Fusion protein and use thereof

Abstract

The present application discloses a fusion protein and a use thereof. The fusion protein comprises an anti-VEGFR2 antibody and an IL-10 monomer, wherein the IL-10 monomer is linked to the anti-VEGFR2 antibody. The fusion protein of the present application has the advantages of strong targeting, small side effects, good efficacy, etc. The fusion protein can be used for preparing a pharmaceutical composition or a drug, and can be used for treating or preventing tumor diseases.

Description

A fusion protein and use thereof

[0001] Priority information

[0002] This application claims priority to and the benefit of Chinese Patent Application No. 202410955537.7, filed on July 16, 2024, and is hereby incorporated by reference in its entirety. TECHNICAL FIELD

[0003] The present application belongs to the technical field of biological medicine, and specifically relates to a fusion protein and use thereof, more specifically to a fusion protein, nucleic acid, vector, cell or host, pharmaceutical composition and use thereof. BACKGROUND

[0004] Vascular endothelial growth factor receptor 2 (VEGFR2) plays an important role in vascular endothelial cell signaling, including participating in cell survival, proliferation, migration, and vascular permeability, etc., promoting tumor angiogenesis and cancer development. Tumor vascular networks are leaky, disorganized, immature, thin-walled, and poorly perfused, and blunting or collapse of tumor vessels often leads to the formation of hypoxic regions in tumors. VEGFR2 is mainly expressed on endothelial cells, is up-regulated on various tumors, and is up-regulated on hypoxic conditions and tumor-infiltrating CD8+ T cells. IL-10R contains two subunits, IL-10Ra and IL-10Rb, and is expressed on various immune cells such as monocytes / macrophages, DC cells, T cells, B cells, NK cells, etc. After IL-10 acts on the receptor, it activates the STAT3 signaling pathway, and has the effect of inhibiting inflammatory cytokines and inhibiting immune activity on monocytes / macrophages and DC cells; IL-10 can promote B cell differentiation and antibody expression; but for CD8 + T cells, IL-10 plays a significant role in promoting activation. In recent years, with the mechanism of the anti-tumor effect of IL-10 being continuously explored, IL-10 is considered to have great potential in tumor immunotherapy. IL-10 can directly act on exhausted CD8+ T cells in the tumor microenvironment, reverse the exhausted state through metabolic reprogramming, reduce CD8 + T cell activation-induced cell death (AICD), promote proliferation, cytokine secretion, and killing activity, and exert anti-tumor activity.

[0005] However, the drugs based on anti-VEGFR2 antibodies and IL-10 need to be further improved in terms of effect on tumors and other diseases. Therefore, it is still necessary to actively explore drugs targeting anti-VEGFR2 antibodies and IL-10 with less side effects and good therapeutic effects. SUMMARY

[0006] The present application aims to solve at least one of the technical problems in the prior art to some extent. To this end, the present application provides a fusion protein and uses thereof.

[0007] The present application is based on the following findings of the inventors:

[0008] Wild-type IL-10 currently exhibits certain hematological toxicity in preclinical monkey toxicity experiments and in the clinic, so that no IL-10 related drugs are currently on the market. Based on this, the inventors found that using a modified reduced activity IL-10 monomer can significantly reduce the activity of wild-type IL-10, thereby reducing toxicity. Further, the present application prepares a VEGFR2 x IL-10M bifunctional fusion protein by combining an anti-VEGFR2 antibody and a reduced activity IL-10 monomer, which targets the tumor microenvironment through the VEGFR2 end, normalizes tumor blood vessels, increases T cell infiltration, and at the same time restores the activity of exhausted CD8 + T cells in the tumor microenvironment through the IL-10 end, enhances CD8 + T cell killing activity, and enhances anti-tumor immune response. Based on this, the VEGFR2 x IL-10M bifunctional fusion protein of the present application, under the action of VEGFR2 antibody targeting enrichment, can further reduce the peripheral toxicity of IL-10 and can improve the local activity.

[0009] Therefore, in a first aspect of the present application, a fusion protein is provided. According to embodiments of the present application, the fusion protein comprises an anti-VEGFR2 antibody and an IL-10 monomer; wherein the IL-10 monomer is connected to the anti-VEGFR2 antibody. The fusion protein of the present application has the advantages of strong targeting, small side effects, good drug efficacy, etc., and in particular strong tumor killing.

[0010] In a second aspect of the present application, a nucleic acid is provided. According to embodiments of the present application, the nucleic acid encodes the fusion protein of the first aspect. The nucleic acid according to embodiments of the present application can encode the aforementioned fusion protein.

[0011] In a third aspect of the present application, a vector is provided. According to embodiments of the present application, the vector comprises the nucleic acid of the second aspect. The vector according to embodiments of the present application carries the aforementioned nucleic acid, thereby encoding the aforementioned fusion protein.

[0012] In a fourth aspect of the present application, a cell or host is provided. According to embodiments of the present application, the cell or host carries the nucleic acid of the second aspect or the vector of the third aspect; or the cell or host expresses the fusion protein of the first aspect. The cell or host can be used to effectively express the aforementioned fusion protein in the cell or host under suitable conditions.

[0013] In a fifth aspect, the present application provides a pharmaceutical composition. According to embodiments of the present application, the pharmaceutical composition comprises the fusion protein of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, or the cell or host of the fourth aspect. As known from the above, the fusion protein of the first aspect or the fusion protein prepared by using the nucleic acid of the second aspect, the vector of the third aspect, or the cell or host of the fourth aspect has the advantages of strong targeting, small side effects, good drug efficacy, etc., especially better anti-cancer activity and higher safety. Therefore, the obtained pharmaceutical composition can effectively prevent and / or treat tumors.

[0014] In a sixth aspect, the present application provides use of the fusion protein of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the cell or host of the fourth aspect, or the pharmaceutical composition of the fifth aspect in the preparation of a medicament for treating or preventing tumors.

[0015] The present application provides use of the fusion protein of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the cell or host of the fourth aspect, or the pharmaceutical composition of the fifth aspect in treating or preventing tumors.

[0016] The present application provides the fusion protein of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the cell or host of the fourth aspect, or the pharmaceutical composition of the fifth aspect for treating or preventing tumors.

[0017] In a seventh aspect, the present application provides a method for treating or preventing tumors. According to embodiments of the present application, the method comprises administering to a subject a pharmaceutically acceptable dose of the fusion protein of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the cell or host of the fourth aspect, or the pharmaceutical composition of the fifth aspect.

[0018] The amino acid sequence table A1 of the present application is as follows:

[0019] Additional aspects and advantages of the present application will be in part apparent and in part pointed out below in the description of the application. BRIEF DESCRIPTION OF DRAWINGS

[0020] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.

[0021] Figure 1 is a structural schematic diagram of the fusion protein in Example 1 of the present application;

[0022] Figure 2 is the binding result of the fusion protein and the control antibody to IL-10R1 in Example 2 of the present application;

[0023] Figure 3 is the binding result of the fusion protein and the control antibody to VEGFR2 in Example 3 of the present application;

[0024] Figure 4 is the blocking activity result of the fusion protein and the control antibody to VEGFR2 and VEGF165 in Example 4 of the present application;

[0025] Figure 5 is the VEGFR2 reporter gene activity result of the fusion protein and the control antibody in Example 5 of the present application;

[0026] Figure 6 is the STAT3 signal pathway activation activity result of the fusion protein and the control antibody in Example 6 of the present application;

[0027] Figure 7 is the binding activity result of the fusion protein and the control antibody to exhausted CD8 + T cells in Example 7 of the present application;

[0028] Figure 8 is the apoptosis activity result of the fusion protein and the control antibody to inhibit exhausted CD8 + T cells in Example 8 of the present application;

[0029] Figure 9 is the binding activity result of the fusion protein and the control antibody to HUVEC cells in Example 9 of the present application;

[0030] Figure 10 is the proliferation activity result of the fusion protein and the control antibody to inhibit HUVEC cells in Example 10 of the present application;

[0031] Figure 11 is the in vivo anti-tumor efficacy result of the fusion protein and the control antibody in Example 11 of the present application;

[0032] Figure 12 is the in vivo anti-tumor efficacy result of the fusion protein and the control antibody in Example 12 of the present application. DETAILED DESCRIPTION

[0033] The embodiments of the present application are described in detail below. The embodiments described below are exemplary only and are not to be construed as limiting the present application.

[0034] It should be noted that the terms "first", "second" are used only for the purpose of description, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, the meaning of "a plurality of" is two or more, unless otherwise specified.

[0035] The present application is explained

[0036] Definitions and general terminology

[0037] The endpoints of the ranges and any values disclosed herein are not limited to the precise values recited as implicitly contained therein all such ranges and values should be construed as being approximate. For numeric values, the endpoints of each range, the endpoints of each range and individual point values, and individual point values can be combined with one another to generate one or more new numeric ranges, which should be considered as being specifically disclosed herein.

[0038] For the purposes of the present application, certain technical and scientific terms are specifically defined below. Unless specifically defined herein, all other technical and scientific terms used in the present document have the meanings that are commonly understood by one of ordinary skill in the art in the field of the present document. Abbreviations for amino acid residues are the standard three letter and / or one letter codes used in the art to designate one of the 20 commonly occurring L-amino acids (i.e., amino acids in the left-handed L-configuration).

[0039] In the present document, the terms "comprising" or "comprise" are open-ended, that is, they mean including, but not limited to, what is specifically recited in the present document.

[0040] In the present document, the terms "optionally", "optional", "optionally", "optional" or "optional" generally mean that the subsequently described event or circumstance can or can not occur, and that the description includes instances where the event or circumstance occurs, as well as instances where it does not.

[0041] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code, such as the twenty amino acids found in proteins, as well as modified amino acids, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Common natural amino acids are, for example: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G); histidine (His; H), isoleucine (lie; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). An amino acid analog refers to a compound having the same basic chemical structure as a naturally occurring amino acid (i.e., an alpha carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group), such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. An amino acid analog typically has a modified R group (e.g., norleucine) or a modified peptide backbone, but retains the same basic chemical structure as a naturally occurring amino acid. An amino acid mimetic refers to a chemical compound having a structure different from the general chemical structure of an amino acid, but which functions in a manner similar to a naturally occurring amino acid.

[0042] In this document, "IL10" and "IL-10" are used generically and have the same meaning, and the terms "wild-type IL-10", "native IL-10 molecule" or "native IL-10" all refer to a dimeric structure (comprising two native IL-10 monomers) having two identical amino acid sequences as shown in SEQ ID NO: 1, the native IL-10 molecule binds to IL-10 receptor (IL-10R), and the IL-10 monomer has 10-fold weaker activity to IL-10Ra than the native IL-10 dimer (see experimental data in the reference: J Biol Chem. 2000 May 5;275(18):13552-7. doi: 10.1074 / jbc.275.18.13552.), and is almost unable to mediate further binding to IL-10Rβ, thus it is difficult to activate downstream signals to cause biological functional responses.

