5-fold serial dilution, 8 dilution points) at 50μl/well, then add 50μl/well of 500nM monoclonal antibody solution, and mix well. The analysis results are shown in FIGS. 11A to 11D and Table 20. The results showed that the binding activity of MB001 and MB048 decreased in the presence of high-concentration NB149-95 monoclonal antibody, while MB065 (MSLN R1/R3) and MB069 (MSLN R1/R3) had no binding activity in the presence of different epitope monoclonal antibodies. Significantly lower, demonstrating that antibodies targeting two epitopes (MSLN R1/R3) can simultaneously bind to MSLN membrane protein.

Table 20 FACS detection of Anti-MSLN bi-epitope multispecific antibody binding

Example 6 Endocytic activity of Anti-MSLN multispecific antibody

OVCAR3 cell preparation and detection methods refer to Example 2.3. Instructions for primary antibody preparation and endocytosis conditions: Add the antibody to be tested or the control antibody (200nM as the initial concentration, 10-fold serial dilution, 3 dilution points) at 50μl/well, mix well and incubate at 4°C for 1 hour, wash with PBS Discard the supernatant after 3 times. Add 50 μl/well of FACS buffer, resuspend the cells, place one at 4°C for 1 hour, and the other at 37°C for 1 hour. After the supernatant was centrifuged, the diluted secondary antibody was added.

The analysis results are shown in Figures 12A to 12C and Tables 21-22. The results showed that both MB065 and MB069 had endocytic activity, and both of them were weaker than that of positive control antibody MB001, among which MB065 had the weakest endocytic activity. If the CD3 multispecific antibody binds to tumor cells, most of the antibody is quickly internalized, which will reduce the chance of binding to T cells, resulting in greatly weakened T cell activation and tumor killing activity.

Table 21 FACS detection of endocytosis of Anti-MSLN multispecific antibody

Table 22 FACS detection of endocytosis rate of Anti-MSLN multispecific antibody

Endocytosis rate = (4℃_MFI-37℃_MFI)/4℃_MFI*100%

Example 7 In vitro activation state detection of T lymphocytes 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, the expression of CD69 is significantly up-regulated. It is a test to detect whether T lymphocytes are effectively induced and activated. of markers.

Primary T cells were isolated and enriched from PBMCs of healthy donors using a T cell isolation kit (purchased from STEMCELL technologies, product number 17951, refer to the kit instructions for usage), and cultured overnight. NCI-H292 cells were digested with trypsin to prepare a single cell suspension. Adjust the tumor cell density to 2×10 ⁵ cells/ml and the T cell density to 1×10 ⁶ cells/ml with complete medium. Take 50 μl of adjusted NCI-H292 cells and 50 μl of primary T cells and mix them evenly, and add them to each well of a flat-bottomed 96-well plate with a micropipette. The volume of the cell mixture is 100 μl/well (tumor cells and The numbers of T cells were 1×10 ⁴ and 5×10 ⁴ respectively). The antibodies were serially diluted with complete medium (the initial antibody concentration was 2nM, 6-fold dilution, 8 concentration gradients), and 100 μl of antibody dilutions of different concentrations were added to each well, so that the final volume of each well was 200 μl. Cultivate in vitro for 24 hours in an incubator. Centrifuge to remove the supernatant, resuspend with flow buffer, add APC-anti-human CD3 (purchased from Biolegend, product number 300312), FITC-anti-human CD69 (purchased from Biolegend, product number 317408), and react in the dark for 30 minutes. Flow cytometry was used to measure the changes in the proportion of CD69+CD3+ T cells after different concentrations of antibody treatment. The results were calculated and graphed using GraphPad Prism 9.0 software, see Figure 13.

MB001 has the strongest T cell activation activity due to the use of high-affinity CD3 antibodies, while MB048, MB065 and MB069 with low-affinity CD3 have weaker T cell activation activities; MB060 negative control has no T cells because it cannot bind to MSLN Activation activity.

Example 8 Detection of In vitro Killing Activity and Cytokine Release of T Cells Mediated by Multispecific Antibodies against Tumor Cells

In this experiment, three tumor cell lines were selected as the target cells of T cell killing experiment in vitro, among which OVCAR3 was human ovarian cancer cells with high expression of MSLN, NCI-H292 was human lung cancer cells with low expression of MSLN, and A431 was human lung cancer cells without MSLN expression. Skin squamous cell carcinoma cells were used as a negative control. Primary T cells were isolated from PBMCs of healthy donors using a T cell isolation kit (purchased from STEMCELL technologies, product number 17951, refer to the kit instructions for usage) and cultured overnight.

