WO2021218895A1 - 针对PD-1和TGF-β的双功能蛋白 - Google Patents

针对PD-1和TGF-β的双功能蛋白 Download PDF

Info

Publication number
WO2021218895A1
WO2021218895A1 PCT/CN2021/089837 CN2021089837W WO2021218895A1 WO 2021218895 A1 WO2021218895 A1 WO 2021218895A1 CN 2021089837 W CN2021089837 W CN 2021089837W WO 2021218895 A1 WO2021218895 A1 WO 2021218895A1
Authority
WO
WIPO (PCT)
Prior art keywords
cancer
seq
amino acid
acid sequence
bifunctional protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2021/089837
Other languages
English (en)
French (fr)
Chinese (zh)
Inventor
赵伟
李盈淳
吕海丽
谢联香
张哲文
秦宇
张喜全
程艳菊
吕鹏
李田田
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chia Tai Tianqing Pharmaceutical Group Co Ltd
Original Assignee
Chia Tai Tianqing Pharmaceutical Group Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to AU2021263650A priority Critical patent/AU2021263650A1/en
Priority to US17/922,122 priority patent/US20230235057A1/en
Priority to EP21795280.3A priority patent/EP4144763A4/en
Priority to JP2022565971A priority patent/JP7711095B2/ja
Priority to MX2022013620A priority patent/MX2022013620A/es
Priority to BR112022022020A priority patent/BR112022022020A2/pt
Application filed by Chia Tai Tianqing Pharmaceutical Group Co Ltd filed Critical Chia Tai Tianqing Pharmaceutical Group Co Ltd
Priority to KR1020227041823A priority patent/KR20230003178A/ko
Priority to CA3181579A priority patent/CA3181579A1/en
Publication of WO2021218895A1 publication Critical patent/WO2021218895A1/zh
Anticipated expiration legal-status Critical
Priority to JP2025115631A priority patent/JP2025148434A/ja
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • This application generally relates to the field of antibody drugs, especially the treatment of malignant tumors.
  • the present application provides a bifunctional protein capable of binding PD-1 (programmed death receptor-1) and TGF- ⁇ (transforming growth factor- ⁇ ), and medical uses of the bifunctional protein.
  • PD-1 programmed death receptor-1
  • TGF- ⁇ transforming growth factor- ⁇
  • T cells express many important membrane protein immune molecules, including PD-1 (Programmed Death-1, also known as CD279) protein, which belongs to the CD28 family of the immunoglobulin superfamily, and its ligand ( PD-L1, PD-L2) belong to the B7 family.
  • PD-1 Programmed Death-1, also known as CD279 protein
  • CD279 CD28 family of the immunoglobulin superfamily
  • PD-L1, PD-L2 its ligand
  • PD-L1 and PD-1 negatively regulates the immune function of T cells and is an important checkpoint for suppressive immunity of peripheral T cells.
  • PD-L1 is low expressed in normal human tissues to maintain immune tolerance and avoid autoimmune reactions.
  • tumor cells can inhibit T cell immune function through high expression of PD-L1 (or release PD-L1 soluble variants, exosomes), and form 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
  • TGF- ⁇ (transforming growth factor- ⁇ , transforming growth factor- ⁇ ) is a type of cytokine with multifunctional biological activity, which can regulate the body by regulating cell proliferation, differentiation, apoptosis, adhesion, invasion and microenvironment Physiological process.
  • the typical TGF- ⁇ signaling pathway is firstly that TGF- ⁇ binds to type II TGF- ⁇ receptor (TGF- ⁇ RII), and then forms a complex with type I TGF- ⁇ receptor (TGF ⁇ RI) to activate it, and TGF ⁇ RI phosphorylation activates R-Smad (Smad1,2,3,5,8) members, R-Smad combines with Co-Smad (Smad4) to form a complex and transfers into the nucleus to regulate the transcription of target genes.
  • TGF- ⁇ Tumor Suppression through a Lethal EMT. [J ].Cell,2016,164(5).
  • Other studies have shown that TGF- ⁇ may destroy the tumor microenvironment by inducing Treg cells and suppressing effector T cells, and accelerate tumor progression (Shen Yinan et al, TGF- ⁇ regulates hepatocellular carcinoma progression by inducing Treg cell polarization.[J] .Cellular physiology and biochemistry,2015,35(4)).
  • TGF- ⁇ signaling is the cause of drug resistance against PD-(L)1 in patients.
  • the present application provides a bifunctional protein comprising a PD-1 (programmed death receptor-1) binding part and a TGF- ⁇ (transforming growth factor- ⁇ ) binding part.
  • PD-1 programmed death receptor-1
  • TGF- ⁇ transforming growth factor- ⁇
  • the PD-1 binding portion is an anti-PD-1 antibody or antigen-binding fragment. In some embodiments, the PD-1 binding portion is a full-length antibody, Fab fragment, F(ab') 2 fragment, Fv fragment, or single chain Fv fragment (scFv) against PD-1.
  • 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 comprising HCDR1 with an amino acid sequence of GFAFSSYD (SEQ ID NO: 1),
  • the amino acid sequence is HCDR2 with the amino acid sequence of ISGGGRYT (SEQ ID NO: 2) and the HCDR3 with the amino acid sequence of ANRYGEAWFAY (SEQ ID NO: 3)
  • the light chain variable region includes LCDR1 with the amino acid sequence of QDINTY (SEQ ID NO: 4), LCDR2 with the amino acid sequence of RAN (SEQ ID NO: 5) and LCDR3 with the amino acid sequence of LQYDEFPLT (SEQ ID NO: 6).
  • 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.
  • 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 shown in SEQ ID NO: 9 or SEQ ID NO: A variant of the amino acid sequence shown in 9, for example, the amino acid sequence shown in SEQ ID NO: 9 where 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 or It is a variant of the amino acid sequence shown in SEQ ID NO: 10.
  • the anti-PD-1 antibody or antigen-binding fragment is selected from: Nivolumab (Nivolumab), Pembrolizumab (Pembrolizumab), Durvalumab, Teriprizumab (JS-001) , Sintilizumab (IBI308), Camrelizumab (Camrelizumab), Tirelizumab (BGB-A317), AK105 (Kangfang Bio), Genolizumab (GB226), Livzumab Anti-(LZM009), HLX-10, BAT-1306, AK103 (HX008), AK104 (Kangfang Bio), CS1003, SCT-I10A, F520, SG001, GLS-010, or antigen-binding fragments of the above antibodies.
  • the TGF- ⁇ binding portion is a TGF- ⁇ receptor or a binding domain of a TGF- ⁇ receptor. In some embodiments, the TGF- ⁇ binding portion is the extracellular domain or a binding fragment of the extracellular domain of the TGF- ⁇ receptor. In some specific embodiments, the TGF- ⁇ binding portion is a human TGF- ⁇ RII isoform B extracellular domain polypeptide, which comprises the amino acid sequence shown in SEQ ID NO: 11. In some specific embodiments, the TGF- ⁇ binding portion is a variant of the human TGF- ⁇ RII isoform B extracellular domain polypeptide, for example, it has at least 80%, 81%, and 80% of the amino acid sequence shown in SEQ ID NO: 11.
  • the TGF- ⁇ binding portion is an anti-TGF- ⁇ antibody or antigen-binding fragment. In some embodiments, the TGF- ⁇ binding portion is a full-length antibody, Fab fragment, F(ab′) 2 fragment, Fv fragment, or single chain Fv fragment (scFv) against TGF- ⁇ .
  • the PD-1 binding portion and the TGF- ⁇ binding portion are connected by a flexible linker.
  • the flexible linker is a GGGS type linker. In some specific embodiments, the flexible linker is the linker shown in SEQ ID NO: 12.
  • the bifunctional protein comprises: (1) two identical first polypeptides, and the amino acid sequence of the first polypeptide is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 13; And (2) two identical second polypeptides, the amino acid sequence of the second polypeptide has at least 80% identity with the amino acid sequence shown in SEQ ID NO: 14.
  • this application provides a nucleic acid molecule encoding the bifunctional protein of the first aspect.
  • the present application provides a pharmaceutical composition, which comprises the bifunctional protein of the first aspect, and a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition is used to prevent or treat malignant tumors.
  • 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 cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, protuberans Dermofibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and/or myelodysplastic syndrome.
  • the malignant tumor is in situ, metastatic, recurrent, and/or refractory.
  • this application provides the use of the bifunctional protein of the first aspect or the nucleic acid molecule of the second aspect in the preparation of a medicine for the prevention or treatment of malignant tumors.
