TGF-β RII mutants and their fusion proteins

Abstract

In view of the technical problem that TGF-β RII and its fusion proteins are prone to degradation and cleavage during recombinant expression, a TGF-β RII mutant and its fusion protein are provided. Compared with the extracellular domain of wild-type TGF-β RII of SEQ ID NO: 6, the TGF-β RII mutant has mutations selected from Gln at position 6, Asp at position 12, and Gly at position 20. The TGF-β RII mutant can bind to TGF-β. Compared with wild-type TGF-β RII, the TGF-β RII mutant is less susceptible to cleavage and / or degradation during recombinant expression. This is advantageous for the mass production of antibody / TGF-β RII bifunctional proteins with more stable quality.

Description

[Technical Field]

[0001] This application claims the benefit of priority from Chinese Patent Application No. 202010721371.4, filed on July 24, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to the field of biopharmaceuticals, particularly to therapeutic protein drugs for tumors. In particular, the present invention relates to TGF-β RII mutants and fusion proteins, and their uses. [Background technology]

[0003] Programmed death factor ligand 1 (PD-L1), also known as cluster of differentiation 274 (CD274) or B7 homologous protein 1 (B7-H1), is a member of the B7 family and is encoded by the CD274 gene. The mature PD-L1 protein is a 40 kDa, 272-amino acid type I transmembrane protein that is inducibly expressed on the surface of activated T cells, B cells, dendritic cells, macrophages, mesenchymal stem cells, bone marrow-derived mast cells, and non-hematopoietic cells. It is also widely expressed in tumor tissues, such as lung cancer, liver cancer, and bladder cancer. PD-L1 expression can be rapidly upregulated in tumor tissues and other tissues in response to stimulation by interferon and other inflammatory factors. The receptor for PD-L1 is programmed death protein 1 (PD-1). PD-1, also known as CD279, is a member of the CD28 family of T cell receptors and is expressed on the surface of various immune cells, such as activated T cells, B cells, and monocytes. Binding of PD-L1 to its receptor PD-1 can induce apoptosis, anergy, and exhaustion of T cells, subsequently inhibiting the activation, proliferation, and antitumor function of tumor antigen-specific T cells, leading to tumor immune evasion. PD-1 / PD-L1-blocking antibodies can alleviate the immunosuppressive effects of PD-L1 and enhance the recognition and killing of tumor cells by in vivo immune cells, such as T cells, thereby achieving tumor-killing effects. To date, several antibody drugs targeting PD-1 / PD-L1 have been marketed worldwide and have shown good clinical therapeutic effects against various tumors, including melanoma, lung cancer, renal cancer, Hodgkin's lymphoma, head and neck squamous cell carcinoma, and urothelial carcinoma. However, therapeutic PD-1 / PD-L1 antibodies still have several problems, the main one being the low efficacy rate when used alone in clinical trials. For most cancers, therapeutic PD-1 / PD-L1 antibodies, when used alone and indiscriminately, achieve an average efficacy rate of only 10% to 20%. Therefore, it is desirable to develop more effective antibody molecules to meet clinical needs.

[0004] Transforming growth factor-β (TGF-β) is a member of the TGF-β superfamily, whose primary function is to regulate cell growth and differentiation. TGF-β possesses high-affinity receptors (TGF-βR) on the cell surface, which are divided into three subtypes: TGF-βRI, TGF-βRII, and TGF-βRIII. TGF-β binds to TGF-βRII, which has serine / threonine protein kinase activity, on the cell membrane to form a complex with TGF-βRII. This complex then transduces signals, activating corresponding signaling pathways and regulating cell proliferation, differentiation, and apoptosis. In the tumor microenvironment, TGF-β regulates the development of regulatory T cells (Tregs), inhibits DC maturation, inhibits IgA production by B cells, and inhibits NK cell activation, thereby causing immunosuppression and promoting tumor cell growth and metastasis. Blocking the binding of TGF-β to its main receptor, TGF-β RII, can inhibit the tumor-promoting activity of TGF-β, and therefore the development of TGF-β blockers is an important direction for tumor therapy.

[0005] Currently, numerous small molecule inhibitors and large protein drugs targeting the TGF-β pathway are under clinical research, and several clinical trials are underway regarding their combination with PD-1 / PD-L1 antibody drugs, and the mechanisms of combination have been thoroughly investigated as reported in the literature (Non-Patent Document 1, Non-Patent Document 2). Therefore, the development of therapies or drugs capable of blocking both the PD-1 / PD-L1 pathway and the TGF-β / TGF-β-R pathway is expected to further resolve the problem of immunosuppression in the tumor microenvironment.

[0006] Based on the combination of TGF-β blockers and PD-1 / PD-L1 inhibitors, drugs that simultaneously block both the PD-1 / PD-L1 pathway and the TGF-β / TGF-β-R pathway are being developed. Clinical data on M7824, a bifunctional fusion protein formed by Merck & GSK's PD-L1 antibody and TGF-β RII, has shown that it has a promising clinical therapeutic effect on various solid tumors (NCT02699515, NCT02517398, and NCT03427411). Merck has filed numerous patent applications for the structure of M7824. See Patent Document 1. Several bifunctional molecules with similar structures, such as SHR-1701 developed by Hengrui Medicine, have entered clinical trials one after another (CTR20182404 / CTR20181823). Currently, many antibody / TGF-β RII fusion proteins are disclosed in patent documents such as Patent Documents 2, 3, 4, 5, and 1. However, some fusion proteins have the problem of instability. For practical production and application, there is still a need to develop more stable and highly expressed antibody / TGF-β R fusion proteins.

[0007] Although M7824 has a good antitumor effect, it suffers from the problem of being easily cleaved by TGF-β RII during production, which poses some difficulties in processing and quality control during production. To solve this problem, Hengrui Medicine has designed a truncated form of TGF-β RII. Research shown in Patent Document 6 found that a truncated form containing a deletion of the first 26 amino acids at the N-terminus of TGF-β RII, particularly amino acids 14 to 21, maintains the physiological function of TGF-β RII and is more stable. However, because the TGF-β RII contained in an antibody / TGF-β RII fusion protein is smaller than an antibody, the function of TGF-β RII in the fusion protein is susceptible to interference by the antibody molecule, even if it is shielded by the antibody molecule. This problem can only be alleviated to a certain extent by using a flexible linker; cleavage at the N-terminus of TGF-β RII further exacerbates the difference in molecular size between the two functional moieties of the antibody / TGF-β RII fusion protein molecule. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] International Publication No. 2015118175 [Patent Document 2] International Publication No. 2006074451 [Patent Document 3] International Publication No. 2009152610 [Patent Document 4] International Publication No. 9309228 [Patent Document 5] International Publication No. 9409815 [Patent Document 6] International Publication No. 2018 / 205985 [Non-patent literature]

[0009] [Non-Patent Document 1] Nature, 2018 Feb 22. doi: 10.1038 / nature 25492 [Non-patent document 2] Nature, 2018 Feb 22. doi: 10.1038 / nature 25501 Summary of the Invention [Problem to be solved by the invention]

[0010] The technical problem that the present invention aims to solve is that antibody / TGF-β RII fusion proteins in the prior art are prone to cleavage and degradation during production, and the difference in molecular size between the two functional parts of an antibody / TGF-β RII fusion protein is further exacerbated when an N-terminally truncated TGF-β RII is used.

[0011] Through research, the inventors have discovered that the main reason for the instability of antibody / TGF-β RII fusion proteins is as follows: after the C-terminus of the antibody is linked to TGF-β RII via a linker peptide, cleavage occurs between the antibody and TGF-β RII, resulting in an incomplete structure of the fusion protein, which may affect the purity and uniformity of the quality of the fusion protein and ultimately raise concerns about the safety and efficacy of the drug. Based on this finding, the present specification provides a technical solution for making mutational modifications to TGF-β RII, which overcomes the disadvantage of antibody / TGF-β RII fusion proteins being prone to cleavage and degradation, balances the molecular sizes of the two functional moieties, and provides antibody / TGF-β RII fusion proteins with more stable quality and are more convenient for mass production. Specifically, the technical solution of the present invention is presented as follows: [Means for solving the problem]

[0012] In one aspect, the present invention provides a TGF-β RII mutant, characterized in that it comprises a mutation at one or more amino acid residue positions selected from the group consisting of positions 6, 12 and 20 relative to wild-type TGF-β RII, wherein the numbering of the amino acid residues in wild-type TGF-β RII refers to SEQ ID NO: 6.