On the second day, OVCAR3, NCI-H292 or A431 cells were digested with trypsin to prepare a single cell suspension. Adjust the tumor cell density to 2×10 ⁵ cells/ml and the T cell density to 1×10 ⁶ cells/ml with complete medium. Take 50 μl of adjusted OVCAR3, NCI-H292 or A431 cells and 50 μl of primary T cells and mix them evenly (the numbers of tumor cells and T cells in each well are 1×10 ⁴ and 5×10 ⁴ ), and use a micropipette Add the pipette to each well of a flat-bottomed 96-well plate, and the volume of the cell mixture is 100 μl/well. The antibodies were serially diluted with complete medium (the initial antibody concentration was 25nM, 6-fold dilution, 8 gradients), and 100 μl of antibody diluents of different concentrations were added to each well, so that the final volume of each well was 200 μl. After culturing for 24 hours, the supernatant was taken to detect the production of IFNγ, TNFα and IL-6 cytokines in T cells by ELISA (the kit was purchased from BD, the article numbers were 555142, 555212 and 555220, respectively, and the method of use was referred to the kit instruction manual). The CellTiter-Glo kit (purchased from Promega, catalog number G9243, refer to the product manual for usage) was used to detect cell viability. Define only the well of T cells as the positive control of killing 100%, define the antibody concentration as 0 as the blank negative control, cell killing rate=(blank well reading value-sample well reading value)/(blank well well reading value-positive control Well reading) x 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 levels, it can be seen that whether it is OVCAR3 cells with high expression of MSLN or NCI-H292 cells with low expression of MSLN, MB069 shows the strongest activation of T cells and tumor cells in vitro. T cell activation and in vitro tumor cell killing activity 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 CD3 multispecific antibody has good target antigen specificity, and will not produce non-specific killing on normal tissues that do not express the target antigen . The release level of cytokines reflects the activity of multispecific antibodies, and generally positively correlates with the activation level of T cells, killing activity in vitro and antitumor activity in vivo. At the same time, the stronger the T cell activation activity of multispecific antibodies, the higher the risk of cytokine storm (CRS) after entering the human body.

Example 9 In vivo anti-tumor activity of multispecific antibody in human ovarian cancer OVCAR-3 (highly expressed MSLN) xenograft model

9.1 In vivo anti-tumor activity of multispecific antibodies in human PBMC-reconstituted human ovarian cancer OVCAR-3 (highly expressed MSLN) xenograft model

In this embodiment, human PBMC cells are injected into immunodeficiency NPG mice (5-6w, female, Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) by tail vein injection to obtain a human PBMC reconstruction model, and then in this animal model Established a human ovarian cancer OVCAR-3 xenograft model (xenograft model) with high MSLN expression and a human lung cancer NCI-H292 xenograft model with low MSLN expression, and validated multispecific antibodies in these two models antitumor activity in vivo.

Expand OVCAR-3 cells (American Type Culture Collection, ATCC) to the required number in T-300 cell culture flasks, and in its logarithmic growth phase (confluence is about 80%; the day before inoculation needs to be given Cells were replaced with fresh medium) to begin collection. First, the medium in the cell culture flask was removed, washed twice with phosphate buffer saline (PBS, Thermo Fisher Scientific Co., Ltd., Hyclone, 29588138), and then an appropriate amount of 0.25% trypsin digestion solution (Thermo Fisher Co., Ltd., Gibco, #25200-072/219085), shake the bottom of the bottle, so that the trypsin digestion solution evenly covers the cell surface, digest at 37°C for 5min, add 20% fetal bovine serum (Fetal Bovine serum, FBS, Sai The complete medium of Mofei Shier Technology Co., Ltd., Gibco, #10099-141C/2261480CP) was used to stop the digestion reaction, and the cells adhered to the bottom of the bottle were gently blown away from the culture bottle, and the digested cell suspension was collected into a 50 mL centrifuge Tube, 350g, centrifuged for 5min, 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 70μm sieve, take 500μL of the cell suspension The liquid was counted in a cell counter, and finally, according to the cell counting results, the cell density was adjusted to 1×10 ⁷ cells/mL using serum-free medium, placed on ice, and transferred to the SPF animal room through the transfer window for inoculation modeling. Before inoculation, the above cell suspension was mixed with Matrigel (Corning Biotechnology Co., Ltd., #356237) in equal proportions, and 200 μL of the above cell mixture was inoculated subcutaneously in the right axilla of each mouse.

