Bifunctional protein against PD-1 and TGF-β

Abstract

To provide a bifunctional protein capable of binding to PD-1 (programmed death receptor-1) and TGF-β (transforming growth factor-β), the medical use of the bifunctional protein, and a method for preparing the same.SOLUTION: A bifunctional protein comprises a PD-1 binding moiety and a TGF-β binding moiety, wherein the PD-1 binding moiety is preferably an anti-PD-1 antibody or an antigen-binding fragment thereof, for example, a full-length antibody, an Fab fragment, an F(ab')2 fragment, an Fv fragment, or a single chain Fv fragment (scFv).SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from Chinese Patent Application No. 202010359751.8 filed on April 29, 2020, the entire contents of which are incorporated herein by reference for all purposes.

[0002] [Technical Field] The present invention relates generally 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 receptor-1) and TGF-β (transforming growth factor-β), and pharmaceutical uses of the bifunctional protein. [Background technology]

[0003] T cells express many important membrane protein immune molecules, including the PD-1 (Programmed Death-1, also known as CD279) protein, which belongs to the CD28 family of the immunoglobulin superfamily, and its ligands (PD-L1 and PD-L2) belong to the B7 family. PD-L1 downregulates T cell immune function upon binding to PD-1, and is an important peripheral T cell inhibitory immune checkpoint. Normal human tissues have low PD-L1 expression to maintain immune tolerance and avoid autoimmune reactions. However, tumor cells suppress T cell immune function through high expression of PD-L1 (or release of soluble PD-L1 mutants and exomes), creating an immunosuppressive tumor immune microenvironment. Blocking the PD-1 / PD-L1 signaling pathway restores T cell immune function, allowing them to identify and kill tumor cells.

[0004] Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates physiological processes in living organisms by regulating cell proliferation, differentiation, apoptosis, adhesion, invasion, and the microenvironment. The typical TGF-β signaling pathway involves TGF-β binding to the type II TGF-β receptor (TGF-βRII), which then activates the type I TGF-β receptor (TGF-βRI). TGF-βRI then phosphorylates and activates R-Smads (Smad1, 2, 3, 5, and 8), which then bind to the co-Smad (Smad4) to form a complex and enter the cell nucleus, where it regulates the transcription of target genes.

[0005] High expression of TGF-β in the tumor microenvironment 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)). Research has also shown that TGF-β disrupts the tumor microenvironment by inducing Treg cells and suppressing effector T cells, likely 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)). Some researchers also believe that TGF-β signaling may be responsible for the development of anti-PD-(L)1 drug resistance in patients.

[0006] Currently, research into the joint targeting of TGF-β and PD-1 / PD-L1 has already been reported, but this research is promising and 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 death receptor-1) binding domain and a TGF-β (transforming growth factor-β) binding domain. 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, a Fab fragment, a F(ab')2 fragment, an Fv fragment, or a single-chain Fv fragment (scFv) against PD-1.

[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, wherein the heavy chain variable region comprises an HCDR1 having the amino acid sequence GFAFSSYD (SEQ ID NO:1), an HCDR2 having the amino acid sequence ISGGGRYT (SEQ ID NO:2), and an HCDR3 having the amino acid sequence ANRYGEAWFAY (SEQ ID NO:3), and the light chain variable region comprises an LCDR1 having the amino acid sequence QDINTY (SEQ ID NO:4), an LCDR2 having the amino acid sequence RAN (SEQ ID NO:5), and an 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 set forth in SEQ ID NO:7, and / or the amino acid sequence of the light chain variable region is set forth in 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 set forth in SEQ ID NO:9 or a variant of the amino acid sequence set forth in SEQ ID NO:9, e.g., the amino acid sequence of SEQ ID NO:9 in which the C-terminal residue A is replaced with K, and / or the amino acid sequence of the light chain constant region is set forth in SEQ ID NO:10 or a variant of the amino acid sequence set forth in 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 (Koho Biosciences), geptanolimab (GB226), LZM009 (Livzon Mabpharm), HLX-10, BAT-1306, AK103 (HX008), AK104 (Koho Biosciences), 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 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 set forth in SEQ ID NO:11. In some specific embodiments, the TGF-β binding moiety can be a variant of a 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 set forth 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, a Fab fragment, a F(ab')2 fragment, an Fv fragment, or a 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 set forth in SEQ ID NO:12.

[0012] In some embodiments, the bifunctional protein comprises (1) two completely identical first polypeptides whose amino acid sequences are at least 80% identical to the amino acid sequence set forth in SEQ ID NO:13, and (2) two completely identical second polypeptides whose amino acid sequences are at least 80% identical to the amino acid sequence set forth in 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 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 malignancies, head and neck cancer, liver cancer, nasopharyngeal carcinoma, 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 a bifunctional protein of the first aspect or a 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 malignancies, head and neck cancer, liver cancer, nasopharyngeal carcinoma, 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 the bifunctional protein of the first aspect or the pharmaceutical composition of the third aspect to an individual suffering from a malignant tumor.

