WO2023208191A1 - Recombinant antibody and use thereof - Google Patents

Recombinant antibody and use thereof Download PDF

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Publication number
WO2023208191A1
WO2023208191A1 PCT/CN2023/091569 CN2023091569W WO2023208191A1 WO 2023208191 A1 WO2023208191 A1 WO 2023208191A1 CN 2023091569 W CN2023091569 W CN 2023091569W WO 2023208191 A1 WO2023208191 A1 WO 2023208191A1
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peptide
terminus
fragment
optionally
wild
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PCT/CN2023/091569
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French (fr)
Chinese (zh)
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张轶博
芦迪
霍永庭
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广东菲鹏制药股份有限公司
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Publication of WO2023208191A1 publication Critical patent/WO2023208191A1/en

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    • 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
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0684—Cells of the urinary tract or kidneys
    • C12N5/0686—Kidney cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/495—Transforming growth factor [TGF]

Definitions

  • the present invention relates to the field of biotechnology. Specifically, the present invention relates to recombinant antibodies and their applications. More specifically, the present invention relates to recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, and the use of the above substances in the preparation of medicines. Drugs, uses of recombinant antibodies in preparation of kits, and kits.
  • cancer treatment options are mainly radiotherapy and chemotherapy. Due to the lack of targeting, chemotherapy and radiotherapy not only kill cancer cells, but also have a strong killing effect on normal tissues. Since then, targeted therapy has been developed based on chemotherapy. Targeted therapy mainly targets overexpressed proteins or proto-oncogenes that are important for tumor survival and growth. Targeted therapy effectively reduces drug resistance, organ toxicity, etc.
  • a series of disadvantages In recent years, people have gradually realized that cancer immunotherapy is an emerging cancer treatment concept. It reorganizes the host immune response, especially the adaptive T cell response, by activating the immune system or removing the suppression of the immune system, providing immune surveillance and killing cancer cells. , has better specificity and can also achieve long-term protective immunity.
  • Transforming growth factor- ⁇ (transforming growth factor- ⁇ , TGF ⁇ ) belongs to the TGF ⁇ superfamily that regulates cell growth and differentiation. It has three forms, including TGF ⁇ 1/2/3. In addition to TGF ⁇ , this family also includes activins, inhibins, Mullerian inhibitor substance (MIS) and bone morpho-genetic proteins (BMPs). TGF ⁇ was originally named because this cytokine can transform normal fibroblasts into cells with anchorage-independent growth and plays an important regulatory role in cell growth, differentiation and immune function.
  • TGF ⁇ transforming growth factor- ⁇
  • TGF ⁇ inactive growth factor ⁇
  • LAP latency associated peptide
  • TGF ⁇ can inhibit the proliferation of T cells stimulated by mitogens and allogeneic antigens or the growth of IL-2-dependent T cells. It can also inhibit the expression of MHC class II antigen in melanoma cells induced by IFN- ⁇ .
  • TGF ⁇ can also inhibit IFN in PBMC. The production of - ⁇ and TNF- ⁇ promotes the expression of IL-6.
  • TGF ⁇ is a potent immunosuppressant, and disruption of TGF ⁇ signaling can lead to autoimmunity, inflammation, and cancer.
  • Increased TGF ⁇ expression is often associated with malignancy in many cancers, as well as defects in the cytostatic response to TGF ⁇ , whose immunosuppressive functions then dominate, contributing to tumorigenesis.
  • TGF ⁇ exerts tumor suppressive or tumor promoting effects in tumors in a cell context-dependent manner.
  • TGF ⁇ can inhibit the expression of proto-oncogene c-myc, but during tumor development, with the introduction of mutations or changes in epigenetic modifications, cancer cells gradually become resistant to the inhibitory effect of TGF ⁇ signaling, ultimately leading to tumor development.
  • TGF ⁇ is produced by cancer cells and several other cell types present in the TME, including Tregs. Fibroblasts, macrophages, and platelets are also important TGF ⁇ producers in tumors. Elevated TGF ⁇ levels prevent naive T cells from differentiating into Th1 effector phenotypes, promote their transformation into Treg subtypes, and inhibit the antigen presentation function of dendritic cells. Tauriello et al. found that TGF ⁇ blockade made a mouse liver metastasis cancer model more sensitive to PD1/PDL1 therapy.
  • the invention provides a recombinant antibody.
  • the recombinant antibody includes two identical peptide chains 1, and each peptide chain 1 includes: 1) a first TGF ⁇ RII fragment; and 2) an antibody heavy chain constant region; wherein, the antibody heavy chain The C-terminus of the constant region is linked to the N-terminus of the first TGF ⁇ RII fragment.
  • Recombinant antibodies according to embodiments of the present invention can effectively bind to TGF ⁇ , block the combination of TGF ⁇ RII and TGF ⁇ on the cell surface, and significantly promote the proliferation of T cells. They have a long half-life in vivo and can effectively treat or prevent tumors.
  • the invention provides a recombinant antibody.
  • the recombinant antibody includes two identical peptide chains 3, each peptide chain 3 includes: 1) a fourth TGF ⁇ RII fragment; 2) an antibody Fc region; and 3) a fifth TGF ⁇ RII fragment or a tumor target Peptide; wherein the C-terminus of the antibody Fc region is connected to the N-terminus of the fourth TGF ⁇ RII fragment, and the N-terminus of the antibody Fc region is connected to the C-terminus of the fifth TGF ⁇ RII fragment or tumor-targeting peptide.
  • Recombinant antibodies according to embodiments of the present invention can effectively bind to TGF ⁇ , block the combination of TGF ⁇ RII and TGF ⁇ on the cell surface, and significantly promote the proliferation of T cells. They have a long half-life in vivo and can effectively treat or prevent tumors.
  • the invention provides a nucleic acid molecule.
  • the nucleic acid molecule encodes the recombinant antibody described in the first aspect or the second aspect.
  • the recombinant antibodies obtained according to the nucleic acid molecules of the embodiments of the present invention can effectively bind to TGF ⁇ , block the combination of TGF ⁇ RII and TGF ⁇ on the cell surface, significantly promote the proliferation of T cells, have a long half-life in vivo, and can effectively treat or prevent tumors. .
  • the invention provides an expression vector.
  • the nucleic acid molecules described above are carried.
  • the expression vector according to the embodiment of the present invention is introduced into a suitable recipient cell, under the mediation of the regulatory system, the expression of the recombinant antibody described above can be effectively realized, thereby achieving large-scale acquisition of the recombinant antibody in vitro.
  • the invention provides a recombinant cell.
  • the recombinant cells carry the aforementioned nucleic acid molecules, expression vectors, or express the aforementioned recombinant antibodies.
  • Recombinant cells according to embodiments of the present invention can be used for the in vitro expression and large-scale acquisition of recombinant antibodies capable of recognizing TGF ⁇ as described above.
  • the invention provides a composition.
  • the above-mentioned recombinant antibodies, nucleic acid molecules, expression vectors or recombinant cells are included.
  • recombinant antibodies according to embodiments of the present invention, or recombinant antibodies obtained by expressing the nucleic acid molecules, expression vectors or recombinant cells under appropriate conditions can effectively bind to TGF ⁇ and block TGF ⁇ RII and TGF ⁇ on the cell surface.
  • the combination, and significantly promotes the proliferation of T cells has a long half-life in vivo, and can effectively treat or prevent tumors.
  • the recombinant antibodies or expressed antibodies contained in the composition can effectively bind to TGF ⁇ , Blocks the combination of TGF ⁇ RII and TGF ⁇ on the cell surface, and significantly promotes the proliferation of T cells. It has a long half-life in vivo and can effectively treat or prevent tumors.
  • the present invention proposes the use of the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells or compositions in the preparation of medicines.
  • the medicament is used to prevent or treat tumors.
  • recombinant antibodies according to some specific embodiments of the present invention, or recombinant antibodies obtained by expressing the nucleic acid molecules, expression vectors or recombinant cells under appropriate conditions, and recombinant antibodies included in the composition can effectively interact with TGF ⁇ Bind, block the combination of TGF ⁇ RII and TGF ⁇ on the cell surface, and significantly promote the proliferation of T cells.
  • the surface TGF ⁇ RII binds to TGF ⁇ and significantly promotes the proliferation of T cells. It has a long half-life in vivo and can effectively treat or prevent tumors.
  • the invention provides a medicament.
  • it includes: the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells or compositions.
  • recombinant antibodies according to some specific embodiments of the present invention, or recombinant antibodies obtained by expressing the nucleic acid molecules, expression vectors or recombinant cells under appropriate conditions, and recombinant antibodies included in the composition can effectively interact with TGF ⁇
  • the combination has strong specificity and can not only neutralize TGF ⁇ , but also exert a better targeting effect, thereby realizing the biological effects of other drugs in the drug, such as inhibiting the activity of TGF ⁇ molecules and killing cells expressing TGF ⁇ molecules.
  • the present invention proposes the use of the aforementioned recombinant antibody in preparing a kit.
  • the kit is used to detect TGF ⁇ .
  • the recombinant antibody can effectively bind to TGF ⁇ and has strong specificity. Therefore, the kit prepared according to the recombinant antibody according to the embodiment of the present invention can effectively detect whether the sample to be tested contains TGF ⁇ and its content.
  • the present invention provides a kit.
  • the kit includes the recombinant antibody described above.
  • the recombinant antibody provided according to the embodiment of the present invention can bind to TGF ⁇ . Therefore, a kit containing the recombinant antibody can be used to effectively diagnose or detect TGF ⁇ .
  • the kit can be used for scientific research, such as qualitative or quantitative detection of TGF ⁇ in biological samples, and can also be used to judge the status of an individual, such as determining whether the TGF ⁇ level of the individual is higher or lower after obtaining the TGF ⁇ level. normal level.
  • the present invention provides a method for diagnosing related diseases caused by TGF ⁇ .
  • the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, and kits are used to detect TGF ⁇ in the sample to be tested; based on the detection results, the content of TGF ⁇ in the sample to be tested is determined.
  • Increased TGF ⁇ in the tumor microenvironment is related to immune escape. Increased TGF ⁇ levels will prevent naive T cells from differentiating into Th1 effector phenotypes, promote their transformation into Treg subtypes, and inhibit the antigen presentation function of dendritic cells.
  • TGF ⁇ is A potent immunosuppressant, increased expression of TGF ⁇ is often associated with malignancy in many cancers, as well as defects in the cell growth inhibitory response to TGF ⁇ , which then dominates its immunosuppressive function and contributes to tumorigenesis, as proposed in this application.
  • the recombinant antibodies, or recombinant antibodies expressed by nucleic acid molecules, expression vectors, recombinant cells, or recombinant antibodies included in compositions and kits can effectively bind to TGF ⁇ . Therefore, the method described in this application can be used to effectively detect The TGF ⁇ content in the sample to be tested is derived from the subject, and can effectively diagnose related diseases caused by TGF ⁇ .
  • the present invention proposes a method for assessing the staging of related diseases caused by TGF ⁇ .
  • the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, and kits are used to detect TGF ⁇ in the sample to be tested; based on the detection results, the content of TGF ⁇ in the sample to be tested is determined.
  • increased TGF ⁇ in the tumor microenvironment is associated with immune escape.
  • Increased TGF ⁇ levels prevent naive T cells from differentiating toward a Th1 effector phenotype, promote their transformation into Treg subtypes, and inhibit dendritic cell antigens.
  • TGF ⁇ is a potent immunosuppressant. Increased expression of TGF ⁇ is often associated with many malignant tumors, as well as defects in the cell growth inhibitory response to TGF ⁇ , and then its immunosuppressive function dominates, contributing to tumorigenesis and disease. At different stages of occurrence, the content of TGF ⁇ in peripheral blood will change.
  • the recombinant antibodies proposed in this application or the recombinant antibodies expressed by nucleic acid molecules, expression vectors, recombinant cells, or the recombinant antibodies included in the compositions and kits
  • the method described in this application can be used to effectively detect the TGF ⁇ content in the test sample derived from the subject individual, and based on the TGF ⁇ content, the period of related diseases caused by TGF ⁇ can be estimated. to evaluate.
  • the present invention proposes a method for evaluating the prognosis of related diseases caused by TGF ⁇ .
  • the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, and combinations are used Materials and kits are used to detect TGF ⁇ in the sample to be tested; based on the detection results, the content of TGF ⁇ in the sample to be tested is determined.
  • TGF ⁇ is a potent immunosuppressant, and increased expression of TGF ⁇ is often associated with many malignant tumors, as well as defects in the cell growth inhibitory response to TGF ⁇ , and then its immunosuppressive function dominates, promoting tumorigenesis.
  • TGF ⁇ tumor necrosis factor ⁇
  • the prognosis of such diseases can be effectively evaluated by monitoring the levels of TGF ⁇ in peripheral blood. For example, comparing the levels of TGF ⁇ in subjects before and after treatment, or comparing subjects after treatment. The content of TGF ⁇ in the body is compared with the normal level or disease level, etc.
  • the recombinant antibody proposed in this application or the recombinant antibody expressed by the nucleic acid molecule, expression vector, recombinant cell, or the recombinant antibody contained in the composition or kit
  • the method described in this application can be used to effectively detect the TGF ⁇ content in the test sample derived from the subject individual, and evaluate the prognosis of related diseases caused by TGF ⁇ based on the TGF ⁇ content.
  • Figure 1 shows the structural diagram of four recombinant antibodies in the embodiment of the present invention, in which Targeting Unit represents the tumor targeting peptide, and ECD represents the extracellular domain of wild-type TGF ⁇ RII.
  • Figure 2 shows the structural diagram of control molecule #5 in the embodiment of the present invention
  • Figure 3 shows the structural diagram of control molecule #6 in the embodiment of the present invention
  • Figure 4 shows an analysis diagram of the ELISA detection results of the in vitro binding of recombinant antibodies to TGF ⁇ 1 and TGF ⁇ 3 in the embodiment of the present invention, in which R1054 represents recombinant antibody #1, R1055 represents recombinant antibody #3, R1056 represents recombinant antibody #2, and R0796 represents Recombinant antibody #4, iso(hIgG1.8) represents the isotype control group,
  • Figure A shows the analysis of the ELISA detection results of the in vitro binding of recombinant antibodies to TGF ⁇ 1 in the embodiment of the present invention.
  • the abscissa represents the log value of the concentration (nM) of each recombinant antibody and control sample, and the ordinate represents the OD value at 450 nm.
  • Figure B shows an analysis chart of the ELISA detection results of the in vitro binding of the recombinant antibody of the present invention to TGF ⁇ 3, in which the abscissa represents the log value of the concentration (nM) of each recombinant antibody and control sample, and the ordinate represents the OD value at 450nm;
  • Figure 5 shows the blocking effect of recombinant antibodies in the embodiment of the present invention on the binding of TGF ⁇ RII and TGF ⁇ on the cell surface.
  • the abscissa represents the log value of the concentration (nM) of each recombinant antibody and control sample, and the ordinate represents the average.
  • Fluorescence intensity R1054 represents recombinant antibody #1, R1055 represents recombinant antibody #3, R1056 represents recombinant antibody #2, R0796 represents recombinant antibody #4, iso(hIgG1.8) represents isotype control group, Noab represents negative control group;
  • Figure 6-A shows the results of the effect of recombinant antibodies on T cell proliferation in the embodiment of the present invention.
  • the abscissa represents different treatment groups, the ordinate (Proliferation%) represents the proliferation ratio of T cells, and R1054 represents the recombinant antibody.
  • #1, R1055 represents recombinant antibody #3, R1056 represents recombinant antibody #2, R0796 represents recombinant antibody #4, iso(hIgG1.8) represents isotype control group, no activated represents inactive, activated represents activated, no Ab represents negative control Group;
  • FIG. 6-B shows the results of the effect of recombinant antibodies on T cell proliferation in the embodiment of the present invention, in which R1054 represents recombinant antibody #1, R1055 represents recombinant antibody #3, R1056 represents recombinant antibody #2, and R0796 represents recombinant antibody #4, no activated means not activated, TGF ⁇ 3ng/mL means the concentration of TGF ⁇ is 3ng/mL, Ab 50nM means the recombinant antibody concentration is 50nM, activated means activated.
  • first”, “second”, “third”, “fourth” or “fifth” etc. are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying a designation.
  • features defined as “first”, “second”, “third”, “fourth”, and “fifth” may explicitly or implicitly include at least one of these features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise expressly and specifically limited.
  • Common natural amino acids include, for example, alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys ;C); Glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine Acid (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser ; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).
  • the "antibody IgG1.8" is a mutant of the wild-type IgG1 antibody. Compared with the wild-type IgG1 antibody, it has L234A, L235A and K447A mutations, wherein the amino acid number of the wild-type IgG1 antibody is For numbering according to the EU numbering system, for example, the "L234A” means that according to the EU numbering system, the leucine numbered at position 234 is replaced by alanine.
  • recombinant antibody is a protein expressed by a host cell containing nucleic acid encoding a full-length antibody or a portion thereof, for example, a protein expressed by a recombinant host cell containing nucleic acid encoding an antibody heavy chain constant region and a TGF ⁇ RII fragment.
  • the invention provides a recombinant antibody comprising two identical peptide chains 1, each peptide chain 1 comprising: 1) a first TGF ⁇ RII fragment; and 2) an antibody heavy chain constant region ; Wherein, the C-terminus of the antibody heavy chain constant region is connected to the N-terminus of the first TGF ⁇ RII fragment.
  • Recombinant antibodies according to some specific embodiments of the present invention can effectively bind to TGF ⁇ , block the binding of TGF ⁇ RII and TGF ⁇ on the cell surface, and significantly Promotes the proliferation of T cells, has a long half-life in the body, and can effectively treat or prevent tumors.
  • the above-mentioned recombinant antibody may further include at least one of the following additional technical features:
  • the first TGF ⁇ RII fragment includes a wild-type TGF ⁇ RII extracellular domain.
  • the first TGF ⁇ RII fragment includes an N-terminal fragment of wild-type TGF ⁇ RII extracellular domain, a first connecting peptide and a C-terminal fragment of wild-type TGF ⁇ RII extracellular domain, wherein, The C-terminal fragment of the wild-type TGF ⁇ RII extracellular domain includes at most 122 amino acids of the C-terminus of the wild-type TGF ⁇ RII extracellular domain.
  • the C-terminus of the N-terminal fragment of the wild-type TGF ⁇ RII extracellular domain is connected to the N-terminus of the first linking peptide, and the C-terminus of the first linking peptide is connected to the N-terminus of the first linking peptide.
  • the C-terminal fragment of wild-type TGF ⁇ RII extracellular domain is linked to the N-terminus.
  • the first TGF ⁇ RII fragment includes an N-terminal fragment of wild-type TGF ⁇ RII extracellular domain, a first connecting peptide and a C-terminal fragment of wild-type TGF ⁇ RII extracellular domain, wherein, The C-terminal fragment includes up to 122 amino acids of the C-terminus of the TGF ⁇ RII extracellular domain (e.g., 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122 amino acids of the TGF ⁇ RII extracellular domain).
  • the C-terminal fragment includes up to 122 amino acids of the C-terminus of the TGF ⁇ RII extracellular domain (e.g., 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122 amino acids of the TGF ⁇ RII extracellular domain).
  • the N-terminus of the first connecting peptide is connected to the C-terminal of the N-terminal fragment of the TGF ⁇ RII extracellular domain, and the C-terminal of the first connecting peptide is connected to the TGF ⁇ RII extracellular domain
  • the C-terminal fragment is connected to the N-terminus.
  • the first TGF ⁇ RII fragment includes an N-terminal fragment of wild-type TGF ⁇ RII extracellular domain, a first connecting peptide and a C-terminal fragment of wild-type TGF ⁇ RII extracellular domain, wherein,
  • the C-terminal fragment of the TGF ⁇ RII extracellular domain is 14-23 consecutive N-terminal truncations of the TGF ⁇ RII extracellular domain (for example, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 ) polypeptides of amino acid residues, preferably truncated polypeptides of 16-23 consecutive amino acid residues.
  • the first TGF ⁇ RII fragment includes an N-terminal fragment of wild-type TGF ⁇ RII extracellular domain, a first connecting peptide and a C-terminal fragment of wild-type TGF ⁇ RII extracellular domain, wherein, wild-type TGF ⁇ RII extracellular domain
  • the N-terminal fragment of the TGF ⁇ RII extracellular domain includes 1 to 6 amino acids of the N-terminus of the TGF ⁇ RII extracellular domain; in some embodiments, the TGF ⁇ RII mutant fragment includes the contiguous N-terminal portion of the TGF ⁇ RII extracellular domain. 1, 2, 3, 4, 5 or 6 amino acids.
  • the first TGF ⁇ RII fragment includes an N-terminal fragment of wild-type TGF ⁇ RII extracellular domain, a first connecting peptide and a C-terminal fragment of wild-type TGF ⁇ RII extracellular domain, wherein,
  • the first connecting peptide is a (GS) ⁇ G polypeptide, wherein is an integer, y is selected from an integer between 0 and 6.
  • each peptide chain 1 further includes: a second TGF ⁇ RII fragment.
  • the second TGF ⁇ RII fragment includes an N-terminal fragment of the wild-type TGF ⁇ RII extracellular domain, a second connecting peptide and a C-terminal fragment of the wild-type TGF ⁇ RII extracellular domain.
  • the C-terminal fragment of the wild-type TGF ⁇ RII extracellular domain includes at most 122 amino acids of the C-terminus of the wild-type TGF ⁇ RII extracellular domain.
  • the C-terminus of the N-terminal fragment of the wild-type TGF ⁇ RII extracellular domain is connected to the N-terminus of the second linking peptide, and the C-terminus of the second linking peptide is connected to the N-terminus of the second linking peptide.
  • the C-terminal fragment of wild-type TGF ⁇ RII extracellular domain is linked to the N-terminus.
  • the C-terminus of the second TGF ⁇ RII fragment is connected to the N-terminus of the antibody heavy chain constant region.
  • the first TGF ⁇ RII fragment includes an N-terminal fragment of wild-type TGF ⁇ RII extracellular domain, a second connecting peptide and a C-terminal fragment of wild-type TGF ⁇ RII extracellular domain, wherein, The C-terminal fragment includes up to 122 amino acids of the C-terminus of the TGF ⁇ RII extracellular domain (e.g., 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122 amino acids of the TGF ⁇ RII extracellular domain).
  • the C-terminal fragment includes up to 122 amino acids of the C-terminus of the TGF ⁇ RII extracellular domain (e.g., 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122 amino acids of the TGF ⁇ RII extracellular domain).
  • the N-terminus of the second linking peptide is connected to the C-terminal of the N-terminal fragment of the TGF ⁇ RII extracellular domain, and the C-terminus of the second linking peptide is connected to the TGF ⁇ RII extracellular domain
  • the C-terminal fragment is connected to the N-terminus.
  • the first TGF ⁇ RII fragment includes an N-terminal fragment of wild-type TGF ⁇ RII extracellular domain, a second connecting peptide and a C-terminal fragment of wild-type TGF ⁇ RII extracellular domain, wherein,
  • the C-terminal fragment of the TGF ⁇ RII extracellular domain is 14-23 consecutive N-terminal truncations of the TGF ⁇ RII extracellular domain (for example, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 ) polypeptides of amino acid residues, preferably truncated polypeptides of 16-23 consecutive amino acid residues.
  • the first TGF ⁇ RII fragment includes an N-terminal fragment of wild-type TGF ⁇ RII extracellular domain, a second connecting peptide and a C-terminal fragment of wild-type TGF ⁇ RII extracellular domain, wherein, wild-type TGF ⁇ RII extracellular domain
  • the N-terminal fragment of the TGF ⁇ RII extracellular domain includes 1 to 6 amino acids of the N-terminus of the TGF ⁇ RII extracellular domain; in some embodiments, the TGF ⁇ RII mutant fragment includes the contiguous N-terminal portion of the TGF ⁇ RII extracellular domain. 1, 2, 3, 4, 5 or 6 amino acids.
  • the first TGF ⁇ RII fragment includes an N-terminal fragment of wild-type TGF ⁇ RII extracellular domain, a second connecting peptide and a C-terminal fragment of wild-type TGF ⁇ RII extracellular domain, wherein,
  • the second connecting peptide is a (GS) ⁇ G polypeptide, wherein X is preferably any integer from 1 to 6; or the second connecting peptide is selected from (GxS)y linker, wherein is an integer, y is selected from an integer between 0 and 6.
  • two identical peptide chains 2 are further included, each of which includes a third TGF ⁇ RII fragment and an antibody light chain constant region.
  • the C-terminus of the third TGF ⁇ RII fragment is connected to the N-terminus of the antibody light chain constant region.
  • each peptide chain 2 is connected to one peptide chain 1 through an inter-chain disulfide bond.
  • the third TGF ⁇ RII fragment includes the wild-type TGF ⁇ RII extracellular domain.
  • the third TGF ⁇ RII fragment includes an N-terminal fragment of the wild-type TGF ⁇ RII extracellular domain, a third connecting peptide and a C-terminal fragment of the wild-type TGF ⁇ RII extracellular domain.
  • the C-terminal fragment of the wild-type TGF ⁇ RII extracellular domain includes at most 122 amino acids of the C-terminus of the wild-type TGF ⁇ RII extracellular domain.
  • the C-terminus of the N-terminal fragment of the wild-type TGF ⁇ RII extracellular domain is connected to the N-terminus of the third linking peptide, and the C-terminus of the third linking peptide is connected to the N-terminus of the third linking peptide.
  • the C-terminal fragment of wild-type TGF ⁇ RII extracellular domain is linked to the N-terminus.
  • the first TGF ⁇ RII fragment includes an N-terminal fragment of wild-type TGF ⁇ RII extracellular domain, a third connecting peptide and a C-terminal fragment of wild-type TGF ⁇ RII extracellular domain, wherein, The C-terminal fragment includes the C-terminus of the TGF ⁇ RII extracellular domain to More than 122 amino acids (e.g., 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, or 122 amino acid residues at the C-terminus of the TGF ⁇ RII extracellular domain, or, for example, including the TGF ⁇ RII extracellular domain
  • the C-terminal 112 to 117 amino acids for example, the 112, 113, 114, 115, 116 or 117 C-terminal amino acid residues of the TGF ⁇ RII extracellular domain
  • the C-terminal 112 to 117 amino acids for example, the 112, 113, 114, 115, 116 or 117 C-terminal amino acid residue
  • the first TGF ⁇ RII fragment includes an N-terminal fragment of wild-type TGF ⁇ RII extracellular domain, a third connecting peptide and a C-terminal fragment of wild-type TGF ⁇ RII extracellular domain, wherein,
  • the C-terminal fragment of the TGF ⁇ RII extracellular domain is 14-23 consecutive N-terminal truncations of the TGF ⁇ RII extracellular domain (for example, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 ) polypeptides of amino acid residues, preferably truncated polypeptides of 16-23 consecutive amino acid residues.
  • the first TGF ⁇ RII fragment includes an N-terminal fragment of wild-type TGF ⁇ RII extracellular domain, a third connecting peptide and a C-terminal fragment of wild-type TGF ⁇ RII extracellular domain, wherein, wild-type TGF ⁇ RII extracellular domain
  • the N-terminal fragment of the TGF ⁇ RII extracellular domain includes 1 to 6 amino acids of the N-terminus of the TGF ⁇ RII extracellular domain; in some embodiments, the TGF ⁇ RII mutant fragment includes the contiguous N-terminal portion of the TGF ⁇ RII extracellular domain. 1, 2, 3, 4, 5 or 6 amino acids.
  • the first TGF ⁇ RII fragment includes an N-terminal fragment of wild-type TGF ⁇ RII extracellular domain, a third connecting peptide and a C-terminal fragment of wild-type TGF ⁇ RII extracellular domain, wherein,
  • the third connecting peptide is a (GS) ⁇ G polypeptide, wherein X is preferably any integer from 1 to 6; or the third connecting peptide is selected from (GxS)y linker, wherein is an integer, y is selected from an integer between 0 and 6.
  • the N-terminal fragment of the wild-type TGF ⁇ RII extracellular domain includes the first 6 amino acids of the N-terminus of the wild-type TGF ⁇ RII extracellular domain.
  • the C-terminal fragment of the wild-type TGF ⁇ RII extracellular domain includes 112 to 117 amino acids of the C-terminus of the wild-type TGF ⁇ RII extracellular domain.
  • the wild-type TGF ⁇ RII extracellular domain includes the amino acid sequence shown in SEQ ID NO:2.
  • the first connecting peptide and/or the second connecting peptide and/or the third connecting peptide are flexible fragments.
  • the first connecting peptide and/or the second connecting peptide and/or the third connecting peptide are short peptides containing G and S amino acids.
  • the first connecting peptide and/or the second connecting peptide and/or the third connecting peptide contain 5 to 15 amino acids.
  • the first connecting peptide and/or the second connecting peptide and/or the third connecting peptide include the amino acid sequence shown in SEQ ID NO:3.
  • the first TGF ⁇ RII fragment, the second TGF ⁇ RII fragment and the third TGF ⁇ RII fragment include the amino acid sequence shown in SEQ ID NO: 2, 4, 28 or 29.
  • a fourth connecting peptide is further included.
  • the N-terminus of the fourth linking peptide is connected to the C-terminus of the antibody heavy chain constant region, and the C-terminus of the fourth linking peptide is connected to the N-terminus of the first TGF ⁇ RII fragment. connected.
  • the fourth connecting peptide is a flexible fragment.
  • the fourth connecting peptide is a short peptide containing G and S amino acids.
  • the fourth connecting peptide includes the amino acid sequence shown in SEQ ID NO:5.
  • a fifth linking peptide is further included.
  • the N-terminus of the fifth linking peptide is connected to the C-terminus of the second TGF ⁇ RII fragment, and the C-terminus of the fifth linking peptide is connected to the N-terminus of the antibody heavy chain constant region. connected.
  • it further includes a sixth connecting peptide, the N-terminus of the sixth connecting peptide is connected to the C-terminal of the third TGF ⁇ RII fragment, and the C-terminal of the sixth connecting peptide is connected to the C-terminal of the antibody
  • the N-terminus of the light chain constant region is linked.
  • the fifth connecting peptide and/or the sixth connecting peptide is a flexible fragment.
  • the fifth connecting peptide and/or the sixth connecting peptide is a short peptide containing G and S amino acids.
  • the fifth connecting peptide and/or the sixth connecting peptide includes the amino acid sequence shown in SEQ ID NO:5.
  • At least a part of the antibody heavy chain constant region or the antibody light chain constant region is from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.
  • At least a portion of the antibody heavy chain constant region or the antibody light chain constant region is derived from a human antibody or a mutant thereof.
  • At least a portion of the antibody heavy chain constant region or the antibody light chain constant region is derived from human antibody IgG or a mutant thereof.
  • At least a portion of the antibody heavy chain constant region or the antibody light chain constant region is derived from human antibody IgG1 or a mutant thereof.
  • the antibody heavy chain constant region has amino acid mutations at positions 234, 235 and 447 compared to wild-type human IgG1.
  • the antibody heavy chain constant region has L234A, L235A and K447A mutations compared to wild-type human IgG1.
  • the antibody heavy chain constant region includes the amino acid sequence shown in SEQ ID NO:7.
  • the antibody light chain constant region includes the amino acid sequence shown in SEQ ID NO:8.
  • the peptide chain 1 includes the amino acid sequence shown in SEQ ID NO: 21, 34, 35 or 58.
  • the peptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 23, 38, 39 or 59.
  • the recombinant antibody includes: 1) the amino acid sequence shown in SEQ ID NO:21 and SEQ ID NO:23; or 2) the amino acid sequence shown in SEQ ID NO:34 and SEQ ID NO:38 The amino acid sequence; or 3) the amino acid sequence shown in SEQ ID NO:35 and SEQ ID NO:39; or 4) the amino acid sequence shown in SEQ ID NO:58 and SEQ ID NO:59.
  • the present invention also provides a recombinant antibody, including two identical peptide chains 3, each peptide chain 3 including: 1) the fourth TGF ⁇ RII fragment; 2) the antibody Fc region; and 3) the fifth TGF ⁇ RII fragment or tumor-targeting peptide; wherein, the C-terminus of the antibody Fc region is connected to the N-terminus of the fourth TGF ⁇ RII fragment, and the N-terminus of the antibody Fc region is connected to the fifth TGF ⁇ RII fragment or tumor-targeting peptide. The C terminal is connected.
  • Recombinant antibodies according to some specific embodiments of the present invention can effectively bind to TGF ⁇ , block the combination of TGF ⁇ RII and TGF ⁇ on the cell surface, and significantly promote the proliferation of T cells. They have a long half-life in vivo and can effectively treat or prevent tumors.
  • the above-mentioned recombinant antibody may further include at least one of the following additional technical features:
  • said fourth TGF ⁇ RII fragment and/or said fifth TGF ⁇ RII fragment comprises a wild-type TGF ⁇ RII extracellular domain.
  • the fourth TGF ⁇ RII fragment includes an N-terminal fragment of wild-type TGF ⁇ RII extracellular domain, a seventh connecting peptide and a C-terminal fragment of wild-type TGF ⁇ RII extracellular domain, wherein, The C-terminal fragment of the wild-type TGF ⁇ RII extracellular domain includes at most 122 amino acids of the C-terminus of the wild-type TGF ⁇ RII extracellular domain.
  • the fifth TGF ⁇ RII fragment includes an N-terminal fragment of wild-type TGF ⁇ RII extracellular domain, an eighth connecting peptide and a C-terminal fragment of wild-type TGF ⁇ RII extracellular domain, wherein, the The C-terminal fragment of the wild-type TGF ⁇ RII extracellular domain includes at most 122 amino acids of the C-terminus of the wild-type TGF ⁇ RII extracellular domain.
  • the C-terminus of the N-terminal fragment of the wild-type TGF ⁇ RII extracellular domain is connected to the N-terminus of the seventh connecting peptide, and the seventh The C-terminus of the linker peptide is connected to the N-terminus of the C-terminal fragment of the wild-type TGF ⁇ RII extracellular domain.
  • the C-terminus of the N-terminal fragment of the wild-type TGF ⁇ RII extracellular domain is connected to the N-terminus of the eighth connecting peptide, and the eighth The C-terminus of the linker peptide is connected to the N-terminus of the C-terminal fragment of the wild-type TGF ⁇ RII extracellular domain.
  • the seventh connecting peptide and/or the eighth connecting peptide is a flexible fragment.
  • the seventh connecting peptide and/or the eighth connecting peptide is a short peptide containing G and S amino acids.
  • the seventh connecting peptide and/or the eighth connecting peptide contains 5 to 15 amino acids.
  • the seventh connecting peptide and/or the eighth connecting peptide includes the amino acid sequence shown in SEQ ID NO:3.
  • the recombinant antibody further includes a ninth linking peptide.
  • the N-terminus of the fourth TGF ⁇ RII fragment is connected to the C-terminus of the ninth linking peptide, and the N-terminus of the ninth linking peptide is connected to the C-terminus of the antibody Fc region.
  • the recombinant antibody further includes a tenth linking peptide.
  • the C-terminus of the fifth TGF ⁇ RII fragment is connected to the N-terminus of the tenth linking peptide, and the C-terminus of the tenth linking peptide is connected to the N-terminus of the antibody Fc region.
  • the ninth connecting peptide and/or the tenth connecting peptide is a flexible fragment.
  • the ninth connecting peptide and/or the tenth connecting peptide is a short peptide containing G and S amino acids.
  • the ninth connecting peptide and/or the tenth connecting peptide includes the amino acid sequence shown in SEQ ID NO:5.
  • the wild-type TGF ⁇ RII extracellular domain includes the amino acid sequence shown in SEQ ID NO:2.
  • the fourth TGF ⁇ RII fragment and the fifth TGF ⁇ RII fragment include the amino acid sequence shown in SEQ ID NO: 2, 4, 28 or 29.
  • the tumor targeting peptide includes the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 26.
  • each of the peptide chains 3 is connected by an inter-chain disulfide bond.
  • At least part of the Fc region of the antibody is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.
  • At least part of the Fc region of the antibody is derived from a human antibody or a mutant thereof.
  • At least a portion of the antibody Fc region is derived from human antibody IgG or a mutant thereof.
  • At least part of the antibody Fc region is derived from human antibody IgG1 or a mutant thereof.
  • the antibody Fc region has amino acid mutations at positions 234, 235 and 447 compared to wild-type human IgG1.
  • the antibody Fc region has L234A, L235A and K447A mutations compared to wild-type human IgG1.
