A fusion protein that simultaneously targets CD3 and CD137, its preparation method and uses

Abstract

The present application provides a fusion protein, which comprises: an anti-CD137 antibody or an antigen-binding fragment thereof that can specifically bind to the CD137 molecule; an anti-CD3 antibody or an antigen-binding fragment thereof that can specifically bind to the CD3 molecule; and a first peptide linker and a second peptide linker. The present application also provides a nucleic acid encoding the fusion protein, an expression vector containing the nucleic acid, a host cell containing the nucleic acid or the expression vector, a method for preparing the fusion protein, a pharmaceutical composition containing the fusion protein, and the use of the fusion protein.

Description

Technical Field

[0001] The present application generally relates to the field of antibodies. More specifically, the present application relates to a fusion protein that simultaneously targets CD3 and CD137, and its preparation method and use. Background of the Invention

[0002] In the mid-1980s, researchers designed antibodies with two binding modules, one that binds to a tumor cell antigen and the other that matches the CD3 protein on the surface of T cells. In 1985, Pilar Perez et al. (Specific targeting of cytotoxic T cells by anti-T3 linked to anti-target cell antibody, Pilar Perez, Robert W. Hoffman, Stephen Shaw, Jeffrey A. Bluestone & David M. Segal Nature volume 316, pages 354-356 (1985) Cite this article) reported that such bispecific antibodies could destroy cancer cells in a culture dish and shrink tumors in mice. In 2000, Peter Kufer and Gert Riethmüller introduced a simplified bispecific antibody in which the two modules are linked by a flexible peptide rather than the traditional antibody backbone. The simplified design made the antibody easier to manufacture, but due to the absence of the antibody backbone, the kidneys cleared it from the blood within 2 hours. This type of molecule is also known as a bispecific T cell engager ( ).

[0003] Although BITE molecules have demonstrated powerful anti-tumor capabilities, their production yield remains low, and such bispecific antibodies sometimes also cause serious side effects, including liver damage and over-immune responses, in which white blood cells secrete large amounts of toxic cytokine signals. Such a cytokine "storm" can cause fever and, in severe cases, organ damage.

[0004] To address the manufacturing problems of bispecific antibodies, a large number of different molecular patterns have emerged in recent years for the molecular design and manufacture of bispecific antibodies. However, there are still many challenges in how to design to enable two or more binding modules to achieve multi-targeting and synergistic effects. Summary of the Invention

[0005] To solve the above technical problems, a new fusion protein is provided in this article, specifically a new multispecific antibody that can simultaneously target CD3 and CD137 on activated T cells, and its affinity for both CD3 and CD137 antigens is relatively low (KD is between 10 - 1000 nM), but the molecular binding affinity to T cells is not lower than that of traditional CD3 bispecific antibodies. Therefore, while improving effective T cell recruitment, the multispecific antibody also reduces the overactivation of T cells caused by high-affinity molecules targeting CD3, thereby reducing the risk of potential adverse reactions; in addition, this new multispecific antibody has a higher molecular weight, which can prevent excessive renal clearance and thus increase its in vivo half-life.

[0006] In a first aspect, the present application provides a fusion protein comprising:

[0007] a) an anti-CD137 antibody or an antigen-binding fragment thereof that can specifically bind to the CD137 molecule;

[0008] b) an anti-CD3 antibody or an antigen-binding fragment thereof that can specifically bind to the CD3 molecule; and

[0009] c) a first peptide linker and a second peptide linker,

[0010] wherein the first peptide linker is used to connect the heavy chain of the anti-CD137 antibody or its antigen-binding fragment and the heavy chain of the anti-CD3 antibody or its antigen-binding fragment, and the second peptide linker is used to connect the light chain of the anti-CD137 antibody or its antigen-binding fragment and the light chain of the anti-CD3 antibody or its antigen-binding fragment,

[0011] and only one disulfide bond can be formed between the first peptide linker and the second peptide linker.

[0012] In some embodiments, the fusion protein further comprises:

[0013] d) a first antibody or an antigen-binding fragment thereof that can specifically bind to a first antigen, and

[0014] e) a third peptide linker and a fourth peptide linker,

[0015] wherein the heavy chain of the first antibody or its antigen-binding fragment is connected to the heavy chain of the anti-CD3 antibody or its antigen-binding fragment through the third peptide linker, and

[0016] the light chain of the first antibody or its antigen-binding fragment is connected to the light chain of the anti-CD3 antibody or its antigen-binding fragment through the fourth peptide linker,

[0017] and only one disulfide bond can be formed between the third peptide linker and the fourth peptide linker.

[0018] In some embodiments, each of the first peptide linker, the second peptide linker, the third peptide linker, and the fourth peptide linker is independently selected from the following: a peptide linker comprising any one of the sequences shown in Seq ID NO.1-2 (Seq ID NO.1: Xaa Pro Pro Cys Pro Ala Pro Glu; Seq ID NO.2: Glu Pro Ala Pro Cys Pro Pro Xaa, where Xaa can be any amino acid other than Cys or absent), where X represents any amino acid other than Cys, or is absent.

[0019] In some embodiments, each of the first to fourth peptide linkers is the hinge region of a natural antibody, where the hinge region has a deletion mutation that retains only one cysteine.

[0020] In some embodiments, any one of the first to fourth peptide linkers is independently selected from the IgG1 hinge region with a C229 deletion mutation or the IgG1 hinge region with a C229 deletion mutation and an inversion of the hinge region D224-S242.

[0021] In some embodiments, the antigen-binding fragment is selected from Fab fragment, Fab’ fragment, F(ab’)2 fragment, Fv fragment, diabody, or single-chain antibody molecule such as sc-Fv.

[0022] In some embodiments, the first antigen is selected from MESOTHELIN, EGFR, PSMA, GD2, CEA, MUC1, FAP, BCMA, EphA2, CD19, CD22, EpCAM, CEA, PD-L1, B7H3, ROR1, c-Met, and GPC3.

[0023] In some embodiments, the first antigen, CD137 molecule, and CD3 molecule are independently derived from a mammal, preferably a non-human primate or a human.

[0024] In some embodiments, the affinity constant of the first antibody or its antigen-binding fragment binding to the first antigen is 10-1000 times the affinity constant of the anti-CD137 antibody or its antigen-binding fragment binding to the CD137 molecule or the anti-CD3 antibody or its antigen-binding fragment binding to the CD3 molecule.

[0025] In a second aspect, the present application provides a fusion protein, which sequentially comprises from the N-terminus to the C-terminus:

[0026] a) The Fab fragment of a first antibody that can specifically bind to a first antigen;

[0027] b) an anti-CD3 antibody or an antigen-binding fragment thereof that can specifically bind to the CD3 molecule;

[0028] c) an anti-CD137 antibody or an antigen-binding fragment thereof that can specifically bind to the CD137 molecule;

[0029] wherein the heavy chains of the Fab fragment, the anti-CD3 antibody or its antigen-binding fragment, and the anti-CD137 antibody or its antigen-binding fragment are sequentially connected by a first peptide linker and a third peptide linker, and the light chains of the Fab fragment, the anti-CD3 antibody or its antigen-binding fragment, and the anti-CD137 antibody or its antigen-binding fragment are sequentially connected by a second peptide linker and a fourth peptide linker,

[0030] wherein only one disulfide bond can be formed between the first peptide linker and the second peptide linker, and only one disulfide bond can be formed between the third peptide linker and the fourth peptide linker, each independently selected from the following: peptide linkers comprising any one of the sequences shown in Seq ID NO.1-2, wherein X represents any amino acid other than Cys, or is absent.

[0031] In some embodiments, each of the first to fourth peptide linkers is the hinge region of a natural antibody, wherein the hinge region has a deletion mutation that retains only one cysteine.

[0032] In some embodiments, any one of the first to fourth peptide linkers is independently selected from the IgG1 hinge region with a C229 deletion mutation or the IgG1 hinge region with a C229 deletion mutation and an inversion of the hinge region D224-S242.

[0033] In some embodiments, the first antigen is selected from MESOTHELIN, EGFR, PSMA, GD2, CEA, MUC1, FAP, BCMA, EphA2, CD19, CD22, EpCAM, CEA, PD-L1, B7H3, ROR1, c-Met, and GPC3.

[0034] In some embodiments, the first antigen, the CD137 molecule, and the CD3 molecule are independently derived from mammals, preferably non-human primates or humans.

[0035] In some embodiments, the affinity constant of the binding of the Fab fragment to the first antigen is 10-1000 times the affinity constant of the anti-CD137 antibody or its antigen-binding fragment to the CD137 molecule or the affinity constant of the anti-CD3 antibody or its antigen-binding fragment to the CD3 molecule.

[0036] In a third aspect, the present application provides a nucleic acid encoding the fusion protein described in the first or second aspect.

[0037] In a fourth aspect, the present application provides an expression vector comprising the nucleic acid described in the third aspect.

[0038] In a fifth aspect, the present application provides a host cell comprising the nucleic acid described in the third aspect or the expression vector described in the fourth aspect.

[0039] In some embodiments, the host cell is a mammalian cell. Mammalian cells can include, but are not limited to, CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, and PER.C6 cells.

[0040] In a sixth aspect, the present application provides a method for preparing the fusion protein described in the first aspect or the second aspect, which comprises:

[0041] a) culturing the host cell described in the fifth aspect; and

[0042] b) recovering the fusion protein from the host cell or the culture supernatant of the host cell.

[0043] In a seventh aspect, the present application provides a pharmaceutical composition comprising the fusion protein described in the first aspect or the second aspect, the nucleic acid described in the third aspect, the expression vector described in the fourth aspect, or the host cell described in the fifth aspect, and a pharmaceutically acceptable carrier.

[0044] In an eighth aspect, the present application provides the use of the fusion protein described in the first aspect or the second aspect, the nucleic acid described in the third aspect, the expression vector described in the fourth aspect, or the host cell described in the fifth aspect in the preparation of a drug for treating, ameliorating, or preventing tumors, autoimmune diseases, or infectious diseases.

[0045] In a ninth aspect, the present application provides a method for treating, ameliorating, or preventing tumors, autoimmune diseases, or infectious diseases, which comprises administering to an individual in need the fusion protein described in the first aspect or the second aspect, the nucleic acid described in the third aspect, the expression vector described in the fourth aspect, or the host cell described in the fifth aspect.

[0046] In a tenth aspect, the present application provides the use of the fusion protein described in the first aspect or the second aspect, the nucleic acid described in the third aspect, the expression vector described in the fourth aspect, or the host cell described in the fifth aspect for treating, ameliorating, or preventing tumors, autoimmune diseases, or infectious diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1Shows the schematic diagram of the fusion protein constructed in this application. Among them, A shows the schematic diagram of the trispecific antibody T1 that simultaneously targets CD3×CD137×CD19, B shows the schematic diagram of the trispecific antibody T2 that simultaneously targets CD3×CD137×GPC3; C shows the schematic diagram of the trispecific antibody Triad19, and D shows the schematic diagram of the bispecific antibody ZWB56.

[0048] Figure 2 Shows the chromatogram of affinity purification of the fusion protein using Protein L. Among them, A: Chromatogram of purification of the trispecific antibody T1; B: Chromatogram of purification of the trispecific antibody T2.

[0049] Figure 3 Shows the SDS-PAGE electrophoresis diagram of the fusion protein expressed by CHO-K1 cells. Among them, M: DNA Marker; 1: Non-reducing condition; 2: Reducing condition; A: SDS-PAGE electrophoresis result of the trispecific antibody T1; B: SDS-PAGE electrophoresis result of the trispecific antibody T2.

[0050] Figure 4 Shows the results of flow cytometry detection of the binding of the fusion protein to cell surface antigens. Among them, A1: Binding of the trispecific antibody T1 to Nalm-6 cells expressing CD19; A2: Binding of the trispecific antibody T1 to Jurkat cells expressing CD3; A3: Binding of the trispecific antibodies T1 and T2 to HEK293-CD137 cells expressing CD137; B1: Binding of the trispecific antibody T2 to Jurkat cells expressing CD3; B2: Binding of the trispecific antibody T2 to HepG2 cells expressing GPC3.

[0051] Figure 5 Shows the SDS-PAGE results of the trispecific antibody Triad19 and the bispecific antibody ZWB56. Among them, A: SDS-PAGE electrophoresis result of the bispecific antibody ZWB56; B: SDS-PAGE electrophoresis result of the trispecific antibody Triad19. The 1st - 3rd lanes of the two electrophoresis diagrams are non-reducing condition; DNA Marker and reducing condition respectively.