[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 malignancies, head and neck cancer, liver cancer, nasopharyngeal carcinoma, 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, said bifunctional protein comprising a PD-1 (programmed death receptor-1) binding portion and a TGF-β (transforming growth factor-β) binding portion, and further comprising the bifunctional protein of the first aspect, said 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 allowing protein expression; harvesting the cell culture and / or supernatant and isolating and purifying said bifunctional protein. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a schematic diagram showing the structure of an exemplary PD-1 / TGFβ bifunctional protein according to the present invention. [Figure 2] FIG. 1 shows the results of detecting the biological activity of the PD-1 terminus of an exemplary PD-1 / TGFβ bifunctional protein according to the present invention using a reporter gene method, with nivolumab serving as a control sample. [Figure 3]1 shows the results of detecting the TGFβ-binding activity of an exemplary PD-1 / TGFβ bifunctional protein according to the present invention by enzyme-linked immunosorbent assay. [Sequence Description] SEQ ID NOs: 1-3 are the sequences of CDR1-CDR3 of the heavy chain variable region of the anti-PD-1 antibody portion of an exemplary PD-1 / TGFβ bifunctional protein according to the present invention. SEQ ID NOs: 4-6 are the sequences of CDR1-CDR3 of the light chain variable region of the anti-PD-1 antibody portion of an exemplary PD-1 / TGFβ bifunctional protein according to the present invention. SEQ ID NOs: 7 and 8 are the sequences of the heavy chain variable region and light chain variable region, respectively, of the anti-PD-1 antibody portion of an exemplary PD-1 / TGFβ bifunctional protein according to the present invention. SEQ ID NOs: 9 and 10 are the sequences of the heavy chain constant region and light chain constant region, respectively, of the anti-PD-1 antibody portion of an exemplary PD-1 / TGFβ bifunctional protein according to the present invention. SEQ ID NO:11 is the TGF-β binding portion of an exemplary PD-1 / TGFβ bifunctional protein of the invention, i.e., the human TGF-βRII isoform B extracellular domain polypeptide. SEQ ID NO:12 is a flexible linker between the anti-PD-1 antibody portion and the TGF-β binding portion of an exemplary PD-1 / TGFβ bifunctional protein of the invention. SEQ ID NO:13 is the heavy chain portion sequence of an exemplary PD-1 / TGFβ bifunctional protein of the invention, where the heavy chain portion consists of the heavy chain of the anti-PD-1 antibody portion, 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 light chain portion sequence of an exemplary PD-1 / TGFβ bifunctional protein of the invention, where the light chain portion consists of the light chain of the anti-PD-1 antibody portion. SEQ ID NO:15 is the coding nucleic acid sequence of SEQ ID NO:13 (excluding the coding sequence for the signal peptide). SEQ ID NO: 16 is the coding nucleic acid sequence (excluding the coding sequence for the signal peptide) of SEQ ID NO: 14. SEQ ID NOs: 17 and 18 are the heavy and light chain sequences, respectively, of the control PD1 monoclonal antibody, Nivolumab.SEQ ID NOS: 19 and 14 are the heavy and light chain sequences, respectively, of another control PD1 monoclonal antibody (derived from Chinese Patent Application No. 201610705763.5 (CN106977602)). SEQ ID NOS: 20 and 18 are the heavy and light chain partial sequences of the bifunctional protein control nivolumab / TGF-βRII bifunctional protein, where the heavy chain portion consists of the nivolumab heavy chain (SEQ ID NOS: 17 with the C-terminal amino acid residue mutated from K to A), a flexible linker (SEQ ID NOS: 12), and a human TGF-βRII isoform B extracellular domain polypeptide (SEQ ID NOS: 11), and the light chain portion consists of the nivolumab light chain. SEQ ID NOS: 21 and 22 are the heavy and light chain sequences of the experimental control IgG1 protein. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description of the Invention <Definition> The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise specified, terms used herein have the meanings commonly understood by those of ordinary skill in the art. All patents, journal articles and other published documents cited herein are hereby incorporated by reference in their entirety.

[0022] As used herein, the term "antibody" refers to an immunoglobulin molecule capable of specifically binding 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, and the like. As used herein, "antibody" includes not only intact (i.e., full-length) antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), variants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified conformation of an immunoglobulin molecule containing an antigen recognition site of a desired specificity, such as antibody sequence variants, antibody amino acid sequence variants, 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). A full-length antibody may be any type of antibody, such as IgD, IgE, IgG, IgA, or IgM (or a subclass thereof), but antibodies 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. There are typically five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different immunoglobulin classes are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0024] As used herein, the term "antigen-binding fragment" refers to a portion of an antibody structure that determines antigen-binding ability. Those skilled in the art will understand that the main portion of an antibody structure that determines antigen-binding ability is the CDR, and therefore, the CDR is also the core component of an antigen-binding fragment. The antigen-binding region may include a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of the VH and VL typically includes three complementarity-determining regions: CDR1, CDR2, and CDR3.