[0013] Furthermore, the TGF-β RII mutant according to the present invention is characterized in that it comprises one or more mutations selected from the group consisting of Q6N, D12T and G20T compared to wild-type TGF-β RII.

[0014] Furthermore, the TGF-β RII mutant according to the present invention is characterized in that it is capable of binding to TGF-β, where TGF-β includes TGF-β1, TGF-β2 and TGF-β3.

[0015] Furthermore, the TGF-β RII mutants according to the invention are characterized in that they have less cleavage and / or degradation when recombinantly expressed compared to wild-type TGF-β RII.

[0016] In a second aspect, the present invention provides a fusion protein comprising two or more functional fragments, characterised in that at least one of the functional fragments has the amino acid sequence of a TGF-β RII mutant according to the first aspect of the invention.

[0017] Furthermore, the fusion protein according to the invention is characterized in that the two or more functional fragments function independently of each other.

[0018] Optionally, the functional fragments are linked to each other via a polypeptide linker (linker).

[0019] Furthermore, the fusion protein according to the present invention is characterized in that the functional fragment further comprises an antibody or an antigen-binding portion thereof, a receptor or a ligand-binding portion thereof, a cytokine or a fragment thereof, a cytotoxin or a mutant thereof, a label or a tracer, etc.

[0020] Furthermore, the fusion protein according to the present invention is characterized in that the functional fragment specifically binds to a target selected from the group consisting of a target for tumor or cancer immunotherapy, a target for chronic infectious disease immunotherapy, and a target for autoimmune disease therapy.

[0021] Furthermore, the fusion proteins of the present invention are characterized in that the targets include epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), insulin receptor (InsR), Bruton's tyrosine kinase (BTK), HER2, CTLA, CD20, CD52, CD30, CD33, CD133, PD-1, PD-L1, Src, Abl, phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB / Akt), target of rapamycin (mTOR), serine-threonine protein kinase Ras, mitogen-activated protein kinase (MAPK), STAT1, STAT3, STAT5, etc.

[0022] In a third aspect, the present invention provides a multifunctionally active molecule having two or more functional activities, characterised in that at least one of the functional activities is TGF-β binding activity conferred by a polypeptide fragment having the amino acid sequence of a TGF-β RII mutant according to the first aspect of the invention.

[0023] Furthermore, the multifunctionally active molecule according to the present invention is characterized in that the functional activity further comprises antigen-binding activity, ligand-binding activity, cytokine activity, cytotoxicity or labeling activity.

[0024] Furthermore, the multifunctionally active molecule according to the present invention is characterized in that its functional activities further include binding activity to the following molecules: epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), insulin receptor (InsR), Bruton's tyrosine kinase (BTK), HER2, CTLA, CD20, CD52, CD30, CD33, CD133, PD-1, PD-L1, Src, Abl, phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB / Akt), target of rapamycin (mTOR), serine-threonine protein kinase Ras, mitogen-activated protein kinase (MAPK), STAT1, STAT3, STAT5, etc.

[0025] In a fourth aspect, the present invention provides an antibody-TGF-β RII conjugate molecule, characterised in that the antibody targets a tumor therapy target and the TGF-β RII has the amino acid sequence of a TGF-β RII mutant according to the first aspect of the invention.

[0026] Furthermore, the antibody-TGF-β RII conjugate molecule according to the invention is characterized in that the antibody specifically targets epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), insulin receptor (InsR), Bruton's tyrosine kinase (BTK), HER2, CTLA, CD20, CD52, CD30, CD33, CD133, PD-1, PD-L1, Src, Abl, phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB / Akt), target of rapamycin (mTOR), serine-threonine protein kinase Ras, mitogen-activated protein kinase (MAPK), STAT1, STAT3 or STAT5, preferably EGFR, VEGFR, PDGFR, FGFR, HER2, CTLA, CD20, CD133, PD-1 or PD-L1.

[0027] Furthermore, the antibody-TGF-β RII conjugate molecule of the present invention is characterized in that the antibody is a murine antibody, chimeric antibody, humanized antibody, Fab antibody, Fab' antibody, F(ab')2 antibody, Fv antibody, scFv antibody, or nanobody. The antibody or fragment thereof provided by the present invention may be in any form, such as a monoclonal antibody, single-chain antibody, single-domain antibody, bifunctional antibody, nanobody, fully humanized antibody, partially humanized antibody, or chimeric antibody. Alternatively, the antibody or fragment thereof may be a half antibody or an antigen-binding fragment of a half antibody, such as scFv, BsFv, dsFv, (dsFv)2, Fab, Fab', F(ab')2, or Fv. Regarding the fragments provided by the present invention, preferably, the fragment may be any fragment of an antibody capable of binding to PD-L1. The antibody or antigen-binding fragment thereof according to the present invention may be a murine antibody, chimeric antibody, humanized antibody, Fab, Fab', F(ab')2, Fv, or scFv.

[0028] Preferably, the antibody provided by the present invention is IgA, IgD, IgE, IgG or IgM, more preferably IgG1. The antibody fragment is selected from the group consisting of scFv, Fab, F(ab')2 and Fv fragments of the antibody.

[0029] Preferably, the antibody or fragment thereof further comprises a human or mouse constant region, preferably a human or mouse light chain constant region (CL) and / or heavy chain constant region (CH). More preferably, the antibody or fragment thereof comprises a heavy chain constant region and / or a kappa-type or lambda-type light chain constant region selected from the group consisting of IgG, IgA, IgM, IgD and IgE. According to certain embodiments of the present invention, the antibody may be a monoclonal antibody, preferably a murine, chimeric or humanized monoclonal antibody. More preferably, the heavy chain constant region of the monoclonal antibody is of the IgG1 or IgG4 subtype.

[0030] Furthermore, an antibody-TGF-β RII conjugate molecule according to the invention is characterized in that the antibody is an anti-human PD-L1 antibody or an antigen-binding fragment thereof, and the anti-human PD-L1 antibody or antigen-binding fragment thereof has a heavy chain comprising CDR1 set forth in SEQ ID NO: 25, a CDR2 set forth in SEQ ID NO: 26, and a CDR3 set forth in SEQ ID NO: 27, and a light chain comprising CDR1 set forth in SEQ ID NO: 28, a CDR2 set forth in SEQ ID NO: 29, and a CDR3 set forth in SEQ ID NO: 30.

[0031] Furthermore, the antibody-TGF-β RII conjugate molecule according to the present invention is characterized in that the antibody is an anti-human PD-L1 Nanobody, and the amino acid sequence of the anti-human PD-L1 Nanobody is set forth in SEQ ID NO:20.

[0032] Furthermore, the antibody-TGF-β RII conjugate molecule of the invention is characterized in that TGF-β RII is linked to the anti-human PD-L1 antibody via a linker peptide, and the linker peptide preferably comprises (G4S)n, where n is an integer between 1 and 4.

[0033] In a fifth aspect, the present invention provides a composition comprising a TGF-β RII mutant according to the first aspect of the invention, a fusion protein according to the second aspect of the invention, a multifunctionally active molecule according to the third aspect of the invention or a conjugated molecule according to the fourth aspect of the invention, and a pharmaceutically acceptable excipient.

[0034] In a sixth aspect, the present invention provides a nucleic acid encoding a TGF-β RII mutant according to the first aspect of the invention, a fusion protein according to the second aspect of the invention, a multifunctionally active molecule according to the third aspect of the invention or a conjugated molecule according to the fourth aspect of the invention.

[0035] In a seventh aspect, the present invention provides a recombinant vector comprising a nucleic acid according to the sixth aspect of the invention.

[0036] In an eighth aspect, the present invention provides a recombinant host cell comprising a nucleic acid according to the sixth aspect of the invention or a recombinant vector according to the seventh aspect of the invention.

