RU2843303C1 - BIFUNCTIONAL PROTEIN AGAINST PD-1 AND TGF-β - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to antitumour bifunctional proteins, and can be used in medicine to prevent or treat a malignant tumour. Disclosed are bifunctional proteins that can bind to PD-1 (programmed cell death-1 receptor) and TGF-β (transforming growth factor-β), their medical application and method of production.

EFFECT: invention enables to obtain fusion proteins based on anti-PD-1 antibodies and TGFβRII, which have activity of binding PD-1 and TGFβ, exceeding activity of fused protein nivolumab/TGF-βRII, as well as tumour inhibitory action of PD1/TGFβRII exceeding action of nivolumab/TGFβRII.

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Description

Reference to related patent applications

The present invention claims priority from Chinese Patent Application No. 202010359751.8, filed on April 29, 2020, incorporated herein by reference for all purposes.

Field of invention

The description of the present invention generally relates to the field of antibody-based drugs, and in particular to the treatment of malignant tumors. In particular, the description of the present invention provides a bifunctional protein that can bind to PD-1 (programmed cell death receptor-1) and TGF-β (transforming growth factor-β), and a medical use of the bifunctional protein.

Prior art

T cells express many important membrane protein immune molecules, including PD-1 (programmed cell death protein-1, programmed cell death protein-1 receptor, also known as CD279), a member of the CD28 family of immunoglobulin superfamily, and its ligands (PD-L1, PD-L2) belong to the B7 family. PD-L1 negatively regulates T cell immune function after binding to PD-1, and is an important inhibitory immune checkpoint of peripheral T cells. Low expression of PD-L1 in normal human tissue can maintain immune tolerance and avoid autoimmune reaction. However, tumor cells inhibit T cell immune function by highly expressing PD-L1 (or releasing soluble PD-L1 and exosomes), thus forming an immune-inhibitory tumor immune microenvironment. T cell immune function can be preserved by blocking the PD-1/PD-L1 signaling pathway, so that tumor cells can be recognized and destroyed.

TGF-β (transforming growth factor-β) is a class of cytokines with multifunctional biological activities that can regulate physiological processes in the body by regulating proliferation, differentiation, apoptosis, adhesion, invasion and microenvironment of cells. In a typical TGF-β signaling pathway, TGF-β first binds to TGF-β receptor type II (TGF-βR II) and then forms a complex with TGF-β receptor type I (TGFβRI) to activate TGFβRI, TGFβRI phosphorylates and activates R-Smad members (Smad 1, 2, 3, 5, 8), and R-Smad then associates with Co-Smad (Smad 4) to form a complex and translocates to the nucleus to regulate the transcription of target genes.

In the tumor microenvironment, high expression of TGF-β shows a tendency to be associated with invasion, metastasis, immune evasion, treatment resistance and worse prognosis (David Charles J et al., TGF-β Tumor Suppression through a Lethal EMT. [J]. Cell, 2016, 164(5)). Studies have also demonstrated that TGF-β likely disrupts the tumor microenvironment by inducing Treg cells (central regulators of the immune response) and inhibiting effector T cells, thus accelerating tumor progression (Shen Yinan et al., TGF-β regulates hepatocellular carcinoma progression by inducing Treg cell polarization. [J]. Cellular physiology and biochemistry, 2015, 35(4)). The researchers also believe that TGF-β signaling is responsible for the development of resistance to the anti-PD-(L)1 antibody drug in patients.

Currently, studies using TGF-β and PD-1/PD-L1 as a combined target have been reported. However, further studies are urgently needed as this research direction has good prospects.

Brief summary of the invention

In a first aspect of the description of the present invention, a bifunctional protein is provided comprising a PD-1 (programmed cell death receptor-1) binding moiety and a TGF-β (transforming growth factor-β) binding moiety.

In some embodiments, the PD-1 binding moiety is an anti-PD-1 antibody or an antigen-binding fragment thereof. In some embodiments, the PD-1 binding moiety is a full-length antibody, a Fab fragment, an F(ab') ₂ fragment, an Fv fragment, or a single-chain Fv fragment (scFv) of an anti-PD-1 antibody.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 having the amino acid sequence GFAFSSYD (SEQ ID NO: 1), HCDR2 having the amino acid sequence ISGGGRYT (SEQ ID NO: 2), and HCDR3 having the amino acid sequence ANRYGEAWFAY (SEQ ID NO: 3), and the light chain variable region comprises LCDR1 having the amino acid sequence QDINTY (SEQ ID NO: 4), LCDR2 having the amino acid sequence RAN (SEQ ID NO: 5), and LCDR3 having the amino acid sequence LQYDEFPLT (SEQ ID NO: 6). In some embodiments, the amino acid sequence of the heavy chain variable region is represented by SEQ ID NO: 7, and/or the amino acid sequence of the light chain variable region is represented by SEQ ID NO: 8. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region, wherein the amino acid sequence of the heavy chain constant region is represented by SEQ ID NO: 9 or is a variant of the amino acid sequence represented by, for example, SEQ ID NO: 9, the amino acid sequence represented by SEQ ID NO: 9, wherein the A residue at the C-terminus is replaced with K, and/or the amino acid sequence of the light chain constant region is represented by SEQ ID NO: 10 or is a variant of the amino acid sequence represented by SEQ ID NO: 10.

In other embodiments, the anti-PD-1 antibody or antigen-binding fragment is selected from: nivolumab, pembrolizumab, durvalumab, toripalimab (JS-001), sintilimab (IBI308), camrelizumab, tislelizumab (BGB-A317), AK105 (Akeso Bioscience), genolimzumab (GB226), Livzon Mabpharm (LZM009), HLX-10, BAT-1306, AK103 (HX008), AK104 (Akeso Bioscience), CS1003, SCT-I10A, F520, SG001, GLS-010 or antigen-binding fragments thereof.

In some embodiments, the TGF-β binding moiety is a TGF-β receptor or a TGF-β receptor binding domain. In some embodiments, the TGF-β binding moiety is an extracellular domain of a TGF-β receptor or a binding fragment of an extracellular domain of a TGF-β receptor. In some specific embodiments, the TGF-β binding moiety is a human TGF-βRII isoform B extracellular domain polypeptide comprising the amino acid sequence shown in SEQ ID NO: 11. In some specific embodiments, the TGF-β binding moiety is a variant of a human TGF-βRII isoform B extracellular domain polypeptide, such as a polypeptide or fragment of a peptide having 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence shown in SEQ ID NO: 11, or any fragment described herein.

In some embodiments, the TGF-β binding moiety is an anti-TGF-β antibody or antigen-binding fragment thereof. In some embodiments, the TGF-β binding moiety is a full-length antibody, a Fab fragment, an F(ab') ₂ fragment, an Fv fragment, or a single-chain Fv fragment (scFv) against TGF-β.

In some embodiments, the PD-1 binding moiety and the TGF-β binding moiety are linked by a flexible linker. In some embodiments, the flexible linker is a GGGS type linker. In some specific embodiments, the flexible linker is a linker represented by SEQ ID NO: 12.

In some embodiments, the bifunctional protein comprises: (1) two identical first polypeptides, the amino acid sequence of the first polypeptide having at least 80% identity to the amino acid sequence presented by SEQ ID NO: 13; and (2) two identical second polypeptides, wherein the amino acid sequence of the second polypeptide is at least 80% identical to the amino acid sequence presented by SEQ ID NO: 14.

In a second aspect, the present invention provides a nucleic acid molecule encoding a bifunctional protein according to the first aspect.

In a third aspect, the description of the present invention provides a pharmaceutical composition comprising a bifunctional protein according to the first aspect and a pharmaceutically acceptable excipient, diluent or carrier.