[0025] Those skilled in the art are aware that complementarity-determining regions (CDRs, usually CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest influence on the affinity and specificity of an antibody. There are two common definitions for the CDR sequences of 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 a given antibody variable region sequence, the CDR sequences in the VH and VL sequences may be determined according to the Chothia definition or the Kabat definition.

[0026] For a given antibody variable region sequence, the CDR sequences in the variable region sequence can be analyzed in a variety of ways, and can be determined, for example, 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) a F(ab')2 fragment, which may be a bivalent fragment having two Fab' fragments linked by disulfide bridges in the hinge region (i.e., a Fab' dimer); (3) an Fv fragment, which has 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) an (scFv)2, which may include two VH domains linked via a peptide linker and two VL domains combined with two VH domains via disulfide bridges.

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

[0029] As used herein, the term "single chain antibody (scfv, single chain fragment variable)" refers to an antibody having a single chain structure, generally constructed using genetic engineering techniques, which is a polypeptide chain comprising a heavy chain variable region (VH) and a light chain variable region (VL). Usually, a flexible linker 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 can fold into the correct conformation for antigen binding.

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

[0031] 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 antigen epitope.

[0032] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for the possible presence of naturally occurring mutations in a few individuals. The monoclonal antibodies described herein specifically include "chimeric" antibodies in which a portion of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and also include fragments of such antibodies so long as they exhibit the desired biological activity.

[0033] As used herein, the term "identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptide sequences. Sequence comparison and percent identity determination between two sequences can be performed using the default settings of the BLASTN / BLASTP algorithm, available on the National Center for Biotechnology Institute website.

[0034] As used herein, the term "treatment" includes therapeutic treatment and prophylactic treatment or preventative measures in which a therapeutic agent is administered to a subject to reduce at least one symptom or slow the progression of a symptom of said disease, disorder, or condition (e.g., cancer or tumor).

[0035] As used herein, the term "EC 50 "Median effective concentration," also known as the median effective concentration, is the concentration at which 50% of the maximum effect is achieved after a given exposure time.

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

[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, a Fab fragment, a F(ab')2 fragment, an Fv fragment, or a 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 having the amino acid sequence GFAFSSYD (SEQ ID NO:1), an HCDR2 having the amino acid sequence ISGGGRYT (SEQ ID NO:2), and an HCDR3 having the amino acid sequence ANRYGEAWFAY (SEQ ID NO:3), and the light chain variable region comprises an LCDR1 having the amino acid sequence QDINTY (SEQ ID NO:4), an LCDR2 having the amino acid sequence RAN (SEQ ID NO:5), and an 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 set forth in SEQ ID NO:7, and / or the amino acid sequence of the light chain variable region is set forth in 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 set forth in SEQ ID NO:9 and / or the amino acid sequence of the light chain constant region is set forth in 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 set forth in SEQ ID NO:9, in which the C-terminal residue A is replaced with K. Modifications to antibody constant regions are known to those skilled in the art. In some embodiments, the heavy chain constant region can be selected from IgG1, IgG2, IgG3, IgG4, or other classes, with IgG1 being preferred. In some embodiments, the antibody constant region may include modifications such as amino acid insertion, deletion, substitution, or chemical modification. In some embodiments, any amino acid residue in the constant region may be replaced 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 contains mutations that alter its function, for example, mutating the lysine residue (K) at the C-terminus of the antibody heavy chain constant region (common in wild-type IgG1 antibodies) to a hydrophobic amino acid such as alanine (A) or leucine (L) to reduce protease hydrolysis and increase serum half-life; such modifications are particularly suitable for situations in which the C-terminus of the antibody heavy chain is fused to another moiety. The C-terminal residue of the heavy chain constant region of the anti-PD-1 antibody portion of an exemplary PD-1 / TGFβ bifunctional protein of the present invention is correspondingly modified.

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

[0042] 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 set forth in SEQ ID NO:12.

[0043] In some embodiments, the bifunctional protein comprises: (1) two completely identical first polypeptides whose amino acid sequences are 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%) identical to the amino acid sequence set forth in SEQ ID NO:13; and (2) two completely identical second polypeptides whose amino acid sequences are 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%) identical to the amino acid sequence set forth in SEQ ID NO:14.

[0044] As a non-limiting example, a bifunctional protein of the present invention (i.e., a PD-1 / TGFβ bifunctional protein, hereinafter also referred to as a "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 set forth 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 Figure 1. As shown in Figure 1, PD1 / TGFβRII is based on a native anti-PD-1 antibody, with a flexible linker and a human TGF-βRII isoform B extracellular domain polypeptide sequentially extended at the CH3 terminus of the heavy chain constant region.