[0043] In the present context, the term "vector" generally refers to a nucleic acid molecule capable of self-replicating in a suitable host into which it has been introduced, which transfers the inserted nucleic acid molecule into and / or between cells or hosts. The vector can include a vector primarily used for inserting DNA or RNA into a cell, a vector primarily used for replicating DNA or RNA, and a vector primarily used for expression of transcription and / or translation of DNA or RNA. The vector also includes a vector having a plurality of the above-mentioned functions. The vector can be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell or host. Generally, the vector can produce a desired expression product by culturing a suitable cell or host containing the vector.

[0044] In the present context, the term "cell" generally refers to a cell having a unique characteristic with stable inheritance, obtained by modifying or recombining the genetic material of a host cell using genetic engineering techniques or cell fusion techniques. Among them, the term "host cell" refers to a prokaryotic cell or eukaryotic cell into which a recombinant vector can be introduced. The term "transformed" or "transfected" used herein means introducing a nucleic acid (e.g., a vector) into a cell by various techniques known in the art. A suitable host cell can be transformed or transfected with the DNA sequence of the present application, and can be used for expression and / or secretion of a target protein. Examples of suitable host cells that can be used in the present application include immortalized hybridoma cells, NS / 0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (human amniotic fluid-derived cells), and CoS cells.

[0045] In the present context, the term "pharmaceutical composition" generally refers to a composition in unit dosage form, and can be prepared by any one of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier that constitutes one or more accessory ingredients.

[0046] In the present context, the term "pharmaceutically acceptable" ingredient is a substance suitable for use with humans and / or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., with reasonably acceptable benefit / risk ratio.

[0047] In the present context, the term "pharmaceutically acceptable excipient" can include any solvent, stabilizer, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. Except for any conventional excipient incompatible with the fusion protein of the present application in the range of, for example, any adverse biological effects produced or interactions with any other components of the pharmaceutically acceptable composition in a deleterious manner, their use is also considered within the scope of the present application.

[0048] In the present context, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The fusion protein or the pharmaceutical composition of the present application can be administered by any common route, as long as it can reach the intended tissue. Various means of administration are contemplated, including intraperitoneal, intravenous, intramuscular, subcutaneous, and the like, but the present application is not limited to these exemplified means of administration. Preferably, the fusion protein or the pharmaceutical composition of the present application is administered by intravenous injection or subcutaneous injection.

[0049] In the present context, the term "treatment" refers to obtaining a desired pharmacological and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure of a disease and / or adverse effect attributable to the disease. "Treatment" as used herein covers the treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or condition from occurring in an individual which can be predisposed to the disease but has not yet developed the disease; (b) inhibiting the disease, i.e., arresting its development; or (c) relieving the disease, i.e., causing the partial or complete regression of the disease. "Treatment" as used herein covers any use of a fusion protein or a pharmaceutical composition to treat, cure, relieve, improve, lessen, or inhibit a disease in an individual, including, but not limited to, administering a medicament comprising a fusion protein described herein to an individual in need thereof.

[0050] As used herein, the term "effective amount" or "effective dose" refers to an amount that is functional or active and acceptable to a human and / or an animal.

[0051] Detailed description of the fusion protein of the present application and uses thereof

[0052] The present application provides a fusion protein, a nucleic acid, a vector, a cell or a host, a pharmaceutical composition, and uses thereof, which will be described in detail below, respectively.

[0053] Fusion protein

[0054] In a first aspect of the present application, a fusion protein is provided. According to embodiments of the present application, the fusion protein comprises an anti-VEGFR2 antibody and an IL-10 monomer; wherein the IL-10 monomer is connected to the anti-VEGFR2 antibody. The fusion protein of the present application has the advantages of strong targeting, small side effects, good drug efficacy, and the like, and in particular, strong tumor killing.

[0055] In the present text, the term "antibody" is used in the broadest sense, and is intended to include immunoglobulin molecules capable of specific binding to an antigen. The antibody can include full-length monoclonal antibodies (or full-length monospecific antibodies), or multispecific antibodies, and the specific structure is not limited as long as they include heavy chains and light chains, and can exhibit the desired biological activity. It generally includes a light chain with a relatively light molecular weight and a heavy chain with a relatively heavy molecular weight, and the antibody molecule is formed by the heavy chain (H chain) and the light chain (L chain) connected by a disulfide bond. Among them, the amino-terminal (N-terminal) amino acid sequence of the peptide chain varies greatly, which is called the variable region (V region); the carboxyl-terminal (C-terminal) is relatively stable and varies little, which is called the constant region (C region). The V region of the L chain and the H chain are called the light chain variable region (VL) and the heavy chain variable region (VH), respectively, and the C region of the L chain and the H chain are called the light chain constant region (CL or CL fragment) and the heavy chain constant region (CH or CH fragment), respectively, wherein the heavy chain constant region includes at least one of CH1 fragment, hinge region and Fc fragment (CH2 and CH3 fragments), and preferably, the heavy chain constant region includes CH1 fragment, hinge region, CH2 fragment and CH3 fragment in sequence from N-terminal to C-terminal.

[0056] According to an embodiment of the present application, the anti-VEGFR2 antibody is selected from VEGFR2 full-length monospecific antibody, Fab antibody, F(ab')2 antibody, Fab' antibody, Fv antibody, scFv antibody, dsFv antibody, nanobody.

[0057] In the present text, the terms "full-length antibody", "full-length monospecific antibody" or "full-length monoclonal antibody" all refer to an antibody formed by connecting at least two identical light chains and at least two identical heavy chains, which can be connected by inter-chain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD) or immunoglobulin E (IgE); or can be connected by knob-into-hole.

[0058] In the present text, the term "Fab antibody" generally refers to an antibody or fragment containing only Fab molecules, which is composed of VH and CH1 of the heavy chain and a complete light chain, and the light chain and the heavy chain are connected by one disulfide bond.

[0059] In the present text, the term "F(ab')2 antibody" has two antigen-binding F(ab') parts connected together by disulfide bonds.

[0060] In the present text, the term "Fab' antibody" includes VH and CH1 of the heavy chain, a complete light chain, and a hinge region, and the light chain and the heavy chain are connected by one disulfide bond.

[0061] In the present specification, the term "Fv antibody" generally refers to an antibody or fragment in which a light chain variable region (VL) and a heavy chain variable region (VH) are connected by a non-covalent bond, and is the smallest functional fragment of an antibody that retains an intact antigen binding site.

[0062] In the present specification, the term "single chain antibody" or "scFv antibody" refers to an antibody or fragment in which a heavy chain variable region and a light chain variable region of an antibody are connected by a short peptide.

[0063] In the present specification, the term "dsFv antibody" refers to an antibody or fragment in which a cysteine mutation point is introduced into each of VH and VL to form a disulfide bond between VH and VL, thereby achieving structural stability, and the stability of a small molecule Fv antibody is enhanced.

[0064] In the present specification, the term "nanobody" generally refers to a heavy chain variable region (VHH) portion of a heavy chain antibody in which a light chain is naturally absent, and the heavy chain antibody is generally present in a camelid, and includes a heavy chain variable region (VHH) and a conventional CH2 and CH3 region, and the heavy chain variable region (VHH) specifically binds to an antigen, and the heavy chain variable region (VHH) alone can exhibit an antigen-specific binding effect.

[0065] According to an embodiment of the present application, the N-terminus of the IL-10 monomer is connected to the C-terminus of the anti-VEGFR2 antibody, or the N-terminus of the anti-VEGFR2 antibody is connected to the C-terminus of the IL-10 monomer.

[0066] According to an embodiment of the present application, the N-terminus of the IL-10 monomer is connected to the C-terminus of the anti-VEGFR2 antibody.

[0067] It should be noted that when the anti-VEGFR2 antibody is selected from an antibody consisting of one chain (for example, an Fv antibody, an scFv antibody, a dsFv antibody, a nanobody), the N-terminus of the IL-10 monomer is connected to the C-terminus of the anti-VEGFR2 antibody. When the anti-VEGFR2 antibody is selected from an antibody consisting of a plurality of chains (for example, a VEGFR2 full-length monoclonal antibody, a Fab antibody, a F(ab')2 antibody, a Fab' antibody), the N-terminus of the IL-10 monomer is connected to the C-terminus of any one of the chains of the anti-VEGFR2 antibody.

[0068] According to an embodiment of the present application, the anti-VEGFR2 antibody is selected from a VEGFR2 full-length monoclonal antibody, a Fab antibody, a F(ab')2 antibody, and a Fab' antibody, and the N-terminus of the IL-10 monomer is connected to the C-terminus of the heavy chain of the anti-VEGFR2 antibody.

[0069] It is to be noted that the heavy chain in the present application includes a heavy chain variable region and optionally a heavy chain constant region, wherein the heavy chain constant region includes at least one of a CH1 fragment, a hinge region and an Fc fragment (CH2 and CH3 fragments). Exemplarily, the anti-VEGFR2 antibody is selected from a Fab antibody or a F(ab')2 antibody, and the heavy chain is the CH1 fragment.

[0070] According to embodiments of the present application, the anti-VEGFR2 antibody is selected from an Fv antibody, an scFv antibody, a dsFv antibody, a nanobody, and the N-terminus of the IL-10 monomer is connected to the C-terminus of the anti-VEGFR2 antibody.

[0071] In some alternative embodiments of the present application, the anti-VEGFR2 antibody is selected from an Fv antibody, an scFv antibody, a dsFv antibody, the C-terminus of the heavy chain in the anti-VEGFR2 antibody is connected to the N-terminus of the light chain, and the N-terminus of the IL-10 monomer is connected to the C-terminus of the light chain.

[0072] In some alternative embodiments of the present application, the anti-VEGFR2 antibody is selected from an Fv antibody, an scFv antibody, a dsFv antibody, the C-terminus of the heavy chain in the anti-VEGFR2 antibody is connected to the N-terminus of the light chain, and the N-terminus of the IL-10 monomer is connected to the C-terminus of the light chain.

[0073] According to embodiments of the present application, the anti-VEGFR2 antibody includes HCDRs and LCDRs, the HCDRs include HCDRs defined in a heavy chain variable region as shown in SEQ ID NO: 1, and the LCDRs include LCDRs defined in a light chain variable region as shown in SEQ ID NO: 1.