[0052] Figure 6 Shows the results of detecting the functional activity of the antibodies Triad19 / ZWB56 using the CD3 functional cell line Jurkat Dual.

[0053] Figure 7 and 8 Respectively show the fitted drug killing curves of the trispecific antibody Triad19 and the bispecific antibody ZWB56.

[0054] Figure 9 The killing rate of the antibody Triad19 / ZWB56 against cells is shown.

[0055] Figure 10 The quantitative results of each factor are shown.

[0056] Figures 11 - 15 The results of T cell subset analysis of T cells after the T cell killing experiment are shown.

[0057] Detailed Description of the Invention

[0058] The following definitions and methods are provided to better define the present application and to guide those of ordinary skill in the art in the practice of the present application. Unless otherwise stated, the terms of the present application are understood in accordance with the conventional usage of those of ordinary skill in the relevant art.

[0059] Definition

[0060] As used herein, the term "about" refers to ±10% of the recited number. For example, about 1% refers to the range of 0.9% to 1.1%.

[0061] As used herein, the term "fusion protein" refers to the purposeful linking together of two or more segments of genes encoding functional proteins, followed by the expression of said proteins. Such a protein product obtained by the head-to-tail linking of the coding regions of two or more genes under artificial conditions and controlled by regulatory sequences is a fusion protein.

[0062] As used herein, the term "peptide linker" in the context of the present application refers to a short peptide used to link two functional proteins, with a length ranging from 3 amino acids (aa) to 76 amino acids. The peptide linker can provide a certain degree of flexibility to each functional protein in the fusion protein, enabling them to perform their respective functions. The peptide linker used in the present application preferably contains only one cysteine, so as to form a stable disulfide bond between two peptide linkers.

[0063] As used herein, the term "antibody" refers to any form of antibody or its fragment that can exhibit the desired biological activity. Therefore, it is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, fusion proteins (such as bispecific antibodies), and antibody fragments, as long as they can exhibit the desired biological activity. Thus, those skilled in the art can also understand that the term "antibody" as used herein can also refer to a fusion protein of any form of the same or different antibodies or their fragments that can exhibit the desired biological activity, thereby achieving the function of a multispecific antibody.

[0064] As used herein, the term "antigen" refers to a molecule or molecular moiety that can be bound by a selective binding agent such as an antibody, and can also be used in an animal to prepare an antibody that binds to an epitope of the antigen. An antigen can have one or more antigenic epitopes. Antigens described herein can include, but are not limited to, most proteins, bacteria, viruses, bacterial exotoxins, polysaccharides (such as the capsular polysaccharide of pneumococcus), and lipids, etc.

[0065] As used herein, the term "specific binding" is a term well-known in the art, and methods for measuring such specific binding of an antibody to an antigen are also well-known in the art. For example, in some embodiments, "specific binding" means that an antibody binds to an intended target, but does not bind significantly to other targets. Compared to binding to other epitopes, the antibody binds to the intended target epitope with a significantly increased affinity and / or for a longer duration.

[0066] As used herein, the term "antigen-binding fragment" includes fragments or derivatives of an antibody that substantially retain their binding activity. Thus, the term "antigen-binding fragment" refers to a portion of a full-length antibody, typically its antigen-binding region or variable region. Examples of antigen-binding fragments include, but are not limited to: Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, diabodies, single-chain antibody molecules such as sc-Fv, and fusion proteins formed from antibody fragments. It is also contemplated that antigen-binding fragments can include conservative amino acid substitutions that do not substantially alter their binding activity.

[0067] As used herein, the term "Fab fragment" contains the light chain and the CH1 and variable regions of the heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule.

[0068] As used herein, the term "Fab' fragment" contains the light chain and a portion or fragment of the heavy chain, said portion or fragment containing the VH domain and the CH1 domain and the region between the CH1 and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0069] As used herein, the term "F(ab')2 fragment" contains two light chains and two heavy chains, said heavy chains containing a portion of the constant region between the CH1 and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. An F(ab')2 fragment thus consists of two Fab' fragments, and the two Fab' fragments are linked together by a disulfide bond between the two heavy chains.

[0070] As used herein, the term "Fv fragment" contains the variable regions from the heavy and light chains, but lacks the constant regions.

[0071] As used herein, the term "single-chain Fv" or "scFv" refers to an antibody fragment that comprises the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding.

[0072] As used herein, the term "diabody" refers to a small antibody fragment that has two antigen-binding sites and that comprises a heavy-chain variable domain (VH) and a linked light-chain variable domain (VL) (VH-VL or VL-VH) in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, each domain is forced to pair with the complementary domain on the other chain, thereby generating two antigen-binding sites.

[0073] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. The hypervariable regions include the amino acid residues from the "complementary determining regions" or "CDRs" (e.g., residues 24-34 (LCDR-1), 50-56 (LCDR-2), and 89-97 (LCDR-3) in the light-chain variable domain and residues 31-35 (HCDR-1), 50-65 (HCDR-2), and 95-102 (HCDR-3) in the heavy-chain variable domain; Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md., and / or the amino acid residues from the "hypervariable loops" (i.e., residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light-chain variable domain and residues 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy-chain variable domain; Chothia and Lesk, (1987) J. Mol. Biol. 196:901-917). "Framework region" or "FR" residues are those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

[0074] As used herein, the term "tumor-associated antigen" refers to any molecule (e.g., protein, peptide, lipid, carbohydrate, etc.) that is expressed alone, predominantly expressed, or overexpressed by tumor cells such that the antigen is associated with the tumor. A tumor-associated antigen can be an antigen expressed by only one type of tumor such that the tumor antigen is associated with or is unique to only one type of tumor. Optionally, the tumor antigen can be a tumor antigen associated with or unique to multiple types of tumors. For example, a tumor-associated antigen can be expressed by both breast cancer cells and colon cancer cells but not by normal, non-tumor or non-cancer cells. Exemplary tumor-associated antigens are tumor cell surface antigens, which are more amenable to recognition by therapeutic and diagnostic antibodies.

[0075] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring variations that may be present in minor amounts. Monoclonal antibodies are highly specific for a single epitope. The monoclonal antibodies disclosed herein are not limited to the source of the antibody or the manner of its preparation (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals, etc.). The term includes intact immunoglobulins and fragments thereof that fall within the definition of "antibody".

[0076] As used herein, the term "expression vector" refers to a vector containing a recombinant polynucleotide that comprises an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression can be provided by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses and adeno-associated viruses) that incorporate the recombinant polynucleotide. Detailed Description

[0077] The present application provides a fusion protein capable of simultaneously targeting CD3 and CD137, which links an anti-CD3 antibody or its antigen-binding fragment and an anti-CD137 antibody or its antigen-binding fragment through a linker such as a peptide linker. The fusion protein comprising an anti-CD3 antibody or its antigen-binding fragment and an anti-CD137 antibody or its antigen-binding fragment can further link another antibody or its antigen-binding fragment at the C-terminus or N-terminus of the fusion protein through a linker such as a peptide linker, thereby preparing a trispecific antibody.

[0078] The present application ingeniously utilizes the stabilizing effect of disulfide bonds in the antibody hinge region, and uses the mutated antibody hinge as a linker to connect the antibodies in the fusion protein of the present application to each other, such that only a pair of disulfide bonds is formed between the heavy chain and the light chain at the mutated antibody hinge region. Through the present application, not only can a stable and highly pure target multispecific antibody, such as a trispecific antibody, be easily obtained, but also the obtained trispecific antibody can bind its target antigens with a higher binding affinity compared to a bispecific antibody.

[0079] In a first aspect, the present application provides a fusion protein comprising:

[0080] a) an anti-CD137 antibody or an antigen-binding fragment thereof that can specifically bind to the CD137 molecule;

[0081] b) an anti-CD3 antibody or an antigen-binding fragment thereof that can specifically bind to the CD3 molecule; and

[0082] c) a first peptide linker and a second peptide linker,

[0083] wherein the first peptide linker is used to connect the heavy chain of the anti-CD137 antibody or an antigen-binding fragment thereof and the heavy chain of the anti-CD3 antibody or an antigen-binding fragment thereof, and the second peptide linker is used to connect the light chain of the anti-CD137 antibody or an antigen-binding fragment thereof and the light chain of the anti-CD3 antibody or an antigen-binding fragment thereof,

[0084] and only one disulfide bond can be formed between the first peptide linker and the second peptide linker.

[0085] In some embodiments, the fusion protein further comprises:

[0086] d) a first antibody or an antigen-binding fragment thereof that can specifically bind to a first antigen, and

[0087] e) a third peptide linker and a fourth peptide linker,

[0088] wherein the heavy chain of the first antibody or an antigen-binding fragment thereof is connected to the heavy chain of the anti-CD3 antibody or an antigen-binding fragment thereof through the third peptide linker, and

[0089] the light chain of the first antibody or an antigen-binding fragment thereof is connected to the light chain of the anti-CD3 antibody or an antigen-binding fragment thereof through the fourth peptide linker,

[0090] and only one disulfide bond can be formed between the third peptide linker and the fourth peptide linker.

[0091] In some embodiments, each of the first peptide linker, the second peptide linker, the third peptide linker, and the fourth peptide linker is independently selected from the following: a peptide linker comprising any one of the sequences shown in Seq ID NO.1-2 (Seq ID NO.1: Xaa Pro Pro Cys Pro Ala Pro Glu; Seq ID NO.2: Glu Pro Ala Pro Cys Pro Pro Xaa, where Xaa can be any amino acid other than Cys or absent), where X represents any amino acid other than Cys, or is absent.

[0092] In some embodiments, each of the first to fourth peptide linkers is the hinge region of a natural antibody, where the hinge region has a deletion mutation that retains only one cysteine.

[0093] In some embodiments, any one of the first to fourth peptide linkers is independently selected from the IgG1 hinge region with a C229 deletion mutation or the IgG1 hinge region with a C229 deletion mutation and an inversion of the hinge region from D224-S242.

[0094] In some embodiments, the antigen-binding fragment is selected from Fab fragment, Fab’ fragment, F(ab’)2 fragment, Fv fragment, diabody, or single-chain antibody molecule such as sc-Fv.

[0095] In some embodiments, the fusion protein is a bispecific antibody or a trispecific antibody.

[0096] In some embodiments, the first antigen is selected from MESOTHELIN, EGFR, PSMA, GD2, CEA, MUC1, FAP, BCMA, EphA2, CD19, CD22, EpCAM, CEA, PD-L1, B7H3, ROR1, c-Met, and GPC3.

[0097] In some embodiments, the first antigen, CD137 molecule, and CD3 molecule are independently derived from mammals, preferably non-human primates or humans.

[0098] In some embodiments, the affinity constant of the first antibody or its antigen-binding fragment binding to the first antigen is 10-1000 times the affinity constant of the anti-CD137 antibody or its antigen-binding fragment binding to the CD137 molecule or the anti-CD3 antibody or its antigen-binding fragment binding to the CD3 molecule.

[0099] In this article, the binding force between an antibody and an antigen is called antibody affinity, which is essentially a non-covalent force. It reflects the ability of an antibody molecule to bind to an antigen. Methods for measuring the affinity of an antibody for a specific antigen are well-known in the art, including but not limited to biolayer interferometry (BLI), solid-phase radioimmunoassay (SP-RIA), equilibrium dialysis, antigen precipitation assay, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), etc. The magnitude of antibody affinity can be represented by the affinity constant K D and the higher the affinity constant K D , the stronger the ability of the antibody to bind to the antigen.

[0100] In some embodiments, the first peptide linker and the second peptide linker are the same.

[0101] In some embodiments, the first peptide linker and the second peptide linker are different.

[0102] In some embodiments, the third peptide linker and the fourth peptide linker are the same.

[0103] In some embodiments, the third peptide linker and the fourth peptide linker are different.

[0104] In preferred embodiments, the first peptide linker and the second peptide linker are the same, the third peptide linker and the fourth peptide linker are the same, however, the first peptide linker is different from the third peptide linker.

[0105] In preferred embodiments, all four of the first to fourth peptide linkers are the same.

[0106] In specific embodiments, the first peptide linker and the second peptide linker are the IgG1 hinge region with a C229 deletion mutation, and the third peptide linker and the fourth peptide linker are the IgG1 hinge region with a C229 deletion mutation and an inversion of the hinge region D224-S242.

[0107] In specific embodiments, the first peptide linker and the second peptide linker are the IgG1 hinge region with a C229 deletion mutation and an inversion of the hinge region D224-S242, and the third peptide linker and the fourth peptide linker are the IgG1 hinge region with a C229 deletion mutation.