[0045] PD1 / TGFβRII is an exemplary bifunctional protein of the present invention, which has higher TGFβ binding activity and PD-1 terminal biological activity, as well as a superior tumor-suppressing effect, compared to the reported nivolumab / TGFβRII fusion protein. Furthermore, it has lower cytotoxicity and side effects than conventional PD-1 antibodies (e.g., nivolumab). Because of its lower cytotoxicity and side effects, PD1 / TGFβRII can be administered at higher doses, which allows for better TGFβ inhibition and depletion, and its more optimal dose safety margins favor 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 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 malignancies, head and neck cancer, liver cancer, nasopharyngeal carcinoma, 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 compositions may further comprise lubricants (such as talc, magnesium stearate, and mineral oil), wetting agents, emulsifying agents, suspending agents, preservatives (such as benzoic acid, sorbic acid, and calcium propionate), sweeteners, and / or flavoring agents. In some embodiments, pharmaceutical compositions of the present invention may be prepared in the form of tablets, pills, powders, tablets, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules.

[0049] In some embodiments, the pharmaceutical compositions of the present invention may be delivered using any physiologically acceptable method of administration, including, but not limited to, oral administration, parenteral administration, nasal administration, rectal administration, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, and the like. In some embodiments, pharmaceutical compositions for therapeutic purposes may be prepared and stored in the form of a lyophilized formulation or aqueous solution by mixing reagents having a desired purity and, if necessary, pharmaceutically acceptable carriers, excipients, etc.

[0050] According to a fourth aspect, the present invention provides the use of a bifunctional protein of the first aspect or a 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 malignancies, head and neck cancer, liver cancer, nasopharyngeal carcinoma, 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 a malignant tumor, comprising 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 malignancies, head and neck cancer, liver cancer, nasopharyngeal carcinoma, 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, said bifunctional protein comprising a PD-1 (programmed death receptor-1) binding portion and a TGF-β (transforming growth factor-β) binding portion, said 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 allowing protein expression; harvesting the cell culture and / or supernatant and isolating and purifying said bifunctional protein.

[0054] Unless inconsistent, the embodiments and technical features described in the first aspect also apply 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 sucrose or glycerin to the eluent is advantageous in reducing degradation of the fusion protein. The following examples are for illustrative purposes only 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, a PD1 / TGFβRII fusion protein according to the present invention was constructed, and its structural schematic is shown in Figure 1. Nucleotide sequences encoding the heavy chain portion (SEQ ID NO: 13) and light chain portion (SEQ ID NO: 14) of the PD1 / TGFβRII fusion protein fused to a signal peptide were synthesized and then cloned into the pcDNA3.1 expression vector. The PD1 / TGFβRII fusion protein expression vector was co-transfected into CHO cells using standard methods of instantaneous or stable transfection, and the transfected cells were cultured in an incubator at 37°C with 8% CO2.

[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 contained in SEQ ID NO:13. GGGGSGGGGSGGGGSGGGGSG Amino acid sequence of the heavy chain of the PD1 / TGFβRII fusion protein (SEQ ID NO:13): EVQLVESGGGLVQPGGSLRLSCAASGFAFSSYDMSWVRQAPGKGLDWVATISGGGRYTYYPDSVKGRFTISRDNSKNNLYLQMNSLRAEDTALYYCANRYGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGS GGGGSGGGGSGGGGSGIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPD

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

[0058] Example 2: Purification of PD1 / TGFβRII fusion protein The cell culture medium obtained in Example 1 was centrifuged, and 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. The column was washed with the equilibration buffer for 3 to 5 column volumes, and then the cell supernatant was injected (sample loading). After the injection was completed, the column was washed with the equilibration buffer. Next, the column was rinsed with a rinse buffer (0.5 mol / L sodium chloride + 25 mmol / L phosphate buffer, pH 7.0), and the column was further equilibrated with the equilibration buffer 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), and the eluted sample was collected. The sample was neutralized with 2 M Tris-HCl buffer (pH 9.5).

[0059] The neutralized eluted sample (pH 6.0) was subjected to anion exchange chromatography using an equilibration buffer of 10 mmol / L citrate buffer + 10 mmol / L phosphate buffer + 10 mmol / L Tris, pH 6.0. After rinsing the column with the equilibration buffer for 3 to 5 column volumes, the neutralized eluted sample was injected (sample loading). The passed sample was collected, and after injection, the column was rinsed with the equilibration buffer. The sample passed through the anion exchange chromatography 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, pH 5.0. The sample passed through the anion exchange chromatography was adjusted to pH 5.0 and then injected. After injection, the column was washed with the equilibration buffer for 3 to 5 column volumes. Next, the sample 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 samples by molecular exclusion chromatography The components of the PD1 / TGFβRII fusion protein sample obtained by the purification in Example 2 were separated using a gel chromatography column. Elution was performed using a neutral pH buffer as the mobile phase, and the molecular weight components were eluted in descending order of molecular weight. The column was Thermo MabPac TM The SEC-1 300Å, 5μm, 7.8*300mm gel column was used, and the mobile phase was (20mmol / L disodium hydrogen phosphate + 300mmol / L sodium chloride + 2% isopropyl alcohol solution, pH=7.4). The sample was diluted to 1mg / mL with the mobile phase to prepare the test solution. 50μL of the test solution was precisely weighed and injected into the liquid chromatograph, and detection was performed at a wavelength of 280nm. Isocratic elution was performed for 35 minutes at a flow rate of 0.5mL / min.

