WO2026016967A1 - Fusion protein and use thereof - Google Patents

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 its uses

Priority information

This application claims priority and benefit to patent application 202410955537.7, filed with the China National Intellectual Property Administration on July 16, 2024, the entire contents of which are incorporated herein by reference.

Technical Field

This application belongs to the field of biomedical technology, specifically relating to a fusion protein and its uses, and more specifically to a fusion protein, nucleic acid, vector, cell or host, pharmaceutical composition and its uses.

Background Technology

Vascular endothelial growth factor receptor 2 (VEGFR2) plays a crucial role in vascular endothelial cell signaling, including involvement in cell survival, proliferation, migration, and vascular permeability, promoting tumor angiogenesis and cancer development. Tumor vascular networks are leaky, disorganized, immature, thin-walled, and poorly perfused; blunting or collapse of tumor vessels often leads to hypoxic areas in tumor formation. VEGFR2 is primarily expressed on endothelial cells, upregulated in various tumors, and further upregulated under hypoxic conditions and on tumor-infiltrating CD8+ T cells. IL-10R comprises two subunits, IL-10Ra and IL-10Rb, and is expressed on various immune cells, including monocytes/macrophages, dendritic cells (DCs), T cells, B cells, and NK cells. After acting on its receptor, IL-10 activates the STAT3 signaling pathway, inhibiting inflammatory cytokines and suppressing immune activity in monocytes/macrophages and DCs; IL-10 can promote B cell differentiation and antibody expression; however, it significantly promotes the activation of CD8 ⁺ T cells. In recent years, as the anti-tumor mechanism of IL-10 has been 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 apoptosis after activation (AICD), promote proliferation, cytokine secretion and killing activity, and exert anti-tumor activity.

However, the efficacy of drugs based on anti-VEGFR2 antibodies and IL-10 for diseases such as tumors needs further improvement. Therefore, it is still necessary to actively explore anti-VEGFR2 antibodies and IL-10 drugs with strong targeting, few side effects, and good therapeutic effects.

Summary of the Invention

This application aims to address at least one of the technical problems existing in the prior art to a certain extent. To this end, this application provides a fusion protein and its uses.

This application is based on the following discoveries of the inventors:

Wild-type IL-10 has demonstrated hematologic toxicity in both preclinical monkey toxicology experiments and clinical trials, resulting in the current lack of marketable IL-10-related drugs. Based on this, the inventors discovered that using a modified, attenuated IL-10 monomer can significantly reduce the activity of wild-type IL-10, thereby reducing toxicity. Furthermore, this application prepares a VEGFR2×IL-10M bifunctional fusion protein by combining an anti-VEGFR2 antibody and an attenuated IL-10 monomer. This fusion protein targets the tumor microenvironment via the VEGFR2 terminus, normalizing tumor angiogenesis and increasing T cell infiltration. Simultaneously, it restores the activity of overactivated and exhausted CD8 ⁺ T cells in the tumor microenvironment via the IL-10 terminus, enhancing CD8 ⁺ T cell killing activity and strengthening the anti-tumor immune response. Therefore, the VEGFR2×IL-10M bifunctional fusion protein of this application, under the targeted enrichment effect of the VEGFR2 antibody, can further reduce the peripheral toxicity of IL-10 and enhance local activity.

Therefore, in a first aspect of this application, a fusion protein is proposed. According to embodiments of this application, 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 this application has advantages such as strong targeting, few side effects, and good efficacy, especially strong tumor-killing activity.

In a second aspect of this application, a nucleic acid is provided. According to an embodiment of this application, the nucleic acid encodes the fusion protein described in the first aspect. The nucleic acid according to an embodiment of this application can encode the aforementioned fusion protein.

In a third aspect, this application provides a vector. According to an embodiment of this application, the vector comprises the nucleic acid described in the second aspect. The vector according to an embodiment of this application carries the aforementioned nucleic acid, thereby encoding the aforementioned fusion protein.

In a fourth aspect, this application provides a cell or host. According to embodiments of this application, the cell or host carries the nucleic acid described in the second aspect or the vector described in the third aspect; or the cell or host expresses the fusion protein described in the first aspect. Using this cell or host, under suitable conditions, the aforementioned fusion protein can be efficiently expressed within the cell or host.

In a fifth aspect, this application provides a pharmaceutical composition. According to embodiments of this application, the pharmaceutical composition comprises the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the carrier described in the third aspect, or the cell or host described in the fourth aspect. As is known, the fusion protein described in the first aspect, or the fusion protein prepared using the nucleic acid described in the second aspect, the carrier described in the third aspect, or the cell or host described in the fourth aspect, has advantages such as strong targeting, few side effects, and good efficacy, especially better anticancer activity and higher safety. Therefore, the obtained pharmaceutical composition can effectively prevent and/or treat tumors.

In a sixth aspect of this application, 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 the treatment or prevention of tumors is provided.

This application proposes the use of the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the vector described in the third aspect, the cell or host described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect in the treatment or prevention of tumors.

This application proposes the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the vector described in the third aspect, the cell or host described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect, for the treatment or prevention of tumors.

In a seventh aspect of this application, a method for treating or preventing tumors is proposed. According to embodiments of this application, the method includes administering to a subject a pharmaceutically acceptable dose of the fusion protein of the first aspect, the nucleic acid of the second aspect, the carrier of the third aspect, the cell or host of the fourth aspect, or the pharmaceutical composition of the fifth aspect.

The amino acid sequence listing A1 of this application is shown below:

Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application.

Attached Figure Description

The above and/or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

Figure 1 is a schematic diagram of the structure of the fusion protein in Embodiment 1 of this application;

Figure 2 shows the binding results of the fusion protein and control antibody to IL-10R1 in Example 2 of this application;

Figure 3 shows the binding results of the fusion protein and control antibody to VEGFR2 in Example 3 of this application;

Figure 4 shows the blocking activity results of the fusion protein and control antibody against VEGFR2 and VEGF165 in Example 4 of this application;

Figure 5 shows the VEGFR2 reporter gene activity results of the fusion protein and control antibody in Example 5 of this application;

Figure 6 shows the activation activity of the fusion protein and control antibody on the STAT3 signaling pathway in Example 6 of this application;

Figure 7 shows the binding activity results of the fusion protein and control antibody with exhausted CD8 ⁺ T cells in Example 7 of this application;

Figure 8 shows the results of the fusion protein and control antibody inhibiting the apoptosis activity of exhausted CD8 ⁺ T cells in Example 8 of this application;

Figure 9 shows the binding activity results of the fusion protein and control antibody with HUVEC cells in Example 9 of this application;

Figure 10 shows the results of the inhibition of HUVEC cell proliferation activity by the fusion protein and control antibody in Example 10 of this application;

Figure 11 shows the in vivo antitumor efficacy results of the fusion protein and control antibody in Example 11 of this application;

Figure 12 shows the in vivo antitumor efficacy results of the fusion protein and control antibody in Example 12 of this application.

Detailed Implementation

The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

This application details

Definitions and General Terms

The endpoints and any values of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of the various ranges, the endpoint values of the various ranges and individual point values, and individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains. Abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids (i.e., amino acids with a levorotatory conformation).

In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

In this document, the terms “optional,” “optional,” “alternatively,” “optional,” or “optional” generally refer to an event or condition that may or may not occur as described below, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code and their modified forms, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common naturally occurring amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; 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). Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. Amino acid analogs typically have modified R groups (e.g., ortholeucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimics are chemical compounds that have a structure different from the general chemical structure of amino acids, but function in a similar manner to naturally occurring amino acids.

In this article, "IL10" and "IL-10" are used interchangeably and have the same meaning. The terms "wild-type IL-10", "natural IL-10 molecule" or "natural IL-10" all refer to the dimer structure (including two natural IL-10 monomers) which have two identical amino acid sequences as shown in SEQ ID NO: 1. The natural IL-10 molecule binds to the IL-10 receptor (IL-10R), while the activity of the IL-10 monomer for IL-10Rα is 10 times weaker than that of the natural IL-10 dimer (for specific experimental data, refer to: J Biol Chem. 2000 May 5; 275(18):13552-7. doi:10.1074/jbc.275.18.13552.) and it can hardly mediate further binding with IL-10Rβ. Therefore, it is difficult to activate downstream signals to cause biological functional responses.

In this document, the term "vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into and/or between cells or hosts. The vector may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and/or translation of DNA or RNA. The vector also includes vectors having multiple of the aforementioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell or host. Typically, by culturing a suitable cell or host containing the vector, the vector can produce the desired expression product.

