Bifunctional protein against PD-1 and TGF-β

Abstract

The present invention provides a bifunctional protein capable of binding to PD-1 (programmed death receptor-1) and TGF-β (transforming growth factor-β), pharmaceutical uses of the bifunctional protein, and methods for producing the same.

Description

Technical Field

[0001] [Cross - reference to Related Applications] This application claims the priority of Chinese Patent Application No. 202010359751.8 filed on April 29, 2020. The entire content of the said application is incorporated herein by reference and used for all purposes.

[0002] [Technical Field] The present invention generally relates to the field of antibody pharmaceuticals, particularly to the treatment of malignant tumors. Specifically, the present invention provides a bifunctional protein capable of binding to PD - 1 (Programmed Death - 1) and TGF - β (Transforming Growth Factor - β), and the pharmaceutical use of the bifunctional protein.

Background Art

[0003] T cells express many important membrane protein immune molecules. Among them, the PD - 1 (Programmed Death - 1, also called Programmed Cell Death Receptor - 1, CD279) protein belongs to the CD28 family of the immunoglobulin superfamily, and its ligands (PD - L1, PD - L2) belong to the B7 family. When PD - L1 binds to PD - 1, it down - regulates T - cell immune function, which is an important peripheral T - cell inhibitory immune checkpoint. In normal human tissues, PD - L1 is low - expressed to maintain immune tolerance and avoid autoimmune reactions. However, tumor cells suppress the immune function of T cells by highly expressing PD - L1 (or releasing soluble variants of PD - L1, exosomes), forming an immunosuppressive tumor immune microenvironment. By blocking the PD - 1 / PD - L1 signaling pathway, the immune function of T cells can be restored, and tumor cells can be identified and killed.

[0004] TGF-β (transforming growth factor-β, transforming growth factor-β) is a cytokine with multifunctional biological activity and can regulate the physiological processes of the organism by regulating cell proliferation, differentiation, apoptosis, adhesion, invasion, and microenvironment. The typical TGF-β signaling pathway is first activated by TGF-β binding to the type II TGF-β receptor (TGF-βRII) and then forming a complex with the type I TGF-β receptor (TGFβRI). TGFβRI phosphorylates and activates R-Smad (Smad1, 2, 3, 5, 8) members. R-Smad binds to Co-Smad (Smad4) to form a complex, which translocates into the cell nucleus to control the transcription of target genes.

[0005] In the tumor microenvironment, high expression of TGF-β leads to invasion, metastasis, immune evasion, treatment resistance, and poor prognosis (David Charles J et al., TGF-β Tumor Suppression through a Lethal EMT. [J]. Cell, 2016, 164(5)). Also, according to research, TGF-β is likely to disrupt the tumor microenvironment and accelerate tumor progression through the induction of Treg cells and the suppression of effector T cells (Shen Yinan et al., TGF-β regulates hepatocellular carcinoma progression by inducing Treg cell polarization. [J]. Cellular physiology and biochemistry, 2015, 35(4)). Additionally, some researchers believe that TGF-β signaling is the cause of the production of anti-PD-(L)1 drug resistance in patients.

[0006] Currently, research on co-targeting TGF-β and PD-1 / PD-L1 has already been reported. However, since this research has potential, further research is urgently needed.

Summary of the Invention

[0007] According to a first aspect, the present invention provides a bifunctional protein comprising a PD-1 (programmed cell death receptor-1) binding portion and a TGF-β (transforming growth factor-β) binding portion. In some embodiments, the PD-1 binding portion is an anti-PD-1 antibody or an antigen-binding fragment. In some embodiments, the PD-1 binding portion is a full-length antibody against PD-1, a Fab fragment, an F(ab')2 fragment, an Fv fragment or a single-chain Fv fragment (scFv).

[0008] In some embodiments, the anti-PD-1 antibody or antigen-binding fragment comprises a heavy-chain variable region and a light-chain variable region. The heavy-chain variable region comprises an HCDR1 with an amino acid sequence of GFAFSSYD (SEQ ID NO:1), an HCDR2 with an amino acid sequence of ISGGGRYT (SEQ ID NO:2), and an HCDR3 with an amino acid sequence of ANRYGEAWFAY (SEQ ID NO:3). The light-chain variable region comprises an LCDR1 with an amino acid sequence of QDINTY (SEQ ID NO:4), an LCDR2 with an amino acid sequence of RAN (SEQ ID NO:5), and an LCDR3 with an amino acid sequence of 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. The amino acid sequence of the heavy-chain constant region is represented by SEQ ID NO:9 or a variant of the amino acid sequence represented by SEQ ID NO:9, for example, the amino acid sequence represented by SEQ ID NO:9 in which the C-terminal residue A is substituted with K, and / or the amino acid sequence of the light-chain constant region is represented by SEQ ID NO:10 or a variant of the amino acid sequence represented by SEQ ID NO:10.

[0009] In some 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 (manufactured by Akeso), gepotidarlimab (GB226), LZM009 (manufactured by Livzon Mabpharm), HLX-10, BAT-1306, AK103 (HX008), AK104 (manufactured by Akeso), CS1003, SCT-I10A, F520, SG001, GLS-010, or an antigen-binding fragment of any of the above antibodies.

[0010] In some embodiments, the TGF-β binding moiety is a TGF-β receptor or a binding domain of a TGF-β receptor. 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. 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 may be a variant of the human TGF-βRII isoform B extracellular domain polypeptide, such as a polypeptide or peptide fragment 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 of the foregoing fragments herein.