[0061] The results were quantitatively analyzed using the area normalization method. The peak area percentages of high-molecular-weight impurities, immunoglobulin monomers, and low-molecular-weight impurities were calculated. The results showed that in the PD1 / TGFβRII fusion protein sample, the high-molecular-weight impurities were 0.19%, the immunoglobulin monomers were 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 in the same manner, but with the C-terminal amino acid residue of the original heavy chain constant region of nivolumab changed from K to A, consistent with the exemplary PD1 / TGFβRII fusion protein of the present invention.

[0062] Example 4: Detection of biological activity of the PD-1 terminus of PD1 / TGFβRII fusion protein by reporter gene method The detection procedure is as follows: CHO-PDL1-CD3L cells (purchased from China Food and Drug Administration) in the logarithmic growth phase were harvested and the viable cell density was adjusted to 5 × 10 in DMED / F12 complete medium. 5The cells were adjusted to a viable cell density of 2 × 10 cells / mL and added to a 96-well white plate at 100 μL per well. The cells were then placed in a cell incubator at 37°C with 5% CO for 16–20 hours. The next day, a Jurkat-PD-1-NFAT cell (purchased from China Food and Drug Administration) suspension was prepared and diluted to a viable cell density of 2 × 10 cells / mL in 1640 basal medium containing 2% FBS. 6 The 96-well white plate containing the CHO-PDL1-CD3L cells was removed from the incubator, the supernatant was aspirated and discarded, and 50 μL of the Jurkat-PD-1-NFAT cell suspension was added to the plate. Next, 50 μL of the control PD-1 antibody (nivolumab; SEQ ID NOs: 17 and 18 are the heavy and light chain sequences, respectively) and the gradient dilutions of the PD1 / TGFβRII fusion proteins prepared in Examples 1 to 3 according to the present invention (initial concentration: 200,000 ng / mL, 3-fold gradient dilutions, a total of 11 dilution gradients) were added to the plate. The plate was then placed in a cell incubator at 37°C containing 5% CO2 and incubated for 4 to 6 hours. During the incubation period, Bio-Lite Luciferase Reagent (Vazyme, DD1201-03) was removed and thawed at room temperature. After the incubation was completed, 100 μL / well of the 96-well white plate was added and incubated at room temperature for 2-3 minutes in the dark. RLU values ​​were read using a multifunction plate reader (Thermo, Varioskan Flash). The experimental data were analyzed using Prism software, and dose-response curves for the reference and test products were plotted. The EC values ​​for the reference and test products were then calculated. 50 The biological activity of the test product was calculated.

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

[0064] [Table 1]

[0065] Example 5: Detection of TGFβ binding activity of PD1 / TGFβRII fusion protein by enzyme-linked immunosorbent assay The detection flow is as follows. 1) 2 μg / mL human TGFβ1 protein (Sinobiological, 10804-H08H) was used as an antigen and adsorbed onto a highly absorbent 96-well plate at 100 μL / well, followed by incubation overnight at 2-8°C. 2) The 96-well plate was washed three times with 250 μL / well of PBST20 (PBS solution containing 0.05% Tween 20), and then 250 μL / well of blocking solution (PBS solution containing 3% BSA) was added and incubated at 25°C for 2 hours. 3) After washing the 96-well plate three times with PBST20 at 250 μL / well, gradient-diluted PD1 / TGFβRII fusion proteins prepared in Examples 1 to 3 according to the present invention (initial concentration 4000 ng / mL, 4-fold gradient dilution, total of 7 dilutions) were added at 100 μL / well, and the plate was incubated at 25°C for 2 hours.

[0066] 6) After washing the 96-well plate three times with 250 μL / well of PBST20, 100 μL of HRP-sheep anti-human IgG antibody (PE, NEF802001EA) diluted 1:3,500 with diluent was added per well, and the plate was incubated at 25°C for 1 hour. 7) The 96-well plate was washed five times with PBST20 at 250 μL / well, and then TMB solution was added at 100 μL / well, followed by incubation at 25° C. in the dark for 5 minutes. 10) The reaction was stopped by adding 100 μL / well of 1 mol / L H2SO4 and leaving it at room temperature for 5 minutes. The OD values ​​were measured at wavelengths of 450 nm and 650 nm using a microplate reader (Thermo Scientific, Varioskan Flash), and the data were analyzed using Graphpad Prism.