In this document, the term "cell" generally refers to a cell whose genetic material has been modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequence of this application and can be used for the expression and/or secretion of target proteins. Examples of suitable host cells that can be used in this application include immortalized hybridoma cells, NS/0 myeloma cells, 293 cells, Chinese hamster ovary (CHO) cells, HeLa cells, Cap cells (cells derived from human amniotic fluid), and CoS cells.

In this document, the term "pharmaceutical composition" generally refers to a composition in unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components.

In this article, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and/or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit/risk ratio.

In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, stabilizer, diluent, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients is also within the scope of consideration for this application, except for any range of incompatibilities with the fusion protein of this application, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition.

In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The fusion protein or pharmaceutical composition of this application may be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but this application is not limited to these exemplified routes of administration. Preferably, the fusion protein or pharmaceutical composition of this application is administered via intravenous or subcutaneous injection.

In this document, the term "treatment" refers to the administration of a drug to achieve a desired pharmacological and/or physiological effect. This effect may be preventative in terms of complete or partial prevention of a disease or its symptoms, and/or therapeutic in terms of partial or complete cure of a disease and/or adverse effects caused by the disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of a condition in individuals susceptible to disease but not yet diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) alleviation of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of a fusion protein or pharmaceutical composition to an individual to treat, cure, alleviate, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing the fusion protein described herein to an individual in need.

As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and/or animals and is acceptable to humans and/or animals.

Detailed description of the fusion protein and its uses in this application

This application discloses a fusion protein, nucleic acid, vector, cell or host, pharmaceutical composition and its uses, which will be described in detail below.

Fusion protein

In a first aspect, this application proposes a fusion protein. According to embodiments of this application, 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 this application has advantages such as strong targeting, few side effects, and good efficacy, especially strong tumor-killing activity.

In this article, the term "antibody" is used in the broadest sense to refer to an immunoglobulin molecule capable of specifically binding to an antigen. This antibody can include full-length monoclonal antibodies (or full-length monoclonal antibodies) or multispecific antibodies, with no specific structural restrictions, as long as they include both heavy and light chains and exhibit the desired biological activity. It typically consists of a lighter light chain and a heavier heavy chain, linked by disulfide bonds. The amino acid sequence at the amino terminus (N-terminus) of the peptide chain varies considerably and is called the variable region (V-terminus); the carboxyl terminus (C-terminus) is relatively stable and changes very little and is called the constant region (C-terminus). The V regions of the L chain and H chain are called the light chain variable region (VL) and the heavy chain variable region (VH), respectively. The C regions of the L chain and H chain are called the light chain constant region (CL or CL segment) and the heavy chain constant region (CH or CH segment), respectively. The heavy chain constant region includes at least one of the CH1 segment, the hinge region, and the Fc segment (CH2 and CH3 segments). Preferably, the heavy chain constant region includes the CH1 segment, the hinge region, the CH2 segment, and the CH3 segment sequentially from the N end to the C end.

According to embodiments of this application, the anti-VEGFR2 antibody is selected from full-length VEGFR2 monoclonal antibodies, Fab antibodies, F(ab') ₂ antibodies, Fab' antibodies, Fv antibodies, scFv antibodies, dsFv antibodies, and nanobodies.

In this article, the terms "full-length antibody," "full-length monoclonal antibody," or "full-length monoclonal antibody" refer to antibodies composed of at least two identical light chains and at least two identical heavy chains linked together, which can be linked by interchain disulfide bonds, such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), immunoglobulin D (IgD), or immunoglobulin E (IgE); they can also be linked by knot-into-hole linkages.

In this article, the term "Fab antibody" generally refers to an antibody or fragment containing only Fab molecules, which consists of the VH and CH1 of the heavy chain and the complete light chain, linked by a disulfide bond.

In this paper, the term “F(ab') ₂ antibody” has two antigen-binding F(ab') parts linked together by disulfide bonds.

In this paper, the term "Fab' antibody" includes the VH and CH1 of the heavy chain, the complete light chain, and the hinge region, with the light and heavy chains linked by a disulfide bond.

In this article, the term "Fv antibody" generally refers to an antibody or fragment consisting only of a light chain variable region (VL) and a heavy chain variable region (VH) linked by non-covalent bonds. It is the smallest functional fragment of an antibody that retains the complete antigen-binding site.

In this paper, the terms "single-chain antibody" and "scFv antibody" refer to antibodies or fragments formed by linking the variable regions of the antibody heavy chain and light chain through short peptides.

In this paper, the term "dsFv antibody" refers to a small molecule Fv antibody that achieves structural stability by introducing a cysteine mutation point on VH and VL respectively, thereby forming a disulfide bond between VH and VL.

In this article, the term "nanobody" generally refers to the heavy chain variable region (VHH) portion of a naturally occurring heavy chain antibody that lacks a light chain. Heavy chain antibodies are commonly found in camel-like animals and consist of a heavy chain variable region (VHH) and conventional CH2 and CH3 regions. They bind specifically to antigens through the heavy chain variable region (VHH), and the heavy chain variable region (VHH) alone can exert an antigen-specific binding effect.

According to an embodiment of this application, 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.

According to an embodiment of this application, the N-terminus of the IL-10 monomer is linked to the C-terminus of the anti-VEGFR2 antibody.

It should be noted that when the anti-VEGFR2 antibody is selected from antibodies composed of a single chain (e.g., Fv antibody, scFv antibody, dsFv antibody, nanobody), the N-terminus of the IL-10 monomer is linked to the C-terminus of the anti-VEGFR2 antibody. When the anti-VEGFR2 antibody is selected from antibodies composed of multiple chains (e.g., full-length VEGFR2 monoclonal antibody, Fab antibody, F(ab') ₂ antibody, Fab' antibody), the N-terminus of the IL-10 monomer is linked to the C-terminus of any one of the chains of the anti-VEGFR2 antibody.

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

It should be noted that the heavy chain in this 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 the CH1 fragment, the hinge region, and the Fc fragment (CH2 and CH3 fragments). For example, the anti-VEGFR2 antibody is selected from Fab antibodies or F(ab') ₂ antibodies, and the heavy chain is the CH1 fragment.

According to an embodiment of this application, the anti-VEGFR2 antibody is selected from Fv antibody, scFv antibody, dsFv antibody, and nanobody, and the N-terminus of the IL-10 monomer is linked to the C-terminus of the anti-VEGFR2 antibody.

In some optional embodiments of the present invention, the anti-VEGFR2 antibody is selected from Fv antibody, scFv antibody, and dsFv antibody, wherein the C-terminus of the heavy chain in the anti-VEGFR2 antibody is linked to the N-terminus of the light chain, and the N-terminus of the IL-10 monomer is linked to the C-terminus of the light chain.

In some optional embodiments of the present invention, the anti-VEGFR2 antibody is selected from Fv antibody, scFv antibody, and dsFv antibody, wherein the C-terminus of the light chain of the anti-VEGFR2 antibody is linked to the N-terminus of the heavy chain, and the N-terminus of the IL-10 monomer is linked to the C-terminus of the heavy chain.

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

In this document, the terms "complementarity-determining region," "CDR," or "CDRs" refer to highly variable regions of the heavy and light chains of an immunoglobulin, specifically regions containing the major amino acid residues responsible for binding affinity to one or more recognized antigens or epitopes. In specific embodiments of this disclosure, CDRs refer to highly variable regions of the heavy and light chains of the antibody.

In this paper, heavy chain complementarity determination regions (heavy chain variable regions CDRs) are referred to as "HCDRs" or "HCDRs", which include HCDR1, HCDR2, and HCDR3; light chain complementarity determination regions (light chain variable regions CDRs) are referred to as "LCDRs" or "LCDRs", which include LCDR1, LCDR2, and LCDR3. Commonly used CDR numbering schemes in this field include: Kabat numbering, Chothia numbering, IMGT numbering, ChothiaMartin numbering, and AHoLesk 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 in Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). For the “Chothia definition”, see Chothia et al., J Mol Biol 196:901-917 (1987). Exemplary defined CDRs are listed in Table A2 below. Given the amino acid sequence of the variable region of an antibody, those skilled in the art can routinely determine which residues contain a particular CDR.

Table A2: CDR Definition ¹

The numbers for all CDRs defined in ^Table A2 are based on the Kabat numbering system (see below).
^2. As shown in Table A2, “AbM” with a lowercase “b” refers to the CDR defined by the “AbM” antibody modeling software of Oxford Molecular.

Kabat et al. also defined a numbering system applicable to the variable region sequence of any antibody. Those skilled in the art can readily map this Kabat numbering system to any variable region sequence without relying on any experimental data outside the sequence itself. As used herein, “Kabat numbering” refers to the numbering system described in Kabat et al., U.S. Dept. of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).