[0011] In some embodiments, the TGF-β binding moiety is an anti-TGF-β antibody or antigen-binding fragment. In some embodiments, the TGF-β binding moiety is a full-length antibody, Fab fragment, F(ab')2 fragment, Fv fragment or single-chain Fv fragment (scFv) against TGF-β. In some embodiments, the PD-1 binding portion and the TGF-β binding portion are linked via a flexible linker. In some embodiments, the flexible linker is a GGGS-type linker. In some specific embodiments, the flexible linker is the linker represented by SEQ ID NO:12.

[0012] In some embodiments, the bifunctional protein comprises (1) two identical first polypeptides having an amino acid sequence with at least 80% identity to the amino acid sequence represented by SEQ ID NO:13, and (2) two identical second polypeptides having an amino acid sequence with at least 80% identity to the amino acid sequence represented by SEQ ID NO:14.

[0013] According to a second aspect, the present invention provides a nucleic acid molecule encoding the bifunctional protein of the first aspect. According to a third aspect, the present invention provides a pharmaceutical composition comprising the bifunctional protein of the first aspect and a pharmaceutically acceptable excipient, diluent or carrier.

[0014] In some embodiments, the pharmaceutical composition is used for the prevention or treatment of malignant tumors. 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 malignant tumor, 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 carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and / or myelodysplastic syndrome. In some specific embodiments, the malignant tumor is primary, metastatic, recurrent, and / or refractory.

[0015] According to a fourth aspect, the present invention provides the use of the bifunctional protein of the first aspect or the nucleic acid molecule of the second aspect in the manufacture of a medicament for preventing or treating malignant tumors.

[0016] 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 malignant tumor, 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 carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and / or myelodysplastic syndrome. In some specific embodiments, the malignant tumor is primary, metastatic, recurrent, and / or refractory.

[0017] According to a fifth aspect, the present invention provides a method for preventing or treating a malignant tumor, comprising administering to an individual suffering from a malignant tumor the bifunctional protein of the first aspect or the pharmaceutical composition of the third aspect.

[0018] 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 malignant tumor, 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 carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and / or myelodysplastic syndrome. In some specific embodiments, the malignant tumor is primary, metastatic, recurrent, and / or refractory.

[0019] According to a sixth aspect, the present invention provides a method for preparing a bifunctional protein, the bifunctional protein comprising a PD-1 (programmed cell death receptor-1) binding portion and a TGF-β (transforming growth factor-β) binding portion, and further comprising the bifunctional protein described in the first aspect, the method comprising: introducing an expression vector containing a nucleic acid molecule encoding the bifunctional protein into a host cell, and culturing the host cell under conditions enabling protein expression; and collecting the cell culture and / or supernatant, and separating and purifying the bifunctional protein.

Brief Description of the Drawings

[0020]

Figure 1

Figure 2

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Mode for Carrying Out the Invention

[0021] 〔Detailed Description of the Invention〕 <Definition> The following definitions and methods are provided to better define the present invention and guide those skilled in the art in the implementation of the present invention. Unless otherwise specified, the terms used in this specification have the meanings commonly understood by those skilled in the art. All patent documents, academic papers, and other published publications cited in this specification are incorporated herein by reference in their entirety.

[0022] As used herein, the term "antibody" means 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. Targets include, but are not limited to, carbohydrates, polynucleotides, lipids, polypeptides, etc. As used herein, "antibody" refers not only to complete (i.e., full-length) antibodies, but also to antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), variants thereof, fusion proteins containing antibody moieties, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and modified conformations of any other immunoglobulin molecule containing an antigen recognition site of the desired specificity, such as sequence variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.

[0023] Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain comprises a heavy chain variable region (VH) and first, second, and third constant regions (CH1, CH2, and CH3). Each light chain comprises a light chain variable region (VL) and a constant region (CL). The full-length antibody may be any type of antibody such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), but the antibody need not belong to any particular class. Immunoglobulins can be classified into different classes based on the antibody amino acid sequence of the heavy chain constant region. Typically, there are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these classes can be further classified into subclasses (isotypes) such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are known.

[0024] 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 understand that the major portion of the antibody structure that determines antigen-binding ability is the CDR, and thus the CDR is also a core component of the antigen-binding fragment. The antigen-binding region may include the heavy-chain variable region (VH), the light-chain variable region (VL), or both. Each of VH and VL typically includes three complementarity-determining regions, CDR1, CDR2, and CDR3.

[0025] Those skilled in the art know that the complementarity-determining regions (CDRs, usually CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of the antibody. There are two common definition methods for the CDR sequences in VH or VL, the Chothia definition and the Kabat definition (see, for example, Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991), A1-Lazikani et al., J. Mol. Biol. 273:927-948 (1997), and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989)). For the variable region sequence of a given antibody, the CDR sequences in the VH and VL sequences may be determined according to the Chothia definition or the Kabat definition.

[0026] For the variable region sequence of a given antibody, the CDR sequences in the variable region sequence can be analyzed in various ways, for example, determined using the online software Abysis (http: / / www.abysis.org / ).

[0027] 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 in the hinge region (i.e., a dimer of Fab'); (3) an Fv fragment having a single-arm VL and VH domain 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) a (scFv)2, which may include two VH domains linked via a peptide linker and two VL domains combined with the two VH domains via a disulfide bridge.

[0028] As used herein, the terms "Fab fragment", "Fab portion" or similar terms mean an antibody fragment capable of binding to an antigen, which is generated after treating a complete antibody with papain, and includes a complete light chain (VL-CL), a heavy chain variable region, and a CH1 fragment (VH-CH1).

[0029] As used herein, the term "single-chain antibody (scfv, single chain fragment variable)" generally means an antibody having a single-chain structure constructed using genetic engineering techniques, which is a polypeptide chain containing a heavy chain variable region (VH) and a light chain variable region (VL). Usually, a flexible linker peptide is designed between the heavy chain variable region and the light chain variable region so that the heavy chain variable region and the light chain variable region are folded into a correct conformation capable of binding to an antigen.