In some embodiments, the pharmaceutical composition is used to prevent or treat a malignant tumor. In some specific embodiments, the malignant tumor is selected from colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine malignancy, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and/or myelodysplastic syndromes. In some specific embodiments, the malignancy is primary, metastatic, recurrent, and/or difficult to treat.

In a fourth aspect of the description of the present invention, there is provided the use of a bifunctional protein according to the first aspect or a nucleic acid molecule according to the second aspect for the preparation of a medicament for the prevention or treatment of a malignant tumor.

In some embodiments, the malignant tumor is selected from colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine malignancy, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and/or myelodysplastic syndromes. In some specific embodiments, the malignant tumor is primary, metastatic, recurrent, and/or difficult to treat.

In a fifth aspect of the description of the present invention, there is provided a method for preventing or treating a malignant tumor, comprising administering a bifunctional protein according to the first aspect or a pharmaceutical composition according to the third aspect to a subject having a malignant tumor.

In some embodiments, the malignant tumor is selected from colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine malignancy, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and/or myelodysplastic syndromes. In some specific embodiments, the malignant tumor is primary, metastatic, recurrent, and/or difficult to treat.

In a sixth aspect of the description of the present invention, there is provided a method for producing a bifunctional protein comprising a PD-1 (programmed cell death protein receptor-1) binding moiety and a TGF-β (transforming growth factor-β) binding moiety and comprising the bifunctional protein according to the first aspect, wherein the method comprises the following steps:

introducing an expression vector containing a nucleic acid molecule encoding a bifunctional protein into a host cell and culturing the host cell under conditions that allow expression of the protein; and

collection of cell culture and/or supernatant, and isolation and purification of the bifunctional protein.

Brief description of graphic materials

Fig. 1 shows a schematic structural diagram of an example of a bifunctional PD-1/TGFβ protein according to the description of the present invention.

Fig. 2 shows the results of a reporter gene assay of the biological activity of a PD-1 binding moiety of an exemplary PD-1/TGFβ bifunctional protein according to the description of the present invention, wherein nivolumab is used as a control sample.

Fig. 3 shows the results of an enzyme-linked immunosorbent assay of the TGF-β binding activity of an exemplary PD-1/TGFβ bifunctional protein in accordance with the description of the present invention.

Description of sequences

SEQ ID NOs: 1-3 are the CDR1-CDR3 sequences of the heavy chain variable region of an anti-PD-1 antibody fragment of an exemplary PD-1/TGFβ bifunctional protein according to the disclosure of the present invention.

SEQ ID NOs: 4-6 are the CDR1-CDR3 sequences of the light chain variable region of an anti-PD-1 antibody fragment of an exemplary PD-1/TGFβ bifunctional protein according to the disclosure of the present invention.

SEQ ID NOs: 7 and 8 are sequences of the variable region of the heavy chain and the variable region of the light chain of a fragment of an anti-PD-1 antibody of an exemplary PD-1/TGFβ bifunctional protein, respectively, according to the description of the present invention.

SEQ ID NOs: 9 and 10 are the sequences of the heavy chain constant region and the light chain constant region of an anti-PD-1 antibody fragment of an exemplary PD-1/TGFβ bifunctional protein, respectively, according to the description of the present invention.

SEQ ID NO: 11 is a TGF-β binding moiety of an exemplary PD-1/TGFβ bifunctional protein according to the disclosure, i.e., a human TGF-βRII isoform B extracellular domain polypeptide.

SEQ ID NO: 12 is a flexible linker between a fragment of an anti-PD-1 antibody and a TGF-β binding moiety of an exemplary PD-1/TGFβ bifunctional protein as described herein.

SEQ ID NO: 13 is the sequence of a heavy chain fragment of an exemplary PD-1/TGFβ bifunctional protein according to the description of the present invention, wherein the heavy chain fragment consists of a heavy chain fragment of an anti-PD-1 antibody, a flexible linker (SEQ ID NO: 12) and a human TGF-βRII isoform B extracellular domain polypeptide (SEQ ID NO: 11).

SEQ ID NO: 14 is the sequence of a light chain fragment of an exemplary PD-1/TGFβ bifunctional protein according to the disclosure of the present invention, wherein the light chain fragment consists of a light chain fragment of an anti-PD-1 antibody.

SEQ ID NO: 15 is the nucleic acid sequence encoding SEQ ID NO: 13 (which does not contain the signal peptide coding sequence).

SEQ ID NO: 16 is the nucleic acid sequence encoding SEQ ID NO: 14 (which does not contain the signal peptide coding sequence).

SEQ ID NO: 17 and 18 are the heavy chain and light chain sequences of the control monoclonal antibody PD1 nivolumab, respectively.

SEQ ID NOs: 19 and 14 are the heavy chain and light chain sequences of another control monoclonal antibody PD1 (from Chinese Patent Application No. 201610705763.5 (CN 106977602)), respectively.

SEQ ID NOs: 20 and 18 are sequences of a heavy chain and a light chain fragment of a nivolumab/TGF-βRII bifunctional protein as a bifunctional control protein, respectively, wherein the heavy chain fragment consists of the nivolumab heavy chain (the C-terminal amino acid residue in SEQ ID NO: 17 has a mutation from K to A), a flexible linker (SEQ ID NO: 12), and the human TGF-βRII isoform B extracellular domain polypeptide (SEQ ID NO: 11), and the light chain fragment consists of the nivolumab light chain.

SEQ ID NO:21 and 22 are the heavy chain and light chain sequences of the experimental control IgG1 protein.

Detailed description of the invention

Definitions

The following definitions and methods are provided to better define the description of the present invention and to guide those skilled in the art in practicing the description of the present invention. Unless otherwise specified, the terms used in the description of the present invention have the meanings commonly understood by those skilled in the art. All patent documents, scientific articles and other publications cited herein are incorporated herein by reference.

As used herein, the term "antibody" refers to an immunoglobulin molecule that can specifically bind to a target via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. The target includes, but is not limited to, a carbohydrate, a polynucleotide, a lipid, a polypeptide, and the like. The term "antibody" as used herein refers not only to an intact (i.e., full-length) antibody, but also to an antigen-binding fragment thereof (e.g., Fab, Fab', F(ab') ₂ , Fv), a variant thereof, a fusion protein comprising an antibody fragment, a humanized antibody, a chimeric antibody, a diabody, a linear antibody, a single-chain antibody, a multispecific antibody (e.g., a bispecific antibody), and any other immunoglobulin molecule of a modified configuration containing an antigen recognition site of the desired specificity, including a glycosylated antibody variant, an amino acid sequence variant of an antibody, and a covalently modified antibody.

An intact or full-length antibody typically contains two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and the first, second, and third constant regions (CH1, CH2, and CH3). Each light chain contains a light chain variable region (VL) and a constant region (CL). A full-length antibody may be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or a subclass thereof), but the antibody does not have to belong to any particular class. Immunoglobulins can be divided into different classes depending on the amino acid sequences of the constant regions of the antibody's heavy chain. Generally, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these classes can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2. The heavy chain constant domains corresponding to the different immunoglobulin classes are named α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of the different immunoglobulin classes are well known.

As used herein, the term "antigen-binding fragment" refers to the portion of the antibody structure that determines antigen-binding ability. Those skilled in the art will appreciate that the major portion of the antibody structure that determines antigen-binding ability is the CDR, which is thus also the major component of the antigen-binding fragment. The antigen-binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of the VH and VL typically comprises three complementarity-determining regions, CDR1, CDR2, and CDR3.

It is well known to those skilled in the art that the complementarity determining regions (CDRs, typically CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of the antibody. For the CDR sequences in VH or VL, there are two general definitions, namely the Chothia definition and the Kabat definition (see, e.g., Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sc/.USA86:9268-9272 (1989)). For the variable region sequences of a given antibody, the CDR sequences in the VH and VL sequences may be defined according to the Chothia definition or the Kabat definition.