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

In an optional embodiment of this application, HCDRs and LCDRs are defined using the same numbering system, such as by Kabat, Chothia, AbM, Contact, or IMGT.

According to embodiments of this application, the anti-VEGFR2 antibody includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:3, 4, and 5, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:6, 7, and 8.

It should be noted that the HCDRs and LCDRs mentioned above in this application (such as the amino acid sequences shown in SEQ ID NO:3-8) are numbered according to the Kabat numbering system. However, those skilled in the art are fully capable of converting the sequences in the sequence listing (such as the amino acid sequences shown in SEQ ID NO:3-8) into HCDRs and LCDRs numbered according to other numbering systems, all of which are within the scope of protection of this application. Specifically, this application exemplarily demonstrates HCDRs and LCDRs of ramucirumab numbered using other numbering systems. HCDRs and LCDRs numbered according to different numbering systems for the heavy chain variable regions and light chain variable regions of other monoclonal antibodies (e.g., the heavy chain variable region (amino acid sequence shown in SEQ ID NO:1) and light chain variable region (amino acid sequence shown in SEQ ID NO:2) of ramucirumab are also within the scope of protection of this application. For example, the amino acid sequences of ramucirumab's HCDRs and LCDRs (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2) are specifically shown in Table A3:

Table A3. HCDRs and LCDRs of ramucirumab under various definitions

According to an embodiment of this application, the anti-VEGFR2 antibody includes a heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:2.

According to an embodiment of this application, the anti-VEGFR2 antibody is selected from full-length VEGFR2 monoclonal antibodies, and the N-terminus of the IL-10 monomer is linked to the C-terminus of the heavy chain of the full-length VEGFR2 monoclonal antibody.

According to an embodiment of this application, the N-terminus of the IL-10 monomer is connected to the C-terminus of the heavy chain of the full-length VEGFR2 monoclonal antibody.

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

According to embodiments of this application, the full-length VEGFR2 monoclonal antibody is ramucirumab, a mutant of ramucirumab, or a chimeric form of ramucirumab.

In this document, the term "chimera" refers to an antibody obtained by splicing the CDRs of a target antibody with other frame regions or constant regions, where other frame regions and/or constant regions are frame regions and/or constant regions of other antibodies besides the frame regions and/or constant regions of the target antibody; for example, an antibody obtained by splicing the variable region of a target antibody with other constant regions, where other constant regions are constant regions of other antibodies besides the constant regions of the target antibody. For example, "ramucirumab chimera" refers to an antibody obtained by splicing the variable region of ramucirumab with other constant regions, where the constant regions are different from the constant regions of ramucirumab; wherein the constant region of ramucirumab originates from the constant region of human IgG1, and the constant region in the ramucirumab chimera is selected from constant regions other than human IgG1, for example, it may originate from the constant region of human IgG2, or it may be selected from mutants of the constant region of human IgG2, the specific source is not limited, and all are within the scope of protection of this application.

In this document, the term "variant" or "mutant" can refer to any naturally occurring or engineered molecule containing one or more nucleotide or amino acid mutations. For example, a "mutant of ramucirumab" can be a mutation of the CDRs of ramucirumab, a mutation of the FRs of ramucirumab, or a mutation of the constant region of ramucirumab. The specific mutation type is not limited and is within the scope of protection of this application.

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

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

In an optional embodiment of this application, the mutant of ramucirumab has HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:3, 4, and 5, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:6, 7, and 8.

In an optional embodiment of this application, the chimera of ramucirumab has HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:3, 4, and 5, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:6, 7, and 8.

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

According to an embodiment of this application, the N-terminus of the IL-10 monomer is linked to the C-terminus of the heavy chain of ramucirumab. This further enhances the therapeutic efficacy of the fusion protein against related diseases, particularly its tumor-killing ability.

In an optional embodiment of this application, the Fab antibody is selected from ramucirumab, and the N-terminus of the IL-10 monomer is linked to the C-terminus of the CH1 fragment of the Fab antibody.

According to embodiments of this application, at least a portion of at least one of the heavy chain constant region and the light chain constant region of the full-length VEGFR2 monoclonal antibody is derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof.

According to embodiments of this application, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or IgD.

According to embodiments of this application, the light chain constant region includes light chain constant regions selected from κ-type or λ-type.

According to embodiments of this application, the light chain constant region and the heavy chain constant region are both derived from at least one of rabbit-derived antibodies or mutants thereof, mouse-derived antibodies or mutants thereof, and human-derived antibodies or mutants thereof.

According to embodiments of this application, the heavy chain constant region is selected from the human IgG1 heavy chain constant region or a mutant thereof.

According to embodiments of this application, mutants of the human IgG1 heavy chain constant region have activity that weakens ADCC effects or activity that weakens CDC effects, compared to wild-type human IgG1 heavy chain constant region.

According to embodiments of this application, compared to the wild-type human IgG1 heavy chain constant region, the mutant of the human IgG1 heavy chain constant region has any of the following sets of mutations: 1) L234A and L235A; 2) L235A, G237A, A327Q, and K447A. All of the above mutation sites are located in the Fc fragment of the human IgG1 heavy chain constant region, and mutations at these sites can weaken the ADCC effect or the CDC effect.

In this document, the amino acid numbers of the heavy chain constant region of IgG1 or the Fc fragment of IgG1 are based on the EU numbering system. For example, position 234 refers to position 234 according to the EU numbering system; "L234A" means that leucine at position 234 according to the EU numbering system is replaced by alanine; "L235A" means that leucine at position 235 according to the EU numbering system is replaced by alanine; "G237A" means that glycine at position 237 according to the EU numbering system is replaced by alanine; "A327Q" means that alanine at position 327 according to the EU numbering system is replaced by glutamine; and "K447A" means that lysine at position 447 according to the EU numbering system is replaced by alanine.

In an optional embodiment of this application, the mutant of the human IgG1 heavy chain constant region has a knock-in-hole structure compared to the wild-type human IgG1 heavy chain constant region.

In this paper, the term "knob into hole structure" refers to the formation of a button (hole) mutation in the CH3 region of the heavy chain constant region, which facilitates heavy chain interlocking and the formation of a heterodimer. For example, this can be achieved by mutating the amino acids in the CH3 domain of the heavy chain constant region of human IgG1 (T366S, L368A, Y407V, Y349C mutation in one chain, i.e., "hole"; and T366W, S354C mutation in the other chain, i.e., "knob").

In one alternative embodiment of this application, the modified IL-10 monomer has at least two amino acids missing from its N-terminus compared to the natural IL-10 monomer, for example, two, three, four, five, six, seven, eight, nine, ten, or eleven amino acids.

According to embodiments of this application, the IL-10 monomer is selected from modified IL-10 monomers; wherein, compared with the natural IL-10 monomer, the modified IL-10 monomer has at least two amino acids missing from its N-terminus, and contains a spacer peptide between two adjacent amino acids, one of which is located at positions 115, 116, 117, 118, and 119, and the spacer peptide is a flexible linker peptide. Therefore, compared with natural IL-10, the modified IL-10 monomer of this application can reduce its activity, thereby reducing the toxicity of the fusion protein.

It should be noted that the "115th, 116th, 117th, 118th, and 119th positions" mentioned above were obtained by numbering the amino acid sequence of the natural IL-10 monomer starting from the N-terminus.

According to embodiments of this application, compared to the natural IL-10 monomer, the modified IL-10 monomer contains a spacer peptide between the 114th and 115th amino acids.

According to embodiments of this application, compared to the natural IL-10 monomer, the modified IL-10 monomer contains a spacer peptide between amino acids at positions 115 and 116.

According to embodiments of this application, compared to the natural IL-10 monomer, the modified IL-10 monomer contains a spacer peptide between amino acids at positions 116 and 117.

According to embodiments of this application, compared to the natural IL-10 monomer, the modified IL-10 monomer contains a spacer peptide between the 117th and 118th amino acids.

According to embodiments of this application, compared to the natural IL-10 monomer, the modified IL-10 monomer contains a spacer peptide between amino acids at positions 118 and 119.

In an optional embodiment of this application, the natural IL-10 monomer has the amino acid sequence shown in SEQ ID NO: 15, wherein the amino acid sequence shown in SEQ ID NO: 15 is specifically described in Table A1.

