WO2018176505A1 - 一种恢复衰竭性免疫细胞功能的融合蛋白及其应用 - Google Patents

一种恢复衰竭性免疫细胞功能的融合蛋白及其应用 Download PDF

Info

Publication number
WO2018176505A1
WO2018176505A1 PCT/CN2017/079679 CN2017079679W WO2018176505A1 WO 2018176505 A1 WO2018176505 A1 WO 2018176505A1 CN 2017079679 W CN2017079679 W CN 2017079679W WO 2018176505 A1 WO2018176505 A1 WO 2018176505A1
Authority
WO
WIPO (PCT)
Prior art keywords
fusion protein
antibody
immune cell
seq
restoring
Prior art date
Application number
PCT/CN2017/079679
Other languages
English (en)
French (fr)
Inventor
岳喜连
刘根桃
吴国祥
Original Assignee
上海科医联创生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 上海科医联创生物科技有限公司 filed Critical 上海科医联创生物科技有限公司
Priority to US16/496,348 priority Critical patent/US10815303B2/en
Publication of WO2018176505A1 publication Critical patent/WO2018176505A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • the invention relates to the field of fusion protein technology, in particular to a fusion protein for restoring the function of depleted immune cells and an application thereof.
  • Cancer is the most serious disease that threatens people's lives and quality of life. Chronic diseases caused by viral infections are even more difficult problems in the world. There are various shortcomings in the current clinical application of drugs for cancer. For example, the side effects of chemotherapy drugs are large, and targeted drugs are prone to drug resistance (Curr Pharm Des. 2010, 16:3-10), and immunological checkpoint inhibitors are inefficient ( N Engl J Med 2012, 366: 2443-2454), chimeric antigen receptor T (Car-T) cell therapy has the disadvantage of high cytokine storms and high recurrence rates (Curr Opin Pediatr. 2017, 29: 27-33) .
  • HBV hepatitis B virus
  • PNAS liver cancer
  • Tumors occur as a result of genetic mutations in the process of cell division, and the growth of mutant cells loses regulatory control. If tumor cells are not cleared in a short period of time, they will gradually cause functional failure of tumor antigen-specific T cells (Nature Medicine 1999, 5: 677-685), forming tolerance to tumors, and the immune system is no longer sensitive to tumor cells. , causing further growth and spread of tumors, forming cancer (PNAS. 2002, 99: 12293-7). The occurrence of chronic diseases caused by viral infection is also a result of antigen-specific cellular failure (Trends Immunol. 2014, 35: 51-60; Blood 2007, 109: 4671-4678; Cell Death and Disease 2015, 6: e1694).
  • TIL tumor invasive lymphocytes
  • PNAS 2010, 107:7875-7880; JI 2007, 178:2714-2720 immunosuppressive receptors
  • Depleted T cells lose the ability to secrete cytokines, such as gamma interference IFN- ⁇ or the like (Blood 2013, 121: 1367-1376; J. Biomed. Biotechnol. 2011, 451694).
  • cytokines such as gamma interference IFN- ⁇ or the like
  • depleted T cells In phenotype, depleted T cells generally express high immunosuppressive checkpoint receptors, such as programmed death receptor (PD-1), programmed death ligand (PD-L), and T cell immunoglobulin 3 (Tim). -3), cytotoxic T lymphocyte antigen 4 (CTLA-4) and lymphocyte activation gene 3 (Lag-3), etc. (Nat Immunol. 2011; 12: 492-9). Activation of these signaling pathways inhibits T cell expansion, reduces the function of effector T cells, promotes T cell apoptosis, and induces immune tolerance (J Clin Invest. 2011, 121: 2350-2360).
  • PD-1 programmed death receptor
  • PD-L programmed death ligand
  • Tim T cell immunoglobulin 3
  • CTLA-4 cytotoxic T lymphocyte antigen 4
  • Lag-3 lymphocyte activation gene 3
  • Antibodies can specifically recognize protein antigens on the surface of target cells, and checkpoint inhibitory antibodies can suppress immunosuppressive signals on the surface of depleted cells, for example, by inhibiting the binding of PD-1 antibody Pembrolizumab and Nivolumab to PD-1.