  • the antibody Fc region includes the amino acid sequence shown in SEQ ID NO: 6.
  • the peptide chain 3 includes the amino acid sequence shown in SEQ ID NO: 17, 19, 30, 31, 32, 33, 54 or 55.
  • the present invention provides a nucleic acid molecule encoding a recombinant antibody as described above.
  • the recombinant antibodies obtained according to the nucleic acid molecules of the embodiments of the present invention can effectively bind to TGF ⁇ , block the combination of TGF ⁇ RII and TGF ⁇ on the cell surface, significantly promote the proliferation of T cells, have a long half-life in vivo, and can effectively treat or prevent tumors. .
  • the above-mentioned nucleic acid molecule may further include at least one of the following additional technical features:
  • the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO: 20, 44, 45 or 52.
  • the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO: 22, 48, 49 or 53.
  • the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO: 16, 18, 40, 41, 42, 43, 56 or 57.
  • nucleic acids mentioned in the description and claims of the present invention actually include either or both complementary double strands.
  • nucleic acid sequence in this application includes DNA form or RNA form, and disclosing one of them means that the other one is also disclosed.
  • the present invention provides an expression vector carrying the nucleic acid molecule described above.
  • the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule.
  • these control elements can come directly from the carrier itself, or they can be exogenous, that is, they do not come from the carrier itself.
  • the nucleic acid is operably connected to the control element.
  • operably connected refers to connecting an exogenous gene to a vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, can exert their intended effects on regulating the transcription and translation of the exogenous gene. function.
  • the nucleic acid molecules used to encode the two identical peptide chains 1 and/or the two identical peptide chains 2 of the recombinant antibody can be independently inserted into different vectors. Commonly, they are inserted into the same vector. Commonly used vectors can be, for example, plasmids, phages, etc.
  • the expression vector according to some specific embodiments of the present invention is introduced into a suitable recipient cell, the expression of the aforementioned recombinant antibody can be effectively realized under the mediation of the regulatory system, thereby achieving large-scale acquisition of the recombinant antibody in vitro.
  • the above-mentioned expression vector may further include at least one of the following additional technical features:
  • the expression vector is a eukaryotic expression vector or a virus. Then, the aforementioned recombinant antibody can be expressed in appropriate recipient cells, such as CHO cells.
  • the virus includes a lentivirus.
  • the present invention provides a recombinant cell that carries the aforementioned nucleic acid molecule, expression vector, or expresses the aforementioned recombinant antibody.
  • Recombinant cells according to some specific embodiments of the present invention can be used for the in vitro expression and large-scale acquisition of recombinant antibodies capable of recognizing TGF ⁇ as described above.
  • the above-mentioned recombinant cells may further include at least one of the following additional technical features:
  • the recombinant cells are eukaryotic cells.
  • the recombinant cells are mammalian cells.
  • the recombinant cells do not include animal germ cells, fertilized eggs or embryonic stem cells.
  • the recombinant cells of the present invention are not particularly limited and can be prokaryotic cells, eukaryotic cells or phages.
  • the prokaryotic cell can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc.
  • the eukaryotic cells may include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Trichoderma, insect cells such as Fall Armyworm, plant cells such as tobacco, BHK cells, CHO cells, COS cells, and myeloma cells. cells and other mammalian cells.
  • the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.
  • suitable conditions refer to conditions suitable for the expression of the recombinant antibodies described in this application.
  • conditions suitable for the expression of recombinant antibodies include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, and suitable cell culture. environment and appropriate cell culture time.
  • suitable conditions are not particularly limited. Those skilled in the art can optimize the most suitable conditions for the expression of the recombinant antibody according to the specific environment of the laboratory.
  • compositions for pharmaceutical uses, drugs, kits
  • the present invention provides a composition including the aforementioned recombinant antibody, nucleic acid molecule, expression vector or recombinant cell.
  • recombinant antibodies according to some specific embodiments of the present invention, or recombinant antibodies obtained by expressing the nucleic acid molecules, expression vectors or recombinant cells under appropriate conditions can effectively bind to TGF ⁇ and block TGF ⁇ RII on the cell surface. It binds to TGF ⁇ and significantly promotes the proliferation of T cells. It has a long half-life in vivo and can effectively treat or prevent tumors.
  • the recombinant antibodies or expressed antibodies contained in the composition can effectively interact with TGF ⁇ . Binding blocks the binding of TGF ⁇ RII to TGF ⁇ on the cell surface, and significantly promotes the proliferation of T cells. It has a long half-life in vivo and can effectively treat or prevent tumors.
  • compositions are food compositions, pharmaceutical compositions, etc., including combinations that are separated in time and/or space, as long as they can function together to achieve the purpose of the present invention.
  • the ingredients contained in the composition can be administered to the subject as a whole or separately.
  • the ingredients contained in the composition are administered to the subject separately, the individual ingredients may be administered to the subject simultaneously or sequentially.
  • the present invention provides the use of the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells or compositions in the preparation of medicines for preventing or treating tumors.
  • recombinant antibodies according to some specific embodiments of the present invention, or recombinant antibodies obtained by expressing the nucleic acid molecules, expression vectors or recombinant cells under appropriate conditions, and recombinant antibodies included in the composition can effectively interact with TGF ⁇ Bind, block the combination of TGF ⁇ RII and TGF ⁇ on the cell surface, and significantly promote the proliferation of T cells. It has a long half-life in the body and can effectively treat or prevent tumors.
  • drugs prepared using the above substances can also block cells.
  • the surface TGF ⁇ RII binds to TGF ⁇ and significantly promotes the proliferation of T cells. It has a long half-life in vivo and can effectively treat or prevent tumors.
  • the present invention provides a medicine, including: the aforementioned recombinant antibody, nucleic acid molecule, expression vector, recombinant cell or composition.
  • recombinant antibodies according to some specific embodiments of the present invention, or recombinant antibodies obtained by expressing the nucleic acid molecules, expression vectors or recombinant cells under appropriate conditions, and recombinant antibodies included in the composition can effectively interact with TGF ⁇
  • the combination has strong specificity and can not only neutralize TGF ⁇ , but also exert a better targeting effect, thereby realizing the biological effects of other drugs in the drug, such as inhibiting the activity of TGF ⁇ molecules and killing cells expressing TGF ⁇ molecules.
  • these drugs further include a pharmaceutically acceptable carrier, including any solvent, solid excipient, diluent, binder, disintegrant or other liquid excipient, dispersant, flavoring or Suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, solid binders, glidants or lubricants, etc., are suitable for the unique target dosage form.
  • a pharmaceutically acceptable carrier including any solvent, solid excipient, diluent, binder, disintegrant or other liquid excipient, dispersant, flavoring or Suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, solid binders, glidants or lubricants, etc.
  • the recombinant antibodies of the invention may be incorporated into pharmaceutical compositions suitable for parenteral administration (eg, intravenous, subcutaneous, intraperitoneal, intramuscular).
  • parenteral administration eg, intravenous, subcutaneous, intraperitoneal, intramuscular
  • These pharmaceutical compositions can be prepared in various forms. Examples include liquid, semi-solid, and solid dosage forms, including, but not limited to, liquid solutions (eg, injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories.
  • Typical pharmaceutical compositions are in the form of injection or infusion solutions.
  • the recombinant antibodies may be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
  • drugs according to embodiments of the present invention can play a diagnostic role by relying on the antibodies that can specifically target and bind TGF ⁇ as proposed above in combination with diagnostic reagents, and then play a role in detecting parts of the organism where TGF ⁇ is abnormally expressed.
  • Diagnosis such as combination with diagnostic nuclides, nanomaterials, etc., enables visual observation of cells, tissues, and organs that abnormally express TGF ⁇ in organisms, thereby assisting medical workers or scientific researchers in making more accurate judgments on lesions.
  • treatment and “prevention” as used herein and words derived therefrom do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art would consider potentially beneficial or therapeutically effective.
  • the methods of the present invention may provide any amount and any level of treatment or prevention of tumors in a mammal.
  • treatment or prevention provided by the methods of the present invention may include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented, such as neoplasia.
  • prevention may encompass delaying the onset of a disease or its symptoms or in a patient.
  • the recombinant antibodies herein can also be made into part of a kit or other diagnostic reagents as needed.
  • the present invention provides the use of the previously described recombinant antibody in the preparation of a kit for detecting TGF ⁇ .
  • the recombinant antibody can be used in combination with any detection reagent or therapeutic preparation, such as with diagnostic nuclide, nanomaterials, etc., to detect the target site through the radioactivity of the nuclide, and thereby obtain information about the target site; it can also be used with treatment
  • the combined use of radionuclides uses the radioactivity of nuclides to specifically kill target cells, tissues, etc.
  • the above-mentioned uses may further include at least one of the following additional technical features:
  • the TGF ⁇ includes TGF ⁇ 1 and TGF ⁇ 3.
  • TGF ⁇ 1 and TGF ⁇ 3 are examples of TGF ⁇ 1 and TGF ⁇ 3.
  • the present invention also provides a kit, which includes the above-mentioned recombinant antibody.
  • the kit provided by the present invention can be used, for example, in Western blotting, immunoprecipitation, and other kits that utilize the specific binding properties of TGF ⁇ antigen and antibody for detection.
  • kits may contain any one or more of the following: antagonists, TGF ⁇ antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc.
  • the recombinant antibodies can be used in different types of diagnostic tests, for example, to detect a variety of diseases or the presence of drugs, toxins or other proteins in vitro or in vivo.
  • the subject's serum or blood can be tested to detect related diseases, such as: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, etc. Cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer, these tumor cells can be any cells whose growth is unregulated.
  • related diseases such as: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, etc.
  • these tumor cells can be any cells whose growth is unregulated.
  • the present invention proposes a method for diagnosing related diseases caused by TGF ⁇ , using the previously described recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, and kits to detect TGF ⁇ in the test sample. Detection; based on the detection results, determine the content of TGF ⁇ in the sample to be tested.
  • increased TGF ⁇ in the tumor microenvironment is associated with immune escape.
  • Increased TGF ⁇ levels prevent naive T cells from differentiating toward a Th1 effector phenotype, promote their transformation into Treg subtypes, and inhibit dendritic cell antigens.
  • TGF ⁇ is a potent immunosuppressant.
  • TGF ⁇ Increased expression of TGF ⁇ is often associated with malignancy in many cancers, as well as defects in the cell growth inhibitory response to TGF ⁇ , and then its immunosuppressive function dominates, contributing to tumorigenesis.
  • the recombinant antibodies proposed in this application, or the recombinant antibodies expressed by nucleic acid molecules, expression vectors, recombinant cells, or the recombinant antibodies included in the compositions and kits can effectively bind to TGF ⁇ . Therefore, using the recombinant antibodies described in this application The method can effectively detect the TGF ⁇ content in test samples derived from test individuals, and can effectively diagnose related diseases caused by TGF ⁇ .
  • the above method may further include at least one of the following additional technical features:
  • the content of TGF ⁇ in the sample to be tested is not lower than the minimum standard for disease, which is an indication that the sample to be tested is derived from patients suffering from related diseases caused by TGF ⁇ .
  • the TGF ⁇ includes TGF ⁇ 1 and TGF ⁇ 3.
  • TGF ⁇ 1 and TGF ⁇ 3 are examples of TGF ⁇ 1 and TGF ⁇ 3.
  • the sample to be tested includes: blood, tissue, cells, feces or urine.
  • the related diseases caused by TGF ⁇ include tumors.
  • the present invention proposes a method for assessing the staging of related diseases caused by TGF ⁇ , using the previously described recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, and kits to detect TGF ⁇ in the test sample. Carry out detection; based on the detection results, determine the content of TGF ⁇ in the sample to be tested.
  • increased TGF ⁇ in the tumor microenvironment is associated with immune escape.
  • Increased TGF ⁇ levels prevent naive T cells from differentiating toward a Th1 effector phenotype, promote their transformation into Treg subtypes, and inhibit dendritic cell antigens.
  • TGF ⁇ is a potent immunosuppressant.
  • TGF ⁇ Increased expression of TGF ⁇ is often associated with many malignant tumors, as well as defects in the cell growth inhibitory response to TGF ⁇ , and then its immunosuppressive function dominates, contributing to tumorigenesis and disease. At different stages of occurrence, the content of TGF ⁇ in peripheral blood will change.
  • the recombinant antibodies proposed in this application, or the recombinant antibodies expressed by nucleic acid molecules, expression vectors, recombinant cells, or the recombinant antibodies included in the compositions and kits On the basis that all can effectively combine with TGF ⁇ , the method described in this application can be used to effectively detect the TGF ⁇ content in the test sample derived from the subject individual, and based on the TGF ⁇ content, the period of related diseases caused by TGF ⁇ can be estimated. to evaluate.
  • the above method may further include at least one of the following additional technical features:
  • the related diseases caused by TGF ⁇ include tumors, and the content of TGF ⁇ in the sample to be tested is not lower than the minimum level for stage IV tumor disease, and the sample to be tested is derived from patients with stage IV tumors.
  • the patient's indication is that the content of TGF ⁇ in the sample to be tested is located in tumor stages IV and III.
  • the level between the stage III disease is an indication that the sample to be tested is derived from a patient with stage III tumor; the content of TGF ⁇ in the sample to be tested is between the standard levels for stage III and stage II tumor disease, which is the sample to be tested.
  • TGF ⁇ in the sample to be tested is between the standard levels for stage I and stage II disease is an indication that the sample to be tested is derived from a patient with stage I tumor.
  • the level of TGF ⁇ changes according to the type of tumor when the tumor is in stages I, II, III, and IV. To judge the stage of the tumor, as long as the sample to be tested is The content of TGF ⁇ can be known by comparing it with the standard level of TGF ⁇ at the tumor stage, or by comparing the content of TGF ⁇ in the sample to be tested with the content of TGF ⁇ in samples derived from known diseased individuals or groups.
  • the TGF ⁇ includes TGF ⁇ 1 and TGF ⁇ 3.
  • TGF ⁇ 1 and TGF ⁇ 3 are examples of TGF ⁇ 1 and TGF ⁇ 3.
  • the sample to be tested includes: blood, tissue, cells, feces or urine.
  • the present invention proposes a method for assessing the prognosis of related diseases caused by TGF ⁇ , using the previously described recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, and kits to detect TGF ⁇ in the test sample. Carry out detection; based on the detection results, determine the content of TGF ⁇ in the sample to be tested.
  • TGF ⁇ is a potent immunosuppressant, and increased expression of TGF ⁇ is often associated with many malignant tumors, as well as defects in the cell growth inhibitory response to TGF ⁇ , and then its immunosuppressive function dominates, promoting tumorigenesis.
  • TGF ⁇ tumor necrosis factor ⁇
  • the prognosis of such diseases can be effectively evaluated by monitoring the levels of TGF ⁇ in peripheral blood. For example, comparing the levels of TGF ⁇ in subjects before and after treatment, or comparing subjects after treatment. The content of TGF ⁇ in the body is compared with the normal level or disease level, etc.
  • the recombinant antibody proposed in this application or the recombinant antibody expressed by the nucleic acid molecule, expression vector, recombinant cell, or the recombinant antibody contained in the composition or kit
  • the method described in this application can be used to effectively detect the TGF ⁇ content in the test sample derived from the subject individual, and evaluate the prognosis of related diseases caused by TGF ⁇ based on the TGF ⁇ content.
  • the above method may further include at least one of the following additional technical features:
  • the sample to be tested is derived from patients suffering from related diseases caused by TGF ⁇ before or after treatment.
  • the prognostic effect of the related diseases caused by TGF ⁇ is determined based on the content of TGF ⁇ in the test sample of the patient suffering from the related diseases caused by TGF ⁇ before or after treatment.
  • a decrease in the content of TGF ⁇ in the test sample of a patient suffering from a related disease caused by TGF ⁇ after treatment is an indicator of a good prognosis of the patient.
  • the TGF ⁇ includes TGF ⁇ 1 and TGF ⁇ 3.
  • the sample to be tested includes: blood, tissue, cells, feces or urine.
  • the related diseases caused by TGF ⁇ include tumors.
  • the N-terminus of the wild-type TGF ⁇ RII extracellular domain is truncated by 14-21 (14, 19, 21) amino acids to obtain the C-terminal fragment of the wild-type TGF ⁇ RII extracellular domain, and then inserted into More than 15 amino acids, in which the inserted amino acids include some of the truncated 14-21 amino acids (N-terminal fragment of wild-type TGF ⁇ RII extracellular domain) and flexible fragments (first connecting peptide, second connecting peptide peptide, the third connecting peptide, the seventh connecting peptide, the eighth connecting peptide), the flexible fragment combines the retained N-terminal fragment of the wild-type TGF ⁇ RII extracellular domain and the C-terminal fragment of the wild-type TGF ⁇ RII extracellular domain.
  • TGF ⁇ RII Trap-1 TGF ⁇ RII Trap-2, TGF ⁇ RII Trap-3.
  • the immunomodulatory molecule TGF ⁇ RII Trap, tumor targeting peptide and wild-type TGF ⁇ RII extracellular domain are separated from different constant region fragments of IgG1 antibodies (Fc region mutants, heavy chain constant region HC, light chain constant region LC) using connecting peptides.
  • the amino acid sequence of the wild-type TGF ⁇ RII is shown in SEQ ID NO: 1
  • the amino acid sequence of the extracellular domain of the wild-type TGF ⁇ RII is shown in SEQ ID NO: 2
  • the first connecting peptide, the second connecting peptide, the third connecting peptide, the seventh connecting peptide, and the eighth connecting peptide are GGSGSGSG (SEQ ID NO: 3)
  • the amino acid sequence of the TGF ⁇ RII Trap is as SEQ ID NO: 4 , 28 and 29, the amino acid sequence of the connecting peptide (the fourth connecting peptide, the fifth connecting peptide, the sixth connecting peptide, the ninth connecting peptide, the tenth connecting peptide) is shown in SEQ ID NO: 5, so
  • the Fc region mutants are Fc mutants of wild-type human IgG1 antibodies, including two types, namely Fc region mutant 1 and Fc region mutant 2 (Fc region of antibody
  • Fc region mutant 2 (the Fc region of the antibody IgG1.8) is obtained by It is obtained by L234A and L235A mutations on the basis of Fc region mutant 1.
  • the amino acid sequence of the Fc region mutant 1 is as shown in SEQ ID NO:50
  • the amino acid sequence of the Fc region mutant 2 is as SEQ ID NO:6.
  • the amino acid sequence of the antibody heavy chain constant region is shown in SEQ ID NO:7
  • the amino acid sequence of the antibody light chain constant region is shown in SEQ ID NO:8
  • the amino acid sequence of the tumor-targeting peptide As shown in SEQ ID NO:9 or SEQ ID NO:26.
  • the different constant regions of the obtained antibodies are respectively connected to the N-terminal or C-terminal of the mutated TGF ⁇ RII extracellular domain and/or tumor-targeting peptide and/or wild-type TGF ⁇ RII extracellular domain to form Fc-hTGF ⁇ RII Trap recombinant Antibodies or Fc-ECD recombinant antibodies.
  • Molecule 1 (recombinant antibody #1): has two identical peptide chains (molecule 1 single peptide chain 1, molecule 1 single peptide chain 2, molecule 1 single peptide chain 3, or molecule 1 single peptide chain 4), each peptide
  • the C-terminus of one TGF ⁇ RII Trap molecule or ECD molecule in the chain is connected to the N-terminus of Fc region mutant 2
  • the C-terminus of Fc region mutant 2 is connected to the N-terminus of another TGF ⁇ RII Trap molecule or ECD molecule, as shown in Figure 1
  • the nucleic acids encoding the four peptide chains of molecule 1 have the nucleotide sequences shown in SEQ ID NO: 16, 40, 41, and 56 respectively.
  • the four peptides of molecule 1 obtained The chains have the amino acid sequences shown in SEQ ID NO: 17, 30, 31, and 54 respectively, and the four molecule numbers are #1-1 (R1054), #1-2, #1-3, and #1-4.
  • the amino acid sequence of #1-1 (R1054) is SEQ ID NO:17
  • the amino acid sequence of #1-2 is SEQ ID NO:30
  • the amino acid sequence of #1-3 is SEQ ID NO:31
  • Molecule 2 (recombinant antibody #2): has two identical peptide chains (molecule 2 single peptide chain 1, molecule 2 single peptide chain 2, molecule 2 single peptide chain 3, or molecule 2 single peptide chain 4), each peptide
  • the chain includes a tumor-targeting peptide, the C-terminus of the tumor-targeting peptide is connected to the N-terminus of the Fc region mutant 2, and the C-terminus of the Fc region mutant 2 is connected to the N-terminus of a TGF ⁇ RII Trap molecule or an ECD molecule.
  • the nucleic acids encoding a single peptide chain of molecule 2 have the nucleotide sequences shown in SEQ ID NO: 18, 42, 43, and 57 respectively.
  • the four obtained The single peptide chains of molecule 2 have the amino acid sequences shown in SEQ ID NO: 19, 32, 33, and 55 respectively.
  • the four types of molecule 2 are numbered #2-1 (R1056), #2-2, #2-3, # 2-4. Among them, the amino acid sequence of #2-1 (R1056) is SEQ ID NO: 19, the amino acid sequence of #2-2 is SEQ ID NO: 32, the amino acid sequence of #2-3 is SEQ ID NO: 33, and the amino acid sequence of #2-4 SEQ ID NO:55.
  • Molecule 3 (Recombinant Antibody #3): Has two identical heavy chains (Molecular 3 Heavy Chain 1, Molecule 3 Heavy Chain 2, Molecule 3 Heavy Chain 3, or Molecule 3 Heavy Chain 4) and two identical light chains ( Molecule 3 light chain 1, Molecule 3 light chain 2, Molecule 3 light chain 3, or Molecule 3 light chain 4), where the C-terminus of one TGF ⁇ RII Trap molecule or ECD molecule in each heavy chain is connected to the heavy chain constant region The N-terminus is connected, and the C-terminus of the heavy chain constant region is connected to the N-terminus of another TGF ⁇ RII Trap molecule or ECD molecule, as shown in 1-E and 1-F in Figure 1, encoding the nuclei of the four heavy chains
  • the nucleotide sequences are shown in SEQ ID NO: 20, 44, 45, and 52 respectively.
  • the amino acid sequences of the four heavy chains of the obtained molecule 3 are shown in SEQ ID NO: 21, 34, 35, and 58 respectively; each light chain The C-terminus of a TGF ⁇ RII Trap molecule or ECD molecule in the chain is connected to the N-terminus of the antibody light chain constant region (CL, Kappa).
  • the nucleotide sequences encoding the four light chains are such as SEQ ID NO: 22, 48, As shown in 49 and 53, the amino acid sequences of the four light chains of the obtained molecule 3 are shown in SEQ ID NO: 23, 38, 39, and 59 respectively.
  • the four kinds of molecule 3 are numbered #3-1 (R1055), #3 -2, #3-3, #3-4.
  • the amino acid sequence of #3-1 (R1055) is SEQ ID NO: 22
  • the amino acid sequence of #3-2 is SEQ ID NO: 48
  • the amino acid sequence of #3-3 is SEQ ID NO: 49
  • the amino acid sequence of #3-4 SEQ ID NO:53.
  • Molecule 4 (recombinant antibody #4): has two identical peptide chains (molecule 4 single peptide chain 1, molecule 4 single peptide chain 2, molecule 4 single peptide chain 3, molecule 4 single peptide chain 4), each peptide chain
  • the C-terminus of the middle Fc region mutant 1 is connected to the N-terminus of a TGF ⁇ RII Trap molecule or ECD molecule, as shown in 1-G and 1-H in Figure 1.
  • the four nucleic acids encoding molecule 4 have SEQ ID NO. : The nucleotide sequences shown in SEQ ID NO: 24, 46, 47, and 63.
  • the four obtained molecules 4 have the amino acid sequences shown in SEQ ID NO: 25, 36, 37, and 64.
  • the four molecules 4 are numbered #4-1 (R0796), #4-2, #4-3, #4-4.
  • the amino acid sequence of #4-1 (R0796) is SEQ ID NO: 25
  • the amino acid sequence of #4-2 is SEQ ID NO: 36
  • the amino acid sequence of #4-3 is SEQ ID NO: 37
  • the amino acid sequence of #4-4 SEQ ID NO:64.
  • Wild-type TGF ⁇ RII has the following amino acid sequence:
  • the extracellular domain (ECD) of wild-type TGF ⁇ RII has the amino acid sequence of SEQ ID NO:2.
  • the nucleic acid encoding the extracellular domain (ECD) of wild-type TGF ⁇ RII has the following nucleotide sequence:
  • the first connecting peptide and/or the second connecting peptide and/or the third connecting peptide and/or the seventh connecting peptide and/or the eighth connecting peptide have the following amino acid sequence:
  • TGF ⁇ RII Trap-1 has the amino acid sequence of SEQ ID NO:4.
  • TGF ⁇ RII Trap-2 has an amino acid sequence such as SEQ ID NO: 28.
  • TGF ⁇ RII Trap-3 has an amino acid sequence such as SEQ ID NO: 29.
  • the fourth connecting peptide, the fifth connecting peptide, the sixth connecting peptide, the ninth connecting peptide, and the tenth connecting peptide have the following amino acid sequences:
  • the antibody Fc region has an amino acid sequence such as SEQ ID NO: 6.
  • the antibody heavy chain constant region has an amino acid sequence such as SEQ ID NO:7.
  • the antibody light chain constant region has an amino acid sequence such as SEQ ID NO:8.
  • the tumor targeting peptide has the following amino acid sequence:
  • the nucleic acid encoding TGF ⁇ RII Trap-1 has the following nucleotide sequence:
  • the nucleic acid encoding TGF ⁇ RII Trap-2 has the following nucleotide sequence:
  • the nucleic acid encoding TGF ⁇ RII Trap-3 has the following nucleotide sequence:
  • the nucleic acid encoding the connecting peptide (the fourth connecting peptide, the fifth connecting peptide, the sixth connecting peptide, the ninth connecting peptide, and the tenth connecting peptide) has the following nucleotide sequence:
  • the nucleic acid encoding Fc region mutant 2 (Fc region of IgG1.8 antibody) has the following nucleotide sequence:
  • the nucleic acid encoding the constant region of the antibody heavy chain has the following nucleotide sequence:
  • the nucleic acid encoding the antibody light chain constant region has the following nucleotide sequence:
  • the nucleic acid encoding the tumor-targeting peptide has the following nucleotide sequence:
  • the nucleic acid encoding single peptide chain 1 of molecule 1 has a nucleotide sequence such as SEQ ID NO: 16.
  • the nucleic acid encoding a single peptide chain 2 of molecule 1 has a nucleotide sequence such as SEQ ID NO: 40.
  • the nucleic acid encoding single peptide chain 3 of molecule 1 has a nucleotide sequence such as SEQ ID NO: 41.
  • the nucleic acid encoding a single peptide chain 4 of molecule 1 has a nucleotide sequence such as SEQ ID NO: 56.
  • Molecule 1 single peptide chain 1 has an amino acid sequence such as SEQ ID NO: 17.
  • Molecule 1 single peptide chain 2 has an amino acid sequence such as SEQ ID NO: 30.
  • Molecule 1 single peptide chain 3 has the amino acid sequence SEQ ID NO: 31.
  • Molecule 1 single peptide chain 4 has the amino acid sequence SEQ ID NO: 54.
  • the nucleic acid encoding single peptide chain 1 of molecule 2 has a nucleotide sequence such as SEQ ID NO: 18.
  • the nucleic acid encoding single peptide chain 2 of molecule 2 has a nucleotide sequence such as SEQ ID NO: 42.
  • the nucleic acid encoding single peptide chain 3 of molecule 2 has a nucleotide sequence such as SEQ ID NO: 43.
  • the nucleic acid encoding single peptide chain 4 of molecule 2 has a nucleotide sequence such as SEQ ID NO: 57.
  • Molecule 2 single peptide chain 1 has the amino acid sequence SEQ ID NO:19.
  • Molecule 2 single peptide chain 2 has an amino acid sequence such as SEQ ID NO: 32.
  • Molecule 2 single peptide chain 3 has the amino acid sequence SEQ ID NO: 33.
  • Molecule 2 single peptide chain 4 has an amino acid sequence such as SEQ ID NO: 55.
  • the nucleic acid encoding molecule 3 heavy chain 1 has a nucleotide sequence such as SEQ ID NO: 20.
  • the nucleic acid encoding molecule 3 heavy chain 2 has a nucleotide sequence such as SEQ ID NO: 44.
  • the nucleic acid encoding molecule 3 heavy chain 3 has a nucleotide sequence such as SEQ ID NO: 45.
  • the nucleic acid encoding molecule 3 heavy chain 4 has a nucleotide sequence such as SEQ ID NO: 52.
  • Molecule 3 heavy chain 1 has an amino acid sequence such as SEQ ID NO: 21.
  • Molecule 3 heavy chain 2 has an amino acid sequence such as SEQ ID NO: 34.
  • Molecule 3 heavy chain 3 has an amino acid sequence such as SEQ ID NO: 35.
  • Molecule 3 heavy chain 4 has an amino acid sequence such as SEQ ID NO: 58.
  • the nucleic acid encoding molecule 3 light chain 1 has a nucleotide sequence such as SEQ ID NO: 22.
  • the nucleic acid encoding molecule 3 light chain 2 has a nucleotide sequence such as SEQ ID NO: 48.
  • the nucleic acid encoding molecule 3 light chain 3 has a nucleotide sequence such as SEQ ID NO: 49.
  • the nucleic acid encoding molecule 3 light chain 4 has a nucleotide sequence such as SEQ ID NO: 53.
  • Molecule 3 light chain 1 has an amino acid sequence such as SEQ ID NO: 23.
  • Molecule 3 light chain 2 has an amino acid sequence such as SEQ ID NO: 38.
  • Molecule 3 light chain 3 has an amino acid sequence such as SEQ ID NO: 39.
  • Molecule 3 light chain 4 has an amino acid sequence such as SEQ ID NO: 59.
  • the nucleic acid encoding single peptide chain 1 of molecule 4 has the following nucleotide sequence:
  • the nucleic acid encoding single peptide chain 2 of molecule 4 has the following nucleotide sequence:
  • the nucleic acid encoding the single peptide chain 3 of molecule 4 has the following nucleotide sequence:
  • the nucleic acid encoding the single peptide chain 4 of molecule 4 has the following nucleotide sequence:
  • Molecule 4 single peptide chain 1 has the following amino acid sequence:
  • Molecule 4 single peptide chain 2 has the following amino acid sequence:
  • Molecule 4 single peptide chain 3 has the following amino acid sequence:
  • Molecule 4 single peptide chain 4 has the following amino acid sequence:
  • Fc region mutant 1 has the following amino acid sequence:
  • the nucleic acid encoding Fc region mutant 1 has the following nucleotide sequence:
  • nucleotide sequences encoding recombinant antibodies #1, #2, #3, and #4 obtained in Example 1 were inserted into the expression plasmid pcDNA3.4 respectively, and transiently transfected into human embryonic kidney HEK 293 cells, and the nucleotide sequences produced by the cells were isolated and purified.
  • Recombinant antibodies, in which transfection and isolation and purification of proteins are routine experimental operations.
  • the recombinant antibody #1-1 (R1054), recombinant antibody #2-1 (R1056), and recombinant antibody #3-1 (R1055) are not
  • the molecular weights of the bands on SDS-PAGE under reducing conditions are approximately 120kD, 105kD and 190kD respectively, and the molecular weights of the bands on SDS-PAGE under reducing conditions are approximately 60kD, 50kD and 95kD respectively.
  • This example detects recombinant antibodies #1-1 (R1054), #1-2, #1-3, #1-4, #2-1 (R1056), #2-2, #2-3, #2- 4.
  • #3-1(R1055), #3-2, #3-3, #3-4, #4-1(R0796), #4-2, #4-3, #4-4 activity and used human antibody IgG1 (iso (hIgG1.8) group) as the isotype control group, molecule #5, and molecule #6 as the control group.
  • the structure of molecule #5 is shown in Figure 2, which has 2 lines.
  • the same peptide chain (single peptide chain of molecule #5), the Fc region contained in molecule #5 is the Fc region of antibody IgG1.8 (Fc region mutant 2), the amino acid sequence of the single peptide chain of molecule #5 is as SEQ ID NO :65; molecule
  • the structure of #6 is shown in Figure 3. It contains two identical heavy chains (molecule #6 heavy chain) and light chain (molecule #6 light chain).
  • the heavy chain constant region contained in molecule #6 is antibody IgG1.8.
  • the heavy chain constant region of molecule #6, the heavy chain and light chain amino acid sequences of molecule #6 are shown in SEQ ID NO: 66 and 67 respectively; the preparation method is the same as Example 1 and Example 2, and the antigen used for detection is human TGF ⁇ 1 (CA59 ), human TGF ⁇ 3 (CJ44) (purchased from Novoprotein), the specific detection process is as follows:
  • step e After the incubation in step e, wash the enzyme plate five times with PBST (300 ⁇ L/well), then add TMB in a volume of 100 ⁇ L/well, and leave it at room temperature for 20 minutes in the dark; add 2M stop solution in a volume of 100 ⁇ L/well. hole.
  • the specific experimental results are shown in Figure 4.
  • the ELISA result analysis shows: recombinant antibodies #1-1 (R1054), #1-2, #1-3, #1-4, #2-1 (R1056), #2 -2, #2-3, #2-4, #3-1(R1055), #3-2, #3-3, and #3-4 all retain the in vitro binding activity to hTGF ⁇ , and recombinant antibody #1 -1, #2-1, #3-1 and TGF ⁇ 1 or TGF ⁇ 3 binding EC50 are all between 0.1-0.3nM.
  • the single peptide chain of molecule #5 has the following amino acid sequence:
  • the single heavy chain of molecule #6 has the following amino acid sequence:
  • the single light chain of molecule #6 has the following amino acid sequence:
  • CHO cells stably transfected to express human TGF ⁇ RII were constructed and monoclonal lines were selected. Use the following method to detect recombinant antibodies #1-1(R1054), #1-2, #1-3, #1-4, #2-1(R1056), #2-2, #2-3, #2- 4. Blocking of #3-1(R1055), #3-2, #3-3, #3-4, #4-1(R0796), #4-2, #4-3, #4-4 The activity was tested, in which the human antibody IgG1 (iso (hIgG1.8) group) was used as the isotype control group, and the No Ab group (no anti- body) as the negative control group, and the above molecule #5 and molecule #6 were used as the control group.
  • human antibody IgG1 iso (hIgG1.8) group
  • a. Use 3% BSA to gradiently dilute R1054, R1055, R1056, and R0796 proteins respectively.
  • the starting concentration is 100nM, and the dilution factor is 3 times.
  • a total of 10 gradients are set, specifically 100nM, 33.33nM, 11.11nM, 3.7nM, and 1.23 nM, 0.41nM, 0.14nM, 0.045nM, 0.015nM, 0.005nM; dilute TGF ⁇ 1.biotin with 3% BSA to a concentration of 0.5 ⁇ g/mL;
  • CHO-TGF ⁇ RII cells into a 96-well plate at 2E5/well, add a volume of 100 ⁇ l to each well; centrifuge the cells at 350G for 5 minutes at room temperature, discard the supernatant, and add the incubated hTGF ⁇ 1.biotin and recombinant antibody obtained in step b to mix solution, add a volume of 100 ⁇ L/well, mix gently, and incubate at 4°C for 1 hour;
  • step d After the incubation in step c, centrifuge the cells at 350G for 5 minutes at room temperature, discard the supernatant, and add SA-PE (purchased from Biolegend, diluted 1:300) in a volume of 100 ⁇ L/well, mix well, and incubate at 4°C. 1 hour;
  • SA-PE purchased from Biolegend, diluted 1:300
  • step d After the incubation in step d, centrifuge the cells at 350G room temperature for 5 minutes, discard the supernatant, add 150 ⁇ L PBS to each well to resuspend the cells, detect with a Beckman Cytoflex flow cytometer, and use Graphpad Prism7 to calculate and graph the data.
  • This example verified the function of the recombinant antibody at the cellular level.
  • the inventor detected the recombinant antibodies #1-1 (R1054), #1-2, #1-3, #1-4, #2- in the presence of TGF ⁇ 1. 1(R1056), #2-2, #2-3, #2-4, #3-1(R1055), #3-2, #3-3, #3-4, #4-1(R0796) , #4-2, #4-3, and #4-4 on T cell proliferation, human antibody IgG1 (iso (hIgG1.8) group) as the isotype control group, no Ab group (no antibody added) as The negative control group, the above-mentioned molecule #5, and molecule #6 served as the control group.
  • the experiment adopts the following exemplary methods:
  • PBMC peripheral blood mononuclear cells
  • STMCELL human T cell enrichment kit
  • CD3/CD28 beads (life, 40203D) to the CFSE-stained T cells obtained in step d at a volume of 50 ⁇ l/well.
  • the ratio of CD3/CD28 beads to T cells is 1:30;
  • step f Add the pre-incubated recombinant antibody and TGF ⁇ 1 mixture obtained in step c to the 96-well plate.
  • the added volume of the mixture is 100 ⁇ L/well, and culture it in the incubator;
  • T cell proliferation rate after -1(R1056), #2-2, #2-3, #2-4, #3-1(R1055), #3-2, #3-3, or #3-4 All increased to varying degrees. Among them, the inhibition was strongest after the addition of R1055. Compared with the group without recombinant antibody, the proliferation rate of the highest group (R1055) increased by 22.13%, which was close to the proliferation rate of the control group without TGF ⁇ 1. (34%), and the proliferation rate showed a dependence on the concentration of each group of R1054, R1055, and R1056.
  • the present invention is based on the following findings of the inventor: research has found that increased TGF ⁇ in the tumor microenvironment is related to immune escape, and increased TGF ⁇ levels will prevent naive T cells from differentiating into Th1 effector phenotypes and promote their differentiation into Treg subtypes. transformation and inhibit the antigen presentation function of dendritic cells. Therefore, the inventor modified TGF ⁇ receptor 2 (TGF ⁇ RII) and constructed a recombinant antibody using the mutated and modified TGF ⁇ RII.
  • TGF ⁇ RII TGF ⁇ receptor 2