[0074] In the present disclosure, the term "complementarity determining region", "CDR" or "CDRs" refers to the highly variable regions of the heavy and light chains of immunoglobulins, which refer to the regions containing one or more, or even all, of the amino acid residues that play a major role in the binding affinity for the recognized antigen or epitope. In the detailed description of the present disclosure, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

[0075] In the present text, heavy chain complementarity determining regions (heavy chain variable region CDRs) are denoted "HCDRs" or "HCDR" and include HCDR1, HCDR2 and HCDR3; light chain complementarity determining regions (light chain variable region CDRs) are denoted "LCDRs" or "LCDR" and include LCDR1, LCDR2 and LCDR3. Commonly used CDR numbering schemes include: Kabat numbering, Chothia numbering, IMGT numbering, Chothia Martin numbering and AHo Lesk numbering. CDR definition schemes include: Kabat definition, Chothia definition, IMGT definition, Contact definition and AbM definition. As described herein, "Kabat numbering" and "Kabat definition" refer to the numbering and definition system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). See Chothia et al., J Mol Biol 196:901-917 (1987) for "Chothia definition". Exemplary defined CDRs are listed in Table A2 below. One of skill in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of a given antibody.

[0076] Table A2: CDR definitions 1 1 The numbering of all CDR definitions in Table A2 is according to the Kabat numbering system (see below). 2 "AbM" as used in Table A2 with lower case "b" refers to CDRs defined by the "AbM" antibody modeling software of Oxford Molecular.

[0077] Kabat et al. also defined a numbering system applicable to variable region sequences of any antibody. One of ordinary skill in the art can unambiguously assign the Kabat numbering system to any variable region sequence, without dependence on any experimental data other than the sequence itself. As described herein, "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0078] According to embodiments of the present application, the HCDRs and / or LCDRs are defined by Kabat, Chothia, AbM, Contact or IMGT.

[0079] In an alternative embodiment of the present application, the HCDRs and LCDRs are defined using the same numbering system, such as defined by Kabat, defined by Chothia, defined by AbM, defined by Contact, or defined by IMGT.

[0080] According to embodiments of the present application, the anti-VEGFR2 antibody comprises a heavy chain variable region having HCDR1, HCDR2, HCDR3 as set forth in SEQ ID NOs: 3, 4, and 5, and a light chain variable region having LCDR1, LCDR2, LCDR3 as set forth in SEQ ID NOs: 6, 7, and 8.

[0081] It is to be noted that the HCDRs and LCDRs (amino acid sequences as set forth in SEQ ID NOs: 3-8) described above in the present application are numbered according to the Kabat numbering system. However, one of ordinary skill in the art is fully capable of converting the sequences of the sequence listing (amino acid sequences as set forth in SEQ ID NOs: 3-8) into HCDRs and LCDRs numbered according to other numbering systems, which are all within the scope of the present application. Among them, the HCDRs and LCDRs of Ramucirumab numbered according to other numbering systems are exemplarily shown in the present application, and the HCDRs and LCDRs numbered according to different numbering systems based on the heavy chain variable region (amino acid sequence as set forth in SEQ ID NO: 1) and the light chain variable region (amino acid sequence as set forth in SEQ ID NO: 2) of other mAbs (such as the heavy chain variable region (amino acid sequence as set forth in SEQ ID NO: 1) and the light chain variable region (amino acid sequence as set forth in SEQ ID NO: 2) of Ramucirumab) are also within the scope of the present application. Exemplarily, the HCDRs and LCDRs of Ramucirumab (amino acid sequences of the heavy chain variable region as set forth in SEQ ID NO: 1, and amino acid sequences of the light chain variable region as set forth in SEQ ID NO: 2) are specifically shown in Table A3:

[0082] Table A3. HCDRs and LCDRs of Ramucirumab under various definitions

[0083] According to embodiments of the present application, the anti-VEGFR2 antibody comprises a heavy chain variable region having HCDR1, HCDR2, HCDR3 as set forth in SEQ ID NOs: 3, 4, and 5, and a light chain variable region having LCDR1, LCDR2, LCDR3 as set forth in SEQ ID NOs: 6, 7, and 8.

[0084] According to embodiments of the present application, the anti-VEGFR2 antibody is selected from a VEGFR2 full-length mAb, and the N terminus of the IL-10 monomer is connected to the C terminus of the heavy chain of the VEGFR2 full-length mAb.

[0085] According to embodiments of the present application, the N terminus of the IL-10 monomer is connected to the C terminus of the heavy chain of the VEGFR2 full-length mAb.

[0086] According to an embodiment of the present application, the heavy chain variable region of the VEGFR2 full-length monoclonal antibody has an amino acid sequence as shown in SEQ ID NO: 1, and the light chain variable region of the VEGFR2 full-length monoclonal antibody has an amino acid sequence as shown in SEQ ID NO: 2.

[0087] According to an embodiment of the present application, the VEGFR2 full-length monoclonal antibody is Ramucirumab, a mutant of Ramucirumab, or a chimera of Ramucirumab.

[0088] In the present application, the term "chimera" refers to an antibody in which the CDRs of a target antibody are spliced with other framework regions or constant regions, which are framework regions and / or constant regions of other antibodies other than the framework regions and / or constant regions of the target antibody; for example, an antibody in which the variable region of a target antibody is spliced with other constant regions, which are constant regions of other antibodies other than the constant regions of the target antibody. Illustratively, "a chimera of Ramucirumab" refers to an antibody in which the variable region of Ramucirumab is spliced with other constant regions, which are different from the constant regions of Ramucirumab; wherein the constant regions of Ramucirumab are derived from the constant regions of human IgG1, and the constant regions in the chimera of Ramucirumab are selected from constant regions other than human IgG1, for example, constant regions derived from human IgG2, and can also be selected from mutants of constant regions derived from human IgG2, and the specific derivation is not limited, all of which are within the protection scope of the present application.

[0089] In the present application, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule containing one or more nucleotide or amino acid mutations. Illustratively, "a mutant of Ramucirumab" can be a mutation to the CDRs of Ramucirumab, a mutation to the FRs of Ramucirumab, or a mutation to the constant regions of Ramucirumab, and the specific mutation type is not limited, all of which are within the protection scope of the present application.

[0090] In an optional embodiment of the present application, the mutant of Ramucirumab has HCDRs defined in the heavy chain variable region as shown in SEQ ID NO: 1, and has LCDRs defined in the light chain variable region as shown in SEQ ID NO: 1.

[0091] In an optional embodiment of the present application, the chimera of Ramucirumab has HCDRs defined in the heavy chain variable region as shown in SEQ ID NO: 1, and has LCDRs defined in the light chain variable region as shown in SEQ ID NO: 1.

[0092] In an alternative embodiment of the present application, the mutant of Ramucirumab has HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 3, 4 and 5, and has LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO: 6, 7 and 8.

[0093] In an alternative embodiment of the present application, the chimeric body of Ramucirumab has HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 3, 4 and 5, and has LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO: 6, 7 and 8.

[0094] According to an embodiment of the present application, the VEGFR2 full-length monoclonal antibody has a heavy chain variable region as shown in SEQ ID NO: 9 or 10, and has a light chain variable region as shown in SEQ ID NO: 11.

[0095] According to an embodiment of the present application, the N-terminal of the IL-10 monomer is connected to the C-terminal of the heavy chain of Ramucirumab. In this way, the therapeutic effect of the fusion protein on related diseases can be further improved, especially the killing ability on tumors.

[0096] In an alternative embodiment of the present application, the Fab antibody is selected from Ramucirumab, and the N-terminal of the IL-10 monomer is connected to the C-terminal of the CH1 fragment of the Fab antibody.

[0097] According to an embodiment of the present application, at least one of the heavy chain constant region and the light chain constant region of the VEGFR2 full-length monoclonal antibody is from at least one of a murine antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, an ass antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof.

[0098] According to an embodiment of the present application, the heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD.

[0099] According to an embodiment of the present application, the light chain constant region comprises a light chain constant region selected from kappa type or lambda type.

[0100] According to an embodiment of the present application, the light chain constant region and the heavy chain constant region are both from at least one of a rabbit antibody or a mutant thereof, a murine antibody or a mutant thereof, and a human antibody or a mutant thereof.

[0101] According to an embodiment of the present application, the heavy chain constant region is selected from a human IgGl heavy chain constant region or a mutant thereof.

[0102] According to an embodiment of the present application, the mutant of the human IgGl heavy chain constant region has an activity of weakening ADCC effect or an activity of weakening CDC effect compared with the wild-type human IgGl heavy chain constant region.

[0103] According to an embodiment of the present application, the mutant of the human IgGl heavy chain constant region has any one of the following mutations: 1) L234A and L235A; 2) L235A, G237A, A327Q and K447A, compared with the wild-type human IgGl heavy chain constant region. The above mutation sites are located in the Fc fragment of the human IgGl heavy chain constant region, and the ADCC effect can be weakened or the CDC effect can be weakened by mutating the above sites.

[0104] In this text, the amino acid number of the heavy chain constant region of IgGl or the Fc fragment of IgGl is numbered according to the EU numbering system, for example, the 234th position refers to the 234th position numbered according to the EU numbering system; the "L234A" refers to the substitution of leucine at the 234th position numbered according to the EU numbering system by alanine; the "L235A" refers to the substitution of leucine at the 235th position numbered according to the EU numbering system by alanine; "G237A" refers to the substitution of glycine at the 237th position numbered according to the EU numbering system by alanine; "A327Q" refers to the substitution of alanine at the 327th position numbered according to the EU numbering system by glutamine; "K447A" refers to the substitution of lysine at the 447th position numbered according to the EU numbering system by alanine.

[0105] In an alternative embodiment of the present application, the mutant of the human IgGl heavy chain constant region has a knob into hole structure compared with the wild-type human IgGl heavy chain constant region.

[0106] In this text, the term "knob into hole structure" is to form knob hole mutations in the CH3 region of the heavy chain constant region, which facilitates heavy chain occlusion to form heterodimers, for example, by mutating the amino acids in the CH3 domain of the human IgGl heavy chain constant region (T366S, L368A, Y407V, Y349C mutations in one chain, i.e. "hole"; T366W, S354C mutations in the other chain, i.e. "knob").

[0107] In an alternative embodiment of the present application, the N-terminal of the modified IL-10 monomer is deleted by at least 2 amino acids, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids, compared with the native IL-10 monomer.

[0108] According to embodiments of the present application, the IL-10 monomer is selected from engineered IL-10 monomers; wherein, compared with the native IL-10 monomer, the engineered IL-10 monomer has a deletion of at least 2 amino acids at the N-terminus and contains a spacer peptide between two adjacent amino acid positions, one of which is position 115, 116, 117, 118, 119, and the spacer peptide is a flexible linker peptide. Thus, compared with native IL-10, the engineered IL-10 monomer of the present application can weaken its activity, thereby reducing the toxicity of the fusion protein.