  • the conduction and activation of the function of restoring immune cells has clinically produced a clinical effect in the treatment of cancer, especially in some patients, the complete disappearance of the tumor (N Engl J Med 2012, 366: 2455-2465; N Engl J Med 2012, 366: 2443-2454).
  • IL-2 interleukin 2
  • cytokines activate all cells expressing their receptors, which may activate non-target cells, which may cause a series of toxic side effects and limit its clinical application (JI 2014, 192: 5451-5458). Therefore, the clinical application range and efficacy of IL-2 have very limited limitations (Immunity 2013, 38: 13-25).
  • the object of the present invention is to overcome the deficiencies in the prior art and to provide a fusion protein which restores the function of depleted immune cells, which can recognize both depleted immune cells, expand the number of immune cells, and restore the function of immune cells.
  • the present invention adopts the following technical solutions:
  • the present invention provides a fusion protein for restoring the function of a failing immune cell, comprising a functional region for identifying a failing immune cell and a functional region for activating attenuating immune cells, and the two functional regions are linked by a non-functional amino acid fragment of a certain length. Therefore, the protein folding of the two functional regions does not interfere with each other, ensuring the dual function of the fusion protein.
  • the technical measures adopted by the present invention further include:
  • the functional region recognizing a depleted immune cell is a phenotype receptor that recognizes a debilitating immune cell using an immunological checkpoint-specific antibody, and the phenotypic receptor of the depleted immune cell is co-suppressive Immune checkpoint.
  • the phenotypic receptors of the depleted immune cells include, but are not limited to, PD-1, PD-L1, TIM-3, CTLA-4, LAG-3, such receptor genes on the surface of immune cells Overexpression leads to the failure of immune cells and lack of function.
  • the amino acid sequence of the PD-1 is the sequence of SEQ ID NO: 1
  • the amino acid sequence of the PD-L1 is the sequence of SEQ ID NO: 2
  • the amino acid sequence of the TIM-3 is SEQ ID.
  • the sequence of NO: 3 the amino acid sequence of CTLA-4 is the sequence of SEQ ID NO: 4
  • the amino acid sequence of LAG-3 is the sequence of SEQ ID NO: 5.
  • the specific antibodies of the phenotype receptor include, but are not limited to, PD-1 antibody, CTLA-4 antibody, PD-L1 antibody, TIM-3 antibody, LAG-3 antibody, and antibodies and receptors.
  • Binding blocks the binding and signaling of the receptor to its ligand, preventing checkpoint ligands in tumor tissue or tumor microenvironment It inhibits the immune system and simultaneously carries the activation function of the fusion protein to the surface of the target cell, making the activation functional zone more prone to effect and preventing side effects caused by the off-target effect.
  • the amino acid sequence of the PD-1 antibody comprises the sequences shown in SEQ ID NOS: 6 to 9
  • the amino acid sequence of the CTLA-4 antibody comprises the sequences shown in SEQ ID NOS: 10 to 11, the PD-L1
  • the amino acid sequence of the antibody includes the sequences shown in SEQ ID NOS: 12-13.
  • said functional region that activates amplifying depleted immune cells is used to activate failing immune cells using cytokines or functionally similar mutants, or ligands or functionally similar mutants of phenotypic receptors, or activating antibody.
  • the cytokine or functionally similar mutants include, but are not limited to, IL-2, IL-15, IL-21; the receptors thereof are predominantly expressed on the surface of T cells and NK cells.
  • a cytokine-like mutant is one that increases its activation response to a target cell by a mutation of the amino acid sequence or simply utilizes the polypeptide of the functional domain without changing its basic function, thereby reducing its effect on non-target cells. For example, by altering the amino acid sequence on IL-2, the mutant may reduce activation of T regulatory cells and reduce the effects on non-target cells and organs, thereby reducing the side effects of clinical application.
  • the ligand or functionally similar mutant of the phenotype receptor includes, but is not limited to, 4-1BBL, OX-40L.
  • the activating receptor on the surface of the immune cell can bind to the corresponding ligand, through activation and signaling, to achieve the purpose of activating and amplifying the target cell; its function similar to the mutant may be through amino acid sequence mutation or just contain the functional part, the purpose It is to change the affinity or part of the function with the receptor.