Abstract

Provided are a recombinant antibody and use thereof. The recombinant antibody comprises two identical peptide chains. Each of the peptide chains comprises a TGFβ RII fragment and an antibody heavy chain constant region. The recombinant antibody has good binding activity with TGFβ, can effectively block the binding of TGFβ RII to TGFβ on the surface of a cell, and can promote the proliferation of T cells, such that the recombinant antibody can effectively treat or prevent TGFβ-related diseases, such as tumors.

Description

一种重组抗体及其应用A recombinant antibody and its application 技术领域Technical field
本发明涉及生物技术领域，具体地，本发明涉及重组抗体及其应用，更具体地，本发明涉及重组抗体，核酸分子，表达载体，重组细胞，组合物，上述物质在制备药物中的用途，药物，重组抗体在制备试剂盒中用途以及试剂盒。The present invention relates to the field of biotechnology. Specifically, the present invention relates to recombinant antibodies and their applications. More specifically, the present invention relates to recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, and the use of the above substances in the preparation of medicines. Drugs, uses of recombinant antibodies in preparation of kits, and kits.
背景技术Background technique
传统的癌症治疗方案以放疗和化疗为主，化疗和放疗因为缺乏靶向性，在杀伤癌细胞的同时，对正常组织也有很强的杀伤作用。此后，在化疗基础上研究发展出了靶向治疗，靶向治疗主要针对对肿瘤存活和生长具有重要性的过表达蛋白或原癌基因，靶向治疗有效减少了诸如耐药、脏器毒性等一系列弊端。近些年来人们逐渐意识到癌症免疫治疗是新兴的癌症治疗概念，其通过激活免疫系统或解除免疫系统的抑制，重整宿主免疫应答，尤其是适应性T细胞应答，提供免疫监视、杀伤癌细胞，特异性更好，还可实现长期保护性免疫。Traditional cancer treatment options are mainly radiotherapy and chemotherapy. Due to the lack of targeting, chemotherapy and radiotherapy not only kill cancer cells, but also have a strong killing effect on normal tissues. Since then, targeted therapy has been developed based on chemotherapy. Targeted therapy mainly targets overexpressed proteins or proto-oncogenes that are important for tumor survival and growth. Targeted therapy effectively reduces drug resistance, organ toxicity, etc. A series of disadvantages. In recent years, people have gradually realized that cancer immunotherapy is an emerging cancer treatment concept. It reorganizes the host immune response, especially the adaptive T cell response, by activating the immune system or removing the suppression of the immune system, providing immune surveillance and killing cancer cells. , has better specificity and can also achieve long-term protective immunity.
转化生长因子-β(transforming growth factor-β,TGFβ)属于调节细胞生长和分化的TGFβ超家族，具有三种形式，包括TGFβ1/2/3。这一家族除TGFβ外，还有活化素(activins)、抑制素(inhibins)、缪勒氏管抑制质(Mullerian inhibitor substance,MIS)和骨形成蛋白(bone morpho-genetic proteins,BMPs)。TGFβ最初因为这种细胞因子能使正常的成纤维细胞转化为非贴壁依赖性生长的细胞而得名，在细胞的生长、分化和免疫功能中都发挥重要的调节作用。Transforming growth factor-β (transforming growth factor-β, TGFβ) belongs to the TGFβ superfamily that regulates cell growth and differentiation. It has three forms, including TGFβ1/2/3. In addition to TGFβ, this family also includes activins, inhibins, Mullerian inhibitor substance (MIS) and bone morpho-genetic proteins (BMPs). TGFβ was originally named because this cytokine can transform normal fibroblasts into cells with anchorage-independent growth and plays an important regulatory role in cell growth, differentiation and immune function.
机体多种细胞均可分泌非活性状态的TGFβ。在体外，非活性状态的TGFβ又称为latency associated peptide(LAP)，通过酸裂解可被活化。在体内，酸性环境可存在于骨折附近和正在愈合的伤口。蛋白本身的裂解作用可使TGFβ复合体变为活化TGFβ。TGFβ可以抑制丝裂原、同种异体抗原刺激的T细胞增殖或IL-2依赖的T细胞生长，也可以抑制IFN-γ诱导黑色素瘤细胞MHCⅡ类抗原表达，并且，TGFβ还可以抑制PBMC中IFN-γ和TNF-α的产生，促进IL-6的表达。Various cells in the body can secrete inactive TGFβ. In vitro, the inactive state of TGFβ, also known as latency associated peptide (LAP), can be activated through acid cleavage. In the body, an acidic environment can exist near fractures and in healing wounds. Cleavage of the protein itself turns the TGFβ complex into activated TGFβ. TGFβ can inhibit the proliferation of T cells stimulated by mitogens and allogeneic antigens or the growth of IL-2-dependent T cells. It can also inhibit the expression of MHC class II antigen in melanoma cells induced by IFN-γ. Moreover, TGFβ can also inhibit IFN in PBMC. The production of -γ and TNF-α promotes the expression of IL-6.
机体多数免疫细胞，包括B细胞、T细胞、树突状细胞和巨噬细胞，都分泌TGFβ，而TGFβ又通过其他细胞因子负调控免疫细胞的增殖、分化和激活。因此，TGFβ是一种有效的免疫抑制剂，TGFβ信号的紊乱会导致机体自身免疫、炎症和癌症出现。TGFβ表达的增加通常与许多癌症的恶性肿瘤，以及对TGFβ的细胞生长抑制反应的缺陷有关，然后其免疫抑制功能占主导地位，促成肿瘤发生。TGFβ在肿瘤中以细胞背景依赖的方式发挥肿瘤抑制或肿瘤促进的作用。TGFβ能够抑制原癌基因c-myc的表达，但在肿瘤发展过程中，随着突变的引入或表观遗传修饰的变化后，癌细胞逐渐耐受TGFβ信号的抑制作用，最终导致肿瘤的发展。Most immune cells in the body, including B cells, T cells, dendritic cells and macrophages, secrete TGFβ, and TGFβ negatively regulates the proliferation, differentiation and activation of immune cells through other cytokines. Therefore, TGFβ is a potent immunosuppressant, and disruption of TGFβ signaling can lead to autoimmunity, inflammation, and cancer. Increased TGFβ expression is often associated with malignancy in many cancers, as well as defects in the cytostatic response to TGFβ, whose immunosuppressive functions then dominate, contributing to tumorigenesis. TGFβ exerts tumor suppressive or tumor promoting effects in tumors in a cell context-dependent manner. TGFβ can inhibit the expression of proto-oncogene c-myc, but during tumor development, with the introduction of mutations or changes in epigenetic modifications, cancer cells gradually become resistant to the inhibitory effect of TGFβ signaling, ultimately leading to tumor development.
近年的研究发现，肿瘤微环境中TGFβ增加与免疫逃逸相关，几种常见的癌症类型均表现出富含TGFβ的TME。TGFβ由癌细胞和TME中存在的其他几种细胞类型(包括Treg)产生。成纤维细胞、巨噬细胞和血小板也是肿瘤中重要的TGFβ生产者。TGFβ水平升高会阻止幼稚T细胞向Th1效应子表型分化，促进其向Treg亚型的转化，并抑制树突状细胞的抗原呈递功能。Tauriello等人发现TGFβ阻断使小鼠肝转移癌症模型对PD1/PDL1疗法更敏感。Recent studies have found that increased TGFβ in the tumor microenvironment is associated with immune escape, and several common cancer types exhibit TME rich in TGFβ. TGFβ is produced by cancer cells and several other cell types present in the TME, including Tregs. Fibroblasts, macrophages, and platelets are also important TGFβ producers in tumors. Elevated TGFβ levels prevent naive T cells from differentiating into Th1 effector phenotypes, promote their transformation into Treg subtypes, and inhibit the antigen presentation function of dendritic cells. Tauriello et al. found that TGFβ blockade made a mouse liver metastasis cancer model more sensitive to PD1/PDL1 therapy.
因此，需进一步开发针对TGFβ的治疗性抗体。Therefore, further development of therapeutic antibodies against TGFβ is needed.
发明内容Contents of the invention
在本发明的第一方面，本发明提出了一种重组抗体。根据本发明的实施例，所述重组抗体包括两条相同的肽链1，每条肽链1包括：1)第一TGFβRII片段；以及2)抗体重链恒定区；其中，所述抗体重链恒定区的C端与所述第一TGFβRII片段的N端相连。根据本发明实施例的重组抗体可以有效与TGFβ进行结合，阻断细胞表面的TGFβRII与TGFβ的结合，并显著促进T细胞的增殖，具有较长的体内半衰期，可以有效治疗或预防肿瘤。In a first aspect of the invention, the invention provides a recombinant antibody. According to an embodiment of the present invention, the recombinant antibody includes two identical peptide chains 1, and each peptide chain 1 includes: 1) a first TGFβRII fragment; and 2) an antibody heavy chain constant region; wherein, the antibody heavy chain The C-terminus of the constant region is linked to the N-terminus of the first TGFβRII fragment. Recombinant antibodies according to embodiments of the present invention can effectively bind to TGFβ, block the combination of TGFβRII and TGFβ on the cell surface, and significantly promote the proliferation of T cells. They have a long half-life in vivo and can effectively treat or prevent tumors.
在本发明的第二方面，本发明提出了一种重组抗体。根据本发明的实施例，所述重组抗体包括两条相同的肽链3，每条肽链3包括：1)第四TGFβRII片段；2)抗体Fc区；以及3)第五TGFβRII片段或肿瘤靶向肽；其中，所述抗体Fc区的C端与所述第四TGFβRII片段的N端相连，所述抗体Fc区的N端与所述第五TGFβRII片段或肿瘤靶向肽的C端相连。根据本发明实施例的重组抗体可以有效与TGFβ进行结合，阻断细胞表面的TGFβRII与TGFβ的结合，并显著促进T细胞的增殖，具有较长的体内半衰期，可以有效治疗或预防肿瘤。In a second aspect of the invention, the invention provides a recombinant antibody. According to an embodiment of the present invention, the recombinant antibody includes two identical peptide chains 3, each peptide chain 3 includes: 1) a fourth TGFβRII fragment; 2) an antibody Fc region; and 3) a fifth TGFβRII fragment or a tumor target Peptide; wherein the C-terminus of the antibody Fc region is connected to the N-terminus of the fourth TGFβRII fragment, and the N-terminus of the antibody Fc region is connected to the C-terminus of the fifth TGFβRII fragment or tumor-targeting peptide. Recombinant antibodies according to embodiments of the present invention can effectively bind to TGFβ, block the combination of TGFβRII and TGFβ on the cell surface, and significantly promote the proliferation of T cells. They have a long half-life in vivo and can effectively treat or prevent tumors.
在本发明的第三方面，本发明提出了一种核酸分子。根据本发明的实施例，所述核酸分子编码第一方面或第二方面所述的重组抗体。根据本发明实施例核酸分子获得的重组抗体可以有效与TGFβ进行结合，阻断细胞表面的TGFβRII与TGFβ的结合，并显著促进T细胞的增殖，具有较长的体内半衰期，可以有效治疗或预防肿瘤。In a third aspect of the invention, the invention provides a nucleic acid molecule. According to an embodiment of the present invention, the nucleic acid molecule encodes the recombinant antibody described in the first aspect or the second aspect. The recombinant antibodies obtained according to the nucleic acid molecules of the embodiments of the present invention can effectively bind to TGFβ, block the combination of TGFβRII and TGFβ on the cell surface, significantly promote the proliferation of T cells, have a long half-life in vivo, and can effectively treat or prevent tumors. .
在本发明的第四方面，本发明提出了一种表达载体。根据本发明的实施例，携带前面所述的核酸分子。根据本发明实施例的表达载体导入合适的受体细胞后，可在调控系统的介导下，有效实现前面所述的重组抗体的表达，进而实现所述重组抗体的体外大量获得。 In a fourth aspect of the invention, the invention provides an expression vector. According to embodiments of the present invention, the nucleic acid molecules described above are carried. After the expression vector according to the embodiment of the present invention is introduced into a suitable recipient cell, under the mediation of the regulatory system, the expression of the recombinant antibody described above can be effectively realized, thereby achieving large-scale acquisition of the recombinant antibody in vitro.
在本发明的第五方面，本发明提出了一种重组细胞。根据本发明的实施例，所述重组细胞携带前面所述的核酸分子、表达载体，或者表达前面所述的重组抗体。根据本发明实施例的重组细胞可用于前面所述的能够识别TGFβ的重组抗体的体外表达和大量获得。In a fifth aspect of the invention, the invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cells carry the aforementioned nucleic acid molecules, expression vectors, or express the aforementioned recombinant antibodies. Recombinant cells according to embodiments of the present invention can be used for the in vitro expression and large-scale acquisition of recombinant antibodies capable of recognizing TGFβ as described above.
在本发明的第六方面，本发明提出了一种组合物。根据本发明的实施例，包括前面所述的重组抗体、核酸分子、表达载体或重组细胞。如前所述，根据本发明实施例的重组抗体，或所述核酸分子、表达载体或重组细胞在合适条件下表达获得的重组抗体均可以有效与TGFβ进行结合，阻断细胞表面的TGFβRII与TGFβ的结合，并显著促进T细胞的增殖，具有较长的体内半衰期，可以有效治疗或预防肿瘤，进一步地，所述组合物中所包含的重组抗体或表达的抗体均能够有效与TGFβ进行结合，阻断细胞表面的TGFβRII与TGFβ的结合，并显著促进T细胞的增殖，具有较长的体内半衰期，可以有效治疗或预防肿瘤。In a sixth aspect of the invention, the invention provides a composition. According to embodiments of the present invention, the above-mentioned recombinant antibodies, nucleic acid molecules, expression vectors or recombinant cells are included. As mentioned above, recombinant antibodies according to embodiments of the present invention, or recombinant antibodies obtained by expressing the nucleic acid molecules, expression vectors or recombinant cells under appropriate conditions, can effectively bind to TGFβ and block TGFβRII and TGFβ on the cell surface. The combination, and significantly promotes the proliferation of T cells, has a long half-life in vivo, and can effectively treat or prevent tumors. Furthermore, the recombinant antibodies or expressed antibodies contained in the composition can effectively bind to TGFβ, Blocks the combination of TGFβRII and TGFβ on the cell surface, and significantly promotes the proliferation of T cells. It has a long half-life in vivo and can effectively treat or prevent tumors.
在本发明的第七方面，本发明提出了前面所述的重组抗体、核酸分子、表达载体、重组细胞或组合物在制备药物中的用途。根据本发明的实施例，所述药物用于预防或治疗肿瘤。如前所述，根据本发明一些具体实施方案的重组抗体，或所述核酸分子、表达载体或重组细胞在合适条件下表达获得的重组抗体，以及组合物中包含的重组抗体均可以有效与TGFβ进行结合，阻断细胞表面的TGFβRII与TGFβ的结合，并显著促进T细胞的增殖，具有较长的体内半衰期，可以有效治疗或预防肿瘤，进一步地，利用上述物质制备的药物同样能够阻断细胞表面的TGFβRII与TGFβ的结合，并显著促进T细胞的增殖，具有较长的体内半衰期，可以有效治疗或预防肿瘤。In the seventh aspect of the present invention, the present invention proposes the use of the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells or compositions in the preparation of medicines. According to embodiments of the present invention, the medicament is used to prevent or treat tumors. As mentioned above, recombinant antibodies according to some specific embodiments of the present invention, or recombinant antibodies obtained by expressing the nucleic acid molecules, expression vectors or recombinant cells under appropriate conditions, and recombinant antibodies included in the composition can effectively interact with TGFβ Bind, block the combination of TGFβRII and TGFβ on the cell surface, and significantly promote the proliferation of T cells. It has a long half-life in the body and can effectively treat or prevent tumors. Furthermore, drugs prepared using the above substances can also block cells. The surface TGFβRII binds to TGFβ and significantly promotes the proliferation of T cells. It has a long half-life in vivo and can effectively treat or prevent tumors.
在本发明的第八方面，本发明提出了一种药物。根据本发明的实施例，包括：前面所述的重组抗体、核酸分子、表达载体、重组细胞或组合物。如前所述，根据本发明一些具体实施方案的重组抗体，或所述核酸分子、表达载体或重组细胞在合适条件下表达获得的重组抗体，以及组合物中包含的重组抗体均可以有效与TGFβ进行结合，特异性强，不仅可以中和TGFβ，而且可以发挥较好的靶向作用，进而实现药物中其他药物的生物学作用，如TGFβ分子活性抑制，表达TGFβ分子的细胞杀伤等作用。In an eighth aspect of the invention, the invention provides a medicament. According to embodiments of the present invention, it includes: the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells or compositions. As mentioned above, recombinant antibodies according to some specific embodiments of the present invention, or recombinant antibodies obtained by expressing the nucleic acid molecules, expression vectors or recombinant cells under appropriate conditions, and recombinant antibodies included in the composition can effectively interact with TGFβ The combination has strong specificity and can not only neutralize TGFβ, but also exert a better targeting effect, thereby realizing the biological effects of other drugs in the drug, such as inhibiting the activity of TGFβ molecules and killing cells expressing TGFβ molecules.
在本发明的第九方面，本发明提出了前面所述的重组抗体在制备试剂盒中的用途。根据本发明的实施例，所述试剂盒用于检测TGFβ。如前所述，所述重组抗体可以有效与TGFβ进行结合，特异性强，因此，根据本发明实施例的重组抗体制备的试剂盒可以有效检测待测样品中是否含有TGFβ及其含量。In the ninth aspect of the present invention, the present invention proposes the use of the aforementioned recombinant antibody in preparing a kit. According to an embodiment of the present invention, the kit is used to detect TGFβ. As mentioned above, the recombinant antibody can effectively bind to TGFβ and has strong specificity. Therefore, the kit prepared according to the recombinant antibody according to the embodiment of the present invention can effectively detect whether the sample to be tested contains TGFβ and its content.
在本发明的第十方面，本发明提出了一种试剂盒。根据本发明的实施例，所述试剂盒包括前面所述的重组抗体。根据本发明实施例提供的所述重组抗体可与TGFβ进行结合，因此，包含所述重组抗体的试剂盒可用于有效诊断或检测TGFβ。所述试剂盒可用于科学研究，如定性或定量检测生物样本中的TGFβ，也可以用于对个体状态进行判断，如获得所述个体的TGFβ水平后，判断其TGFβ水平是否高于或低于正常水平。In a tenth aspect of the present invention, the present invention provides a kit. According to an embodiment of the present invention, the kit includes the recombinant antibody described above. The recombinant antibody provided according to the embodiment of the present invention can bind to TGFβ. Therefore, a kit containing the recombinant antibody can be used to effectively diagnose or detect TGFβ. The kit can be used for scientific research, such as qualitative or quantitative detection of TGFβ in biological samples, and can also be used to judge the status of an individual, such as determining whether the TGFβ level of the individual is higher or lower after obtaining the TGFβ level. normal level.
在本发明的第十一方面，本发明提出了一种诊断TGFβ引起的相关疾病的方法。根据本发明的实施例，采用前面所述的重组抗体、核酸分子、表达载体、重组细胞、组合物、试剂盒对待测样品中的TGFβ进行检测；基于检测结果，确定待测样品中TGFβ的含量。肿瘤微环境中TGFβ增加与免疫逃逸相关，TGFβ水平升高会阻止幼稚T细胞向Th1效应子表型分化，促进其向Treg亚型的转化，并抑制树突状细胞的抗原呈递功能，TGFβ是一种有效的免疫抑制剂，TGFβ表达的增加通常与许多癌症的恶性肿瘤，以及对TGFβ的细胞生长抑制反应的缺陷有关，然后其免疫抑制功能占主导地位，促成肿瘤发生，而本申请提出的所述重组抗体，或核酸分子、表达载体、重组细胞表达的重组抗体，或组合物、试剂盒中包含的重组抗体均可以有效与TGFβ进行结合，因此，采用本申请所述的方法可以有效检测来源于受试个体的待测样品中TGFβ含量，并可以对TGFβ引起的相关疾病进行有效诊断。In an eleventh aspect of the present invention, the present invention provides a method for diagnosing related diseases caused by TGFβ. According to embodiments of the present invention, the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, and kits are used to detect TGFβ in the sample to be tested; based on the detection results, the content of TGFβ in the sample to be tested is determined. . Increased TGFβ in the tumor microenvironment is related to immune escape. Increased TGFβ levels will prevent naive T cells from differentiating into Th1 effector phenotypes, promote their transformation into Treg subtypes, and inhibit the antigen presentation function of dendritic cells. TGFβ is A potent immunosuppressant, increased expression of TGFβ is often associated with malignancy in many cancers, as well as defects in the cell growth inhibitory response to TGFβ, which then dominates its immunosuppressive function and contributes to tumorigenesis, as proposed in this application. The recombinant antibodies, or recombinant antibodies expressed by nucleic acid molecules, expression vectors, recombinant cells, or recombinant antibodies included in compositions and kits can effectively bind to TGFβ. Therefore, the method described in this application can be used to effectively detect The TGFβ content in the sample to be tested is derived from the subject, and can effectively diagnose related diseases caused by TGFβ.
在本发明的第十二方面，本发明提出了一种评估TGFβ引起的相关疾病分期的方法。根据本发明的实施例，采用前面所述的重组抗体、核酸分子、表达载体、重组细胞、组合物、试剂盒对待测样品中的TGFβ进行检测；基于检测结果，确定待测样品中TGFβ的含量。如前所述，肿瘤微环境中TGFβ增加与免疫逃逸相关，TGFβ水平升高会阻止幼稚T细胞向Th1效应子表型分化，促进其向Treg亚型的转化，并抑制树突状细胞的抗原呈递功能，TGFβ是一种有效的免疫抑制剂，TGFβ表达的增加通常与许多恶性肿瘤，以及对TGFβ的细胞生长抑制反应的缺陷有关，然后其免疫抑制功能占主导地位，促成肿瘤发生，处于疾病发生的不同时期，外周血内TGFβ的含量会发生变化，在本申请提出的所述重组抗体，或核酸分子、表达载体、重组细胞表达的重组抗体，或组合物、试剂盒中包含的重组抗体均可以有效与TGFβ进行结合的基础上，采用本申请所述的方法可以有效检测来源于受试个体的待测样品中TGFβ含量，并基于所述TGFβ含量对TGFβ引起的相关疾病所处的时期进行评估。In a twelfth aspect of the present invention, the present invention proposes a method for assessing the staging of related diseases caused by TGFβ. According to embodiments of the present invention, the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, and kits are used to detect TGFβ in the sample to be tested; based on the detection results, the content of TGFβ in the sample to be tested is determined. . As mentioned before, increased TGFβ in the tumor microenvironment is associated with immune escape. Increased TGFβ levels prevent naive T cells from differentiating toward a Th1 effector phenotype, promote their transformation into Treg subtypes, and inhibit dendritic cell antigens. Presenting function, TGFβ is a potent immunosuppressant. Increased expression of TGFβ is often associated with many malignant tumors, as well as defects in the cell growth inhibitory response to TGFβ, and then its immunosuppressive function dominates, contributing to tumorigenesis and disease. At different stages of occurrence, the content of TGFβ in peripheral blood will change. The recombinant antibodies proposed in this application, or the recombinant antibodies expressed by nucleic acid molecules, expression vectors, recombinant cells, or the recombinant antibodies included in the compositions and kits On the basis that all can effectively combine with TGFβ, the method described in this application can be used to effectively detect the TGFβ content in the test sample derived from the subject individual, and based on the TGFβ content, the period of related diseases caused by TGFβ can be estimated. to evaluate.
在本发明的第十三方面，本发明提出了一种评估TGFβ引起的相关疾病的预后方法，根据本发明的实施例，采用前面所述的重组抗体、核酸分子、表达载体、重组细胞、组合物、试剂盒对待测样品中的TGFβ进行检测；基于检测结果，确定待测样品中TGFβ的含量。如前所述，TGFβ是一种有效的免疫抑制剂，TGFβ表达的增加通常与许多恶性肿瘤，以及对TGFβ的细胞生长抑制反应的缺陷有关，然后其免疫抑制功能占主导地位，促成肿瘤发生，TGFβ引起的相关疾病进行治疗后，通过监测外周血内TGFβ的含量可以有效评估该类疾病的预后，例如，将治疗前后的受试者体内TGFβ的含量进行比较，或将治疗后的受试者体内TGFβ的含量与正常水平或患病水平进行比较等，在本申请提出的所述重组抗体，或核酸分子、表达载体、重组细胞表达的重组抗体，或组合物、试剂盒中包含的重组抗体均可以有效与TGFβ进行结合的基础上，采用本申请所述的方法可以有效检测来源于受试个体的待测样品中TGFβ含量，并基于所述TGFβ含量对TGFβ引起的相关疾病的预后进行评估。In the thirteenth aspect of the present invention, the present invention proposes a method for evaluating the prognosis of related diseases caused by TGFβ. According to embodiments of the present invention, the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, and combinations are used Materials and kits are used to detect TGFβ in the sample to be tested; based on the detection results, the content of TGFβ in the sample to be tested is determined. As mentioned before, TGFβ is a potent immunosuppressant, and increased expression of TGFβ is often associated with many malignant tumors, as well as defects in the cell growth inhibitory response to TGFβ, and then its immunosuppressive function dominates, promoting tumorigenesis. After treatment of related diseases caused by TGFβ, the prognosis of such diseases can be effectively evaluated by monitoring the levels of TGFβ in peripheral blood. For example, comparing the levels of TGFβ in subjects before and after treatment, or comparing subjects after treatment. The content of TGFβ in the body is compared with the normal level or disease level, etc. The recombinant antibody proposed in this application, or the recombinant antibody expressed by the nucleic acid molecule, expression vector, recombinant cell, or the recombinant antibody contained in the composition or kit On the basis that all can effectively combine with TGFβ, the method described in this application can be used to effectively detect the TGFβ content in the test sample derived from the subject individual, and evaluate the prognosis of related diseases caused by TGFβ based on the TGFβ content. .
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described one by one here.
附图说明Description of the drawings
图1显示了本发明实施例中的4种重组抗体的结构图，其中，Targeting Unit(靶标单元)表示肿瘤靶向肽，ECD表示野生型TGFβRII的胞外结构域， Figure 1 shows the structural diagram of four recombinant antibodies in the embodiment of the present invention, in which Targeting Unit represents the tumor targeting peptide, and ECD represents the extracellular domain of wild-type TGFβRII.
1-A和1-B显示了重组抗体#1的结构图，1-A and 1-B show the structural diagram of recombinant antibody #1,
1-C和1-D显示了重组抗体#2的结构图，1-C and 1-D show the structural diagram of recombinant antibody #2,
1-E和1-F显示了重组抗体#3的结构图，1-E and 1-F show the structural diagram of recombinant antibody #3,
1-G和1-H显示了重组抗体#4的结构图；1-G and 1-H show the structural diagram of recombinant antibody #4;
图2显示了本发明实施例中的对照组分子#5的结构图；Figure 2 shows the structural diagram of control molecule #5 in the embodiment of the present invention;
图3显示了本发明实施例中的对照组分子#6的结构图；Figure 3 shows the structural diagram of control molecule #6 in the embodiment of the present invention;
图4显示了本发明实施例中的重组抗体与TGFβ1和TGFβ3体外结合的ELISA检测结果分析图，其中，R1054表示重组抗体#1，R1055表示重组抗体#3，R1056表示重组抗体#2，R0796表示重组抗体#4，iso(hIgG1.8)表示同型对照组，Figure 4 shows an analysis diagram of the ELISA detection results of the in vitro binding of recombinant antibodies to TGFβ1 and TGFβ3 in the embodiment of the present invention, in which R1054 represents recombinant antibody #1, R1055 represents recombinant antibody #3, R1056 represents recombinant antibody #2, and R0796 represents Recombinant antibody #4, iso(hIgG1.8) represents the isotype control group,
A图显示了本发明实施例中的重组抗体与TGFβ1体外结合的ELISA检测结果分析图，其中横坐标表示各重组抗体、对照样品的浓度(nM)的log值，纵坐标表示450nm下的OD值，Figure A shows the analysis of the ELISA detection results of the in vitro binding of recombinant antibodies to TGFβ1 in the embodiment of the present invention. The abscissa represents the log value of the concentration (nM) of each recombinant antibody and control sample, and the ordinate represents the OD value at 450 nm. ,
B图显示了本发明的重组抗体与TGFβ3体外结合的ELISA检测结果分析图，其中横坐标表示各重组抗体、对照样品的浓度(nM)的log值，纵坐标表示450nm下的OD值；Figure B shows an analysis chart of the ELISA detection results of the in vitro binding of the recombinant antibody of the present invention to TGFβ3, in which the abscissa represents the log value of the concentration (nM) of each recombinant antibody and control sample, and the ordinate represents the OD value at 450nm;
图5显示了本发明实施例中的重组抗体对细胞表面TGFβRII与TGFβ结合的阻断效果结果图，其中，横坐标表示各重组抗体、对照样品的浓度(nM)的log值，纵坐标表示平均荧光强度，R1054表示重组抗体#1，R1055表示重组抗体#3，R1056表示重组抗体#2，R0796表示重组抗体#4，iso(hIgG1.8)表示同型对照组，Noab表示阴性对照组；Figure 5 shows the blocking effect of recombinant antibodies in the embodiment of the present invention on the binding of TGFβRII and TGFβ on the cell surface. The abscissa represents the log value of the concentration (nM) of each recombinant antibody and control sample, and the ordinate represents the average. Fluorescence intensity, R1054 represents recombinant antibody #1, R1055 represents recombinant antibody #3, R1056 represents recombinant antibody #2, R0796 represents recombinant antibody #4, iso(hIgG1.8) represents isotype control group, Noab represents negative control group;
图6-A显示了本发明实施例中的重组抗体对T细胞增殖影响的结果图，其中，横坐标表示不同的处理组，纵坐标(Proliferation％)表示T细胞的增殖比例，R1054表示重组抗体#1，R1055表示重组抗体#3，R1056表示重组抗体#2，R0796表示重组抗体#4，iso(hIgG1.8)表示同型对照组，no activated表示未激活，activated表示激活，no Ab表示阴性对照组；Figure 6-A shows the results of the effect of recombinant antibodies on T cell proliferation in the embodiment of the present invention. The abscissa represents different treatment groups, the ordinate (Proliferation%) represents the proliferation ratio of T cells, and R1054 represents the recombinant antibody. #1, R1055 represents recombinant antibody #3, R1056 represents recombinant antibody #2, R0796 represents recombinant antibody #4, iso(hIgG1.8) represents isotype control group, no activated represents inactive, activated represents activated, no Ab represents negative control Group;
图6-B显示了本发明实施例中的重组抗体对T细胞增殖影响的结果图，其中，R1054表示重组抗体#1，R1055表示重组抗体#3，R1056表示重组抗体#2，R0796表示重组抗体#4，no activated表示未激活，TGFβ3ng/mL表示TGFβ的浓度为3ng/mL，Ab 50nM表示重组抗体浓度为50nM，activated表示激活。Figure 6-B shows the results of the effect of recombinant antibodies on T cell proliferation in the embodiment of the present invention, in which R1054 represents recombinant antibody #1, R1055 represents recombinant antibody #3, R1056 represents recombinant antibody #2, and R0796 represents recombinant antibody #4, no activated means not activated, TGFβ3ng/mL means the concentration of TGFβ is 3ng/mL, Ab 50nM means the recombinant antibody concentration is 50nM, activated means activated.
具体实施方式Detailed ways
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。Embodiments of the invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are exemplary and are intended to explain the present invention and are not to be construed as limiting the present invention.