[0037] In a ninth aspect, the present invention provides a method for producing a product, characterized in that the method comprises producing a TGF-β RII mutant, a fusion protein thereof, a multifunctionally active molecule thereof or a conjugated molecule thereof using a nucleic acid according to the sixth aspect of the invention, a recombinant vector according to the seventh aspect or a recombinant host cell according to the eighth aspect of the invention.

[0038] In a tenth aspect, the present invention provides a method for reducing or eliminating degradation or cleavage of a recombinant protein comprising a TGF-β RII fragment, comprising subjecting a coding region encoding a TGF-β RII fragment in the recombinant protein to mutagenesis so that the TGF-β RII encoded by the coding region contains mutations at one or more amino acid residue positions selected from the group consisting of positions 6, 12 and 20, compared to wild-type TGF-β RII, wherein the numbering of amino acid residues in wild-type TGF-β RII refers to SEQ ID NO: 6.

[0039] Furthermore, a method of reducing or eliminating recombinant protein degradation or cleavage according to the present invention is characterized in that the TGF-β RII fragment comprises one or more mutations selected from the group consisting of Q6N, D12T, and G20T, compared to wild-type TGF-β RII.

[0040] In an eleventh aspect, the present invention provides a method for treating a disease, characterized in that it comprises administering to a subject in need thereof an effective amount of a product selected from the group consisting of a TGF-β RII mutant according to the first aspect of the invention, a fusion protein according to the second aspect, a multifunctionally active molecule according to the third aspect, a conjugated molecule according to the fourth aspect, a composition according to the fifth aspect, a nucleic acid according to the sixth aspect, a recombinant vector according to the seventh aspect, or a recombinant cell according to the eighth aspect.

[0041] Furthermore, the method according to the present invention is characterized in that it is a method for preventing or treating tumors or cancer, chronic infectious diseases, or autoimmune diseases.

[0042] Furthermore, the method according to the present invention is characterized in that the tumor or cancer is preferably selected from the group consisting of pharyngeal squamous cell carcinoma, non-small cell lung cancer, pancreatic cancer, liver cancer, urothelial cancer, colon cancer and gastric cancer.

[0043] In a twelfth aspect, the present invention provides the use of a product in the manufacture of a medicament, characterised in that the product comprises a TGF-β RII mutant according to the first aspect of the invention, a fusion protein according to the second aspect, a multifunctionally active molecule according to the third aspect, a conjugated molecule according to the fourth aspect, a composition according to the fifth aspect, a nucleic acid according to the sixth aspect, a recombinant vector according to the seventh aspect or a recombinant cell according to the eighth aspect.

[0044] Furthermore, the use of the product in the manufacture of a medicament according to the invention is characterized in that the medicament is for the prevention or treatment of tumors or cancer, chronic infectious diseases, or autoimmune diseases.

[0045] Furthermore, the use of the product in the manufacture of a medicament according to the present invention is characterized in that the tumor or cancer is preferably selected from the group consisting of pharyngeal squamous cell carcinoma, non-small cell lung cancer, pancreatic cancer, liver cancer, urothelial cancer, colon cancer and gastric cancer.

[0046] To facilitate a better understanding of the present invention, definitions of several terms are provided below. Additional definitions are set forth throughout the Detailed Description section below.

[0047] As used herein, the term "antibody" is intended to encompass full-length antibodies and any antigen-binding fragments (i.e., antigen-binding portions) or single chains thereof. A full-length antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, with the heavy and light chains linked by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further divided into hypervariable regions called complementarity-determining regions (CDRs) and more conserved framework regions (FRs) that separate the CDRs. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to tissues or factors within the host, including various immune system cells (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0048] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having specificities for different antigens.

[0049] As used herein, the term "antigen-binding fragment" of an antibody (or abbreviated as "portion of an antibody") refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen-binding function of an antibody can be realized by fragments of a full-length antibody. Examples of binding fragments encompassed by the "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of a VL, a VH, a CL, and a CH1; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of a VH and a CH1; (iv) an Fv fragment consisting of a single antibody arm, the VL and the VH; (v) a dAb fragment consisting of a VH (Ward et al., (1989) Nature 341: 544-546); (vi) an isolated complementarity-determining region (CDR); and (vii) a nanobody, which is a heavy chain variable region comprising a single variable domain and two constant domains. Furthermore, the two domains, VL and VH, of an Fv fragment are encoded by different genes but can be recombinantly joined via a synthetic linker to form a single protein chain in which the VL and VH regions pair to form a monovalent molecule (referred to as single-chain Fc (scFv); see, e.g., Bird et al., (1988) Science 242: 423-426 and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). These single-chain antibodies are also intended to be encompassed within the meaning of this term. These antibody fragments can be obtained using conventional techniques known to those skilled in the art and can be functionally screened in the same manner as intact antibodies.

[0050] Antigen-binding fragments of the present invention include those capable of specifically binding to an antigen. Examples of antibody-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv (scFv), and single-domain fragments.

[0051] Fab fragments contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments in that several residues, including one or more cysteines from the antibody hinge region, are added to the carboxyl terminus of the heavy chain CH1 domain. F(ab') fragments are generated by cleavage of the disulfide bond at the hinge cysteines of the F(ab')2 pepsin digestion product. Additional chemical couplings of antibody fragments are known to those skilled in the art. Fab and F(ab')2 fragments lack the fragment crystallizable (Fc) region of intact antibodies, are cleared more rapidly from an animal's circulation, and may exhibit less nonspecific tissue binding than intact antibodies (see, e.g., Wahl et al., 1983, J. Nucl. Med. 24:316).

[0052] As is generally understood in the art, the "Fc" region is the fragment crystallizable constant region of an antibody that does not contain the antigen-specific binding region. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments derived from the second and third constant domains (CH2 and CH3 domains, respectively) of the antibody's two heavy chains. The Fc region of IgM and IgE contains three heavy chain constant domains (CH2, CH3, and CH4 domains) on each polypeptide chain.

[0053] An "Fv" fragment is the minimum fragment of an antibody which contains a complete target recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association (VH-VL dimer). In this configuration, the three CDRs from each variable domain interact to define a target binding site on the surface of the VH-VL dimer. Generally, the six CDRs confer target binding specificity to the antibody. However, in some cases, even a single variable domain (or half of an Fv containing only three target-specific CDRs) may have the ability to recognize and bind to a target, albeit with lower affinity than the entire binding site.

[0054] "Single-chain Fv" or "scFv" antibody-binding fragments comprise the VH and VL domains of antibody present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for target binding.

[0055] A "single domain fragment" consists of a single VH or VL domain that exhibits sufficient affinity for an antigen. In certain embodiments, the single domain fragment is camelized (see, e.g., Riechmann, 1999, Journal of Immunological Methods 231:25-38).

[0056] The antibodies of the present invention include derivatized antibodies. For example, derivatized antibodies are typically modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or attachment to a cellular ligand or other protein. Any of a number of chemical modifications can be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, derivatives can contain one or more unnatural amino acids, for example, using the ambrx technique (see, e.g., Wolfson, 2006, Chem. Biol. 13 (10): 1011-2).

[0057] A polypeptide linker, also known as a polypeptide linking arm or polypeptide linker, is a polypeptide molecule used to link two functionally active molecules. The linker sequence connecting the two components in the fusion protein is closely related to whether the two active components in the fusion protein can form a correct spatial structure and better perform their biological functions. Recombinantly produced fusion proteins require that the linker inserted into the fusion protein does not interfere with the functions of the respective target proteins.

[0058] Much related research has been conducted on the design and selection of linker sequences. Currently, two types of linkers have been mainly studied: (1) helix-form linkers such as [A(EAAAK)nA], and (2) linkers with low hydrophobicity and low charge effect, including peptide chains of different lengths that can form helices, flexible linkers, Staphylococcus protein A, etc. The length of the linker is another important factor. If the linker is too long, the fusion protein may become sensitive to proteases, reducing the yield of active fusion protein during production. If a shorter linker is used, the two molecules may be fused too closely, affecting the function of the protein.

[0059] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0060] First, the inventors identified the cleavage and degradation sites of the antibody / TGF-β RII fusion protein molecule through mass spectrometry analysis of the recombinant antibody / TGF-β RII fusion protein combined with bioinformatics analysis, and further verified the cleavage and degradation sites by designing amino acid mutations, thus overcoming the technical problem that the antibody / TGF-β RII fusion protein is prone to degradation upon recombinant expression.