[0030] As used herein, the terms "Fc fragment", "Fc domain", "Fc portion", or similar terms mean a part of the heavy chain constant region of an antibody, including a hinge region, a CH2 fragment and a CH3 fragment of the constant region.

[0031] As used herein, the term "specific binding" means a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope.

[0032] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations in a small number of them. Monoclonal antibodies described herein include, in particular, "chimeric" antibodies, where a portion of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies as long as they exhibit the desired biological activity.

[0033] As used herein, the term "identity" refers to sequence similarity between two polynucleotide sequences or between two polypeptide sequences. Comparison of sequences between two sequences and measurement of the percentage of identity can be carried out with the default settings of the BLASTN / BLASTP algorithms available on the website of the National Center for Biotechnology Information.

[0034] As used herein, the term "treatment" includes therapeutic treatment and prophylactic treatment or prophylactic measures, which reduce at least one symptom of the disease, disorder, or medical condition (e.g., cancer or tumor) or alleviate the progression of the symptoms by administering a therapeutic agent to a subject.

[0035] As used herein, the term "EC 50 " is also referred to as the half-maximal effective concentration and is the concentration at which 50% of the maximum effect is obtained after a predetermined exposure time.

[0036] According to a first aspect, the present invention provides a bifunctional protein comprising a PD-1 (programmed cell death receptor-1) binding portion and a TGF-β (transforming growth factor-β) binding portion.

[0037] In some embodiments, the PD-1 binding moiety is an anti-PD-1 antibody or antigen-binding fragment. In some embodiments, the PD-1 binding moiety is a full-length antibody, Fab fragment, F(ab')2 fragment, Fv fragment or single-chain Fv fragment (scFv) against PD-1.

[0038] 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 an HCDR1 with an amino acid sequence of GFAFSSYD (SEQ ID NO:1), an HCDR2 with an amino acid sequence of ISGGGRYT (SEQ ID NO:2), and an HCDR3 with an amino acid sequence of ANRYGEAWFAY (SEQ ID NO:3), and the light chain variable region comprises an LCDR1 with an amino acid sequence of QDINTY (SEQ ID NO:4), an LCDR2 with an amino acid sequence of RAN (SEQ ID NO:5) and an LCDR3 with an amino acid sequence of 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, and 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.

[0039] 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 in which the C-terminal residue A is substituted with K. Modifications to the constant region of an antibody are known to those skilled in the art. In some embodiments, the heavy chain constant region can be selected from IgG1, IgG2, IgG3 and IgG4 or other classes, with IgG1 being preferred. In some embodiments, the constant region of the antibody may include modifications such as amino acid insertions, deletions, substitutions, or chemical modifications. In some embodiments, any amino acid residue of the constant region may be substituted with an amino acid residue of any allotype, preferably an amino acid residue of G1m(3) and / or nG1m(1). In some embodiments, the constant region includes mutations that alter the function of the effect, for example, mutating the lysine residue (K) at the C-terminus of the heavy chain constant region of the antibody (commonly found in wild-type IgG1-type antibodies) to a hydrophobic amino acid, such as alanine (A) or leucine (L), to reduce proteolytic cleavage and increase the serum half-life, and such modifications are also particularly suitable for situations where the C-terminus of the antibody heavy chain is further fused to other parts. The C-terminal residue of the heavy chain constant region of the anti-PD-1 antibody portion in the exemplary PD-1 / TGFβ bifunctional protein according to the present invention has been subjected to the corresponding treatment.

[0040] In some embodiments, the TGF-β binding moiety is a TGF-β receptor or a binding domain of a TGF-β receptor. In some embodiments, the TGF-β binding moiety is an extracellular domain of a TGF-β receptor or a binding fragment of the extracellular domain. 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 the human TGF-βRII isoform B extracellular domain polypeptide, for example, a polypeptide or peptide fragment 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 fragment of the above.

[0041] In some embodiments, the TGF-β binding moiety is an anti-TGF-β antibody or an antigen-binding fragment. In some embodiments, the TGF-β binding moiety is a full-length antibody against TGF-β, a Fab fragment, an F(ab’)2 fragment, an Fv fragment, or a single-chain Fv fragment (scFv).

[0042] In some embodiments, the PD-1 binding moiety and the TGF-β binding moiety are linked via a flexible linker. In some embodiments, the flexible linker is a GGGS-type linker. In some specific embodiments, the flexible linker is the linker shown in SEQ ID NO:12.

[0043] In some embodiments, the bifunctional protein comprises: (1) two identical first polypeptides having an amino acid sequence with 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 shown in SEQ ID NO: 13; and (2) two identical second polypeptides having an amino acid sequence with 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 shown in SEQ ID NO: 14.

[0044] As a non-limiting example, the bifunctional protein according to the present invention (i.e., PD-1 / TGFβ bifunctional protein, hereinafter also referred to as "PD1 / TGFβRII fusion protein" or "PD1 / TGFβRII") may be composed 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 shown in 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 its molecular structure is shown in FIG. 1. As shown in FIG. 1, based on the natural anti-PD-1 antibody, PD1 / TGFβRII sequentially extends a flexible linker and a human TGF-βRII isoform B extracellular domain polypeptide at the CH3 terminus of the heavy chain constant region.