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

[0068] [Table 2]

[0069] Example 6: Therapeutic effect of PD1 / TGFβRII fusion protein on subcutaneous colon cancer cell MC38 / hPD-L1 mouse xenografts C57 / PD-1 transgenic mice (purchased from Jiangsu Jisui Yaokang Biotechnology Co., Ltd.) were used as experimental mice, with 3 × 10 5 MC38 / hPD-L1 cells were subcutaneously inoculated, and tumors were 40-70 mm 3 Once tumors reached 1000 mm, they were divided into groups based on tumor volume and administered the drug intraperitoneally (ip) at an injection volume of 0.1 mL / 10 g body weight once every two days for a total of six doses. The administration schedule is shown in Table 3, and the day of administration was designated D0. Tumor diameter was measured twice a week with a vernier caliper, and the effect of the drug on tumor growth was evaluated based on T / C% or tumor inhibition rate TGI (%) calculated using the following formula. The experiment was terminated when the tumor volume reached 1500 mm or the end point. 3 When the tumor size reached 100 mg / kg, the animals were euthanized by CO2 anesthesia and then dissected to collect and photograph the tumors.

[0070] The formula for calculating tumor volume (V) is V = 1 / 2 × a × b 2where a and b represent length and width, respectively. T / C (%) = (T - T0) / (C - C0) × 100, where T and C are the tumor volumes of the treated and negative control mice at the end of the experiment, and T0 and C0 are the tumor volumes of the treated and negative control mice at the start of the experiment. The T / C values ​​of the treated and negative control mice were calculated based on the T / C values ​​of the treated and negative control mice. Tumor inhibition rate (TGI) (%) = 100 - T / C (%). The results are shown in Table 4. The PD1 / TGFβRII (3.7 mg / kg, IP, twice daily, a total of six doses) prepared in Examples 1 to 3 of the present invention achieved a tumor inhibition rate of 74% against subcutaneously implanted tumors in MC38 / hPD-L1 mice at day 19, which was superior to the control PD-1 monoclonal antibody. The tumor-bearing mice developed good tolerance to the drug and did not experience any obvious symptoms such as weight loss.

[0071] [Table 3]

[0072] [Table 4] Note: hIgG4 (HG4K, Sino Biological Inc.) was used as a negative control. The control PD-1 monoclonal antibody was antibody 14C12H1L1, described in Chinese Patent Application No. 201610705763.5 (CN106977602), whose heavy and light chain amino acid sequences are set forth in SEQ ID NOs: 19 and 14, respectively, of the present invention.

[0073] Example 7: In vitro activity detection of PD1 / TGFβRII fusion protein Using the methods of Examples 1 to 3, exemplary PD1 / TGFβRII fusion proteins and nivolumab / TGF-βRII fusion proteins according to the present invention were prepared in the same batch. Using the reporter gene method of Example 4, the biological activity of the PD-1 end of the PD1 / TGFβRII fusion protein according to the present invention was detected and compared. The results were as shown in Table 5. Using the enzyme-linked immunosorbent assay of Example 5, the TGFβ binding activity of the PD1 / TGFβRII fusion protein according to the present invention was detected and compared. The results were as shown in Table 6. The nivolumab / TGF-βRII fusion protein in this and the following examples was self-produced and has heavy and light chain sequences set forth in SEQ ID NOs: 20 and 18.

[0074] [Table 5]

[0075] [Table 6]

[0076] Example 8: Therapeutic effect of PD1 / TGFβRII fusion protein on MC38 / hPD-L1 mouse xenografts Humanized PD-1 mice (purchased from Biocytogen) were used as experimental mice, and 4 × 10 5 MC38 / hPD-L1 cells were inoculated, and tumors were 100–300 mm 3Once the mice reached a normal growth stage, they were randomly divided into three groups and administered the drugs via intraperitoneal (ip) injection a total of eight times. The dosing schedule is shown in Table 7, and the day of administration was designated D0. Tumor volume was measured two to three times a week, and mouse weights were recorded. Tumor diameter was measured using a vernier caliper, and the T / C (%) or tumor volume inhibition rate (1-T / C) was calculated using the following formula to determine the effect of the drugs on tumor growth. At the end of the experiment, the animals were anesthetized with CO2 and sacrificed. The tumors were then dissected, collected, and photographed.

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

[0078] As shown in Tables 8 and 9, PD1 / TGFβRII and nivolumab / TGF-βRII (prepared from the same lot as in Example 7) achieved tumor volume inhibition rates of 46.8% and 32.3%, respectively, in MC38 / hPD-L1-implanted mice at D23, indicating that the tumor-inhibitory effect of PD1 / TGFβRII according to the present invention is superior to that of nivolumab / TGF-βRII. Furthermore, the body weights of mice in the PD1 / TGFβRII and nivolumab / TGF-βRII groups increased variably, indicating that neither of the two fusion proteins caused any obvious toxic reactions.