[0061] Second, the inventors modified TGF-β RII by site-directed mutagenesis. This modification does not alter the number of amino acids in TGF-β RII or the length of TGF-β RII, thereby avoiding the loss of function or masking caused by excessively large molecular weight differences when TGF-β RII is fused / conjugated to a multisubunit protein such as an antibody. In the present disclosure, site-directed mutagenesis was performed on the amino acids at positions 6, 12, and 20 of TGF-β RII. When the resulting TGF-β RII is used as a component of a fusion protein, it not only maintains its specific binding activity to TGF-β, thereby effectively binding to TGF-β1, TGF-β2, and TGF-β3, but also retains the biological function of TGF-β R, thereby blocking the binding of TGF-β to TGF-β R in vivo, inhibiting the tumor-promoting activity of TGF-β, and providing anti-tumor effects and functions.

[0062] Third, we also engineered and screened antibodies fused to TGF-β RII. By using a nanobody with a relatively small molecular weight and fusing it to TGF-β RII via a linker, we were able to reduce both the difference in molecular size between the two components of the bifunctional fusion protein and the structural complexity of the recombinant bifunctional fusion protein, providing an antibody / TGF-β RII bifunctional fusion protein and a method for its preparation that are more convenient for mass production with more consistent quality.

[0063] Embodiments of the present invention will be described in detail below with reference to the accompanying drawings. [Brief explanation of the drawings]

[0064] [Figure 1] 1 is a graph showing the results of culture supernatants of cells expressing the fusion protein H182-MUT4-TGF-β RII for 9 to 14 days, as detected by reducing 12% polyacrylamide gel electrophoresis (SDS-PAGE). [Figure 2a]This figure shows the supernatants of the fusion protein H182-MUT4-TGF-β RII and its mutants, detected by reducing 12% polyacrylamide gel electrophoresis: parental: culture supernatant of cells expressing H182-MUT4-TGF-β RII, m1: culture supernatant of cells expressing H182-MUT4-TGF-β RIIm1, m2: culture supernatant of cells expressing H182-MUT4-TGF-β RIIm2, and m3: culture supernatant of cells expressing H182-MUT4-TGF-β RIIm3. [Figure 2b] Figure 1 shows purified proteins of the fusion protein H182-MUT4-TGF-β RII and its mutants detected by reducing 12% polyacrylamide gel electrophoresis: Parent: purified protein of H182-MUT4-TGF-β RII, m1: purified protein of H182-MUT4-TGF-β RIIm1, m2: purified protein of H182-MUT4-TGF-β RIIm2, m3: purified protein of H182-MUT4-TGF-β RIIm3. [Figure 3] 1 is a graph showing the results of recombinant protein H182-MUT4-TGF-β RII analyzed by SEC-HPLC. [Figure 4a] 1 is a graph showing the results of recombinant protein H182-MUT4-TGF-β RIIm1 analyzed by SEC-HPLC. [Figure 4b] 1 is a graph showing the results of recombinant protein H182-MUT4-TGF-β RIIm2 analyzed by SEC-HPLC. [Figure 4c] 1 is a graph showing the results of recombinant protein H182-MUT4-TGF-β RIIm3 analyzed by SEC-HPLC. [Figure 5]10 is a graph showing the results of a sample of the protein H182-MUT4-TGF-β RIIm2 exposed to high temperatures, analyzed by SEC-HPLC: red line: H182-MUT4-TGF-β RIIm2-40C, sample bottle 1:A,1; loading 1: channel 2489 ChA; green line: H182-MUT4-TGF-β RIIm2-40C-1W, sample bottle 1:A,2; loading 1: channel 2489 ChA; blue line: H182-MUT4-TGF-β RIIm2-40C-2W, sample bottle 1:A,3; loading 1: channel 2489 ChA; gray line: H182-MUT4-TGF-β RIIm2-40C-3W, sample bottle 1:A,4; loading 1: channel 2489 ChA. [Figure 6] 1 shows the results of repeatedly freeze-thawed samples of the protein H182-MUT4-TGF-β RIIm2 analyzed by SEC-HPLC. Red line: H182-MUT4-TGF-β RIIm2-20C, sample bottle 1:A,5; loading 1: channel 2489 ChA. Green line: H182-MUT4-TGF-β RIIm2-20C-1 freeze-thaw, sample bottle 1:A,6; loading 1: channel 2489 ChA. Blue line: H182-MUT4-TGF-β RIIm2-20C-2 freeze-thaw, sample bottle 1:A,7; loading 1: channel 2489 ChA. Gray line: H182-MUT4-TGF-β RIIm2-20C-3 freeze-thaw, sample bottle 1:A,8; loading 1: channel 2489 ChA. [Figure 7] Figure 1 shows the culture supernatants of anti-human PD-L1 antibody / TGF-β RII fusion protein cultures for 10 and 15 days, as detected by reducing 12% polyacrylamide gel electrophoresis. [Figure 8] 1 is a graph showing the results of purified proteins from 15-day M7824 culture supernatant analyzed by SEC-HPLC. [Figure 9] 10 is a graph showing the results of purified proteins from the culture supernatant of 15-day hzF2-TGF-β RIIm2 analyzed by SEC-HPLC. [Figure 10] Figure 10 is a graph showing the results of affinity analysis of hzF2-TGF-β RIIm2 for recombinant human PD-L1 extracellular domain protein. [Figure 11] Figure 10 is a graph showing the results of affinity analysis of M7824 to recombinant human PD-L1 extracellular domain protein. [Figure 12] 1 is a graph showing the results of affinity analysis of hzF2-TGF-β RIIm2 for recombinant human TGF-β1 protein. [Figure 13] 1 is a graph showing the results of an affinity analysis of M7824 for recombinant human TGF-β1 protein. [Figure 14] Figure 10 shows the binding activity of the fusion protein hzF2-TGF-β RIIm2 to recombinant human PD-L1 extracellular domain protein, as detected by ELISA. [Figure 15] 1 is a graph showing the results of the binding activity of the fusion protein hzF2-TGF-β RIIm2 to recombinant human TGF-β1 protein, detected by ELISA. [Figure 16] 1 is a graph showing the results of the binding activity of the fusion protein hzF2-TGF-β RIIm2 to recombinant human TGF-β2 protein, as detected by ELISA. [Figure 17] 1 is a graph showing the results of the binding activity of the fusion protein hzF2-TGF-β RIIm2 to recombinant human TGF-β3 protein, detected by ELISA. [Figure 18] Figure 10 shows the binding activity of the fusion protein hzF2-TGF-β RIIm2 to native PD-L1 protein on the cell surface, as detected by FACS. [Figure 19] Figure 1 shows the inhibitory effect of the fusion protein hzF2-TGF-β RIIm2 on the binding of human PD-L1 to its receptor PD-1, as detected by ELISA. [Figure 20] FIG. 1 shows the detection of the biological activity of the fusion protein hzF2-TGF-β RIIm2. [Figure 21] FIG. 1 shows statistics of tumor volume of subcutaneously implanted tumors in PBMC-immune reconstituted mice. [Figure 22] FIG. 1 shows statistics of tumor weights of subcutaneously implanted tumors in PBMC-immune reconstituted mice. [Figure 23] 1 is a graph showing the results of evaluating the antitumor activity of the fusion protein hzF2-TGF-β RIIm2 in a tumor model of human CD34+ cord blood stem cell-humanized mice subcutaneously inoculated with HCC827 cells. DETAILED DESCRIPTION OF THE INVENTION

[0065] To solve the technical problem that TGF-β RII and its fusion proteins are prone to degradation and cleavage upon recombinant expression, the present invention provides a TGF-β RII mutant and its fusion protein, wherein the TGF-β RII mutant has mutation(s) at position(s) selected from position 6 (Gln), position 12 (Asp), and position 20 (Gly) compared to the extracellular domain of wild-type TGF-β RII shown in SEQ ID NO: 6. The TGF-β RII mutant can bind to TGF-β. The TGF-β RII mutant has less cleavage and / or degradation when recombinantly expressed compared to wild-type TGF-β RII. An antibody / TGF-β RII bifunctional protein and its fusion protein are provided that are more convenient for mass production with more stable quality.