此外，术语“第一”、“第二”、“第三”、“第四”或“第五”等仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”、“第三”、“第四”、“第五”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。In addition, the terms "first", "second", "third", "fourth" or "fifth" etc. are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implying a designation. The number of technical features. Thus, features defined as "first", "second", "third", "fourth", and "fifth" may explicitly or implicitly include at least one of these features. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise expressly and specifically limited.
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。The endpoints of ranges and any values disclosed herein are not limited to the precise range or value, but these ranges or values are to be understood to include values approaching such ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges. These values The scope shall be deemed to be specifically disclosed herein.
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文中使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。In order to make the present invention easier to understand, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Abbreviations for amino acid residues are the standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.
常见的天然氨基酸包括例如，丙氨酸(Ala；A)、精氨酸(Arg；R)、天冬酰胺(Asn；N)、天冬氨酸(Asp；D)、半胱氨酸(Cys；C)；谷氨酸(Glu；E)、谷氨酰胺(Gln；Q)、甘氨酸(Gly；G)；组氨酸(His；H)、异亮氨酸(Ile；I)、亮氨酸(Leu；L)、赖氨酸(Lys；K)、甲硫氨酸(Met；M)、苯丙氨酸(Phe；F)、脯氨酸(Pro；P)、丝胺酸(Ser；S)、苏氨酸(Thr；T)、色氨酸(Trp；W)、酪氨酸(Tyr；Y)和缬氨酸(Val；V)。Common natural amino acids include, for example, alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys ;C); Glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine Acid (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser ; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).
本申请中，所述“抗体IgG1.8”为所述野生型IgG1抗体的突变体，相较于野生型IgG1抗体，具有L234A、L235A和K447A突变，其中，所述野生型IgG1抗体的氨基酸编号为根据EU编号系统编号，例如，所述“L234A”是指按EU编号系统，编号为第234位的亮氨酸被丙氨酸替代。In this application, the "antibody IgG1.8" is a mutant of the wild-type IgG1 antibody. Compared with the wild-type IgG1 antibody, it has L234A, L235A and K447A mutations, wherein the amino acid number of the wild-type IgG1 antibody is For numbering according to the EU numbering system, for example, the "L234A" means that according to the EU numbering system, the leucine numbered at position 234 is replaced by alanine.
术语“重组抗体”是由包含编码全长抗体或其部分的核酸的宿主细胞表达的蛋白，例如由编码抗体重链恒定区和TGFβRII片段的核酸的重组宿主细胞表达的蛋白。The term "recombinant antibody" is a protein expressed by a host cell containing nucleic acid encoding a full-length antibody or a portion thereof, for example, a protein expressed by a recombinant host cell containing nucleic acid encoding an antibody heavy chain constant region and a TGFβRII fragment.
重组抗体Recombinant antibodies
在一些实施方案中，本发明提出了一种重组抗体，所述重组抗体包括两条相同的肽链1，每条肽链1包括：1)第一TGFβRII片段；以及2)抗体重链恒定区；其中，所述抗体重链恒定区的C端与所述第一TGFβRII片段的N端相连。根据本发明一些具体实施例的重组抗体可以有效与TGFβ进行结合，阻断细胞表面的TGFβRII与TGFβ的结合，并显著 促进T细胞的增殖，具有较长的体内半衰期，可以有效治疗或预防肿瘤。In some embodiments, the invention provides a recombinant antibody comprising two identical peptide chains 1, each peptide chain 1 comprising: 1) a first TGFβRII fragment; and 2) an antibody heavy chain constant region ; Wherein, the C-terminus of the antibody heavy chain constant region is connected to the N-terminus of the first TGFβRII fragment. Recombinant antibodies according to some specific embodiments of the present invention can effectively bind to TGFβ, block the binding of TGFβRII and TGFβ on the cell surface, and significantly Promotes the proliferation of T cells, has a long half-life in the body, and can effectively treat or prevent tumors.
根据本发明的一些具体实施方案，上述重组抗体还可以进一步包括如下附加技术特征的至少之一：According to some specific embodiments of the present invention, the above-mentioned recombinant antibody may further include at least one of the following additional technical features:
根据本发明的一些具体实施方案，所述第一TGFβRII片段包括野生型TGFβRII胞外结构域。According to some specific embodiments of the invention, the first TGFβRII fragment includes a wild-type TGFβRII extracellular domain.
根据本发明的一些具体实施方案，所述第一TGFβRII片段包括野生型TGFβRII胞外结构域的N-末端片段、第一连接肽和野生型TGFβRII胞外结构域的C-末端片段，其中，所述野生型TGFβRII胞外结构域的C-末端片段包括野生型TGFβRII胞外结构域C-末端的至多122个氨基酸。根据本发明的一些具体实施方案，所述野生型TGFβRII胞外结构域的N-末端片段的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述野生型TGFβRII胞外结构域的C-末端片段的N端相连。According to some specific embodiments of the present invention, the first TGFβRII fragment includes an N-terminal fragment of wild-type TGFβRII extracellular domain, a first connecting peptide and a C-terminal fragment of wild-type TGFβRII extracellular domain, wherein, The C-terminal fragment of the wild-type TGFβRII extracellular domain includes at most 122 amino acids of the C-terminus of the wild-type TGFβRII extracellular domain. According to some specific embodiments of the invention, the C-terminus of the N-terminal fragment of the wild-type TGFβRII extracellular domain is connected to the N-terminus of the first linking peptide, and the C-terminus of the first linking peptide is connected to the N-terminus of the first linking peptide. The C-terminal fragment of wild-type TGFβRII extracellular domain is linked to the N-terminus.
根据本发明的一些具体实施方案，所述第一TGFβRII片段包括野生型TGFβRII胞外结构域的N-末端片段、第一连接肽和野生型TGFβRII胞外结构域的C-末端片段，其中，所述C-末端片段包括TGFβRII胞外结构域的C-末端的至多122个氨基酸(例如112、113、114、115、116、117、118、119、120、121或122个TGFβRII胞外结构域的C-末端的氨基酸残基，或例如包括TGFβRII胞外结构域的C-末端的112～117个氨基酸(例如112、113、114、115、116或117个TGFβRII胞外结构域的C-末端的氨基酸残基))；所述第一连接肽的N端与所述TGFβRII胞外结构域的N-末端片段的C端相连，所述第一连接肽的C端与所述TGFβRII胞外结构域的C-末端片段的N端相连。According to some specific embodiments of the present invention, the first TGFβRII fragment includes an N-terminal fragment of wild-type TGFβRII extracellular domain, a first connecting peptide and a C-terminal fragment of wild-type TGFβRII extracellular domain, wherein, The C-terminal fragment includes up to 122 amino acids of the C-terminus of the TGFβRII extracellular domain (e.g., 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122 amino acids of the TGFβRII extracellular domain). C-terminal amino acid residues, or, for example, including the C-terminal 112 to 117 amino acids of the TGFβRII extracellular domain (e.g., 112, 113, 114, 115, 116, or 117 amino acids of the C-terminal of the TGFβRII extracellular domain) amino acid residue)); the N-terminus of the first connecting peptide is connected to the C-terminal of the N-terminal fragment of the TGFβRII extracellular domain, and the C-terminal of the first connecting peptide is connected to the TGFβRII extracellular domain The C-terminal fragment is connected to the N-terminus.
根据本发明的一些具体实施方案，所述第一TGFβRII片段包括野生型TGFβRII胞外结构域的N-末端片段、第一连接肽和野生型TGFβRII胞外结构域的C-末端片段，其中，所述TGFβRII胞外结构域的C-末端片段为TGFβRII胞外结构域的N-端截短连续14-23个(例如14、15、16、17、18、19、20、21、22或23个)氨基酸残基的多肽，优选截短连续16-23个氨基酸残基的多肽。According to some specific embodiments of the present invention, the first TGFβRII fragment includes an N-terminal fragment of wild-type TGFβRII extracellular domain, a first connecting peptide and a C-terminal fragment of wild-type TGFβRII extracellular domain, wherein, The C-terminal fragment of the TGFβRII extracellular domain is 14-23 consecutive N-terminal truncations of the TGFβRII extracellular domain (for example, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 ) polypeptides of amino acid residues, preferably truncated polypeptides of 16-23 consecutive amino acid residues.
根据本发明的一些具体实施方案，所述第一TGFβRII片段包括野生型TGFβRII胞外结构域的N-末端片段、第一连接肽和野生型TGFβRII胞外结构域的C-末端片段，其中，野生型TGFβRII胞外结构域的N-末端片段包括TGFβRII胞外结构域的N末端的1～6个氨基酸；在一些实施方案中，所述TGFβRII突变体片段包括TGFβRII胞外结构域的N末端的连续1个、2个、3个、4个、5个或6个氨基酸。According to some specific embodiments of the present invention, the first TGFβRII fragment includes an N-terminal fragment of wild-type TGFβRII extracellular domain, a first connecting peptide and a C-terminal fragment of wild-type TGFβRII extracellular domain, wherein, wild-type TGFβRII extracellular domain The N-terminal fragment of the TGFβRII extracellular domain includes 1 to 6 amino acids of the N-terminus of the TGFβRII extracellular domain; in some embodiments, the TGFβRII mutant fragment includes the contiguous N-terminal portion of the TGFβRII extracellular domain. 1, 2, 3, 4, 5 or 6 amino acids.
根据本发明的一些具体实施方案，所述第一TGFβRII片段包括野生型TGFβRII胞外结构域的N-末端片段、第一连接肽和野生型TGFβRII胞外结构域的C-末端片段，其中，所述第一连接肽为(GS)XG多肽，其中，X优选为1至6的任意整数；或者，所述第一连接肽选自(GxS)y连接子，其中，x选自1-5的整数，y选自0-6的整数。According to some specific embodiments of the present invention, the first TGFβRII fragment includes an N-terminal fragment of wild-type TGFβRII extracellular domain, a first connecting peptide and a C-terminal fragment of wild-type TGFβRII extracellular domain, wherein, The first connecting peptide is a (GS) × G polypeptide, wherein is an integer, y is selected from an integer between 0 and 6.
根据本发明的一些具体实施方案，每条肽链1进一步包括：第二TGFβRII片段。According to some specific embodiments of the invention, each peptide chain 1 further includes: a second TGFβRII fragment.
根据本发明的一些具体实施方案，所述第二TGFβRII片段包括所述野生型TGFβRII胞外结构域的N-末端片段、第二连接肽和所述野生型TGFβRII胞外结构域的C-末端片段，其中，所述野生型TGFβRII胞外结构域的C-末端片段包括所述野生型TGFβRII胞外结构域C-末端的至多122个氨基酸。According to some specific embodiments of the invention, the second TGFβRII fragment includes an N-terminal fragment of the wild-type TGFβRII extracellular domain, a second connecting peptide and a C-terminal fragment of the wild-type TGFβRII extracellular domain. , wherein the C-terminal fragment of the wild-type TGFβRII extracellular domain includes at most 122 amino acids of the C-terminus of the wild-type TGFβRII extracellular domain.
根据本发明的一些具体实施方案，所述野生型TGFβRII胞外结构域的N-末端片段的C端与所述第二连接肽的N端相连，所述第二连接肽的C端与所述野生型TGFβRII胞外结构域的C-末端片段的N端相连。According to some specific embodiments of the invention, the C-terminus of the N-terminal fragment of the wild-type TGFβRII extracellular domain is connected to the N-terminus of the second linking peptide, and the C-terminus of the second linking peptide is connected to the N-terminus of the second linking peptide. The C-terminal fragment of wild-type TGFβRII extracellular domain is linked to the N-terminus.
根据本发明的一些具体实施方案，所述第二TGFβRII片段的C端与所述抗体重链恒定区的N端相连。According to some specific embodiments of the invention, the C-terminus of the second TGFβRII fragment is connected to the N-terminus of the antibody heavy chain constant region.
根据本发明的一些具体实施方案，所述第一TGFβRII片段包括野生型TGFβRII胞外结构域的N-末端片段、第二连接肽和野生型TGFβRII胞外结构域的C-末端片段，其中，所述C-末端片段包括TGFβRII胞外结构域的C-末端的至多122个氨基酸(例如112、113、114、115、116、117、118、119、120、121或122个TGFβRII胞外结构域的C-末端的氨基酸残基，或例如包括TGFβRII胞外结构域的C-末端的112～117个氨基酸(例如112、113、114、115、116或117个TGFβRII胞外结构域的C-末端的氨基酸残基))；所述第二连接肽的N端与所述TGFβRII胞外结构域的N-末端片段的C端相连，所述第二连接肽的C端与所述TGFβRII胞外结构域的C-末端片段的N端相连。According to some specific embodiments of the present invention, the first TGFβRII fragment includes an N-terminal fragment of wild-type TGFβRII extracellular domain, a second connecting peptide and a C-terminal fragment of wild-type TGFβRII extracellular domain, wherein, The C-terminal fragment includes up to 122 amino acids of the C-terminus of the TGFβRII extracellular domain (e.g., 112, 113, 114, 115, 116, 117, 118, 119, 120, 121 or 122 amino acids of the TGFβRII extracellular domain). C-terminal amino acid residues, or, for example, including the C-terminal 112 to 117 amino acids of the TGFβRII extracellular domain (e.g., 112, 113, 114, 115, 116, or 117 amino acids of the C-terminal of the TGFβRII extracellular domain) Amino acid residue)); the N-terminus of the second linking peptide is connected to the C-terminal of the N-terminal fragment of the TGFβRII extracellular domain, and the C-terminus of the second linking peptide is connected to the TGFβRII extracellular domain The C-terminal fragment is connected to the N-terminus.
根据本发明的一些具体实施方案，所述第一TGFβRII片段包括野生型TGFβRII胞外结构域的N-末端片段、第二连接肽和野生型TGFβRII胞外结构域的C-末端片段，其中，所述TGFβRII胞外结构域的C-末端片段为TGFβRII胞外结构域的N-端截短连续14-23个(例如14、15、16、17、18、19、20、21、22或23个)氨基酸残基的多肽，优选截短连续16-23个氨基酸残基的多肽。According to some specific embodiments of the present invention, the first TGFβRII fragment includes an N-terminal fragment of wild-type TGFβRII extracellular domain, a second connecting peptide and a C-terminal fragment of wild-type TGFβRII extracellular domain, wherein, The C-terminal fragment of the TGFβRII extracellular domain is 14-23 consecutive N-terminal truncations of the TGFβRII extracellular domain (for example, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 ) polypeptides of amino acid residues, preferably truncated polypeptides of 16-23 consecutive amino acid residues.
根据本发明的一些具体实施方案，所述第一TGFβRII片段包括野生型TGFβRII胞外结构域的N-末端片段、第二连接肽和野生型TGFβRII胞外结构域的C-末端片段，其中，野生型TGFβRII胞外结构域的N-末端片段包括TGFβRII胞外结构域的N末端的1～6个氨基酸；在一些实施方案中，所述TGFβRII突变体片段包括TGFβRII胞外结构域的N末端的连续1个、2个、3个、4个、5个或6个氨基酸。According to some specific embodiments of the present invention, the first TGFβRII fragment includes an N-terminal fragment of wild-type TGFβRII extracellular domain, a second connecting peptide and a C-terminal fragment of wild-type TGFβRII extracellular domain, wherein, wild-type TGFβRII extracellular domain The N-terminal fragment of the TGFβRII extracellular domain includes 1 to 6 amino acids of the N-terminus of the TGFβRII extracellular domain; in some embodiments, the TGFβRII mutant fragment includes the contiguous N-terminal portion of the TGFβRII extracellular domain. 1, 2, 3, 4, 5 or 6 amino acids.
根据本发明的一些具体实施方案，所述第一TGFβRII片段包括野生型TGFβRII胞外结构域的N-末端片段、第二连接肽和野生型TGFβRII胞外结构域的C-末端片段，其中，所述第二连接肽为(GS)XG多肽，其中，X优选为1至6的任意整数；或者，所述第二连接肽选自(GxS)y连接子，其中，x选自1-5的整数，y选自0-6的整数。According to some specific embodiments of the present invention, the first TGFβRII fragment includes an N-terminal fragment of wild-type TGFβRII extracellular domain, a second connecting peptide and a C-terminal fragment of wild-type TGFβRII extracellular domain, wherein, The second connecting peptide is a (GS) × G polypeptide, wherein X is preferably any integer from 1 to 6; or the second connecting peptide is selected from (GxS)y linker, wherein is an integer, y is selected from an integer between 0 and 6.
根据本发明的一些具体实施方案，进一步包括两条相同的肽链2，所述每条肽链2包括第三TGFβRII片段和抗体轻链恒定区。According to some specific embodiments of the invention, two identical peptide chains 2 are further included, each of which includes a third TGFβRII fragment and an antibody light chain constant region.
根据本发明的一些具体实施方案，所述第三TGFβRII片段的C端与所述抗体轻链恒定区的N端相连。According to some specific embodiments of the invention, the C-terminus of the third TGFβRII fragment is connected to the N-terminus of the antibody light chain constant region.
根据本发明的一些具体实施方案，所述每条肽链2与1条肽链1通过链间二硫键相连。According to some specific embodiments of the present invention, each peptide chain 2 is connected to one peptide chain 1 through an inter-chain disulfide bond.
根据本发明的一些具体实施方案，所述第三TGFβRII片段包括野生型TGFβRII胞外结构域。According to some specific embodiments of the invention, the third TGFβRII fragment includes the wild-type TGFβRII extracellular domain.
根据本发明的一些具体实施方案，所述第三TGFβRII片段包括所述野生型TGFβRII胞外结构域的N-末端片段、第三连接肽和所述野生型TGFβRII胞外结构域的C-末端片段，其中，所述野生型TGFβRII胞外结构域C-末端片段包括野生型TGFβRII胞外结构域C-末端的至多122个氨基酸。According to some specific embodiments of the invention, the third TGFβRII fragment includes an N-terminal fragment of the wild-type TGFβRII extracellular domain, a third connecting peptide and a C-terminal fragment of the wild-type TGFβRII extracellular domain. , wherein the C-terminal fragment of the wild-type TGFβRII extracellular domain includes at most 122 amino acids of the C-terminus of the wild-type TGFβRII extracellular domain.
根据本发明的一些具体实施方案，所述野生型TGFβRII胞外结构域的N-末端片段的C端与所述第三连接肽的N端相连，所述第三连接肽的C端与所述野生型TGFβRII胞外结构域的C-末端片段的N端相连。According to some specific embodiments of the invention, the C-terminus of the N-terminal fragment of the wild-type TGFβRII extracellular domain is connected to the N-terminus of the third linking peptide, and the C-terminus of the third linking peptide is connected to the N-terminus of the third linking peptide. The C-terminal fragment of wild-type TGFβRII extracellular domain is linked to the N-terminus.
根据本发明的一些具体实施方案，所述第一TGFβRII片段包括野生型TGFβRII胞外结构域的N-末端片段、第三连接肽和野生型TGFβRII胞外结构域的C-末端片段，其中，所述C-末端片段包括TGFβRII胞外结构域的C-末端的至 多122个氨基酸(例如112、113、114、115、116、117、118、119、120、121或122个TGFβRII胞外结构域的C-末端的氨基酸残基，或例如包括TGFβRII胞外结构域的C-末端的112～117个氨基酸(例如112、113、114、115、116或117个TGFβRII胞外结构域的C-末端的氨基酸残基))；所述第三连接肽的N端与所述TGFβRII胞外结构域的N-末端片段的C端相连，所述第三连接肽的C端与所述TGFβRII胞外结构域的C-末端片段的N端相连。According to some specific embodiments of the present invention, the first TGFβRII fragment includes an N-terminal fragment of wild-type TGFβRII extracellular domain, a third connecting peptide and a C-terminal fragment of wild-type TGFβRII extracellular domain, wherein, The C-terminal fragment includes the C-terminus of the TGFβRII extracellular domain to More than 122 amino acids (e.g., 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, or 122 amino acid residues at the C-terminus of the TGFβRII extracellular domain, or, for example, including the TGFβRII extracellular domain The C-terminal 112 to 117 amino acids (for example, the 112, 113, 114, 115, 116 or 117 C-terminal amino acid residues of the TGFβRII extracellular domain)); the N-terminal of the third connecting peptide and The C-terminal of the N-terminal fragment of the TGFβRII extracellular domain is connected, and the C-terminal of the third connecting peptide is connected to the N-terminal of the C-terminal fragment of the TGFβRII extracellular domain.
根据本发明的一些具体实施方案，所述第一TGFβRII片段包括野生型TGFβRII胞外结构域的N-末端片段、第三连接肽和野生型TGFβRII胞外结构域的C-末端片段，其中，所述TGFβRII胞外结构域的C-末端片段为TGFβRII胞外结构域的N-端截短连续14-23个(例如14、15、16、17、18、19、20、21、22或23个)氨基酸残基的多肽，优选截短连续16-23个氨基酸残基的多肽。According to some specific embodiments of the present invention, the first TGFβRII fragment includes an N-terminal fragment of wild-type TGFβRII extracellular domain, a third connecting peptide and a C-terminal fragment of wild-type TGFβRII extracellular domain, wherein, The C-terminal fragment of the TGFβRII extracellular domain is 14-23 consecutive N-terminal truncations of the TGFβRII extracellular domain (for example, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 ) polypeptides of amino acid residues, preferably truncated polypeptides of 16-23 consecutive amino acid residues.
根据本发明的一些具体实施方案，所述第一TGFβRII片段包括野生型TGFβRII胞外结构域的N-末端片段、第三连接肽和野生型TGFβRII胞外结构域的C-末端片段，其中，野生型TGFβRII胞外结构域的N-末端片段包括TGFβRII胞外结构域的N末端的1～6个氨基酸；在一些实施方案中，所述TGFβRII突变体片段包括TGFβRII胞外结构域的N末端的连续1个、2个、3个、4个、5个或6个氨基酸。According to some specific embodiments of the present invention, the first TGFβRII fragment includes an N-terminal fragment of wild-type TGFβRII extracellular domain, a third connecting peptide and a C-terminal fragment of wild-type TGFβRII extracellular domain, wherein, wild-type TGFβRII extracellular domain The N-terminal fragment of the TGFβRII extracellular domain includes 1 to 6 amino acids of the N-terminus of the TGFβRII extracellular domain; in some embodiments, the TGFβRII mutant fragment includes the contiguous N-terminal portion of the TGFβRII extracellular domain. 1, 2, 3, 4, 5 or 6 amino acids.
根据本发明的一些具体实施方案，所述第一TGFβRII片段包括野生型TGFβRII胞外结构域的N-末端片段、第三连接肽和野生型TGFβRII胞外结构域的C-末端片段，其中，所述第三连接肽为(GS)XG多肽，其中，X优选为1至6的任意整数；或者，所述第三连接肽选自(GxS)y连接子，其中，x选自1-5的整数，y选自0-6的整数。According to some specific embodiments of the present invention, the first TGFβRII fragment includes an N-terminal fragment of wild-type TGFβRII extracellular domain, a third connecting peptide and a C-terminal fragment of wild-type TGFβRII extracellular domain, wherein, The third connecting peptide is a (GS) × G polypeptide, wherein X is preferably any integer from 1 to 6; or the third connecting peptide is selected from (GxS)y linker, wherein is an integer, y is selected from an integer between 0 and 6.
根据本发明的一些具体实施方案，所述野生型TGFβRII胞外结构域的N-末端片段包括野生型TGFβRII胞外结构域的N-末端前6位氨基酸。According to some specific embodiments of the present invention, the N-terminal fragment of the wild-type TGFβRII extracellular domain includes the first 6 amino acids of the N-terminus of the wild-type TGFβRII extracellular domain.
根据本发明的一些具体实施方案，所述野生型TGFβRII胞外结构域的C-末端片段包括野生型TGFβRII胞外结构域C-末端的112～117个氨基酸。According to some specific embodiments of the present invention, the C-terminal fragment of the wild-type TGFβRII extracellular domain includes 112 to 117 amino acids of the C-terminus of the wild-type TGFβRII extracellular domain.
根据本发明的一些具体实施方案，所述野生型TGFβRII胞外结构域包括SEQ ID NO:2所示的氨基酸序列。
According to some specific embodiments of the present invention, the wild-type TGFβRII extracellular domain includes the amino acid sequence shown in SEQ ID NO:2.
根据本发明的一些具体实施方案，所述第一连接肽和/或所述第二连接肽和/或第三连接肽为柔性片段。According to some specific embodiments of the present invention, the first connecting peptide and/or the second connecting peptide and/or the third connecting peptide are flexible fragments.
根据本发明的一些具体实施方案，所述第一连接肽和/或所述第二连接肽和/或第三连接肽为含有G、S氨基酸的短肽。According to some specific embodiments of the present invention, the first connecting peptide and/or the second connecting peptide and/or the third connecting peptide are short peptides containing G and S amino acids.
根据本发明的一些具体实施方案，所述第一连接肽和/或所述第二连接肽和/或第三连接肽含有5～15个氨基酸。According to some specific embodiments of the present invention, the first connecting peptide and/or the second connecting peptide and/or the third connecting peptide contain 5 to 15 amino acids.
根据本发明的一些具体实施方案，所述第一连接肽和/或所述第二连接肽和/或第三连接肽包括SEQ ID NO:3所示的氨基酸序列。
According to some specific embodiments of the present invention, the first connecting peptide and/or the second connecting peptide and/or the third connecting peptide include the amino acid sequence shown in SEQ ID NO:3.
根据本发明的一些具体实施方案，所述第一TGFβRII片段、所述第二TGFβRII片段和所述第三TGFβRII片段包括SEQ ID NO:2、4、28或29所示的氨基酸序列。
According to some specific embodiments of the present invention, the first TGFβRII fragment, the second TGFβRII fragment and the third TGFβRII fragment include the amino acid sequence shown in SEQ ID NO: 2, 4, 28 or 29.
根据本发明的一些具体实施方案，进一步包括第四连接肽。According to some specific embodiments of the invention, a fourth connecting peptide is further included.
根据本发明的一些具体实施方案，所述第四连接肽的N端与所述抗体重链恒定区的C端相连，所述第四连接肽的C端与所述第一TGFβRII片段的N端相连。According to some specific embodiments of the present invention, the N-terminus of the fourth linking peptide is connected to the C-terminus of the antibody heavy chain constant region, and the C-terminus of the fourth linking peptide is connected to the N-terminus of the first TGFβRII fragment. connected.
根据本发明的一些具体实施方案，所述第四连接肽为柔性片段。According to some specific embodiments of the invention, the fourth connecting peptide is a flexible fragment.
根据本发明的一些具体实施方案，所述第四连接肽为含有G、S氨基酸的短肽。According to some specific embodiments of the present invention, the fourth connecting peptide is a short peptide containing G and S amino acids.
根据本发明的一些具体实施方案，所述第四连接肽包括SEQ ID NO:5所示的氨基酸序列。
According to some specific embodiments of the present invention, the fourth connecting peptide includes the amino acid sequence shown in SEQ ID NO:5.
根据本发明的一些具体实施方案，进一步包括第五连接肽。According to some specific embodiments of the invention, a fifth linking peptide is further included.
根据本发明的一些具体实施方案，所述第五连接肽的N端与所述第二TGFβRII片段的C端相连，所述第五连接肽的C端与所述抗体重链恒定区的N端相连。According to some specific embodiments of the present invention, the N-terminus of the fifth linking peptide is connected to the C-terminus of the second TGFβRII fragment, and the C-terminus of the fifth linking peptide is connected to the N-terminus of the antibody heavy chain constant region. connected.
根据本发明的一些具体实施方案，进一步包括第六连接肽，所述第六连接肽的N端与所述第三TGFβRII片段的C端相连，所述第六连接肽的C端与所述抗体轻链恒定区的N端相连。According to some specific embodiments of the present invention, it further includes a sixth connecting peptide, the N-terminus of the sixth connecting peptide is connected to the C-terminal of the third TGFβRII fragment, and the C-terminal of the sixth connecting peptide is connected to the C-terminal of the antibody The N-terminus of the light chain constant region is linked.
根据本发明的一些具体实施方案，所述第五连接肽和/或第六连接肽为柔性片段。According to some specific embodiments of the present invention, the fifth connecting peptide and/or the sixth connecting peptide is a flexible fragment.
根据本发明的一些具体实施方案，所述第五连接肽和/或第六连接肽为含有G、S氨基酸的短肽。According to some specific embodiments of the present invention, the fifth connecting peptide and/or the sixth connecting peptide is a short peptide containing G and S amino acids.
根据本发明的一些具体实施方案，所述第五连接肽和/或第六连接肽包括SEQ ID NO:5所示的氨基酸序列。According to some specific embodiments of the present invention, the fifth connecting peptide and/or the sixth connecting peptide includes the amino acid sequence shown in SEQ ID NO:5.
根据本发明的一些具体实施方案，所述抗体重链恒定区或所述抗体轻链恒定区的至少一部分来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。According to some specific embodiments of the present invention, at least a part of the antibody heavy chain constant region or the antibody light chain constant region is from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.
根据本发明的一些具体实施方案，所述抗体重链恒定区或所述抗体轻链恒定区的至少一部分来自于人源抗体或其突变体。According to some specific embodiments of the invention, at least a portion of the antibody heavy chain constant region or the antibody light chain constant region is derived from a human antibody or a mutant thereof.
根据本发明的一些具体实施方案，所述抗体重链恒定区或所述抗体轻链恒定区的至少一部分来自于人源抗体IgG或其突变体。According to some specific embodiments of the invention, at least a portion of the antibody heavy chain constant region or the antibody light chain constant region is derived from human antibody IgG or a mutant thereof.
根据本发明的一些具体实施方案，所述抗体重链恒定区或所述抗体轻链恒定区的至少一部分来自于人源抗体IgG1或其突变体。According to some specific embodiments of the invention, at least a portion of the antibody heavy chain constant region or the antibody light chain constant region is derived from human antibody IgG1 or a mutant thereof.
根据本发明的一些具体实施方案，所述抗体重链恒定区相较于野生型人源IgG1具有第234、235和447位氨基酸突变。 According to some specific embodiments of the invention, the antibody heavy chain constant region has amino acid mutations at positions 234, 235 and 447 compared to wild-type human IgG1.
根据本发明的一些具体实施方案，所述抗体重链恒定区相较于野生型人源IgG1具有L234A、L235A和K447A突变。According to some specific embodiments of the invention, the antibody heavy chain constant region has L234A, L235A and K447A mutations compared to wild-type human IgG1.
根据本发明的一些具体实施方案，所述抗体重链恒定区包括SEQ ID NO:7所示的氨基酸序列。
According to some specific embodiments of the invention, the antibody heavy chain constant region includes the amino acid sequence shown in SEQ ID NO:7.
根据本发明的一些具体实施方案，所述抗体轻链恒定区包括SEQ ID NO:8所示的氨基酸序列。
According to some specific embodiments of the invention, the antibody light chain constant region includes the amino acid sequence shown in SEQ ID NO:8.
根据本发明的一些具体实施方案，所述肽链1包括SEQ ID NO:21、34、35或58所示氨基酸序列。
According to some specific embodiments of the present invention, the peptide chain 1 includes the amino acid sequence shown in SEQ ID NO: 21, 34, 35 or 58.
根据本发明的一些具体实施方案，所述肽链2包括SEQ ID NO:23、38、39或59所示氨基酸序列。