[0079] [Table 7]

[0080] [Table 8]

[0081] [Table 9]

[0082] Example 9: Electrochemiluminescence detection of stimulation of cytokine secretion by PD1 / TGFβRII fusion protein PBMC cell concentration was adjusted to approximately 2 x 10 using RPMI 1640 complete medium. 6 The solution was adjusted to 900 μg / mL of IgG1 protein (SEQ ID NO:21 and SEQ ID NO:22 are the heavy and light chain sequences, respectively, prepared in-house), LPS (SIGMA, L4391-1MG), and the PD1 / TGFβRII fusion protein of the present invention were each diluted with RPMI 1640 complete medium to prepare 900 μg / mL of IgG1 protein, 1 μg / mL of LPS, and 10 μg / mL, 100 μg / mL, and 900 μg / mL of PD1 / TGFβRII fusion protein. RPMI 1640 complete medium served as a negative control. 100 μL of each of the solutions prepared above was added to a 96-well cell culture plate, mixed thoroughly, and cultured in a 37°C, 5% CO2 cell incubator. After 48 hours, the cell supernatants in the 96-well plate were taken and the contents of cytokines IL-2, IL-6, IL-8, IL-10, TNF-α and IFN-γ were detected using the V-PLEX Proinflammatory Panel 1 (human) kit (MSD, K15049D-2) and the high-sensitivity immunoassay system (MSD, QuickPlexSQ120). The results are shown in Table 10.

[0083] [Table 10]

[0084] The results in the above table demonstrate that the PD1 / TGFβRII fusion protein of the present invention is unlikely to cause a cytokine storm, and therefore poses little risk of overactivating the immune system and causing systemic inflammation when administered to a subject.

[0085] Example 10: Toxicity test of PD1 / TGFβRII fusion protein in cynomolgus monkeys Single-dose toxicity: In this study, four cynomolgus monkeys were divided into two groups, two in each group, half male and half female. They were intravenously injected with 200 and 500 mg / kg of the PD1 / TGFβRII fusion protein of the present invention, respectively, and observed for 14 days. During the study, general observations were performed and indicators such as body weight, food intake, body temperature, lead II electrocardiogram and blood pressure, hematology, blood biochemistry, and urine were monitored. At the end of the study, a rough necropsy was performed.

[0086] After administration, male monkeys in each group showed a transient decrease in food intake, which recovered by day 8 or 9 of the study. On day 14 of the study, male monkeys in each group showed decreases in RBC, HGB, and HCT. No obvious abnormal changes were observed in any of the other indicators. In a single-dose toxicity study, cynomolgus monkeys were intravenously administered a single dose of 200 or 500 mg / kg of the PD1 / TGFβRII fusion protein of the present invention, with the MTD set at 500 mg / kg. Multiple-dose toxicity: This study involved 40 cynomolgus monkeys, divided into four groups of 10 monkeys, each half male and half female. These groups were a blank control group and the PD1 / TGFβRII fusion protein of the present invention at 15, 50, or 150 mg / kg. The monkeys were administered once weekly for four consecutive weeks (a total of five doses), followed by a 4-week recovery period and observation period.

[0087] On the 15th day and at the end of administration, decreases in RBC, HGB, and HCT, and compensatory increases in RET and RET% were observed in male monkeys in the 50 mg / kg group and in both males and females in the 150 mg / kg group; these changes were also observed in female monkeys in the 50 mg / kg group on the 15th day of administration.

[0088] At the end of the treatment period, macroscopic dissection of one male monkey in the 50 mg / kg group revealed cardiac pericardial adhesions. Histopathological examination: In cynomolgus monkeys in the 150 mg / kg group, minimal to mild cerebral meninges and choroid plexus, cerebellar meninges and choroid plexus, spinal cord, thyroid gland, heart, and pituitary gland monocytic infiltration, and minimal to moderate vascular / perivasculitis were observed in the heart, liver, bladder, epididymis, and seminal vesicles. In cynomolgus monkeys in the 50 mg / kg group, minimal to moderate cerebral meninges, sciatic nerve, thyroid gland, heart, and pituitary gland monocytic infiltration, and minimal to moderate vascular / perivasculitis were observed in the heart, bladder, duodenum, ileum, rectum, fallopian tube, vagina, and uterus. In cynomolgus monkeys in the 15 mg / kg group, minimal to mild cerebral meninges and choroid plexus, cerebellar choroid plexus, sciatic nerve, thyroid gland, and heart monocytic infiltration, and mild cardiac vascular / perivasculitis were observed. In addition, in the 50 and 150 mg / kg groups, mild femoral osteonecrosis, thickening of the epiphyseal plate, slight to mild increases in femoral metaphyseal trabeculae, and osteoclasts were observed in one cynomolgus monkey each.