[0066] The present invention will now be described with reference to specific examples, which will be understood by those skilled in the art as being merely illustrative of the present invention and are not intended to limit the scope of the present invention in any way.

[0067] All experimental procedures in the following examples are conventional unless otherwise specified. All raw materials and reagents used in the following examples are commercially available unless otherwise specified. [Example]

[0068] Example 1: Preparation of anti-human PD-L1 antibody / TGF-β RII fusion protein and control samples 1.1 Preparation of anti-human PD-L1 antibody / TGF-β RII fusion protein Using PCR, the nucleotide sequence (SEQ ID NO: 1) encoding the heavy chain of the PD-L1 antibody H182-MUT4 (MW22, from Chinese Patent Application No. 201911419802.5) was C-terminally linked to the nucleotide sequence (SEQ ID NO: 5) encoding the TGF-β RII extracellular domain via a nucleotide sequence (SEQ ID NO: 3) encoding a linker peptide, yielding the H182-MUT4-H-TGF-β RII encoding sequence (SEQ ID NO: 9), which comprises the PD-L1 antibody heavy chain and TGF-β RII. The nucleotide sequence encoding H182-MUT4-H-TGF-β RII and the nucleotide sequence (SEQ ID NO: 7) encoding the light chain of the PD-L1 antibody (H182-MUT4-L) were ligated by enzymatic digestion and cloned into a stable expression vector containing the glutamine synthetase (GS) gene to construct a eukaryotic expression vector of H182-MUT4-TGF-β RII for stable transfection. The vector was transformed into Escherichia coli cells and propagated, and the eukaryotic expression plasmid for H182-MUT4-TGF-β RII was isolated and obtained in large quantities. The prepared eukaryotic expression plasmid for H182-MUT4-TGF-β RII was electrotransfected into CHO-K1 cells in suspension culture (Nucleofector IIb, Lonza), and cells stably expressing the fusion protein H182-MUT4-TGF-β RII were obtained by MSX pressure screening. The cells were grown in fed-batch culture and monitored daily for cell density and viability. A portion of the cell culture supernatant was harvested daily from day 9, and when cell viability reached less than 20%, the entire cell culture broth was centrifuged at high speed to collect the supernatant. A portion of the expression supernatant was purified using a protein A affinity chromatography column to obtain the anti-human PD-L1 antibody / TGF-β RII fusion protein H182-MUT4-TGF-β RII.

[0069] 1.2 Preparation of anti-human PD-L1 antibody / TGF-β RII fusion protein control The light and heavy chain-TGF-β RII genes of M7824 (see Patent Document 1 for the sequence) were artificially synthesized, ligated by enzymatic digestion, and cloned into a stable expression vector containing the glutamine synthetase (GS) gene to construct a eukaryotic expression vector for stable transfection. The vector was electrotransfected into CHO-K1 cells in suspension culture (Nucleofector IIb, Lonza), and cells stably expressing the recombinant protein M7824 were obtained by MSX pressure screening. The cells were cultured to express the protein, and the recombinant protein M7824 was obtained and used as a control.

[0070] Example 2: Analysis of cleavage sites in H182-MUT4-TGF-β RII 2.1 Analysis of H182-MUT4-TGF-β RII by SDS-PAGE electrophoresis The supernatant from the H182-MUT4-TGF-β RII expression medium was subjected to electrophoretic analysis by reducing SDS-PAGE. The results showed three distinct bands on the electrophoresis gel. The band with a molecular weight greater than 70 kDa was the H182-MUT4-TGF-β RII heavy chain fusion protein chain. The band with a molecular weight between 20 kDa and 30 kDa was the anti-PD-L1 antibody H182-MUT4 light chain. The distinct protein bands between 50 kDa and 70 kDa, which were protein markers, were presumably the H182-MUT4 heavy chain shed from TGF-β RII (Figure 1). The proportion of cleaved protein bands increased with increasing incubation time. The analysis results for M7824 were similar to those for H182-MUT4-TGF-β RII, and similar cleavage-related band problems were also observed.

[0071] 2.2 Analysis of the cleavage site in H182-MUT4-TGF-β RII by mass spectrometry Bands with molecular weights between 50 kDa and 70 kDa were collected from the SDS-PAGE electrophoresis gel. A destaining buffer was added to the sample to thoroughly destain it, and a reducing buffer was added for reduction. Trypsin was then added, and enzymatic digestion was carried out overnight at 37°C. The enzymatic digestion products were then extracted and desalted. Finally, the peptide fragments were redissolved in 0.1% formic acid aqueous solution for subsequent mass spectrometry analysis. For mass spectrometry analysis, an appropriate amount of the peptide fragment sample was subjected to chromatographic separation using an Easy nLC 1200 system (Thermo Scientific) at nanoliter flow rates. After separation of the peptide fragments, they were analyzed by DDA (data-dependent acquisition) mass spectrometry using a Q-exactive Plus mass spectrometer (Thermo Scientific). Finally, MaxQuant 1.6.1.0 software for mass spectral database search was used for parallel analysis. The mass spectrometry analysis results are presented in Table 1 below, and indicate that the sample lacks amino acids from position 478 onward. After comprehensive analysis, together with the speculation that the trypsin digestion site may be after the amino acids Lys and Arg, we considered that the heavy chain of H182-MUT4-TGF-β RII may be cleaved at amino acid position 477 / 478.

[0072] [Table 1]

[0073] Example 3: Design, expression, and analysis of anti-human PD-L1 antibody / TGF-β RII fusion protein mutants 3.1 Design and Expression of Anti-Human PD-L1 Antibody / TGF-β RII Fusion Protein Mutants Based on speculation about the site where cleavage would occur, glycosylation sites before and after the N-terminal enzyme cleavage site of TGF-β RII were designed to protect the cleavage site and prevent cleavage from occurring. The mutant designs are listed in Table 2. The nucleotide sequence encoding the TGF-β RII extracellular domain in the expression vector for the anti-human PD-L1 antibody / TGF-β RII fusion protein H182-MUT4-TGF-β RII was subjected to site-directed mutagenesis using the StarMut Gene Site-Directed Mutagenesis Kit (Cat. No.: T111-01, GenStar) to obtain mutant expression plasmids. The plasmids were then transformed into E. coli cells and propagated to obtain the plasmids for the anti-human PD-L1 antibody / TGF-β RII fusion protein mutants, namely H182-MUT4-TGF-β RIIm1, H182-MUT4-TGF-β RIIm2, and H182-MUT4-TGF-β RIIm3. The prepared H182-MUT4-TGF-β RIIm1, H182-MUT4-TGF-β RIIm2, and H182-MUT4-TGF-β RIIm3 plasmids were electrotransfected into CHO-K1 cells in suspension culture (Nucleofector IIb, Lonza), and cells stably expressing the PD-L1 antibody / TGF-β RII fusion protein mutants were isolated by MSX pressure screening. Cells expressing H182-MUT4-TGF-β RII and its mutants H182-MUT4-TGF-β RIIm1, H182-MUT4-TGF-β RIIm2, and H182-MUT4-TGF-β RIIm3 were cultured in fed-batch culture and monitored daily for cell density and viability. When cell viability reached less than 20%, the entire cell culture broth was centrifuged at high speed to collect the supernatant. A portion of the supernatant was purified using a protein A affinity chromatography column to obtain the fusion protein H182-MUT4-TGF-β RII and its mutants.

[0074] [Table 2]

[0075] 3.2 Detection of H182-MUT4-TGF-β RII and its mutants by SDS-PAGE electrophoresis Samples of the expression supernatant and purified protein of the fusion protein H182-MUT4-TGF-β RII and its mutants were subjected to reducing SDS-PAGE electrophoresis. The results showed that the electrophoresis gel of the H182-MUT4-TGF-β RII mutant protein showed two distinct bands: one with a molecular weight greater than 70 kDa, which represented the protein chain fused to the respective H182-MUT4-TGF-β RII mutant heavy chain; one with a molecular weight between 20 kDa and 30 kDa, which represented the light chain of the anti-PD-L1 antibody H182-MUT4; and the disappearance of the 50 kDa to 70 kDa protein marker band, which represented the heavy chain of H182-MUT4 shed from TGF-β RII (Figure 2a and Figure 2b).