[0045] PD1 / TGFβRII is an exemplary bifunctional protein according to the present invention, which has higher TGFβ binding activity and biological activity at the PD-1 terminus compared to the reported nivolumab / TGF-βRII fusion protein, and further has a more excellent tumor suppression effect. Furthermore, it has lower cytotoxicity and side effects than conventional PD-1 antibodies (for example, nivolumab). Since conventional PD-1 antibodies have high cytotoxicity and side effects, PD1 / TGFβRII can be administered at a higher dose due to its lower cytotoxicity and side effects, thereby enabling better suppression and consumption of TGFβ, and the safety limit of the dose is more ideal and advantageous for high-dose administration and clinical application.

[0046] According to a second aspect, the present invention provides a nucleic acid molecule encoding the bifunctional protein of the first aspect. According to a third aspect, the present invention provides a pharmaceutical composition comprising the bifunctional protein of the first aspect and a pharmaceutically acceptable excipient, diluent or carrier.

[0047] In some embodiments, the pharmaceutical composition is used for the prevention or treatment of malignant tumors. 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 malignant tumor, 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 carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and / or myelodysplastic syndrome. In some specific embodiments, the malignant tumor is primary, metastatic, recurrent, and / or refractory.

[0048] In some embodiments, the pharmaceutical composition may further contain a lubricant (such as talc, magnesium stearate and mineral oil, etc.), a wetting agent, an emulsifier, a suspending agent, a preservative (such as benzoic acid, sorbic acid and calcium propionate, etc.), a sweetening enhancer and / or a flavoring agent, etc. In some embodiments, the pharmaceutical composition according to the present invention may be manufactured in the form of tablets, pills, powders, tablets, elixirs, suspensions, emulsions, solutions, syrups, suppositories or capsules.

[0049] In some embodiments, the pharmaceutical composition according to the present invention can be delivered using any physiologically acceptable administration method including, but not limited to, oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravenous injection, subcutaneous administration, transdermal administration, inhalation administration, etc. In some embodiments, the pharmaceutical composition for therapeutic purposes may be prepared and stored in the form of a lyophilized preparation or an aqueous solution by mixing a reagent having a desired purity with a pharmaceutically acceptable carrier, excipient, etc. as necessary.

[0050] According to a fourth aspect, the present invention provides the use of the bifunctional protein of the first aspect or the nucleic acid molecule of the second aspect in the manufacture of a medicament for preventing or treating malignant tumors. 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 malignant tumors, 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 carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and / or myelodysplastic syndrome. In some specific embodiments, the malignant tumor is primary, metastatic, recurrent, and / or refractory.

[0051] According to a fifth aspect, the present invention provides a method for preventing or treating malignant tumors, which includes administering the bifunctional protein of the first aspect or the pharmaceutical composition of the third aspect to an individual suffering from a malignant tumor.

[0052] 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 malignant tumor, 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 carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and / or myelodysplastic syndrome. In some specific embodiments, the malignant tumor is primary, metastatic, recurrent, and / or refractory.

[0053] According to a sixth aspect, the present invention provides a method for preparing a bifunctional protein, the bifunctional protein comprising a PD-1 (programmed cell death receptor-1) binding portion and a TGF-β (transforming growth factor-β) binding portion, the method comprising: introducing an expression vector containing a nucleic acid molecule encoding the bifunctional protein into a host cell, and culturing the host cell under conditions enabling protein expression; and collecting the cell culture and / or supernatant, and separating and purifying the bifunctional protein.

[0054] Unless inconsistent, the embodiments and technical features described in the first aspect are also applicable to the sixth aspect. In some embodiments, the host cell is a mammalian cell, such as a CHO cell. In some embodiments, the supernatant after centrifugation of the cell culture is collected. In some embodiments, the purification of the bifunctional protein uses one or more of affinity chromatography, anion exchange chromatography, and cation exchange chromatography. In some embodiments of affinity chromatography, the eluent contains sucrose or glycerin. The inventors of the present invention have found that adding an eluent of sucrose or glycerin is advantageous for reducing the degradation of the fusion protein. The following examples are only used for illustration and are not intended to limit the scope of the present invention.

Example

[0055] Example 1: Expression of PD1 / TGFβRII Fusion Protein In this example, the PD1 / TGFβRII fusion protein according to the present invention was constructed, and the structural schematic diagram is shown in FIG. 1. After synthesizing the nucleotide sequences encoding the heavy chain portion (SEQ ID NO: 13) and the light chain portion (SEQ ID NO: 14) of the PD1 / TGFβRII fusion protein fused with a signal peptide, they were cloned into the pcDNA3.1 expression vector. The expression vector of the PD1 / TGFβRII fusion protein was co-transfected into CHO cells using standard forms of transient or stable transfection, and the transfected cells were placed in an incubator at 37 °C containing 8% CO2 and cultured.

[0056] The human TGF-βRII isoform B extracellular domain polypeptide (SEQ ID NO: 11) is included in SEQ ID NO: 13. IPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD The amino acid sequence of the linker (SEQ ID NO: 12) is included in SEQ ID NO: 13. GGGGSGGGGSGGGGSGGGGSG Amino acid sequence of the heavy chain of PD1 / TGFβRII fusion protein (SEQ ID NO: 13): EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

[0057] Amino acid sequence of the light chain of the PD1 / TGFβRII fusion protein (SEQ ID NO:14): DIQMTQSPSSMSASVGDRVTFTCRASQDINTYLSWFQQKPGKSPKTLIYRANRLVSGVPSRFSGSGSGQDYTLTISSLQPEDMATYYCLQYDEFPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0058] Example 2: Purification of the PD1 / TGFβRII fusion protein After centrifuging the cell culture solution obtained in Example 1, the supernatant was collected and subjected to the first stage of purification using Protein A affinity chromatography. The equilibration buffer was 10 mmol / L phosphate buffer at pH 6.0. After washing the column with 3 to 5 column volumes of the equilibration buffer, the cell supernatant was injected (sample loading). After the injection was completed, the column was washed with the equilibration buffer. Next, it was rinsed with a rinse buffer (0.5 mol / L sodium chloride + 25 mmol / L phosphate buffer, pH 7.0), and the column was equilibrated with the equilibration buffer again for 3 to 5 column volumes. Finally, the column was washed with an elution buffer (20 mmol / L citrate buffer + 5% sucrose, pH 3.6), the eluted sample was collected, and the sample was neutralized with 2 M Tris-hydrochloride buffer (pH 9.5).