According to some specific embodiments of the present invention, the peptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 23, 38, 39 or 59.

根据本发明的一些具体实施方案，所述重组抗体包括：1)SEQ ID NO:21和SEQ ID NO:23所示的氨基酸序列；或2)SEQ ID NO:34和SEQ ID NO:38所示的氨基酸序列；或3)SEQ ID NO:35和SEQ ID NO:39所示的氨基酸序列；或4)SEQ ID NO:58和SEQ ID NO:59所示的氨基酸序列。According to some specific embodiments of the present invention, the recombinant antibody includes: 1) the amino acid sequence shown in SEQ ID NO:21 and SEQ ID NO:23; or 2) the amino acid sequence shown in SEQ ID NO:34 and SEQ ID NO:38 The amino acid sequence; or 3) the amino acid sequence shown in SEQ ID NO:35 and SEQ ID NO:39; or 4) the amino acid sequence shown in SEQ ID NO:58 and SEQ ID NO:59.
在一些实施方案中，本发明又提出了一种重组抗体，包括两条相同的肽链3，每条肽链3包括：1)第四TGFβRII片段；2)抗体Fc区；以及3)第五TGFβRII片段或肿瘤靶向肽；其中，所述抗体Fc区的C端与所述第四TGFβRII片段的N端相连，所述抗体Fc区的N端与所述第五TGFβRII片段或肿瘤靶向肽的C端相连。根据本发明一些具体实施例的重组抗体可以有效与TGFβ进行结合，阻断细胞表面的TGFβRII与TGFβ的结合，并显著促进T细胞的增殖，具有较长的体内半衰期，可以有效治疗或预防肿瘤。In some embodiments, the present invention also provides a recombinant antibody, including two identical peptide chains 3, each peptide chain 3 including: 1) the fourth TGFβRII fragment; 2) the antibody Fc region; and 3) the fifth TGFβRII fragment or tumor-targeting peptide; wherein, the C-terminus of the antibody Fc region is connected to the N-terminus of the fourth TGFβRII fragment, and the N-terminus of the antibody Fc region is connected to the fifth TGFβRII fragment or tumor-targeting peptide. The C terminal is connected. Recombinant antibodies according to some specific embodiments of the present invention can effectively bind to TGFβ, block the combination of TGFβRII and TGFβ on the cell surface, and significantly promote the proliferation of T cells. They have a long half-life in vivo and can effectively treat or prevent tumors.
根据本发明的一些具体实施方案，上述重组抗体还可以进一步包括如下附加技术特征的至少之一：According to some specific embodiments of the present invention, the above-mentioned recombinant antibody may further include at least one of the following additional technical features:
根据本发明的一些具体实施方案，所述第四TGFβRII片段和/或所述第五TGFβRII片段包括野生型TGFβRII胞外结构域。According to some specific embodiments of the invention, said fourth TGFβRII fragment and/or said fifth TGFβRII fragment comprises a wild-type TGFβRII extracellular domain.
根据本发明的一些具体实施方案，所述第四TGFβRII片段包括野生型TGFβRII胞外结构域的N-末端片段、第七连接肽和野生型TGFβRII胞外结构域的C-末端片段，其中，所述野生型TGFβRII胞外结构域的C-末端片段包括野生型TGFβRII胞外结构域C-末端的至多122个氨基酸。According to some specific embodiments of the present invention, the fourth TGFβRII fragment includes an N-terminal fragment of wild-type TGFβRII extracellular domain, a seventh connecting peptide and a C-terminal fragment of wild-type TGFβRII extracellular domain, wherein, The C-terminal fragment of the wild-type TGFβRII extracellular domain includes at most 122 amino acids of the C-terminus of the wild-type TGFβRII extracellular domain.
根据本发明的一些具体实施方案，所述第五TGFβRII片段包括野生型TGFβRII胞外结构域的N-末端片段、第八连接肽和野生型TGFβRII胞外结构域的C-末端片段，其中，所述野生型TGFβRII胞外结构域的C-末端片段包括野生型TGFβRII胞外结构域C-末端的至多122个氨基酸。According to some specific embodiments of the present invention, the fifth TGFβRII fragment includes an N-terminal fragment of wild-type TGFβRII extracellular domain, an eighth connecting peptide and a C-terminal fragment of wild-type TGFβRII extracellular domain, wherein, the The C-terminal fragment of the wild-type TGFβRII extracellular domain includes at most 122 amino acids of the C-terminus of the wild-type TGFβRII extracellular domain.
根据本发明的一些具体实施方案，所述第四TGFβRII片段中，所述野生型TGFβRII胞外结构域的N-末端片段的C端与所述第七连接肽的N端相连，所述第七连接肽的C端与所述野生型TGFβRII胞外结构域的C-末端片段的N端相连。According to some specific embodiments of the present invention, in the fourth TGFβRII fragment, the C-terminus of the N-terminal fragment of the wild-type TGFβRII extracellular domain is connected to the N-terminus of the seventh connecting peptide, and the seventh The C-terminus of the linker peptide is connected to the N-terminus of the C-terminal fragment of the wild-type TGFβRII extracellular domain.
根据本发明的一些具体实施方案，所述第五TGFβRII片段中，所述野生型TGFβRII胞外结构域的N-末端片段的C端与所述第八连接肽的N端相连，所述第八连接肽的C端与所述野生型TGFβRII胞外结构域的C-末端片段的N端相连。According to some specific embodiments of the present invention, in the fifth TGFβRII fragment, the C-terminus of the N-terminal fragment of the wild-type TGFβRII extracellular domain is connected to the N-terminus of the eighth connecting peptide, and the eighth The C-terminus of the linker peptide is connected to the N-terminus of the C-terminal fragment of the wild-type TGFβRII extracellular domain.
根据本发明的一些具体实施方案，所述第七连接肽和/或所述第八连接肽为柔性片段。According to some specific embodiments of the present invention, the seventh connecting peptide and/or the eighth connecting peptide is a flexible fragment.
根据本发明的一些具体实施方案，所述第七连接肽和/或所述第八连接肽为含有G、S氨基酸的短肽。According to some specific embodiments of the present invention, the seventh connecting peptide and/or the eighth connecting peptide is a short peptide containing G and S amino acids.
根据本发明的一些具体实施方案，所述第七连接肽和/或所述第八连接肽含有5～15个氨基酸。According to some specific embodiments of the present invention, the seventh connecting peptide and/or the eighth connecting peptide contains 5 to 15 amino acids.
根据本发明的一些具体实施方案，所述第七连接肽和/或所述第八连接肽包括SEQ ID NO:3所示的氨基酸序列。According to some specific embodiments of the present invention, the seventh connecting peptide and/or the eighth connecting peptide includes the amino acid sequence shown in SEQ ID NO:3.
根据本发明的一些具体实施方案，所述重组抗体进一步包括第九连接肽。According to some specific embodiments of the invention, the recombinant antibody further includes a ninth linking peptide.
根据本发明的一些具体实施方案，所述第四TGFβRII片段的N端与所述第九连接肽的C端相连，所述第九连接肽的N端与所述抗体Fc区的C端相连。According to some specific embodiments of the invention, the N-terminus of the fourth TGFβRII fragment is connected to the C-terminus of the ninth linking peptide, and the N-terminus of the ninth linking peptide is connected to the C-terminus of the antibody Fc region.
根据本发明的一些具体实施方案，所述重组抗体进一步包括第十连接肽。According to some specific embodiments of the invention, the recombinant antibody further includes a tenth linking peptide.
根据本发明的一些具体实施方案，所述第五TGFβRII片段的C端与所述第十连接肽的N端相连，所述第十连接肽的C端与所述抗体Fc区的N端相连。According to some specific embodiments of the invention, the C-terminus of the fifth TGFβRII fragment is connected to the N-terminus of the tenth linking peptide, and the C-terminus of the tenth linking peptide is connected to the N-terminus of the antibody Fc region.
根据本发明的一些具体实施方案，所述第九连接肽和/或第十连接肽为柔性片段。According to some specific embodiments of the present invention, the ninth connecting peptide and/or the tenth connecting peptide is a flexible fragment.
根据本发明的一些具体实施方案，所述第九连接肽和/或第十连接肽为含有G、S氨基酸的短肽。According to some specific embodiments of the present invention, the ninth connecting peptide and/or the tenth connecting peptide is a short peptide containing G and S amino acids.
根据本发明的一些具体实施方案，所述第九连接肽和/或第十连接肽包括SEQ ID NO:5所示的氨基酸序列。According to some specific embodiments of the present invention, the ninth connecting peptide and/or the tenth connecting peptide includes the amino acid sequence shown in SEQ ID NO:5.
根据本发明的一些具体实施方案，所述野生型TGFβRII胞外结构域包括SEQ ID NO:2所示的氨基酸序列。According to some specific embodiments of the invention, the wild-type TGFβRII extracellular domain includes the amino acid sequence shown in SEQ ID NO:2.
根据本发明的一些具体实施方案，所述第四TGFβRII片段和第五TGFβRII片段包括SEQ ID NO:2、4、28或29所示的氨基酸序列。According to some specific embodiments of the present invention, the fourth TGFβRII fragment and the fifth TGFβRII fragment include the amino acid sequence shown in SEQ ID NO: 2, 4, 28 or 29.
根据本发明的一些具体实施方案，所述肿瘤靶向肽包括SEQ ID NO:9或SEQ ID NO:26所示的氨基酸序列。
According to some specific embodiments of the present invention, the tumor targeting peptide includes the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 26.
根据本发明的一些具体实施方案，所述每条肽链3通过链间二硫键相连。According to some specific embodiments of the present invention, each of the peptide chains 3 is connected by an inter-chain disulfide bond.
根据本发明的一些具体实施方案，所述抗体Fc区的至少一部分来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一。According to some specific embodiments of the present invention, at least part of the Fc region of the antibody is derived from at least one of a murine antibody, a human antibody, a primate antibody or a mutant thereof.
根据本发明的一些具体实施方案，所述抗体Fc区的至少一部分来自于人源抗体或其突变体。According to some specific embodiments of the invention, at least part of the Fc region of the antibody is derived from a human antibody or a mutant thereof.
根据本发明的一些具体实施方案，所述抗体Fc区的至少一部分来自于人源抗体IgG或其突变体。 According to some specific embodiments of the invention, at least a portion of the antibody Fc region is derived from human antibody IgG or a mutant thereof.
根据本发明的一些具体实施方案，所述抗体Fc区的至少一部分来自于人源抗体IgG1或其突变体。According to some specific embodiments of the invention, at least part of the antibody Fc region is derived from human antibody IgG1 or a mutant thereof.
根据本发明的一些具体实施方案，所述抗体Fc区相较于野生型人源IgG1具有第234、235和447位氨基酸突变。According to some specific embodiments of the invention, the antibody Fc region has amino acid mutations at positions 234, 235 and 447 compared to wild-type human IgG1.
根据本发明的一些具体实施方案，所述抗体Fc区相较于野生型人源IgG1具有L234A、L235A和K447A突变。According to some specific embodiments of the invention, the antibody Fc region has L234A, L235A and K447A mutations compared to wild-type human IgG1.
根据本发明的一些具体实施方案，所述抗体Fc区包括SEQ ID NO:6所示的氨基酸序列。
According to some specific embodiments of the invention, the antibody Fc region includes the amino acid sequence shown in SEQ ID NO: 6.
根据本发明的一些具体实施方案，所述肽链3包括SEQ ID NO:17、19、30、31、32、33、54或55所示的氨基酸序列。

According to some specific embodiments of the present invention, the peptide chain 3 includes the amino acid sequence shown in SEQ ID NO: 17, 19, 30, 31, 32, 33, 54 or 55.

核酸分子、表达载体、重组细胞Nucleic acid molecules, expression vectors, recombinant cells
在一些实施方案中，本发明提出了一种核酸分子，所述核酸分子编码前面所述的重组抗体。根据本发明实施例核酸分子获得的重组抗体可以有效与TGFβ进行结合，阻断细胞表面的TGFβRII与TGFβ的结合，并显著促进T细胞的增殖，具有较长的体内半衰期，可以有效治疗或预防肿瘤。In some embodiments, the present invention provides a nucleic acid molecule encoding a recombinant antibody as described above. The recombinant antibodies obtained according to the nucleic acid molecules of the embodiments of the present invention can effectively bind to TGFβ, block the combination of TGFβRII and TGFβ on the cell surface, significantly promote the proliferation of T cells, have a long half-life in vivo, and can effectively treat or prevent tumors. .
根据本发明的一些具体实施方案，上述核酸分子还可以进一步包括如下附加技术特征至少之一：According to some specific embodiments of the present invention, the above-mentioned nucleic acid molecule may further include at least one of the following additional technical features:
根据本发明的一些具体实施方案，所述核酸分子包括SEQ ID NO:20、44、45或52所示的核苷酸序列。


According to some specific embodiments of the invention, the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO: 20, 44, 45 or 52.


根据本发明的一些具体实施方案，所述核酸分子包括SEQ ID NO:22、48、49或53所示的核苷酸序列。

According to some specific embodiments of the invention, the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO: 22, 48, 49 or 53.

根据本发明的一些具体实施方案，所述核酸分子包括SEQ ID NO:16、18、40、41、42、43、56或57所示的核苷酸序列。



According to some specific embodiments of the invention, the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO: 16, 18, 40, 41, 42, 43, 56 or 57.