[0076] 3.3 Detection of H182-MUT4-TGF-β RII and its mutants by SEC-HPLC Samples of the protein H182-MUT4-TGF-β RII and its mutants H182-MUT4-TGF-β RIIm1, H182-MUT4-TGF-β RIIm2, and H182-MUT4-TGF-β RIIm3 were subjected to SEC-HPLC analysis. The results showed that H182-MUT4-TGF-β RII had clear characteristic peaks of degradation fragments (Figure 3 and Table 3). After the mutational modification, the degradation problem was resolved, the characteristic peaks of degradation fragments disappeared, and the purity of the main HPLC peak was significantly increased (Figures 4a, 4b, and 4c, and Table 3).

[0077] [Table 3]

[0078] Example 4: Stability detection of anti-human PD-L1 antibody / TGF-β RII fusion protein mutants Samples of the bifunctional protein H182-MUT4-TGF-β RIIm2 were stored at 40°C for three weeks, with one tube removed each week for stability testing. Additionally, protein samples were repeatedly frozen and thawed at -20°C three times, with one sample tested for freeze-thaw stability each time. The purity of the treated samples and a control sample, which was always kept at 4°C, was tested by SEC-HPLC to verify their stability. The results are shown in Figures 5 and 6. As shown, the purity of the sample subjected to repeated freeze-thawing at high temperatures was essentially consistent with that of the control sample, indicating that the product was stable and no cleavage occurred.

[0079] Example 5: Affinity detection of anti-human PD-L1 antibody / TGF-β RII fusion protein Antibody affinity was detected by an assay involving capturing antibody Fc fragments on an anti-human IgG Fc capture (AHC) biosensor on a Fortebio Octet QKe system instrument. For the assay, the bifunctional proteins H182-MUT4-TGF-β RII, H182-MUT4-TGF-β RIIm1, H182-MUT4-TGF-β RIIm2, and H182-MUT4-TGF-β RIIm3, as well as H182-MUT4 and TGF-β RII-hFc (Cat. No. CC10, Novoprotein Scientific Inc.), were each diluted to 5 μg / ml in PBS and flowed over the surface of an AHC biosensor (Cat. No. 18-0015, PALL) for 120 seconds. Recombinant human PD-L1-his protein and recombinant human TGF-β1 protein (Catalog No. CA59, Novoprotein Scientific Inc.) were used as the mobile phase at a concentration of 60 nM. The association time was 300 seconds, and the dissociation time was 300 seconds. After the assay was completed, the response value of the blank control was subtracted from the data, and the data was fitted to a 1:1 Langmuir binding model using software to calculate the kinetic parameters of antigen-antibody binding. The results are shown in Table 4. As shown, the affinities of the bifunctional proteins H182-MUT4-TGF-β RII, H182-MUT4-TGF-β RIIm1, H182-MUT4-TGF-β RIIm2, and H182-MUT4-TGF-β RIIm3 for PD-L1 and TGF-β1 were not significantly different from those of H182-MUT4 and TGF-β RII-hFc for PD-L1 and TGF-β1.

[0080] [Table 4]

[0081] Example 6: Design and expression of anti-human PD-L1 nanobody / TGF-β RII fusion protein mutants Using PCR, the nucleotide sequence (SEQ ID NO: 19) encoding the humanized anti-human PD-L1 nanobody hzF2 (from Chinese Patent Application No. 202010324761.8) was cloned into the nucleotide sequence (SEQ ID NO: 19) encoding the linker peptide. 21 The nucleotide sequence was linked at the C-terminus to the nucleotide sequence encoding TGF-β RIIm2 (SEQ ID NO: 13) via a nucleotide sequence encoding hzF2-TGF-β RIIm2 (SEQ ID NO: 23), which contained the PD-L1 Nanobody-TGF-β RIIm2. The nucleotide sequence was then cloned into a stable expression vector containing the glutamine synthetase (GS) gene via an enzyme digestion site to construct a eukaryotic expression vector for hzF2-TGF-β RIIm2 for stable transfection. The vector was transformed into E. coli cells and propagated, and the eukaryotic expression plasmid for the fusion protein hzF2-TGF-β RIIm2 was isolated in large quantities. The prepared eukaryotic expression plasmid was electrotransfected into CHO-K1 cells in suspension culture (Nucleofector IIb, Lonza), and cells stably expressing the fusion protein hzF2-TGF-β RIIm2 were identified by MSX pressure screening. The cells were cultured, expressed, and purified to obtain the fusion protein mutant hzF2-TGF-β RIIm2.

[0082] Example 7: Purity detection of fusion protein hzF2 TGF-β RIIm2 Cells expressing M7824 and hzF2-TGF-β RIIm2 were cultured in a fed-batch culture, and cell density and activity were monitored daily. A portion of the cell culture broth was harvested mid-culture (day 10), and the remaining cell culture broth was harvested late in culture when cell activity reached less than 20% (day 15). The culture broth was centrifuged at high speed to collect the supernatant, which was then purified using a protein A affinity chromatography column. The purified expression supernatant was then subjected to protein quantification and subpackaged for use. The expression supernatant was detected by reducing SDS-PAGE electrophoresis and SEC-HPLC. The SDS-PAGE results (Figure 7) showed three distinct bands in the electrophoresis gel of M7824. The band with a molecular weight greater than 70 kDa was the protein chain fused to the M7824 heavy chain, the band with a molecular weight between 20 and 30 kDa was the M7824 light chain, and the distinct protein band between 50 and 70 kDa, a protein marker, was presumably the M7824 heavy chain shed from TGF-β RII. The proportion of cleaved protein bands increased with increasing incubation time. In contrast, the electrophoresis gel of hzF2-TGF-β RIIm2 showed one clear major band between 50 and 70 kDa, with no other obvious protein bands. The results of SEC-HPLC analysis are shown in Figures 8 and 9 and Table 5. As shown, M7824 had a clear peak of low molecular weight fragments, whereas hzF2-TGF-β RIIm2 clearly showed reduced degradation.

[0083] [Table 5]

[0084] Example 8: Affinity analysis of the fusion protein hzF2-TGF-β RIIm2 Antibody affinity was detected using an assay involving capturing antibody Fc fragments with an anti-human IgG Fc capture (AHC) biosensor on a Fortebio Octet QKe system instrument. For the assay, each of the proteins (hzF2-TGF-β RIIm2 and M7824) was diluted to 4 μg / ml in PBS and flowed over the surface of an AHC biosensor (catalog number: 18-0015, PALL) for 120 seconds. Recombinant human PD-L1-his protein (accession number: NP_054862.1, 19aa-238aa) and human TGF-β1 (catalog number: CA59, Novoprotein Scientific Inc.) were used as the mobile phase. The association time was 300 seconds, and the dissociation time was 300 seconds. After the assay was completed, the data, after subtracting the response value of the blank control, were fitted to a 1:1 Langmuir binding model using software to calculate the kinetic constants of antigen-antibody binding.

[0085] The response curves of hzF2-TGF-β RIIm2 and the control protein M7824 to recombinant human PD-L1 protein are shown in Figures 10 and 11, and their response curves to recombinant human TGF-β1 protein are shown in Figures 12 and 13. The curves were fitted and the affinities calculated. The results showed that hzF2-TGF-β RIIm2 had affinity for PD-L1 with a KD of 1.58E-09M and for TGF-β1 with a KD of 2.46E-09M, while M7824 had affinity for PD-L1 with a KD of 3.21E-09M and for TGF-β1 with a KD of 2.81E-09M. Detailed kinetic parameters are shown in Table 6 below. The results showed that hzF2-TGF-β RIIm2 had high affinity for both human PD-L1 and TGF-β1.