In this document, the terms "linker peptide,""linkingpolypeptide,""linker,""linker," or "connector" refer to the linking unit that connects two polypeptide fragments. Linkers are typically flexible, and their use does not cause the original function of the protein domains to be lost. Linkers can be peptide linkers containing one or more amino acids, for example, about 1-30, 2-24, or 3-15 amino acids. In some embodiments, the linker is selected from the (GxSy)z GmSn linker, where x, y, z, m, and n are independently selected from integers between 0 and 6; alternatively, x is selected from integers between 1 and 5, and y, z, m, and n are independently selected from integers between 0 and 6 (e.g., when x = 4, y = 1, z = 4, m = 1, n = 0, i.e., (G ₄ S ₁ ) ₄ G ₁ S _0, which represents the amino acid sequence: GGGGSGGGGGSGGGGGGGG). In some implementations, the connector is selected from (GxS)zG connector, where x is selected from an integer between 1 and 5, and z is selected from an integer between 1 and 6.

In this document, the term "spacer peptide" refers to a peptide that, when inserted into a specific protein, can alter the structure and/or function of the protein. In this application, any spacer peptide that can alter the structure and/or function of the native IL-10 monomer is acceptable, and its specific sequence is not limited; all are within the scope of this application. The spacer peptide of this application is distinct from the linker peptides that connect other fusion couplers, but conventional linker peptides can also serve as spacers for altering the structure and/or function of the native IL-10 monomer. In an optional example of this application, the insertion of the spacer peptide can prevent the formation of a dimer from two truncated IL-10 monomers.

According to embodiments of this application, the spacer peptide is selected from at least one of GGGSGG, (GS)n, (GGGS)n, (GSGGS)n, (GGGGS)nG, and (GGS)n, where n is any integer between 1 and 10.

According to some optional embodiments of this application, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any two values therebetween as any integer between the endpoint values.

According to embodiments of this application, the spacer peptide has an amino acid sequence as shown in SEQ ID NO:12.

According to embodiments of this application, the modified IL-10 monomer has an amino acid sequence as shown in SEQ ID NO: 13, 14 or 48.

In an optional embodiment of this application, the modified IL-10 monomer has the amino acid sequence shown in SEQ ID NO:13. It can be seen that the modified IL-10 monomer (amino acid sequence as shown in SEQ ID NO:13) compared to the natural IL-10 monomer (amino acid sequence as shown in SEQ ID NO:15) lacks the first two N-terminal amino acids SP (the first two underlined amino acids in the amino acid sequence shown in SEQ ID NO:15 in Table A1), and has a spacer peptide inserted between amino acids at positions 116 and 117 (the bolded and underlined amino acid sequence in the amino acid sequence shown in SEQ ID NO:13 in Table A1).

In an optional embodiment of this application, the modified IL-10 monomer has the amino acid sequence shown in SEQ ID NO:14. It can be seen that, compared to the natural IL-10 monomer (amino acid sequence as shown in SEQ ID NO:14), the modified IL-10 monomer lacks the three N-terminal amino acids SPG (the three underlined amino acids in the amino acid sequence shown in SEQ ID NO:15 in Table A1), and has a spacer peptide inserted between amino acids at positions 116 and 117 (the bolded and underlined amino acid sequence in the amino acid sequence shown in SEQ ID NO:14 in Table A1).

In an optional embodiment of this application, the modified IL-10 monomer has the amino acid sequence shown in SEQ ID NO:48. It can be seen that the modified IL-10 monomer (amino acid sequence as shown in SEQ ID NO:48) has a spacer peptide inserted between amino acids 116 and 117 (the bolded and underlined amino acid sequence in the amino acid sequence shown in SEQ ID NO:48 in Table A1) compared to the natural IL-10 monomer (amino acid sequence as shown in SEQ ID NO:15).

In one embodiment of this application, compared to the natural IL-10 monomer, the modified IL-10 monomer has a deletion of 2 or 3 amino acids at its N-terminus. Therefore, the fusion protein prepared using the modified IL-10 monomer can further reduce the toxicity of the fusion protein. The inventors discovered that removing the first 2 or 3 amino acids from the N-terminus of the natural IL-10 monomer improves the stability of the IL-10 monomer. Furthermore, by inserting a spacer peptide between the 116th and 117th amino acids of the IL-10 monomer, the IL-10 monomer molecule in the fusion protein becomes a closed-loop monomer molecule (the two IL10 chains of the natural IL10 dimer are interleaved; after the linker insertion modification, the IL10 monomer can independently form a domain, i.e., a closed-loop monomer molecule), and the two IL-10 monomers in the same fusion protein will not interfere with each other.

According to an embodiment of this application, the fusion protein further includes a linker peptide, through which the IL-10 monomer is linked to the full-length VEGFR2 monoclonal antibody.

According to some alternative embodiments of this application, the N-terminus of the IL-10 monomer is linked to the C-terminus of the linker peptide, and the N-terminus of the linker peptide is linked to the C-terminus of the heavy chain of the full-length VEGFR2 monoclonal antibody.

According to some alternative embodiments of this application, the N-terminus of the IL-10 monomer is linked to the C-terminus of the linker peptide, and the N-terminus of the linker peptide is linked to the C-terminus of the heavy chain of the ramucirumab.

According to embodiments of this application, the linker peptide is selected from at least one of (GGGGS)nG, (GGGGS)n, (GSGGG)n, (GS)n, (GGGS)n, (GSGGS)n, and (GGGGSG)n, where n is any integer between 1 and 10.

According to some optional embodiments of this application, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or any two values therebetween as any integer between the endpoint values.

According to embodiments of this application, the linker peptide has an amino acid sequence as shown in SEQ ID NO:16 or SEQ ID NO:17.

According to embodiments of this 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

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.

It should be noted that, based on the amino acid sequence of the fusion protein disclosed herein, those skilled in the art will readily conceive of using genetic engineering or other techniques (chemical synthesis, recombinant expression) to prepare the fusion protein, for example, by isolating and purifying the fusion protein from the culture product of recombinant cells capable of recombinantly expressing the antibodies described in any of the preceding claims. This is easily achievable by those skilled in the art. Therefore, regardless of the technique used to prepare the fusion protein disclosed herein, it falls within the scope of protection of this application.

Nucleic acid, vector, cell or host

In the process of preparing or obtaining the fusion proteins described in the first aspect, the nucleic acids expressing these fusion proteins can be linked to different vectors and then expressed in different cells to obtain the corresponding fusion proteins.

In a second aspect of this application, a nucleic acid is provided. According to an embodiment of this application, the nucleic acid encodes the fusion protein described in the first aspect. The nucleic acid according to an embodiment of this application can encode the aforementioned fusion protein.

According to embodiments of this application, the nucleic acid includes DNA or RNA.

It should be noted that, for the nucleic acids mentioned herein, those skilled in the art should understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is given in most cases herein, the other complementary strand is also disclosed. Furthermore, the molecular sequences in this application include DNA or RNA forms; disclosure of one implies that the other is also disclosed.

Those skilled in the art will understand that the features and advantages described above for the fusion protein also apply to this nucleic acid, and will not be repeated here.

In a third aspect, this application proposes a vector. According to an embodiment of this application, the vector comprises the nucleic acid described in the second aspect. When linking the nucleic acid described in the second aspect to the vector, the nucleic acid can be directly or indirectly connected to control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid. Of course, these control elements can be directly derived from the vector itself, or they can be exogenous, i.e., not derived from the vector itself. Naturally, the nucleic acid and the control elements only need to be operably linked.

In this article, "operably ligated" refers to ligating a foreign gene to a vector, enabling the control elements within the vector, such as transcriptional and translational control sequences, to perform their intended functions of regulating the transcription and translation of the foreign gene. Commonly used vectors include plasmids and bacteriophages. According to some specific embodiments of this application, after the vector is introduced into suitable recipient cells, the expression of the aforementioned fusion protein can be effectively achieved under the mediation of a regulatory system, thereby enabling the large-scale in vitro production of the fusion protein.

According to embodiments of this application, the vector may refer to a cloning vector, which can be obtained by operatively ligating the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector in this application is not particularly limited; commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

In some optional embodiments of this application, the vector is a eukaryotic expression vector, a prokaryotic expression vector, a virus, or a bacteriophage.

In some optional embodiments of this application, the expression vector is a plasmid expression vector or a lentiviral expression vector.

Those skilled in the art will understand that the features and advantages described above for fusion proteins and nucleic acids also apply to this vector, and will not be repeated here.

In a fourth aspect, this application provides a cell or host. According to embodiments of this application, the cell or host carries the nucleic acid described in the second aspect or the vector described in the third aspect; or the cell or host expresses the fusion protein described in the first aspect. Using this cell or host, under suitable conditions, the aforementioned fusion protein can be efficiently expressed within the cell or host.

According to embodiments of this application, the cells are obtained by introducing the vector described in the third aspect into a cell or host.