[0108] The amino acid sequences of antibodies are numbered to identify equivalent positions, and there are currently multiple different numbering schemes for antibodies. The Kabat scheme (Kabat et al., 1991) was developed based on the positions of highly variable regions between sequences of the same domain type. It numbers the variable domains of the antibody heavy chain (VH) and light chains (Vλ and Vκ) differently. The Chothia scheme (Al-Lazikani, 1997) is the same as the Kabat scheme, but corrects the positions of the inserted annotations around the first VH complementarity-determining region (CDR) so that they correspond to structural loops. The antibodies in the present application are numbered according to the Kabat scheme.

[0109] In some embodiments, the fusion protein described above can be a trispecific antibody, which comprises a heavy chain and a light chain, wherein the heavy chain sequentially comprises, from the N-terminus to the C-terminus, the heavy chain of an anti-CD137 antibody or its antigen-binding fragment, a first peptide linker, the heavy chain of an anti-CD3 antibody or its antigen-binding fragment, a third peptide linker, and the heavy chain of a first antibody or its antigen-binding fragment, and the light chain sequentially comprises, from the N-terminus to the C-terminus, the light chain of an anti-CD137 antibody or its antigen-binding fragment, a second peptide linker, the light chain of an anti-CD3 antibody or its antigen-binding fragment, a fourth peptide linker, and the light chain of a first antibody or its antigen-binding fragment.

[0110] In some embodiments, the fusion protein described above can be a trispecific antibody, which comprises a heavy chain and a light chain, wherein the heavy chain sequentially comprises, from the N-terminus to the C-terminus, the heavy chain of a first antibody or its antigen-binding fragment, a first peptide linker, the heavy chain of an anti-CD137 antibody or its antigen-binding fragment, a third peptide linker, and the heavy chain of an anti-CD3 antibody or its antigen-binding fragment, and the light chain sequentially comprises, from the N-terminus to the C-terminus, the light chain of a first antibody or its antigen-binding fragment, a second peptide linker, the light chain of an anti-CD137 antibody or its antigen-binding fragment, a fourth peptide linker, and the light chain of an anti-CD3 antibody or its antigen-binding fragment.

[0111] In some embodiments, the fusion protein described above can be a trispecific antibody, which comprises a heavy chain and a light chain, wherein the heavy chain sequentially comprises, from the N-terminus to the C-terminus, the heavy chain of a first antibody or its antigen-binding fragment, a first peptide linker, the heavy chain of an anti-CD3 antibody or its antigen-binding fragment, a third peptide linker, and the heavy chain of an anti-CD137 antibody or its antigen-binding fragment, and the light chain sequentially comprises, from the N-terminus to the C-terminus, the light chain of a first antibody or its antigen-binding fragment, a second peptide linker, the light chain of an anti-CD3 antibody or its antigen-binding fragment, a fourth peptide linker, and the light chain of an anti-CD137 antibody or its antigen-binding fragment.

[0112] In some embodiments, the fusion protein described above can be a trispecific antibody, which comprises a heavy chain and a light chain. The heavy chain sequentially comprises, from the N-terminus to the C-terminus, the heavy chain of an anti-CD3 antibody or its antigen-binding fragment, a first peptide linker, the heavy chain of an anti-CD137 antibody or its antigen-binding fragment, a third peptide linker, and the heavy chain of a first antibody or its antigen-binding fragment. The light chain sequentially comprises, from the N-terminus to the C-terminus, the light chain of an anti-CD3 antibody or its antigen-binding fragment, a second peptide linker, the light chain of an anti-CD137 antibody or its antigen-binding fragment, a fourth peptide linker, and the light chain of a first antibody or its antigen-binding fragment.

[0113] In preferred embodiments, the peptide linker described herein can be selected from the IgG1 hinge region with C229 deletion mutation or the IgG1 hinge region with C229 deletion mutation and D224-S242 inversion in the hinge region.

[0114] In preferred embodiments, the peptide linkers expected to be cross-linked by disulfide bonds are the same. For example, in the final fusion protein, if the first peptide linker and the second peptide linker are expected to be cross-linked by disulfide bonds, they are selected as the same peptide linker. Similarly, if the third peptide linker and the fourth peptide linker are expected to be cross-linked by disulfide bonds, they are selected as the same peptide linker.

[0115] In the present application, the first antibody or its antigen-binding fragment, the anti-CD137 antibody or its antigen-binding fragment, and the anti-CD3 antibody or its antigen-binding fragment can each independently be derived from a monoclonal antibody.

[0116] In some embodiments, the monoclonal antibodies used in the present application may be selected from one or more of the following: blincyto, adalimumab, secukinumab, Rituximab, Trastuzumab, Gemtuzumab ozogamicin, Alemtuzumab, Bevacizumab, Cetuximab, Panitumumab, Ofatumumab, Ipilimumab, Brentuximab vedotin, Denosumab, Pertuzumab, Obinutuzumab, Ramucirumab, 3F8, abagovomab, adecatumumab, afutuzumab, alacizumab (pegol), amatuximab, apolizumab, bavituximab, bectumomab, belimumab, bivatuzumab, cantuzumabmertansine), cantuzumab (ravtansine), capromab (pendetide), catumaxomab, citatuzumab (bogatox), cixutumumab, clivatuzumab (tetraxetan), conatumumab, dacetuzumab, dalotuzumab, Detumomab, drozitumab, ecromeximab, edrecolomab, elotuzumab, enavatuzumab, ensituximab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, FBTA05, flanvotumab, galiximab, gemtuzumab, ganitumab, girentuximab, glembatumumab (vedotin), ibritumomab tiuxetan, icrucumab, igovomab, indatuximab ravtansine, intetumumab, inotuzumabozogamicin), ipilimumab (MDX-101), iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab (mertansine), lucatumumab, lumiliximab, mapatumumab, matuzumab, milatuzumab, mitumomab, mogamulizumab, moxetumomab (pasudotox), nacolomab (tafenatox), naptumomab (estafenatox), narnatumab, necitumumab, nimotuzumab, nivolumab, NR-LU-10, olaratumab, oportuzumab (monatox), oregovomab, panitumumab, pertuzumab, pritumumab, racotumomab, radretumab, robatumumab, omalizumab, sibrotuzumab, siltuximab, taplitumomab (paptox), tenatumomab, teprotumumab, ticilimumab, tremelimumab, tigatuzumab, tucotuzumab (celmoleukin), ublituximab, urelumab, veltuzumab, volociximab, votumumab, and zalutumumab.

[0117] The antigen that the fusion protein of the present application can bind can be a cell-associated protein, such as a cell surface protein on the membrane of a cell (T cell, endothelial cell or tumor cell), or a soluble protein. The antigen can also be any medically relevant protein, such as those proteins upregulated during a disease or infection, such as receptors and / or their corresponding ligands. Specific examples of cell surface proteins include, but are not limited to, adhesion molecules such as integrin, E-selectin, P-selectin or L-selectin, CD2, CD3, CD4, CD5, CD7, CD8, CD11a, CD11b, CD18, CD19, CD20, CD23, CD25, CD33, CD38, CD40, CD45, CD69, CD134, ICOS, CD137, CD27, carcinoembryonic antigen (CEA), TCR, MHC class I and MHC class II antigens, VEGF, and receptors for these proteins. Soluble proteins include interleukins (such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-12, IL-16 or IL-17), viral antigens (such as respiratory syncytial virus or cytomegalovirus antigens), immunoglobulins (such as IgE), interferons (such as interferon α, interferon β or interferon γ), tumor necrosis factor-α (TNFα), tumor necrosis factor-β, colony stimulating factors (such as G-CSF or GM-CSF) and platelet-derived growth factors (such as PDGF-α and PDGF-β) and their receptors (where appropriate). Other antigens include bacterial cell surface antigens, bacterial toxins, viruses (such as influenza virus, EBV, HepA, B and C), bioterrorism agents, radionuclides and heavy metals, and snake and spider venoms and toxins.

[0118] Other antigens that can be bound by the fusion protein of the present application include serum carrier proteins, polypeptides that allow recruitment of cell-mediated effector functions, and radionuclide chelating proteins.

[0119] In some embodiments, the antigen that can be bound by the fusion protein of the present application is a tumor-associated antigen, which includes any one or more of the following: CD20, MESOTHELIN, EGFR, CD33, CD52, VEGF, ROR1, CD30, RANKL, MESOTHELIN, VEGF-R2, Her3, A33 antigen, CD5, CD19, CD22, CD23 (IgE receptor), CA242 antigen, 5T4, VEGFR-1, CD33, CD37, CD40, CD44, CD51, CD52, CD56, CD74, CD80, CD152, CD200, CD221, CCR4, NPC-1C, vimentin, insulin-like growth factor-1 receptor (IGF-1R), alpha-fetoprotein, carcinoembryonic antigen (CEA), integrin αv β3, integrin α5β1, fibroblast activation protein, FAP-α, TAG-72, MUC1, MUC16, prostate-specific membrane antigen (PMSA), EGP40 pan-carcinoma antigen, glycoprotein EpCAM, programmed death-1, phosphatase of regenerating liver 3 (PRL-3), Lewis-Y antigen, GD2, phosphatidylinositol glycan-3 (GPC3), and mesothelin.

[0120] The CD3 molecule consists of 4 subunits: δ, ε, γ, and ζ, with molecular weights of 18.9 kDa, 23.1 kDa, 20.5 kDa, and 18.7 kDa respectively, and lengths of 171, 207, 182, and 164 amino acid residues respectively. Together, they form 6 peptide chains, which often bind tightly to the T cell receptor (TCR) to form a TCR-CD3 complex composed of 8 peptide chains. This complex has the functions of transducing T cell activation signals and stabilizing the TCR structure. The cytoplasmic segment of CD3 contains immunoreceptor tyrosine-based activation motifs (ITAMs). The TCR recognizes and binds to the antigen peptide presented by MHC (major histo-compatibility complex) molecules, resulting in the phosphorylation of tyrosine residues in the conserved sequences of the ITAMs of CD3 by the tyrosine protein kinase p56LCK in T cells. Then, other tyrosine protein kinases with SH2 (Scrhomology2) domains (such as ZAP-70) can be recruited. The phosphorylation of ITAMs and the binding to ZAP-70 are one of the important biochemical reactions in the early stage of the T cell activation signal transduction process. Therefore, the function of the CD3 molecule is to transduce the activation signals generated by TCR recognition of antigens.

[0121] CD137 (TNFRSF9 or 4-1BB) belongs to the members of the tumor necrosis factor (TNF) receptor family and is a type I transmembrane protein composed of 255 amino acids. The relative molecular mass of the CD137 monomer is 30 kDa, and it can also exist in the form of a dimer with a relative molecular mass of approximately 55 kDa. After the cell surface membrane CD137 molecule detaches and enters the blood, it can form soluble CD137 (sCD137), which is almost absent in normal humans. In the immune response, the activation of T lymphocytes requires not only the binding of major MHC / Ag to TCR but also the participation of a second signal, such as the interaction of B7-CD28, CD137-CD137L, etc. The role of CD137 is different from that of CD28. CD28 is expressed on the surface of unstimulated T lymphocytes and mainly affects the early immune response of cells, while CD137 is mainly expressed on activated T lymphocytes and thus mainly exerts its effects in the middle and late stages of T lymphocyte responses. The expression level of CD137 on naive T cells is very low, but when T cells are activated, the expression of CD137 is upregulated and is mainly distributed on the surface of CD4+ T and CD8+ T cells. The binding of CD137 to CD137L can produce a series of biological effects, including inducing T cell activation, releasing chemokines and cytokines, and exacerbating the immune response. In addition, the CD137-CD137L signal also participates in autoimmune diseases, such as type I autoimmune diabetes, etc.

[0122] CD19 is one of the important membrane antigens involved in B cell activation and proliferation. It is a surface marker common to all B cells and does not disappear after B cell activation. It is the most important B cell marker. At the same time, CD19 is also a component of the signal transduction complex on the surface of B cells. The extracellular part of CD19 binds to other membrane antigens for signal transduction. An increase in CD19-positive cells is seen in malignant tumors of the B lymphocyte system. For example, CD19 is expressed in 95% of acute pre-B lymphocyte leukemia cells and 94% of acute mature B lymphocyte leukemia cells, and is also seen in chronic lymphocytic leukemia and Burkitt lymphoma, etc. A decrease in CD19-positive cells is seen in humoral immunodeficiency diseases, such as agammaglobulinemia, those who have long-term used immunosuppressants, etc. Therefore, the detection of CD19 can make a clear etiological diagnosis of the above diseases and provide a basis for differential diagnosis. In addition, because CD19 is widely present on the surface of malignant tumor cells of the B lymphocyte system, it can be used as a cell surface target in the immunotherapy of leukemia and lymphoma.