For the variable region sequences of a given antibody, the CDR sequences in the variable region sequence can be analyzed in various ways, for example, they can be determined using the Abysis interactive software (http://www.abysis.org/).

Examples of antigen-binding fragments include, but are not limited to: (1) a Fab fragment, which may be a monovalent fragment having a VL-CL chain and a VH-CH1 chain; (2) an F(ab') ₂ fragment, which may be a divalent fragment having two Fab' fragments linked by a disulfide bridge (e.g., a Fab' dimer) at the hinge region; (3) an Fv fragment having the VL and VH domains of a single arm of an antibody; (4) a single-chain Fv (scFv), which may be a single polypeptide chain consisting of a VH domain and a VL domain via a peptide linker; and (5) (scFv) ₂ , which may comprise two VH domains linked by a peptide linker and two VL domains combined with two VH domains via a disulfide bridge.

As used herein, the terms "Fab fragment", "Fab portion" or similar terms refer to a fragment of an antibody that can bind to an antigen and that is obtained by treating an intact antibody with the protease papain, including the intact light chain (VL-CL), the variable region of the heavy chain and the CH1 fragment (VH-CH1).

As used herein, the term "single chain variable fragment (scfv)" refers to an antibody having a single chain structure, typically constructed using genetic engineering techniques, comprising one polypeptide chain of a heavy chain variable region (VH) and a light chain variable region (VL). A flexible linker peptide is typically constructed between the heavy chain variable region and the light chain variable region such that the heavy chain variable region and the light chain variable region can be folded into the correct conformation that can bind to an antigen.

As used herein, the terms "Fc fragment", "Fc domain", "Fc region" or similar terms refer to a fragment of the constant region of an antibody heavy chain, including the hinge region and the CH2 and CH3 portions of the constant region.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigenic epitope.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for naturally occurring mutations that may be present in a small number of individuals. The monoclonal antibody described herein specifically includes a "chimeric" antibody in which a portion of the heavy chain and/or light chain is identical to or homologous to corresponding sequences in an antibody obtained from a particular species or belonging to a particular class or subclass of a particular antibody, while the remainder of the heavy chain and/or light chain is identical to or homologous to corresponding sequences in an antibody obtained from another species or belonging to a different class or subclass of antibody, and also includes fragments of such antibodies as long as they exhibit the desired biological activity.

As used herein, the term "identity" refers to the sequence similarity between two polynucleotide sequences or between two polypeptide sequences. Sequence comparison and determination of percent identity between two sequences can be accomplished using the default settings of the BLASTN/BLASTP algorithm available on the National Center for Biotechnology website.

As used herein, the term "treatment" includes therapeutic treatment and prophylactic treatment or preventive measures in which a therapeutic agent is administered to a subject to reduce at least one symptom of a disease, disorder, or condition (e.g., cancer or tumor), or to alleviate the development of symptoms.

The term "EC ₅₀ ", also known as the mean effective concentration, as used herein, refers to the concentration that will achieve 50% of the maximum effect after a specified exposure time.

In a first aspect of the description of the present invention, a bifunctional protein is provided comprising a PD-1 (programmed cell death protein receptor-1) binding moiety and a TGF-β (transforming growth factor-β) binding moiety.

In some embodiments, the PD-1 binding moiety is an anti-PD-1 antibody or antigen-binding fragment thereof. In some embodiments, the PD-1 binding moiety is a full-length antibody, a Fab fragment, an F(ab') ₂ fragment, an Fv fragment, or a single-chain Fv fragment (scFv) against PD-1.

In some embodiments, the anti-PD-1 antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1 having the amino acid sequence GFAFSSYD (SEQ ID NO: 1), HCDR2 having the amino acid sequence ISGGGRYT (SEQ ID NO: 2), and HCDR3 having the amino acid sequence ANRYGEAWFAY (SEQ ID NO: 3), and the light chain variable region comprises LCDR1 having the amino acid sequence QDINTY (SEQ ID NO: 4), LCDR2 having the amino acid sequence RAN (SEQ ID NO: 5), and LCDR3 having the amino acid sequence LQYDEFPLT (SEQ ID NO: 6). In some embodiments, the amino acid sequence of the heavy chain variable region is represented by SEQ ID NO: 7 and/or the amino acid sequence of the light chain variable region is represented by SEQ ID NO: 8. In some embodiments, the anti-PD-1 antibody or antigen-binding fragment further comprises a heavy chain constant region and a light chain constant region, wherein the amino acid sequence of the heavy chain constant region is represented by SEQ ID NO: 9 and/or the amino acid sequence of the light chain constant region is represented by SEQ ID NO: 10.

In some embodiments, the amino acid sequence of the heavy chain constant region is a variant of SEQ ID NO: 9 and/or the amino acid sequence of the light chain constant region is a variant of SEQ ID NO: 10. In some specific embodiments, the amino acid sequence of the heavy chain constant region is the amino acid sequence represented by SEQ ID NO: 9, wherein the A residue at the C-terminus is replaced with a K. Modifications of the constant region of an antibody are known to those skilled in the art. In some embodiments, the heavy chain constant region may be selected from IgG1, IgG2, IgG3, IgG4 or other classes, although IgG1 is preferred. In some embodiments, the constant region of the antibody may comprise modifications, such as insertions, deletions, substitutions or chemical modifications of amino acids. In some embodiments, any amino acid residue of the constant region may be replaced by an amino acid residue of any allotype, preferably by an amino acid residue of Glm(3) and/or nGlm(1). In some embodiments, the constant region comprises a mutation that alters the effector function. For example, a mutation of the lysine (K) residue at the C-terminus of the constant region of the heavy chain of the antibody (usually found in wild-type IgG1 antibodies) is replaced by a hydrophobic amino acid such as alanine (A) or leucine (L), thereby reducing hydrolytic cleavage by proteases and prolonging the half-life in serum, and this modification is also particularly suitable for the case where the C-terminus of the heavy chain of the antibody is further fused to another moiety. The residue at the C-terminus of the constant region of the heavy chain of the anti-PD-1 antibody fragment in the example of the PD-1/TGFβ bifunctional proteins according to the description of the present invention is treated accordingly.

In some embodiments, the TGF-β binding moiety is a TGF-β receptor or a TGF-β receptor binding domain. In some embodiments, the TGF-β binding moiety is an extracellular domain of a TGF-β receptor or a binding fragment of an extracellular domain of a TGF-β receptor. In some specific embodiments, the TGF-β binding moiety is an isoform B of the human TGF-βRII extracellular domain polypeptide comprising the amino acid sequence shown in SEQ ID NO: 11. In some specific embodiments, the TGF-β binding moiety is a variant of isoform B of the human TGF-βRII extracellular domain polypeptide, such as a fragment of a polypeptide or peptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence shown in SEQ ID NO: 11, or any the fragment described here.

In some embodiments, the TGF-β binding moiety is an anti-TGF-β antibody or antigen-binding fragment thereof. In some embodiments, the TGF-β binding moiety is a full-length antibody, a Fab fragment, an F(ab') ₂ fragment, an Fv fragment, or a single-chain Fv fragment (scFv) against TGF-β.

In some embodiments, the PD-1 binding moiety and the TGF-β binding moiety are linked by a flexible linker. In some embodiments, the flexible linker is a linker having the GGGS type. In some specific embodiments, the flexible linker is a linker represented by SEQ ID NO: 12.

In some embodiments, the bifunctional protein comprises: (1) two identical first polypeptides, wherein the amino acid sequence of the first polypeptide has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence set forth in SEQ ID NO: 13; and (2) two identical second polypeptides, wherein the amino acid sequence of the second polypeptide has at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence set forth in SEQ ID NO: 14.