On the second day after inoculation of the above tumor cells, resuscitate the corresponding number of human peripheral blood mononuclear cells (PBMC, Shanghai Saili Biotechnology Co., Ltd., #200256), filter through a 70 μm sieve, take 100 μL of cell suspension, add 400 μL RPMI-1640 without Serum culture medium was counted, and according to the counting results, the PBMC cell concentration was adjusted to 2.5×10 ⁷ cells/mL using RPMI-1640 serum-free medium, 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 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 50-100 mm ³ , orbital blood was collected for the detection of human immune system reconstitution. Mice with appropriate tumor volume and immune reconstitution results were selected for grouping (n=5) and given multiple doses. For specific antibody drug treatment, the dosage regimen is shown in Table 23, wherein MB060 is a negative control, MB001 is a positive control, and MB048, MB065 and MB069 are antibodies to be tested. The dosage is based on the molar weight per unit body weight of the positive molecule MB001, and the weight per kilogram of mouse is converted into the weight per unit weight of the drug according to the formula m=n*M, and finally the weight per unit weight is obtained. Rat administration mass, where m is the administration mass, n is the administration molarity, and M is the molecular weight of the drug, and the administration doses in this article are calculated according to this principle.

Table 23 Drug administration regimen in vivo in humanized ovarian cancer tumor-bearing mice

The results are shown in Figure 15, MB065 (tumor inhibition rate = 69.98%) exhibited better tumor inhibitory 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 is calculated by this formula in this paper.

9.2 In vivo anti-tumor activity of multi-specific antibodies in a xenograft model in which human PBMC and human ovarian cancer cell OVCAR-3 (highly expressed in MSLN) were subcutaneously inoculated into tumors

The treatment method of OVCAR-3 cells is the same as that in 9.1. Use serum-free medium to adjust the cell density to 12×10 ⁷ cells/mL, and store on ice for later use. At the same time, the treatment method of PBMC is the same as that in 9.1. Use RPMI-1640 serum-free medium to adjust the concentration of PBMC cells to 3× ¹⁰⁷ cells/mL, put them on ice, and transfer the above-mentioned cell suspensions and Matrigel matrigel ( Corning Biotechnology Co., Ltd., #356237) was transferred to the SPF animal room for inoculation modeling, mixed in the ultra-clean bench of the SPF animal room according to Table 24, and then subcutaneously inoculated 200 μL of the above cell mixture in the right axilla of each mouse. After inoculation, the tumor growth was monitored. When the overall tumor volume was about ^50-100mm3 , they were divided into groups (n=5) and given multispecific antibody drug therapy. The dosage regimen was shown in Table 23, where MB060 was the negative control and MB001 was the negative control. Positive controls, MB048, MB065 and MB069 are the antibodies to be tested.

Table 24 OVCAR-3 and PBMC mixed subcutaneous inoculation cell mixing scheme

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 than positive molecule MB001 (tumor inhibition rate = 89.09%) %) antitumor activity.

Example 10 In vivo anti-tumor activity of multispecific antibody in human PBMC reconstituted human lung cancer NCI-H292 (low expression of MSLN) xenograft model

In this example, the human lung cancer cell NCI-H292 with low expression of MSLN was selected to establish a mouse (NPG, 5-6w, female, Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) in vivo model, and the multi-specificity was verified on this model. anti-tumor activity of antibodies in vivo.

Human lung cancer cells NCI-H292 (American Type Culture Collection, ATCC) were processed according to the method described in Example 9.1 (the complete medium containing 10% fetal bovine serum was terminated digestion), and finally according to the cell counting results, use Serum-free medium was used to adjust the cell density to 5×10 ⁷ cells/mL, placed on ice, and transferred to the SPF animal room through the transfer window for inoculation modeling, and 200 μL of the above cell suspension was inoculated subcutaneously in the right axilla of each mouse.