[0109] It should be noted that the "115th, 116th, 117th, 118th, 119th" mentioned above are numbered from the N-terminus based on the amino acid sequence of the native IL-10 monomer.

[0110] According to embodiments of the present application, compared with the native IL-10 monomer, the engineered IL-10 monomer contains a spacer peptide between the 114th and 115th amino acids.

[0111] According to embodiments of the present application, compared with the native IL-10 monomer, the engineered IL-10 monomer contains a spacer peptide between the 115th and 116th amino acids.

[0112] According to embodiments of the present application, compared with the native IL-10 monomer, the engineered IL-10 monomer contains a spacer peptide between the 116th and 117th amino acids.

[0113] According to embodiments of the present application, compared with the native IL-10 monomer, the engineered IL-10 monomer contains a spacer peptide between the 117th and 118th amino acids.

[0114] According to embodiments of the present application, compared with the native IL-10 monomer, the engineered IL-10 monomer contains a spacer peptide between the 118th and 119th amino acids.

[0115] In an optional embodiment of the present application, the native IL-10 monomer has an amino acid sequence shown in SEQ ID NO: 15, wherein the amino acid sequence shown in SEQ ID NO: 15 is specifically shown in Table A1.

[0116] In the present disclosure, the term "linker peptide", "linker polypeptide", "linker", "Linker" or "linker" refers to a linking unit connecting two polypeptide fragments, usually with a certain flexibility, the use of the linker will not cause the original function of the protein domain to be lost. The linker can be a peptide linker, which comprises one or more amino acids, such as about 1-30, 2-24 or 3-15 amino acids. In some embodiments, the linker is selected from (GxSy)z GmSn linker, wherein x, y, z, m, n are independently selected from an integer between 0-6; alternatively, x is selected from an integer between 1-5, y, z, m, n are independently selected from an integer between 0-6 (for example, when x = 4, y = 1, z = 4, m = 1, n = 0, that is, (G4S1)4G1S0, which represents the amino acid sequence: GGGGSGGGGSGGGGSGGGGSG). In some embodiments, the linker is selected from (GxS)zG linker, wherein x is selected from an integer between 1-5, z is selected from an integer between 1-6.

[0117] In the present disclosure, the term "spacer peptide" refers to a peptide inserted into a specific protein, which can change the structure and / or function of the protein. The spacer peptide in the present disclosure is not limited in specific sequence as long as the inserted peptide can change the structure and / or function of the native IL-10 monomer. The spacer peptide in the present disclosure is different from the linker peptide connecting other fusion partners, but the conventional linker peptide can also be used as the spacer peptide in the present disclosure to change the structure and / or function of the native IL-10 monomer. In an alternative example of the present disclosure, the insertion of the spacer peptide in the present disclosure can avoid the formation of dimers of two IL-10 truncates.

[0118] According to an embodiment of the present disclosure, the spacer peptide is selected from at least one of GGGSGG, (GS)n, (GGGS)n, (GSGGS)n, (GGGGS)nG, (GGS)n, n is any integer between 1-10.

[0119] According to some alternative embodiments of the present disclosure, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or any integer between any two of them as an end value.

[0120] According to an embodiment of the present disclosure, the spacer peptide has an amino acid sequence as shown in SEQ ID NO: 12.

[0121] According to an embodiment of the present disclosure, the engineered IL-10 monomer has an amino acid sequence as shown in SEQ ID NO: 13, 14 or 48.

[0122] In an alternative embodiment of the present application, the engineered IL-10 monomer has an amino acid sequence as set forth in SEQ ID NO: 13. As can be seen, the engineered IL-10 monomer (having an amino acid sequence as set forth in SEQ ID NO: 13) has a deletion of the first two N-terminal amino acids SP (the first two amino acids underlined in the amino acid sequence set forth in SEQ ID NO: 15 in Table A1) and has an insertion of a spacer peptide between the 116th amino acid and the 117th amino acid (the amino acid sequence in bold and underlined in the amino acid sequence set forth in SEQ ID NO: 13 in Table A1).

[0123] In an alternative embodiment of the present application, the engineered IL-10 monomer has an amino acid sequence as set forth in SEQ ID NO: 14. As can be seen, the engineered IL-10 monomer (having an amino acid sequence as set forth in SEQ ID NO: 14) has a deletion of the first three N-terminal amino acids SPG (the three amino acids underlined in the amino acid sequence set forth in SEQ ID NO: 15 in Table A1) and has an insertion of a spacer peptide between the 116th amino acid and the 117th amino acid (the amino acid sequence in bold and underlined in the amino acid sequence set forth in SEQ ID NO: 14 in Table A1).

[0124] In an alternative embodiment of the present application, the engineered IL-10 monomer has an amino acid sequence as set forth in SEQ ID NO: 48. As can be seen, the engineered IL-10 monomer (having an amino acid sequence as set forth in SEQ ID NO: 48) has an insertion of a spacer peptide between the 116th amino acid and the 117th amino acid (the amino acid sequence in bold and underlined in the amino acid sequence set forth in SEQ ID NO: 48 in Table A1) relative to the native IL-10 monomer (having an amino acid sequence as set forth in SEQ ID NO: 15).

[0125] In an embodiment of the present application, the N-terminal of the modified IL-10 monomer is deleted by 2 or 3 amino acids compared with the native IL-10 monomer. Thus, the fusion protein prepared by using the modified IL-10 monomer described above can further reduce the toxicity of the fusion protein. The inventors found that the stability of the IL-10 monomer is improved after removing the first 2 or 3 amino acids at the N-terminal of the native IL-10 monomer, and the IL-10 monomer molecule in the fusion protein becomes a closed-loop monomer molecule after inserting a spacer peptide between the 116th and 117th amino acids of the IL-10 monomer (the two IL-10 chains of the native IL-10 dimer are intertwined to form, after modification by inserting a linker, the IL-10 monomer can form a domain independently, i.e. a closed-loop monomer molecule), and the two IL-10 monomers in the same fusion protein do not interfere with each other.

[0126] According to an embodiment of the present application, the fusion protein further comprises a connecting peptide, and the IL-10 monomer is connected to the VEGFR2 full-length antibody via the connecting peptide.

[0127] According to some optional embodiments of the present application, the N-terminal of the IL-10 monomer is connected to the C-terminal of the connecting peptide, and the N-terminal of the connecting peptide is connected to the C-terminal of the heavy chain of the VEGFR2 full-length antibody.

[0128] According to some optional embodiments of the present application, the N-terminal of the IL-10 monomer is connected to the C-terminal of the connecting peptide, and the N-terminal of the connecting peptide is connected to the C-terminal of the heavy chain of the Ramucirumab.

[0129] According to an embodiment of the present application, the connecting peptide is selected from at least one of (GGGGS)nG, (GGGGS)n, (GSGGG)n, (GS)n, (GGGS)n, (GSGGS)n, (GGGGSG)n, and n is any integer between 1 and 10.

[0130] According to some optional embodiments of the present application, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any integer between any two of them as an end value.

[0131] According to an embodiment of the present application, the connecting peptide has an amino acid sequence as shown in SEQ ID NO: 16 or SEQ ID NO: 17.

[0132] According to an embodiment of the present application, the fusion protein has a heavy chain as shown in the amino acid sequence of SEQ ID NO: 18 and a light chain as shown in the amino acid sequence of SEQ ID NO: 11; or

[0133] The fusion protein has a heavy chain shown as the amino acid sequence of SEQ ID NO: 19 and a light chain shown as the amino acid sequence of SEQ ID NO: 11.

[0134] It should be noted that based on the amino acid sequence of the fusion protein of the present disclosure, a person skilled in the art can easily conceive of using genetic engineering technology or other technologies (chemical synthesis, recombinant expression) to prepare the fusion protein, for example, the fusion protein can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the antibody as described in any one of the above, which is easy for a person skilled in the art to achieve. Therefore, no matter what technology is used to prepare the fusion protein of the present disclosure, it falls within the scope of the present application.

[0135] Nucleic acid, vector, cell or host

[0136] In the process of preparing or obtaining the fusion protein of the first aspect, the nucleic acid expressing the fusion protein can be used, which is connected with different vectors and then expressed in different cells to obtain the corresponding fusion protein.

[0137] In the second aspect of the present application, a nucleic acid is provided. According to an embodiment of the present application, the nucleic acid encodes the fusion protein of the first aspect. The nucleic acid according to the embodiment of the present application can encode the fusion protein as described above.

[0138] According to an embodiment of the present application, the nucleic acid comprises DNA or RNA.

[0139] It should be noted that for the nucleic acid mentioned herein, a person skilled in the art should understand that it actually includes any one of the complementary double strands, or both. For convenience, in this text, although only one strand is given in most cases, the other complementary strand is actually disclosed. In addition, the sequence of molecules in the present application includes DNA or RNA form, and the disclosure of one means the disclosure of the other.

[0140] A person skilled in the art can understand that the features and advantages described above for the fusion protein also apply to the nucleic acid, which will not be repeated here.

[0141] In the third aspect of the present application, a vector is provided. According to an embodiment of the present application, the vector comprises the nucleic acid of the second aspect. When the nucleic acid of the second aspect is connected to the above-mentioned vector, the nucleic acid can be directly or indirectly connected with the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid, etc. Of course, these control elements can be directly from the vector itself, or can be exogenous, i.e. not from the vector itself. Of course, the nucleic acid can be operably linked to the control elements.

[0142] As used herein, "operably linked" refers to the linkage of a foreign gene to a vector, so that the control elements within the vector, such as transcription control sequences and translation control sequences, etc., can exert their expected functions of regulating the transcription and translation of the foreign gene. Commonly used vectors can be, for example, plasmids, bacteriophages, etc. After the vector according to some embodiments of the present application is introduced into a suitable recipient cell, the expression of the aforementioned fusion protein can be effectively realized under the mediation of the regulation system, and thus the in vitro mass acquisition of the fusion protein can be realized.

[0143] According to embodiments of the present application, the vector can refer to a cloning vector, which can be obtained by operably linking the nucleic acid to a commercially available vector, such as a plasmid or a viral vector. The vector in the present application is not particularly limited, and commonly used plasmids can be used, such as pSeTag2, PEE14, pMH3, etc.

[0144] In some alternative embodiments of the present application, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus or a bacteriophage.

[0145] In some alternative embodiments of the present application, the expression vector is a plasmid expression vector or a lentivirus expression vector.

[0146] Those skilled in the art can understand that the features and advantages described above for the fusion protein, the nucleic acid also apply to the vector, which will not be repeated here.