As a non-limiting example, a bifunctional protein (i.e., a PD-1/TGFβ bifunctional protein, hereinafter also referred to as a "PD1/TGFβRII fusion protein" or "PD1/TGFβRII") according to the disclosure of the present invention may be comprised of an anti-PD-1 antibody (the amino acid sequences of its heavy chain variable region, light chain variable region, heavy chain constant region, and light chain constant region are represented by SEQ ID NOs: 7, 8, 9, and 10, respectively), a flexible linker (SEQ ID NO: 12), and a human TGF-βRII isoform B extracellular domain polypeptide (SEQ ID NO: 11), and a schematic diagram of the molecular structure of the bifunctional protein is shown in Fig. 1. In accordance with the present disclosure, 1 PD1/TGFβRII, based on the natural anti-PD-1 antibody, continues with a flexible linker and the extracellular domain polypeptide of human TGF-βRII isoform B in the CH3-terminal sequence of the heavy chain constant region.

PD1/TGFβRII is an example of a bifunctional protein according to the present invention, and has a higher TGFβ binding activity and a biological activity of a PD-1 binding moiety, and even better tumor suppressive effect than that reported for the nivolumab/TGF-βRII fusion protein. In addition, PD1/TGFβRII has less toxicity and side effects than existing anti-PD-1 antibodies such as nivolumab. Considering that existing anti-PD-1 antibodies have greater cytotoxicity and side effects, the fact that PD1/TGFβRII has less cytotoxicity and side effects allows for higher dose administration for better TGFβ suppression and use with a more desirable dose safety window, and facilitates higher dose administration and clinical application.

In a second aspect of the description of the present invention, there is provided a nucleic acid molecule encoding a bifunctional protein according to the first aspect.

In a third aspect of the description of the present invention, there is provided a pharmaceutical composition comprising a bifunctional protein according to the first aspect and a pharmaceutically acceptable excipient, diluent or carrier.

In some embodiments, the pharmaceutical composition is used to prevent or treat a malignant tumor. In some specific embodiments, the malignant tumor is selected from colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine malignancy, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and/or myelodysplastic syndromes. In some specific embodiments, the malignant tumor is a primary, metastatic, recurrent, and/or difficult-to-treat tumor.

In some embodiments, the pharmaceutical composition may further comprise a lubricant such as talc, magnesium stearate and mineral oil; a humectant; an emulsifier; a suspending agent; a preservative such as benzoic acid, sorbic acid and calcium propionate; a sweetener and/or flavoring agent, etc.

In some embodiments, the pharmaceutical composition according to the description of the present invention can be prepared in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules, etc.

In some embodiments, the pharmaceutical composition according to the description of the present invention can be delivered using any physiologically acceptable route of administration, including, but not limited to: oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, administration by inhalation, etc.

In some embodiments, a pharmaceutical composition for therapeutic use can be prepared for storage in lyophilized compositions or aqueous solutions by mixing an agent having the desired purity with possible pharmaceutically acceptable carriers, excipients, etc.

In a fourth aspect of the description of the present invention, there is provided the use of a bifunctional protein according to the first aspect or a nucleic acid molecule according to the second aspect for the preparation of a medicament for the prevention or treatment of a malignant tumor.

In some embodiments, the malignant tumor is selected from colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine malignancy, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and/or myelodysplastic syndromes. In some specific embodiments, the malignant tumor is a primary, metastatic, recurrent and/or difficult to treat tumor.

In a fifth aspect of the description of the present invention, there is provided a method for preventing or treating a malignant tumor, comprising administering a bifunctional protein according to the first aspect or a pharmaceutical composition according to the third aspect to a subject having a malignant tumor.

In some embodiments, the malignant tumor is selected from colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, prostate cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, thyroid cancer, endometrial cancer, uterine cancer, bladder cancer, neuroendocrine malignancy, head and neck cancer, liver cancer, nasopharyngeal cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and/or myelodysplastic syndromes. In some specific embodiments, the malignant tumor is a primary, metastatic, recurrent and/or difficult to treat tumor.

In a sixth aspect of the description of the present invention, a method for preparing a bifunctional protein comprising a PD-1 (programmed cell death receptor-1) binding moiety and a TGF-β (transforming growth factor-β) binding moiety is provided, wherein the method comprises the following steps:

introducing an expression vector containing a nucleic acid molecule encoding a bifunctional protein into a host cell and culturing the host cell under conditions that allow expression of the protein; and

collection of cell culture and/or supernatant, and isolation and purification of the bifunctional protein.

Without contradiction, the embodiments and technical features described in the first aspect also apply in the sixth aspect.

In some embodiments, the host cell is a mammalian cell, such as a CHO cell (Chinese hamster ovary cell).

In some embodiments, the supernatant is collected after centrifugation of the cell culture.

In some embodiments, purification of the bifunctional protein is accomplished by using one or more of affinity chromatography, anion exchange chromatography, and cation exchange chromatography. In some aspects of affinity chromatography, the elution solution comprises sucrose or glycerol. The inventors of the present disclosure have found that an elution solution to which sucrose or glycerol is added is beneficial for reducing degradation of the fusion protein.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the description of the present invention.

Examples

Example 1: Expression of PD1/TGFβRII fusion protein

In this example, a PD1/TGFβRII fusion protein was constructed according to the description of the present invention, and its schematic structural diagram is shown in Fig. 1. A nucleotide sequence encoding a heavy chain fragment (SEQ ID NO: 13) and a light chain fragment (SEQ ID NO: 14) of the PD1/TGFβRII fusion protein fused to a signal peptide was synthesized and cloned into the pcDNA3.1 expression vector. The PD1/TGFβRII fusion protein expression vector was co-transfected into CHO cells using standard protocols for transient or permanent transfection, and the transfected cells were cultured in an incubator at 37°C with 8% CO ₂ .

Amino acid sequence (SEQ ID NO: 11) of the extracellular domain polypeptide of human TGF-βRII isoform B included in SEQ ID NO: 13:

The amino acid sequence (SEQ ID NO: 12) of the linker included in SEQ ID NO: 13:

Amino acid sequence (SEQ ID NO: 13) of the heavy chain of the PD1/TGFβRII fusion protein:

Amino acid sequence (SEQ ID NO: 14) of the light chain of the PD1/TGFβRII fusion protein:

Example 2: Purification of PD1/TGFβRII fusion protein

The cell culture obtained in Example 1 was centrifuged, and the supernatant was collected and subjected to the first step of purification using protein A affinity chromatography. The equilibration buffer was 10 mmol/L phosphate-buffered saline at pH 6.0. After the chromatography column was washed with the equilibration buffer by 3-5 column volumes, the cell supernatant was loaded. After the loading was completed, the chromatography column was washed with the equilibration buffer. Then, the chromatography column was washed with a washing buffer (0.5 mol/L sodium chloride + 25 mmol/L phosphate-buffered saline at pH 7.0), and then equilibrated by 3-5 column volumes with the equilibration buffer. Finally, the chromatography column was washed with elution buffer (20 mmol/L citrate-buffered saline +5% sucrose, pH 3.6), and the eluted sample was collected and neutralized with 2 M Tris-HCl buffer (pH 9.5).

The above eluted sample (pH 6.0) was subjected to anion exchange chromatography after neutralization. The equilibration buffer was 10 mmol/L citrate-buffered saline +10 mmol/L phosphate-buffered saline +10 mmol/L Tris at pH 6.0. After washing the chromatographic column with the equilibration buffer in an amount of 3-5 column volumes, the above eluted sample after neutralization was applied, the sample passed through the column was collected, and the chromatographic column was washed with the equilibration buffer after completion of the application.