  • 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 cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, bulging skin Fibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and/or myelodysplastic syndrome.
  • the malignant tumor is in situ, metastatic, recurrent, and/or refractory.
  • the present application provides a method for preventing or treating malignant tumors, including administering the bifunctional protein of the first aspect or the pharmaceutical composition of the third aspect to an individual suffering from a malignant tumor.
  • 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 cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, bulging skin Fibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and/or myelodysplastic syndrome.
  • the malignant tumor is in situ, metastatic, recurrent, and/or refractory.
  • the present application provides a method for preparing a bifunctional protein comprising a PD-1 (programmed death receptor-1) binding part and a TGF- ⁇ (transforming growth factor- ⁇ ) binding part, and Including the bifunctional protein described in the first aspect, the method includes the following steps:
  • Figure 1 shows a schematic diagram of the structure of an exemplary PD-1/TGF ⁇ bifunctional protein of the present application.
  • Figure 2 shows the results of the reporter gene method for detecting the biological activity of the PD-1 end of the exemplary PD-1/TGF ⁇ bifunctional protein of the present application, in which Nivolumab was used as a control sample.
  • Figure 3 shows the results of the enzyme-linked immunosorbent assay method for testing the TGF ⁇ binding activity of the exemplary PD-1/TGF ⁇ bifunctional protein of the present application.
  • SEQ ID NO: 1-3 are the sequences of the CDR1-CDR3 of the heavy chain variable region of the anti-PD-1 antibody portion of the exemplary PD-1/TGF ⁇ bifunctional protein of the application.
  • SEQ ID NOs: 4-6 are the sequences of the CDR1-CDR3 of the light chain variable region of the anti-PD-1 antibody portion of the exemplary PD-1/TGF ⁇ bifunctional protein of the application.
  • SEQ ID NOs: 7 and 8 are respectively the sequences of the heavy chain variable region and the light chain variable region of the anti-PD-1 antibody portion of the exemplary PD-1/TGF ⁇ bifunctional protein of the application.
  • SEQ ID NOs: 9 and 10 are respectively the sequences of the heavy chain constant region and the light chain constant region of the anti-PD-1 antibody portion of the exemplary PD-1/TGF ⁇ bifunctional protein of the application.
  • SEQ ID NO: 11 is the TGF- ⁇ binding part of the exemplary PD-1/TGF ⁇ bifunctional protein of the application, that is, the human TGF- ⁇ RII isoform B extracellular domain polypeptide.
  • SEQ ID NO: 12 is a flexible linker between the anti-PD-1 antibody portion of the exemplary PD-1/TGF ⁇ bifunctional protein of the application and the TGF- ⁇ binding part.
  • SEQ ID NO: 13 is the heavy chain partial sequence of the exemplary PD-1/TGF ⁇ bifunctional protein of the application.
  • the heavy chain part is composed of the heavy chain of the anti-PD-1 antibody part, the flexible linker (SEQ ID NO: 12) and Human TGF- ⁇ RII isoform B extracellular domain polypeptide (SEQ ID NO: 11).
  • SEQ ID NO: 14 is the partial light chain sequence of the exemplary PD-1/TGF ⁇ bifunctional protein of the application, and the light chain part is composed of the light chain of the anti-PD-1 antibody part.
  • SEQ ID NO: 15 is the coding nucleic acid sequence of SEQ ID NO: 13 (not including the coding sequence of the signal peptide).
  • SEQ ID NO: 16 is the coding nucleic acid sequence of SEQ ID NO: 14 (not including the coding sequence of the signal peptide).
  • SEQ ID NO: 17 and 18 are the sequences of the heavy chain and light chain of the control PD1 monoclonal antibody Nivolumab (Nivolumab), respectively.
  • SEQ ID NOs: 19 and 14 are the heavy chain and light chain sequences of another control PD1 monoclonal antibody (from Chinese Patent Application No. 201610705763.5 (CN106977602)), respectively.
  • SEQ ID NOs: 20 and 18 are the heavy chain partial sequence and the light chain partial sequence of Nivolumab/TGF- ⁇ RII bifunctional protein as the bifunctional protein control substance, and the heavy chain part is composed of the heavy chain of Nivolumab (SEQ ID NO: 17 C-terminal amino acid residue is mutated from K to A), flexible linker (SEQ ID NO: 12) and human TGF- ⁇ RII isoform B extracellular domain polypeptide (SEQ ID NO: 11)
  • the light chain part is composed of the light chain of nivolumab.
  • SEQ ID NO: 21 and 22 are the sequences of the heavy chain and light chain of the experimental control IgG1 protein.
  • 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.
  • antibody not only includes intact (ie, full-length) antibodies, but also includes antigen-binding fragments (such as Fab, Fab', F(ab') 2 , Fv), variants thereof, and antibody portions. Fusion proteins, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g. bispecific antibodies) and any other immunoglobulin containing antigen recognition sites of the desired specificity Modified configuration of protein molecules, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies.
  • a complete or full-length antibody contains two heavy chains and two light chains.
  • Each heavy chain contains a heavy chain variable region (VH) and first, second, and third constant regions (CH1, CH2, and CH3).
  • Each light chain contains a light chain variable region (VL) and constant region (CL).
  • the full-length antibody can be any kind of antibody, such as IgD, IgE, IgG, IgA, or IgM (or the above subclasses), but the antibody does not need to belong to any specific class.
  • immunoglobulins can be assigned to different classes.
  • immunoglobulins there are five main classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several 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 different immunoglobulin classes are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.
  • antigen-binding fragment refers to the part of the antibody structure that determines the antigen-binding ability.
  • the antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both.
  • VH and VL usually contains three complementarity determining regions CDR1, CDR2, and CDR3.
  • CDR complementarity determining region
  • variable region sequence of a given antibody the CDR sequence in the variable region sequence can be analyzed in a variety of ways, for example, it can be determined using the online software Abysis (http://www.abysis.org/).
  • antigen-binding fragments include, but are not limited to: (1) Fab fragments, which can be monovalent fragments with VL-CL chains and VH-CH1 chains; (2) F(ab') 2 fragments, which can have two A bivalent fragment of the Fab' fragment, the two Fab' fragments are connected by a disulfide bridge in the hinge region (ie, a dimer of Fab'); (3) Fv fragments with VL and VH domains with one arm of an antibody; ( 4) Single-chain Fv (scFv), which can be a single multi-peptide chain composed of a VH domain and a VL domain via a peptide linker; and (5)(scFv) 2 , which can include two peptides connected The VH domain and the two VL domains connected by the symbol, the two VL domains are combined with the two VH domains via a disulfide bridge.
  • Fab fragments which can be monovalent fragments with VL-CL chains and VH-CH
  • Fab fragment refers to antibody fragments that are capable of binding to the antigen produced by treating a complete antibody with papain, including complete light chain (VL-CL), heavy chain Variable regions and CH1 fragments (VH-CH1).
  • single chain antibody scfv, single chain fragment variable
  • VH heavy chain variable region
  • VL light chain variable region
  • a flexible linker is usually designed between the variable region of the heavy chain and the variable region of the light chain so that the variable region of the heavy chain and the variable region of the light chain can be folded into the correct conformation that can bind the antigen.
  • Fc fragment refers to a part of the constant region of an antibody heavy chain, including the hinge region, the CH2 fragment and the CH3 fragment of the constant region.
  • binding refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an epitope.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, each antibody constituting the population is the same except that there may be naturally occurring mutations in a small number of individuals.