[0147] In a fourth aspect of the present application, a cell or host is provided. According to embodiments of the present application, the cell or host carries the nucleic acid of the second aspect or the vector of the third aspect; or the cell or host expresses the fusion protein of the first aspect. Using the cell or host under suitable conditions, the aforementioned fusion protein can be effectively expressed in the cell or host.

[0148] According to embodiments of the present application, the cell is obtained by introducing the vector of the third aspect into the cell or host.

[0149] It should be noted that the cell or host of the present application is not particularly limited, and can be a prokaryotic cell, a eukaryotic cell or a bacteriophage. The prokaryotic cell can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cell includes Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Trichoderma, etc. fungal cells, grasshopper cells, tobacco cells, BHK cells, CHO cells, COS cells, myeloma cells, etc. mammalian cells.

[0150] In an alternative embodiment of the present application, the cell is a mammalian cell, including BHK cells, CHO cells, NSO cells or COS cells, and does not include animal reproductive cells, fertilized eggs or embryonic stem cells.

[0151] It should be noted that the "suitable condition" described in the present application refers to a condition suitable for the expression of the fusion protein described in the present application. It is easily understood by those skilled in the art that the condition suitable for the expression of the fusion protein includes but is not limited to a suitable transformation or transfection method, a suitable transformation or transfection condition, a healthy cell state, a suitable cell density, a suitable cell culture environment, and a suitable cell culture time. The "suitable condition" is not particularly limited, and those skilled in the art can optimize the most suitable condition for the expression of the fusion protein according to the specific environment of the laboratory.

[0152] It is understood by those skilled in the art that the features and advantages described above for the fusion protein, the nucleic acid, and the vector also apply to the cell or the host, which will not be repeated here.

[0153] Pharmaceutical composition

[0154] In a fifth aspect of the present application, a pharmaceutical composition is provided. According to embodiments of the present application, the pharmaceutical composition comprises the fusion protein of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, or the cell or host of the fourth aspect. As known from the foregoing, the fusion protein of the first aspect or the fusion protein prepared using the nucleic acid of the second aspect, the vector of the third aspect, or the cell or host of the fourth aspect has the advantages of strong targeting, small side effects, good drug efficacy, and the like, and in particular has better anti-cancer activity and higher safety. Therefore, the pharmaceutical composition obtained can effectively prevent and / or treat tumors.

[0155] According to embodiments of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0156] According to embodiments of the present application, the excipient includes but is not limited to one or more pharmaceutically acceptable diluents, stabilizers, or pH adjusting agents, and the like.

[0157] According to embodiments of the present application, the pharmaceutical composition is an injection.

[0158] It should be noted that the pharmaceutical composition comprises a combination separated in time and / or space, as long as it can act together to achieve the purpose of the present application. For example, the components contained in the composition can be administered to the subject as a whole, or administered to the subject separately. When the components contained in the composition are administered to the subject separately, each component can be administered to the subject simultaneously or sequentially.

[0159] The pharmaceutical composition of the present application contains a safe and effective amount of the active ingredient of the present application and pharmaceutically acceptable adjuvants. Such adjuvants include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should be matched with the mode of administration, and the dosage form of the pharmaceutical composition of the present application is injection, oral preparation (tablet, capsule, oral solution), transdermal preparation, sustained release preparation. For example, it is prepared by conventional methods using physiological saline or aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

[0160] The effective amount of the active ingredient described in the present application can vary depending on the mode of administration and the severity of the disease to be treated. The selection of the preferred effective amount can be determined by a person of ordinary skill in the art according to various factors (for example, by clinical trials). The factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, etc. For example, several separate doses can be given daily, or the dose can be proportionally reduced, according to the exigencies of the therapeutic situation.

[0161] The pharmaceutically acceptable adjuvants described in the present application include, but are not limited to, water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptide substances, cellulose, nanogel, or combinations thereof. The selection of the carrier should be matched with the mode of administration, which is well known to a person of ordinary skill in the art.

[0162] A person skilled in the art can understand that the features and advantages described above for the fusion protein, nucleic acid, vector and cell or host also apply to the pharmaceutical composition, which will not be repeated here.

[0163] Use

[0164] In the sixth aspect of the present application, the present application provides the use of the fusion protein of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the cell or host of the fourth aspect, or the pharmaceutical composition of the fifth aspect in the preparation of a medicament for treating or preventing tumors.

[0165] In the seventh aspect of the present application, the present application provides the use of the fusion protein of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the cell or host of the fourth aspect, or the pharmaceutical composition of the fifth aspect in the treatment or prevention of tumors.

[0166] In an eighth aspect, the present application provides the fusion protein of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the cell or host of the fourth aspect, or the pharmaceutical composition of the fifth aspect for use in treating or preventing a tumor.

[0167] The terms "treat" and "prevent" and words stemming therefrom, as used herein, do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention, as appreciated by those of ordinary skill in the art, which have potential benefit or therapeutic effect. Moreover, the treatment or prevention provided by the present application can include treatment or prevention of one or more of the conditions or symptoms of the disease being treated or prevented, such as cancer. Additionally, for purposes herein, "prevention" can encompass delaying the onset of a disease or its symptoms or conditions.

[0168] According to embodiments of the present application, the use of the sixth aspect, the seventh aspect, the eighth aspect described above can further include at least one of the following technical features:

[0169] In some optional embodiments of the present application, the tumor comprises at least one of lung cancer, colorectal cancer, head and neck cancer, melanoma, nasopharyngeal carcinoma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, liver cancer, testicular cancer, endometrial cancer, skin cancer, bladder cancer, glioma, kidney cancer, esophageal cancer, oral squamous cell carcinoma.

[0170] In some optional embodiments of the present application, the tumor comprises gastric cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer.

[0171] Those skilled in the art can understand that the features and advantages described above for the fusion protein, the nucleic acid, the vector and the cell or host also apply to the use, which will not be repeated here.

[0172] Method

[0173] In a seventh aspect, the present application provides a method for treating or preventing a tumor. According to embodiments of the present application, the method comprises: administering to a subject a pharmaceutically acceptable dose of the fusion protein of the first aspect, the nucleic acid of the second aspect, the vector of the third aspect, the cell or host of the fourth aspect, or the pharmaceutical composition of the fifth aspect.

[0174] It is noted that the terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal that is being assessed for treatment and / or treated. In one embodiment, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals having cancer, individuals having autoimmune diseases, individuals having pathogen infections, and the like. The subject can be a human, but also includes other mammals, particularly mammals useful as laboratory models of human disease, e.g., mice, rats, and the like.

[0175] The effective amount of the fusion protein, nucleic acid, vector, cell or host, or pharmaceutical composition described herein can vary according to the mode of administration and the severity of the disease to be treated, etc. The selection of an optimal effective amount can be determined by one of ordinary skill in the art (e.g., through clinical trials) according to various factors. The factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated, the body weight of the patient, the immune status of the patient, the route of administration, etc. For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0176] In some optional embodiments of the present application, the tumor comprises at least one of lung cancer, colorectal cancer, head and neck cancer, melanoma, nasopharyngeal cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, liver cancer, testicular cancer, endometrial cancer, skin cancer, bladder cancer, glioma, kidney cancer, esophageal cancer, oral squamous cell carcinoma.

[0177] In some optional embodiments of the present application, the tumor comprises gastric cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer.

[0178] It will be understood by those skilled in the art that the features and advantages described above for the fusion protein, nucleic acid, vector, cell or host, and pharmaceutical composition also apply to the method, which will not be repeated here.

[0179] The solutions of the present application will be explained below in connection with examples. Those skilled in the art will understand that the following examples are only illustrative of the present application and should not be viewed as limiting the scope of the present application. If a specific technique or condition is not specified in the examples, it is performed according to the technique or condition described in the literature in the art or according to the product manual. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained on the market.

[0180] Example 1. Construction and expression of Cyramza x IL-10M(HC)-hIgG1 and Cyramza x IL-10M(HC)-hIgG1 (LALA) molecules

[0181] The plasmid of the fusion protein Cyramza x IL-10M(HC)-hIgGl molecule of Ramucirumab (i.e. Cyramza) and IL-10M (the amino acid sequence of IL-10M is shown as SEQ ID NO: 13) was designed according to the structure of Figure 1. Specifically, the antibody variable region sequence adopts the variable region sequence of Ramucirumab, the heavy chain constant region is the sequence of human IgGl subtype, and the light chain constant region is the sequence of human kappa subtype. The IL-10 monomer is connected to the heavy chain of Ramucirumab through the linker 1 (the amino acid sequence is shown as SEQ ID NO: 16). The constructed plasmid was transfected into the EXPI293 cell line, and the supernatant was collected after 7 days. After protein A purification, the fusion protein (or recombinant antibody) was obtained, named R3207 (the amino acid sequence of the chain is shown as SEQ ID NO: 19, and the amino acid sequence of the light chain is shown as SEQ ID NO: 11).

[0182] In order to compare whether the function of Fc in the fusion protein will affect the anti-tumor efficacy in vivo, the human IgGl Fc of R3207 was further subjected to L234A / L235A mutation to remove the ADCC function and other functions of Fc, i.e. Cyramza x IL-10M(HC)-hIgGl(LALA), named R3201 (the amino acid sequence of the heavy chain is shown as SEQ ID NO: 18, and the amino acid sequence of the light chain is shown as SEQ ID NO: 11).

[0183] IL-10M (the amino acid sequence is shown as SEQ ID NO: 13) was replaced by wild-type IL-10 (the amino acid sequence is shown as SEQ ID NO: 15) to construct and express human IgGl and its L234A / L235A mutant, i.e. Cyramza x wtIL-10(HC)-hIgGl and Cyramza x wtIL-10(HC)-hIgGl(L234A / L235A), respectively named R0628 (the amino acid sequence of the heavy chain is shown as SEQ ID NO: 47, and the amino acid sequence of the light chain is shown as SEQ ID NO: 11) and R4070 (the amino acid sequence of the heavy chain is shown as SEQ ID NO: 45, and the amino acid sequence of the light chain is shown as SEQ ID NO: 11), as positive control antibodies.

[0184] Patent molecule (CN114316064A) R0467, i.e. Cyramza x wtIL-10(HC)-hIgG1(K447A / A327Q / G237A / L235A) as a patent control antibody (the amino acid sequence of the heavy chain thereof is shown as SEQ ID NO: 46, and the amino acid sequence of the light chain thereof is shown as SEQ ID NO: 11), i.e. Cyramza x wtIL-10(HC)-hIgG1(K447A / A327Q / G237A / L235A) was constructed and expressed.