[0123] GPC3 (Glypican-3) is a member of the Glypican family and belongs to membrane-bound heparan sulfate proteoglycans. During mammalian embryogenesis, it can play an important regulatory role in the development and growth of tissues and organs by affecting multiple molecular signaling pathways. Gene mutations and loss of function lead to overgrowth and dysplasia syndromes. Abnormal expression of GPC3 is closely related to the occurrence and development of various tumors. It is considered to play an important role in the diagnosis and treatment of hepatocellular carcinoma (HCC).

[0124] PD-1 (programmed death receptor 1) is an important immunosuppressive molecule, belonging to the immunoglobulin superfamily, and is a membrane protein with 268 amino acid residues. Immunomodulation targeting PD-1 is of great significance for anti-tumor, anti-infection, anti-autoimmune diseases, and organ transplantation survival. Its ligand PD-L1 can also be used as a target, and the corresponding antibody can also play the same role. PD-1 or PD-L1 can act as the binding moiety in the present application, such as the first binding moiety and / or the second binding moiety. Preferably, the extracellular domain of PD-1, i.e., PD-1ECD, acts as the binding moiety in the present application.

[0125] c-Met is a protein product encoded by the c-met proto-oncogene, which is a hepatocyte growth factor receptor with tyrosine kinase activity. It is related to a variety of oncogene products and regulatory proteins and is involved in the regulation of cell signal transduction and cytoskeleton rearrangement, and is an important factor in cell proliferation, differentiation, and motility. Currently, it is considered that c-Met is closely related to the occurrence and metastasis of various cancers.

[0126] The fusion protein of the present application can introduce the CH2-CH3 domain. The CH2-CH3 domain can be linked to the heavy chain of the first antibody or its antigen-binding fragment, anti-CD137 antibody or its antigen-binding fragment, or anti-CD3 antibody or its antigen-binding fragment. Optionally, the CH2-CH3 domain undergoes KiH mutations, introduction of cysteine residues, or introduction of one or more salt bridge mutations to promote heterodimerization, and such additions result in increased stability of the heterodimer. Salt bridges herein include hydrogen bonds and electrostatic interactions, such as salt bridges that can occur between glutamate and lysine residues.

[0127] The heavy and light chains of natural antibodies each include a variable region (i.e., the V region) and a constant region (i.e., the C region). The constant regions of the heavy chain and the light chain are called CH and CL, respectively. The CL lengths of different isotypes (κ or λ) of Ig are basically the same, but the CH lengths of different classes of Ig are different. For example, IgG, IgA, and IgD include CH1, CH2, and CH3, while IgM and IgE include CH1, CH2, CH3, and CH4.

[0128] In a second aspect, the present application provides a fusion protein which sequentially comprises, from the N-terminus to the C-terminus:

[0129] a) a Fab fragment of a first antibody that can specifically bind to a first antigen;

[0130] b) an anti-CD3 antibody or an antigen-binding fragment thereof that can specifically bind to the CD3 molecule;

[0131] c) an anti-CD137 antibody or an antigen-binding fragment thereof that can specifically bind to the CD137 molecule;

[0132] wherein the heavy chains of the Fab fragment, the anti-CD3 antibody or its antigen-binding fragment, and the anti-CD137 antibody or its antigen-binding fragment are sequentially connected by a first peptide linker and a third peptide linker, and the light chains of the Fab fragment, the anti-CD3 antibody or its antigen-binding fragment, and the anti-CD137 antibody or its antigen-binding fragment are sequentially connected by a second peptide linker and a fourth peptide linker,

[0133] wherein only one disulfide bond can be formed between the first peptide linker and the second peptide linker, and only one disulfide bond can be formed between the third peptide linker and the fourth peptide linker, each independently selected from: peptide linkers comprising any one of the sequences shown in Seq ID NO.1-2, wherein X represents any amino acid other than Cys, or is absent.

[0134] In some embodiments, each of the first to fourth peptide linkers is a hinge region of a natural antibody, wherein the hinge region has a deletion mutation that retains only one cysteine.

[0135] In some embodiments, any one of the first to fourth peptide linkers is independently selected from the IgG1 hinge region with a C229 deletion mutation or the IgG1 hinge region with a C229 deletion mutation and an inversion of the hinge region D224-S242.

[0136] In some embodiments, the first antigen is selected from MESOTHELIN, EGFR, PSMA, GD2, CEA, MUC1, FAP, BCMA, EphA2, CD19, CD22, EpCAM, CEA, PD-L1, B7H3, ROR1, c-Met, and GPC3.

[0137] In some embodiments, the first antigen, the CD137 molecule, and the CD3 molecule are independently derived from mammals, preferably non-human primates or humans.

[0138] In some embodiments, the affinity constant of the Fab fragment for binding to the first antigen is 10 - 1000 times the affinity constant of the anti-CD137 antibody or its antigen-binding fragment for the CD137 molecule or the anti-CD3 antibody or its antigen-binding fragment for the CD3 molecule.

[0139] In some embodiments, the antigen-binding fragment is selected from Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fv fragments, diabodies or single-chain antibody molecules such as sc-Fv.

[0140] In a third aspect, the present application provides a nucleic acid encoding the fusion protein described in the first or second aspect.

[0141] In a preferred embodiment, the nucleic acid may be a codon-optimized nucleic acid suitable for expression in a host cell. For example, according to the degeneracy of codons, it still encodes the same protein. Methods for codon optimization according to the host cell used are well known to those skilled in the art.

[0142] In a fourth aspect, the present application provides an expression vector comprising the nucleic acid described in the third aspect.

[0143] Any suitable expression vector can be used. For example, prokaryotic cloning vectors include plasmids from Escherichia coli such as colE1, pCR1, pBR322, pMB9, pUC, pKSM, and RP4. Prokaryotic vectors also include phage DNA such as M13 and derivatives of other filamentous single-stranded DNA phages. Examples of vectors useful for yeast are 2μ plasmids. Suitable vectors for expression in mammalian cells include the following well-known derivatives: SV-40, adenovirus, retrovirus-derived DNA sequences, and shuttle vectors derived from combinations of functional mammalian vectors (such as those described above) and functional plasmids and phage DNA.

[0144] Additional eukaryotic expression vectors are known in the art (e.g., P J. Southern & P. Berg, J. Mol. Appl. Genet, 1:327-341 (1982); Subramani et al., Mol. Cell. Biol, 1:854-864 (1981); Kaufmann & Sharp, "Amplification And Expression of Sequences Cotransfected with a Modular Dihydrofolate Reductase Complementary DNA Gene," J. Mol. Biol, 159:601-621 (1982); Kaufhiann & Sharp, Mol. Cell. Biol, 159:601-664 (1982); Scahill et al., "Expression And Characterization Of The Product Of A Human Immune Interferon DNA Gene In Chinese Hamster Ovary Cells," Proc. Nat'l Acad. Sci USA, 80:4654-4659 (1983); Urlaub & Chasin, Proc. Nat'l Acad. Sci USA, 77:4216-4220, (1980), which are hereby incorporated by reference in their entirety).

[0145] Expression vectors useful in the present application contain at least one expression control sequence operably linked to a DNA sequence or fragment to be expressed. The control sequence is inserted into the vector to control and regulate the expression of the cloned DNA sequence. Examples of useful expression control sequences are the lac system, the trp system, the tac system, the trc system, the major operator and promoter regions of bacteriophage λ, the control region of fd coat protein, the glycolytic promoter of yeast, such as the promoter of 3-phosphoglycerate kinase, the promoter of yeast acid phosphatase, such as Pho5, the promoter of yeast α-mating factor, and promoters derived from polyomavirus, adenovirus, retrovirus, and simian virus, such as the early and late promoters of SV40 and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells and their viruses or combinations thereof.

[0146] In a fifth aspect, the present application provides a host cell comprising the nucleic acid described in the third aspect or the expression vector described in the fourth aspect.

[0147] In some embodiments, the host cell is a mammalian cell. Mammalian cells can include, but are not limited to, CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, and PER.C6 cells. Those skilled in the art can select a suitable host cell according to needs.

[0148] In a sixth aspect, the present application provides a method for preparing the fusion protein described in the first aspect or the second aspect, which includes:

[0149] a) culturing the host cell described in the fifth aspect; and

[0150] b) recovering the fusion protein from the host cell or the culture supernatant of the host cell.

[0151] In a seventh aspect, the present application provides a pharmaceutical composition, which comprises the fusion protein described in the first aspect or the second aspect, the nucleic acid described in the third aspect, the expression vector described in the fourth aspect, or the host cell described in the fifth aspect, and a pharmaceutically acceptable carrier.

[0152] The pharmaceutical composition of the seventh aspect can be prepared into a required dosage form by conventional methods in the pharmaceutical field. In some embodiments, the pharmaceutical composition is preferably in the form of a liquid or suspension.

[0153] In some embodiments, the pharmaceutically acceptable carrier is a carrier that does not weaken the viability and function of immune cells and does not affect the specific binding of an antibody or its antigen-binding fragment to an antigen, including but not limited to cell culture media, buffers, physiological saline, and balanced salt solutions, etc. Examples of buffers include isotonic phosphate, acetate, citrate, borate, and carbonate, etc. In a specific embodiment, the pharmaceutically acceptable carrier is a phosphate buffer containing 1% serum.

[0154] The fusion proteins and pharmaceutical compositions disclosed herein can be used to treat, ameliorate, or prevent tumors, autoimmune diseases, or infectious diseases in an individual.

[0155] The pharmaceutical composition described in the seventh aspect may further comprise a second medicament for treating, ameliorating, or preventing tumors, autoimmune diseases, or infectious diseases in an individual.

[0156] In an eighth aspect, the present application provides the use of the fusion protein described in the first aspect or the second aspect, the nucleic acid described in the third aspect, the expression vector described in the fourth aspect, or the host cell described in the fifth aspect in the preparation of a drug for treating, ameliorating, or preventing tumors, autoimmune diseases, or infectious diseases.

[0157] In a ninth aspect, the present application provides a method for treating, ameliorating or preventing a tumor, an autoimmune disease or an infectious disease in an individual, which comprises administering to the individual a therapeutically effective amount of the fusion protein described in the first aspect or the second aspect, the nucleic acid described in the third aspect, the expression vector described in the fourth aspect or the host cell described in the fifth aspect.

[0158] In some embodiments, the method further comprises administering a second agent for treating, ameliorating or preventing a tumor, an autoimmune disease or an infectious disease.

[0159] In a tenth aspect, the present application provides the use of the fusion protein described in the first aspect or the second aspect, the nucleic acid described in the third aspect, the expression vector described in the fourth aspect or the host cell described in the fifth aspect for treating, ameliorating or preventing a tumor, an autoimmune disease or an infectious disease.

[0160] "Treatment" refers to both therapeutic treatment and prophylactic or preventive measures, the purpose of which is to prevent or slow down (reduce) the target pathological condition or disease. Individuals in need of treatment include those who already have the disease, as well as those who will develop the disease or want to prevent their disease. Thus, the individuals to be treated herein have been diagnosed with the disease or are predisposed to or susceptible to the disease.

[0161] As used herein, the term "individual" refers to a mammal, including but not limited to primates, cattle, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice. Preferably, the mammal is a non-human primate or a human. Particularly preferred mammals are humans.

[0162] In certain embodiments, the tumor is a primary cancer or a metastatic cancer. In specific embodiments, the tumor is selected from lung cancer such as non-small cell lung cancer, colorectal cancer, bladder cancer, hematopoietic system cancers such as leukemia, breast cancer, gastric cancer, gastroesophageal junction adenocarcinoma, B-lymphocyte non-Hodgkin lymphoma, Hodgkin lymphoma, anaplastic large cell lymphoma, head and neck cancers such as head and neck squamous cell carcinoma, malignant glioma, renal cancer, melanoma, prostate cancer, bone cancer, giant cell tumor of bone, pancreatic cancer, sarcoma, liver cancer, cutaneous squamous cell carcinoma, thyroid cancer, cervical cancer, nasopharyngeal cancer, endometrial cancer, or metastatic cancers of the above tumors.