  • the monoclonal antibodies described herein particularly include “chimeric" antibodies, in which a part of the heavy chain and/or light chain is the same or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, and The remaining part of the chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, as long as they can exhibit the desired The biological activity.
  • identity refers to the sequence similarity between two polynucleic acid sequences or between two polypeptide sequences. Sequence comparison and determination of the percent identity between the two sequences can be performed through the default settings of the BLASTN/BLASTP algorithm available on the national center for biotechnology institute website.
  • treatment includes therapeutic treatment as well as preventive treatment or preventive measures, by administering a therapeutic agent to a subject to reduce at least one symptom of the disease, disorder, or condition (e.g., cancer or tumor) Or relieve the development of symptoms.
  • EC 50 also known as the median effective concentration, refers to a predetermined time after exposure, the concentration can reach 50% of maximal effect.
  • the present application provides a bifunctional protein, which includes a PD-1 (programmed death receptor-1) binding part and a TGF- ⁇ (transforming growth factor- ⁇ ) binding part.
  • PD-1 programmed death receptor-1
  • TGF- ⁇ transforming growth factor- ⁇
  • the PD-1 binding portion is an anti-PD-1 antibody or antigen-binding fragment. In some embodiments, the PD-1 binding portion is a full-length antibody, Fab fragment, F(ab') 2 fragment, Fv fragment, or single chain Fv fragment (scFv) against PD-1.
  • 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 comprising HCDR1 with an amino acid sequence of GFAFSSYD (SEQ ID NO: 1),
  • the amino acid sequence is HCDR2 with the amino acid sequence of ISGGGRYT (SEQ ID NO: 2) and the HCDR3 with the amino acid sequence of ANRYGEAWFAY (SEQ ID NO: 3)
  • the light chain variable region includes LCDR1 with the amino acid sequence of QDINTY (SEQ ID NO: 4), LCDR2 with the amino acid sequence of RAN (SEQ ID NO: 5) and LCDR3 with the amino acid sequence of LQYDEFPLT (SEQ ID NO: 6).
  • 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.
  • 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 shown in SEQ ID NO: 9, and/or the The amino acid sequence of the light chain constant region is shown in SEQ ID NO: 10.
  • 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.
  • the amino acid sequence of the heavy chain constant region is the amino acid sequence shown in SEQ ID NO: 9 in which the C-terminal residue A is replaced with K.
  • the modification of antibody constant regions is known to those skilled in the art.
  • the heavy chain constant region can be selected from IgG1, IgG2, IgG3, and IgG4 or other classes, although IgG1 is preferred.
  • the antibody constant region may contain modifications, such as amino acid insertions, deletions, substitutions, or chemical modifications.
  • any amino acid residue in the constant region can be substituted with any allotype amino acid residue, preferably, G1m(3) and/or nG1m(1) amino acid residues.
  • the constant region contains mutations that change the effector function, for example, mutating a lysine residue (K) at the C-terminus of the constant region of an antibody heavy chain (commonly found in wild-type IgG1 antibodies) to a hydrophobic amino acid, such as Alanine (A) or Leucine (L) can reduce the hydrolytic cleavage of proteases and increase the serum half-life. This modification is also particularly suitable for the case where the C-terminus of the antibody heavy chain is further fused with other parts.
  • the C-terminal residues of the heavy chain constant region of the anti-PD-1 antibody portion of the exemplary PD-1/TGF ⁇ bifunctional protein of the present application have been processed accordingly.
  • the TGF- ⁇ binding portion is a TGF- ⁇ receptor or a binding domain of a TGF- ⁇ receptor. In some embodiments, the TGF- ⁇ binding portion is the extracellular domain or a binding fragment of the extracellular domain of the TGF- ⁇ receptor. In some specific embodiments, the TGF- ⁇ binding portion is a human TGF- ⁇ RII isoform B extracellular domain polypeptide, which comprises the amino acid sequence shown in SEQ ID NO: 11. In some specific embodiments, the TGF- ⁇ binding portion is a variant of the human TGF- ⁇ RII isoform B extracellular domain polypeptide, for example, it has at least 80%, 81%, and 80% of the amino acid sequence shown in SEQ ID NO: 11.
  • the TGF- ⁇ binding portion is an anti-TGF- ⁇ antibody or antigen-binding fragment. In some embodiments, the TGF- ⁇ binding portion is a full-length antibody, Fab fragment, F(ab′) 2 fragment, Fv fragment, or single chain Fv fragment (scFv) against TGF- ⁇ .
  • the PD-1 binding portion and the TGF- ⁇ binding portion are connected by a flexible linker.
  • the flexible linker is a GGGS type linker. In some specific embodiments, the flexible linker is the linker shown in SEQ ID NO: 12.
  • the bifunctional protein comprises: (1) two identical first polypeptides, and the amino acid sequence of the first polypeptide has at least 80% of the amino acid sequence shown in SEQ ID NO: 13 ( For example, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99%, or 100%); and (2) Two identical second polypeptides, the amino acid sequence of the second polypeptide is at least 80% with the amino acid sequence shown in SEQ ID NO: 14 % (E.g. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or 100%) identity.
  • the bifunctional protein of the present application ie, PD-1/TGF ⁇ bifunctional protein, hereinafter also referred to as “PD1/TGF ⁇ RII fusion protein” or “PD1/TGF ⁇ RII”
  • PD1/TGF ⁇ RII fusion protein can be produced by anti-PD-1 antibody
  • the amino acid sequences of the heavy chain variable region, light chain variable region, heavy chain constant region, and light chain constant region are shown in SEQ ID NO: 7, 8, 9, 10, respectively), flexible linker (SEQ ID NO : 12) and human TGF- ⁇ RII isoform B extracellular domain polypeptide (SEQ ID NO: 11), and its molecular structure diagram can be seen in Figure 1.
  • PD1/TGF ⁇ RII is based on a natural anti-PD-1 antibody, and a flexible linker and a human TGF- ⁇ RII isoform B extracellular domain polypeptide are successively extended at the CH3 end of the heavy chain constant region.
  • PD1/TGF ⁇ RII is an exemplary bifunctional protein of the present application, which has higher TGF ⁇ binding activity and PD-1 end biological activity than the reported nivolumab/TGF- ⁇ RII fusion protein, even better Tumor suppression effect.
  • it has lower cytotoxicity and side effects than existing PD-1 antibodies (such as Nivolumab).
  • existing PD-1 antibodies such as Nivolumab
  • the lower cytotoxicity and side effects of PD1/TGF ⁇ RII make it possible to use higher doses to better inhibit and consume TGF ⁇ , and the dose safety window is more ideal. , which is conducive to high-dose administration and clinical application.
  • this application provides a nucleic acid molecule encoding the bifunctional protein of the first aspect.
  • this application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the bifunctional protein of the first aspect, and a pharmaceutically acceptable excipient, diluent or carrier.
  • the pharmaceutical composition is used to prevent or treat malignant tumors.
  • 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 cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, protuberans Dermofibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and/or myelodysplastic syndrome.
  • the malignant tumor is in situ, metastatic, recurrent, and/or refractory.
  • the pharmaceutical composition may also include lubricants, such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers; suspending agents; preservatives, such as benzoic acid, sorbic acid, and calcium propionate ; Sweeteners and/or flavoring agents, etc.
  • lubricants such as talc, magnesium stearate, and mineral oil
  • wetting agents such as talc, magnesium stearate, and mineral oil
  • emulsifiers such as talc, magnesium stearate, and mineral oil