The above-described anion exchange chromatography column sample was subjected to cation exchange chromatography. The equilibration buffer was 10 mmol/L citric acid + 10 mmol/L sodium dihydrogen phosphate + 10 mmol/L Tris buffer at pH 5.0. The above-described anion chromatography column sample was adjusted to pH 5.0 and loaded, and the chromatography column was washed with 3 to 5 column volumes of the equilibration buffer after completion of loading. Then, the chromatography column was eluted with an elution buffer (10 mmol/L citrate + 10 mmol/L phosphate + 10 mmol/L Tris buffer, pH 9.0), and the eluate was collected.

Example 3: Detection of PD1/TGFβRII Fusion Protein Sample by Gel Filtration Chromatography The components of the purified PD1/TGFβRII fusion protein sample in Example 2 were separated using gel filtration column chromatography. Elution was performed using a neutral buffer as the mobile phase, and components with different molecular weights were eluted in descending order according to their molecular weights. The column used for gel filtration chromatography was Thermo MabPac™ SEC-1 300A (30 nm), 5 μm, 7.8 × 300 mm with a mobile phase (20 mmol/L disodium hydrogen phosphate + 300 mmol/L sodium chloride + 2% isopropanol solution pH=7.4). The sample was eluted with the mobile phase to obtain 1 mg/mL of the test solution, 50 µL of which were accurately measured and injected into the liquid chromatograph with detection at 280 nm. The flow rate was 0.5 mL/min, and isocratic elution was performed for 35 min.

The results were quantitatively analyzed using the peak area normalization method. The peak area percentages of high molecular weight impurities, immunoglobulin monomers, and low molecular weight impurities were calculated. After detection, the peak area percentage of high molecular weight impurities in the PD1/TGFβRII fusion protein sample was 0.19%, the peak area percentage of immunoglobulin monomers was 99.81%, and no low molecular weight impurities were detected.

The control bifunctional protein (nivolumab/TGF-βRII fusion protein) used in the following examples was prepared according to the same method, wherein the amino acid residue at the C-terminus of the original nivolumab heavy chain constant region was changed from K to A, and the control bifunctional protein was matched with the exemplary PD1/TGFβRII fusion protein as described herein.

Example 4: Biological activity of the PD-1 binding moiety of the PD1/TGFβRII fusion protein according to a reporter gene assay

The assay process is described as follows: CHO-PDL1-CD3L cells (purchased from the National Institutes of Food and Drug Administration) were harvested at logarithmic growth phase, and the viable cell density was adjusted to 5× ¹⁰⁵ cells/mL using complete DMED (Dulbecco's modified Eagle's medium)/P12 medium. The cells were added to a 96-well all-white plate at 100 μl/well, and the plate was incubated in a cell incubator with 5% _CO2 at 37°C for 16 to 20 h. The Jurkat-PD-1-NFAT cell suspension (purchased from the National Institutes of Food and Drug Administration) was prepared the following day, and the viable cell density was adjusted to 2 × 10 ⁶ cells/ml using 1640 basal medium containing 2% FBS (fetal bovine serum). The 96-well all-white plate to which the CHO-PDL1-CD3L cells were added was removed from the incubator, the supernatant was discarded, and the Jurkat-PD-1-NFAT cell suspension was added to the plate at 50 μl/well. Then, serial dilutions (with an initial concentration of 200,000 ng/mL, 3-fold serial dilutions, a total of 11 dilutions) of the control anti-PD-1 antibody (nivolumab, the amino acid sequences of the heavy chain and light chain are shown in SEQ ID NOs: 17 and 18, respectively) or the fusion protein, PD1/TGFβRII, prepared in Examples 1 to 3 according to the description of the present invention were added in an amount of 50 μL/well to the above-mentioned 96-well all-white plate, and the plate was incubated in a cell incubator with 5% CO ₂ at 37°C for 4 to 6 h. During the incubation, Bio-Lite luciferase reagent (Vazyme, DDI201-03) was exposed, melted at room temperature, and added to the above-mentioned 96-well all-white plate in an amount of 100 μl/well after completion of incubation. The plate was incubated in the dark at room temperature for 2 to 3 min, and the RLU (relative light units) value was recorded using a multifunctional microplate reader (Thermo, Varioskan Flash). The experimental data were analyzed using Prism software, dose response curves were plotted for the control and test sample to obtain _EC50 (median effective concentration) values for the control and test sample, and the biological activity of the test sample was calculated.

Biological activity (%) of test sample = (EC ₅₀ value for control/EC ₅₀ value for test sample) × 100%

The biological activity results for the PD-1 binding moiety of the PD1/TGFβRII fusion protein in the examples according to the description of the present invention are presented in Table 1 and Fig. 2, and the results demonstrate that the fusion protein retains the ability to bind to human PD-1.

Example 5: TGFβ binding activity of PD1/TGFβRII fusion protein according to enzyme immunoassay

The analysis process is as follows:

1) A 96-well high absorbance plate was coated with 2 μg/ml human TGFpl protein (Sinobiological, 10804-H08H) serving as an antigen at 100 μl/well and incubated overnight at 2-8°C.

2) After washing the 96-well plate three times with PBST20 (PBS (phosphate-buffered saline) solution containing 0.05% Tween 20) at 250 μl/well, blocking solution (PBS solution containing 3% BSA (bovine serum albumin)) was added to the plate at 250 μl/well and incubated at 25°C for 2 h.

3) After washing the 96-well plate three times with PBST20 at 250 μl/well, serial dilutions of the PD1/TGFβRII fusion protein obtained in Examples 1-3 according to the description of the present invention (with an initial concentration of 4000 ng/ml, 4-fold serial dilutions, a total of 7 dilutions) were added thereto at 100 μl/well and incubated at 25°C for 2 h.

6) After washing the 96-well plate three times with PBST20 at 250 μl/well, 100 μl of goat anti-human IgG antibody with HRP (horseradish peroxidase) (PE, NEF802001EA) diluted to 1:3500 were added to each well and incubated at 25°C for 1 h.

7) After washing the 96-well plate five times with PBST20 in an amount of 250 μl/well, TMB (tetramethylbenzidine) solution was added to it in an amount of 100 μl/well and incubated at 25°C for 5 min in the dark.

10) The reaction was stopped by adding 1 mol/L _H2SO4 at ₁₀₀ μL/well, and the mixture was left at room temperature for 5 min. The OD values at 450 nm/650 nm were measured using a microplate reader (Thermo Scientific, Varioskan Flash), and the data were analyzed using Graphpad Prism.

Binding activity (%) of test sample = (EC ₅₀ value for control/EC ₅₀ value for test sample) × 100%

The in vitro binding results of the PD1/TGFβRII fusion protein in the examples according to the description of the present invention with TGFβ1 are presented in Table 2 and Fig. 3, and the ELISA results demonstrate that the fusion protein retains binding activity to human TGFβ.

Example 6: Efficacy of PD1/TGFβRII Fusion Protein against Murine Subcutaneously Transplanted MC38/hPD-L1 Colon Cancer Cell Tumor

C57/PD-1 transgenic mice (purchased from Jiangsu GemPharmatech Co., Ltd.) were used as experimental mice, and each mouse was subcutaneously inoculated with 3× ¹⁰⁵ MC38/hPD-L1 cells. When the tumor size reached 40-70 mm3, the ^mice were grouped according to the tumor volume and injected with the drug intraperitoneally (ip) once every 2 days for a total of 6 times, and the injected volume was 0.1 ml/10 g body weight. The dosing schedule is shown in Table 3, and the administration day was designated as D0. The tumor diameters were measured twice a week using a vernier caliper. The effect of the drug on tumor growth was examined based on the obtained T/C% or tumor growth inhibition TGI (%) calculated using the following formulas. At the end of the experiment, when it was stopped or when the tumor volume reached 1500 mm ³ , the animals were killed using CO ₂ anesthesia and dissected to remove the tumors. The tumors were photographed.