[0163] In certain embodiments, the autoimmune disease may include systemic lupus erythematosus, rheumatoid arthritis, scleroderma, systemic vasculitis, dermatomyositis, and autoimmune hemolytic anemia, etc.

[0164] In certain embodiments, the infectious diseases include respiratory infectious diseases, digestive tract infectious diseases, blood infectious diseases, surface infectious diseases, sexually transmitted diseases, etc. In specific embodiments, the infectious diseases may include, but are not limited to, influenza, tuberculosis, mumps, measles, pertussis, ascariasis, bacillary dysentery, hepatitis A, hepatitis B, malaria, epidemic encephalitis B, filariasis, schistosomiasis, trachoma, rabies, tetanus, gonorrhea, syphilis, AIDS, etc.

[0165] The "therapeutically effective amount" used herein can be determined according to specific circumstances, and those of ordinary skill in the art can easily master it according to the actual required drug dosage. For example, it can be determined according to the patient's weight, age, and disease condition.

[0166] In this specification and the claims, the words "comprising", "including", and "containing" mean "including but not limited to", and are not intended to exclude other parts, additives, components, or steps.

[0167] It should be understood that the features, characteristics, components, or steps described in a particular aspect, embodiment, or example of the present application can be applied to any other aspect, embodiment, or example described herein, unless there is a contradiction.

[0168] The above disclosure generally describes the present application, and the present application is further exemplified by the following examples. These examples are described only to illustrate the present application and not to limit the scope of the present application. Although special terms and values are used herein, these terms and values are also understood to be exemplary and do not limit the scope of the present application. Unless otherwise specified, the experimental methods and techniques in this specification are conventional methods and techniques in the art. For other materials and equipment that are not specifically indicated by the manufacturer, they are usually commercially available through conventional channels.

[0169] Examples

[0170] The following examples are used to illustrate the present application, but not to limit the scope of the present application. Without departing from the spirit and essence of the present application, any modification or replacement of the methods, steps, or conditions of the present application belongs to the scope of the present application.

[0171] Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0172] Example 1: Preparation, Expression, and Identification of a Trispecific Antibody Targeting CD3×CD137×CD19 Materials

[0173] The VH and VL encoding nucleic acid sequences of the anti-CD3 antibody and the anti-CD19 antibody are derived from Blincyto (Amgen). Among them, the VH encoding nucleic acid sequence of anti-CD19, the VH encoding nucleic acid sequence of anti-CD3, and the encoding nucleic acid sequence of IgG1 CH1-Hinge mut (i.e., the IgG1 hinge region including the C229 deletion mutation) are constructed into the plasmid pQKE3H (General Biosystems (Anhui) Co., Ltd.) by total synthesis. The VL encoding nucleic acid sequence of anti-CD19, the VL encoding nucleic acid sequence of anti-CD3, and the encoding nucleic acid sequence of Kappa-Hinge mut (the IgG1 hinge region including the C229 deletion mutation) are constructed into the plasmid pQKE3L (General Biosystems (Anhui) Co., Ltd.) by total synthesis. The synthetic vector pUC57 IgG1 Hinge mut R-CD137VH, which contains the encoding nucleic acid sequence of IgG1 Hinge mut R (C229 deletion mutation, and the hinge region D224-S242 inverted) synthesized by DNA and the anti-CD137 VH encoding nucleic acid sequence in the anti-CD137 monoclonal antibody (the sequence is referred to the patent, patent number: US20190284292A1) (General Biosystems (Anhui) Co., Ltd.); the synthetic vector pUC57 IgG1 Hinge mut R-CD137 VL, which contains the encoding nucleic acid sequence of IgG1 Hinge mut R (C229 deletion mutation, and the hinge region D224-S242 inverted) synthesized by DNA and the anti-CD137 VL encoding nucleic acid sequence in the anti-CD137 monoclonal antibody (the sequence is referred to the patent, patent number: US20190284292A1) (General Biosystems (Anhui) Co., Ltd.).

[0174] The nucleotide sequences of IgG1 CH1-Hinge mut and Kappa-Hinge mut are as follows:

[0175]

[0176] The nucleotide sequence of IgG1 Hinge mut R is as follows:

[0177]

[0178] 1.1 Preparation of the trispecific antibody expression vector targeting CD3×CD137×CD19 1.1.1 Construction of the trispecific antibody heavy chain expression vector pQKTriad1H

[0179] Using pUC57 IgG1 CH1 Hinge mut R-CD137 VH as a template, the gold medal Mix PCR kit (TSINGKE) was used to amplify the IgG1 Hinge mut R-CD137 VH fragment according to the kit's instructions. The size of the amplified product was approximately 0.4 kb. At the same time, the synthesized vector plasmid pQKE3H was digested with the restriction endonuclease EcoRI (NEB, R3101S). The obtained PCR amplification product and the digested vector pQKE3H were recombinantly ligated using the BM seamless cloning kit (Bomed) according to the kit's instructions to obtain the heavy chain expression vector pQKTriad1H (the ligation order is from 5' to 3'): anti-CD19 antibody VH-CH1-Hinge mut-anti-CD3 antibody VH-IgG1 Hinge mut R-anti-CD137 antibody VH).

[0180] The PCR amplification primer pairs are as follows:

[0181]

[0182] 1.1.2 Construction of the trispecific antibody light chain expression vector pQKTriad1 L

[0183] Using pUC57 IgG1 Hinge mut R-CD137 VL as a template, the gold medal Mix PCR kit (TSINGKE) was used to amplify the IgG1 Hinge mut R-CD137 VL fragment according to the kit's instructions. The size of the amplified product was approximately 0.4 kb. At the same time, the synthesized vector plasmid pQKE3L was digested with the restriction endonuclease EcoRI (NEB, R3101S). The obtained PCR amplification product and the digested vector pQKE3L were recombinantly ligated using the BM seamless cloning kit (Bomed) according to the kit's instructions to obtain the light chain expression vector pQKTriad1L (the ligation order is from 5' to 3' as: anti-CD19 antibody VL-Kappa-Hinge mut-anti-CD3 antibody VL-IgG1 Hinge mut R-anti-CD137 antibody VL).

[0184] The PCR amplification primer pairs are as follows:

[0185]

[0186] 1.1.3 Amplification and preparation of the recombinant plasmid

[0187] The heavy-chain expression vector pQKTriad1H and the light-chain expression vector pQKTriad1L obtained as above were separately transformed into Escherichia coli (E. coli) TOP10. After picking monoclonal colonies and identification, they were cultured in LB medium containing ampicillin (final concentration: 100 mg / L) for 16 hours at 37 °C with shaking at 200 rpm. The bacteria were collected by centrifugation at 8000×g for 20 minutes. The plasmid was isolated and extracted using the NucleoBond Xtra Midi kit (Macherey-nagel) according to the kit instructions, eluted with 1 mL of sterile ultrapure water, and finally the plasmid concentration was measured using a Nanodrop micro-spectrophotometer.

[0188] 1.2 Expression of the antibody

[0189] The heavy-chain expression vector pQKTriad1H and the light-chain expression vector pQKTriad1L were co-transfected into HEK293 cells for expression. 24 hours before transfection, 1.5×10 6 HEK293 (ATCC, No.: CRL-1573) cells were seeded into a 500 mL shake flask containing 100 mL of OPM-293CD05 serum-free medium (OPM, Cat: 81075-001), and cultured under the conditions of 36.5 °C, 7.5% CO2, and suspension culture at 120 rpm. At the time of transfection, the recombinant plasmids pQKTriad1H and pQKTriad1L were mixed in a 1:1 weight ratio (total DNA amount: 100 μg) in 10 mL of OPM-293CD05 medium, then 100 μL of PEI (concentration: 3 mg / mL) was added, and quickly vortexed and mixed evenly, and incubated at room temperature for 15 minutes. Then this mixture was added to the above cell culture. The cells were continuously cultured for 7 days at 36.5 °C, 7.5% CO2, and 120 rpm / min to harvest the expressed antibody. This antibody refers to the anti-CD19×anti-CD3×anti-CD137 trispecific antibody expressed by the plasmids pQKTriad1H and pQKTriad1L, and the antibody was named T1, and the structure is as shown in Figure 1 A as shown in.

[0190] 1.3 Purification of the antibody

[0191] The harvested cell culture was centrifuged at 3000×g for 20 min, and the supernatant was collected and filtered through a 0.45 μm filter. A 5 mL Capto L affinity chromatography column (GE) was equilibrated with a mixed buffer of 20 mM PB and 150 mM NaCl (pH 7.4) at a flow rate of 5 mL / min, and the volume was greater than 5 CV. The filtered sample solution was loaded at a flow rate of 5 mL / min. After loading, the Capto L affinity chromatography column was washed with a mixed buffer of 20 mM PB and 150 mM NaCl (pH 7.4) at a flow rate of 5 mL / min. Elution was performed with 50 mM citric acid (pH 3.0) buffer at a flow rate of 5 mL / min, and the complete elution peak was collected. At the same time, the pH of the collected eluate was adjusted to about 7.0 with 1 M Tris HCl (pH 9.0) buffer ( Figure 2 A in). The purified product was ultrafiltered through an ultrafiltration tube to replace the Tris-citric acid buffer with a commercial PBS buffer. The obtained protein was detected by SDS-PAGE and Coomassie brilliant blue staining ( Figure 3 A in). The protein concentration was measured using a Nanodrop micro-spectrophotometer, and the protein yield was calculated to be 230 mg / L.

[0192] 1.4 Identification of antibodies

[0193] 1.4.1 HPLC determination of antibody purity

[0194] The antibody purified by Capto L was detected for its purity by HPLC (Agilent 1260II) SEC. The chromatographic column was a Sepax hydrophilic size exclusion chromatography column, the mobile phase was 50 mM PB + 300 mM NaCl pH 7.0, the sample loading amount was 10 μg, the flow rate was 1 mL / min, and isocratic elution was performed for 20 min. The results showed that the monomer purity after one-step purification was about 86%.

[0195] 1.4.2 Fortebio determination of antibody affinity

[0196] The purified antibody was used to determine its affinity constant K using a molecular interaction instrument Fortebio Octet QK (Molecular Devices). D . The T1 antibody was immobilized by the sensor of Fab-Ch1 at a fixed concentration of 0.25 μM. Antigens human CD19 (Sino Biological, Cat: 10084-HNAH), human CD3 (Sino Biological, Cat: 10977-H02H) and human CD137 (Sino Biological, Cat: 10041-H002H) were loaded at concentrations of 600 nM, 300 nM, 150 nM and 75 nM respectively. The results of the determination of the affinity constant are shown in Table 1.

[0197] Table 1: Results of determination of affinity constant

[0198] Antibody - Antigen <![CDATA[Affinity constant K D (M)]]> T1 / CD19Ag 6.76E-09 T1 / CD3εAg 1.12E-07 T1 / CD137Ag 7.2E-08

[0199] 1.5 Detection of antibody-cell binding activity by flow cytometry

[0200] 1.5.1 Antibody-Nalm-6 cell binding activity

[0201] The Nalm-6 cells (ATCC number: CRL-3273) that were normally resuscitated and passaged at least 3 times were pipetted evenly and collected. The cells were centrifuged at 400 g / min for 5 min, resuspended and counted, and the cell density was adjusted to 1×10 6 cells / mL. The groups were set as blank group, positive control group, secondary antibody group and experimental group (T1). First, 100 μl of the above cell suspension was added to the tubes of each group. 100 ng of T1 antibody was added to the tube of the T1 group, incubated at room temperature for 30 min, washed with 3 mL of PBS buffer containing 2% FBS, centrifuged at 400 g / min for 5 min, and resuspended with 50 μl of PBS buffer containing 2% FBS; 0.5 μl of water was added to the blank group; 0.5 μl of FITC anti-human CD19 (BioLegend, clone number: HIB19) was added to the positive control group; 0.5 μl of APC anti-human Ig light chain κ (BioLegend, clone number: TB28-2) was added to the T1 group and the secondary antibody group respectively. After mixing each group, they were incubated at room temperature in the dark for 30 min. After incubation, the tubes of each group were washed with PBS buffer containing 2% FBS, centrifuged at 400 g / min for 5 min, resuspended with 100 μl of PBS buffer containing 2% FBS, and detected by a flow cytometer (ACEA, instrument model: NovoCyte). The results are shown as Figure 4 shown in A1 below.