It should be noted that the cells or hosts used in this application are not particularly limited and can be prokaryotic cells, eukaryotic cells, or bacteriophages. The prokaryotic cells can be Escherichia coli, Bacillus subtilis, Streptomyces, or Proteus mirabilis, etc. The eukaryotic cells include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosoma, and Trichoderma; insect cells such as armyworms; plant cells such as tobacco; and mammalian cells such as BHK cells, CHO cells, COS cells, and myeloma cells.

In one optional embodiment of this application, the cells are mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, but do not include animal germ cells, fertilized eggs or embryonic stem cells.

It should be noted that the "suitable conditions" mentioned in this application refer to conditions suitable for the expression of the fusion protein described in this application. Those skilled in the art will readily understand that suitable conditions for the expression of the fusion protein include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy cell state, suitable cell density, suitable cell culture environment, and suitable cell culture time. The term "suitable conditions" is not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the fusion protein according to the specific environment of their laboratory.

Those skilled in the art will understand that the features and advantages described above for fusion proteins, nucleic acids, and vectors also apply to this cell or host, and will not be repeated here.

Pharmaceutical Composition

In a fifth aspect, this application provides a pharmaceutical composition. According to embodiments of this application, the pharmaceutical composition comprises the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the carrier described in the third aspect, or the cell or host described in the fourth aspect. As is known, the fusion protein described in the first aspect, or the fusion protein prepared using the nucleic acid described in the second aspect, the carrier described in the third aspect, or the cell or host described in the fourth aspect, has advantages such as strong targeting, few side effects, and good efficacy, especially better anticancer activity and higher safety. Therefore, the obtained pharmaceutical composition can effectively prevent and/or treat tumors.

According to embodiments of this application, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

According to embodiments of this application, the excipients include, but are not limited to, one or more pharmaceutically acceptable diluents, stabilizers, or pH adjusters.

According to embodiments of this application, the pharmaceutical composition is an injection.

It should be noted that the pharmaceutical composition includes combinations that are separate in time and/or space, as long as they can work together to achieve the purpose of this application. For example, the components contained in the composition may be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components may be administered to the subject simultaneously or sequentially.

The pharmaceutical composition of this application contains a safe and effective amount of the active ingredient of this application and pharmaceutically acceptable excipients. Such excipients include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. Generally, pharmaceutical formulations should be matched to the route of administration; the dosage forms of the pharmaceutical composition of this application are injections, oral formulations (tablets, capsules, oral liquids), transdermal formulations, and sustained-release formulations. For example, it can be prepared using physiological saline or an aqueous solution containing glucose and other excipients by conventional methods. The pharmaceutical composition is preferably manufactured under aseptic conditions.

The effective amount of the active ingredient described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

Pharmaceutically acceptable excipients described in this application include (but are not limited to): water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched to the route of administration, as is well known to those skilled in the art.

Those skilled in the art will understand that the features and advantages described above for fusion proteins, nucleic acids, vectors, and cells or hosts also apply to this pharmaceutical composition, and will not be repeated here.

use

In a sixth aspect of this application, 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 the treatment or prevention of tumors is provided.

In a seventh aspect of this application, the use of the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the vector described in the third aspect, the cell or host described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect in the treatment or prevention of tumors is proposed.

In an eighth aspect of this application, the application proposes the use of the fusion protein described in the first aspect, the nucleic acid described in the second aspect, the vector described in the third aspect, the cell or host described in the fourth aspect, or the pharmaceutical composition described in the fifth aspect, for the treatment or prevention of tumors.

The terms “treatment” and “prevention” as used herein, and words derived therefrom, do not necessarily imply 100% or complete treatment or prevention. Rather, different degrees of treatment or prevention exist, and those skilled in the art will recognize that such treatment or prevention has potential benefit or therapeutic effect. Furthermore, the treatment or prevention provided in this application may include treatment or prevention of one or more diseases, such as cancer, or symptoms of a patient. Additionally, for the purposes of this document, “prevention” may encompass delaying the onset of a disease or its symptoms or the patient's condition.

According to embodiments of this application, the uses described in the sixth, seventh, and eighth aspects above may further include at least one of the following technical features:

In some optional embodiments of this application, the tumor includes 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, and oral squamous cell carcinoma.

In some optional embodiments of this application, the tumor includes gastric cancer, lung cancer, liver cancer, breast cancer, prostate cancer, and ovarian cancer.

Those skilled in the art will understand that the features and advantages described above for fusion proteins, nucleic acids, vectors, and cells or hosts also apply to this use, and will not be repeated here.

method

In a seventh aspect of this application, a method for treating or preventing tumors is proposed. According to embodiments of this application, the method includes administering to a subject a pharmaceutically acceptable dose of the fusion protein of the first aspect, the nucleic acid of the second aspect, the carrier of the third aspect, the cell or host of the fourth aspect, or the pharmaceutical composition of the fifth aspect.

It should be noted that the terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal being evaluated for treatment and/or being treated. In one implementation, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infections, etc. Subjects can be humans, but also include other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, etc.

The effective amount of the fusion protein, nucleic acid, carrier, cell or host, or pharmaceutical composition described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

In some optional embodiments of this application, the tumor includes 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, and oral squamous cell carcinoma.

In some optional embodiments of this application, the tumor includes gastric cancer, lung cancer, liver cancer, breast cancer, prostate cancer, and ovarian cancer.

Those skilled in the art will understand that the features and advantages described above for fusion proteins, nucleic acids, vectors, cells or hosts, and pharmaceutical compositions also apply to this method, and will not be repeated here.

The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

Example 1. Construction and expression of Cyramza×IL-10M(HC)-hIgG1 and Cyramza×IL-10M(HC)-hIgG1(LALA) molecules

A plasmid for the fusion protein Cyramza×IL-10M(HC)-hIgG1, consisting of ramucirum antibody (Cyramza) and IL-10M (the amino acid sequence of IL-10M is shown in SEQ ID NO:13), was designed according to the structure shown in Figure 1. Specifically, the variable region sequence of the antibody adopted the variable region sequence of ramucirum antibody, the heavy chain constant region was the human IgG1 subtype sequence, and the light chain constant region was the human κ subtype sequence. The IL-10 monomer and the ramucirum antibody heavy chain were linked by linker peptide 1 (the amino acid sequence of which is shown in SEQ ID NO:16). The constructed plasmid was transfected into the EXPI293 cell line. After 7 days, the supernatant was collected and purified by protein A to obtain the fusion protein (or recombinant antibody), named R3207 (the amino acid sequence of its chain is shown in SEQ ID NO:19, and the amino acid sequence of its light chain is shown in SEQ ID NO:11).

To compare whether the function of Fc in the fusion protein affects the efficacy of antitumor drugs in vivo, this embodiment further mutated the human IgG1 Fc of R3207 by L234A/L235A to remove the ADCC and other functions of Fc, which is Cyramza×IL-10M(HC)-hIgG1(LALA), named R3201 (its heavy chain amino acid sequence is shown in SEQ ID NO:18, and its light chain amino acid sequence is shown in SEQ ID NO:11).

IL-10M (amino acid sequence as shown in SEQ ID NO:13) was replaced with wild-type IL-10 (amino acid sequence as shown in SEQ ID NO:15) to construct and express human IgG1 and its L234A/L235A mutants, namely Cyramza×wtIL-10(HC)-hIgG1 and Cyramza×wtIL-10(HC)-hIgG1(L234A/L235A), which were named R0628 (the amino acid sequence of its heavy chain is shown in SEQ ID NO:47 and the amino acid sequence of its light chain is shown in SEQ ID NO:11) and R4070 (the amino acid sequence of its heavy chain is shown in SEQ ID NO:45 and the amino acid sequence of its light chain is shown in SEQ ID NO:11), respectively, as positive control antibodies.

The patented molecule (CN114316064A)R0467, namely Cyramza×wtIL-10(HC)-hIgG1(K447A/A327Q/G237A/L235A), was constructed and expressed as a patented control antibody (the amino acid sequence of its heavy chain is shown in SEQ ID NO:46, and the amino acid sequence of its light chain is shown in SEQ ID NO:11), namely Cyramza×wtIL-10(HC)-hIgG1(K447A/A327Q/G237A/L235A).

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

Fc-IL10WT, named R0674, was constructed and expressed. It consists of an Fc region and an IL10 WT fused to its C-terminus. It comprises two identical peptide chains, one of which is shown in SEQ ID NO:36.

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

An hIgG1 antibody targeting hemocyanin and an hIgG1 antibody with L234A/L235A mutation were constructed and expressed as isotype control antibodies, named R0861 (the amino acid sequence of its heavy chain is shown in SEQ ID NO:39, and the amino acid sequence of its light chain is shown in SEQ ID NO:40) and R0862 (the amino acid sequence of its heavy chain is shown in SEQ ID NO:41, and the amino acid sequence of its light chain is shown in SEQ ID NO:42), respectively.