  • suspending agents such as benzoic acid, sorbic acid, and calcium propionate
  • preservatives such as benzoic acid, sorbic acid, and calcium propionate
  • Sweeteners and/or flavoring agents etc.
  • the pharmaceutical composition in this application can be formulated in the form of tablets, pills, powders, lozenges, elixirs, suspensions, emulsions, solutions, syrups, suppositories, or capsules.
  • any physiologically acceptable mode of administration may be used to deliver the pharmaceutical composition of the present application.
  • These modes of administration include, but are not limited to: oral administration, parenteral administration, nasal administration, rectal administration Medicine, intraperitoneal administration, intravascular injection, subcutaneous administration, transdermal administration, inhalation administration, etc.
  • a pharmaceutical composition for therapeutic use can be formulated in the form of a lyophilized preparation or an aqueous solution by mixing reagents with the required purity with pharmaceutically acceptable carriers, excipients, etc., as appropriate. storage.
  • this application provides the use of the bifunctional protein of the first aspect or the nucleic acid molecule of the second aspect in the preparation of a medicine for the prevention or treatment of malignant tumors.
  • 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 cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, bulging skin Fibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and/or myelodysplastic syndrome.
  • the malignant tumor is in situ, metastatic, recurrent, and/or refractory.
  • the present application provides a method for preventing or treating malignant tumors, including administering the bifunctional protein of the first aspect or the pharmaceutical composition of the third aspect to an individual suffering from a malignant tumor.
  • 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 cancer, testicular cancer, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, bulging skin Fibrosarcoma, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and/or myelodysplastic syndrome.
  • the malignant tumor is in situ, metastatic, recurrent, and/or refractory.
  • the present application provides a method for preparing a bifunctional protein comprising a PD-1 (programmed death receptor-1) binding part and a TGF- ⁇ (transforming growth factor- ⁇ ) binding part, so The method includes the following steps:
  • the host cell is a mammalian cell, such as a CHO cell.
  • the centrifuged supernatant of the cell culture is collected.
  • purifying the bifunctional protein includes using one or more of affinity chromatography, anion exchange chromatography, and cation exchange chromatography.
  • affinity chromatography sucrose or glycerol is included in the eluent. The inventor of the present application found that the addition of sucrose or glycerol eluent is beneficial to reduce the degradation of the fusion protein.
  • Example 1 Expression of PD1/TGF ⁇ RII fusion protein
  • the PD1/TGF ⁇ RII fusion protein of the present application was constructed, and the schematic diagram of the structure is shown in FIG. 1.
  • the nucleotide sequences encoding the heavy chain part (SEQ ID NO: 13) and light chain part (SEQ ID NO: 14) of the signal peptide-fused PD1/TGF ⁇ RII fusion protein were synthesized and cloned into the pcDNA3.1 expression vector.
  • the PD1/TGF ⁇ RII fusion protein expression vector was co-transfected into CHO cells by the standard protocol of transient or stable transfection, and the transfected cells were placed in a 37°C incubator containing 8% CO 2 for culture.
  • amino acid sequence of the linker (SEQ ID NO: 12) is contained in SEQ ID NO: 13:
  • amino acid sequence of the heavy chain of the PD1/TGF ⁇ RII fusion protein (SEQ ID NO: 13):
  • amino acid sequence of the light chain of the PD1/TGF ⁇ RII fusion protein (SEQ ID NO: 14):
  • the cell culture solution obtained in Example 1 was centrifuged and the supernatant was collected, and the protein A affinity chromatography was used for the first step of purification.
  • the balance buffer is 10mmol/L phosphate buffer, pH 6.0. After rinsing the chromatography column with an equilibration buffer for 3 to 5 column volumes, the cell supernatant is loaded. After loading the sample, flush the column with equilibration buffer. Then rinse with elution buffer (0.5 mol/L sodium chloride + 25 mmol/L phosphate buffer, pH 7.0), and then balance 3 to 5 column volumes with equilibration buffer. Finally, wash the chromatography column with elution buffer (20mmol/L citrate buffer + 5% sucrose, pH 3.6), collect the eluted sample, and neutralize the sample with 2M Tris-hydrochloric acid buffer (pH 9.5).
  • the neutralized eluted sample (pH 6.0) was subjected to anion exchange chromatography, and the balance buffer was 10 mmol/L citrate buffer + 10 mmol/L phosphate buffer + 10 mmol/L Tris, pH 6.0. After rinsing the chromatography column with the equilibrium buffer for 3 to 5 column volumes, load the neutralized eluted sample, collect the flow-through sample, and rinse the chromatography column with the equilibration buffer after loading.
  • the flow-through sample of the anion exchange chromatography described above is subjected to cation exchange chromatography.
  • the balance buffer is 10mmol/L citric acid+10mmol/L sodium dihydrogen phosphate+10mmol/L Tris buffer, pH 5.0.
  • the flow-through sample of the anion chromatography is adjusted to pH 5.0 and then loaded. After the sample is loaded, the chromatographic column is flushed with a balance solution for 3 to 5 column volumes. Then elution was performed with an elution buffer (10mmol/L citrate+10mmol/L phosphate+10mmol/L Tris buffer, pH 9.0), and the eluate was collected.
  • Example 3 Detection of PD1/TGF ⁇ RII fusion protein samples by size exclusion chromatography
  • the components of the PD1/TGF ⁇ RII fusion protein sample purified in Example 2 were separated using a gel chromatography column.
  • a neutral pH buffer is used as the mobile phase for elution, and each molecular weight component is washed out in order of molecular weight from largest to smallest.
  • the area normalization method was used to quantitatively analyze the results. Calculate the peak area percentages of high molecular weight impurities, immunoglobulin monomers, and low molecular weight impurities. After detection, the high molecular weight impurities of the PD1/TGF ⁇ RII fusion protein sample were 0.19%, and the immunoglobulin monomer was 99.81%, and no low molecular weight impurities were detected.
  • control bifunctional protein used in the following examples was prepared according to the same process, in which the C-terminal amino acid residues of the original heavy chain constant region of nivoliumab were determined by K Change to A, which is consistent with the exemplary PD1/TGF ⁇ RII fusion protein of the present application.
  • Example 4 The reporter gene method detects the biological activity of the PD-1 end of the PD1/TGF ⁇ RII fusion protein
  • the detection process is described as follows: Take logarithmic growth phase CHO-PDL1-CD3L cells (purchased from China Institute for Food and Drug Control), adjust the density of viable cells to 5 ⁇ 10 5 cells/mL with DMED/F12 complete medium, press 100 ⁇ L /Well is added to a 96-well white plate, placed in a 37°C, 5% CO 2 cell incubator, and incubated for 16-20 hours. On the second day, a suspension of Jurkat-PD-1-NFAT cells (purchased from China Institute 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 with CHO-PDL1-CD3L cells Take out the 96-well white plate with CHO-PDL1-CD3L cells from the incubator, aspirate the supernatant, add Jurkat-PD-1-NFAT cell suspension at 50 ⁇ L/well, and then add the reference PD-1 antibody (Navuli Yuumab, SEQ ID NOs: 17 and 18 are the heavy chain and light chain sequences respectively), the PD1/TGF ⁇ RII fusion protein prepared in Examples 1-3 of the present application (initial concentration is 200,000 ng/mL, 3 times Gradient dilution, 11 dilution gradients) were added to the above 96-well white plate at a rate of 50 ⁇ L/well , cultured in a 37°C, 5% CO 2 cell incubator, and incubated for 4-6 hours.
  • the reference PD-1 antibody (Navuli Yuumab, SEQ ID NOs: 17 and 18 are the heavy chain and light chain sequences respectively)