[0059] The eluted sample after the above neutralization (pH 6.0) was subjected to anion exchange chromatography. The equilibration buffer was 10 mmol / L citrate buffer + 10 mmol / L phosphate buffer + 10 mmol / L Tris, and the pH was adjusted to 6.0. After rinsing the column with 3 to 5 column volumes of the equilibration buffer, the above neutralized eluted sample was injected (sample loading), the passed sample was collected, and after the injection was completed, the column was rinsed with the equilibration buffer. Cation exchange chromatography was performed on the sample passed through the above anion exchange chromatography. The equilibration buffer was 10 mmol / L citric acid + 10 mmol / L sodium dihydrogen phosphate + 10 mmol / L Tris buffer, pH 5.0. After adjusting the sample passed through the above anion chromatography to pH 5.0, it was injected. After the injection was completed, the column was washed with 3 to 5 column volumes of the equilibration solution. Next, it 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.

[0060] Example 3: Detection of PD1 / TGFβRII fusion protein sample by size exclusion chromatography Each component of the PD1 / TGFβRII fusion protein sample obtained by the purification of Example 2 was separated using a gel chromatography column. Elution was performed using a buffer at neutral pH as the mobile phase, and each molecular weight component was eluted in descending order of molecular weight. The column was a Thermo MabPac TM gel column with specifications of SEC-1 300Å, 5μm, 7.8 * 300mm. The mobile phase was (20 mmol / L disodium hydrogen phosphate + 300 mmol / L sodium chloride + 2% isopropyl alcohol solution, pH = 7.4). The sample was diluted to 1 mg / mL with the mobile phase to obtain a test article solution. 50 μL of the test article solution was precisely weighed and injected into a liquid chromatograph, and detection was performed at a wavelength of 280 nm. With a flow rate of 0.5 mL / min, isocratic elution was performed for 35 min.

[0061] The results were quantitatively analyzed by the area normalization method. The peak area ratios of high molecular weight impurities, immunoglobulin monomers, and low molecular weight impurities were calculated respectively. As a result of the detection, in the PD1 / TGFβRII fusion protein sample, the high molecular weight impurities were 0.19%, the immunoglobulin monomers were 99.81%, and the low molecular weight impurities were not detected. The control bifunctional protein (nivolumab / TGF-βRII fusion protein) used in the following examples was prepared in the same process, but the C-terminal amino acid residue of the original heavy chain constant region of nivolumab was changed from K to A, which is consistent with the exemplary PD1 / TGFβRII fusion protein according to the present invention.

[0062] Example 4: Detection of the biological activity of the PD-1 terminal of the PD1 / TGFβRII fusion protein by the reporter gene method The detection process is as follows. CHO-PDL1-CD3L cells in the logarithmic growth phase (purchased from the National Institutes for Food and Drug Control) were taken, and the viable cell density was adjusted to 5×10 5Adjusted to cells / mL, added to a 96-well white plate at 100 μL / well, placed in a cell incubator containing 37 °C and 5% CO2, and cultured for 16 - 20 hours. The next day, a suspension of Jurkat-PD-1-NFAT cells (purchased from the National Institutes for Food and Drug Control) was prepared, and the viable cell density was adjusted to 2×10 6 cells / mL with 1640 basal medium containing 2% FBS. The 96-well white plate containing CHO-PDL1-CD3L cells was taken out of the incubator, the supernatant was aspirated and discarded, the Jurkat-PD-1-NFAT cell suspension was added at 50 μL / well, and then a control PD-1 antibody (nivolumab, SEQ ID NOs: 17 and 18 are the sequences of the heavy chain and light chain respectively), a serial dilution of the PD1 / TGFβRII fusion protein prepared in Examples 1 - 3 of the present invention (initial concentration is 200,000 ng / mL, 3-fold serial dilution, a total of 11 dilution gradients) was added to the above 96-well white plate at 50 μL / well, placed in a cell incubator containing 37 °C and 5% CO2, and cultured for 4 - 6 hours. During the incubation period, Bio-Lite luciferase reagent (Vazyme, DD1201-03) was taken out and thawed at room temperature. After the incubation, it was added to the above 96-well white plate at 100 μL / well, incubated at room temperature in the dark for 2 - 3 minutes, and the RLU value was read using a multifunctional plate reader (Thermo, Varioskan Flash). The experimental data was analyzed using Prism software, the dose-response curves of the reference product and the test product were plotted, and the EC 50 of the reference product and the test product was obtained, and the biological activity of the test product was calculated.

[0063] Biological activity (%) of the test product = (EC 50 value of the reference product / EC 50 value of the test product) × 100% The results of the biological activity of the PD-1 terminus of the PD1 / TGFβRII fusion protein of the examples according to the present invention are shown in Table 1 and Figure 2, and the results showed that the binding ability to human-derived PD-1 was retained.