需要说明的是，对于本发明说明书和权利要求书中所提及的核酸，本领域技术人员应当理解，实际包括互补双链的任意一条，或者两条。为了方便，在本说明书和权利要求书中，虽然多数情况下只给出了一条链，但实际上也公开了与之互补的另一条链。另外，本申请中的核酸序列包括DNA形式或RNA形式，公开其中一种，意味着另一种也被公开。It should be noted that those skilled in the art will understand that the nucleic acids mentioned in the description and claims of the present invention actually include either or both complementary double strands. For convenience, in this description and claims, although in most cases only one strand is given, another strand complementary to it is actually disclosed. In addition, the nucleic acid sequence in this application includes DNA form or RNA form, and disclosing one of them means that the other one is also disclosed.
在一些实施方案中，本发明提出了一种表达载体，所述表达载体携带前面所述的核酸分子。在将上述核酸分子连接 到载体上时，可以将所述核酸分子与载体上的控制元件直接或者间接相连，只要这些控制元件能够控制所述核酸分子的翻译和表达等即可。当然这些控制元件可以直接来自于载体本身，也可以是外源性的，即并非来自于载体本身。当然，所述核酸与控制元件进行可操作地连接即可。本文中“可操作地连接”是指将外源基因连接到载体上，使得载体内的控制元件，例如转录控制序列和翻译控制序列等等，能够发挥其预期的调节外源基因的转录和翻译的功能。当然用来编码重组抗体的两条相同的肽链1和/或两条相同的肽链2的所述核酸分子，可以分别独立的插入到不同的载体上，常见的是插入到同一载体上。常用的载体例如可以为质粒、噬菌体等等。根据本发明的一些具体实施方案的表达载体导入合适的受体细胞后，可在调控系统的介导下，有效实现前面所述的重组抗体的表达，进而实现所述重组抗体的体外大量获得。In some embodiments, the present invention provides an expression vector carrying the nucleic acid molecule described above. After connecting the above nucleic acid molecules When placed on a vector, the nucleic acid molecule can be directly or indirectly connected to the control elements on the vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can come directly from the carrier itself, or they can be exogenous, that is, they do not come from the carrier itself. Of course, it suffices that the nucleic acid is operably connected to the control element. In this article, "operably connected" refers to connecting an exogenous gene to a vector so that the control elements in the vector, such as transcription control sequences and translation control sequences, can exert their intended effects on regulating the transcription and translation of the exogenous gene. function. Of course, the nucleic acid molecules used to encode the two identical peptide chains 1 and/or the two identical peptide chains 2 of the recombinant antibody can be independently inserted into different vectors. Commonly, they are inserted into the same vector. Commonly used vectors can be, for example, plasmids, phages, etc. After the expression vector according to some specific embodiments of the present invention is introduced into a suitable recipient cell, the expression of the aforementioned recombinant antibody can be effectively realized under the mediation of the regulatory system, thereby achieving large-scale acquisition of the recombinant antibody in vitro.
根据本发明的一些具体实施方案，上述表达载体还可以进一步包括如下附加技术特征至少之一：According to some specific embodiments of the present invention, the above-mentioned expression vector may further include at least one of the following additional technical features:
根据本发明的一些具体实施方案，所述表达载体为真核表达载体或病毒。进而实现前面所述的重组抗体在合适受体细胞中的表达，如CHO细胞。According to some specific embodiments of the invention, the expression vector is a eukaryotic expression vector or a virus. Then, the aforementioned recombinant antibody can be expressed in appropriate recipient cells, such as CHO cells.
根据本发明的一些具体实施方案，所述病毒包括慢病毒。According to some specific embodiments of the invention, the virus includes a lentivirus.
在一些实施方案中，本发明提出了一种重组细胞，所述重组细胞携带前面所述的核酸分子、表达载体，或者表达前面所述的重组抗体。根据本发明一些具体实施方案的重组细胞可用于前面所述的能够识别TGFβ的重组抗体的体外表达和大量获得。In some embodiments, the present invention provides a recombinant cell that carries the aforementioned nucleic acid molecule, expression vector, or expresses the aforementioned recombinant antibody. Recombinant cells according to some specific embodiments of the present invention can be used for the in vitro expression and large-scale acquisition of recombinant antibodies capable of recognizing TGFβ as described above.
根据本发明的一些具体实施方案，上述重组细胞还可以进一步包括如下附加技术特征至少之一：According to some specific embodiments of the present invention, the above-mentioned recombinant cells may further include at least one of the following additional technical features:
根据本发明的一些具体实施方案，所述重组细胞为真核细胞。According to some specific embodiments of the invention, the recombinant cells are eukaryotic cells.
根据本发明的一些具体实施方案，所述重组细胞为哺乳动物细胞。According to some specific embodiments of the invention, the recombinant cells are mammalian cells.
根据本发明的一些具体实施方案，所述重组细胞不包括动物生殖细胞、受精卵或胚胎干细胞。According to some specific embodiments of the invention, the recombinant cells do not include animal germ cells, fertilized eggs or embryonic stem cells.
需要注意的是，本发明所述重组细胞不受特别限制，可以为原核细胞、真核细胞或噬菌体。所述原核细胞可以为大肠杆菌、枯草杆菌、链霉菌或奇异变形菌等。所述真核细胞可以为包括巴斯德毕赤酵母、酿酒酵母、裂殖酵母、木霉等真菌，草地粘虫等昆虫细胞，烟草等植物细胞，BHK细胞、CHO细胞、COS细胞、骨髓瘤细胞等哺乳动物细胞。在一些实施例中，本发明所述重组细胞优选为哺乳动物细胞，包括BHK细胞、CHO细胞、NSO细胞或COS细胞，且不包括动物生殖细胞、受精卵或胚胎干细胞。It should be noted that the recombinant cells of the present invention are not particularly limited and can be prokaryotic cells, eukaryotic cells or phages. The prokaryotic cell can be Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis, etc. The eukaryotic cells may include fungi such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Trichoderma, insect cells such as Fall Armyworm, plant cells such as tobacco, BHK cells, CHO cells, COS cells, and myeloma cells. cells and other mammalian cells. In some embodiments, the recombinant cells of the present invention are preferably mammalian cells, including BHK cells, CHO cells, NSO cells or COS cells, and do not include animal germ cells, fertilized eggs or embryonic stem cells.
需要说明的是，本申请说明书中所述的“适合条件”，是指适合本申请所述重组抗体表达的条件。本领域技术人员容易理解的是，适合重组抗体表达的条件包括但不限于合适的转化或转染方式、合适的转化或转条件、健康的宿主细胞状态、合适的宿主细胞密度、适宜的细胞培养环境、适宜的细胞培养时间。“适合条件”不受特别限制，本领域技术人员可根据实验室的具体环境，优化最适的所述重组抗体表达的条件。It should be noted that the "suitable conditions" mentioned in the specification of this application refer to conditions suitable for the expression of the recombinant antibodies described in this application. Those skilled in the art can easily understand that conditions suitable for the expression of recombinant antibodies include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, and suitable cell culture. environment and appropriate cell culture time. "Suitable conditions" are not particularly limited. Those skilled in the art can optimize the most suitable conditions for the expression of the recombinant antibody according to the specific environment of the laboratory.
组合物、制药用途、药物、试剂盒Compositions, pharmaceutical uses, drugs, kits
在一些实施方式中，本发明提出了一种组合物，包括前面所述的重组抗体、核酸分子、表达载体或重组细胞。如前所述，根据本发明一些具体实施方案的重组抗体，或所述核酸分子、表达载体或重组细胞在合适条件下表达获得的重组抗体均可以有效与TGFβ进行结合，阻断细胞表面的TGFβRII与TGFβ的结合，并显著促进T细胞的增殖，具有较长的体内半衰期，可以有效治疗或预防肿瘤，进一步地，所述组合物中所包含的重组抗体或表达的抗体均能够有效与TGFβ进行结合，阻断细胞表面的TGFβRII与TGFβ的结合，并显著促进T细胞的增殖，具有较长的体内半衰期，可以有效治疗或预防肿瘤。In some embodiments, the present invention provides a composition including the aforementioned recombinant antibody, nucleic acid molecule, expression vector or recombinant cell. As mentioned above, recombinant antibodies according to some specific embodiments of the present invention, or recombinant antibodies obtained by expressing the nucleic acid molecules, expression vectors or recombinant cells under appropriate conditions, can effectively bind to TGFβ and block TGFβRII on the cell surface. It binds to TGFβ and significantly promotes the proliferation of T cells. It has a long half-life in vivo and can effectively treat or prevent tumors. Furthermore, the recombinant antibodies or expressed antibodies contained in the composition can effectively interact with TGFβ. Binding blocks the binding of TGFβRII to TGFβ on the cell surface, and significantly promotes the proliferation of T cells. It has a long half-life in vivo and can effectively treat or prevent tumors.
需要注意的是，所述组合物为食品组合物、药物组合物等，包括在时间和/或空间上分开的组合，只要其能够共同作用以实现本发明的目的。例如，所述组合物中所含的成分可以以整体施用于受试者，或者分开施用于受试者。当所述组合物中所含的成分分开地施用于受试者时，各个成分可以同时或依次施用于受试者。It should be noted that the compositions are food compositions, pharmaceutical compositions, etc., including combinations that are separated in time and/or space, as long as they can function together to achieve the purpose of the present invention. For example, the ingredients contained in the composition can be administered to the subject as a whole or separately. When the ingredients contained in the composition are administered to the subject separately, the individual ingredients may be administered to the subject simultaneously or sequentially.
在一些实施方式中，本发明提出了前面所述的重组抗体、核酸分子、表达载体、重组细胞或组合物在制备药物中的用途，所述药物用于预防或治疗肿瘤。如前所述，根据本发明一些具体实施方案的重组抗体，或所述核酸分子、表达载体或重组细胞在合适条件下表达获得的重组抗体，以及组合物中包含的重组抗体均可以有效与TGFβ进行结合，阻断细胞表面的TGFβRII与TGFβ的结合，并显著促进T细胞的增殖，具有较长的体内半衰期，可以有效治疗或预防肿瘤，进一步地，利用上述物质制备的药物同样能够阻断细胞表面的TGFβRII与TGFβ的结合，并显著促进T细胞的增殖，具有较长的体内半衰期，可以有效治疗或预防肿瘤。In some embodiments, the present invention provides the use of the aforementioned recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells or compositions in the preparation of medicines for preventing or treating tumors. As mentioned above, recombinant antibodies according to some specific embodiments of the present invention, or recombinant antibodies obtained by expressing the nucleic acid molecules, expression vectors or recombinant cells under appropriate conditions, and recombinant antibodies included in the composition can effectively interact with TGFβ Bind, block the combination of TGFβRII and TGFβ on the cell surface, and significantly promote the proliferation of T cells. It has a long half-life in the body and can effectively treat or prevent tumors. Furthermore, drugs prepared using the above substances can also block cells. The surface TGFβRII binds to TGFβ and significantly promotes the proliferation of T cells. It has a long half-life in vivo and can effectively treat or prevent tumors.
在一些实施方案中，本发明提出了一种药物，包括：前面所述的重组抗体、核酸分子、表达载体、重组细胞或组合物。如前所述，根据本发明一些具体实施方案的重组抗体，或所述核酸分子、表达载体或重组细胞在合适条件下表达获得的重组抗体，以及组合物中包含的重组抗体均可以有效与TGFβ进行结合，特异性强，不仅可以中和TGFβ，而且可以发挥较好的靶向作用，进而实现药物中其他药物的生物学作用，如TGFβ分子活性抑制，表达TGFβ分子的细胞杀伤等作用。In some embodiments, the present invention provides a medicine, including: the aforementioned recombinant antibody, nucleic acid molecule, expression vector, recombinant cell or composition. As mentioned above, recombinant antibodies according to some specific embodiments of the present invention, or recombinant antibodies obtained by expressing the nucleic acid molecules, expression vectors or recombinant cells under appropriate conditions, and recombinant antibodies included in the composition can effectively interact with TGFβ The combination has strong specificity and can not only neutralize TGFβ, but also exert a better targeting effect, thereby realizing the biological effects of other drugs in the drug, such as inhibiting the activity of TGFβ molecules and killing cells expressing TGFβ molecules.
在一些实施方案中，这些药物进一步包括药学上可接受的载体，包括任何溶剂、固体赋形剂、稀释剂、粘合剂、崩解剂或其他液体赋形剂、分散剂、矫味剂或悬浮剂、表面活性剂、等渗剂、增稠剂、乳化剂、防腐剂、固体粘合剂、助流剂或润滑剂，等等，适合于特有的目标剂型。除了任何常规的辅料与本发明的化合物不相容的范围，例如所产生的任何不良的生物效应或与药学上可接受的组合物的任何其他组分以有害的方式产生的相互作用，它们的用途也是本发明所 考虑的范围。In some embodiments, these drugs further include a pharmaceutically acceptable carrier, including any solvent, solid excipient, diluent, binder, disintegrant or other liquid excipient, dispersant, flavoring or Suspending agents, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, solid binders, glidants or lubricants, etc., are suitable for the unique target dosage form. Except to the extent that any conventional excipients are incompatible with the compounds of the invention, such as by producing any adverse biological effects or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their The purpose is also the invention scope of consideration.
例如，本发明的重组抗体可掺入适用于胃肠外施用(例如静脉内、皮下、腹膜内、肌肉内)的药物组合物中。这些药物组合物可以被制备成各种形式。例如液体、半固体和固体剂型等，包括但不限于液体溶液(例如，注射溶液和输注溶液)、分散剂或悬浮剂、片剂、丸剂、粉末、脂质体和栓剂。典型的药物组合物为注射溶液或输注溶液形式。所述重组抗体可通过静脉输注或注射或肌肉内或皮下注射来施用。For example, the recombinant antibodies of the invention may be incorporated into pharmaceutical compositions suitable for parenteral administration (eg, intravenous, subcutaneous, intraperitoneal, intramuscular). These pharmaceutical compositions can be prepared in various forms. Examples include liquid, semi-solid, and solid dosage forms, including, but not limited to, liquid solutions (eg, injection solutions and infusion solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. Typical pharmaceutical compositions are in the form of injection or infusion solutions. The recombinant antibodies may be administered by intravenous infusion or injection or intramuscular or subcutaneous injection.
此外，根据本发明实施例的药物可以发挥诊断作用，依赖于本发明前面所提出的能够特异性的靶向结合TGFβ的抗体，与诊断试剂进行组合，进而发挥对生物体异常表达TGFβ的部位进行诊断，如与诊断性核素、纳米材料等进行组合，实现对生物体异常表达TGFβ的细胞、组织、器官进行可视化观察，进而辅助医疗工作者或科研工作者对病灶进行更准确的判断。In addition, drugs according to embodiments of the present invention can play a diagnostic role by relying on the antibodies that can specifically target and bind TGFβ as proposed above in combination with diagnostic reagents, and then play a role in detecting parts of the organism where TGFβ is abnormally expressed. Diagnosis, such as combination with diagnostic nuclides, nanomaterials, etc., enables visual observation of cells, tissues, and organs that abnormally express TGFβ in organisms, thereby assisting medical workers or scientific researchers in making more accurate judgments on lesions.
本文使用的术语“治疗”和“预防”以及源自于此的词不必暗示100％或完全治疗或预防。相反，存在不同程度的治疗或预防，本领域普通技术人员认为所述治疗或预防具有潜在的益处或治疗效果。在这方面，本发明方法可提供任何量的任何水平的治疗或预防哺乳动物的肿瘤。而且，本发明方法提供的治疗或预防可包括正在治疗或预防的疾病，如肿瘤的一种或多种病患或症状的治疗或预防。另外，为了本文的目的，“预防”可涵盖延缓疾病或其症状或病患的发作。The terms "treatment" and "prevention" as used herein and words derived therefrom do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention that one of ordinary skill in the art would consider potentially beneficial or therapeutically effective. In this regard, the methods of the present invention may provide any amount and any level of treatment or prevention of tumors in a mammal. Furthermore, treatment or prevention provided by the methods of the present invention may include treatment or prevention of one or more conditions or symptoms of the disease being treated or prevented, such as neoplasia. Additionally, for the purposes herein, "prevention" may encompass delaying the onset of a disease or its symptoms or in a patient.
当然，本文中的重组抗体还可以根据需要被制成试剂盒或者其他诊断性试剂的一部分。Of course, the recombinant antibodies herein can also be made into part of a kit or other diagnostic reagents as needed.
在一些实施方案中，本发明提出了前面所述的重组抗体在制备试剂盒中的用途，所述试剂盒用于检测TGFβ。所述重组抗体可以与任何检测试剂或治疗制剂联用，例如与诊断性核素、纳米材料等联合使用，通过核素的放射性对靶标部位进行探测，进而获取靶标部位的信息；也可以与治疗性核素联用，利用核素的放射性，特异性杀伤靶标细胞、组织等。In some embodiments, the present invention provides the use of the previously described recombinant antibody in the preparation of a kit for detecting TGFβ. The recombinant antibody can be used in combination with any detection reagent or therapeutic preparation, such as with diagnostic nuclide, nanomaterials, etc., to detect the target site through the radioactivity of the nuclide, and thereby obtain information about the target site; it can also be used with treatment The combined use of radionuclides uses the radioactivity of nuclides to specifically kill target cells, tissues, etc.
根据本发明的一些具体实施方案，上述用途还可以进一步包括如下附加技术特征至少之一：According to some specific embodiments of the present invention, the above-mentioned uses may further include at least one of the following additional technical features:
根据本发明的一些具体实施方案，所述TGFβ包括TGFβ1和TGFβ3。本领域技术人员可以理解，所述重组抗体可以与任何一种TGFβ进行有效结合，并不仅限于TGFβ1和TGFβ3。According to some specific embodiments of the invention, the TGFβ includes TGFβ1 and TGFβ3. Those skilled in the art can understand that the recombinant antibody can effectively bind to any TGFβ, and is not limited to TGFβ1 and TGFβ3.
在一些实施方案，本发明还提供了一种试剂盒，所述试剂盒包括上述重组抗体。应用本发明提供的试剂盒，例如可以用于免疫印迹、免疫沉淀等涉及到利用TGFβ抗原和抗体特异性结合性能，来检测的试剂盒等。这些试剂盒可包含下列中的任意一种或多种：拮抗剂、TGFβ抗体或者药物参照材料；蛋白纯化柱；免疫球蛋白亲和纯化缓冲剂；细胞的测定稀释剂；说明书或者文献等。所述重组抗体可被用于不同类型的诊断测试，例如可以在体外或者体内检测各种各样的疾病或者药物、毒素或者其他蛋白等的存在。例如可以通过对受试者的血清或者血液进行检测，用来测试相关疾病，如：肺癌、肝癌、卵巢癌、宫颈癌、皮肤癌、膀胱癌、结肠癌、乳腺癌、神经胶质瘤、肾癌、胃癌、食道癌、口腔鳞状细胞癌和头颈癌，这些肿瘤细胞可以是任何生长不受调控的细胞。In some embodiments, the present invention also provides a kit, which includes the above-mentioned recombinant antibody. The kit provided by the present invention can be used, for example, in Western blotting, immunoprecipitation, and other kits that utilize the specific binding properties of TGFβ antigen and antibody for detection. These kits may contain any one or more of the following: antagonists, TGFβ antibodies or drug reference materials; protein purification columns; immunoglobulin affinity purification buffers; cell assay diluents; instructions or literature, etc. The recombinant antibodies can be used in different types of diagnostic tests, for example, to detect a variety of diseases or the presence of drugs, toxins or other proteins in vitro or in vivo. For example, the subject's serum or blood can be tested to detect related diseases, such as: lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, bladder cancer, colon cancer, breast cancer, glioma, kidney cancer, etc. Cancer, stomach cancer, esophageal cancer, oral squamous cell carcinoma and head and neck cancer, these tumor cells can be any cells whose growth is unregulated.
诊断疾病、疾病分期、预后评估的方法Methods for diagnosing disease, staging disease, and assessing prognosis
在一些实施方案中，本发明提出了一种诊断TGFβ引起的相关疾病的方法，采用前面所述的重组抗体、核酸分子、表达载体、重组细胞、组合物、试剂盒对待测样品中的TGFβ进行检测；基于检测结果，确定待测样品中TGFβ的含量。如前所述，肿瘤微环境中TGFβ增加与免疫逃逸相关，TGFβ水平升高会阻止幼稚T细胞向Th1效应子表型分化，促进其向Treg亚型的转化，并抑制树突状细胞的抗原呈递功能，TGFβ是一种有效的免疫抑制剂，TGFβ表达的增加通常与许多癌症的恶性肿瘤，以及对TGFβ的细胞生长抑制反应的缺陷有关，然后其免疫抑制功能占主导地位，促成肿瘤发生，而本申请提出的所述重组抗体，或核酸分子、表达载体、重组细胞表达的重组抗体，或组合物、试剂盒中包含的重组抗体均可以有效与TGFβ进行结合，因此，采用本申请所述的方法可以有效检测来源于受试个体的待测样品中TGFβ含量，并可以对TGFβ引起的相关疾病进行有效诊断。In some embodiments, the present invention proposes a method for diagnosing related diseases caused by TGFβ, using the previously described recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, and kits to detect TGFβ in the test sample. Detection; based on the detection results, determine the content of TGFβ in the sample to be tested. As mentioned before, increased TGFβ in the tumor microenvironment is associated with immune escape. Increased TGFβ levels prevent naive T cells from differentiating toward a Th1 effector phenotype, promote their transformation into Treg subtypes, and inhibit dendritic cell antigens. Presenting function, TGFβ is a potent immunosuppressant. Increased expression of TGFβ is often associated with malignancy in many cancers, as well as defects in the cell growth inhibitory response to TGFβ, and then its immunosuppressive function dominates, contributing to tumorigenesis. The recombinant antibodies proposed in this application, or the recombinant antibodies expressed by nucleic acid molecules, expression vectors, recombinant cells, or the recombinant antibodies included in the compositions and kits can effectively bind to TGFβ. Therefore, using the recombinant antibodies described in this application The method can effectively detect the TGFβ content in test samples derived from test individuals, and can effectively diagnose related diseases caused by TGFβ.
根据本发明的一些具体实施方案，上述方法还可以进一步包括如下附加技术特征至少之一：According to some specific embodiments of the present invention, the above method may further include at least one of the following additional technical features:
根据本发明的一些具体实施方案，所述待测样品中TGFβ的含量不低于患病的最低标准是待测样品来源于患有TGFβ引起的相关疾病的患者的指示。According to some specific embodiments of the present invention, the content of TGFβ in the sample to be tested is not lower than the minimum standard for disease, which is an indication that the sample to be tested is derived from patients suffering from related diseases caused by TGFβ.
根据本发明的一些具体实施方案，所述TGFβ包括TGFβ1和TGFβ3。本领域技术人员可以理解，所述重组抗体可以与任何一种TGFβ进行有效结合，并不仅限于TGFβ1和TGFβ3。According to some specific embodiments of the invention, the TGFβ includes TGFβ1 and TGFβ3. Those skilled in the art can understand that the recombinant antibody can effectively bind to any TGFβ, and is not limited to TGFβ1 and TGFβ3.
根据本发明的一些具体实施方案，所述待测样品包括：血液、组织、细胞、粪便或尿液。According to some specific embodiments of the present invention, the sample to be tested includes: blood, tissue, cells, feces or urine.
根据本发明的一些具体实施方案，所述TGFβ引起的相关疾病包括肿瘤。According to some specific embodiments of the invention, the related diseases caused by TGFβ include tumors.
在一些实施方案中，本发明提出了一种评估TGFβ引起的相关疾病分期的方法，采用前面所述的重组抗体、核酸分子、表达载体、重组细胞、组合物、试剂盒对待测样品中的TGFβ进行检测；基于检测结果，确定待测样品中TGFβ的含量。如前所述，肿瘤微环境中TGFβ增加与免疫逃逸相关，TGFβ水平升高会阻止幼稚T细胞向Th1效应子表型分化，促进其向Treg亚型的转化，并抑制树突状细胞的抗原呈递功能，TGFβ是一种有效的免疫抑制剂，TGFβ表达的增加通常与许多恶性肿瘤，以及对TGFβ的细胞生长抑制反应的缺陷有关，然后其免疫抑制功能占主导地位，促成肿瘤发生，处于疾病发生的不同时期，外周血内TGFβ的含量会发生变化，在本申请提出的所述重组抗体，或核酸分子、表达载体、重组细胞表达的重组抗体，或组合物、试剂盒中包含的重组抗体均可以有效与TGFβ进行结合的基础上，采用本申请所述的方法可以有效检测来源于受试个体的待测样品中TGFβ含量，并基于所述TGFβ含量对TGFβ引起的相关疾病所处的时期进行评估。In some embodiments, the present invention proposes a method for assessing the staging of related diseases caused by TGFβ, using the previously described recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, and kits to detect TGFβ in the test sample. Carry out detection; based on the detection results, determine the content of TGFβ in the sample to be tested. As mentioned before, increased TGFβ in the tumor microenvironment is associated with immune escape. Increased TGFβ levels prevent naive T cells from differentiating toward a Th1 effector phenotype, promote their transformation into Treg subtypes, and inhibit dendritic cell antigens. Presenting function, TGFβ is a potent immunosuppressant. Increased expression of TGFβ is often associated with many malignant tumors, as well as defects in the cell growth inhibitory response to TGFβ, and then its immunosuppressive function dominates, contributing to tumorigenesis and disease. At different stages of occurrence, the content of TGFβ in peripheral blood will change. The recombinant antibodies proposed in this application, or the recombinant antibodies expressed by nucleic acid molecules, expression vectors, recombinant cells, or the recombinant antibodies included in the compositions and kits On the basis that all can effectively combine with TGFβ, the method described in this application can be used to effectively detect the TGFβ content in the test sample derived from the subject individual, and based on the TGFβ content, the period of related diseases caused by TGFβ can be estimated. to evaluate.
根据本发明的一些具体实施方案，上述方法还可以进一步包括如下附加技术特征至少之一：According to some specific embodiments of the present invention, the above method may further include at least one of the following additional technical features:
根据本发明的一些具体实施方案，所述TGFβ引起的相关疾病包括肿瘤，所述待测样品中TGFβ的含量不低于肿瘤IV期患病的最低水平是待测样品来源于患有肿瘤IV期的患者的指示，所述待测样品中TGFβ的含量位于肿瘤IV期和III 期患病的水平之间是待测样品来源于患有肿瘤III期的患者的指示；所述待测样品中TGFβ的含量处于肿瘤III期和II期患病的标准水平之间是待测样品来源于患有肿瘤II期的患者的指示；所述待测样品中TGFβ的含量处于I期和II期患病的标准水平之间是待测样品来源于患有肿瘤I期的患者的指示。本领域技术人员可以理解，所述的肿瘤I期、II期、III期、IV期患病时TGFβ的水平根据肿瘤种类的不同而变化，判断肿瘤所述时期，只要将所述待测样品中TGFβ的含量与该肿瘤阶段TGFβ的标准水平进行对比即可知晓，或将所述待测样品中TGFβ的含量与已知患病个体或群体来源的样品TGFβ的含量进行对比。According to some specific embodiments of the present invention, the related diseases caused by TGFβ include tumors, and the content of TGFβ in the sample to be tested is not lower than the minimum level for stage IV tumor disease, and the sample to be tested is derived from patients with stage IV tumors. The patient's indication is that the content of TGFβ in the sample to be tested is located in tumor stages IV and III. The level between the stage III disease is an indication that the sample to be tested is derived from a patient with stage III tumor; the content of TGFβ in the sample to be tested is between the standard levels for stage III and stage II tumor disease, which is the sample to be tested. An indication that the sample to be tested is derived from a patient with stage II tumor; the content of TGFβ in the sample to be tested is between the standard levels for stage I and stage II disease is an indication that the sample to be tested is derived from a patient with stage I tumor. Those skilled in the art can understand that the level of TGFβ changes according to the type of tumor when the tumor is in stages I, II, III, and IV. To judge the stage of the tumor, as long as the sample to be tested is The content of TGFβ can be known by comparing it with the standard level of TGFβ at the tumor stage, or by comparing the content of TGFβ in the sample to be tested with the content of TGFβ in samples derived from known diseased individuals or groups.
根据本发明的一些具体实施方案，所述TGFβ包括TGFβ1和TGFβ3。本领域技术人员可以理解，所述重组抗体可以与任何一种TGFβ进行有效结合，并不仅限于TGFβ1和TGFβ3。According to some specific embodiments of the invention, the TGFβ includes TGFβ1 and TGFβ3. Those skilled in the art can understand that the recombinant antibody can effectively bind to any TGFβ, and is not limited to TGFβ1 and TGFβ3.
根据本发明的一些具体实施方案，所述待测样品包括：血液、组织、细胞、粪便或尿液。According to some specific embodiments of the present invention, the sample to be tested includes: blood, tissue, cells, feces or urine.
在一些实施方案中，本发明提出了一种评估TGFβ引起的相关疾病的预后方法，采用前面所述的重组抗体、核酸分子、表达载体、重组细胞、组合物、试剂盒对待测样品中的TGFβ进行检测；基于检测结果，确定待测样品中TGFβ的含量。如前所述，TGFβ是一种有效的免疫抑制剂，TGFβ表达的增加通常与许多恶性肿瘤，以及对TGFβ的细胞生长抑制反应的缺陷有关，然后其免疫抑制功能占主导地位，促成肿瘤发生，TGFβ引起的相关疾病进行治疗后，通过监测外周血内TGFβ的含量可以有效评估该类疾病的预后，例如，将治疗前后的受试者体内TGFβ的含量进行比较，或将治疗后的受试者体内TGFβ的含量与正常水平或患病水平进行比较等，在本申请提出的所述重组抗体，或核酸分子、表达载体、重组细胞表达的重组抗体，或组合物、试剂盒中包含的重组抗体均可以有效与TGFβ进行结合的基础上，采用本申请所述的方法可以有效检测来源于受试个体的待测样品中TGFβ含量，并基于所述TGFβ含量对TGFβ引起的相关疾病的预后进行评估。In some embodiments, the present invention proposes a method for assessing the prognosis of related diseases caused by TGFβ, using the previously described recombinant antibodies, nucleic acid molecules, expression vectors, recombinant cells, compositions, and kits to detect TGFβ in the test sample. Carry out detection; based on the detection results, determine the content of TGFβ in the sample to be tested. As mentioned before, TGFβ is a potent immunosuppressant, and increased expression of TGFβ is often associated with many malignant tumors, as well as defects in the cell growth inhibitory response to TGFβ, and then its immunosuppressive function dominates, promoting tumorigenesis. After treatment of related diseases caused by TGFβ, the prognosis of such diseases can be effectively evaluated by monitoring the levels of TGFβ in peripheral blood. For example, comparing the levels of TGFβ in subjects before and after treatment, or comparing subjects after treatment. The content of TGFβ in the body is compared with the normal level or disease level, etc. The recombinant antibody proposed in this application, or the recombinant antibody expressed by the nucleic acid molecule, expression vector, recombinant cell, or the recombinant antibody contained in the composition or kit On the basis that all can effectively combine with TGFβ, the method described in this application can be used to effectively detect the TGFβ content in the test sample derived from the subject individual, and evaluate the prognosis of related diseases caused by TGFβ based on the TGFβ content. .
根据本发明的一些具体实施方案，上述方法还可以进一步包括如下附加技术特征至少之一：According to some specific embodiments of the present invention, the above method may further include at least one of the following additional technical features:
根据本发明的一些具体实施方案，所述待测样品来源于治疗前或治疗后的患有TGFβ引起的相关疾病的患者。According to some specific embodiments of the present invention, the sample to be tested is derived from patients suffering from related diseases caused by TGFβ before or after treatment.
根据本发明的一些具体实施方案，基于治疗前或治疗后的患有TGFβ引起的相关疾病的患者的待测样品中的TGFβ的含量，确定TGFβ引起的相关疾病的预后效果。According to some specific embodiments of the present invention, the prognostic effect of the related diseases caused by TGFβ is determined based on the content of TGFβ in the test sample of the patient suffering from the related diseases caused by TGFβ before or after treatment.
根据本发明的一些具体实施方案，治疗后的患有TGFβ引起的相关疾病的患者的待测样品中的TGFβ的含量下降是患者预后良好的指示。According to some specific embodiments of the present invention, a decrease in the content of TGFβ in the test sample of a patient suffering from a related disease caused by TGFβ after treatment is an indicator of a good prognosis of the patient.
根据本发明的一些具体实施方案，所述TGFβ包括TGFβ1和TGFβ3。According to some specific embodiments of the invention, the TGFβ includes TGFβ1 and TGFβ3.
根据本发明的一些具体实施方案，所述待测样品包括：血液、组织、细胞、粪便或尿液。According to some specific embodiments of the present invention, the sample to be tested includes: blood, tissue, cells, feces or urine.
根据本发明的一些具体实施方案，所述TGFβ引起的相关疾病包括肿瘤。According to some specific embodiments of the invention, the related diseases caused by TGFβ include tumors.
下面参考具体实施例，对本发明进行描述，需要说明的是，这些实施例仅仅是描述性的，而不以任何方式限制本发明。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are only illustrative and do not limit the present invention in any way. If no specific techniques or conditions are specified in the examples, the techniques or conditions described in literature in the field shall be followed (for example, refer to "Molecular Cloning Experimental Guide" translated by J. Sambrook et al., Huang Peitang et al., third edition, Science Press) or follow the product instructions. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.
实施例1重组抗体Fc-hTGFβRII Trap的构建Example 1 Construction of recombinant antibody Fc-hTGFβRII Trap
本实施例将野生型TGFβRII胞外结构域的N-末端截短14-21个(14个、19个、21个)氨基酸，获得野生型TGFβRII胞外结构域的C-末端片段，然后插入不超过15个氨基酸，其中，插入的氨基酸中包括截短的14-21个氨基酸中的部分氨基酸(野生型TGFβRII胞外结构域的N-末端片段)以及柔性片段(第一连接肽、第二连接肽、第三连接肽、第七连接肽、第八连接肽)，所述柔性片段将保留的野生型TGFβRII胞外结构域的N末端片段和野生型TGFβRII胞外结构域的C-末端片段进行连接，获得突变后的TGFβRII胞外结构域-TGFβRII Trap(TGFβRII Trap-1、TGFβRII Trap-2、TGFβRII Trap-3)。采用连接肽将免疫调节分子TGFβRII Trap、肿瘤靶向肽和野生型TGFβRII胞外结构域，与IgG1抗体不同恒定区片段(Fc区突变体、重链恒定区HC、轻链恒定区LC)进行不同方式的连接，构建多种重组抗体，其中，所述野生型TGFβRII的氨基酸序列如SEQ ID NO:1所示，所述野生型TGFβRII的胞外结构域的氨基酸序列如SEQ ID NO:2所示，所述第一连接肽、第二连接肽、第三连接肽、第七连接肽、第八连接肽为GSGSGSGSG(SEQ ID NO:3)，所述TGFβRII Trap的氨基酸序列如SEQ ID NO:4、28、29所示，所述连接肽(第四连接肽、第五连接肽、第六连接肽、第九连接肽、第十连接肽)的氨基酸序列如SEQ ID NO:5所示，所述Fc区突变体为野生型人源IgG1抗体的Fc突变体，包括2种，分别为Fc区突变体1和Fc区突变体2(抗体IgG1.8的Fc区)，其中，Fc区突变体1是将野生型IgG1抗体的Fc区C末端赖氨酸残基(K)被突变成丙氨酸(A)获得的，Fc区突变体2(抗体IgG1.8的Fc区)是在Fc区突变体1的基础上发生L234A和L235A突变获得的，所述Fc区突变体1的氨基酸序列如SEQ ID NO:50所示，所述Fc区突变体2的氨基酸序列如SEQ ID NO:6所示，所述抗体重链恒定区的氨基酸序列如SEQ ID NO:7所示，所述抗体轻链恒定区的氨基酸序列如SEQ ID NO:8所示，所述肿瘤靶向肽的氨基酸序列如SEQ ID NO:9或SEQ ID NO:26所示。In this example, the N-terminus of the wild-type TGFβRII extracellular domain is truncated by 14-21 (14, 19, 21) amino acids to obtain the C-terminal fragment of the wild-type TGFβRII extracellular domain, and then inserted into More than 15 amino acids, in which the inserted amino acids include some of the truncated 14-21 amino acids (N-terminal fragment of wild-type TGFβRII extracellular domain) and flexible fragments (first connecting peptide, second connecting peptide peptide, the third connecting peptide, the seventh connecting peptide, the eighth connecting peptide), the flexible fragment combines the retained N-terminal fragment of the wild-type TGFβRII extracellular domain and the C-terminal fragment of the wild-type TGFβRII extracellular domain. Connect to obtain the mutated TGFβRII extracellular domain-TGFβRII Trap (TGFβRII Trap-1, TGFβRII Trap-2, TGFβRII Trap-3). The immunomodulatory molecule TGFβRII Trap, tumor targeting peptide and wild-type TGFβRII extracellular domain are separated from different constant region fragments of IgG1 antibodies (Fc region mutants, heavy chain constant region HC, light chain constant region LC) using connecting peptides. By connecting in a manner, a variety of recombinant antibodies are constructed, wherein the amino acid sequence of the wild-type TGFβRII is shown in SEQ ID NO: 1, and the amino acid sequence of the extracellular domain of the wild-type TGFβRII is shown in SEQ ID NO: 2 , the first connecting peptide, the second connecting peptide, the third connecting peptide, the seventh connecting peptide, and the eighth connecting peptide are GGSGSGSG (SEQ ID NO: 3), and the amino acid sequence of the TGFβRII Trap is as SEQ ID NO: 4 , 28 and 29, the amino acid sequence of the connecting peptide (the fourth connecting peptide, the fifth connecting peptide, the sixth connecting peptide, the ninth connecting peptide, the tenth connecting peptide) is shown in SEQ ID NO: 5, so The Fc region mutants are Fc mutants of wild-type human IgG1 antibodies, including two types, namely Fc region mutant 1 and Fc region mutant 2 (Fc region of antibody IgG1.8), wherein the Fc region mutant 1 is obtained by mutating the C-terminal lysine residue (K) of the Fc region of the wild-type IgG1 antibody to alanine (A). Fc region mutant 2 (the Fc region of the antibody IgG1.8) is obtained by It is obtained by L234A and L235A mutations on the basis of Fc region mutant 1. The amino acid sequence of the Fc region mutant 1 is as shown in SEQ ID NO:50, and the amino acid sequence of the Fc region mutant 2 is as SEQ ID NO:6. As shown, the amino acid sequence of the antibody heavy chain constant region is shown in SEQ ID NO:7, the amino acid sequence of the antibody light chain constant region is shown in SEQ ID NO:8, and the amino acid sequence of the tumor-targeting peptide As shown in SEQ ID NO:9 or SEQ ID NO:26.
具体的实验操作如下：The specific experimental operations are as follows:
合成编码上述野生型TGFβRII胞外结构域(ECD)的核苷酸序列(SEQ ID NO:60)、突变后的TGFβRII胞外结构域(TGFβRII Trap-1、TGFβRII Trap-2、TGFβRII Trap-3)的核苷酸序列(SEQ ID NO:10、61、62)、编码上述连接肽(第四连接肽、第五连接肽、第六连接肽、第九连接肽、第十连接肽)的核苷酸序列(SEQ ID NO:11)、编码上述Fc区突变 体1的核苷酸序列(SEQ ID NO:51)、编码上述Fc区突变体2的核苷酸序列(SEQ ID NO:12)、编码上述抗体重链恒定区的核苷酸序列(SEQ ID NO:13)、编码所述抗体轻链恒定区的核苷酸序列(SEQ ID NO:14)，编码上述肿瘤靶向肽的核苷酸序列(SEQ ID NO:15或SEQ ID NO:27)。将获得的所述抗体不同恒定区分别与突变后的TGFβRII胞外结构域和/或肿瘤靶向肽和/或野生型TGFβRII胞外结构域的N端或C端连接，形成Fc-hTGFβRII Trap重组抗体或Fc-ECD重组抗体。Synthesize the nucleotide sequence encoding the wild-type TGFβRII extracellular domain (ECD) (SEQ ID NO: 60) and the mutated TGFβRII extracellular domain (TGFβRII Trap-1, TGFβRII Trap-2, TGFβRII Trap-3) The nucleotide sequence (SEQ ID NO: 10, 61, 62), the nucleoside encoding the above-mentioned connecting peptide (the fourth connecting peptide, the fifth connecting peptide, the sixth connecting peptide, the ninth connecting peptide, and the tenth connecting peptide) Acid sequence (SEQ ID NO:11), encoding the above Fc region mutation The nucleotide sequence of entity 1 (SEQ ID NO:51), the nucleotide sequence encoding the above-mentioned Fc region mutant 2 (SEQ ID NO:12), and the nucleotide sequence encoding the above-mentioned antibody heavy chain constant region (SEQ ID NO: 13), the nucleotide sequence encoding the antibody light chain constant region (SEQ ID NO: 14), the nucleotide sequence encoding the above-mentioned tumor targeting peptide (SEQ ID NO: 15 or SEQ ID NO: 27) . The different constant regions of the obtained antibodies are respectively connected to the N-terminal or C-terminal of the mutated TGFβRII extracellular domain and/or tumor-targeting peptide and/or wild-type TGFβRII extracellular domain to form Fc-hTGFβRII Trap recombinant Antibodies or Fc-ECD recombinant antibodies.
本申请利用上述合成序列构建4种结构的重组抗体(#1、#2、#3、#4)，具体的分子结构如图1所示，其中：This application uses the above-mentioned synthetic sequences to construct four kinds of recombinant antibodies (#1, #2, #3, #4). The specific molecular structures are shown in Figure 1, in which:
分子1(重组抗体#1)：具有两条相同的肽链(分子1单条肽链1、分子1单条肽链2、分子1单条肽链3、或分子1单条肽链4)，每条肽链中1个TGFβRII Trap分子或ECD分子的C端与Fc区突变体2的N端相连，Fc区突变体2的C端与另外1个TGFβRII Trap分子或ECD分子的N端相连，如图1中1-A和1-B所示，编码分子1的4种肽链的核酸分别具有SEQ ID NO:16、40、41、56所示的核苷酸序列，获得的分子1的4种肽链分别具有SEQ ID NO:17、30、31、54所示的氨基酸序列，4种分子1编号为#1-1(R1054)、#1-2、#1-3、#1-4。其中#1-1(R1054)的氨基酸序列SEQ ID NO:17，#1-2的氨基酸序列SEQ ID NO:30，#1-3的氨基酸序列SEQ ID NO:31，#1-4的氨基酸序列SEQ ID NO:54。Molecule 1 (recombinant antibody #1): has two identical peptide chains (molecule 1 single peptide chain 1, molecule 1 single peptide chain 2, molecule 1 single peptide chain 3, or molecule 1 single peptide chain 4), each peptide The C-terminus of one TGFβRII Trap molecule or ECD molecule in the chain is connected to the N-terminus of Fc region mutant 2, and the C-terminus of Fc region mutant 2 is connected to the N-terminus of another TGFβRII Trap molecule or ECD molecule, as shown in Figure 1 As shown in 1-A and 1-B, the nucleic acids encoding the four peptide chains of molecule 1 have the nucleotide sequences shown in SEQ ID NO: 16, 40, 41, and 56 respectively. The four peptides of molecule 1 obtained The chains have the amino acid sequences shown in SEQ ID NO: 17, 30, 31, and 54 respectively, and the four molecule numbers are #1-1 (R1054), #1-2, #1-3, and #1-4. Among them, the amino acid sequence of #1-1 (R1054) is SEQ ID NO:17, the amino acid sequence of #1-2 is SEQ ID NO:30, the amino acid sequence of #1-3 is SEQ ID NO:31, and the amino acid sequence of #1-4 SEQ ID NO:54.
分子2(重组抗体#2)：具有两条相同的肽链(分子2单条肽链1、分子2单条肽链2、分子2单条肽链3、或分子2单条肽链4)，每条肽链中包括1个肿瘤靶向肽，所述肿瘤靶向肽的C端与Fc区突变体2的N端相连，Fc区突变体2的C端与1个TGFβRII Trap分子或ECD分子的N端相连，如图1中1-C和1-D所示，编码分子2的单条肽链的核酸分别具有SEQ ID NO:18、42、43、57所示的核苷酸序列，获得的4种分子2的单条肽链分别具有SEQ ID NO:19、32、33、55所示的氨基酸序列，4种分子2编号为#2-1(R1056)、#2-2、#2-3、#2-4。其中#2-1(R1056)的氨基酸序列SEQ ID NO:19，#2-2的氨基酸序列SEQ ID NO:32，#2-3的氨基酸序列SEQ ID NO:33，#2-4的氨基酸序列SEQ ID NO:55。Molecule 2 (recombinant antibody #2): has two identical peptide chains (molecule 2 single peptide chain 1, molecule 2 single peptide chain 2, molecule 2 single peptide chain 3, or molecule 2 single peptide chain 4), each peptide The chain includes a tumor-targeting peptide, the C-terminus of the tumor-targeting peptide is connected to the N-terminus of the Fc region mutant 2, and the C-terminus of the Fc region mutant 2 is connected to the N-terminus of a TGFβRII Trap molecule or an ECD molecule. Connected, as shown in 1-C and 1-D in Figure 1, the nucleic acids encoding a single peptide chain of molecule 2 have the nucleotide sequences shown in SEQ ID NO: 18, 42, 43, and 57 respectively. The four obtained The single peptide chains of molecule 2 have the amino acid sequences shown in SEQ ID NO: 19, 32, 33, and 55 respectively. The four types of molecule 2 are numbered #2-1 (R1056), #2-2, #2-3, # 2-4. Among them, the amino acid sequence of #2-1 (R1056) is SEQ ID NO: 19, the amino acid sequence of #2-2 is SEQ ID NO: 32, the amino acid sequence of #2-3 is SEQ ID NO: 33, and the amino acid sequence of #2-4 SEQ ID NO:55.
分子3(重组抗体#3)：具有两条相同的重链(分子3重链1、分子3重链2、分子3重链3、或分子3重链4)和两条相同的轻链(分子3轻链1、分子3轻链2、分子3轻链3、或分子3轻链4)，其中，每条重链中1个TGFβRII Trap分子或ECD分子的C端与重链恒定区的N端相连，重链恒定区的C端与另外1个TGFβRII Trap分子或ECD分子的N端相连，如图1中的1-E和1-F所示，编码所述4种重链的核苷酸序列分别如SEQ ID NO:20、44、45、52所示，获得的分子3的4种重链的氨基酸序列分别如SEQ ID NO:21、34、35、58所示；每条轻链中1个TGFβRII Trap分子或ECD分子的C端与抗体轻链恒定区(CL，Kappa)的N端相连，编码所述4种轻链的核苷酸序列如SEQ ID NO:22、48、49、53所示，获得的分子3的4种轻链的氨基酸序列分别如SEQ ID NO:23、38、39、59所示，4种分子3编号为#3-1(R1055)、#3-2、#3-3、#3-4。其中#3-1(R1055)的氨基酸序列SEQ ID NO:22，#3-2的氨基酸序列SEQ ID NO:48，#3-3的氨基酸序列SEQ ID NO:49，#3-4的氨基酸序列SEQ ID NO:53。Molecule 3 (Recombinant Antibody #3): Has two identical heavy chains (Molecular 3 Heavy Chain 1, Molecule 3 Heavy Chain 2, Molecule 3 Heavy Chain 3, or Molecule 3 Heavy Chain 4) and two identical light chains ( Molecule 3 light chain 1, Molecule 3 light chain 2, Molecule 3 light chain 3, or Molecule 3 light chain 4), where the C-terminus of one TGFβRII Trap molecule or ECD molecule in each heavy chain is connected to the heavy chain constant region The N-terminus is connected, and the C-terminus of the heavy chain constant region is connected to the N-terminus of another TGFβRII Trap molecule or ECD molecule, as shown in 1-E and 1-F in Figure 1, encoding the nuclei of the four heavy chains The nucleotide sequences are shown in SEQ ID NO: 20, 44, 45, and 52 respectively. The amino acid sequences of the four heavy chains of the obtained molecule 3 are shown in SEQ ID NO: 21, 34, 35, and 58 respectively; each light chain The C-terminus of a TGFβRII Trap molecule or ECD molecule in the chain is connected to the N-terminus of the antibody light chain constant region (CL, Kappa). The nucleotide sequences encoding the four light chains are such as SEQ ID NO: 22, 48, As shown in 49 and 53, the amino acid sequences of the four light chains of the obtained molecule 3 are shown in SEQ ID NO: 23, 38, 39, and 59 respectively. The four kinds of molecule 3 are numbered #3-1 (R1055), #3 -2, #3-3, #3-4. Among them, the amino acid sequence of #3-1 (R1055) is SEQ ID NO: 22, the amino acid sequence of #3-2 is SEQ ID NO: 48, the amino acid sequence of #3-3 is SEQ ID NO: 49, and the amino acid sequence of #3-4 SEQ ID NO:53.
分子4(重组抗体#4)：具有两条相同的肽链(分子4单条肽链1、分子4单条肽链2、分子4单条肽链3、分子4单条肽链4)，每条肽链中Fc区突变体1的C端与1个TGFβRII Trap分子或ECD分子的N端相连，如图1中的1-G和1-H所示，编码分子4的4种核酸分别具有SEQ ID NO:24、46、47、63所示的核苷酸序列，获得的4种分子4具有SEQ ID NO:25、36、37、64所示的氨基酸序列，4种分子4编号为#4-1(R0796)、#4-2、#4-3、#4-4。其中#4-1(R0796)的氨基酸序列SEQ ID NO:25，#4-2的氨基酸序列SEQ ID NO:36，#4-3的氨基酸序列SEQ ID NO:37，#4-4的氨基酸序列SEQ ID NO:64。Molecule 4 (recombinant antibody #4): has two identical peptide chains (molecule 4 single peptide chain 1, molecule 4 single peptide chain 2, molecule 4 single peptide chain 3, molecule 4 single peptide chain 4), each peptide chain The C-terminus of the middle Fc region mutant 1 is connected to the N-terminus of a TGFβRII Trap molecule or ECD molecule, as shown in 1-G and 1-H in Figure 1. The four nucleic acids encoding molecule 4 have SEQ ID NO. : The nucleotide sequences shown in SEQ ID NO: 24, 46, 47, and 63. The four obtained molecules 4 have the amino acid sequences shown in SEQ ID NO: 25, 36, 37, and 64. The four molecules 4 are numbered #4-1 (R0796), #4-2, #4-3, #4-4. Among them, the amino acid sequence of #4-1 (R0796) is SEQ ID NO: 25, the amino acid sequence of #4-2 is SEQ ID NO: 36, the amino acid sequence of #4-3 is SEQ ID NO: 37, and the amino acid sequence of #4-4 SEQ ID NO:64.
野生型TGFβRII具有如下氨基酸序列：
Wild-type TGFβRII has the following amino acid sequence:
野生型TGFβRII的胞外结构域(ECD)具有如SEQ ID NO:2氨基酸序列。The extracellular domain (ECD) of wild-type TGFβRII has the amino acid sequence of SEQ ID NO:2.
编码野生型TGFβRII的胞外结构域(ECD)的核酸具有如下核苷酸序列：