  • Test article biological activity (%) (reference product EC 50 value / EC 50 value of the test article) ⁇ 100%
  • Table 1 The biological activity of the PD-1 end of the PD1/TGF ⁇ RII fusion protein
  • Example 5 Detection of TGF ⁇ binding activity of PD1/TGF ⁇ RII fusion protein by enzyme-linked immunosorbent assay method
  • the detection process is as follows:
  • Test product binding activity (%) (reference product EC 50 value/test product EC 50 value) ⁇ 100%
  • Example 6 The effect of PD1/TGF ⁇ RII fusion protein on colon cancer cell MC38/hPD-L1 mice subcutaneously transplanted tumor
  • C57/PD-1 transgenic mice purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.
  • Each mouse was subcutaneously inoculated with 3 ⁇ 10 5 MC38/hPD-L1 cells, and the tumor grew to 40 ⁇ 70mm. 3.
  • ip intraperitoneal injection
  • the dosing schedule is shown in Table 3, and the day of dosing is D0.
  • the tumor diameter was measured with a vernier caliper twice a week, and the T/C% or tumor inhibition rate TGI (%) calculated by the following formula was used to investigate the effect of the drug on tumor growth.
  • TGI tumor inhibition rate
  • T/C(%) (TT 0 )/(CC 0 ) ⁇ 100, where T and C are the tumor volumes of the mice in the treatment group and the negative control group at the end of the experiment. T 0 and C 0 are the tumor volumes of the mice in the treatment group and the negative control group at the beginning of the experiment.
  • the results are shown in Table 4, the PD1/TGF ⁇ RII (3.7 mg/kg, IP, 2 times a day, 6 times in total) prepared in Examples 1-3 of the present application inhibited subcutaneous transplantation tumors in MC38/hPD-L1 mice on D19
  • the tumor rate is 74%, which is better than the control PD-1 monoclonal antibody; tumor-bearing mice can tolerate the drug well, and there is no significant weight loss and other symptoms.
  • Table 4 The efficacy of PD1/TGF ⁇ RII on colon cancer cell MC38/hPD-L1 subcutaneously transplanted tumor
  • hIgG4 (Sino Biological Inc, HG4K) is a negative control.
  • the control PD1 monoclonal antibody is the antibody 14C12H1L1 in Chinese Patent Application No. 201610705763.5 (CN106977602), and the base acid sequences of the heavy chain and light chain amino acids are shown in SEQ ID NO: 19 and 14 of this application, respectively.
  • Example 7 In vitro activity detection of PD1/TGF ⁇ RII fusion protein
  • the exemplary PD1/TGF ⁇ RII fusion protein and nivolumab/TGF- ⁇ RII fusion protein of the present application were prepared in the same batch by referring to the methods in Examples 1-3.
  • the results show that the PD-1 end biological activity of the PD1/TGF ⁇ RII fusion protein of the present application is better than that of Navoli Yumab/TGF- ⁇ RII fusion protein, the results are shown in Table 5.
  • Refer to the enzyme-linked immunosorbent assay method of Example 5 to detect the TGF ⁇ binding activity of the PD1/TGF ⁇ RII fusion protein, and compare them.
  • the results show that the TGF ⁇ binding activity of the PD1/TGF ⁇ RII fusion protein of the present application is better than that of nivolumab/TGF - ⁇ RII fusion protein, the results are shown in Table 6.
  • the nivolumab/TGF- ⁇ RII fusion protein in this example and the following examples is prepared in-house and has the heavy chain and light chain sequences shown in SEQ ID NOs: 20 and 18.
  • Example 8 Efficacy of PD1/TGF ⁇ RII fusion protein on MC38/hPD-L1 mouse xenograft tumor
  • Humanized PD-1 mice purchased from Biosaito
  • mice were used as experimental mice.
  • Each mouse was inoculated with 4 ⁇ 10 5 MC38/hPD-L1 cells under the right armpit, and the tumor grew to 100-300mm 3 , Randomly divided into 3 groups, through intraperitoneal injection (ip) drugs, a total of 8 administrations, the dosing schedule is shown in Table 7, the day of dosing is D0.
  • the tumor volume was measured 2-3 times a week, and the weight of the mice was recorded. Measure the diameter of the tumor with a vernier caliper, and calculate the T/C (%) or tumor volume inhibition rate (1-T/C) by the following formula to investigate the effect of the drug on tumor growth.
  • the animals were killed by CO 2 anesthesia, and then the tumors were dissected and photographed.
  • Table 8 The effect of PD1/TGF ⁇ RII on the tumor volume (TV) of MC38/hPD-L1 transplanted tumor mice
  • Example 9 Detection of PD1/TGF ⁇ RII fusion protein to stimulate cytokine secretion by electrochemiluminescence method
  • PBMC cell concentration to about 2 ⁇ 10 6 cells/mL with RPMI1640 complete medium, and add 100 ⁇ L/well to the 96-well cell culture plate.
  • Dilute IgG1 protein with RPMI1640 complete medium (SEQ ID NO: 21 and SEQ ID NO: 22 are the sequences of heavy and light chains, respectively, prepared in-house), LPS (SIGMA, L4391-1MG), PD1/TGF ⁇ RII of the application
  • the fusion protein was prepared into 900 ⁇ g/mL IgG1 protein; 1 ⁇ g/mL LPS; 10 ⁇ g/mL, 100 ⁇ g/mL, 900 ⁇ g/mL PD1/TGF ⁇ RII fusion protein, and RPMI1640 complete medium was used as a negative control.
  • the PD1/TGF ⁇ RII fusion protein of the present application shows a low possibility of causing a cytokine storm. Therefore, when administered to a subject, there is basically no excessive activation of the immune system to cause systemic inflammation. risks of.
  • Example 10 Toxicity test of PD1/TGF ⁇ RII fusion protein to cynomolgus monkey
  • Single-dose toxicity There are 4 cynomolgus monkeys in this experiment, 2 in each group, half male and half male. Two groups were set up. A single intravenous injection of 200 and 500 mg/kg of the PD1/TGF ⁇ RII fusion protein of this application was performed. Observation 14 sky. During the test, general observation and weight, food intake, body temperature, lead II electrocardiogram, blood pressure, hematology, blood biochemistry, urine and other indicators were tested. At the end of the test, general anatomical observations were performed.
  • the male monkeys in each group showed a transient decrease in food intake, and they could recover from the 8th to 9th day of the experiment.
  • the male monkeys in each group showed a decrease in RBC, HGB, and HCT. Apart from this, there were no obvious abnormal changes in the other indicators.
  • a single-dose toxicity test cynomolgus monkeys were injected intravenously with 200 and 500 mg/kg of the PD1/TGF ⁇ RII fusion protein of the present application, and the MTD was 500 mg/kg.
  • the male and female monkeys in the 50 mg/kg group and the male and female monkeys in the 150 mg/kg group showed a decrease in RBC, HGB, and HCT, and a compensatory increase in RET and RET%;
  • the female monkeys in the 50mg/kg group also saw the above changes.
  • a male monkey in the 50 mg/kg group showed cardiac pericardial adhesions on gross anatomy. Histopathological examination: In the 150mg/kg group of cynomolgus monkeys, mild to mild cerebral meninges and choroid plexus, cerebellar meninges and choroid plexus, spinal meninges, thyroid, heart, and pituitary mononuclear cell infiltration, mild to moderate heart and liver , Bladder, epididymis, seminal vesicle gland vascular/perivascular inflammation; 50mg/kg group of cynomolgus monkeys showed mild to moderate cerebral meninges, sciatic nerve, thyroid, heart, pituitary mononuclear cell infiltration, mild to moderate heart, bladder, twelve Duct, ileum, rectum, fallopian tube, vagina, uterine vascular/perivascular inflammation; 15mg/kg group of cynomolgus monkeys can see mild to mild
  • the cynomolgus monkeys of each group were generally in good condition, weight, food intake, body temperature, lead II electrocardiogram, respiratory rate, blood biochemistry, eye examination, urine examination, bone marrow smear, organs There were no obvious abnormal changes in weight and coefficients, IgA, IgM, IgG, C3, C4, CIC, lymphocyte subsets and other immune-related indicators, TSH, T3, T4, organ weights and coefficients.
  • the HNSTD of Nivolumab should be 50mg/kg, which is much lower than the HNSTD of the PD1/TGF ⁇ RII fusion protein of this application. It can be seen that the PD1/TGF ⁇ RII fusion protein of the present application exhibits low toxicity, and therefore, it can be expected that the fusion protein can exhibit good safety clinically.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Preparation (AREA)
PCT/CN2021/089837 2020-04-29 2021-04-26 针对PD-1和TGF-β的双功能蛋白 Ceased WO2021218895A1 (zh)