[0185] Isotype x IL-10M(HC)-hIgG1 and isotype x IL-10M(HC)-hIgG1(L234A / L235A) were constructed and expressed, named R3208 (the amino acid sequence of the heavy chain thereof is shown as SEQ ID NO: 37, and the amino acid sequence of the light chain thereof is shown as SEQ ID NO: 38) and R3202 (the amino acid sequence of the heavy chain thereof is shown as SEQ ID NO: 44, and the amino acid sequence of the light chain thereof is shown as SEQ ID NO: 38) respectively, as IL-10M isotype control molecules.

[0186] Fc-IL10WT was constructed and expressed, named R0674, which consists of Fc region and IL10 WT fused at the C terminal thereof, and includes two identical peptide chains, wherein one peptide chain is shown as SEQ ID NO: 36.

[0187] Ramucirumab antibody and L234A / L235A mutant antibody were constructed and expressed as positive control antibodies, named R2496 (the amino acid sequence of the heavy chain thereof is shown as SEQ ID NO: 9, and the amino acid sequence of the light chain thereof is shown as SEQ ID NO: 11) and R2494 (the amino acid sequence of the heavy chain thereof is shown as SEQ ID NO: 10, and the amino acid sequence of the light chain thereof is shown as SEQ ID NO: 11) respectively.

[0188] hIgG1 antibody targeting hemocyanin and L234A / L235A mutant hIgG1 antibody were constructed and expressed as isotype control antibodies, named R0861 (the amino acid sequence of the heavy chain thereof is shown as SEQ ID NO: 39, and the amino acid sequence of the light chain thereof is shown as SEQ ID NO: 40) and R0862 (the amino acid sequence of the heavy chain thereof is shown as SEQ ID NO: 41, and the amino acid sequence of the light chain thereof is shown as SEQ ID NO: 42) respectively.

[0189] The amino acid sequences in the present embodiment are shown in Table A1.

[0190] Example 2. Analysis of the cell binding activity of the Cyramza x IL-10M fusion protein at the IL-10 end

[0191] IL-10R1 was overexpressed in a CHO cell line (Chinese hamster ovary cell line), i.e. CHO-IL-10R1 (the amino acid sequence of which is shown as SEQ ID NO: 43, see Table A1 for details). The binding activity of R3201 and R3207 prepared in Example 1 to CHO-IL-10R1 was detected by flow cytometry. The specific steps are as follows:

[0192] First, the CHO-IL-10R1 cells were counted, and an appropriate amount of cells was centrifuged at 350 x g for 5 minutes, and the supernatant was discarded. The cell density was adjusted to 2 x 10 6 6 cells / mL with 3% BSA. The concentration of the test recombinant antibody and the control antibody was 240 nM, and a 3-fold gradient dilution was performed. 100 μL of the diluted antibody was added to the cells, and incubated at 4°C for 30 minutes. After washing once with 3% BSA, 100 μL of PE-labeled goat anti-Fab antibody (Jackson ImmunoResearch, 109-116-097) diluted 1:500 was added to resuspend the cells, and incubated at 4°C for 30 minutes. After washing once with 3% BSA, 150 μL / well of PBS was added to resuspend the cells, and the fluorescence intensity was analyzed by flow cytometry. The experimental results are shown in Figure 2. Compared with R0862, R3201 and R3207 have better binding activity to IL-10R1.

[0193] Example 3. Analysis of the cell binding activity of the Cyramza x IL-10M fusion protein at the VEGFR2 end

[0194] hVEGFR2 was overexpressed in a 293T cell line to construct a recombinant monoclonal cell line, i.e. 293T-hVEGFR2. The binding activity of Cyramza x IL-10M fusion protein molecules R3201 and R3207 and control Cyramza monoclonal antibodies R2494 and R2496 to 293T-hVEGFR2 cells was detected by flow cytometry. The specific steps are as follows:

[0195] First, the 293T-hVEGFR2 cells were counted, and an appropriate amount of cells was centrifuged at 350 x g for 5 minutes, and the supernatant was discarded. The cell density was adjusted to 2 x 10 6 / mL, and added to 96-well V-bottom plates. The concentration of the test recombinant antibody and the control antibody was set to 120 nM, and 3-fold gradient dilution was performed. 100 μL of the diluted antibody was added to the cells, and incubated at 4°C for 30 minutes. After washing once with 3% BSA, 100 μL of PE-labeled goat anti-human Fab antibody (Jackson ImmunoResearch, 109-116-097) diluted 1:500 was added to resuspend the cells, and incubated at 4°C for 30 minutes. After washing once with 3% BSA, 150 μL / well of PBS was added to resuspend the cells, and the fluorescence intensity was analyzed by flow cytometry. The experimental results are shown in Figure 3, and the Cyramza x IL-10 fusion protein molecules R3201 and R3207 had similar binding activity to the control Cyramza monoclonal antibodies R2496 and R2494, respectively.

[0196] Example 4. Analysis of the blocking activity of Cyramza x IL-10M fusion protein on the binding of VEGFR2 to VEGF165

[0197] Vascular endothelial growth factor (VEGF) is an important angiogenesis regulator, and VEGF165 is a more important member of the family, which regulates the biological functions of endothelial cells, such as proliferation, migration, and tube formation, by interacting with VEGFR2. A recombinant monoclonal cell line was constructed by overexpressing hVEGFR2 in the 293T cell line, which was 293T-hVEGFR2. The ability of the Cyramza x IL-10M fusion protein molecules R3201 and R3207 and the control Cyramza monoclonal antibodies R2494 and R2496 to block the binding of VEGF165-his to 293T-hVEGFR2 cells was detected by flow cytometry. The specific steps are as follows:

[0198] First, the 293T-hVEGFR2 cells were counted, and an appropriate amount of cells was centrifuged at 350 x g for 5 minutes, and the supernatant was discarded. The cell density was adjusted to 2 x 10 6 / mL, 100 μL / well cell suspension was added to 96-well V-bottom plate. The concentration of the test recombinant antibody and control antibody was configured as 240 nM, and 3-fold gradient dilution was performed. 50 μL of the diluted antibody was added to the cells, and incubated at 4°C for 30 min. 8 μg / mL of VEGF165-his (Acro, VE5-H5248) protein diluted with 3% BSA was added to the cells at 50 μL / well, and incubated at 4°C for 30 min, centrifuged at 350 x g for 5 min, and the supernatant was discarded. After washing once with 3% BSA, the cells were resuspended with 100 μL of APC-labeled anti-his tag antibody (Biolegend, 362605) diluted 1:300, and incubated at 4°C for 30 min. After washing once with 3% BSA, the cells were resuspended with 150 μL / well PBS, and the fluorescence intensity was analyzed by flow cytometry. The experimental results are shown in Figure 4, and the Cyramza x IL-10M fusion protein molecules R3201 and R3207 have similar blocking activities to the control Cyramza monoclonal antibodies R2496 and R2494.

[0199] Example 5. VEGFR2 terminal reporter gene activity analysis of Cyramza x IL-10M fusion protein

[0200] The luciferase reporter gene system was transferred into 293T-hVEGFR2 cells to construct a reporter gene monoclonal cell line, 293T-hVEGFR2-Luc, and the activity of Cyramza x IL-10M fusion protein molecules R3201 and R3207 and control Cyramza monoclonal antibodies R2494 and R2496 in blocking VEGF165-activated VEGFR2 signaling pathway was detected by chemiluminescence. The specific steps are as follows:

[0201] The concentration of the test recombinant antibody and control antibody was configured as 240 nM, and 3-fold gradient dilution was performed. The ligand VEGF165-his (Acro, VE5-H5248) was diluted to 54.8 ng / mL, and 25 μL of antibody diluent and 25 μL of ligand diluent were added to each well of a 96-well white flat-bottom plate. The 293T-hVEGFR2-Luc cells in good culture condition were digested, centrifuged, and the supernatant was discarded, and the cells were adjusted to 4 x 10 6 / mL. The cell suspension was added to the antibody and ligand mixture at 50 μL / well, and the cell plate was placed in a 37°C, 5% CO2 incubator for 6 h. The cell culture plate was removed and equilibrated at room temperature for 5-10 min to return to room temperature. 100 μL of thawed luciferase detection reagent (Biotium, RG051M) was added to each well. Incubate at room temperature for 5-10 min to allow the luminescent signal to stabilize, and then detect the chemiluminescence signal using a microplate reader. The data are shown in Figure 5, and the Cyramza x IL-10M fusion protein molecules R3201 and R3207 have similar VEGFR2 reporter gene activity compared with the control Cyramza monoclonal antibodies R2496 and R2494.

[0202] Example 6. Analysis of the STAT3 signal pathway activation activity of Cyramza x IL-10M fusion protein

[0203] STAT3 is an important signal pathway downstream of IL-10. IL-10 plays a strong function in promoting cytotoxicity, preventing apoptosis, and enhancing the adaptability of CD8+ T cells through STAT3 signaling. The STAT3 signal pathway activation experiment was used to analyze the STAT3 signal pathway activation ability of IL-10 before and after modification with or without antigen enrichment. The 293-IL-10R-STAT3-Luc reporter gene cell line was purchased from Jiman Biotechnology. The specific steps are as follows:

[0204] The hVEGFR2-hFc Tag protein was diluted to 2 μg / mL with DPBS the day before, and the protein enrichment group was added to the 96-well whole white cell culture plate at 100 μL / well, and the non-enrichment group was added with 100 μL of DPBS as a control. The culture plate was coated overnight in a 4°C refrigerator. The next morning, the antibody was prepared using DMEM+10% FBS, and the antibody was prepared into a 120 nM working solution and diluted by 3 times. Eight concentration points were prepared. The coated cell plate was removed, and the coating solution was aspirated using a syringe. The prepared antibody working solution was added to the cell plate at 50 μL / well, and the antigen was pre-incubated at 37°C, 5% CO2 in an incubator for 30 min.

[0205] 293-IL-10R-STAT3-Luc cells in good condition with a confluence of about 80-90% were digested into single cells with 0.25% trypsin, centrifuged at 300 x g for 5 min to discard the supernatant, and then the cell density was adjusted to 1 x 10 6 / mL, and 50 μL per well was added to the incubated 96-well whole white flat-bottom plate, i.e., 5 x 10 4Cells were cultured at 37°C, 5% CO2 incubator for 6 hours. The cell plate was taken out of the incubator and equilibrated at room temperature for 5-10 minutes to recover to room temperature, and the luciferase reporter assay reagent (Promega) was added at 100 μL / well, which was thawed at room temperature in advance, and incubated for 10-15 minutes in the dark. The fluorescence value was detected using the Ensight multifunctional microplate reader. The data were analyzed by plotting, and the antibody concentration was taken as the abscissa and the chemiluminescence value was taken as the ordinate for nonlinear fitting using GraphPad Prism 8 software to calculate the EC 50 value, and the results are shown in Figure 6. The activity of the modified IL-10 (R3201, R3207, R3202 and R3208) in activating the STAT3 signaling pathway was much lower than that of the wild type, and the reporter gene activity of R3201 and R3207 was significantly improved relative to the IL-10M isotype control molecules (R3202 and R3208) in the presence of antigen enrichment.