The nucleic acid encoding the extracellular domain (ECD) of wild-type TGFβRII has the following nucleotide sequence:

第一连接肽和/或所述第二连接肽和/或第三连接肽和/或第七连接肽和/或第八连接肽具有如下氨基酸序列：
The first connecting peptide and/or the second connecting peptide and/or the third connecting peptide and/or the seventh connecting peptide and/or the eighth connecting peptide have the following amino acid sequence:
TGFβRII Trap-1具有SEQ ID NO:4氨基酸序列。TGFβRII Trap-1 has the amino acid sequence of SEQ ID NO:4.
TGFβRII Trap-2具有如SEQ ID NO:28氨基酸序列。TGFβRII Trap-2 has an amino acid sequence such as SEQ ID NO: 28.
TGFβRII Trap-3具有如SEQ ID NO:29氨基酸序列。TGFβRII Trap-3 has an amino acid sequence such as SEQ ID NO: 29.
第四连接肽、第五连接肽、第六连接肽、第九连接肽、第十连接肽具有如下氨基酸序列：
The fourth connecting peptide, the fifth connecting peptide, the sixth connecting peptide, the ninth connecting peptide, and the tenth connecting peptide have the following amino acid sequences:
抗体Fc区具有如SEQ ID NO:6氨基酸序列。The antibody Fc region has an amino acid sequence such as SEQ ID NO: 6.
抗体重链恒定区具有如SEQ ID NO:7氨基酸序列。The antibody heavy chain constant region has an amino acid sequence such as SEQ ID NO:7.
抗体轻链恒定区具有如SEQ ID NO:8氨基酸序列。The antibody light chain constant region has an amino acid sequence such as SEQ ID NO:8.
肿瘤靶向肽具有如下氨基酸序列：The tumor targeting peptide has the following amino acid sequence:
RGDRGD(SEQ ID NO:9)；或RGDRGD(SEQ ID NO:9); or
NGRNGR(SEQ ID NO:26)。NGRNGR(SEQ ID NO:26).
编码TGFβRII Trap-1的核酸具有如下核苷酸序列：
The nucleic acid encoding TGFβRII Trap-1 has the following nucleotide sequence:
编码TGFβRII Trap-2的核酸具有如下核苷酸序列：
The nucleic acid encoding TGFβRII Trap-2 has the following nucleotide sequence:
编码TGFβRII Trap-3的核酸具有如下核苷酸序列：
The nucleic acid encoding TGFβRII Trap-3 has the following nucleotide sequence:
编码连接肽(第四连接肽、第五连接肽、第六连肽、第九连接肽、第十连接肽)的核酸具有如下核苷酸序列：
The nucleic acid encoding the connecting peptide (the fourth connecting peptide, the fifth connecting peptide, the sixth connecting peptide, the ninth connecting peptide, and the tenth connecting peptide) has the following nucleotide sequence:
编码Fc区突变体2(IgG1.8抗体的Fc区)的核酸具有如下核苷酸序列：

The nucleic acid encoding Fc region mutant 2 (Fc region of IgG1.8 antibody) has the following nucleotide sequence:

编码抗体重链恒定区的核酸具有如下核苷酸序列：
The nucleic acid encoding the constant region of the antibody heavy chain has the following nucleotide sequence:
编码抗体轻链恒定区的核酸具有如下核苷酸序列：
The nucleic acid encoding the antibody light chain constant region has the following nucleotide sequence:
编码肿瘤靶向肽的核酸具有如下核苷酸序列：The nucleic acid encoding the tumor-targeting peptide has the following nucleotide sequence:
AGGGGTGATAGGGGTGAT(SEQ ID NO:15)；或AGGGGTGATAGGGGTGAT(SEQ ID NO:15); or
AACGGCCGCAACGGCCGC(SEQ ID NO:27)。AACGGCCGCAACGGCCGC (SEQ ID NO: 27).
编码分子1单条肽链1的核酸具有如SEQ ID NO:16核苷酸序列。The nucleic acid encoding single peptide chain 1 of molecule 1 has a nucleotide sequence such as SEQ ID NO: 16.
编码分子1单条肽链2的核酸具有如SEQ ID NO:40核苷酸序列。The nucleic acid encoding a single peptide chain 2 of molecule 1 has a nucleotide sequence such as SEQ ID NO: 40.
编码分子1单条肽链3的核酸具有如SEQ ID NO:41核苷酸序列。The nucleic acid encoding single peptide chain 3 of molecule 1 has a nucleotide sequence such as SEQ ID NO: 41.
编码分子1单条肽链4的核酸具有如SEQ ID NO:56核苷酸序列。The nucleic acid encoding a single peptide chain 4 of molecule 1 has a nucleotide sequence such as SEQ ID NO: 56.
分子1单条肽链1具有如SEQ ID NO:17氨基酸序列。Molecule 1 single peptide chain 1 has an amino acid sequence such as SEQ ID NO: 17.
分子1单条肽链2具有如SEQ ID NO:30氨基酸序列。Molecule 1 single peptide chain 2 has an amino acid sequence such as SEQ ID NO: 30.
分子1单条肽链3具有如SEQ ID NO:31氨基酸序列。Molecule 1 single peptide chain 3 has the amino acid sequence SEQ ID NO: 31.
分子1单条肽链4具有如SEQ ID NO:54氨基酸序列。Molecule 1 single peptide chain 4 has the amino acid sequence SEQ ID NO: 54.
编码分子2单条肽链1的核酸具有如SEQ ID NO:18核苷酸序列。The nucleic acid encoding single peptide chain 1 of molecule 2 has a nucleotide sequence such as SEQ ID NO: 18.
编码分子2单条肽链2的核酸具有如SEQ ID NO:42核苷酸序列。The nucleic acid encoding single peptide chain 2 of molecule 2 has a nucleotide sequence such as SEQ ID NO: 42.
编码分子2单条肽链3的核酸具有如SEQ ID NO:43核苷酸序列。The nucleic acid encoding single peptide chain 3 of molecule 2 has a nucleotide sequence such as SEQ ID NO: 43.
编码分子2单条肽链4的核酸具有如SEQ ID NO:57核苷酸序列。The nucleic acid encoding single peptide chain 4 of molecule 2 has a nucleotide sequence such as SEQ ID NO: 57.
分子2单条肽链1具有如SEQ ID NO:19氨基酸序列。Molecule 2 single peptide chain 1 has the amino acid sequence SEQ ID NO:19.
分子2单条肽链2具有如SEQ ID NO:32氨基酸序列。Molecule 2 single peptide chain 2 has an amino acid sequence such as SEQ ID NO: 32.
分子2单条肽链3具有如SEQ ID NO:33氨基酸序列。Molecule 2 single peptide chain 3 has the amino acid sequence SEQ ID NO: 33.
分子2单条肽链4具有如SEQ ID NO:55氨基酸序列。Molecule 2 single peptide chain 4 has an amino acid sequence such as SEQ ID NO: 55.
编码分子3重链1的核酸具有如SEQ ID NO:20核苷酸序列。The nucleic acid encoding molecule 3 heavy chain 1 has a nucleotide sequence such as SEQ ID NO: 20.
编码分子3重链2的核酸具有如SEQ ID NO:44核苷酸序列。 The nucleic acid encoding molecule 3 heavy chain 2 has a nucleotide sequence such as SEQ ID NO: 44.
编码分子3重链3的核酸具有如SEQ ID NO:45核苷酸序列。The nucleic acid encoding molecule 3 heavy chain 3 has a nucleotide sequence such as SEQ ID NO: 45.
编码分子3重链4的核酸具有如SEQ ID NO:52核苷酸序列。The nucleic acid encoding molecule 3 heavy chain 4 has a nucleotide sequence such as SEQ ID NO: 52.
分子3重链1具有如SEQ ID NO:21氨基酸序列。Molecule 3 heavy chain 1 has an amino acid sequence such as SEQ ID NO: 21.
分子3重链2具有如SEQ ID NO:34氨基酸序列。Molecule 3 heavy chain 2 has an amino acid sequence such as SEQ ID NO: 34.
分子3重链3具有如SEQ ID NO:35氨基酸序列。Molecule 3 heavy chain 3 has an amino acid sequence such as SEQ ID NO: 35.
分子3重链4具有如SEQ ID NO:58氨基酸序列。Molecule 3 heavy chain 4 has an amino acid sequence such as SEQ ID NO: 58.
编码分子3轻链1的核酸具有如SEQ ID NO:22核苷酸序列。The nucleic acid encoding molecule 3 light chain 1 has a nucleotide sequence such as SEQ ID NO: 22.
编码分子3轻链2的核酸具有如SEQ ID NO:48核苷酸序列。The nucleic acid encoding molecule 3 light chain 2 has a nucleotide sequence such as SEQ ID NO: 48.
编码分子3轻链3的核酸具有如SEQ ID NO:49核苷酸序列。The nucleic acid encoding molecule 3 light chain 3 has a nucleotide sequence such as SEQ ID NO: 49.
编码分子3轻链4的核酸具有如SEQ ID NO:53核苷酸序列。The nucleic acid encoding molecule 3 light chain 4 has a nucleotide sequence such as SEQ ID NO: 53.
分子3轻链1具有如SEQ ID NO:23氨基酸序列。Molecule 3 light chain 1 has an amino acid sequence such as SEQ ID NO: 23.
分子3轻链2具有如SEQ ID NO:38氨基酸序列。Molecule 3 light chain 2 has an amino acid sequence such as SEQ ID NO: 38.
分子3轻链3具有如SEQ ID NO:39氨基酸序列。Molecule 3 light chain 3 has an amino acid sequence such as SEQ ID NO: 39.
分子3轻链4具有如SEQ ID NO:59氨基酸序列。Molecule 3 light chain 4 has an amino acid sequence such as SEQ ID NO: 59.
编码分子4单条肽链1的核酸具有如下核苷酸序列：
The nucleic acid encoding single peptide chain 1 of molecule 4 has the following nucleotide sequence:
编码分子4单条肽链2的核酸具有如下核苷酸序列：

The nucleic acid encoding single peptide chain 2 of molecule 4 has the following nucleotide sequence:

编码分子4单条肽链3的核酸具有如下核苷酸序列：
The nucleic acid encoding the single peptide chain 3 of molecule 4 has the following nucleotide sequence:
编码分子4单条肽链4的核酸具有如下核苷酸序列：
The nucleic acid encoding the single peptide chain 4 of molecule 4 has the following nucleotide sequence:
分子4单条肽链1具有如下氨基酸序列：
Molecule 4 single peptide chain 1 has the following amino acid sequence:
分子4单条肽链2具有如下氨基酸序列：

Molecule 4 single peptide chain 2 has the following amino acid sequence:

分子4单条肽链3具有如下氨基酸序列：
Molecule 4 single peptide chain 3 has the following amino acid sequence:
分子4单条肽链4具有如下氨基酸序列：
Molecule 4 single peptide chain 4 has the following amino acid sequence:
Fc区突变体1具有如下氨基酸序列：
Fc region mutant 1 has the following amino acid sequence:
编码Fc区突变体1的核酸具有如下核苷酸序列：
The nucleic acid encoding Fc region mutant 1 has the following nucleotide sequence:
实施例2重组抗体的表达Example 2 Expression of recombinant antibodies
将实施例1获得的编码重组抗体#1、#2、#3、#4的核苷酸序列分别插入表达质粒pcDNA3.4中，并瞬时转染人胚肾HEK 293细胞，分离纯化细胞产生的重组抗体，其中，转染及分离纯化蛋白均为常规实验操作，所述重组抗体#1-1(R1054)、重组抗体#2-1(R1056)、重组抗体#3-1(R1055)在非还原条件下于SDS-PAGE上的条带分子量分别约为120kD、105kD和190kD，在还原条件下于SDS-PAGE上的条带分子量分别约为60kD、50kD、95kD。The nucleotide sequences encoding recombinant antibodies #1, #2, #3, and #4 obtained in Example 1 were inserted into the expression plasmid pcDNA3.4 respectively, and transiently transfected into human embryonic kidney HEK 293 cells, and the nucleotide sequences produced by the cells were isolated and purified. Recombinant antibodies, in which transfection and isolation and purification of proteins are routine experimental operations. The recombinant antibody #1-1 (R1054), recombinant antibody #2-1 (R1056), and recombinant antibody #3-1 (R1055) are not The molecular weights of the bands on SDS-PAGE under reducing conditions are approximately 120kD, 105kD and 190kD respectively, and the molecular weights of the bands on SDS-PAGE under reducing conditions are approximately 60kD, 50kD and 95kD respectively.
实施例3 ELISA检测重组抗体与hTGFβ的体外结合活性Example 3 ELISA detection of in vitro binding activity of recombinant antibodies and hTGFβ
本实施例检测重组抗体#1-1(R1054)、#1-2、#1-3、#1-4、#2-1(R1056)、#2-2、#2-3、#2-4、#3-1(R1055)、#3-2、#3-3、#3-4、#4-1(R0796)、#4-2、#4-3、#4-4的体外结合活性，并以人源抗体IgG1(iso(hIgG1.8)组)作为同型对照组、分子#5、分子#6作为对照组，其中，分子#5的结构如图2所示，其具有2条相同的肽链(分子#5单条肽链)，分子#5所包含的Fc区为抗体IgG1.8的Fc区(Fc区突变体2)，分子#5单条肽链的氨基酸序列如SEQ ID NO:65所示；分子 #6的结构如图3所示，包含2条相同的重链(分子#6重链)和轻链(分子#6轻链)，分子#6所包含的重链恒定区为抗体IgG1.8的重链恒定区，分子#6的重链、轻链氨基酸序列分别如SEQ ID NO:66、67所示；其制备方法同实施例1和实施例2，检测使用的抗原为human TGFβ1(CA59)、human TGFβ3(CJ44)(购自Novoprotein)，具体的检测流程如下：This example detects recombinant antibodies #1-1 (R1054), #1-2, #1-3, #1-4, #2-1 (R1056), #2-2, #2-3, #2- 4. In vitro binding of #3-1(R1055), #3-2, #3-3, #3-4, #4-1(R0796), #4-2, #4-3, #4-4 activity, and used human antibody IgG1 (iso (hIgG1.8) group) as the isotype control group, molecule #5, and molecule #6 as the control group. Among them, the structure of molecule #5 is shown in Figure 2, which has 2 lines. The same peptide chain (single peptide chain of molecule #5), the Fc region contained in molecule #5 is the Fc region of antibody IgG1.8 (Fc region mutant 2), the amino acid sequence of the single peptide chain of molecule #5 is as SEQ ID NO :65; molecule The structure of #6 is shown in Figure 3. It contains two identical heavy chains (molecule #6 heavy chain) and light chain (molecule #6 light chain). The heavy chain constant region contained in molecule #6 is antibody IgG1.8. The heavy chain constant region of molecule #6, the heavy chain and light chain amino acid sequences of molecule #6 are shown in SEQ ID NO: 66 and 67 respectively; the preparation method is the same as Example 1 and Example 2, and the antigen used for detection is human TGFβ1 (CA59 ), human TGFβ3 (CJ44) (purchased from Novoprotein), the specific detection process is as follows:
a.配备pH为9.6的包被缓冲液(Na2CO3/NaHCO3)，稀释候选蛋白浓度至0.5μg/mL，添加体积为50μL/孔，加到酶标板中，放置到4℃过夜。a. Prepare coating buffer (Na 2 CO 3 /NaHCO 3 ) with a pH of 9.6, dilute the candidate protein concentration to 0.5 μg/mL, add the volume to 50 μL/well, add it to the enzyme plate, and place it at 4°C overnight. .
b.配备PBST(5‰的吐温20)，使用PBST(300μL/孔)洗涤酶标板3次，加入2％的BSA溶液进行封闭，添加体积为200μL/孔，室温放置1小时。b. Prepare PBST (5‰ Tween 20), wash the enzyme plate three times with PBST (300 μL/well), add 2% BSA solution for blocking, add a volume of 200 μL/well, and leave it at room temperature for 1 hour.
c.室温反应后，使用PBST(200μL/孔)洗涤酶标板3次；c. After the reaction at room temperature, wash the enzyme plate three times with PBST (200 μL/well);
d.用2％BSA梯度稀释抗体样品至8个浓度梯度(100nM、33.33nM、11.11nM、3.7nM、1.23nM、0.41nM、0.14nM、0.045nM)；将稀释样品以100μL/孔的体积添加至酶标板中，于室温孵育2小时。d. Use 2% BSA to dilute the antibody sample to 8 concentration gradients (100nM, 33.33nM, 11.11nM, 3.7nM, 1.23nM, 0.41nM, 0.14nM, 0.045nM); add the diluted sample in a volume of 100μL/well Transfer to a microtiter plate and incubate at room temperature for 2 hours.
e.孵育结束后使用PBST(300μL/孔)洗涤酶标板三次；用2％BSA稀释Goat anti hIgG-HRP，稀释倍数为1:15K，100μL/孔，室温孵育1小时；e. After incubation, wash the enzyme plate three times with PBST (300 μL/well); dilute Goat anti hIgG-HRP with 2% BSA to a dilution factor of 1:15K, 100 μL/well, and incubate at room temperature for 1 hour;
f.步骤e孵育结束后，使用PBST(300μL/孔)洗涤酶标板五次，然后加入TMB，添加体积为100μL/孔，避光室温放置20min；加入2M的终止液，添加体积为100μL/孔。f. After the incubation in step e, wash the enzyme plate five times with PBST (300 μL/well), then add TMB in a volume of 100 μL/well, and leave it at room temperature for 20 minutes in the dark; add 2M stop solution in a volume of 100 μL/well. hole.
g.将步骤f获得的产物用iX3酶标仪(Molecular Device公司)读取450nM处的吸收值，用GraphPad7.0分析作图。g. Use an iX3 microplate reader (Molecular Device Company) to read the absorbance value at 450nM of the product obtained in step f, and use GraphPad7.0 to analyze and draw the graph.
具体的实验结果如图4所示，ELISA结果分析显示：重组抗体#1-1(R1054)、#1-2、#1-3、#1-4、#2-1(R1056)、#2-2、#2-3、#2-4、#3-1(R1055)、#3-2、#3-3、#3-4均保留了与hTGFβ的体外结合活性，且重组抗体#1-1、#2-1、#3-1与TGFβ1或TGFβ3结合EC50均在0.1-0.3nM之间。The specific experimental results are shown in Figure 4. The ELISA result analysis shows: recombinant antibodies #1-1 (R1054), #1-2, #1-3, #1-4, #2-1 (R1056), #2 -2, #2-3, #2-4, #3-1(R1055), #3-2, #3-3, and #3-4 all retain the in vitro binding activity to hTGFβ, and recombinant antibody #1 -1, #2-1, #3-1 and TGFβ1 or TGFβ3 binding EC50 are all between 0.1-0.3nM.
分子#5单条肽链具有如下氨基酸序列：
The single peptide chain of molecule #5 has the following amino acid sequence:
分子#6的单条重链具有如下氨基酸序列：
The single heavy chain of molecule #6 has the following amino acid sequence:
分子#6的单条轻链具有如下氨基酸序列：
The single light chain of molecule #6 has the following amino acid sequence:
实施例4重组抗体对细胞表面TGFβRII与TGFβ结合的阻断效果检测Example 4 Detection of the blocking effect of recombinant antibodies on the binding of TGFβRII and TGFβ on the cell surface
分别构建稳定转染表达人TGFβRII的CHO细胞(CHO-hTGFβRII)，挑取单克隆建系。采用如下方法对重组抗体#1-1(R1054)、#1-2、#1-3、#1-4、#2-1(R1056)、#2-2、#2-3、#2-4、#3-1(R1055)、#3-2、#3-3、#3-4、#4-1(R0796)、#4-2、#4-3、#4-4的阻断活性进行检测，其中，人源抗体IgG1(iso(hIgG1.8)组)作为同型对照组、No Ab组(不添加任何抗 体)作为阴性对照组、上述分子#5、分子#6作为对照组。CHO cells stably transfected to express human TGFβRII (CHO-hTGFβRII) were constructed and monoclonal lines were selected. Use the following method to detect recombinant antibodies #1-1(R1054), #1-2, #1-3, #1-4, #2-1(R1056), #2-2, #2-3, #2- 4. Blocking of #3-1(R1055), #3-2, #3-3, #3-4, #4-1(R0796), #4-2, #4-3, #4-4 The activity was tested, in which the human antibody IgG1 (iso (hIgG1.8) group) was used as the isotype control group, and the No Ab group (no anti- body) as the negative control group, and the above molecule #5 and molecule #6 were used as the control group.
a.用3％BSA分别梯度稀释R1054、R1055、R1056、R0796蛋白，起始浓度为100nM，稀释倍数为3倍，共设置10个梯度，具体为100nM、33.33nM、11.11nM、3.7nM、1.23nM、0.41nM、0.14nM、0.045nM、0.015nM、0.005nM；3％BSA稀释TGFβ1.biotin至浓度为0.5μg/mL；a. Use 3% BSA to gradiently dilute R1054, R1055, R1056, and R0796 proteins respectively. The starting concentration is 100nM, and the dilution factor is 3 times. A total of 10 gradients are set, specifically 100nM, 33.33nM, 11.11nM, 3.7nM, and 1.23 nM, 0.41nM, 0.14nM, 0.045nM, 0.015nM, 0.005nM; dilute TGFβ1.biotin with 3% BSA to a concentration of 0.5μg/mL;
b.将TGFβ1.biotin稀释液和重组抗体的稀释液按1:1体积比混匀，4℃孵育30min；b. Mix the TGFβ1.biotin diluent and the recombinant antibody diluent at a volume ratio of 1:1, and incubate at 4°C for 30 minutes;
c.将CHO-TGFβRⅡ细胞按2E5/孔铺至96孔板，每孔添加体积100μl；细胞350G室温离心5min，弃去上清液，加入步骤b获得的孵育后的hTGFβ1.biotin和重组抗体混合液，添加体积为100μL/孔，轻轻混匀，4℃孵育1小时；c. Spread CHO-TGFβRⅡ cells into a 96-well plate at 2E5/well, add a volume of 100 μl to each well; centrifuge the cells at 350G for 5 minutes at room temperature, discard the supernatant, and add the incubated hTGFβ1.biotin and recombinant antibody obtained in step b to mix solution, add a volume of 100 μL/well, mix gently, and incubate at 4°C for 1 hour;
d.步骤c孵育结束后，将细胞于350G室温离心5min，弃去上清液，加入SA-PE(购自Biolegend，1:300稀释)，添加体积为100μL/孔，混匀，4℃孵育1小时；d. After the incubation in step c, centrifuge the cells at 350G for 5 minutes at room temperature, discard the supernatant, and add SA-PE (purchased from Biolegend, diluted 1:300) in a volume of 100 μL/well, mix well, and incubate at 4°C. 1 hour;
e.步骤d孵育结束后，将细胞于350G室温离心5min，弃去上清液，每孔加入150μL PBS重悬细胞，用Beckman公司Cytoflex流式细胞仪检测，用Graphpad Prism7计算数据并作图。e. After the incubation in step d, centrifuge the cells at 350G room temperature for 5 minutes, discard the supernatant, add 150 μL PBS to each well to resuspend the cells, detect with a Beckman Cytoflex flow cytometer, and use Graphpad Prism7 to calculate and graph the data.
具体的实验结果如图5所示，对CHO-hTGFβRII细胞与hTGFβ1.biotin的结合，重组抗体#1-1(R1054)、#1-2、#1-3、#1-4、#2-1(R1056)、#2-2、#2-3、#2-4、#3-1(R1055)、#3-2、#3-3、#3-4展示出与R0796类似的阻断效果，阻断活性的IC50值在0.05-0.25nM之间。阴性对照组对TGFβRII与hTGFβ1.biotin的结合无阻断效果。The specific experimental results are shown in Figure 5. For the binding of CHO-hTGFβRII cells to hTGFβ1.biotin, recombinant antibodies #1-1 (R1054), #1-2, #1-3, #1-4, #2- 1(R1056), #2-2, #2-3, #2-4, #3-1(R1055), #3-2, #3-3, #3-4 exhibit similar blocking to R0796 Effect, the IC 50 value of blocking activity is between 0.05-0.25nM. The negative control group had no blocking effect on the combination of TGFβRII and hTGFβ1.biotin.
实施例5 T细胞增殖抑制实验Example 5 T cell proliferation inhibition experiment
本实施例验证了重组抗体在细胞水平的功能，发明人检测了TGFβ1存在情况下，重组抗体#1-1(R1054)、#1-2、#1-3、#1-4、#2-1(R1056)、#2-2、#2-3、#2-4、#3-1(R1055)、#3-2、#3-3、#3-4、#4-1(R0796)、#4-2、#4-3、#4-4对T细胞增殖的影响，人源抗体IgG1(iso(hIgG1.8)组)作为同型对照组、no Ab组(不添加任何抗体)作为阴性对照组、上述分子#5、分子#6作为对照组。实验采用如下示例性方法：This example verified the function of the recombinant antibody at the cellular level. The inventor detected the recombinant antibodies #1-1 (R1054), #1-2, #1-3, #1-4, #2- in the presence of TGFβ1. 1(R1056), #2-2, #2-3, #2-4, #3-1(R1055), #3-2, #3-3, #3-4, #4-1(R0796) , #4-2, #4-3, and #4-4 on T cell proliferation, human antibody IgG1 (iso (hIgG1.8) group) as the isotype control group, no Ab group (no antibody added) as The negative control group, the above-mentioned molecule #5, and molecule #6 served as the control group. The experiment adopts the following exemplary methods:
a.从健康个体的全血中分离外周血单个核细胞(PBMC)，使用人T细胞富集试剂盒(STEMCELL，10951)，从PBMC中分离T细胞，具体实验操作按照说明书进行；a. Isolate peripheral blood mononuclear cells (PBMC) from the whole blood of healthy individuals. Use the human T cell enrichment kit (STEMCELL, 10951) to isolate T cells from PBMC. The specific experimental operations are performed according to the instructions;
b.利用DPBS将上述T细胞密度调整为5E6/mL，使用CFSE(eBioscience，85-65-0850-84)对T细胞进行染色，具体操作参考试剂说明书，CFSE的使用浓度为0.5μM；b. Use DPBS to adjust the density of the above T cells to 5E6/mL, and use CFSE (eBioscience, 85-65-0850-84) to stain the T cells. For specific operations, refer to the reagent instructions. The concentration of CFSE used is 0.5 μM;
c.用细胞培养基稀释上述重组抗体，配置浓度为200nM，并3倍梯度稀释，设4个浓度梯度；同时稀释TGFβ1为20ng/mL，两者均加入U形板中，混合均匀，室温共孵育30min；c. Dilute the above recombinant antibody with cell culture medium to a concentration of 200nM, and dilute it 3 times, setting up 4 concentration gradients; at the same time dilute TGFβ1 to 20ng/mL, add both to the U-shaped plate, mix evenly, and incubate at room temperature. Incubate for 30 minutes;
d.调整T细胞密度为2E6/mL，将调整好密度的细胞以50μL/孔的体积加入96孔U形板，即最终细胞密度为1E5/孔；d. Adjust the T cell density to 2E6/mL, and add the cells with the adjusted density into the 96-well U-shaped plate at a volume of 50 μL/well, that is, the final cell density is 1E5/well;
e.将CD3/CD28 beads(life，40203D)按50μl/孔体积加入到步骤d获得的CFSE染色后的T细胞中，CD3/CD28 beads与T细胞的比例为1:30；e. Add CD3/CD28 beads (life, 40203D) to the CFSE-stained T cells obtained in step d at a volume of 50 μl/well. The ratio of CD3/CD28 beads to T cells is 1:30;
f.将步骤c获得的提前孵育好的重组抗体和TGFβ1混合液加入至96孔板，混合液的添加体积为100μL/孔，在培养箱进行培养；f. Add the pre-incubated recombinant antibody and TGFβ1 mixture obtained in step c to the 96-well plate. The added volume of the mixture is 100 μL/well, and culture it in the incubator;
g.培养第五天后，流式检测T细胞的CFSE值，算出T细胞在每一代中所占的比例。g. After the fifth day of culture, detect the CFSE value of T cells by flow cytometry and calculate the proportion of T cells in each generation.
具体的实验结果如图6-A和6-B所示，其中，no actived表示未添加CD3/CD28 beads未激活，TGFβ3ng/mL表示添加3ng/mL TGFβ，Ab 50nM表示添加50nM重组抗体组，actived表示添加CD3/CD28 beads激活。不加CD3/CD28 beads时，T细胞无增殖，在加入CD3/CD28 beads下，T细胞增殖率可以达到34％。而TGFβ加入体系后，T细胞的增殖比例显著下降，从34％降至10.8％，如果同时加入#1-1(R1054)、#1-2、#1-3、#1-4、#2-1(R1056)、#2-2、#2-3、#2-4、#3-1(R1055)、#3-2、#3-3、或#3-4后，T细胞增殖率均有不同程度的增加，其中，R1055加入后解除抑制最强，最高的一组(R1055)相较于不加重组抗体组，增殖率增加了22.13％，接近未加TGFβ1的对照组的增殖率(34％)，且增殖率与R1054、R1055、R1056各组的浓度呈现依赖关系。The specific experimental results are shown in Figures 6-A and 6-B. Among them, no actived means that CD3/CD28 beads are not activated, TGFβ3ng/mL means that 3ng/mL TGFβ is added, Ab 50nM means that 50nM recombinant antibody group is added, and actived Indicates adding CD3/CD28 beads for activation. When CD3/CD28 beads are not added, T cells do not proliferate. With the addition of CD3/CD28 beads, the T cell proliferation rate can reach 34%. After TGFβ was added to the system, the proliferation ratio of T cells decreased significantly, from 34% to 10.8%. If #1-1 (R1054), #1-2, #1-3, #1-4, and #2 were added at the same time, T cell proliferation rate after -1(R1056), #2-2, #2-3, #2-4, #3-1(R1055), #3-2, #3-3, or #3-4 All increased to varying degrees. Among them, the inhibition was strongest after the addition of R1055. Compared with the group without recombinant antibody, the proliferation rate of the highest group (R1055) increased by 22.13%, which was close to the proliferation rate of the control group without TGFβ1. (34%), and the proliferation rate showed a dependence on the concentration of each group of R1054, R1055, and R1056.
本发明是基于发明人的下列发现而完成的：研究发现，肿瘤微环境中TGFβ增加与免疫逃逸相关，TGFβ水平升高会阻止幼稚T细胞向Th1效应子表型分化，促进其向Treg亚型的转化，并抑制树突状细胞的抗原呈递功能，因此，发明人对TGFβ受体2(TGFβRII)进行了改造，并利用突变改造后的TGFβRII构建了重组抗体，通过对重组抗体进行体外结合活性、细胞表面TGFβRII与TGFβ结合阻断、T细胞增殖抑制等实验，获得了三种与TGFβ具有良好的结合活性，可以有效阻断细胞表面TGFβRII与TGFβ结合，并促进T细胞增殖的重组抗体，所述抗体可以有效治疗或预防肿瘤。The present invention is based on the following findings of the inventor: research has found that increased TGFβ in the tumor microenvironment is related to immune escape, and increased TGFβ levels will prevent naive T cells from differentiating into Th1 effector phenotypes and promote their differentiation into Treg subtypes. transformation and inhibit the antigen presentation function of dendritic cells. Therefore, the inventor modified TGFβ receptor 2 (TGFβRII) and constructed a recombinant antibody using the mutated and modified TGFβRII. By performing in vitro binding activity on the recombinant antibody , cell surface TGFβRII and TGFβ binding blockade, T cell proliferation inhibition and other experiments, we obtained three recombinant antibodies that have good binding activity with TGFβ, can effectively block the binding of TGFβRII and TGFβ on the cell surface, and promote T cell proliferation. Therefore, The above-mentioned antibodies can effectively treat or prevent tumors.
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制， 本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。 Although the embodiments of the present invention have been shown and described above, it can be understood that the above-mentioned embodiments are illustrative and should not be construed as limitations of the present invention. Those of ordinary skill in the art may make changes, modifications, substitutions and variations to the above-described embodiments within the scope of the present invention.