[0086] [Table 6]

[0087] Example 9: Binding activity of the fusion protein hzF2-TGF-β RIIm2 detected by ELISA Plates were coated with recombinant human PD-L1-his protein (accession number: NP_054861.2, 19aa-238aa), human TGF-β1 (catalog number: CA59, Novoprotein Scientific Inc.), human TGF-β2 (catalog number: CJ79, Novoprotein Scientific Inc.), and human TGF-β3 (catalog number: CJ44, Novoprotein Scientific Inc.) at a concentration of 1μg / ml each at 4°C overnight. The plates were then blocked with 5% BSA in a constant-temperature incubator at 37°C for 60 minutes. HzF2-TGF-β RIIm2 and control protein M7824, as well as the isotype control NC-hIgG1 (12 dilutions obtained by serial 3-fold dilutions from an initial concentration of 10 μg / ml) were added to the plate and incubated at 37°C for 60 minutes. The plate was then washed four times with PBST. A 5000-fold dilution of HRP-anti-human Fc (catalog number: 109-035-098, Jackson ImmunoResearch) was added to the plate and incubated for 45 minutes. The plate was then washed four times with PBST. TMB substrate (catalog number: ME142, GalaxyBio, Beijing) was added and developed for 15 minutes. The reaction was stopped by adding 2M HCl, and the plate absorbance at 450 nm was read and recorded.

[0088] The results showed that hzF2-TGF-β RIIm2 had binding activity to human PD-L1, TGF-β1, TGF-β2, and TGF-β3, which was comparable to that of M7824, with EC50 values ​​of 0.261 nM, 0.394 nM, 18.045 nM, and 1.121 nM, respectively, and M7824 had EC50 values ​​of 0.209 nM, 0.472 nM, 18.172 nM, and 0.981 nM, respectively, to human PD-L1, TGF-β1, TGF-β2, and TGF-β3 (Figures 14 to 17).

[0089] Example 10: Binding activity of the fusion protein hzF2-TGF-β RIIm2 detected by FACS Human breast cancer cells MDA-MB-231, which naturally express human PD-L1, were plated in a 96-well plate at 2 × 10 4 After cells / well were added, 5% BSA was added and blocking was performed for 30 minutes at room temperature. hzF2-TGF-β RIIm2, control protein M7824, and isotype control NC-hIgG1 (12 dilutions obtained by serial 3-fold dilutions from an initial concentration of 10 nM) were then added to the plate, followed by incubation on ice for 1 hour. After washing the cells twice with ice-cold PBS (containing 0.05% Tween), 200-fold diluted goat anti-human IgG Fc-FITC (catalog number: F9512, Sigma) was added to the plate and incubated on ice for 45 minutes. The cells were then washed twice with ice-cold PBS (containing 0.05% Tween) and resuspended in 200 μL of PBS. Mean fluorescence intensity (MFI) values ​​were measured using a flow cytometer.

[0090] The results showed that the 50% effective concentration (EC50) values ​​of hzF2-TGF-β RIIm2 and M7824 upon binding to PD-L1 on the cell surface were 0.0717 nM and 0.197 nM, respectively (Figure 18).

[0091] Example 11: Blocking activity of the fusion protein hzF2-TGF-β RIIm2 detected by ELISA Plates were coated with recombinant human PD-1-hFc protein (accession number: NP_005009.2, 21aa-167aa) at a concentration of 1μg / ml overnight at 4°C. The plates were then blocked with 5% BSA in a constant-temperature incubator at 37°C for 60 minutes. 50μl of hzF2-TGF-β RIIm2 and control protein M7824, as well as the isotype control NC-hIgG1 (12 dilutions obtained by 1.5-fold serial dilutions from an initial concentration of 60nM), were added to the plates, followed by the addition of 50μL of PD-L1-mFc (accession number: NP_054862.1, 19aa-238aa) at a concentration of 1μg / ml. The plates were then incubated in a constant-temperature incubator at 37°C for 60 minutes and washed four times with PBST. HRP-anti-mouse Fc (catalog number: 115-035-071, Jackson ImmunoResearch) diluted 5000 times was added to the plate and incubated for 45 minutes, after which the plate was washed four times with PBST. TMB substrate (catalog number: ME142, GalaxyBio, Beijing) was added and color development was allowed for 15 minutes. The reaction was stopped by adding 2 M HCl, and the plate absorbance at 450 nm was read and recorded.

[0092] The results showed that hzF2-TGF-β RIIm2 could effectively block the binding of recombinant human PD-L1 to its receptor PD-1. The competitive inhibitory effects of hzF2m9-TGF-β RIIm2 and M7824 on the binding of human PD-L1 to its receptor PD-1 were detected by ELISA, and their 50% inhibitory concentration (IC50) values ​​were 7.533 nM and 6.935 nM, respectively (Figure 19).

[0093] Example 12: Observation of the cytological activity of the fusion protein hzF2-TGF-β RIIm2, which blocks the binding of PD-L1 to its receptor PD1 CHO cells recombinantly expressing human PD-L1 and anti-CD3-ScFv (CHO-PD-L1-CD3L, Jiangsu T-Mab Biopharma Co., Ltd.) were seeded at 5000 cells / well in a 96-well plate (Cat. No. 3917, Corning) and incubated overnight in a cell incubator. The supernatant was then discarded. HzF2-TGF-β RIIm2 and control protein M7824 (8 dilutions obtained by 2.5-fold serial dilutions from an initial concentration of 5 μg / ml) (25 μL / well) and 50 μL of a suspension of Jurkat cells recombinantly expressing human PD-1 and luciferase (Jurkat-PD1-NFAT, Jiangsu T-Mab Biopharma Co., Ltd.) (1 × 10 6 1000 cells / mL) was added to the plate, which was then incubated in a cell incubator for 6 hours. Bio-Turbo firefly luciferase substrate (catalog number: RA-GL03, RHINOZYME BIOTECHNOLOGY) was added to the plate at 125 μL / well, and the plate was placed in a microplate thermostat and incubated at 800 rpm in the dark for 5 minutes. The multifunction microplate reader was set to operate in luminescence mode, with an interpretation of 500 (the instrument's default value). The RLU values ​​were read, and the detection results are shown in Figure 20. As shown, hzF2-TGF-β RIIm2 and the control protein M7824 exhibited blocking effects on the PD-L1 / PD1 pathway with EC50 values ​​of 10.122 nM and 8.537 nM, respectively.

[0094] [Table 7]

[0095] Example 13: Evaluation of the antitumor efficacy of the fusion protein hzF2-TGF-β RIIm2 in a PBMC-immune reconstituted mouse model subcutaneously implanted with human pharyngeal squamous cell carcinoma Fadu cells 5×10 human pharyngeal squamous cell carcinoma (FaDu) cells were inoculated into the right flank of 5- to 6-week-old male NCG mice. 6Mice were inoculated subcutaneously with human PBMCs at a concentration of 2 × 10 cells / 0.1 mL. 6 Cells / mouse were inoculated. Tumors were 40 mm 3 ~60mm 3 When tumors reached the required volume, mice with tumors of 6 mice per group were randomly divided into two groups and administered hzF2-TGF-β RIIm2 or the isotype control hIgG1, respectively. The administration schedule is shown in Table 8. The results are shown in Figures 21 and 22. As shown, hzF2-TGF-β RIIm2 significantly inhibited tumor growth and demonstrated clear antitumor efficacy, achieving a tumor growth inhibition (TGI; tumor weight) rate of 53%.

[0096] [Table 8]

[0097] Example 14: Evaluation of the antitumor activity of the fusion protein hzF2-TGF-β RIIm2 in a human CD34+ cord blood stem cell-humanized mouse tumor model subcutaneously inoculated with HCC827 cells Human non-small cell lung cancer HCC827 cell line was collected and cultured, then cultured and passaged in RMPI 1640 medium (supplemented with 10% inactivated FBS) at 37°C in a 5% CO2 incubator. Logarithmic growth phase tumor cells were used for in vivo tumor inoculation. Twenty qualified female human CD34+ umbilical cord blood stem cell-humanized mice were inoculated with human non-small cell lung cancer HCC827 cells after one week of adaptive feeding, and the tumor volume and body weight after tumor cell inoculation were observed. When the tumor volume was 120 mm 3 ~200mm 3Mice were selected and randomly divided into three groups, with five mice per group, according to tumor volume and body weight. Treatment began on the day of grouping, which was considered day 0. The treatment schedule and grouping information are presented in Table 9. In this experiment, the antitumor efficacy of equimolar doses of the test hzF2-TGF-β RIIm2 and the control drug atezolizumab, administered alone, was examined in a human CD34+ umbilical cord blood stem cell-humanized mouse tumor model subcutaneously inoculated with HCC827 cells. Results showed that both 8 mg / kg hzF2-TGF-β RIIm2 and 10 mg / kg atezolizumab showed strong efficacy when administered alone. The tumor growth inhibition (TGI; tumor volume) rate in the hzF2-TGF-β RIIm2-treated group reached over 80%, superior to that of atezolizumab (Figure 23 and Table 10).