[0206] Example 7. Cyramza x IL-10M fusion protein binding activity analysis with exhausted CD8 + T cells

[0207] To evaluate the binding activity of the fusion protein molecule with exhausted CD8 + T cells, the exhausted CD8 + T cells were induced in vitro, and the binding activity of R3201, R3207 and control Cyramza monoclonal antibodies R2496, R2494 with exhausted CD8 + T cells was detected by flow cytometry. The specific steps are as follows:

[0208] CD8 + T cells were sorted from the peripheral blood of healthy people using human CD8 magnetic beads (StemCell, 17953), and the CD8 + T cells were resuspended at a density of 1 x 10 6 / mL using RPMI 1640 (containing 10% FBS and 100 U / mL IL2). CD3 / CD28 activation magnetic beads (Dingli Haiyuan) were added at a ratio of 1:1 according to the cell amount, and then inoculated in a 10 cm cell culture dish after mixing gently. After incubation at 37°C, 5% CO2 incubator for 72 hours, the medium was supplemented and incubated for another 48 hours.

[0209] The induced CD8 + T cells were counted, and an appropriate amount of cells was centrifuged at 350 x g for 5 minutes, and the supernatant was discarded. The cell density was adjusted to 2 x 10 6The cells were resuspended in 100 μL / well of cell suspension in 96-well V-bottom plate. The concentration of the test recombinant antibody and the control antibody was configured to be 400 nM, and 3-fold gradient dilution was performed. 100 μL of the diluted antibody was added to the cells, and incubated at 4°C for 30 minutes. After washing once with 3% BSA, 100 μL of APC-labeled goat anti-Fab antibody (Jackson ImmunoResearch, 109-605-097) diluted 1:600 was added to resuspend the cells, and incubated at 4°C for 30 minutes. After washing once with 3% BSA, 150 μL / well of PBS was added to resuspend the cells, and the fluorescence intensity was analyzed by flow cytometry. The experimental results are shown in FIG. 7, and the binding activity of R3201 and R3207 was better than that of the control Cyramza monoclonal antibodies R2496, R2494 and the isotype controls R0862 and R0861.

[0210] Example 8. Cyramza x IL-10M fusion protein inhibits exhausted CD8 + T cell apoptosis activity analysis

[0211] To evaluate the activity of IL-10 in inhibiting CD8 + T cell apoptosis and reversing the activity of exhausted CD8 + T cells, CD8 + T cells were sorted from the peripheral blood of healthy people using human CD8 magnetic beads (StemCell, 17953). The CD8 + T cells were resuspended at a density of 1 x 10 6 / mL using RPMI 1640 containing 10% FBS and 100 U / mL IL2. The CD3 / CD28 activation magnetic beads (TOMO Biosciences) were added at a ratio of 1:1 based on the amount of cells, mixed gently by blowing, and inoculated in a 10 cm cell culture dish. After incubation in a 37°C, 5% CO2 incubator for 72 hours, the medium was supplemented and incubated for another 48 hours. The hVEGFR2-his protein (Sino Biological, 10012-H08H) was diluted to 4 μg / mL with DPBS one day before cell plating, and 100 μL was added to each well of a 96-well flat-bottom plate and placed in a 4°C refrigerator for overnight coating.

[0212] Dilute the antibody to 200nM working solution with RPMI 1640 medium (without IL-2) and dilute 5 times gradient, 3 concentration points. Discard the coating solution, wash the coated plate with DPBS once, add the prepared antibody at 100μL / well, incubate at 37℃ for 1 hour. Collect the cells in the culture dish, mix well by blowing, and then distribute into 15ml centrifuge tubes. Place on the magnetic stand for about 10 minutes to remove the old magnetic beads. Centrifuge the cells, discard the old medium, resuspend and count. Resuspend the medium with RPMI 1640 medium (without IL-2), add new CD3 / CD28 activated magnetic beads, mix well by blowing, and then inoculate 1×10 5 / well cells, 100μL in the incubated 96-well flat-bottom cell plate, continue to culture for 4 days.

[0213] Transfer the cells to be tested to a 96-well V-bottom cell plate, centrifuge and discard the supernatant, wash once with Annexin-V binding buffer, and prepare the staining solution with Annexin-V binding buffer (mix 1μL APC annexin-V and 1μL Propidium Iodide Solution per well), add 100μL / well (mix gently), incubate at 4℃ for 15 minutes in the dark, centrifuge and discard the supernatant, and use a flow cytometer to detect cell apoptosis, analyze the data of APC and PE fluorescence channel double positive, the results are shown in Figure 8, R3201 and R3207 have significant inhibition of exhausted CD8 + T cell apoptosis activity.

[0214] Example 9. Cyramza x IL-10M fusion protein binding activity analysis with HUVEC cells

[0215] To evaluate the binding activity of the fusion protein molecule to cells naturally expressing VEGFR2 (primary HUVEC cells), the binding activity of R3201, R3207, and control Cyramza monoclonal antibodies R2496, R2494 to primary HUVEC was detected by flow cytometry. The specific steps are as follows:

[0216] Count the primary HUVEC cells, centrifuge 350xg for 5 minutes, and discard the supernatant. Adjust the cell density to 2x10 6Cells were added at a concentration of 100 μL / well to a 96-well V plate. The recombinant antibody and control antibody at a concentration of 66 nM were prepared and serially diluted 3-fold. 100 μL of the diluted antibody was added to the cells and incubated at 4°C for 30 minutes. After washing once with 3% BSA, the cells were resuspended in 100 μL of APC-labeled goat anti-Fab antibody (Jackson ImmunoResearch, 109-605-097) diluted 1:500 and incubated at 4°C for 30 minutes. After washing once with 3% BSA, the cells were resuspended in 150 μL / well PBS. Fluorescence intensity was analyzed by flow cytometry, and the experimental data were plotted. The results are shown in Figure 9. The Cyramza×IL-10M fusion protein molecules R3201 and R3207 showed similar binding activity to the control Cyramza monoclonal antibodies R2496 and R2494, respectively.

[0217] Example 10. Analysis of the inhibitory activity of Cyramza×IL-10M fusion protein on HUVEC cell proliferation.

[0218] Primary HUVEC cells (isolated from human umbilical cord) that were growing well in complete HUVEC medium and had a confluence of approximately 80% were digested into single cells using TrypLE™ Express enzyme (Gibco), and then washed once with HUVEC basal medium. The cells were resuspended in HUVEC basal medium, filtered through a 40 μm cell filter, and then counted. The cell density was adjusted to 4 × 10⁶ cells / year. 4 Cells were added at 100 μL per well to a 96-well plate. The cells were starved overnight at 37°C with 5% CO2. Unused wells were replenished with 200 μL of DPBS. The next day, the antibody was diluted to 800 nM with HUVEC basal medium containing 20% ​​FBS and added at 50 μL per well to the cell culture plate. The plates were incubated at 37°C for 30 minutes. The VEGF165 (Acro, VE5-H5248) concentration was adjusted to 200 ng / mL with HUVEC basal medium containing 20% ​​FBS, and added at 50 μL per well to the 96-well plate. Wells without VEGF165 were replenished with 50 μL of basal medium containing 20% ​​FBS. The cell culture plates were then incubated statically for 4 days. Cells were used after 4 days. Fluorescence values ​​were detected using a luminescent cell viability assay kit (Promega), and the experimental results are shown in Figure 10. Cyramza×IL-10M fusion protein molecules R3201 and R3207 have similar inhibitory activity against HUVEC proliferation as control Cyramza monoclonal antibodies R2496 and R2494, and can effectively inhibit angiogenesis.

[0219] Example 11. Anti-tumor efficacy analysis of Cyramza x IL-10M(HC)-hIgG1 and Cyramza-hIgG1 molecules in vivo

[0220] To verify the anti-tumor activity of R3207 in vivo, the B16F1-KDR humanized mouse tumor model was used. The specific experimental steps are as follows:

[0221] B16F1 was inoculated into hKDR humanized mice at a density of 2 x 10 5 B16F1 was inoculated into hKDR humanized mice at a density of 2 x 10 2 The tumor volume was measured twice a week, and the tumor volume was calculated according to the formula: tumor volume = tumor long diameter * tumor short diameter / 2. The experimental results are shown in Figure 11 and Table 1. R3207 had a significant anti-tumor effect in vivo, and the efficacy was significantly better than R2496. Therefore, it can be further illustrated that the anti-tumor activity of Cyramza x IL-10M fusion protein molecule R3207 is significantly better than the control monoclonal antibody R2496.

[0222] Table 1 * The average TV of each treatment group was compared with the R0861 group; One-way ANONVA, Dunnett's Multiple Comparison Test

[0223] Example 12. Anti-tumor efficacy analysis of Cyramza x IL-10M(HC)-hIgG1 and Cyramza x IL-10M(HC)-hIgG1(L234A / L235A) molecules in vivo

[0224] To further compare the anti-tumor activity of R3207 and R3201 in vivo, a pharmacodynamic comparison experiment was performed in the B16F1-KDR humanized mouse tumor model. The specific experimental steps are as follows:

[0225] B16F1 was inoculated into hKDR humanized mice at a density of 2 x 10 5Individual density of B16F1 was inoculated into hKDR humanized mice to construct tumor model (D0). Recombinant antibodies R2496, R3207, R3201 and negative control antibody R0862 were intravenously injected at D0 / D2 / D4. The same molar concentration was used in each group, and the dosages of R2496 and R3207, R3201 were 8.2 mg / kg and 10.5 mg / kg, and the dosage of R0861 was 8.3 mg / kg. The tumor volume of mice was measured twice a week, and the tumor volume was calculated according to the formula: tumor volume = tumor long diameter * tumor short diameter 2 The experimental results are shown in Figure 12 and Table 2. R3201 and R3207 have significant anti-tumor effect in vivo, and the drug efficacy is significantly better than R2496.