[0064]

Table 1

[0065] Example 5: Detection of TGFβ binding activity of PD1 / TGFβRII fusion protein by enzyme-linked immunosorbent assay The detection process is as follows. 1) Using 2 μg / mL of human-derived TGFβ1 protein (Sinobiological, 10804-H08H) as an antigen, adsorb it onto a high-binding 96-well plate at 100 μL / well and incubate overnight at 2 - 8°C. 2) Wash the 96-well plate three times with PBST20 (PBS solution containing 0.05% Tween 20) at 250 μL / well, then add 250 μL / well of blocking solution (PBS solution containing 3% BSA) and incubate at 25°C for 2 hours. 3) Wash the 96-well plate three times with PBST20 at 250 μL / well, then add the PD1 / TGFβRII fusion protein prepared in Examples 1 - 3 of the present invention diluted in a gradient at 100 μL / well (initial concentration 4000 ng / mL, 4-fold gradient dilution, a total of 7 dilution degrees) and incubate at 25°C for 2 hours.

[0066] 6) Wash the 96-well plate three times with PBST20 at 250 μL / well, then add 100 μL per well of HRP-goat anti-human IgG antibody (PE, NEF802001EA) diluted 1:3,500 with the dilution solution and incubate at 25°C for 1 hour. 7) Wash the 96-well plate five times with PBST20 at 250 μL / well, then add 100 μL / well of TMB solution and incubate at 25°C in the dark for 5 minutes. 10) Add 1 mol / L of H2SO4 at 100 μL / well to stop the reaction and leave it at room temperature for 5 minutes. Measure the OD value at a wavelength of 450 nm / 650 nm using a microplate reader (Thermo Scientific, Varioskan Flash) and analyze the data with Graphpad Prism.

[0067] Test article binding activity (%) = (Reference product EC 50 value / Test article EC 50 value) × 100% The results of the binding of the PD1 / TGFβRII fusion protein of the examples according to the present invention to TGFβ1 in vitro are shown in Table 2 and FIG. 3, and the ELISA results showed that the binding activity to TGFβ was retained.

[0068]

Table 2

[0069] Example 6: Therapeutic effect of PD1 / TGFβRII fusion protein on subcutaneous transplanted tumors of colon cancer cells MC38 / hPD-L1 in mice C57 / PD-1 transgenic mice (purchased from Jiangsu Jicui Yakang Biotechnology Co., Ltd.) were used as experimental mice, and 3×10 5 individual MC38 / hPD-L1 cells were subcutaneously inoculated. When the tumor grew to 40 - 70 mm 3 in size, they were grouped according to tumor volume, and the drug was intraperitoneally injected (ip) once every 2 days at an injection volume of 0.1 mL / 10 g body weight for a total of 6 times. The dosing schedule is shown in Table 3, and the dosing day was D0. The tumor diameter was measured with calipers twice a week, and the effect of the drug on tumor growth was considered by T / C% or tumor growth inhibition rate TGI (%) calculated by the following formula. When the experiment was completed, the experimental end point was reached, or the tumor volume reached 1500 mm 3 in size, the animals were anesthetized with CO2 and euthanized, then dissected to collect the tumors and photographed.

[0070] The formula for calculating the tumor volume (V) is V = 1 / 2 × a × b 2wherein, a and b represent the length and width respectively. T / C(%) = (T - T0) / (C - C0)×100, wherein T and C are the tumor volumes of the treated group mice and the negative control group mice at the end of the experiment, and T0 and C0 are the tumor volumes of the treated group mice and the negative control group mice at the start of the experiment. The T / C values of the treated group and the negative control group were calculated based on the T / C values of the treated group mice and the negative control group mice respectively. Tumor growth inhibition rate (TGI)(%) = 100 - T / C(%). The results are shown in Table 4. PD1 / TGFβRII (3.7 mg / kg, IP, twice a day, for a total of 6 times) prepared in Examples 1 to 3 according to the present invention had a tumor growth inhibition rate of 74% against subcutaneous transplanted tumors of MC38 / hPD-L1 mice on D19, which was superior to the control PD-1 monoclonal antibody. The cancer-bearing mice developed good resistance to the drug and did not show obvious symptoms such as weight loss.

[0071]

Table 3

[0072]

Table 4

[0073] Example 7: Detection of in vitro activity of PD1 / TGFβRII fusion protein Referring to the methods of Examples 1 to 3, exemplary PD1 / TGFβRII fusion protein and nivolumab / TGF-βRII fusion protein according to the present invention were prepared in the same lot. Referring to the reporter gene method of Example 4, the biological activity of the PD-1 terminal of the PD1 / TGFβRII fusion protein was detected and compared. As a result, the biological activity of the PD-1 terminal of the PD1 / TGFβRII fusion protein according to the present invention was superior to that of the nivolumab / TGF-βRII fusion protein, and the results shown in Table 5 were obtained. Referring to the enzyme-linked immunosorbent assay of Example 5, the TGFβ binding activity of the PD1 / TGFβRII fusion protein was detected and compared. As a result, the TGFβ binding activity of the PD1 / TGFβRII fusion protein according to the present invention was superior to that of the nivolumab / TGF-βRII fusion protein, and the results shown in Table 6 were obtained. The nivolumab / TGF-βRII fusion protein in this example and the following examples was self-made and had heavy and light chain sequences shown by SEQ ID NO:20 and 18.

[0074]

Table 5

[0075]

Table 6

[0076] Example 8: Therapeutic effect of PD1 / TGFβRII fusion protein on MC38 / hPD-L1 mouse transplanted tumors Humanized PD-1 mice (purchased from Biocytogen) were used as experimental mice, and 4×10 5 cells of MC38 / hPD-L1 were inoculated under the right axilla of each mouse, and the tumor was 100 - 300 mm 3When it grew to a certain size, it was randomly divided into three groups, and a total of 8 intraperitoneal injections (ip) of the drug were administered. The administration plan is shown in Table 7, and the administration day was designated as D0. The tumor volume was measured 2 - 3 times a week, and the body weight of the mice was recorded. The tumor diameter was measured using calipers, and the T / C(%) or tumor volume inhibition rate (1 - T / C) was calculated according to the following formula to examine the effect of the drug on tumor growth. At the end of the experiment, the animals were anesthetized with CO2 and killed, then dissected to collect the tumors and photographed.