On the second day after the above tumor cells were inoculated, the PBMCs were treated in the same way as in 9.1. Use RPMI-1640 serum-free medium to adjust the PBMC cell concentration to 2.5×10 ⁷ cells/mL, place them on ice, and transfer them to the SPF animal room through the transfer window. For inoculation modeling, each mouse was inoculated with 200 μL of the above cell suspension in the tail vein for immune reconstitution. After inoculation, the tumor growth was monitored. When the overall tumor volume was around 100 mm ³ , blood was collected from the orbit to detect the reconstitution of the human immune system. Mice with appropriate tumor volume and immune reconstitution results were selected for grouping (n=8) and given multispecific For antibody drug treatment, the dosage regimen is shown in Table 25, wherein MB060 is a negative control, MB001 is a positive control, and MB048, MB065 and MB069 are antibodies to be tested.

Table 25 Drug administration regimen in vivo in humanized lung cancer tumor-bearing mice

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 tumor suppression 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 were equivalent, and were 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 = 54.48%) were basically the same tumor inhibition rate, both were significantly weaker than MB069 (tumor inhibition rate = 84.05%), and MB048 was significantly weaker than MB065.

Example 11 Pharmacokinetic experiment of multispecific antibody in cynomolgus monkey

Seven cynomolgus monkeys aged 3-5 weeks, weighing 3.7-5.7kg, were grouped according to different test products and doses, and the drug was diluted with a preparation solvent, and different individuals were given single intravenous injections of MB001, MB069 and MB065, and their pharmacokinetics were compared. Kinetic difference. Blood samples were collected from the hindlimb vein, and the blood collection time points were before administration, 0.25 hours, 2 hours, 4 hours, 8 hours, 24 hours, 48 hours, 72 hours, 120 hours, 168 hours, 240 hours, 336 hours, 504 hours after administration hours, 672 hours and 744 hours. After blood samples were collected, they were stored on ice for blood sample separation. Then, centrifuge at 1500×g at 4°C for 10 minutes to separate the serum into a low-adsorption centrifuge tube, mark the compound code and time point, and store it at -80°C before analysis. Blood samples should be collected and frozen within 2 hours after centrifugation. The hMSLN-FL-his protein was coated, and the concentrations of the compounds in the control and test groups of the present invention in the serum were determined by indirect enzyme-linked immunosorbent assay. Pharmacokinetic parameters were calculated based on the plasma concentrations of each animal at different time points. It can be seen from the results that the half-lives of MB001, MB069 and MB065 can all support clinical administration once a week.

Table 26 In vivo pharmacokinetic detection in cynomolgus monkeys

Example 12 Stability experiment of multispecific antibody in human serum and cynomolgus monkey serum

12.1 Stability test of multispecific antibody 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 that of NCI-H292 cells in Example 7. Antibody treatment is divided into two types: 1. The antibody is not pretreated; 2. The antibody is pre-incubated in human serum at 37°C for two days. Differently treated antibodies were serially diluted with complete medium (the initial antibody concentration was 25 nM, 6-fold dilution, 10 concentration gradients). 100 μl of antibody diluents of different concentrations were added to each well, so that the final volume of each well was 200 μl. The in vitro cytotoxic activity test treatment is divided into two types: 1. The in vitro killing test is carried out in 10% FBS complete medium; 2. The in vitro killing test system is additionally added with 20% human serum.

After culturing for 24 hours, the CellTiter-Glo kit (purchased from Promega, refer to the product instruction manual) was used to measure the cell viability, and the effect of human serum on the in vitro cytotoxic activity of T cells mediated by multispecific antibodies was detected. The results are shown in Figure 19A . After two days of incubation at 37°C in human serum, the activities of MB001 and MB048 decreased more, while the activities of MB065 and MB069 hardly decreased. However, comparing the killing activity in the no-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 multispecific antibodies, especially MB065 and MB069, EC50 increased several ten times. It may be due to the change in the spatial conformation of the anti-HSA in the multispecific antibody combined with the HSA protein in human serum, which leads to changes in the binding activity of the multispecific antibody to tumor cells and T cells, resulting in T cell activation Activity weakened. According to 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 practice in the future.