Claims (30)

  1. 一种重组抗体，其包括两条相同的肽链1，每条肽链1包括：A recombinant antibody comprising two identical peptide chains 1, each peptide chain 1 comprising:
    1)第一TGFβ RII片段；以及1) The first TGFβ RII fragment; and
    2)抗体重链恒定区；2) Antibody heavy chain constant region;
    其中，所述抗体重链恒定区的C端与所述第一TGFβ RII片段的N端相连。Wherein, the C-terminus of the antibody heavy chain constant region is connected to the N-terminus of the first TGFβ RII fragment.
  2. 根据权利要求1所述的重组抗体，其中所述第一TGFβ RII片段包括野生型TGFβ RII胞外结构域；The recombinant antibody of claim 1, wherein the first TGFβ RII fragment includes a wild-type TGFβ RII extracellular domain;
    任选地，所述第一TGFβ RII片段包括所述野生型TGFβ RII胞外结构域的N-末端片段、第一连接肽和所述野生型TGFβ RII胞外结构域的C-末端片段，其中，所述野生型TGFβ RII胞外结构域的C-末端片段包括野生型TGFβ RII胞外结构域C-末端的至多122个氨基酸。Optionally, the first TGFβ RII fragment includes an N-terminal fragment of the wild-type TGFβ RII extracellular domain, a first connecting peptide and a C-terminal fragment of the wild-type TGFβ RII extracellular domain, wherein , the C-terminal fragment of the wild-type TGFβ RII extracellular domain includes at most 122 amino acids of the C-terminus of the wild-type TGFβ RII extracellular domain.
  3. 根据权利要求2所述的重组抗体，其中所述野生型TGFβRII胞外结构域的N-末端片段的C端与所述第一连接肽的N端相连，所述第一连接肽的C端与所述野生型TGFβRII胞外结构域的C-末端片段的N端相连。The recombinant antibody according to claim 2, wherein the C-terminus of the N-terminal fragment of the wild-type TGFβRII extracellular domain is connected to the N-terminus of the first linking peptide, and the C-terminus of the first linking peptide is connected to The N-terminus of the C-terminal fragment of the wild-type TGFβRII extracellular domain is linked.
  4. 根据权利要求1至3任一项所述的重组抗体，其中每条肽链1进一步包括：第二TGFβRII片段；The recombinant antibody according to any one of claims 1 to 3, wherein each peptide chain 1 further includes: a second TGFβRII fragment;
    任选地，所述第二TGFβ RII片段包括野生型TGFβRII胞外结构域；Optionally, the second TGFβRII fragment includes a wild-type TGFβRII extracellular domain;
    任选地，所述第二TGFβRII片段包括所述野生型TGFβRII胞外结构域的N-末端片段、第二连接肽和所述野生型TGFβRII胞外结构域的C-末端片段，其中，所述野生型TGFβRII胞外结构域的C-末端片段包括所述野生型TGFβRII胞外结构域C-末端的至多122个氨基酸；Optionally, the second TGFβRII fragment includes an N-terminal fragment of the wild-type TGFβRII extracellular domain, a second connecting peptide and a C-terminal fragment of the wild-type TGFβRII extracellular domain, wherein, The C-terminal fragment of the wild-type TGFβRII extracellular domain includes up to 122 amino acids of the C-terminus of the wild-type TGFβRII extracellular domain;
    任选地，所述野生型TGFβRII胞外结构域的N-末端片段的C端与所述第二连接肽的N端相连，所述第二连接肽的C端与所述野生型TGFβRII胞外结构域的C-末端片段的N端相连；Optionally, the C-terminus of the N-terminal fragment of the wild-type TGFβRII extracellular domain is connected to the N-terminus of the second linking peptide, and the C-terminus of the second linking peptide is connected to the wild-type TGFβRII extracellular domain. The C-terminal fragment of the domain is linked to the N-terminus;
    任选地，所述第二TGFβRII片段的C端与所述抗体重链恒定区的N端相连。Optionally, the C-terminus of the second TGFβRII fragment is linked to the N-terminus of the antibody heavy chain constant region.
  5. 根据权利要求1至4任一项所述的重组抗体，其进一步包括两条相同的肽链2，所述每条肽链2包括第三TGFβRII片段和抗体轻链恒定区；The recombinant antibody according to any one of claims 1 to 4, further comprising two identical peptide chains 2, each of which includes a third TGFβRII fragment and an antibody light chain constant region;
    任选地，所述第三TGFβRII片段的C端与所述抗体轻链恒定区的N端相连；Optionally, the C-terminus of the third TGFβRII fragment is connected to the N-terminus of the antibody light chain constant region;
    任选地，所述每条肽链2与1条肽链1通过链间二硫键相连；Optionally, each of the peptide chains 2 is connected to one peptide chain 1 through an inter-chain disulfide bond;
    任选地，所述第三TGFβ RII片段包括野生型TGFβRII胞外结构域；Optionally, the third TGFβRII fragment includes a wild-type TGFβRII extracellular domain;
    任选地，所述第三TGFβRII片段包括所述野生型TGFβRII胞外结构域的N-末端片段、第三连接肽和所述野生型TGFβRII胞外结构域的C-末端片段，其中，所述野生型TGFβ RII胞外结构域的C-末端片段包括野生型TGFβ RII胞外结构域C-末端的至多122个氨基酸；Optionally, the third TGFβRII fragment includes an N-terminal fragment of the wild-type TGFβRII extracellular domain, a third connecting peptide and a C-terminal fragment of the wild-type TGFβRII extracellular domain, wherein, The C-terminal fragment of the wild-type TGFβ RII extracellular domain includes up to 122 amino acids of the C-terminus of the wild-type TGFβ RII extracellular domain;
    任选地，所述野生型TGFβRII胞外结构域的N-末端片段的C端与所述第三连接肽的N端相连，所述第三连接肽的C端与所述野生型TGFβRII胞外结构域的C-末端片段的N端相连。Optionally, the C-terminus of the N-terminal fragment of the wild-type TGFβRII extracellular domain is connected to the N-terminus of the third linking peptide, and the C-terminus of the third linking peptide is connected to the wild-type TGFβRII extracellular domain. The C-terminal fragment of the domain is linked to the N-terminus.
  6. 根据权利要求1至5任一项所述的重组抗体，其中所述野生型TGFβRII胞外结构域的N-末端片段包括野生型TGFβ RII胞外结构域N-末端的前6位氨基酸；The recombinant antibody according to any one of claims 1 to 5, wherein the N-terminal fragment of the wild-type TGFβRII extracellular domain includes the first 6 amino acids of the N-terminus of the wild-type TGFβRII extracellular domain;
    任选地，所述野生型TGFβ RII胞外结构域的C-末端片段包括野生型TGFβ RII胞外结构域C-末端的112～117个氨基酸；Optionally, the C-terminal fragment of the wild-type TGFβ RII extracellular domain includes 112 to 117 amino acids of the C-terminus of the wild-type TGFβ RII extracellular domain;
    任选地，所述野生型TGFβRII胞外结构域包括SEQ ID NO:2所示的氨基酸序列；Optionally, the wild-type TGFβRII extracellular domain includes the amino acid sequence shown in SEQ ID NO: 2;
    任选地，所述第一连接肽和/或所述第二连接肽和/或第三连接肽为柔性片段；Optionally, the first connecting peptide and/or the second connecting peptide and/or the third connecting peptide are flexible fragments;
    任选地，所述第一连接肽和/或第二连接肽和/或第三连接肽为含有G、S氨基酸的短肽；Optionally, the first connecting peptide and/or the second connecting peptide and/or the third connecting peptide are short peptides containing G and S amino acids;
    任选地，所述第一连接肽和/或第二连接肽和/或第三连接肽含有5～15个氨基酸；Optionally, the first connecting peptide and/or the second connecting peptide and/or the third connecting peptide contain 5 to 15 amino acids;
    任选地，所述第一连接肽和/或第二连接肽和/或第三连接肽包括SEQ ID NO:3所示的氨基酸序列。Optionally, the first connecting peptide and/or the second connecting peptide and/or the third connecting peptide include the amino acid sequence shown in SEQ ID NO:3.
  7. 根据权利要求1至6任一项所述的重组抗体，其中所述第一TGFβRII片段、所述第二TGFβRII片段和所述第三TGFβRII片段包括SEQ ID NO:2、4、28或29所示的氨基酸序列。The recombinant antibody according to any one of claims 1 to 6, wherein the first TGFβRII fragment, the second TGFβRII fragment and the third TGFβRII fragment include those shown in SEQ ID NO: 2, 4, 28 or 29 amino acid sequence.
  8. 根据权利要求1至7任一项所述的重组抗体，其进一步包括第四连接肽；The recombinant antibody according to any one of claims 1 to 7, further comprising a fourth connecting peptide;
    任选地，所述第四连接肽的N端与所述抗体重链恒定区的C端相连，所述第四连接肽的C端与所述第一TGFβ RII片段的N端相连；Optionally, the N-terminus of the fourth linking peptide is connected to the C-terminus of the antibody heavy chain constant region, and the C-terminus of the fourth linking peptide is connected to the N-terminus of the first TGFβ RII fragment;
    任选地，所述第四连接肽为柔性片段；Optionally, the fourth connecting peptide is a flexible fragment;
    任选地，所述第四连接肽为含有G、S氨基酸的短肽；Optionally, the fourth connecting peptide is a short peptide containing G and S amino acids;
    任选地，所述第四连接肽包括SEQ ID NO:5所示的氨基酸序列。Optionally, the fourth connecting peptide includes the amino acid sequence shown in SEQ ID NO:5.
  9. 根据权利要求4至8任一项所述的重组抗体，其进一步包括第五连接肽；The recombinant antibody according to any one of claims 4 to 8, further comprising a fifth connecting peptide;
    任选地，所述第五连接肽的N端与所述第二TGFβ RII片段的C端相连，所述第五连接肽的C端与所述抗体重链恒定区的N端相连；Optionally, the N-terminus of the fifth linking peptide is connected to the C-terminus of the second TGFβ RII fragment, and the C-terminus of the fifth linking peptide is connected to the N-terminus of the antibody heavy chain constant region;
    任选地，进一步包括第六连接肽，所述第六连接肽的N端与所述第三TGFβ RII片段的C端相连，所述第六连接肽的C端与所述抗体轻链恒定区的N端相连；Optionally, further comprising a sixth connecting peptide, the N-terminus of the sixth connecting peptide is connected to the C-terminus of the third TGFβ RII fragment, and the C-terminus of the sixth connecting peptide is connected to the antibody light chain constant region The N-terminal is connected;
    任选地，所述第五连接肽和/或所述第六连接肽为柔性片段；Optionally, the fifth connecting peptide and/or the sixth connecting peptide is a flexible fragment;
    任选地，所述第五连接肽和/或所述第六连接肽为含有G、S氨基酸的短肽；Optionally, the fifth connecting peptide and/or the sixth connecting peptide is a short peptide containing G and S amino acids;
    任选地，所述第五连接肽和/或所述第六连接肽包括SEQ ID NO:5所示的氨基酸序列。Optionally, the fifth connecting peptide and/or the sixth connecting peptide includes the amino acid sequence shown in SEQ ID NO:5.
  10. 根据权利要求1至9任一项所述的重组抗体，其中所述抗体重链恒定区或所述抗体轻链恒定区的至少一部分来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一；The recombinant antibody according to any one of claims 1 to 9, wherein at least a part of the antibody heavy chain constant region or the antibody light chain constant region is from a murine antibody, a human antibody, a primate antibody or a mutation thereof at least one of the entities;
    任选地，所述抗体重链恒定区或所述抗体轻链恒定区的至少一部分来自于人源抗体或其突变体；Optionally, at least a portion of the antibody heavy chain constant region or the antibody light chain constant region is derived from a human antibody or a mutant thereof;
    任选地，所述抗体重链恒定区或所述抗体轻链恒定区的至少一部分来自于人源抗体IgG或其突变体；Optionally, at least a portion of the antibody heavy chain constant region or the antibody light chain constant region is derived from human antibody IgG or a mutant thereof;
    任选地，所述抗体重链恒定区或所述抗体轻链恒定区的至少一部分来自于人源抗体IgG1或其突变体；Optionally, at least a portion of the antibody heavy chain constant region or the antibody light chain constant region is derived from human antibody IgG1 or a mutant thereof;
    任选地，所述抗体重链恒定区相较于野生型人源IgG1具有第234位、第235位和第447位氨基酸突变； Optionally, the antibody heavy chain constant region has amino acid mutations at positions 234, 235 and 447 compared to wild-type human IgG1;
    任选地，所述抗体重链恒定区相较于野生型人源IgG1具有L234A、L235A和K447A突变；Optionally, the antibody heavy chain constant region has L234A, L235A and K447A mutations compared to wild-type human IgG1;
    任选地，所述抗体重链恒定区包括SEQ ID NO:7所示的氨基酸序列；Optionally, the antibody heavy chain constant region includes the amino acid sequence shown in SEQ ID NO:7;
    任选地，所述抗体轻链恒定区包括SEQ ID NO:8所示的氨基酸序列。Optionally, the antibody light chain constant region includes the amino acid sequence shown in SEQ ID NO:8.
  11. 根据权利要求1至10任一项所述的重组抗体，其中所述肽链1包括SEQ ID NO:21、34、35或58所示的氨基酸序列；The recombinant antibody according to any one of claims 1 to 10, wherein said peptide chain 1 includes the amino acid sequence shown in SEQ ID NO: 21, 34, 35 or 58;
    所述肽链2包括SEQ ID NO:23、38、39或59所示氨基酸序列。The peptide chain 2 includes the amino acid sequence shown in SEQ ID NO: 23, 38, 39 or 59.
  12. 根据权利要求11所述的重组抗体，其包括：The recombinant antibody according to claim 11, comprising:
    1)SEQ ID NO:21和SEQ ID NO:23所示的氨基酸序列；或1) The amino acid sequence shown in SEQ ID NO:21 and SEQ ID NO:23; or
    2)SEQ ID NO:34和SEQ ID NO:38所示的氨基酸序列；或2) The amino acid sequence shown in SEQ ID NO:34 and SEQ ID NO:38; or
    3)SEQ ID NO:35和SEQ ID NO:39所示的氨基酸序列；或3) The amino acid sequence shown in SEQ ID NO:35 and SEQ ID NO:39; or
    4)SEQ ID NO:58和SEQ ID NO:59所示的氨基酸序列。4) The amino acid sequence shown in SEQ ID NO:58 and SEQ ID NO:59.
  13. 一种重组抗体，其包括两条相同的肽链3，每条肽链3包括：A recombinant antibody including two identical peptide chains 3, each peptide chain 3 including:
    1)第四TGFβ RII片段；1) The fourth TGFβ RII fragment;
    2)抗体Fc区；以及2) Antibody Fc region; and
    3)第五TGFβ RII片段或肿瘤靶向肽；3) The fifth TGFβ RII fragment or tumor-targeting peptide;
    其中，所述抗体Fc区的C端与所述第四TGFβ RII片段的N端相连，所述抗体Fc区的N端与所述第五TGFβ RII片段或肿瘤靶向肽的C端相连。Wherein, the C-terminus of the Fc region of the antibody is connected to the N-terminus of the fourth TGFβ RII fragment, and the N-terminus of the Fc region of the antibody is connected to the C-terminus of the fifth TGFβ RII fragment or the tumor-targeting peptide.
  14. 根据权利要求13所述的重组抗体，其中所述第四TGFβ RII片段和/或所述第五TGFβ RII片段包括野生型TGFβ RII胞外结构域；The recombinant antibody of claim 13, wherein the fourth TGFβ RII fragment and/or the fifth TGFβ RII fragment comprises a wild-type TGFβ RII extracellular domain;
    任选地，所述第四TGFβ RII片段包括野生型TGFβ RII胞外结构域的N-末端片段、第七连接肽和野生型TGFβ RII胞外结构域的C-末端片段，其中，所述野生型TGFβ RII胞外结构域的C-末端片段包括野生型TGFβ RII胞外结构域C-末端的至多122个氨基酸；Optionally, the fourth TGFβ RII fragment includes an N-terminal fragment of wild-type TGFβ RII extracellular domain, a seventh connecting peptide and a C-terminal fragment of wild-type TGFβ RII extracellular domain, wherein the wild-type The C-terminal fragment of the extracellular domain of TGFβ RII includes up to 122 amino acids of the C-terminus of the extracellular domain of wild-type TGFβ RII;
    任选地，所述第五TGFβ RII片段包括野生型TGFβ RII胞外结构域的N-末端片段、第八连接肽和野生型TGFβ RII胞外结构域的C-末端片段，其中，所述野生型TGFβ RII胞外结构域的C-末端片段包括野生型TGFβ RII胞外结构域C-末端的至多122个氨基酸。Optionally, the fifth TGFβ RII fragment includes an N-terminal fragment of wild-type TGFβ RII extracellular domain, an eighth linker peptide and a C-terminal fragment of wild-type TGFβ RII extracellular domain, wherein the wild-type The C-terminal fragment of the wild-type TGFβ RII extracellular domain includes up to 122 amino acids of the C-terminus of the wild-type TGFβ RII extracellular domain.
  15. 根据权利要求14所述的重组抗体，其中所述第四TGFβ RII片段中，所述野生型TGFβRII胞外结构域的N-末端片段的C端与所述第七连接肽的N端相连，所述第七连接肽的C端与所述野生型TGFβRII胞外结构域的C-末端片段的N端相连；The recombinant antibody according to claim 14, wherein in the fourth TGFβRII fragment, the C-terminus of the N-terminal fragment of the wild-type TGFβRII extracellular domain is connected to the N-terminus of the seventh connecting peptide, so The C-terminus of the seventh connecting peptide is connected to the N-terminus of the C-terminal fragment of the wild-type TGFβRII extracellular domain;
    任选地，所述第五TGFβ RII片段中，所述野生型TGFβRII胞外结构域的N-末端片段的C端与所述第八连接肽的N端相连，所述第八连接肽的C端与所述野生型TGFβRII胞外结构域的C-末端片段的N端相连；Optionally, in the fifth TGFβRII fragment, the C-terminus of the N-terminal fragment of the wild-type TGFβRII extracellular domain is connected to the N-terminus of the eighth linking peptide, and the C-terminus of the eighth linking peptide is The end is connected to the N-terminus of the C-terminal fragment of the wild-type TGFβRII extracellular domain;
    任选地，所述第七连接肽和/或所述第八连接肽为柔性片段；Optionally, the seventh connecting peptide and/or the eighth connecting peptide are flexible fragments;
    任选地，所述第七连接肽和/或所述第八连接肽为含有G、S氨基酸的短肽；Optionally, the seventh connecting peptide and/or the eighth connecting peptide is a short peptide containing G and S amino acids;
    任选地，所述第七连接肽和/或所述第八连接肽含有5～15个氨基酸；Optionally, the seventh connecting peptide and/or the eighth connecting peptide contains 5 to 15 amino acids;
    任选地，所述第七连接肽和/或所述第八连接肽包括SEQ ID NO:3所示的氨基酸序列。Optionally, the seventh connecting peptide and/or the eighth connecting peptide includes the amino acid sequence shown in SEQ ID NO:3.
  16. 根据权利要求13至15任一项所述的重组抗体，其进一步包括第九连接肽；The recombinant antibody according to any one of claims 13 to 15, further comprising a ninth connecting peptide;
    任选地，所述第四TGFβ RII片段的N端与所述第九连接肽的C端相连，所述第九连接肽的N端与所述抗体Fc区的C端相连。Optionally, the N-terminus of the fourth TGFβ RII fragment is connected to the C-terminus of the ninth linking peptide, and the N-terminus of the ninth linking peptide is connected to the C-terminus of the antibody Fc region.
  17. 根据权利要求16所述的重组抗体，其进一步包括第十连接肽；The recombinant antibody according to claim 16, further comprising a tenth connecting peptide;
    任选地，所述第五TGFβ RII片段的C端与所述第十连接肽的N端相连，所述第十连接肽的C端与所述抗体Fc区的N端相连；Optionally, the C-terminal of the fifth TGFβ RII fragment is connected to the N-terminal of the tenth connecting peptide, and the C-terminal of the tenth connecting peptide is connected to the N-terminal of the Fc region of the antibody;
    任选地，所述第九连接肽和/或第十连接肽为柔性片段；Optionally, the ninth connecting peptide and/or the tenth connecting peptide is a flexible fragment;
    任选地，所述第九连接肽和/或第十连接肽为含有G、S氨基酸的短肽；Optionally, the ninth connecting peptide and/or the tenth connecting peptide is a short peptide containing G and S amino acids;
    任选地，所述第九连接肽和/或第十连接肽包括SEQ ID NO:5所示的氨基酸序列。Optionally, the ninth connecting peptide and/or the tenth connecting peptide includes the amino acid sequence shown in SEQ ID NO:5.
  18. 根据权利要求13至17任一项所述的重组抗体，其中所述野生型TGFβ RII胞外结构域包括SEQ ID NO:2所示的氨基酸序列；The recombinant antibody according to any one of claims 13 to 17, wherein the wild-type TGFβ RII extracellular domain includes the amino acid sequence shown in SEQ ID NO: 2;
    任选地，所述第四TGFβ RII片段和第五TGFβ RII片段包括SEQ ID NO:2、4、28或29所示的氨基酸序列；Optionally, the fourth TGFβ RII fragment and the fifth TGFβ RII fragment include the amino acid sequence shown in SEQ ID NO: 2, 4, 28 or 29;
    任选地，所述肿瘤靶向肽包括SEQ ID NO:9或SEQ ID NO:26所示的氨基酸序列；Optionally, the tumor targeting peptide includes the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 26;
    任选地，所述每条肽链3通过链间二硫键相连；Optionally, each of the peptide chains 3 is connected through inter-chain disulfide bonds;
    任选地，所述抗体Fc区的至少一部分来自于鼠源抗体、人源抗体、灵长目源抗体或其突变体的至少之一；Optionally, at least a portion of the Fc region of the antibody is derived from at least one of a murine antibody, a human antibody, a primate antibody, or a mutant thereof;
    任选地，所述抗体Fc区的至少一部分来自于人源抗体或其突变体；Optionally, at least a portion of the Fc region of the antibody is derived from a human antibody or a mutant thereof;
    任选地，所述抗体Fc区的至少一部分来自于人源抗体IgG或其突变体；Optionally, at least a portion of the antibody Fc region is derived from human antibody IgG or a mutant thereof;
    任选地，所述抗体Fc区的至少一部分来自于人源抗体IgG1或其突变体；Optionally, at least a portion of the antibody Fc region is derived from human antibody IgG1 or a mutant thereof;
    任选地，所述抗体Fc区相较于野生型人源IgG1具有第234位、第235位和第447位氨基酸突变；Optionally, the antibody Fc region has amino acid mutations at positions 234, 235 and 447 compared to wild-type human IgG1;
    任选地，所述抗体Fc区相较于野生型人源IgG1具有L234A、L235A和K447A突变；Optionally, the antibody Fc region has L234A, L235A and K447A mutations compared to wild-type human IgG1;
    任选地，所述抗体Fc区包括SEQ ID NO:6所示的氨基酸序列。Optionally, the antibody Fc region includes the amino acid sequence shown in SEQ ID NO: 6.
  19. 根据权利要求12至17任一项所述的重组抗体，其中所述肽链3包括SEQ ID NO:17、19、30、31、32、33、54或55所示的氨基酸序列。The recombinant antibody according to any one of claims 12 to 17, wherein said peptide chain 3 includes the amino acid sequence shown in SEQ ID NO: 17, 19, 30, 31, 32, 33, 54 or 55.
  20. 一种核酸分子，其中所述核酸分子编码权利要求1至19任一项所述的重组抗体。A nucleic acid molecule encoding the recombinant antibody of any one of claims 1 to 19.
  21. 根据权利要求20所述的核酸分子，其中所述核酸分子包括SEQ ID NO:20、44、45或52所示的核苷酸序列；The nucleic acid molecule according to claim 20, wherein the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO: 20, 44, 45 or 52;
    任选地，所述核酸分子包括SEQ ID NO:22、48、49或53所示的核苷酸序列。Optionally, the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO: 22, 48, 49 or 53.
  22. 根据权利要求20所述的核酸分子，其中所述核酸分子包括SEQ ID NO:16、18、40、41、42、43、56或57所示 的核苷酸序列。The nucleic acid molecule according to claim 20, wherein the nucleic acid molecule comprises SEQ ID NO: 16, 18, 40, 41, 42, 43, 56 or 57 nucleotide sequence.
  23. 一种表达载体，其携带权利要求20至22任一项所述的核酸分子。An expression vector carrying the nucleic acid molecule of any one of claims 20 to 22.
  24. 一种重组细胞，其携带权利要求20至22任一项所述的核酸分子、权利要求23所述的表达载体或者表达权利要求1～19任一项所述的重组抗体。A recombinant cell carrying the nucleic acid molecule of any one of claims 20 to 22, the expression vector of claim 23, or expressing the recombinant antibody of any one of claims 1 to 19.
  25. 根据权利要求24所述的重组细胞，其中所述重组细胞为真核细胞；The recombinant cell according to claim 24, wherein the recombinant cell is a eukaryotic cell;
    任选地，所述重组细胞为哺乳动物细胞。Optionally, the recombinant cells are mammalian cells.
  26. 一种组合物，其包括权利要求1至19任一项所述的重组抗体、权利要求20至22任一项所述的核酸分子、权利要求23所述的表达载体或权利要求24至25任一项所述的重组细胞。A composition comprising the recombinant antibody of any one of claims 1 to 19, the nucleic acid molecule of any one of claims 20 to 22, the expression vector of claim 23, or any of claims 24 to 25. The recombinant cell described in one item.
  27. 权利要求1～19任一项所述的重组抗体、权利要求20至22任一项所述的核酸分子、权利要求23所述的表达载体、权利要求24～25任一项所述的重组细胞或权利要求26所述的组合物在制备药物中的用途，所述药物用于预防或治疗肿瘤。The recombinant antibody according to any one of claims 1 to 19, the nucleic acid molecule according to any one of claims 20 to 22, the expression vector according to claim 23, and the recombinant cell according to any one of claims 24 to 25. Or the use of the composition of claim 26 in the preparation of medicaments for preventing or treating tumors.
  28. 一种药物，其包括：权利要求1至19任一项所述的重组抗体、权利要求20至22任一项所述的核酸分子、权利要求23所述的表达载体、权利要求24至25任一项所述的重组细胞或权利要求26所述的组合物。A medicine, which includes: the recombinant antibody described in any one of claims 1 to 19, the nucleic acid molecule described in any one of claims 20 to 22, the expression vector described in claim 23, any of claims 24 to 25 The recombinant cell of claim 26 or the composition of claim 26.
  29. 权利要求1至19任一项所述的重组抗体在制备试剂盒中的用途，所述试剂盒用于检测TGFβ；The use of the recombinant antibody according to any one of claims 1 to 19 in the preparation of a kit for detecting TGFβ;
    任选的，所述TGFβ包括TGFβ1和TGFβ3。Optionally, the TGFβ includes TGFβ1 and TGFβ3.
  30. 一种试剂盒，其所述试剂盒包括权利要求1至19任一项所述的重组抗体。 A kit comprising the recombinant antibody according to any one of claims 1 to 19.
PCT/CN2023/091569 2022-04-29 2023-04-28 Recombinant antibody and use thereof WO2023208191A1 (en)

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