Priority Applications (9)

Application Number Priority Date Filing Date Title
US17/922,122 US20230235057A1 (en) 2020-04-29 2021-04-26 Bifunctional protein against pd-1 and tgf-beta
EP21795280.3A EP4144763A4 (en) 2020-04-29 2021-04-26 BIFUNCTIONAL PROTEIN AGAINST PD-1 AND TGF-BETA
JP2022565971A JP7711095B2 (ja) 2020-04-29 2021-04-26 ＰＤ－１とＴＧＦ－βに対する二機能性タンパク質
MX2022013620A MX2022013620A (es) 2020-04-29 2021-04-26 Proteina bifuncional frente a pd-1 y tgf-beta.
BR112022022020A BR112022022020A2 (pt) 2020-04-29 2021-04-26 Proteína bifuncional contra pd-1 e tgf-ss
AU2021263650A AU2021263650A1 (en) 2020-04-29 2021-04-26 Bifunctional protein against PD-1 and TGF-beta
KR1020227041823A KR20230003178A (ko) 2020-04-29 2021-04-26 PD-1 및 TGF-β에 대한 이중 기능성 단백질
CA3181579A CA3181579A1 (en) 2020-04-29 2021-04-26 Bifunctional protein against pd-1 and tgf-.beta.
JP2025115631A JP2025148434A (ja) 2020-04-29 2025-07-09 ＰＤ－１とＴＧＦ－βに対する二機能性タンパク質