[0077] The calculation formula for tumor volume (TV) is TV = 1 / 2 × a × b 2 where a and b represent the length and width respectively. The calculation formula for relative tumor volume (RTV) is RTV=(TV t ) / (TV0), where TV0 represents the tumor volume of the mice on D0, and TV t represents the tumor volume of the mice measured each time. The calculation formula for relative tumor growth rate T / C(%) is T / C(%) = T RTV / C RTV ×100%, where T RTV represents the RTV of the treatment group and C RTV represents the RTV of the PBS group.

[0078] As shown in the results in Tables 8 and 9, PD1 / TGFβRII and nivolumab / TGF-βRII (prepared in the same lot as shown in Example 7) had tumor volume inhibition rates of 46.8% and 32.3% respectively against MC38 / hPD-L1 transplanted tumor mice on D23, indicating that the tumor suppression effect of PD1 / TGFβRII according to the present invention is superior to that of nivolumab / TGF-βRII. Furthermore, the body weights of the mice in the PD1 / TGFβRII group and the nivolumab / TGF-βRII group increased fluctuantly, showing that neither of the two fusion proteins caused obvious toxic reactions.

[0079]

Table 7

[0080]

Table 8

[0081]

Table 9

[0082] Example 9: Detection by electrochemiluminescence method of the stimulation of cytokine secretion by PD1 / TGFβRII fusion protein The PBMC cell concentration was adjusted to about 2×10 6 cells / mL using RPMI1640 complete medium and added to a 96-well cell culture plate at 100 μL / well. Using RPMI1640 complete medium, the IgG1 protein (SEQ ID NO:21 and SEQ ID NO:22 are the sequences of the heavy chain and light chain respectively, self-made), LPS (SIGMA, L4391-1MG), and the PD1 / TGFβRII fusion protein according to the present invention were each diluted to prepare 900 μg / mL of IgG1 protein, 1 μg / mL of LPS, 10 μg / mL, 100 μg / mL, and 900 μg / mL of PD1 / TGFβRII fusion protein, and RPMI1640 complete medium was used as a negative control. 100 μL of each of the solutions prepared above was added to a 96-well cell culture plate and mixed well, and cultured in a 37°C, 5% CO2 cell incubator. After 48 hours, the cell supernatant in the 96-well plate was taken, and the contents of cytokines IL-2, IL-6, IL-8, IL-10, TNF-α, and IFN-γ were detected using a V-PLEX Proinflammatory Panel 1 (human) kit (MSD, K15049D-2), detected by a high-sensitivity immunoassay system (MSD, QuickPlex SQ120), and the results are shown in Table 10.

[0083]

Table 10

[0084] From the results in the above table, it was found that the PD1 / TGFβRII fusion protein according to the present invention is unlikely to cause a cytokine storm, and thus there is almost no risk of over-activating the immune system and causing systemic inflammation when administered to a subject.

[0085] Example 10: Toxicity Test of PD1 / TGFβRII Fusion Protein on Cynomolgus Monkeys Single-dose toxicity: In this test, 4 cynomolgus monkeys were divided into 2 groups of 2 each, with an equal number of males and females in each group. The PD1 / TGFβRII fusion protein according to the present invention was intravenously injected at 200 and 500 mg / kg respectively, and the monkeys were observed for 14 days. During the test period, general observations, body weight, food intake, body temperature, II-induced electrocardiogram and blood pressure, hematology, blood biochemistry, urine and other indicators were detected, and gross anatomical observations were performed at the end of the test.

[0086] After administration, in the male monkeys of each group, a transient decrease in food intake was observed, which recovered by the 8th to 9th day of the test. On the 14th day of the test, a decrease in RBC, HGB, and HCT was observed in the male monkeys of each group. No obvious abnormal changes were observed in other indicators. In the single-dose toxicity test, cynomolgus monkeys were intravenously administered the PD1 / TGFβRII fusion protein according to the present invention at 200 and 500 mg / kg respectively, and the MTD was 500 mg / kg. Multiple-dose toxicity: In this test, a total of 40 cynomolgus monkeys were used and divided into 4 groups of 10 each, with an equal number of males and females in each group, as a blank control group and 15, 50, and 150 mg / kg groups of the PD1 / TGFβRII fusion protein according to the present invention. They were administered once a week for 4 consecutive weeks (a total of 5 times), then the dosing was stopped for recovery, and they were observed for 4 weeks.

[0087] On the 15th day of administration and at the end of administration, a decrease in RBC, HGB, and HCT and a compensatory increase in RET and RET% were observed in male monkeys in the 50 mg / kg group and male and female monkeys in the 150 mg / kg group. On the 15th day of administration, such changes were also observed in female monkeys in the 50 mg / kg group.