  • the activating antibody comprises an antibody against 4-1BB, an antibody against XO-40L, an antibody against CD28, an antibody against CD3, an antibody against CD27.
  • the activating antibody can achieve a ligand-like function, stimulate the activation signal on the target cell, and achieve the purpose of amplifying the target cell.
  • amino acid sequence of IL-2 is the sequence shown in SEQ ID NO: 14
  • amino acid sequence of IL-15 is the sequence shown in SEQ ID NO: 15
  • amino acid sequence of IL-21 is SEQ ID. NO: sequence shown in 16.
  • amino acid sequence of 4-1BBL is the sequence of SEQ ID NO: 17
  • amino acid sequence of OX-40L is the sequence of SEQ ID NO: 18.
  • the amino acid sequence of the antibody of 4-1BB includes the sequences shown in SEQ ID NOS: 19 to 20, and the amino acid sequence of the antibody of XO-40L includes the sequences shown in SEQ ID NOS: 21-22.
  • the functional region recognizing the depleted immune cells is a phenotype receptor PD-1 that recognizes a failing immune cell using a PD-1 antibody, and activates a functional region that amplifies the depleted immune cells to activate debilitating immunity using IL-2. cell.
  • the preparation step of a fusion protein for restoring the function of depleted immune cells is as follows:
  • Step 1) The C-terminus of the human PD-1 antibody heavy chain and the N-terminal amino acid sequence of interleukin 2 are linked by a non-functional amino acid to form a fusion protein heavy chain construct gene;
  • Step 2 co-transforming the fusion protein construct gene of step 1) and the light chain gene of the synthesized PD-1 antibody into an expression vector, and transfecting into hamster ovary cells;
  • Step 3) The hamster ovary cells described in the step 2) are placed in an incubator for a period of time, and the supernatant is taken and purified to obtain a recombinant fusion protein.
  • the human PD-1 antibody heavy chain is the sequence shown in SEQ ID NO: 6, the light chain of the synthetic PD-1 antibody is the sequence shown in SEQ ID NO: 7, and the interleukin 2 is the SEQ ID.
  • the depleted immune cells are at least one of a depleted specific T cell and a depleted NK cell.
  • the invention also provides the use of a fusion protein as described above for restoring debilitating immune cell function, the medical use of the fusion protein being used for non-diagnostic or non-therapeutic purposes.
  • the application is a single application of the fusion protein or a combination of a fusion protein and a composition of chemotherapy, a targeted drug, an antibody drug, and a cell therapy for the preparation of a medicament for treating a disease caused by immune cell failure.
  • the disease comprises cancer and a chronic viral infection.
  • the cancer comprises renal cell carcinoma, melanoma, lymphoma, colorectal cancer, liver cancer, soft nest cancer, head and neck squamous cell carcinoma, bladder cancer, lung cancer; and the virus in the chronic viral infection disease includes HIV, HBV, HCV, EBV, HPV, CMV.
  • the present invention has the following beneficial effects:
  • the fusion protein of the invention can meet the needs of the patient's immune recovery, and the recombinant fusion protein can recognize the depleted immune cells, expand the quantity thereof and restore its function; and its clinical application can enhance the inhibition.
  • the function of tumor growth and control of viral infection has a good clinical prospect and a wide range of applications.
  • a band is a protein in a reduced state
  • B band is a protein in an original state
  • MK is a protein standard molecular weight marker.
  • FIG. 2 is a schematic diagram showing the ratio of CD8+ T cells in vitro in a recombinant fusion protein human PBMC prepared according to an embodiment of the present invention: wherein the fusion protein group is the first part of FIG. 2, and the PD-1 antibody group is the second part of FIG. In part, the interleukin 2 group is the third part of Fig. 2.
  • Figure 3 is a schematic diagram showing the ratio of PD-1+ cells in the CD8+ T cells in vitro by the recombinant fusion protein human PBMC prepared in one embodiment of the present invention: wherein the fusion protein group is the first part of Figure 3, the PD-1 antibody The group is the second part of Fig. 3, and the group of interleukin 2 is the third part of Fig. 3.