The formula for calculating the tumor volume (V) is: V=1/2×a× ^b2 , where a and b represent the length and width, respectively; T/C(%)=(T- _T0 )/(C- _C0 )×100, where T and C are the tumor volumes of the treated mice and the negative control mice at the end of the experiment, respectively; To and Co are the tumor volumes of the treated mice and the negative control mice, respectively, at the beginning of the experiment; and the T/C values of the treated group and the negative control group are calculated according to the T/C values of the treated mice and the negative control mice, respectively; tumor growth inhibition (TGI)(%)=100-T/C(%). According to the data shown in Table 4, the tumor growth suppression rate of PD1/TGFβRII (3.7 mg/kg, IP, twice a day, 6 times in total) obtained in Examples 1-3 according to the description of the present invention against subcutaneously transplanted MC38/hPD-L1 tumors at D19 was 74%, which was superior to that of the control anti-PD-1 monoclonal antibody; the tumor-bearing mice could tolerate the drug well and did not show symptoms such as significant weight loss.

Note: hIgG4 (Sino Biological Inc, HG4K) was used as a negative control. The control anti-PD1 monoclonal antibody is 14C12H1L1 antibody described in Chinese Patent Application No. 201610705763.5 (CN 106977602), and the amino acid sequences of the heavy chain and the light chain are shown in SEQ ID NOs: 19 and 14, respectively, in the description of the present invention.

Example 7: In vitro activity assay of PD1/TGFβRII fusion protein An example of PD1/TGFβRII fusion protein according to the description of the present invention and nivolumab/TGF-βRII fusion protein were prepared in batch according to the method in Examples 1-3. The biological activity of the PD-1 binding moiety of the PD1/TGFβRII fusion protein was examined by the reporter gene assay according to Example 4 and compared with that of the nivolumab/TGF-βRII fusion protein, and the results demonstrated that the biological activity of the PD-1 binding moiety of the PD1/TGFβRII fusion protein was superior to that of the nivolumab/TGF-βRII fusion protein. The results are shown in Table 5. The TGFβ-binding activity of the PD1/TGFβRII fusion protein was examined by the enzyme-linked immunosorbent assay according to Example 5 and compared with that of the nivolumab/TGF-βRII fusion protein, and the results showed that the TGFβ-binding activity of the PD1/TGFβRII fusion protein was superior to that of the nivolumab/TGF-βRII fusion protein. The results are shown in Table 6. The nivolumab/TGF-βRII fusion proteins in this example and the following examples were prepared by themselves, and they had the heavy chain and light chain sequences shown in SEQ ID NOs: 20 and 18, respectively.

Example 8: Efficacy of PD1/TGFβRII fusion protein against MC38/hPD-L1 tumor transplanted into mice

Humanized PD-1 mice (purchased from Biocytogen Beijing Co., Ltd.) were used as experimental mice, and 4× ¹⁰⁵ MC38/hPD-L1 cells were inoculated into the right axillae of each mouse. When the tumors grew to ^100-300mm3 , the mice were randomly divided into 3 groups and injected with the drug intraperitoneally (i.p.) for a total of 8 times. The drug administration schedule is shown in Table 7, and the administration day was designated as D0. Tumor volumes were measured 2-3 times a week, and the body weights of mice were recorded 2-3 times a week. Tumor diameters were measured using a vernier caliper. The effect of the drug on tumor growth was examined based on T/C% or tumor growth inhibition (1-T/C) calculated according to the formulas. At the end of the experiment, the animals were euthanized using _CO2 anesthesia and dissected to remove the tumors. The tumors were photographed.

The formula for calculating the tumor volume (TV) is: TV=1/2 × a × ^b2 , where a and b are the length and width, respectively; the formula for calculating the relative tumor volume (RTV) is: RTV=(TV _t ) / (TV ₀ ), where TV ₀ is the tumor volume of the mouse at D0, and TV _t is the tumor volume of the mouse at each measurement; the formula for calculating the relative tumor proliferation rate T/C (%) is: T/C (%)= _TRTV / _CRTV × 100%, where _TRTV represents the RTV for the treated group and _CRTV represents the RTV for the PBS group.

The results are shown in Tables 8 and 9, and the tumor volume inhibition rates of PD1/TGFβRII and nivolumab/TGFβRII (prepared in the same batches as shown in Example 7) on mice bearing MC38/hPD-L1 tumors transplanted at D23 were 46.8% and 32.3%, respectively, demonstrating that the tumor inhibitory effect of PD1/TGFβRII according to the present invention was superior to that of nivolumab/TGFβRII. In addition, the body weight of mice in the PD1/TGFβRII group and the nivolumab/TGF-βRII group increased with variations, indicating that neither fusion protein caused obvious toxic responses.

Example 9: Stimulation of cytokine secretion measured by electrochemiluminescence by PD1/TGFβRII fusion protein

PBMCs (peripheral blood mononuclear cells) were adjusted to a concentration of approximately 2× ¹⁰⁶ cells/mL with RPMI1640 complete medium and then added to a 96-well cell culture plate at 100 µL/well. IgG1 protein (the amino acid sequences of the heavy chain and the light chain represented by SEQ ID NO: 21 and SEQ ID NO: 22, respectively, were prepared in house), LPS (SIGMA, L4391-1MG), and PD1/TGFβRII fusion protein according to the description of the present invention were diluted in RPMI1640 complete medium to prepare, respectively, 900 μg/ml IgG1 protein, 1 μg/ml LPS, and 10 μg/ml, 100 μg/ml, and 900 μg/ml PD1/TGFβRII fusion proteins, and RPMI1640 complete medium was used as a negative control. The above prepared solutions were added to a 96-well cell culture plate at 100 μl/well and mixed well. The plate contents were cultured in a cell incubator with 5% _CO2 at 37°C. The cell supernatant was collected from the 96-well plate after 48 h. The levels of IL-2, IL-6, IL-8, IL-10, TNF-α, and IFN-γ were measured using the V-PLEX Proinflammatory Panel 1 (human) kit (MSD, K15049D-2) and an ultrasensitive multifactorial electrochemiluminescence analyzer (MSD, QuickPlexSQ120), and the results are presented in Table 10.

As can be seen from the results in the above table, the PD1/TGFβRII fusion protein according to the present invention shows a low probability of causing a cytokine storm. Thus, the subject is essentially free of the risk of systemic inflammation caused by overactivation of the immune system after administration.

Example 10: Toxicity analysis of PD1/TGFβRII fusion protein in cynomolgus monkey

Single Dose Toxicity: In this test, 4 cynomolgus macaques were divided into two groups of 2 animals, half male and half female in each group. The two groups of macaques were intravenously administered a single dose of 200 mg/kg or 500 mg/kg of the PD1/TGFβRII fusion protein according to the description of the present invention, respectively, and observed for 14 days. During the test, general observation was performed, and parameters such as body weight, food intake, body temperature, two-lead ECG (electrocardiogram) and blood pressure, hematology, blood biochemistry, and urine were recorded. General anatomical observation was performed at the end of the study.

After administration, the food intake of the male macaques in each group decreased in a short time and recovered on the 8th to 9th day of testing. On the 14th day of testing, RBC (red blood cells), HGB (hemoglobin), and HCT (hematocrit) decreased in the male macaques in each group. In addition, no obvious abnormal change was found in other indices. In this single dose toxicity test, cynomolgus macaques were intravenously administered a single dose of 200 mg/kg or 500 mg/kg of the PD1/TGFβRII fusion protein according to the description of the present invention, and the MTD (maximum tolerated dose) was 500 mg/kg.

Repeated Dose Toxicity: In this test, 40 cynomolgus macaques were divided into 4 groups of 10 animals, half male and half female in each group, i.e., control group and treatment groups, received 15 mg/kg, 50 mg/kg and 150 mg/kg of the PD1/TGFβRII fusion protein as described herein. The drug was administered once a week for 4 weeks (a total of 5 doses). After completion of the administration, the macaques were observed for 4 weeks.