[0202] 1.5.2 Antibody-Jurkat cell binding activity

[0203] The Jurkat cells (Union Cell Resource Center, resource number: 3111C0001CCC000075) that were normally resuscitated and passaged at least 3 times were pipetted evenly and collected. The cells were centrifuged at 400 g / min for 5 min, resuspended and counted, and the cell density was adjusted to 1×10 6cells / mL. The groups were set as the blank group, the secondary antibody group, and the experimental group (T1). First, add 100 μl of the above cell suspension to the tubes of each group. Add 100 ng of T1 antibody to the tubes of the T1 group, incubate at room temperature for 30 min, wash with 3 mL of PBS buffer containing 2% FBS, centrifuge at 400 g / min for 5 min, and resuspend with 50 μl of PBS buffer containing 2% FBS; add 0.5 μl of water to the blank group; add 0.5 μl of APC anti-human Ig light chain K (BioLegend, clone number: TB28-2) to the T1 group and the secondary antibody group respectively. After mixing each group, incubate in the dark at room temperature for 30 min. After incubation, wash the tubes of each group with PBS buffer containing 2% FBS, centrifuge at 400 g / min for 5 min, resuspend with 100 μl of PBS buffer containing 2% FBS, and detect with a flow cytometer (ACEA, instrument model: NovoCyte). The results are as Figure 4 shown in A2 of

[0204] 1.5.3 Binding Activity of Antibody to HEK293-CD137 Cells

[0205] Normal resuscitated HEK293 cells (Peking Union Medical College Cell Resource Center, Resource No.: 3111C0001CCC000010) were passaged and cultured for at least 3 generations. Cells were passaged 24 h before transfection and seeded into 6-well plates. On the day of transfection, PEI (Sigma, Cat: 764647) and synthetic plasmid pENTER CD137 (General Biosystems (Anhui) Co., Ltd.) were thawed to room temperature. 5 μg of plasmid was added to 500 μl of DMEM medium (gibco, REF: 11965-092), and then 15 μg of PEI was added. The mixture was immediately vortexed for 15 min and then gently added dropwise to the cell culture medium. The cells were placed in an incubator and cultured for another 24 - 48 h. After 48 h, the DMEM medium containing 10% FBS with 2 μg / mL puromycin was replaced. After 3 days, a large number of cells died. The cell culture flask was gently tapped, and the supernatant was discarded. The cells that adhered well were the potential stable transfected cell lines. After 8 - 10 days, depending on the cell growth status, the cells were digested and seeded into 96-well plates to screen for monoclonal cell lines. During this period, the cells were continuously cultured under pressure with DMEM medium containing 2 μg / mL puromycin and 10% FBS. The finally obtained cell line was named HEK293-CD137. The extracellular region gene of human CD137 was transfected into the genome of this cell line, which could stably express the extracellular region protein of human CD137 and display it on the cell membrane. The HEK293-CD137 cells that had been resuscitated and cultured for more than 3 generations were washed once with 10 mL of PBS, digested with 1 mL of 0.05% trypsin for 1 min, 4 mL of DMEM medium containing 10% FBS was added, and the cells were collected after pipetting evenly. The cells were centrifuged at 1000 rpm / min for 5 min, resuspended and counted, and the cell density was adjusted to 1×10 6 cells / mL. The groups were set as blank group, positive control group, secondary antibody group and experimental group (T1). First, 10 μl of the above cell suspension was added to the tubes of each group. 100 ng of Triad1TsAb was added to the tubes of the T1 group and incubated at room temperature for 30 min, washed with 3 mL of PBS buffer containing 2% FBS, centrifuged at 1000 rpm / min for 5 min, and resuspended with 50 μl of PBS buffer containing 2% FBS; 0.5 μl of water was added to the blank group; 0.5 μl of anti-CD137 antibody (the antibody is described in the patent, Patent No.: US-2019-0284292-A1) was added to the positive control group; 0.5 μl of APC anti-human Ig light chain K (BioLegend, clone number: TB28-2) was added to the T1 group and the secondary antibody group respectively. After mixing each group, they were incubated in the dark at room temperature for 30 min. After incubation, the tubes of each group were washed with PBS buffer containing 2% FBS, centrifuged at 1000 rpm / min for 5 min, resuspended with 100 μl of PBS buffer containing 2% FBS, and detected by flow cytometry (ACEA, instrument model: NovoCyte). The results are as followsFigure 4 as shown in A3 in

[0206] Example 2: Preparation, Expression and Identification of a Trispecific Antibody Targeting CD3×CD137×GPC3

[0207] 2.1 Preparation of an Expression Vector for a Trispecific Antibody Targeting CD3×CD137×GPC3

[0208] For the specific operating procedures of expression vector construction and plasmid amplification, refer to Example 1. Among them, the synthetic vector pUC57GPC3 scFv (sequence see patent, patent number: US7919086B2) (General Biosystems (Anhui) Co., Ltd.) was synthesized, which contains the anti-GPC3 scFv-encoding nucleic acid sequence synthesized by DNA and is used to amplify the anti-GPC3 VH fragment; the plasmid pQKTriad1 H (see Example 1) was used as a template to amplify the CH1-Hinge mut-CD3 VH-CD137 VH fragment. The fully synthetic vector pQKX1 (General Biosystems (Anhui) Co., Ltd.) was double-digested with SapI and EcoRI, and the corresponding PCR products and enzyme digestion products were recovered and ligated recombinantly to obtain the final recombinant plasmid named pQKTriad2H (the ligation order from 5' to 3' is: anti-GPC3 antibody VH-CH1-Hinge mut-anti-CD3 antibody VH-IgG1 Hinge mut R-anti-CD137 antibody VH). The synthetic vector pUC57 GPC3 scFv (sequence see patent, patent number: US7919086B2) (General Biosystems (Anhui) Co., Ltd.) was synthesized, which contains the anti-GPC3 scFv-encoding nucleic acid sequence synthesized by DNA and is used to amplify the anti-GPC3 VL fragment; the plasmid pQKTriad1 L (see Example 1) was used as a template to amplify the Kappa-Hinge mut-CD3 VL-CD137 VL fragment. The fully synthetic vector pQKX2 (General Biosystems (Anhui) Co., Ltd.) was double-digested with SapI and EcoRI, and the corresponding PCR products and enzyme digestion products were recovered and ligated recombinantly to obtain the final recombinant plasmid named pQKTriad2L (the ligation order from 5' to 3' is: anti-GPC3 antibody VL-Kappa-Hinge mut-anti-CD3 antibody VL-IgG1 Hinge mut R-anti-CD137 antibody VL).

[0209] The PCR amplification primer pairs are as follows:

[0210]

[0211] 2.2 Expression of the Antibody

[0212] Transfection was carried out according to the procedure described in Example 1, where the transfected cells were HEK293 (ATCC number: CRL-1573) and the transfection volume was 100 mL. The transfected cells were suspended and cultured in a 500 mL shake flask for 7 days to harvest the antibody. The culture conditions were 36.5 °C, 7.5% CO2, and 120 rpm / min. The obtained antibody refers to the anti-GPC3 × anti-CD3 × anti-CD137 trispecific antibody expressed by plasmids pQKTriad2H and pQKTriad2L, and the antibody is named T2. The structure is as shown in Figure 1 B in

[0213] 2.3 Purification of the antibody

[0214] Purification was carried out according to the procedure described in Example 1 ( Figure 2 B in Figure 3 B in ). After buffer replacement, the protein concentration was measured using a Nanodrop micro-spectrophotometer, and the protein yield was calculated to be 50 mg / L.

[0215] 2.4 Identification of the antibody

[0216] 2.4.1 HPLC determination of antibody purity

[0217] The purity of antibody T2 was determined by HPLC with reference to Example 1. After one-step purification, the monomer purity was approximately 76%.

[0218] 2.4.2 Fortebio determination of antibody affinity

[0219] The specific procedure was referred to Example 1. The T2 antibody was immobilized through the Fab-Ch1 sensor at a fixed concentration of 0.25 μM. The antigens human GPC3 (Sino Biological, Cat: 10088-H08H), human CD3 (Sino Biological, Cat: 10977-H02H), and human CD137 (Sino Biological, Cat: 10041-H002H) were loaded at concentrations of 600 nM, 300 nM, 150 nM, and 75 nM, respectively. The results of the affinity constant determination are shown in Table 2.

[0220] Table 2: Results of the affinity constant determination

[0221] Antibody - Antigen <![CDATA[Affinity constant K D (M)]]> T2 / GPC3Ag 1.04E-08 T2 / CD3εAg 7.4E-08 T2 / CD137Ag 4.4E-08

[0222] 2.5 Flow cytometry detection of antibody-cell binding activity

[0223] 2.5.1 Antibody-Jurkat cell binding activity

[0224] The specific materials and operation procedures are referred to 1.5.2 in Example 1, where the antibody in the experimental group (T2) was T2. The results are as shown in Figure 4 B1 in

[0225] 2.5.2 Binding Activity of Antibody to HEK293-CD137 Cells

[0226] For the specific materials and operation procedures, refer to 1.5.3 in Example 1, where the antibody in the experimental group (T2) is T2. The results are as shown in Figure 4 A3 in

[0227] 2.5.3 Binding Activity of Antibody to HepG2 Cells

[0228] For HepG2 cells (Peking Union Medical College Cell Resource Center, Resource No.: 3111C0001CCC000035) that are normally resuscitated and passaged for at least 3 generations, pipette them evenly and collect the cells. Centrifuge at 1000 rpm / min for 5 min, resuspend and count, and adjust the cell density to 1×10 6 cells / mL. The groups are set as the blank group, the secondary antibody group, and the experimental group (T2). First, add 100 μl of the above cell suspension to the tubes of each group. Add 100 ng of T2 TsAb to the tube of the T2 group, incubate at room temperature for 30 min, wash with 3 mL of PBS buffer containing 2% FBS, centrifuge at 1000 rpm / min for 5 min, and resuspend with 50 μl of PBS buffer containing 2% FBS; add 0.5 μl of water to the blank group; add 0.5 μl of APC anti-human Ig light chain K (BioLegend, clone number: TB28-2) to the T2 group and the secondary antibody group respectively. After mixing each group, incubate in the dark at room temperature for 30 min. After incubation, wash the tubes of each group with PBS buffer containing 2% FBS, centrifuge at 1000 rpm / min for 5 min, resuspend with 100 μl of PBS buffer containing 2% FBS, and detect with a flow cytometer (ACEA, instrument model: NovoCyte). The results are as shown in Figure 4 B2 in

[0229] Example 3: Preparation, Expression and Identification of Tri-specific Antibody Targeting CD3×CD137×c-Met and Bispecific Antibody Targeting CD3×c-Met

[0230] 3.1.1 Preparation of Expression Vector of Tri-specific Antibody Triad19 Targeting CD3×CD137×c-Met

[0231] The specific operation procedures for expression vector construction and plasmid amplification refer to Example 1. The VH and VL coding nucleic acid sequences of the anti-c-Met antibody are derived from Onartuzumab (MetMAb) (Patent No.: US7615529B2). The synthetic vector pUC57 c-MetscFv (General Biosystems (Anhui) Co., Ltd.) contains the anti-c-Met scFv coding nucleic acid sequence synthesized by DNA and is used to amplify the anti-c-Met VH fragment; the plasmid pQKTriad1 H (see Example 1) is used as a template to amplify the IgG 1CH1-Hinge mut-CD3 VH-IgG1 Hinge mut R-CD137 VH fragment. The fully synthetic vector pQKX1 (General Biosystems (Anhui) Co., Ltd.) is digested with SapI and EcoRI, and the corresponding PCR products and digestion products are recovered and ligated by recombinant enzymes to obtain the final recombinant plasmid named pQKTriad19H (the ligation order from 5' to 3' is: anti-c-Met antibody VH-IgG1CH1-Hinge-mut-anti-CD3 antibody VH-IgG1 Hinge mut R-anti-CD137 antibody VH). The synthetic vector pUC57 c-MetscFv (General Biosystems (Anhui) Co., Ltd.) contains the anti-c-Met scFv coding nucleic acid sequence synthesized by DNA and is used to amplify the anti-c-Met VL fragment; the plasmid pQKTriad1 L (see Example 1) is used as a template to amplify the Kappa-Hingemut-CD3 VL-IgG1 Hinge mut R-CD137 VL fragment. The fully synthetic vector pQKX2 (General Biosystems (Anhui) Co., Ltd.) is digested with SapI and EcoRI, and the corresponding PCR products and digestion products are recovered and ligated by recombinant enzymes to obtain the final recombinant plasmid named pQKTriad19L (the ligation order from 5' to 3' is: anti-c-Met antibody VL-Kappa-Hingemut-anti-CD3 antibody VL-IgG1 Hinge mut R-anti-CD137 antibody VL).