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

[0090] In a multiple-dose toxicity study, 15, 50, and 150 mg / kg of the PD1 / TGFβRII fusion protein of the present invention were intravenously administered to cynomolgus monkeys (once weekly for four consecutive weeks, a total of five doses), with an HNSTD of 150 mg / kg. Based on the information disclosed in the European Medicines Agency's assessment report for nivolumab, the HNSTD of nivolumab was 50 mg / kg, which was significantly lower than the HNSTD of the PD1 / TGFβRII fusion protein of the present invention. Therefore, the PD1 / TGFβRII fusion protein of the present invention is expected to exhibit good clinical safety due to its low toxicity.

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

Claims

1. A bifunctional protein containing a PD-1 (programmed death receptor-1) binding site and a TGF-β (transforming growth factor-β) binding site.

2. The PD-1 binding portion may be an anti-PD-1 antibody or antigen-binding fragment, e.g., a full-length antibody, a Fab fragment, or an F(ab') ２ The bifunctional protein of claim 1, which is a fragment, an Fv fragment or a single chain Fv fragment (scFv).

3. 3. The bifunctional protein of claim 2, wherein 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 having the amino acid sequence GFAFSSYD (SEQ ID NO: 1), an HCDR2 having the amino acid sequence ISGGGRYT (SEQ ID NO: 2), and an HCDR3 having the amino acid sequence ANRYGEAWFAY (SEQ ID NO: 3), and the light chain variable region comprises an LCDR1 having the amino acid sequence QDINTY (SEQ ID NO: 4), an LCDR2 having the amino acid sequence RAN (SEQ ID NO: 5), and an LCDR3 having the amino acid sequence LQYDEFPLT (SEQ ID NO: 6).

4. The bifunctional protein of claim 3 , wherein the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO: 7 and / or the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:

8.

5. 5. The bifunctional protein of claim 3 or 4, wherein 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 set forth in SEQ ID NO: 9 or a variant of the amino acid sequence set forth in SEQ ID NO: 9, e.g., the amino acid sequence set forth in SEQ ID NO: 9 in which the C-terminal residue A is replaced with K, and / or the amino acid sequence of the light chain constant region is set forth in SEQ ID NO: 10 or a variant of the amino acid sequence set forth in SEQ ID NO:

10.

6. The TGF-β binding part is a TGF-β receptor or a binding domain of a TGF-β receptor, such as the extracellular domain or a binding fragment of the extracellular domain of a TGF-β receptor; Antibodies or antigen-binding fragments against TGF-β, e.g., full-length antibodies, Fab fragments, F(ab') ２ The bifunctional protein of any one of claims 1 to 5, which is a fragment, an Fv fragment or a single chain Fv fragment (scFv).

7. 7. The bifunctional protein of claim 6, wherein the TGF-β binding portion is a human TGF-β RII isoform B extracellular domain polypeptide comprising the amino acid sequence shown in SEQ ID NO:

11.

8. The bifunctional protein of any one of claims 1 to 7, wherein the PD-1-binding portion and the TGF-β-binding portion are linked via a flexible linker, for example, a GGGGS-type linker, for example, a linker shown in SEQ ID NO:

12.

9. (1) two completely identical first polypeptides whose amino acid sequences have at least 80% identity with the amino acid sequence set forth in SEQ ID NO: 13; (2) two completely identical second polypeptides whose amino acid sequences have at least 80% identity with the amino acid sequence set forth in SEQ ID NO: 14; The bifunctional protein according to any one of claims 1 to 8.

10. A nucleic acid molecule encoding the bifunctional protein of any one of claims 1 to 9.

11. A pharmaceutical composition comprising the bifunctional protein of any one of claims 1 to 9 and a pharmaceutically acceptable excipient, diluent or carrier.

12. 12. The pharmaceutical composition of claim 11, for use in the prevention or treatment of a malignant tumor 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 malignancies, head and neck cancer, liver cancer, nasopharyngeal carcinoma, 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, optionally wherein the malignant tumor is primary, metastatic, recurrent and / or refractory.

13. Use of the bifunctional protein according to any one of claims 1 to 9 or the nucleic acid molecule according to claim 10 in the manufacture of a medicament for preventing or treating malignant tumors, comprising: 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 malignancies, head and neck cancer, liver cancer, nasopharyngeal carcinoma, 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, and optionally, the malignant tumor is primary, metastatic, recurrent, and / or refractory.

14. A method for preventing or treating a malignant tumor, comprising: Administering the bifunctional protein of any one of claims 1 to 9 or the pharmaceutical composition of claim 11 to an individual suffering from a malignant tumor; 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 malignancies, head and neck cancer, liver cancer, nasopharyngeal carcinoma, 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, and optionally, the malignant tumor is primary, metastatic, recurrent, and / or refractory.

15. A method for producing the bifunctional protein according to any one of claims 1 to 9 or the pharmaceutical composition according to claim 11, 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; harvesting the cell culture and / or supernatant and isolating and purifying the bifunctional protein; Optionally, combining the isolated and purified bifunctional protein with a pharmaceutically acceptable excipient, diluent, or carrier to prepare the product.

Images