A decrease in RBC, HGB and HCT, and a compensatory increase in RET (reticulocytes) and RET% could be found in the male macaques in the 50 mg/kg group and in the male and female macaques in the 150 mg/kg group on the 15th day of administration and at the end of administration; on the 15th day of administration, the above changes could also be found in the female macaques in the 50 mg/kg group.

At the end of administration, cardiac pericardial adhesions could be found in the general anatomy of 1 male macaque monkey in the 50 mg/kg group. Histopathological examination: very weak to weak mononuclear cell infiltration in the pia mater of the brain and choroid plexus, pia mater of the cerebellum and choroid plexus, splenium membrane, thyroid gland, heart and pituitary gland, and very moderate to moderate vascular and/or perivascular inflammation in the heart, liver, urinary bladder, epididymis, seminal vesicles could be found in the cynomolgus macaque monkeys in the 150 mg/kg group; Very mild to moderate mononuclear cell infiltration in the meninges, sciatic nerve, thyroid gland, heart and pituitary gland, and very mild to moderate vascular and/or perivascular inflammation in the heart, bladder, duodenum, ileum, rectum, fallopian tube, vagina, uterus could be found in cynomolgus monkeys in the 50 mg/kg group; very mild to moderate mononuclear cell infiltration in the meninges and choroid plexus, cerebellar choroid plexus, sciatic nerve, thyroid gland and heart, and mild vascular and/or perivascular inflammation in the heart could be found in cynomolgus monkeys in the 15 mg/kg group. In addition, 1 cynomolgus monkey in the 50 mg/kg and 150 mg/kg groups, respectively, had mild femoral necrosis and epiphyseal plate thickening, and very mild to moderate increases in femoral metaphyseal trabeculae and osteoclasts.

In addition, no dead or dying animals were found, the cynomolgus macaques in each group were in good general condition, and no obvious abnormal changes were found in body weight, food intake, body temperature, two-lead electrocardiogram, respiratory rate, serum chemistry, ophthalmology, urine examination, bone marrow smear, organ ratio and weight, immune indices such as IgA, IgM, IgG, C3, C4, CIC (circulating immune complex) and lymphocyte subset, and TSH (thyroid stimulating hormone), T3, T4 and organ ratio and weight.

In the repeated dose toxicity test, cynomolgus monkeys were intravenously administered 15 mg/kg, 50 mg/kg or 150 mg/kg of the PD1/TGFβRII fusion protein as described herein (once a week for 4 weeks, 5 doses in total), and the HNSTD (highest no-no toxic dose) was 150 mg/kg. According to the information disclosed in the nivolumab evaluation report in the European Medicines Agency, the HNSTD of nivolumab was determined to be 50 mg/kg, which is much lower than the HNSTD of the PD1/TGFβRII fusion protein as described herein. As can be seen, the PD1/TGFβRII fusion protein as described herein exhibits low toxicity. Therefore, it can be expected that this fusion protein can exhibit good safety in clinical use.

Although the description of the present invention has been described in detail with respect to the general description and the specific embodiments given above, it is obvious to those skilled in the art that modifications and improvements can be made based on the description of the present invention. Accordingly, these modifications or improvements made without departing from the spirit of the description of the present invention are within the scope of protection of the description of the present invention.

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SEQUENCE LIST

<110> Chia Tai Tianqing Pharmaceutical Group Co., Ltd.

<120> BIFUNCTIONAL PROTEIN AGAINST PD-1 AND TGF-β

<150>CN202010359751.8

<151> 2020-04-29

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Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Asn Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Asn Arg Tyr Gly Glu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala

435 440 445

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

450 455 460

Gly Gly Gly Ser Gly Ile Pro Pro His Val Gln Lys Ser Val Asn Asn

465 470 475 480

Asp Met Ile Val Thr Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu

485 490 495

Cys Lys Phe Cys Asp Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser

500 505 510

Cys Met Ser Asn Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu

515 520 525

Val Cys Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu

530 535 540

Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu

545 550 555 560

Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly

565 570 575

Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn

580 585 590

Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn Pro Asp

595 600 605

<210> 14

<211> 214

<212> PRT

<213> Artificial sequence

<400> 14

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Met Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Phe Thr Cys Arg Ala Ser Gln Asp Ile Asn Thr Tyr

20 25 30

Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile

35 40 45

Tyr Arg Ala Asn Arg Leu Val Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Gln Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Met Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Leu