12.2 Stability experiment of multispecific antibody in cynomolgus monkey serum

The experimental method is the same as 12.1, only human serum is partially replaced with cynomolgus monkey serum. After culturing for 24 hours, the cell viability was measured with the CellTiter-Glo kit (purchased from Promega, refer to the product manual for the usage method), and the results are shown in FIG. 19B . After two days of incubation at 37°C in cynomolgus monkey serum, the activities of MB001 and MB048 decreased slightly, while those of MB065 and MB069 hardly decreased. However, comparing the killing activity in the no-cynomolgus monkey serum system and the 20% cynomolgus monkey serum system, it can be seen that the addition of 20% cynomolgus monkey serum has little effect on MB001, but it will significantly weaken MB048, MB065 and MB069 The killing activity of multispecific antibody, EC50 increased more than ten times. It may be due to the change in the spatial conformation of the anti-HSA in the multispecific antibody combined with the CSA protein in the monkey serum, resulting in a change in the binding activity of the multispecific antibody to tumor cells and T cells, resulting in T cell activation Activity weakened. According to the experimental results, it is inferred that after MB065 and MB069 enter monkeys, the T cell activation activity will be lower than the results of current in vitro tests.

Example 13 Identification of binding ability of Anti-CD33×CD70 multispecific antibody to protein or cell

13.1 Flow cytometry (FACS) to detect the binding of multispecific antibodies to tumor cells

Tumor cells THP-1 (purchased from the Cell Bank of Chinese Academy of Sciences, article number: TCHu 57), U937 (purchased from ATCC, article number: CRL-1593.2), 786-O (purchased from ATCC, article number: CRL-1932), and Jurkat (purchased from the Cell Bank of Chinese Academy of Sciences, article number: TCHU123). Expand the desired cells in T-175 cell culture flasks to the logarithmic growth phase, aspirate the medium, wash twice with PBS buffer, digest the cells with trypsin, then stop the digestion with complete medium, and blow the cells to a single cell suspension. After counting the cells, centrifuge, wash the cell pellet twice with PBS, resuspend the cell pellet with FACS buffer (PBS+2% fetal calf serum) to 2×10 ⁶ cells/mL, add 50 μl per well to 96-well FACS In the reaction plate, add the antibody to be tested (400nM or 100nM as the initial concentration, 3-fold or 5-fold gradient dilution) at 50 μl/well, mix with the cell suspension, and incubate at 4°C for 1 hour. After centrifugation and washing with PBS buffer for 3 times, 50 μl of iFluor647-labeled Anti-His secondary antibody (purchased from Genscript, catalog number: A01802-100) was added to each well, and incubated at 4° C. for 1 hour. Centrifuge and wash 3 times with PBS buffer, resuspend in 100 μl PBS, detect and analyze the results with FACS (FACS CantoTM, purchased from BD Company). Data analysis was performed by software (CellQuest) to obtain the mean fluorescence intensity (MFI) of the cells. Then analyze by software (GraphPad Prism8), perform data fitting, and calculate EC50. The analysis results are shown in Figures 20A-20D and Table 27, in which MB060 is the negative control; the results show that the Anti-CD33×CD70 multispecific antibody has binding activity to tumor cells with different expression levels, indicating that the Anti-CD33×CD70 multispecific antibody Antibodies can specifically bind tumor cells expressing human CD33 protein.

Table 27 The binding reaction of CD33×CD70 multispecific antibody to tumor cells

13.2 Flow cytometry (FACS) detection of antibody binding 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) was cloned into pcDNA3.1 Vector and plasmid preparation (completed by General Biosystems (Anhui) Co., Ltd.). Plasmid transfection was carried out on CHO-K1 cell line (purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) (

3000 Transfection Kit, purchased from Invitrogen, item number: L3000-015), and puromycin was used for pressure screening. Select the cell line B8 with better growth, higher fluorescence intensity, and monoclonal cell line to continue to expand and culture and freeze in liquid nitrogen). The preparation of detection cells and antibodies to be tested and detection methods refer to Example 13.1. The analysis results are shown in Figure 21, where MB060 is the negative control. The results showed that the Anti-CD33×CD70 multispecific antibody had binding activity to the recombinant cells expressing human CD33 protein, indicating that the combination of Anti-CD33 monoclonal antibody into multispecific antibody retained the characteristics of monoclonal antibody.