The amino acid sequences in this embodiment are shown in Table A1.

Example 2. Cell-binding activity analysis of the IL-10 terminus of the Cyramza×IL-10M fusion protein

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

First, CHO-IL-10R1 cells were counted. An appropriate amount of cells was centrifuged at 350×g for 5 minutes, and the supernatant was discarded. The cell density was adjusted to 2× ^10⁶ cells/mL with 3% BSA, and 100 μL/well of cell suspension was added to a 96-well plate. The recombinant antibody to be tested and the control antibody at a concentration of 240 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 PE-labeled goat anti-Fab antibody (Jackson ImmunoResearch, 109-116-097) 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 of PBS. Fluorescence intensity was analyzed by flow cytometry, and the experimental data were plotted. The experimental results are shown in Figure 2. Compared with R0862, R3201 and R3207 have better binding activity to IL-10R1.

Example 3. Cell binding activity analysis of the VEGFR2 terminus of the Cyramza×IL-10M fusion protein

A recombinant monoclonal cell line, 293T-hVEGFR2, was constructed by overexpressing hVEGFR2 in the 293T cell line. The binding activity of Cyramza×IL-10M fusion protein molecules R3201 and R3207, as well as control Cyramza monoclonal antibodies R2494 and R2496, to 293T-hVEGFR2 cells was detected by flow cytometry. The specific steps are as follows:

First, 293T-hVEGFR2 cells were counted. An appropriate amount of cells was centrifuged at 350×g for 5 minutes, and the supernatant was discarded. The cell density was adjusted to 2× ^10⁶ /mL with 3% BSA, and 100 μL of cell suspension was added to each well of a 96-well V plate. The recombinant antibody and control antibody at a concentration of 120 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 PE-labeled goat anti-human Fab antibody (Jackson Immuno Research, 109-116-097) 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 of PBS. Fluorescence intensity was analyzed by flow cytometry, and the experimental data were plotted. The experimental results are shown in Figure 3. The Cyramza×IL-10 fusion protein molecules R3201 and R3207 have similar binding activities to the control Cyramza monoclonal antibodies R2496 and R2494, respectively.

Example 4. Analysis of the activity of Cyramza×IL-10M fusion protein in blocking the binding of VEGFR2 and VEGF165.

Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis, with VEGF165 being a key member. VEGF165 regulates endothelial cell proliferation, migration, and tubule formation through its interaction with VEGFR2. A recombinant monoclonal cell line, 293T-hVEGFR2, was constructed by overexpressing hVEGFR2 in the 293T cell line. Flow cytometry was used to detect the ability of Cyramza×IL-10M fusion protein molecules R3201 and R3207, as well as the control Cyramza monoclonal antibodies R2494 and R2496, to block the binding of VEGF165-his to 293T-hVEGFR2 cells. The specific steps are as follows:

First, count the 293T-hVEGFR2 cells. Centrifuge an appropriate amount of cells at 350×g for 5 minutes and discard the supernatant. Adjust the cell density to 2× ^10⁶ /mL with 3% BSA, and add 100 μL of cell suspension to each well of a 96-well V plate. Prepare the 240 nM recombinant antibody and control antibody, and perform 3-fold serial dilutions. Add 50 μL of the diluted antibody to the cells and incubate at 4°C for 30 minutes. Prepare an 8 μg/mL VEGF165-his (Acro, VE5-H5248) protein dilution with 3% BSA and add 50 μL of the solution to each well of the cells. Incubate at 4°C for 30 minutes, centrifuge at 350×g for 5 minutes, and discard the supernatant. Wash once with 3% BSA, resuspend the cells in 100 μL of APC-labeled anti-his tag antibody (Biolegend, 362605) diluted 1:300, and incubate at 4°C for 30 minutes. After washing once with 3% BSA, cells were resuspended in 150 μL/well PBS. Fluorescence intensity was analyzed by flow cytometry, and experimental data were plotted. The experimental results are shown in Figure 4. The Cyramza×IL-10M fusion protein molecules R3201 and R3207 have similar blocking activities to the control Cyramza monoclonal antibodies R2496 and R2494, respectively.

Example 5. Activity analysis of the VEGFR 2-terminal reporter gene of Cyramza×IL-10M fusion protein

A reporter gene monoclonal cell line, 293T-hVEGFR2-Luc, was constructed by transfecting luciferase reporter gene systems into 293T-hVEGFR2 cells. The activity of Cyramza×IL-10M fusion protein molecules R3201 and R3207, as well as the control Cyramza monoclonal antibodies R2494 and R2496, in blocking the VEGF165-activated VEGFR2 signaling pathway was detected by chemiluminescence immunoassay. The specific steps are as follows:

Prepare the recombinant antibody and control antibody at a concentration of 240 nM and perform 3-fold serial dilutions. Dilute the ligand VEGF165-his (Acro, VE5-H5248) to 54.8 ng/mL, and add 25 μL of antibody dilution and 25 μL of ligand dilution to each well of a 96-well white plate. Take well-cultured 293T-hVEGFR2-Luc cells, digest them, centrifuge and discard the supernatant, and adjust the cell count to 4 × ^10⁶ /mL using DMEM + 10% FBS medium. Add 50 μL of the cell suspension to the antibody and ligand mixture to each well, and incubate the cell culture plate at 37°C in a 5% _CO₂ incubator for 6 h. Remove the cell culture plate and allow it to equilibrate to room temperature for 5-10 minutes. Add 100 μL of pre-thawed firefly luciferase assay reagent (Beyotime, RG051M) to each well. Incubate at room temperature for 5-10 minutes to stabilize the luminescence signal, and then detect the chemiluminescence signal using an ELISA reader. The analysis data is shown in Figure 5. The Cyramza×IL-10M fusion protein molecules R3201 and R3207 have similar VEGFR2 reporter gene activities to the control Cyramza monoclonal antibodies R2496 and R2494, respectively.

Example 6. Analysis of the activation activity of Cyramza×IL-10M fusion protein on the STAT3 signaling pathway

STAT3 is an important downstream signaling pathway of IL-10. IL-10 plays a powerful role in promoting cytotoxicity, preventing apoptosis, and enhancing the adaptability of CD8+ T cells through STAT3 signaling. This study analyzed the activation capacity of modified IL-10 on the STAT3 signaling pathway under antigen enrichment and non-enrichment conditions using a STAT3 signaling pathway activation assay. The 293-IL-10R-STAT3-Luc reporter gene cell line used in the experiment was purchased from Jimon Biotechnology. The specific steps are as follows:

The previous afternoon, hVEGFR2-hFc Tag protein was diluted to 2 μg/mL with DPBS. 100 μL/well was added to each well of a 96-well whole leukocyte culture plate for the protein enrichment group, and 100 μL of DPBS was added to the non-enriched group as a control. The culture plates were incubated overnight at 4°C. The next morning, antibodies were prepared using DMEM + 10% FBS to a working solution of 120 nM, and then serially diluted 3-fold to 8 concentration points. The coated cell plates were removed, the coating solution was aspirated with a multipipe, and 50 μL/well of the prepared antibody working solution was added to each well. The plates were pre-incubated with the antigen at 37°C, 5% _CO2 for 30 minutes.

293-IL-10R-STAT3-Luc cells with good growth and confluence (approximately 80-90%) were digested into single cells with 0.25% trypsin, centrifuged at 300×g for 5 minutes, and the supernatant was discarded. The cell density was then adjusted to 1× ^10⁶ /mL, and 50 μL was added per well to each well of a pre-incubated 96-well white flat-bottomed plate (5× ^10⁴ cells per well). The cells were incubated at 37℃ in a 5% _CO₂ incubator for 6 hours. After removing the cell culture plate from the incubator, it was allowed to equilibrate to room temperature for 5-10 minutes. 100 μL of pre-thawed luciferase reporter gene assay reagent (Beyotime) was added to each well, and the plate was incubated in the dark for 10-15 minutes. Fluorescence values were detected using an Ensight microplate reader. Data were plotted and analyzed using GraphPad Prism 8 software. A nonlinear fitting was performed with antibody concentration as the x-axis and chemiluminescence value as the y-axis to calculate the _EC⁵⁰ value. The results are shown in Figure 6. The modified IL-10 (R3201, R3207, R3202, and R3208) showed significantly lower activity in activating the STAT3 signaling pathway compared to the wild type. In the presence of antigen enrichment, the reporter gene activity of R3201 and R3207 was significantly enhanced compared to the IL-10M isotype control molecules (R3202 and R3208).