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010359751 2020-04-29
CN202010359751.8 2020-04-29

Publications (1)

Publication Number Publication Date
WO2021218895A1 true WO2021218895A1 (zh) 2021-11-04

Family

ID=78331777

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/089837 Ceased WO2021218895A1 (zh) 2020-04-29 2021-04-26 针对PD-1和TGF-β的双功能蛋白

Country Status (9)

Country Link
US (1) US20230235057A1 (https=)
EP (1) EP4144763A4 (https=)
JP (2) JP7711095B2 (https=)
KR (1) KR20230003178A (https=)
AU (1) AU2021263650A1 (https=)
BR (1) BR112022022020A2 (https=)
CA (1) CA3181579A1 (https=)
MX (1) MX2022013620A (https=)
WO (1) WO2021218895A1 (https=)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023208191A1 (zh) * 2022-04-29 2023-11-02 广东菲鹏制药股份有限公司 一种重组抗体及其应用
WO2025251034A1 (en) * 2024-05-31 2025-12-04 Amgen Inc. Purification method

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109053895B (zh) 2018-08-30 2020-06-09 中山康方生物医药有限公司 抗pd-1-抗vegfa的双功能抗体、其药物组合物及其用途

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DD120103A1 (https=) 1975-07-07 1976-05-20
CN106977602A (zh) 2016-08-23 2017-07-25 中山康方生物医药有限公司 一种抗 pd1 单克隆抗体、其药物组合物及其用途
WO2019211489A1 (en) * 2018-05-04 2019-11-07 Merck Patent Gmbh COMBINED INHIBITION OF PD-1/PD-L1, TGFβ AND DNA-PK FOR THE TREATMENT OF CANCER
WO2020014285A2 (en) * 2018-07-09 2020-01-16 Intrexon Corporation Fusion constructs and methods of using thereof
CN110734498A (zh) * 2019-10-15 2020-01-31 上海科棋药业科技有限公司 一种用于解除免疫抑制的融合蛋白及其应用

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112016014952A2 (pt) * 2014-02-10 2017-09-19 Merck Patent Gmbh Inibição direcionada de tgfbeta
EP3770171A1 (en) * 2015-04-03 2021-01-27 XOMA Technology Ltd. Treatment of cancer using inhibitors of tgf-beta and pd-1
CN106967172B (zh) * 2016-08-23 2019-01-08 康方药业有限公司 抗ctla4-抗pd-1 双功能抗体、其药物组合物及其用途
MY202480A (en) * 2017-05-12 2024-04-30 Shanghai hengrui pharmaceutical co ltd Fusion protein containing tgf- receptor and medicinal uses thereof
US20200140547A1 (en) * 2017-05-26 2020-05-07 The Johns Hopkins University Multifunctional antibody-ligand traps to modulate immune tolerance
CN112585165B8 (zh) * 2018-04-25 2025-02-14 普罗米修斯生物科学公司 优化的抗tl1a抗体
US10597453B2 (en) * 2018-06-29 2020-03-24 Gensun Biopharma, Inc. Antitumor immune checkpoint regulator antagonists
CN109053895B (zh) * 2018-08-30 2020-06-09 中山康方生物医药有限公司 抗pd-1-抗vegfa的双功能抗体、其药物组合物及其用途
CN114340735B (zh) * 2019-06-28 2024-11-12 璟尚生物制药公司 突变的TGFβ1-RII胞外域和免疫球蛋白支架组成的抗肿瘤拮抗剂
CN114616251B (zh) * 2019-10-21 2024-05-24 南京维立志博生物科技有限公司 靶向PD-1和TGFβ的重组蛋白