13.3 Flow cytometry (FACS) detection of cross-binding of antibodies to CHO-K1-human CD70

The recombinant cell line CHO-K1-human CD70 expressing human CD70 protein was purchased from Kangyuan Biotech (KC-1267) and the preparation and detection methods of the detection cells and antibodies to be tested refer to Example 13.1. The analysis results are shown in Figure 22, where MB060 is the negative control, and BDD20-09-9# has only CD33 monoclonal antibody but no CD70 monoclonal antibody. The results showed that the Anti-CD33×CD70 multispecific antibody had binding activity to the recombinant cells expressing human CD70 protein, indicating that the combination of Anti-CD70 monoclonal antibody into multispecific antibody retained the characteristics of monoclonal antibody.

Example 14 Different formats of polyclonal antibodies mediate differences in the killing activity of tumor cells and normal cells expressing CD33

In this experiment, the AML3 cell line Molm13 with high expression of CD33 was added to PBMC of healthy donors to simulate the in vivo environment of AML patients, and to verify that different formats of polyclonal antibodies mediate tumor cells and normal cells expressing CD33 (mainly CD14+ monocytes in peripheral blood) Differences in killing activity.

Resuscitate frozen healthy donor PBMC cells, label the revived PBMC cells with CellTrace ^TM Violet dye, use complete medium to adjust the Molm13 tumor cells to 2.5×10 ⁶ cells/ml, 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 numbers of Molm13 cells and PBMC cells in each well are 1.25× ¹⁰⁵ and 5× ¹⁰⁴ , respectively), and use a micropipette The device was added to each well of a flat-bottomed 96-well plate, and the volume of the cell mixture was 100 μl/well. The antibodies were serially diluted with complete medium (the initial antibody concentration was 5 μg/ml, 20-fold dilution, 8 gradients), and 100 μl of antibody diluents of different concentrations were 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 with flow buffer, add Fc Receptor Blocking Solution (purchased from Biolegend, Cat. Cat. No. 367110) staining, react in the dark 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 perform flow cytometry Detection (FACS Canto Plus, purchased from BD Company), to determine the ratio of PI+ cells in CellTrace ^™ Violet+CD14+ double-positive cells and the ratio of PI+ cells in CellTrace ^™ Violet-negative cells were different format polyclonal antibodies against normal cells and tumor cells expressing CD33 The killing activity was calculated and drawn using GraphPad Prism 9.0 software. The results are shown in Figure 23-24, different formats of antibodies have killing activity on tumor cells and monocytes, but BDD20-09-15# has better killing activity on tumor cells than BDD20-09-09#.

Claims

A multispecific antibody, characterized in that the multispecific antibody comprises at least three parts: (A) a target antigen binding part, (B) a half-life extending part, and (C) a T cell engaging part;

(A) Target antigen-binding portion: preferably a target antigen-binding antibody or a target antigen-binding ligand; the target antigen-binding antibody may be selected from any antigen-binding fragment;

(B) half-life extension moiety: preferably anti-HSA antibody;

(C) T cell engaging portion: wherein the T cell engaging portion is preferably an anti-CD3 antibody or an antigen-binding fragment;

The antigen-binding fragment is preferably Fd, Fv, scFv, diabody or single domain antibody (VHH);

The fragments of the three parts (A), (B) and (C) can be connected through a linker, or can be directly connected without a linker.
The multispecific antibody according to claim 1, wherein the (A) target antigen binding portion or (C) T cell engaging portion can be repeated or comprise multiple portions;

Preferably, the multispecific antibody contains two target antigen-binding parts, A1 and A2, and A1 and A2 can be the same or different; preferably, A1 and A2 bind to different antigen targets; preferably, A1 and A2 bind to the same antigen different epitopes of the target;

Preferably, part (C) is scFv binding to CD3;

More preferably, part (C) is a scFv that binds to human CD3.
The multispecific antibody according to claim 1, wherein the structural order of the multispecific antibody from the N-terminal to the C-terminal 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 the (C) T cell engaging portion 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;

Preferably, said (C)T cell engaging moiety comprises heavy chain CDRs and/or light chain CDRs selected from:

The heavy chain CDR1 is shown in SEQ ID NO.34, 39, 42, 47;