Example 7. Analysis of the binding activity of Cyramza×IL-10M fusion protein with exhausted CD8 ⁺ T cells

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

CD8 ⁺ T cells were isolated from peripheral blood of healthy individuals using human CD8 magnetic beads (StemCell, 17953). The CD8 ⁺ T cells were resuspended at a density of 1× ^10⁶ /mL using RPMI 1640 (containing 10% FBS and 100 U/mL IL2). CD3/CD28 activating magnetic beads (Tongli Haiyuan) were added at a 1:1 cell ratio, gently mixed, and seeded into 10 cm cell culture dishes. The cells were incubated at 37°C in a 5% _CO₂ incubator for 72 hours, followed by culture replenishment for another 48 hours.

After induction, CD8 ⁺ T cells were counted, and an appropriate amount of cells were centrifuged at 350×g for 5 minutes, discarding the supernatant. The cell density was adjusted to 2× ^10⁶ cells/mL with 3% BSA, and 100 μL/well of cell suspension was added to a 96-well V plate. The recombinant antibody to be tested and the control antibody at a concentration of 400 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 1:600 and incubated at 4°C for 30 minutes. After washing once with 3% BSA, the cells were resuspended in 150 μL/well of PBS, and the fluorescence intensity was analyzed by flow cytometry, and the experimental data were plotted. The experimental results are shown in Figure 7. Compared with the control Cyramza monoclonal antibodies R2496 and R2494 and the isotype controls R0862 and R0861, R3201 and R3207 have better binding activity.

Example 8. Analysis of Cyramza×IL-10M fusion protein's inhibitory activity against apoptosis in exhausted CD8 ⁺ T cells

To evaluate the activity of IL-10 in inhibiting CD8 ⁺ T cell apoptosis and reversing exhausted CD8 ⁺ T cells, CD8 ⁺ T cells were sorted from peripheral blood of healthy individuals using human CD8 magnetic beads (StemCell, 17953). CD8 ⁺ T cells were resuspended at a density of 1× ^10⁶ /mL in RPMI 1640 (containing 10% FBS and 100 U/mL IL2). CD3/CD28 activating magnetic beads (Tongli Haiyuan) were added at a 1:1 cell ratio, gently mixed, and seeded into 10 cm cell culture dishes. The cells were incubated at 37°C in a 5% _CO₂ incubator for 72 hours, followed by culture replenishment for another 48 hours. One day before cell plating, hVEGFR2-his protein (Sino Biological, 10012-H08H) was diluted to 4 μg/mL with DPBS, and 100 μL was added to each well of a 96-well plate and incubated overnight at 4°C.

The antibody was diluted to a working solution of 200 nM using RPMI 1640 medium (IL-2-free) and serially diluted 5-fold to three concentration points. The coating solution was discarded using a pipette, and the coated plate was washed once with DPBS. The prepared antibody was added at 100 μL/well, and the plate was incubated at 37°C for 1 hour. Cells were collected from the culture dishes, mixed thoroughly by pipetting, and aliquoted into 15 ml centrifuge tubes. The tubes were placed on a magnetic rack for approximately 10 minutes to remove the old magnetic beads. After centrifugation, the old medium was discarded, and the cells were resuspended and counted. The resuspended medium was RPMI 1640 medium (IL-2-free), with fresh CD3/CD28 activation beads added. The cells were gently mixed by pipetting and seeded at 1× ^10⁵ cells/well, 100 μL, into 96-well flat-bottom cell plates and cultured for 4 days.

The cells to be tested were resuspended and transferred to 96-well V-bottom cell plates. After centrifugation, the supernatant was discarded. The cells were washed once with Annexin-V binding buffer, and staining solution was prepared with Annexin-V binding buffer (1 μL of APC annexin-V and 1 μL of Propidium Iodide Solution were added to each well). The solution was added at a rate of 100 μL/well (mixed gently). After incubation at 4°C in the dark for 15 minutes, the cells were centrifuged and the supernatant was discarded. Cell apoptosis was detected by flow cytometry. The data of double positivity of APC and PE fluorescence channels were analyzed. The results are shown in Figure 8. R3201 and R3207 showed significant inhibitory activity against apoptosis of exhausted CD8 ⁺ T cells.

Example 9. Analysis of the binding activity of Cyramza×IL-10M fusion protein to HUVEC cells

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

Primary HUVEC cells were counted, and an appropriate amount of cells was centrifuged at 350×g for 5 minutes, discarding the supernatant. The cell density was adjusted to 2× ^10⁶ cells/mL with 3% BSA, and 100 μL/well of cell suspension was added 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 of PBS, and the fluorescence intensity was analyzed by flow cytometry, and the experimental data were plotted. The experimental results are shown in Figure 9. The Cyramza×IL-10M fusion protein molecules R3201 and R3207 have similar binding activities to the control Cyramza monoclonal antibodies R2496 and R2494, respectively.

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

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 and filtered through a 40 μm cell filter before cell counting. The cell density was adjusted to 4 × ^10⁴ /mL, and 100 μL was added to each well of a 96-well plate. The cells were starved overnight at 37°C in a 5% _CO₂ incubator. Unused wells were supplemented with 200 μL of DPBS. The next day, the antibody was diluted to 800 nM in HUVEC basal medium containing 20% FBS and added to each well at 50 μL. The plates were incubated at 37°C for 30 minutes. The concentration of VEGF165 (Acro, VE5-H5248) was adjusted to 200 ng/mL using HUVEC basal medium containing 20% FBS. 50 μL of this medium was added to each well of a 96-well plate. Wells without VEGF165 were supplemented with 50 μL of basal medium containing 20% FBS. The plates were then incubated statically for 4 days. The 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.

Example 11. In vivo antitumor efficacy analysis of Cyramza×IL-10M(HC)-hIgG1 and Cyramza-hIgG1 molecules

To verify the in vivo antitumor activity of R3207, it was validated in a B16F1-KDR humanized mouse tumor model. The specific experimental steps are as follows:

B16F1 was inoculated into hKDR humanized mice at a density of 2 × ^10⁵ cells/mouse to construct a tumor model (D0). Recombinant antibodies R2496, R3207, and the negative control antibody R0861 were administered intravenously at D0/D2/D4, respectively. Equimolar concentrations were used across groups, with R2496 and R3207 doses of 8.2 mg/kg and 10.5 mg/kg, and R0861 dose of 8.3 mg/kg. Tumor volume was measured twice weekly and calculated as tumor volume = tumor long axis * tumor short ^axis² / 2. The experimental results are shown in Figure 11 and Table 1. R3207 exhibited significant antitumor activity in vivo, and its efficacy was significantly superior to that of R2496. Therefore, this further demonstrates that the Cyramza×IL-10M fusion protein molecule R3207 has significantly better antitumor activity than the control monoclonal antibody R2496.

Table 1

*Comparison of mean TV in each treatment group with R0861 group; One-way ANONVA, Dunnett's Multiple Comparison Test

Example 12. In vivo antitumor efficacy analysis of Cyramza×IL-10M(HC)-hIgG1 and Cyramza×IL-10M(HC)-hIgG1 (L234A/L235A) molecules.

To further compare the in vivo antitumor activities of R3207 and R3201, a pharmacodynamic comparison experiment was conducted in a B16F1-KDR humanized mouse tumor model. The specific experimental steps are as follows:

B16F1 was inoculated into hKDR humanized mice at a density of 2 × ^10⁵ cells/mouse to construct a tumor model (D0). Recombinant antibodies R2496, R3207, R3201, and the negative control antibody R0862 were administered intravenously at D0/D2/D4, respectively. Equimolar concentrations were used across groups, with R2496 and R3207/R3201 administered at doses of 8.2 mg/kg and 10.5 mg/kg, respectively, and R0861 at a dose of 8.3 mg/kg. Tumor volume was measured twice weekly and calculated as tumor volume = tumor long axis * tumor short axis²/ ² . The experimental results are shown in Figure 12 and Table 2. R3201 and R3207 showed significant antitumor activity in vivo, with significantly better efficacy than R2496.

Table 2

*Comparison of mean TV in each treatment group with R0862 group; One-way ANONVA, Dunnett's Multiple Comparison Test

Example 13: Early safety assessment of the anti-Cyramza×IL-10M fusion protein

Experiment 1: This experiment aimed to administer R0674 intravenously to cynomolgus monkeys, observe the toxic reactions caused by R0674, and study its toxicokinetics to provide a reference for subsequent experimental design. During the experiment, the following observations were performed on the animals: death and near-death experiences, clinical observation, body weight, food intake, body temperature, hematological and coagulation parameters, serum biochemical parameters, and immune function. After euthanasia, gross dissection was performed, and major organs (adrenal glands, brain, epididymis, heart, kidneys, liver, spleen, testes, thymus, thyroid gland, and parathyroid glands) were weighed. Histopathological examinations were performed on the liver, spleen, and kidneys.