[0098] [Table 9]

[0099] [Table 10]

[0100] The above description of the embodiments of the present invention is not intended to limit the present invention, and those skilled in the art can make various changes and modifications to the present invention without departing from the spirit of the present invention, which should be included in the scope of the appended claims.

Claims

1. a TGF-β RII mutant comprising a single mutation at one amino acid residue position selected from the group consisting of positions 6, 12 and 20 relative to wild-type TGF-β RII; the single mutation is selected from the group consisting of Q6N, D12T, and G20T; A TGF-β RII mutant, wherein the numbering of the amino acid residues of said wild-type TGF-β RII is based on SEQ ID NO:

6.

2. 2. The TGF-β RII mutant of claim 1, which is capable of binding to TGF-β, said TGF-β including TGF-β1, TGF-β2 and TGF-β3.

3. 2. The TGF-β RII mutant of claim 1, characterized in that it has less cleavage and / or degradation when recombinantly expressed compared to the wild-type TGF-β RII.

4. A fusion protein comprising two or more functional fragments, characterized in that at least one of said functional fragments has the amino acid sequence of a TGF-β RII mutant according to any one of claims 1 to 3.

5. The fusion protein of claim 4, wherein the two or more functional fragments function independently of each other, and optionally the functional fragments are linked to each other via a polypeptide linker (linker).

6. The fusion protein of claim 4, wherein the functional fragment further comprises an antibody or an antigen-binding portion thereof, a receptor or a ligand-binding portion thereof, a cytokine, a cytotoxin, a label, or a tracer.

7. The fusion protein according to any one of claims 4 to 6, characterized in that the functional fragment specifically binds to a target selected from the group consisting of a target for tumor or cancer immunotherapy, a target for chronic infectious disease immunotherapy, and a target for autoimmune disease therapy.

8. 8. The fusion protein of claim 7, wherein the target is epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), insulin receptor (InsR), Bruton's tyrosine kinase (BTK), HER2, CTLA, CD20, CD52, CD30, CD33, CD133, PD-1, PD-L1, Src, Abl, phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB / Akt), target of rapamycin (mTOR), serine-threonine protein kinase Ras, mitogen-activated protein kinase (MAPK), STAT1, STAT3, or STAT5.

9. 10. A multifunctionally active molecule having two or more functional activities, characterized in that at least one of the functional activities is TGF-β binding activity conferred by a polypeptide fragment having the amino acid sequence of a TGF-β RII mutant according to any one of claims 1 to 3.

10. 10. The multifunctionally active molecule of claim 9, wherein the functional activity further comprises antigen-binding activity, ligand-binding activity, cytokine activity, cytotoxicity, or labeling activity.

11. 11. The multifunctionally active molecule of claim 10, wherein the functional activity comprises binding activity to the following molecules: epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), insulin receptor (InsR), Bruton's tyrosine kinase (BTK), HER2, CTLA, CD20, CD52, CD30, CD33, CD133, PD-1, PD-L1, Src, Abl, phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB / Akt), target of rapamycin (mTOR), serine-threonine protein kinase Ras, mitogen-activated protein kinase (MAPK), STAT1, STAT3, or STAT5.

12. 10. An antibody-TGF-β RII conjugate molecule, characterized in that the antibody targets a target for tumor therapy and the TGF-β RII has the amino acid sequence of a TGF-β RII mutant according to any one of claims 1 to 3.

13. 13. The antibody-TGF-β RII conjugate molecule of claim 12, wherein the antibody specifically targets epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), insulin receptor (InsR), Bruton's tyrosine kinase (BTK), HER2, CTLA, CD20, CD52, CD30, CD33, CD133, PD-1, PD-L1, Src, Abl, phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB / Akt), target of rapamycin (mTOR), serine-threonine protein kinase Ras, mitogen-activated protein kinase (MAPK), STAT1, STAT3, or STAT5.

14. 13. The antibody-TGF-β RII conjugate molecule of claim 12, wherein the antibody is a murine antibody, a chimeric antibody, a humanized antibody, a Fab antibody, a Fab′ antibody, a F(ab′)2 antibody, an Fv antibody, a scFv antibody, or a nanobody.

15. The antibody-TGF-β RII conjugate molecule of claim 12, wherein the antibody is an anti-human PD-L1 antibody or an antigen-binding fragment thereof, and the anti-human PD-L1 antibody or antigen-binding fragment thereof has a heavy chain comprising CDR1 set forth in SEQ ID NO:25, a CDR2 set forth in SEQ ID NO:26, and a CDR3 set forth in SEQ ID NO:27, and a light chain comprising CDR1 set forth in SEQ ID NO:28, a CDR2 set forth in SEQ ID NO:29, and a CDR3 set forth in SEQ ID NO:

30.

16. The antibody-TGF-β RII conjugate molecule of claim 12, wherein the antibody is an anti-human PD-L1 nanobody, and the amino acid sequence of the anti-human PD-L1 nanobody is SEQ ID NO:

20.

17. 17. The antibody-TGF-β RII conjugate molecule of claim 15 or 16, wherein the TGF-β RII is linked to the anti-human PD-L1 antibody via a linker peptide.

18. The antibody-TGF-β RII conjugate molecule of claim 17, wherein the linker peptide comprises (G 4 S) n , where n is an integer from 1 to 4.

19. A composition comprising a TGF-β RII mutant according to any one of claims 1 to 3, a fusion protein according to any one of claims 4 to 8, a multifunctionally active molecule according to any one of claims 9 to 11 or a conjugated molecule according to any one of claims 12 to 18, and a pharmaceutically acceptable excipient.

20. A nucleic acid encoding a TGF-β RII mutant according to any one of claims 1 to 3, a fusion protein according to any one of claims 4 to 8, a multifunctionally active molecule according to any one of claims 9 to 11 or a conjugate molecule according to any one of claims 12 to 18.

21. 21. A recombinant vector or recombinant host cell comprising the nucleic acid of claim 20.

22. 22. A method for producing a product, comprising using a nucleic acid according to claim 20, or a recombinant vector or recombinant host cell according to claim 21, to produce a TGF-β RII mutant, a fusion protein thereof, a multifunctionally active molecule thereof, or a conjugated molecule thereof.

23. 1. A method of reducing or eliminating degradation or cleavage of a recombinant protein comprising a TGF-β RII fragment, comprising subjecting a coding region encoding the TGF-β RII fragment in the recombinant protein to mutagenesis so that the TGF-β RII encoded by the coding region contains a single mutation at one amino acid residue position selected from the group consisting of positions 6, 12 and 20 compared to wild-type TGF-β RII, the single mutation being selected from the group consisting of Q6N, D12T and G20T, wherein the numbering of amino acid residues in the wild-type TGF-β RII is based on SEQ ID NO:

6.

24. 21. Use of a product in the manufacture of a medicament for therapy, characterized in that the product comprises a TGF-β RII mutant according to any one of claims 1 to 3, a fusion protein according to any one of claims 4 to 8, a multifunctionally active molecule according to any one of claims 9 to 11, a conjugated molecule according to any one of claims 12 to 18, a composition according to claim 19, a nucleic acid according to claim 20, or a recombinant vector or recombinant host cell according to claim 21.

25. 25. The use according to claim 24, characterized in that the medicament is for the prevention or treatment of tumors or cancer, chronic infectious diseases, or autoimmune diseases.

26. 26. The use according to claim 25, characterized in that the tumor or cancer is selected from the group consisting of pharyngeal squamous cell carcinoma, non-small cell lung cancer, pancreatic cancer, liver cancer, urothelial carcinoma, colon cancer and gastric cancer.

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