The heavy chain CDR2 is shown in SEQ ID NO.35, 40, 43, 45, 48;

The 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, 64;

The light chain CDR2 is shown in SEQ ID NO.51, 56, 59, 62, 65;

The light chain CDR3 is shown in SEQ ID NO.52, 53, 54, 57, 60, 63, 66;

Preferably, said (C)T cell engaging portion comprises 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% identity with the above heavy chain CDRs and/or light chain CDRs the sequence of.
The multispecific antibody according to any one of claims 1-3, wherein the target antigen 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, said target antigen binding portion comprises complementarity determining regions (CDRs) selected from the following fragments: SEQ ID NO.18-28, 103-106;

Preferably, said (A) target antigen binding portion comprises heavy chain CDRs and/or light chain CDRs selected from:

The heavy chain CDR1 is shown in SEQ ID NO.76, 80, 89, 92, 110, 113, 116;

The heavy chain CDR2 is shown in SEQ ID NO.77, 79, 81, 90, 93, 111, 114, 117;

The 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, 108;

The light chain CDR3 is shown in SEQ ID NO.85, 88, 109;

Preferably, said (A) target antigen binding portion comprises 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% identity with the above heavy chain CDRs and/or light chain CDRs the sequence of.
The multispecific antibody according to any one of claims 1-3, wherein part (B) comprises complementarity determining regions (CDRs) selected from the following fragments: SEQ ID NO.15-17;

Preferably, said part (B) comprises CDRs selected from:

CDR1 is shown in SEQ ID NO.67, 70, 73;

CDR2 as shown in SEQ ID NO. 68, 71, 74; and

CDR3 is shown in SEQ ID NO.69, 72, 75;

Preferably, said part (B) comprises a sequence having 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80% identity with the above-mentioned CDRs.
The multispecific antibody according to any one of claims 1-3, characterized in that, the multispecific antibody according to any one of claims 1-3, wherein (A) and (B) are connected by a linker ), (C) each fragment of the three parts; 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 GGGGSGGGGSGGGGSGGGGS.
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) a humanized antibody or fragment thereof; or,

(3) Fully human antibodies or fragments thereof;

Preferably, the multispecific antibody comprises the sequence shown in SEQ ID NO.97, 98, 99, 119, 120, 121, or 122, or has 99%, 98%, 97%, 96%, 95% of the above sequence %, 94%, 93%, 92%, 91%, 90%, 85%, 80% identical sequences.
An isolated nucleic acid fragment, characterized in that the nucleic acid fragment encodes the multispecific antibody according to any one of claims 1-8.
A carrier (vector), characterized in that the carrier comprises the nucleic acid fragment according to claim 9.
A kind of host cell, it is characterized in that, described host cell comprises the vector described in claim 10; Preferably, described cell is prokaryotic cell or eukaryotic cell, such as bacteria (Escherichia coli), fungus (yeast), insect cell or mammalian cells (CHO cell line or 293T cell line).
A method for preparing the multispecific antibody according to any one of claims 1-8, characterized in that the method comprises culturing the cell according to claim 11, and isolating the multispecific antibody expressed by the cell.
A pharmaceutical composition, characterized in that the pharmaceutical composition comprises the multispecific antibody according to any one of claims 1-8, or the nucleic acid fragment according to claim 9, or the carrier according to claim 10; Or the product prepared by the method described in claim 12; Optionally, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier (carrier), diluent or auxiliary agent; Optionally, the pharmaceutical composition also includes Contains additional antineoplastic agents.
A method for preventing and/or treating proliferative diseases, neoplastic diseases, inflammatory diseases, immune disorders, autoimmune diseases, infectious diseases, viral diseases, allergies, parasitic reactions, graft-versus-host disease or host-versus-transplantation A method for a physical disease, comprising administering an effective amount of the multispecific antibody of any one of claims 1-8, or the nucleic acid fragment of claim 9, or the carrier of claim 10, to a patient in need thereof, Or the product prepared by the method according to claim 12 or the pharmaceutical composition according to 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;

More preferably mesothelioma, lung cancer, breast cancer, esophageal cancer, pancreatic cancer, ovarian cancer, pleural cancer, cholangiocarcinoma, cervical cancer, gastric cancer, leukemia, histiocytic lymphoma, or renal cancer;

More preferably 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 myeloid leukemia.