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DD120103A1 (https=) 1975-07-07 1976-05-20
CN106977602A (zh) 2016-08-23 2017-07-25 中山康方生物医药有限公司 一种抗 pd1 单克隆抗体、其药物组合物及其用途
WO2019211489A1 (en) * 2018-05-04 2019-11-07 Merck Patent Gmbh COMBINED INHIBITION OF PD-1/PD-L1, TGFβ AND DNA-PK FOR THE TREATMENT OF CANCER
WO2020014285A2 (en) * 2018-07-09 2020-01-16 Intrexon Corporation Fusion constructs and methods of using thereof
CN110734498A (zh) * 2019-10-15 2020-01-31 上海科棋药业科技有限公司 一种用于解除免疫抑制的融合蛋白及其应用

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AL-LAZIKAM ET AL., J. MOL. BIOL., vol. 273, 1997, pages 927 - 948
DAVID CHARLES J ET AL.: "TGF-β Tumor Suppression through a Lethal EMT. [J", CELL, vol. 164, no. 5, 2016, XP029433852, DOI: 10.1016/j.cell.2016.01.009
KABAT: "Sequences of Proteins of Immunological Interest", 1991, NATIONAL INSTITUTES OF HEALTH
MARTIN ET AL., PROC. NATL. ACAD. SCI.USA, vol. 86, 1989, pages 9268 - 9272
See also references of EP4144763A4
SHEN YINAN ET AL.: "TGF-β regulates hepatocellular carcinoma progression by inducing Treg cell polarization. [J", CELLULAR PHYSIOLOGY AND BIOCHEMISTRY, vol. 35, no. 4, 2015

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023208191A1 (zh) * 2022-04-29 2023-11-02 广东菲鹏制药股份有限公司 一种重组抗体及其应用
WO2025251034A1 (en) * 2024-05-31 2025-12-04 Amgen Inc. Purification method

Also Published As

Publication number Publication date
MX2022013620A (es) 2023-01-30
US20230235057A1 (en) 2023-07-27
AU2021263650A1 (en) 2023-01-19
JP7711095B2 (ja) 2025-07-22
JP2025148434A (ja) 2025-10-07
JP2023523083A (ja) 2023-06-01
EP4144763A1 (en) 2023-03-08
KR20230003178A (ko) 2023-01-05
BR112022022020A2 (pt) 2023-01-31
EP4144763A4 (en) 2024-05-29
CA3181579A1 (en) 2021-11-04

Similar Documents

Publication Publication Date Title
CN112940134B (zh) 针对PD-1和TGF-β的双功能蛋白
JP2021516952A (ja) 新規な抗体分子、その調製方法及びその使用
CN109988240B (zh) 抗gpc-3抗体及其用途
US11525009B2 (en) Bispecific antibody and antibody conjugate for tumor therapy and use thereof
US11421029B2 (en) Recombinant bispecific antibodies to PD-L1 and CTLA-4
JP2025148434A (ja) ＰＤ－１とＴＧＦ－βに対する二機能性タンパク質
CN117500833A (zh) 一种双抗组合及其应用
CN112125975A (zh) Pd-l1和cd47双特异性融合蛋白及其医药用途
JP2022511311A (ja) ヒトｐｄ‐ｌ１抗体
US20220289825A1 (en) FUSION PROTEIN TARGETING PD-L1 AND TGF-ß AND USE THEREOF
EP4410834A1 (en) Ctla-4 binding molecule and use thereof
TW202602921A (zh) 多功能ｎｋ細胞接合器
CN114616247B (zh) Ox40/pd-l1双特异性抗体
RU2843303C1 (ru) БИФУНКЦИОНАЛЬНЫЙ БЕЛОК ПРОТИВ PD-1 И TGF-β
CN118742565A (zh) 逆转treml1诱导的免疫抑制的方法
WO2021244553A1 (zh) 一种抗pd-l1和egfr的四价双特异性抗体
WO2021022037A1 (en) Il-38-specific antiobodies
HK40081562A (en) Bifunctional protein aiming at pd-1 and tgf-beta
TWI919022B (zh) 逆轉treml1誘導之免疫抑制之方法
CA3215850A1 (en) Composition comprising an ige antibody
HK40066344B (zh) Ox40/pd-l1双特异性抗体
HK40066344A (zh) Ox40/pd-l1双特异性抗体
CN118725133A (zh) 双特异性抗体及其用途
CA3260711A1 (en) Fusion protein
HK40076695A (en) FUSION PROTEIN TARGETING PD-L1 AND TGF-β AND USE THEREOF

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21795280

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2022565971

Country of ref document: JP

Kind code of ref document: A

Ref document number: 3181579

Country of ref document: CA

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112022022020

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 20227041823

Country of ref document: KR

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2021795280

Country of ref document: EP

Effective date: 20221129

REG Reference to national code

Ref country code: BR

Ref legal event code: B01E

Ref document number: 112022022020

Country of ref document: BR

Free format text: COM BASE NA PORTARIA INPI 48/2022, APRESENTAR NOVO CONTEUDO ELETRONICO DE LISTAGEM DE SEQUENCIAS, POIS O CONTEUDO APRESENTADO NA PETICAO INICIAL NAO POSSUI TODOS OS CAMPOS OBRIGATORIOS (CAMPO 130). INCLUIR CAMPOS 140-141 EM RESPOSTA A EXIGENCIA, UMA VEZ QUE O DEPOSITANTE JA POSSUI O NUMERO DO PEDIDO NA FASE NACIONAL.

ENP Entry into the national phase

Ref document number: 2021263650

Country of ref document: AU

Date of ref document: 20210426

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 112022022020

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20221028

WWG Wipo information: grant in national office

Ref document number: 2022130875

Country of ref document: RU