[0226] Table 2 * The average TV of each treatment group was compared with the R0862 group; One-way ANONVA, Dunnett's Multiple Comparison Test

[0227] Example 13, Early safety evaluation of anti-Cyramza x IL-10M fusion protein

[0228] Test 1: This example aims to observe the toxicity reaction caused by R0674 and study the toxicokinetic characteristics of R0674 by intravenous injection of R0674 in cynomolgus monkeys, and to provide a reference for subsequent test design. During the test period, the following observations were made on the animals: death and near-death, clinical observation, body weight, food intake, body temperature, hematology and coagulation index detection, serum biochemical index and immune function detection. After euthanasia of the animals, gross autopsy was performed, and the weights of the main organs (adrenal gland, brain, epididymis, heart, kidney, liver, spleen, testis, thymus, thyroid and parathyroid) were measured, and histopathological examination was performed on the liver, spleen and kidney.

[0229] The results show that during the experiment, no obvious abnormalities were found in animal death and near-death, clinical observation, body weight, food intake, body temperature, coagulation, immune function detection, and organ weight. However, hematology toxicity was observed in the experimental group (R0674), and the serum biochemical index was significantly abnormal.

[0230] Test 2: This example aims to observe the toxicity reaction caused by anti-Cyramza-IL10M fusion protein R3201 and study the toxicokinetic characteristics of R3201 by intravenous injection of R3201 in cynomolgus monkeys at different doses, and to provide a reference for subsequent test design. During the test period, the following observations were made on the animals: death and near-death, clinical observation, body weight, body temperature, hematology and coagulation index detection, serum biochemical index and immune function detection.

[0231] Results showed that: during the experiment, no obvious abnormality was found in animal death and near-death, clinical observation, body weight, body temperature, coagulation, and immune function detection. Hematology, serum biochemistry, lymphocyte subtype, and cytokine were also not obviously abnormal.

[0232] In summary, the anti-Cyramza-IL10M fusion protein R3201 showed better safety in the cynomolgus monkey experiment.

[0233] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any appropriate manner in any one or more embodiments or examples. Furthermore, different embodiments or examples described in the present specification and the features of different embodiments or examples can be combined and combined by those skilled in the art without contradiction.

[0234] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A fusion protein, characterized in that, comprises an anti-VEGFR2 antibody and an IL-10 monomer; wherein the IL-10 monomer is linked to the anti-VEGFR2 antibody.

2. The fusion protein of claim 1, wherein, the anti-VEGFR2 antibody is selected from the group consisting of a VEGFR2 full-length mAb, a Fab antibody, a F(ab’)2 antibody, a Fab’ antibody, a Fv antibody, a scFv antibody, a dsFv antibody, a nanobody; optionally, the N-terminus of the IL-10 monomer is linked to the C-terminus of the anti-VEGFR2 antibody, or the N-terminus of the anti-VEGFR2 antibody is linked to the C-terminus of the IL-10 monomer; optionally, the N-terminus of the IL-10 monomer is linked to the C-terminus of the anti-VEGFR2 antibody; optionally, the anti-VEGFR2 antibody is selected from the group consisting of a VEGFR2 full-length mAb, a Fab antibody, a F(ab’)2 antibody, a Fab’ antibody, the N-terminus of the IL-10 monomer is linked to the C-terminus of the heavy chain of the anti-VEGFR2 antibody; optionally, the anti-VEGFR2 antibody is selected from the group consisting of a Fv antibody, a scFv antibody, a dsFv antibody, a nanobody, the N-terminus of the IL-10 monomer is linked to the C-terminus of the anti-VEGFR2 antibody; optionally, the anti-VEGFR2 antibody comprises HCDRs and LCDRs, the HCDRs comprise HCDRs as defined in a heavy chain variable region of SEQ ID NO: 1, the LCDRs comprise LCDRs as defined in a light chain variable region of SEQ ID NO: 1; optionally, the HCDRs and / or LCDRs are defined by Kabat, Chothia, AbM, Contact, or IMGT; optionally, the anti-VEGFR2 antibody comprises HCDR1, HCDR2, HCDR3 having the sequences of SEQ ID NOs: 3, 4, and 5, and LCDR1, LCDR2, LCDR3 having the sequences of SEQ ID NOs: 6, 7, and 8; optionally, the anti-VEGFR2 antibody comprises a heavy chain variable region having the sequence of SEQ ID NO: 1, and a light chain variable region having the sequence of SEQ ID NO: 2; optionally, the anti-VEGFR2 antibody is selected from the group consisting of a VEGFR2 full-length mAb, the N-terminus of the IL-10 monomer is linked to the C-terminus of the heavy chain of the VEGFR2 full-length mAb; optionally, the heavy chain variable region of the VEGFR2 full-length mAb has the amino acid sequence of SEQ ID NO: 1, the light chain variable region of the VEGFR2 full-length mAb has the amino acid sequence of SEQ ID NO: 2; optionally, the VEGFR2 full-length mAb is Ramucirumab, a mutant of Ramucirumab, or a chimera of Ramucirumab; optionally, the mutant of Ramucirumab or the chimera of Ramucirumab has HCDRs as defined in a heavy chain variable region of SEQ ID NO: 1, and has LCDRs as defined in a light chain variable region of SEQ ID NO: 1; Optionally, the mutant of Ramucirumab or the chimera of Ramucirumab has HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO: 3, 4 and 5, and has LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO: 6, 7 and 8; Optionally, the VEGFR2 full-length monoclonal antibody has a heavy chain variable region as shown in SEQ ID NO: 9 or 10 and has a light chain variable region as shown in SEQ ID NO: 11; Optionally, at least a portion of at least one of the heavy chain constant region and the light chain constant region of the VEGFR2 full-length monoclonal antibody is from at least one of a murine antibody, a primate antibody, a bovine antibody, a horse antibody, a dairy cow antibody, a pig antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, an ass antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof; Optionally, the heavy chain constant region comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; Optionally, the light chain constant region comprises a light chain constant region selected from kappa type or lambda type; Optionally, the light chain constant region and the heavy chain constant region are both from at least one of a rabbit antibody or a mutant thereof, a murine antibody or a mutant thereof, and a human antibody or a mutant thereof; Optionally, the heavy chain constant region is selected from a human IgG1 heavy chain constant region or a mutant thereof; Optionally, the mutant of the human IgG1 heavy chain constant region has an activity of attenuating ADCC effect or an activity of attenuating CDC effect compared with a wild-type human IgG1 heavy chain constant region; Optionally, the mutant of the human IgG1 heavy chain constant region has any one of the following mutations compared with a wild-type human IgG1 heavy chain constant region: 1) L234A and L235A; 2) L235A, G237A, A327Q and K447A.

3. The fusion protein according to any one of claims 1-2, characterized in that, The IL-10 monomer is selected from an engineered IL-10 monomer; Optionally, the engineered IL-10 monomer has a deletion of at least 2 amino acids at the N-terminus compared with a native IL-10 monomer, preferably a deletion of 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids; Optionally, the engineered IL-10 monomer has a spacer peptide between two adjacent amino acids compared with a native IL-10 monomer; Optionally, one of the two adjacent amino acids is at position 115, 116, 117, 118 or 119, and the spacer peptide is a flexible linker peptide; Optionally, the engineered IL-10 monomer has a deletion of at least 2 amino acids at the N-terminus compared with a native IL-10 monomer, and has a spacer peptide between two adjacent amino acids, one of the two adjacent amino acids is at position 115, 116, 117, 118 or 119, and the spacer peptide is a flexible linker peptide; Optionally, the N-terminal of the engineered IL-10 monomer is deleted by 2 amino acids compared with the native IL-10 monomer, and contains a spacer peptide between the 116th and 117th amino acids; Optionally, the spacer peptide is selected from at least one of GGGSGG, (GS)n, (GGGS)n, (GSGGS)n, (GGGGS)nG, (GGS)n, n is any integer between 1 and 10; Optionally, the spacer peptide has an amino acid sequence as shown in SEQ ID NO: 12; Optionally, the engineered IL-10 monomer has an amino acid sequence as shown in SEQ ID NO: 13, 14 or 48.

4. The fusion protein according to any one of claims 1 to 3, characterized in that, further comprising a linker peptide, the IL-10 monomer is connected to the VEGFR2 full-length monoclonal antibody through the linker peptide; Optionally, the linker peptide is selected from at least one of (GGGGS)nG, (GGGGS)n, (GSGGG)n, (GS)n, (GGGS)n, (GSGGS)n, (GGGGSG)n, n is any integer between 1 and 10; Optionally, the linker peptide has an amino acid sequence as shown in SEQ ID NO: 16 or 17.

5. The fusion protein according to any one of claims 1 to 4, characterized in that, the fusion protein has a heavy chain as shown in the amino acid sequence of SEQ ID NO: 18 and a light chain as shown in the amino acid sequence of SEQ ID NO: 11; or the fusion protein has a heavy chain as shown in the amino acid sequence of SEQ ID NO: 19 and a light chain as shown in the amino acid sequence of SEQ ID NO:

11.

6. A nucleic acid, characterized in that, the nucleic acid encodes the fusion protein of any one of claims 1-5.

7. A vector, characterized in that, the vector comprises the nucleic acid of claim 6.

8. A cell or host, characterized in that, the cell or host carries the nucleic acid of claim 6 or the vector of claim 7; or the cell or host expresses the fusion protein of any one of claims 1-5.

9. A pharmaceutical composition, characterized by, the fusion protein of any one of claims 1-5, the nucleic acid of claim 6, the vector of claim 7, or the cell or host of claim 8; Optionally, further comprising a pharmaceutically acceptable excipient.

10. Use of the fusion protein of any one of claims 1-5, the nucleic acid of claim 6, the vector of claim 7, the cell or host of claim 8, or the pharmaceutical composition of claim 9 in the manufacture of a medicament for treating or preventing a tumor.

11. Use of the fusion protein of any one of claims 1-5, the nucleic acid of claim 6, the vector of claim 7, the cell or host of claim 8, or the pharmaceutical composition of claim 9 in treating or preventing a tumor.

12. The fusion protein of any one of claims 1-5, the nucleic acid of claim 6, the vector of claim 7, the cell or host of claim 8, or the pharmaceutical composition of claim 9 for treating or preventing a tumor.

13. A method of treating or preventing a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-12. comprising: administering to the subject a pharmaceutically acceptable dose of the fusion protein of any one of claims 1-5, the nucleic acid of claim 6, the vector of claim 7, the cell or host of claim 8, or the pharmaceutical composition of claim 9.

14. Use according to any one of claims 10 to 12 or method according to claim 13, characterized in that, The tumor is selected from at least one of gastric cancer, gastric cancer, lung cancer, colorectal cancer, head and neck cancer, melanoma, nasopharyngeal cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, liver cancer, testicular cancer, endometrial cancer, skin cancer, bladder cancer, glioma, kidney cancer, esophageal cancer, oral squamous cell carcinoma; Optionally, the tumor is selected from at least one of gastric cancer, lung cancer, liver cancer, breast cancer, prostate cancer, ovarian cancer.

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