[0232] The PCR amplification primer pairs are as follows:

[0233]

[0234] 3.1.2 Preparation of the expression vector of the bispecific antibody ZWB56 targeting CD3×c-Met

[0235] The specific operation procedures for expression vector construction and plasmid amplification refer to Example 1. The VH and VL coding nucleic acid sequences of the anti-c-Met antibody are derived from Onartuzumab (MetMAb) (Patent No.: US7615529B2). Using the pQKTriad19H plasmid (see Example 3.1.1) as a template, it is used to amplify the c-Met VH-IgG1 CH1-Hinge mut-CD3 VH fragment. The full-synthetic vector pQKX1 (General Biosystems (Anhui) Co., Ltd.) is double-digested with SapI and EcoRI, and the corresponding PCR products and enzyme digestion products are recovered, and then recombinant ligation is carried out to obtain the final recombinant plasmid named pQKZWB56H (the ligation order from 5' to 3' is: anti-c-Met antibody VH-IgG1 CH1-Hinge-mut-anti-CD3 antibody VH); using the pQKTriad19L plasmid (see Example 3.1.1) as a template, it is used to amplify the c-Met VL-Kappa-Hinge mut-CD3 VL fragment. The full-synthetic vector pQKX2 (General Biosystems (Anhui) Co., Ltd.) is double-digested with SapI and EcoRI, and the corresponding PCR products and enzyme digestion products are recovered, and then recombinant ligation is carried out to obtain the final recombinant plasmid named pQKZWB56L (the ligation order from 5' to 3' is: anti-c-Met antibody VL-Kappa-Hinge-mut-anti-CD3 antibody VL). Compared with the trispecific antibody Triad19, the bispecific antibody ZWB56 only lacks the IgG1 Hinge mut R-anti-CD137 antibody VH and IgG1 Hinge mut R-anti-CD137 antibody VL( Figure 1 C and Figure 1 D).

[0236] The following are the PCR amplification primer pairs:

[0237]

[0238] 3.2 Antibody Expression

[0239] Transfection is carried out according to the procedure described in Example 1, where the transfected cells are HEK293 (ATCC number: CRL-1573) and the transfection volume is 100 mL. The transfected cells are suspended and cultured in a 500 mL shake flask for 7 days to harvest the antibody. The culture conditions are 36.5 °C, 7.5% CO2, and 120 rpm / min. When the obtained antibody is the anti-c-Met × anti-CD3 × anti-CD137 trispecific antibody expressed by the plasmids pQKTriad19H and pQKTriad19L, the antibody is named Triad19, and the structure is as Figure 1as shown in C; when the obtained antibody is the anti-c-Met × anti-CD3 bispecific antibody expressed by plasmids pQKZWB56H and pQKZWB56L, the antibody is named ZWB56, and the structure is as Figure 1 shown in D.

[0240] 3.3 Purification of the antibody

[0241] Purification was carried out with reference to the procedure described in Example 1. After buffer replacement, the protein concentration was measured using a Nanodrop micro-spectrophotometer, and the protein yield was calculated. Among them, Triad19 was 9.8 mg / L and ZWB56 was 125 mg / L.

[0242] 3.4 Identification of the antibody

[0243] 3.4.1 Determination of the antibody size and purity by SDS-PAGE

[0244] SDS-PAGE gel was selected for protein electrophoresis, and reducing and non-reducing buffers were added respectively to identify the molecular band sizes of the protein in the reduced and non-reduced states. After Coomassie Brilliant Blue staining, the accuracy and purity of the protein were analyzed according to the protein Marker. The results are as Figure 5 shown. Analyzing the protein electrophoresis, the bands showed that the electrophoresis was consistent with the expected protein size, and the protein purity was high.

[0245] 3.4.2 Determination of the antibody affinity by Fortebio

[0246] The kinetic parameters of the binding of ZWB56 / Triad19 to c-Met protein, CD3 protein, and CD137 protein were measured using a Fortebio molecular interaction instrument. Specifically, His or Fc sensors were used to capture and immobilize c-Met protein, CD3 protein, and CD137 protein. After equilibration in PBS, they were combined with ZWB56 / Triad19. ZWB56 / Triad19 was diluted to 4 concentrations with PBS, namely 500 nM, 250 nM, 125 nM, and 62.5 nM, and dissociated in PBS. The results of the determination of the affinity constant are shown in Table 3 and Table 4.

[0247] Table 3: Results of the determination of the affinity constant of T19 and each target

[0248] Antibody - Antigen <![CDATA[Affinity constant K D (M)]]> T19 / c - Met Ag 3.73E-09 T19 / CD3εAg 1.04E-07 T19 / CD137Ag 1.02E-07

[0249] Table 4: Results of the determination of the affinity constant of B56 and each target

[0250] Antibody - Antigen <![CDATA[Affinity constant K D (M)]]> B56 / c - Met Ag 2.37E-09 B56 / CD3εAg 1.16E-07

[0251] 3.5 Detection of the antibody functional activity by the CD3 functional cell line Jurkat Dual

[0252] 1). Target cell treatment: Prepare a bottle of target cells U87 MG in T75 (Xiehe Cell Resource Center, resource number: 3111C0001CCC000208), digest with 0.25% trypsin, centrifuge at 850 rpm for 5 min at room temperature. After discarding the supernatant, resuspend the cells with working medium (DMEM medium containing 10% FBS (inactivated)), and count the cell density and viability with an automatic cell counter. Then dilute the cell suspension to 5×10 4 cells / mL.

[0253] 2). Add the diluted U87 MG cells into a 96-well plate at 100 μL / well. Place it in a 37 °C cell incubator with 5% CO2 and incubate for 6 h until the cells adhere to the wall.

[0254] 3). Jurkat Dual cell treatment: Take a bottle of Jurkat Dual cells in T75 (Xiehe Cell Resource Center, resource number: 3111C0001CCC000075), centrifuge at 850 rpm for 5 min, discard the supernatant, resuspend and count with working medium (1640 medium containing 10% FBS (inactivated)), and adjust the cells to 2×10 6 cells / mL.

[0255] 4). Dilute Triad19 / ZWB56. The highest concentration point of Triad19 is 20 μg / mL, add it at 50 μL / well, and the corresponding final drug concentration is 10 μg / mL, that is, the final concentration is 100 nM. The highest concentration point of ZWB56 is 15 μg / mL, add it at 50 μL / well, and the corresponding final drug concentration is 7.5 μg / mL, that is, the final concentration is 100 nM. Dilute in three-fold gradient, with a total of 10 concentration points.

[0256] 5). According to the well plate design, add 50 μL of different concentration Triad19 / ZWB56 diluent and 50 μL of diluted Jurkat Dual cells into each well of the 96-well plate, so that each well contains 1×10 5 Jurkat Dual cells.

[0257] 6). Fill the control wells or the wells without target cells with complete medium to 200 μl.

[0258] 7). Transfer the 96-well plate to a constant temperature cell incubator (37 °C, 5% CO2) and culture for 18 - 24 h.

[0259] 8). After the culture is completed, centrifuge at 300 - 400 g for 5 min at room temperature.

[0260] 9). Take a new 96-well plate (black transparent bottom), aspirate 40 μl of cell culture supernatant, add 50 μl of QUANTI-Luc substrate to each well, mix well, and then detect the signal intensity value on a multifunctional microplate reader.

[0261] 10). Analyze the test results using GraphPad Prism 8 software and statistically analyze the results.

[0262] The results are as Figure 6 shown. When the target cells U87 MG expressing c-Met are added, the CD3 partial functional signal of Triad19 / ZWB56 is significantly enhanced, and at the same time, the CD3 function of Triad19 is stronger than that of ZWB56. Without the action of target cells, the simple antibody drug has no function of activating Jurkat Dual cells.

[0263] Example 4: RTCA analysis of T cell killing experiment under the action of antibodies

[0264] The experimental steps are as follows:

[0265] 1. Take U87 MG cells expressing c-Met, digest them and centrifuge, resuspend and count them with complete medium (DMEM / 1640 + 10% FBS + 1% GluMax), and dilute them at a concentration of 5×10 4 cells / mL.

[0266] 2. Take the RTCA (ACEA) supporting plate Eplate16, operate according to the RTCA instrument. First, add 50 μL of complete medium to the RTCA plate, and place the plate on the instrument to perform the pre-activation step.

[0267] 3. After the plate is pre-activated, add U87 MG cells to the plate at 100 μL / well, and start the program after standing for half an hour.

[0268] 4. Observe the cell growth curve. When the cells are in the growth exponential phase, resuscitate PBMC (Ausbian Biotechnology (Shanghai) Co., Ltd.), and use the EasySep TM Human T Cell Isolation Kit from Stemcell company to sort T cells from human PBMC, resuspend the cells with working medium (1640 medium containing 1% GluMax and 10% inactivated FBS) and count them.

[0269] 5. According to the effector-to-target ratio of 5:1, that is, the target cells are 5000 cells / well and the effector T cells are 25000 cells / well, dilute the sorted T cells to 5×10 5 cells / mL.

[0270] 6. Triad19 and ZWB56 were diluted in a gradient manner, with the highest drug concentration point being 5 nM, corresponding to a final concentration of 0.5 μg / mL for Triad19 and 0.375 μg / mL for ZWB56. The drug preparation concentration was 4 times the final concentration, i.e. 2 μg / mL for Triad19 and 1.5 μg / mL for ZWB56.

[0271] 7. Pause the RTCA program, discard 50 μL of supernatant from each cell in the plate well, add 50 μL of diluted T cells to each well, add 50 μL of Triad19 / ZWB56 according to the corresponding concentration, and add complete culture medium to the control well to make up to 200 μL.

[0272] 8. After the plate wells have been left on the RTCA instrument for 30 minutes, start the program. Observe the cell growth curve in the killing experiment, and fit the drug killing curve after analysis. Figure 7 , Figure 8 As shown in the figure, the IC50 value of Triad19 is at the sub-nM level, and the EC50 value of ZWB56 is at the nM level. GraphPad Prism 8 was used to analyze the cell killing rate at each drug concentration, as shown in the figure. Figure 9 shown.

[0273] 9. Take a 96-well plate and conduct parallel experiments simultaneously. The purpose is to take the cell supernatant 16-20 hours after the drug administration to detect IL-2 / IL-6 cytokines. When the killing experiment is terminated, take the cell supernatant of the parallel wells to detect IFN-γ factor.

[0274] 10. Human IL-2 / IL-6 / IFN-γ ELISA kit (Lianke Bio) was used to perform cytokine ELISA detection, and the factors of each sample were quantified. After the OD value was detected by microplate reader, the relevant data were analyzed by GraphPad Prism 8, and the results were summarized in Figure 10 .

[0275] 11. Collect T cells after T cell killing experiment and perform T cell grouping analysis, including CD4, CD8, CD45RO, CCR7, PD-1, Granzyme B, analyze the ratio of CD4 and CD8 cells, the ratio of central memory T cells and effector memory T cells, and the change of Granzyme B with drug concentration, such as Figures 11 - 15 GraphPad Prism 8 was used for all the above-mentioned graphics and analyses.

[0276] Result analysis: The results of the T cell killing experiment showed that the drug killing curve was fitted to obtain the drug IC50, and the killing ability of Triad19 was one order of magnitude higher than that of ZWB56. Analyzing the fitting curves of the cell killing rate at each concentration point, it can be seen that at each corresponding concentration point, the killing power of ZWB56 is weaker than that of Triad19. The analysis results of IL2, IL6, and IFN-γ cytokines showed that the expression levels of each cytokine showed an obvious drug dose relationship, and the higher the drug concentration, the higher the expression level of each factor. Similarly, at the same drug concentration, ZWB56 is weaker.

[0277] The results of flow cytometry analysis showed that after administration of Triad19 / ZWB56, as the drug dose increased, the proportion of CD8 in CD3 T cells increased, and the corresponding proportion of CD4 decreased. The proportion of CD4 or CD8 effector memory T cells (TEM) tended to increase with the increase of drug concentration, and the central memory T cells (TCM) showed basically no change. For the Granzyme B index, Triad19 was significantly higher than ZWB56 at high concentration points, which also exactly corresponded to the reason for the weaker killing ability of ZWB56. PD-1 can also be used as an indicator of T cell activation to a certain extent. Analyzing, as the drug concentration increased, PD-1 on both CD4 T cells and CD8 T cells showed an increasing trend and had a certain regularity.