The results showed that during the experiment, no significant abnormalities were found in animal mortality and near-death experiences, clinical observation, body weight, food intake, body temperature, coagulation, immune function tests, or organ weight. However, the experimental group (R0674) exhibited hematological toxicity, with significantly abnormal serum biochemical indicators.

Experiment 2: This experiment aimed to observe the toxic reactions and pharmacokinetic characteristics of cynomolgus monkeys after intravenous injection of the anti-Cyramza-IL10M fusion protein R3201 at different doses, providing a reference for subsequent experimental design. During the experiment, the following observations were performed on the animals: death and near-death experiences, clinical observation, body weight, body temperature, hematological and coagulation parameters, serum biochemical parameters, and immune function.

The results showed that during the experiment, no significant abnormalities were found in animal mortality and near-death experiences, clinical observation, body weight, body temperature, coagulation, or immune function tests. No significant abnormalities were also observed in hematology, serum biochemistry, lymphocyte subtypes, or cytokines.

In conclusion, the anti-Cyramza-IL10M fusion protein R3201 demonstrated superior safety in cynomolgus monkey experiments.

In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims (14)

A fusion protein, characterized in that it comprises an anti-VEGFR2 antibody and an IL-10 monomer; wherein the IL-10 monomer is linked to the anti-VEGFR2 antibody. According to claim 1, the fusion protein is characterized in that the anti-VEGFR2 antibody is selected from VEGFR2 full-length monoclonal antibody, Fab antibody, F(ab') ₂ antibody, Fab' antibody, Fv antibody, scFv antibody, dsFv antibody, and 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 full-length VEGFR2 monoclonal antibody, Fab antibody, F(ab') ₂ antibody, and Fab' antibody, wherein 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 Fv antibody, scFv antibody, dsFv antibody, and nanobody, and the N-terminus of the IL-10 monomer is linked to the C-terminus of the anti-VEGFR2 antibody; Optionally, the anti-VEGFR2 antibody includes HCDRs and LCDRs, wherein the HCDRs include HCDRs defined in the heavy chain variable region as shown in SEQ ID NO:1, and the LCDRs include LCDRs defined in the light chain variable region as shown in SEQ ID NO:1. Optionally, the HCDRs and/or LCDRs are defined by Kabat, Chothia, AbM, Contact, or IMGT; Optionally, the anti-VEGFR2 antibody includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO:3, 4, and 5, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO:6, 7, and 8; Optionally, the anti-VEGFR2 antibody includes a heavy chain variable region as shown in SEQ ID NO:1 and a light chain variable region as shown in SEQ ID NO:2; Optionally, the anti-VEGFR2 antibody is selected from full-length VEGFR2 monoclonal antibodies, and the N-terminus of the IL-10 monomer is linked to the C-terminus of the heavy chain of the full-length VEGFR2 monoclonal antibody; Optionally, the heavy chain variable region of the full-length VEGFR2 monoclonal antibody has the amino acid sequence shown in SEQ ID NO:1, and the light chain variable region of the full-length VEGFR2 monoclonal antibody has the amino acid sequence shown in SEQ ID NO:2; Optionally, the full-length VEGFR2 monoclonal antibody is ramucirumab, a mutant of ramucirumab, or a chimera of ramucirumab; Optionally, the mutant or chimeric form of ramucirumab has HCDRs defined in the heavy chain variable region as shown in SEQ ID NO:1 and LCDRs defined in the light chain variable region as shown in SEQ ID NO:1. Optionally, the mutant or chimeric form of ramucirumab has HCDR1, HCDR2, HCDR3 as shown in SEQ ID NO:3, 4 and 5, and LCDR1, LCDR2, LCDR3 as shown in SEQ ID NO:6, 7 and 8. Optionally, the full-length VEGFR2 monoclonal antibody has a heavy chain variable region as shown in SEQ ID NO:9 or 10 and 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 full-length VEGFR2 monoclonal antibody is derived from at least one of mouse antibodies, primate antibodies, bovine antibodies, equine antibodies, dairy bovine antibodies, porcine antibodies, sheep antibodies, goat antibodies, canine antibodies, feline antibodies, rabbit antibodies, camel antibodies, donkey antibodies, deer antibodies, mink antibodies, chicken antibodies, duck antibodies, goose antibodies, turkey antibodies, fighting rooster antibodies, or mutants thereof; Optionally, the heavy chain constant region includes a heavy chain constant region selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; Optionally, the light chain constant region includes a light chain constant region selected from κ-type or λ-type; Optionally, the light chain constant region and the heavy chain constant region are both derived from at least one of rabbit antibody or its mutant, mouse antibody or its mutant, and human antibody or its mutant; Optionally, the heavy chain constant region is selected from the human IgG1 heavy chain constant region or a mutant thereof; Optionally, compared with the wild-type human IgG1 heavy chain constant region, the mutant of the human IgG1 heavy chain constant region has activity that weakens ADCC effect or activity that weakens CDC effect; Optionally, compared with the wild-type human IgG1 heavy chain constant region, the mutant of the human IgG1 heavy chain constant region has any of the following sets of mutations: 1) L234A and L235A; 2) L235A, G237A, A327Q and K447A. The fusion protein according to any one of claims 1-2 is characterized in that the IL-10 monomer is selected from a modified IL-10 monomer; Optionally, compared with the natural IL-10 monomer, the modified IL-10 monomer has at least 2 amino acids missing at its N-terminus, preferably 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acids missing. Optionally, compared to the natural IL-10 monomer, the modified IL-10 monomer contains a spacer peptide between two adjacent amino acids; Optionally, one of the two adjacent amino acids is located at position 115, 116, 117, 118, or 119, and the spacer peptide is a flexible linker peptide. Optionally, compared with the natural IL-10 monomer, the modified IL-10 monomer has at least two amino acids missing from its N-terminus, and contains a spacer peptide between two adjacent amino acids, wherein one of the two adjacent amino acids is located at position 115, 116, 117, 118, or 119, and the spacer peptide is a flexible linker peptide. Optionally, compared with the natural IL-10 monomer, the modified IL-10 monomer has two amino acids missing from its N-terminus and contains a spacer peptide between amino acids at positions 116 and 117. Optionally, the spacer peptide is selected from at least one of GGGSGG, (GS)n, (GGGS)n, (GSGGS)n, (GGGGS)nG, and (GGS)n, where 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 modified IL-10 monomer has an amino acid sequence as shown in SEQ ID NO: 13, 14 or 48. The fusion protein according to any one of claims 1-3 is characterized in that it further comprises a linker peptide, wherein the IL-10 monomer is linked to the full-length VEGFR2 monoclonal antibody via 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, and (GGGGSG)n, where 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. The fusion protein according to any one of claims 1-4 is 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. A nucleic acid, characterized in that the nucleic acid encodes the fusion protein according to any one of claims 1 to 5. A vector, characterized in that the vector comprises the nucleic acid as described in claim 6. 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 according to any one of claims 1 to 5. A pharmaceutical composition, characterized in that it comprises the fusion protein according to any one of claims 1 to 5, the nucleic acid according to claim 6, the vector according to claim 7, or the cell or host according to claim 8; Optionally, pharmaceutically acceptable excipients may be further included. Use of the fusion protein of any one of claims 1 to 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 preparation of a medicament for the treatment or prevention of tumors. Use of the fusion protein according to any one of claims 1 to 5, the nucleic acid according to claim 6, the vector according to claim 7, the cell or host according to claim 8, or the pharmaceutical composition according to claim 9 in the treatment or prevention of tumors. The fusion protein according to any one of claims 1 to 5, the nucleic acid according to claim 6, the vector according to claim 7, the cell or host according to claim 8, or the pharmaceutical composition according to claim 9, are used for the treatment or prevention of tumors. A method for treating or preventing tumors, characterized in that it comprises administering to a subject a pharmaceutically acceptable dose of the fusion protein of any one of claims 1 to 5, the nucleic acid of claim 6, the carrier of claim 7, the cell or host of claim 8, or the pharmaceutical composition of claim 9. The method according to any one of claims 10 to 12 or claim 13 is characterized in that the tumor is selected from at least one of gastric cancer, 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, and oral squamous cell carcinoma; Optionally, the tumor is selected from gastric cancer, lung cancer, liver cancer, breast cancer, prostate cancer, and ovarian cancer.