[0088] At the end of the administration, macroscopic dissection of one male monkey in the 50 mg / kg group revealed adhesion of the pericardium of the heart. Histopathological examination: In cynomolgus monkeys in the 150 mg / kg group, mild to moderate mononuclear cell infiltration was observed in the leptomeninges and choroid plexus of the brain, leptomeninges and choroid plexus of the cerebellum, spinal meninges, thyroid gland, heart, and pituitary gland, and mild to moderate vasculitis / perivascular inflammation was observed in the heart, liver, bladder, epididymis, and seminal vesicle. In cynomolgus monkeys in the 50 mg / kg group, mild to moderate mononuclear cell infiltration was observed in the leptomeninges of the cerebrum, sciatic nerve, thyroid gland, heart, and pituitary gland, and mild to moderate vasculitis / perivascular inflammation was observed in the heart, bladder, duodenum, ileum, rectum, fallopian tube, vagina, and uterus. In cynomolgus monkeys in the 15 mg / kg group, mild to slight mononuclear cell infiltration was observed in the leptomeninges and choroid plexus of the cerebrum, choroid plexus of the cerebellum, sciatic nerve, thyroid gland, and heart, and slight vasculitis / perivascular inflammation of the heart was observed. In addition, in the 50 and 150 mg / kg groups, mild femoral necrosis, thickening of the epiphyseal plate, and mild to slight trabeculae of the femoral diaphysis and an increase in osteoclasts were observed in one cynomolgus monkey each.

[0089] Furthermore, no dead or dying animals were observed, and the general condition of the cynomolgus monkeys in each group was good. There were no obvious abnormal changes in body weight, food intake, body temperature, II-induced electrocardiogram, respiratory rate, blood biochemistry, ophthalmological examination, urine examination, bone marrow smear specimens, organ weights and coefficients, immune-related indices such as IgA, IgM, IgG, C3, C4, CIC, lymphocyte subsets, TSH, T3, T4, organ weights and coefficients.

[0090] In the repeated-dose toxicity test, the PD1 / TGFβRII fusion protein according to the present invention at 15, 50, and 150 mg / kg was intravenously administered to cynomolgus monkeys (once a week for 4 consecutive weeks, a total of 5 doses), and the HNSTD was set at 150 mg / kg. Inferring from the disclosed information in the Assessment report of Nivolumab by the European Medicines Agency, the HNSTD of nivolumab is 50 mg / kg, which is much lower than the HNSTD of the PD1 / TGFβRII fusion protein according to the present invention. From this, since the PD1 / TGFβRII fusion protein according to the present invention has low toxicity, it is expected to show good clinical safety.

[0091] Although the present invention has been described in detail using the above general description and specific examples, some corrections or improvements may be made based on the present invention, which will be obvious to those skilled in the art. Therefore, any of these corrections or improvements made without departing from the spirit of the present invention fall within the scope of the claims of the present invention.

Claims

1. A bifunctional protein comprising a PD-1 (programmed cell death receptor-1) binding portion and a TGF-β (transforming growth factor-β) binding portion, wherein the PD-1 binding portion 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, and the heavy chain variable region comprises an HCDR1 consisting of the amino acid sequence GFAFSSYD (SEQ ID NO: 1), an HCDR2 consisting of the amino acid sequence ISGGGRYT (SEQ ID NO: 2), and an HCDR3 consisting of the amino acid sequence ANRYGEAWFAY (SEQ ID NO: 3), and the light chain variable region comprises an LCDR1 consisting of the amino acid sequence QDINTY (SEQ ID NO: 4), an LCDR2 consisting of the amino acid sequence RAN (SEQ ID NO: 5), and an LCDR3 consisting of the amino acid sequence LQYDEFPLT (SEQ ID NO: 6), wherein the TGF-β binding portion is a human TGF-βRII isoform B extracellular domain polypeptide comprising the amino acid sequence shown in SEQ ID NO: 11, and wherein the PD-1 binding portion and the TGF-β binding portion are linked by a GGGGS-type linker shown in SEQ ID NO: 12, a bifunctional protein.

2. The bifunctional protein according to claim 1, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 7 and / or the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

3. The bifunctional protein according to claim 1, wherein the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 9 or is a variant of the amino acid sequence shown in SEQ ID NO: 9, and the variant is the amino acid sequence shown in SEQ ID NO: 9 in which the C-terminal residue A is replaced by K, and / or the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

10.

4. A bifunctional protein comprising a PD-1 (programmed cell death receptor-1) binding portion and a TGF-β (transforming growth factor-β) binding portion, (1) two identical first polypeptides, wherein the amino acid sequence of the first polypeptide is the amino acid sequence shown in SEQ ID NO: 13, and (2) two identical second polypeptides, wherein the amino acid sequence of the second polypeptide is the amino acid sequence shown in SEQ ID NO: 14, a bifunctional protein.

5. A nucleic acid molecule encoding the bifunctional protein according to claim 1.

6. A pharmaceutical composition comprising the bifunctional protein according to claim 1 and a pharmaceutically acceptable excipient, diluent or carrier.

7. A pharmaceutical composition comprising the bifunctional protein according to claim 4 and a pharmaceutically acceptable excipient, diluent or carrier.

8. The pharmaceutical composition according to claim 6 or 7 for use in the prevention or treatment of malignant tumors.

9. The pharmaceutical composition according to claim 8, wherein 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 malignant tumor, 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 carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and / or myelodysplastic syndrome.

10. The pharmaceutical composition according to claim 8, wherein the malignant tumor is primary, metastatic, recurrent or refractory.

11. A pharmaceutical composition for use in the prevention or treatment of malignant tumors, comprising the bifunctional protein according to claim 1 or 4.

12. The pharmaceutical composition according to claim 11, wherein 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 malignant tumor, 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 carcinoma, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma and / or myelodysplastic syndrome.

13. The pharmaceutical composition according to claim 11, wherein the malignant tumor is primary, metastatic, recurrent or refractory.

14. A method for preparing the bifunctional protein according to claim 1 or 4, comprising: introducing an expression vector containing a nucleic acid molecule encoding the bifunctional protein into a host cell and culturing the host cell under conditions allowing protein expression; and collecting the cell culture and / or supernatant and separating and purifying the bifunctional protein. A method comprising the above steps.

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