[0278] Generally speaking, under the comparison of equimolar administration, Triad19 has stronger abilities of T cell activation and tumor cell killing than ZWB56, including the comparison of each cytokine and the analysis of each cell subset by flow cytometry.

[0279] Although the technical solutions of this application have been described in detail in the above text with general descriptions and specific implementation examples, based on these technical solutions, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of this application all fall within the scope protected by this application.

[0280] Informal Sequence Listing

[0281] Anti-CD137 VH (SEQ ID NO:26):

[0282] Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala SerVal Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asp TrpVal Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Asn Ile Tyr Pro Asp SerGly Gly Thr Asn Tyr Ala Glu Lys Phe Lys Ser Arg Val Thr Leu Thr Val Asp ThrSer Ile Ser Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala ValTyr Tyr Cys Ala Arg Glu Glu Ala Leu Gly Gly Tyr Tyr Glu Leu Thr Tyr Trp GlyGln Gly Thr Leu Val Thr Val Ser Ser

[0283] Anti-CD137 VL (SEQ ID NO:27):

[0284] Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly GluArg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Ser Thr Ser Ser Tyr Ser TyrMet His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Lys Tyr AlaSer Asn Leu Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys GlnHis Ser Trp Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

[0285] Anti-CD3 VH (SEQ ID NO:28):

[0286] Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala SerVal Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His TrpVal Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser ArgGly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp LysSer Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala ValTyr Tyr Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln GlyThr Thr Leu Thr Val Ser Ser

[0287] Anti-CD3 VL (SEQ ID NO:29):

[0288] Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

[0289] Adapter sequence of C229 deletion mutation (SEQ ID NO:30):

[0290] Asp Lys Thr His Thr Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

[0291] Adapter sequence of C229 deletion mutation and inversion of hinge region D224 - S242 (SEQ ID NO:31):

[0292] Ser Pro Gly Gly Leu Leu Glu Pro Ala Pro Cys Pro Pro Thr His Thr Lys Asp Sequence Listing <110> Beijing ImmunArk Pharmaceutical Technology Co., Ltd. <120> A fusion protein simultaneously targeting CD3 and CD137, its preparation method and uses <160> 31 <170> SIPOSequenceListing 1.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (1)..(1) <223> Xaa can be any amino acid other than Cys or absent <220> <221> UNSURE <222> (1)..(1) <223> The 'Xaa' at position 1 represents Gln, Arg, Pro, or Leu. <400> 1 Xaa Pro Pro Cys Pro Ala Pro Gly 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <221> SITE <222> (8)..(8) <223> Xaa can be any amino acid other than Cys or absent <220> <221> UNSURE <222> (8)..(8) <223> The 'Xaa' at position 8 represents Gln, Arg, Pro, or Leu. <400> 2 Gly Pro Ala Pro Cys Pro Pro Xaa 1 5 <210> 3 <211> 363 <212> DNA / RNA <213> Artificial Sequence <400> 3 gctagcacca agggcccatc cgtcttcccc ctggcaccct cctccaagag cacctctggg 60 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacaccaccg tgcccagcac ctgaactcct ggggggaccg 360 tca 363 <210> 4 <211> 375 <212> DNA / RNA <213> Artificial Sequence <400> 4 cgaactgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg tgacaaaact cacacaccac cgtgcccagc acctgaactc 360 ctggggggac cgtca 375 <210> 5 <211> 54 <212> DNA / RNA <213> Artificial Sequence <400> 5 tcaccgggag ggctgctcga acctgcacca tgcccgccaa cacacactaa agac 54 <210> 6 <211> 47 <212> DNA / RNA <213> Artificial Sequence <400> 6 ggcacactgg tcacagtttc tagctcaccg ggagggctgc tcgaacc 47 <210> 7 <211> 47 <212> DNA / RNA <213> Artificial Sequence <400> 7 ctgattatga tcaatgaatt ctcaagagga cactgtgacc agggtgc 47 <210> 8 <211> 46 <212> DNA / RNA <213> Artificial Sequence <400> 8 ggaggcacca agctgacagt tttgtcaccg ggagggctgc tcgaac 46 <210> 9 <211> 49 <212> DNA / RNA <213> Artificial Sequence <400> 9 ctgattatga tcaatgaatt ctcacttgat ttccaccttg gtgcctccg 49 <210> 10 <211> 44 <212> DNA / RNA <213> Artificial Sequence <400> 10 tgtggctgag aggtgccaga tgtcaagttc agctggttca gtct 44 <210> 11 <211> 47 <212> DNA / RNA <213> Artificial Sequence <400> 11 gacggatggg cccttggtgc tagcagaaga gactgtgacc agggttc 47 <210> 12 <211> 47 <212> DNA / RNA <213> Artificial Sequence <400> 12 gaaccctggt cacagtctct tctgctagca ccaagggccc atccgtc 47 <210> 13 <211> 47 <212> DNA / RNA <213> Artificial Sequence <400> 13 ctgattatga tcaatgaatt ctcaagagga cactgtgacc agggtgc 47 <210> 14 <211> 47 <212> DNA / RNA <213> Artificial Sequence <400> 14 ctgtggctga gaggtgccag atgtgacgtg gtcatgacac agagccc 47 <210> 15 <211> 48 <212> DNA / RNA <213> Artificial Sequence <400> 15 gacagatggt gcagccacag ttcgcttgat ttccagcttg gtgccctg 48 <210> 16 <211> 48 <212> DNA / RNA <213> Artificial Sequence <400> 16 agggcaccaa gctggaaatc aagcgaactg tggctgcacc atctgtct 48 <210> 17 <211> 49 <212> DNA / RNA <213> Artificial Sequence <400> 17 ctgattatga tcaatgaatt ctcacttgat ttccaccttg gtgcctccg 49 <210> 18 <211> 44 <212> DNA / RNA <213> Artificial Sequence <400> 18 gtggctgaga ggtgccagat gtgaggtcca gctcgtcgag tccg 44 <210> 19 <211> 43 <212> DNA / RNA <213> Artificial Sequence <400> 19 ggatgggccc ttggtgctag cggcggagga gacggtgacg agg 43 <210> 20 <211> 43 <212> DNA / RNA <213> Artificial Sequence <400> 20 ctcgtcaccg tctcctccgc cgctagcacc aagggcccat ccg 43 <210> 21 <211> 42 <212> DNA / RNA <213> Artificial Sequence <400> 21 gtggctgaga ggtgccagat gtgacatcca gatgacccag tc 42 <210> 22 <211> 42 <212> DNA / RNA <213> Artificial Sequence <400> 22 gatggtgcag ccacagttcg cttgatctcg accttggtgc cc 42 <210> 23 <211> 44 <212> DNA / RNA <213> Artificial Sequence <400> 23 gggcaccaag gtcgagatca agcgaactgt ggctgcacca tctg 44 <210> 24 <211> 42 <212> DNA / RNA <213> Artificial Sequence <400> 24 gtggctgaga ggtgccagat gtgacatcca gatgacccag tc 42 <210> 25 <211> 42 <212> DNA / RNA <213> Artificial Sequence <400> 25 gctcttgggt gaccactgtc tgacactctc ccctgttgaa gc 42 <210> 26 <211> 121 <212> PRT <213> Artificial sequence <400> 26 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Met Asp Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Asn Ile Tyr Pro Asp Ser Gly Gly Thr Asn Tyr Ala Glu Lys Phe 50 55 60 Lys Ser Arg Val Thr Leu Thr Val Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Glu Ala Leu Gly Gly Tyr Tyr Glu Leu Thr Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 27 <211> 111 <212> PRT <213> Artificial sequence <400> 27 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Ser Thr Ser 20 25 30 Ser Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln His Ser Trp 85 90 95 Glu Ile Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 <210> 28 <211> 119 <212> PRT <213> Artificial sequence <400> 28 Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 29 <211> 106 <212> PRT <213> Artificial sequence <400> 29 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 30 <211> 18 <212> PRT <213> Artificial sequence <400> 30 Asp Lys Thr His Thr Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser <210> 31 <211> 18 <212> PRT <213> Artificial sequence <400> 31 Ser Pro Gly Gly Leu Leu Glu Pro Ala Pro Cys Pro Pro Thr His 1 5 10 15 Thr Lys Asp

Claims

1. A fusion protein, which from the N-terminus to the C-terminus is as follows: a) The Fab fragment of a first antibody that can specifically bind to a first antigen; b) The antigen-binding fragment of an anti-CD3 antibody that can specifically bind to the CD3 molecule, and the antigen-binding fragment is an Fv fragment, wherein the Fv fragment is composed of VH shown in SEQ ID NO: 28 and VL shown in SEQ ID NO: 29; c) The antigen-binding fragment of an anti-CD137 antibody that can specifically bind to the CD137 molecule, and the antigen-binding fragment is an Fv fragment, wherein the Fv fragment is composed of VH shown in SEQ ID NO: 26 and VL shown in SEQ ID NO: 27; wherein the heavy chains of the Fab fragment, the antigen-binding fragment of the anti-CD3 antibody, and the antigen-binding fragment of the anti-CD137 antibody are sequentially connected by a first peptide linker and a third peptide linker, and the light chains of the Fab fragment, the antigen-binding fragment of the anti-CD3 antibody, and the antigen-binding fragment of the anti-CD137 antibody are sequentially connected by a second peptide linker and a fourth peptide linker, wherein only one disulfide bond can be formed and is the same between the first peptide linker and the second peptide linker, only one disulfide bond can be formed and is the same between the third peptide linker and the fourth peptide linker, and the first peptide linker to the fourth peptide linker are each independently selected from peptide linkers of any one of the sequences shown in Seq ID NO: 30 and 31.

2. The fusion protein according to claim 1, wherein the first antigen, the CD137 molecule, and the CD3 molecule are independently derived from mammals.

3. The fusion protein according to claim 2, wherein the first antigen, the CD137 molecule, and the CD3 molecule are independently derived from non-human primates or humans.

4. The fusion protein according to claim 1, wherein the affinity constant of the Fab fragment binding to the first antigen is 10 - 1000 times the affinity constant of the antigen-binding fragment of the anti-CD137 antibody binding to the CD137 molecule or the affinity constant of the antigen-binding fragment of the anti-CD3 antibody binding to the CD3 molecule.

5. The fusion protein according to any one of claims 1 - 3, wherein the first antigen is MESOTHELIN.

6. The fusion protein according to any one of claims 1 - 3, wherein the first antigen is EGFR.

7. The fusion protein according to any one of claims 1 - 3, wherein the first antigen is PSMA.

8. The fusion protein according to any one of claims 1 - 3, wherein the first antigen is GD2.

9. The fusion protein according to any one of claims 1 - 3, wherein the first antigen is MUC1.

10. The fusion protein according to any one of claims 1 - 3, wherein the first antigen is FAP.

11. The fusion protein according to any one of claims 1 - 3, wherein the first antigen is BCMA.

12. The fusion protein according to any one of claims 1 - 3, wherein the first antigen is EphA2.

13. The fusion protein according to any one of claims 1-3, wherein the first antigen is CD19.

14. The fusion protein according to any one of claims 1-3, wherein the first antigen is CD22.

15. The fusion protein according to any one of claims 1-3, wherein the first antigen is EpCAM.

16. The fusion protein according to any one of claims 1-3, wherein the first antigen is CEA.

17. The fusion protein according to any one of claims 1-3, wherein the first antigen is PD-L1.

18. The fusion protein according to any one of claims 1-3, wherein the first antigen is B7H3.

19. The fusion protein according to any one of claims 1-3, wherein the first antigen is ROR1.

20. The fusion protein according to any one of claims 1-3, wherein the first antigen is c-Met.

21. The fusion protein according to any one of claims 1-3, wherein the first antigen is GPC3.

22. A nucleic acid encoding the fusion protein according to any one of claims 1-21.

23. An expression vector comprising the nucleic acid according to claim 22.

24. A host cell comprising the nucleic acid according to claim 22 or the expression vector according to claim 23, wherein the host cell is a mammalian cell.

25. The host cell according to claim 24, wherein the mammalian cell is selected from CHO cells, NS0 cells, SP2 / 0 cells, HEK293 cells, COS cells, and PER.C6 cells.

26. A method for preparing the fusion protein according to any one of claims 1-21, comprising: a) culturing the host cell according to claim 24 or 25; and b) recovering the fusion protein from the host cell or the culture supernatant of the host cell.

27. A pharmaceutical composition comprising the fusion protein according to any one of claims 1-21, the nucleic acid according to claim 22, the expression vector according to claim 23, or the host cell according to claim 24 or 25, and a pharmaceutically acceptable carrier.

Images