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 15

<211> 1815

<212> DNA/RNA

<213> Artificial sequence

<400> 15

gaggtacaac tggtggaatc cggtggcggt ctcgtgcagc ctggcggcag cctgagactg 60

agttgtgctg cttctggctt tgccttctcc tcctacgaca tgtcctgggt gcggcaggcc 120

cctggaaagg gcctggattg ggtggccacc atttccggag gcggcagata cacctactac 180

cctgactctg tcaagggccg gttcaccatc tccagagaca actccaagaa caacctgtac 240

ctgcagatga actccctgag agccgaggac accgccctgt actactgcgc caaccggtac 300

ggcgaggctt ggttcgccta ctggggccaa ggcaccctcg tgaccgtatc atccgcctcc 360

acaaagggcc cttctgtgtt ccctctggcc ccttcctcca agtctacaag cggaggcacc 420

gctgctctgg gctgcctggt caaggactac ttccccgaac ccgtgaccgt gtcttggaac 480

tccggcgctc tgacctctgg agtgcatacc ttccctgccg tgctgcagtc ctccggcctg 540

tactctctgt ccagcgtggt caccgtgcct agcagcagtc tgggaaccca gacatacatc 600

tgcaacgtga accacaagcc ctccaataca aaagtggaca agaaggtgga gcctaaatcc 660

tgcgacaaga cccacacctg tcctccttgc cctgctcctg aggccgctgg cgccccttct 720

gtgtttctgt tcccccctaa gcccaaggac accctgatga tctcccggac ccccgaggtg 780

acctgcgtgg tggtggacgt gtcccacgag gaccctgaag tgaagttcaa ctggtacgtg 840

gatggcgtgg aagtgcacaa cgccaagacc aagcctagag aggagcagta caacagcacc 900

tacagagtgg tctccgtgct gaccgttctg caccaggact ggctgaacgg caaggagtac 960

aagtgcaagg tgtccaacaa ggccctgccc gcccctatcg agaagaccat ctctaaggct 1020

aagggccagc ctagagaacc tcaagtgtac accctgcctc catctcggga tgagctgaca 1080

aagaatcagg tgtctctgac ctgtctggtg aagggcttct acccctctga catcgccgtg 1140

gagtgggagt ctaacggcca gcccgagaac aactacaaga ccacccctcc tgtgctggac 1200

tccgacggct ccttcttcct gtactccaag ctgaccgtgg acaagtctag atggcagcag 1260

ggcaacgtgt tctcctgctc cgtgatgcac gaggccctgc acaaccacta cacacagaaa 1320

tccctgtccc tgtcccctgg cgctggaggc ggtggctccg gcggcggcgg ctccggtggc 1380

ggaggctccg gaggcggcgg ctctggcatc ccccctcacg tgcagaagag cgtcaacaac 1440

gatatgatcg tgaccgacaa caacggagct gtgaagtttc ctcaactgtg caagttctgc 1500

gacgtcagat tctctacctg tgataaccag aagtcctgca tgtccaactg cagcatcacc 1560

tccatctgcg agaaacctca agaggtgtgc gtggctgtgt ggcggaagaa cgacgaaaac 1620

atcaccctgg aaaccgtatg tcacgatcct aagctgcctt accacgactt catcctggaa 1680

gatgccgcct ctcccaagtg catcatgaaa gagaagaaga aacccggcga gacctttttc 1740

atgtgctctt gctccagcga cgagtgcaac gacaatatca tcttcagcga ggaatacaac 1800

accagcaacc ctgac 1815

<210> 16

<211> 642

<212> DNA/RNA

<213> Artificial sequence

<400> 16

gatatccaga tgacccagtc cccctcctcc atgtccgcct ccgtgggcga cagagtgacc 60

ttcacctgca gagcttctca ggacatcaac acctacctgt cctggttcca gcagaagcct 120

ggcaagtctc ctaagaccct gatctacaga gccaaccggc tggtgtccgg cgtgccttct 180

cggttctccg gatctggctc tggccaggat tacaccctga ccatctcctc tctgcagcct 240

gaggacatgg ccacctacta ctgcctgcag tacgacgagt tccctctgac attcggcgct 300

ggcaccaagc tggaactgaa gcggaccgtg gccgctccta gcgtgttcat cttccctcct 360

tccgacgaac aactgaagtc cggcaccgcc tctgtggtgt gcctgctgaa caacttctac 420

cctagagagg ccaaggtgca gtggaaggtg gacaacgccc tgcaaagcgg caactcccaa 480

gagtccgtca ccgagcagga cagcaaggac tccacctact ccctgtcttc tacactgacc 540

ctgtccaagg ccgactacga gaagcacaag gtgtacgcct gcgaggtgac acaccagggc 600

ctgagctccc ctgtgaccaa gtccttcaac agaggcgagt gc 642

<210> 17

<211> 440

<212> PRT

<213> Artificial sequence

<400> 17

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser

115 120 125

Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys

180 185 190

Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

305 310 315 320

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

340 345 350

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 18

<211> 214

<212> PRT

<213> Artificial sequence

<400> 18

Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

35 40 45

Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 19

<211> 448

<212> PRT

<213> Artificial sequence

<400> 19

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val

35 40 45

Ala Thr Ile Ser Gly Gly Gly Arg Tyr Thr Tyr Tyr Pro Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Asn Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Asn Arg Tyr Gly Glu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 20

<211> 597

<212> PRT

<213> Artificial sequence

<400> 20

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser

115 120 125

Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys

180 185 190

Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

290 295 300

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly

305 310 315 320

Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

340 345 350

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Leu Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly

435 440 445

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ile Pro Pro

450 455 460

His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr Asp Asn Asn

465 470 475 480

Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe

485 490 495

Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr

500 505 510

Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys

515 520 525

Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp Pro Lys Leu

530 535 540

Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile

545 550 555 560

Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys

565 570 575

Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn

580 585 590

Thr Ser Asn Pro Asp

595

<210> 21

<211> 446

<212> PRT

<213> Artificial sequence

<400> 21

Glu Val Gln Leu Glu Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Thr Thr Tyr

20 25 30

Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His Ser Leu Glu Trp Ile

35 40 45

Gly Glu Ile Leu Pro Gly Ser Asp Ser Thr Tyr Tyr Asn Glu Lys Val

50 55 60

Lys Gly Lys Val Thr Phe Thr Ala Asp Ala Ser Ser Asn Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Asp Gly Phe Tyr Val Tyr Trp Gly Gln Gly Thr Thr Leu

100 105 110

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

115 120 125

Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu

130 135 140

Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly

145 150 155 160

Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser

165 170 175

Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu

180 185 190

Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr

195 200 205

Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr

210 215 220

Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe

225 230 235 240

Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro

245 250 255

Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val

260 265 270

Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr

275 280 285

Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val

290 295 300

Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys

305 310 315 320

Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser

325 330 335

Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro

340 345 350

Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val

355 360 365

Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly

370 375 380

Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp

385 390 395 400

Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp

405 410 415

Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His

420 425 430

Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 22

<211> 214

<212> PRT

<213> Artificial sequence

<400> 22

Asp Ile Glu Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly

1 5 10 15

Asp Ser Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn

20 25 30

Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Thr Ser Gln Ser Met Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Thr

65 70 75 80

Glu Asp Phe Gly Val Tyr Phe Cys Gln Gln Ser Gly Ser Trp Pro Arg

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Asp Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<---

Claims (18)

1. A bifunctional protein capable of binding to PD-1 (programmed cell death receptor-1) and TGF-β (transforming growth factor-β), containing a PD-1-binding moiety and a TGF-β-binding moiety, wherein the PD-1 binding moiety is an anti-PD-1 antibody comprising a heavy chain variable region, a light chain variable region, a heavy chain constant region and a light chain constant region, wherein the amino acid sequence of the heavy chain variable region is represented by SEQ ID NO: 7, the amino acid sequence of the light chain variable region is represented by SEQ ID NO: 8, the amino acid sequence of the heavy chain constant region is represented by SEQ ID NO: 9, or is a variant of the amino acid sequence represented by SEQ ID NO: 9, wherein the variant is the amino acid sequence represented by SEQ ID NO: 9, wherein the A residue at the C-terminus is replaced with K, and the amino acid sequence of the light chain constant region is represented by SEQ ID NO: 10; wherein the TGF-β binding moiety is a human TGF-βRII isoform B extracellular domain polypeptide comprising the amino acid sequence represented by SEQ ID NO: 11; and wherein the PD-1 binding moiety and the TGF-β binding moiety are connected by a GGGGS type linker represented by SEQ ID NO: 12, and said linker extends from the CH3 end of the heavy chain constant region of the PD-1 binding moiety to the TGF-β binding moiety. 2. A bifunctional protein capable of binding to PD-1 (programmed cell death receptor-1) and TGF-β (transforming growth factor-β), comprising: (1) two identical first polypeptides, wherein the amino acid sequence of the first polypeptide has the amino acid sequence presented by SEQ ID NO: 13; and (2) two identical second polypeptides, wherein the amino acid sequence of the second polypeptide has the amino acid sequence presented by SEQ ID NO: 14. 3. A nucleic acid molecule encoding a bifunctional protein according to any one of claims 1, 2. 4. A pharmaceutical composition for the prevention or treatment of a malignant tumor, comprising an effective amount of a bifunctional protein according to any one of claims 1, 2 and a pharmaceutically acceptable excipient, diluent or carrier, wherein the malignant tumor is selected from colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, endometrial cancer, uterine cancer, bladder cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, small cell lung cancer, non-small cell lung cancer, melanoma and/or myelodysplastic syndromes. 5. A method for preventing or treating a malignant tumor, comprising administering an effective amount of a bifunctional protein according to any one of claims 1, 2 or a pharmaceutical composition according to claim 4 to a subject having a malignant tumor, wherein the malignant tumor is selected from colorectal cancer, breast cancer, ovarian cancer, pancreatic cancer, gastric cancer, kidney cancer, cervical cancer, myeloma, lymphoma, leukemia, endometrial cancer, uterine cancer, bladder cancer, head and neck cancer, liver cancer, nasopharyngeal cancer, small cell lung cancer, non-small cell lung cancer, melanoma and/or myelodysplastic syndromes. 6. The method according to claim 5, wherein the malignant tumor is primary, metastatic, recurrent, or difficult to treat. 7. A method for producing a bifunctional protein according to any one of claims 1, 2, comprising introducing an expression vector containing a nucleic acid molecule encoding a bifunctional protein according to any one of claims 1, 2 into a host cell and culturing the host cell under conditions that ensure expression of the protein; collection of cell culture and/or supernatant; and isolation and purification of the bifunctional protein according to any of claims 1, 2. 8. A method for producing a pharmaceutical composition according to claim 4, comprising introducing an expression vector containing a nucleic acid molecule encoding a bifunctional protein according to any one of claims 1, 2 into a host cell and culturing the host cell under conditions that ensure expression of the protein; collection of cell culture and/or supernatant; isolation and purification of the bifunctional protein according to any one of claims 1, 2; and combining the isolated and purified bifunctional protein according to any one of claims 1, 2 with a pharmaceutically acceptable excipient, diluent or carrier.