  • FIG. 4 is a schematic diagram showing the ratio of CD8+ T cells and NK cells in peripheral blood lymphocytes of a recombinant fusion protein mouse in an embodiment of the present invention: wherein the fusion protein group is the third part of FIG. 4, and the interleukin 2 group In the second part of Figure 4, the PBS control group is the first part of Figure 4.
  • the present invention provides a fusion protein for restoring the function of a failing immune cell, comprising a functional region for recognizing a failing immune cell and a functional region for activating attenuating immune cells, and the two functional regions are passed through a non-functional amino acid fragment of a certain length.
  • the invention also provides the use of the above fusion protein for the preparation of a medicament for treating a disease caused by immune cell failure.
  • This example is the genetic construction and production purification of recombinant fusion proteins.
  • N-terminal amino acid sequence of the human PD-1 antibody heavy chain (SEQ ID NO: 6) C-terminus and interleukin 2 (SEQ ID NO: 14)
  • the functional amino acid (SGGGGSGGGGSGGGGSG) was ligated to form a fusion protein heavy chain construct gene (SEQ ID NO: 23), which was then transferred into the eukaryotic expression vector pcDNA3.1.
  • the light chain (SEQ ID NO: 7) gene of the PD-1 antibody was transferred into the eukaryotic expression vector pcDNA3.1 by gene synthesis, digestion and further cloning.
  • the heavy chain expression vector and the light chain expression vector of the fusion protein were simultaneously transfected into Chinese hamster ovary cells (CHO).
  • the transfected cells were cultured in a 37 ° C, 5% CO 2 incubator. After 72 hours, the supernatant was taken and further purified by Protein A affinity chromatography.
  • the final purified protein was a bifunctional recombinant fusion protein.
  • a non-fusion protein was prepared as described above according to the PD-1 antibody sequence (SEQ ID NO: 6, SEQ ID NO: 7) and the interleukin 2 sequence (SEQ ID NO: 14) by the above method.
  • the purified protein was confirmed by electrophoresis to confirm the molecular weight (Fig.
  • This example is a measure of the activity and function of a bifunctional recombinant fusion protein in vitro culture of human PBMC cells.
  • Peripheral blood was isolated and purified by lymphocyte density gradient centrifugation (Ficoll).
  • the cell density was diluted to 5 ⁇ 10 6 /ml in X-Vivo15 medium in a 24-well plate, and the test protein was added to a final concentration of 2 ⁇ g/ml. .
  • Figure 2 shows that fusion protein treatment (part 1 of Figure 2) significantly increased culture compared to unfused PD-1 antibody (part 2 of Figure 2) and interleukin 2 (part 3 of Figure 2).
  • the proportion of CD8+ cells in the cells 58.8%, 52.9%, 51.4%).
  • the ratio of PD-1+ cells in the fusion protein-treated group to CD8+ T cells was 3.16% (part 1 of Fig. 3), and that of the PD-1 antibody-treated group was 1.53% (Fig. 3).
  • the second part of Fig. 3) was 1.31% in the interleukin 2 treated group (part 3 of Fig. 3), so that the fusion protein significantly enhanced the expansion of the depleted cells (PD-1+) in CD8+ T cells.
  • the recombinant fusion protein designed according to the present invention can not only selectively amplify CD8+ T cells, but also directionally amplify depleted CD8+PD-1+ cells to achieve the intended purpose of the fusion protein.
  • This example is the effect of a bifunctional recombinant fusion protein on in vivo effector cells.
  • mice were injected with fusion protein (4 ⁇ g/day/day) or interleukin 2 (40 ⁇ g/day/day) or PBS (control group) through the abdominal cavity for 3 consecutive days.
  • fusion protein 4 ⁇ g/day/day
  • interleukin 2 40 ⁇ g/day/day
  • PBS control group
  • peripheral blood was taken from the tail, and anti-mouse CD8 was used.
  • NK1.1 flow The antibody was detected after staining, and the data analysis is shown in Fig. 4.
  • recombinant fusion protein significantly increased the proportion of NK cells in lymphocytes (Figure 4 Part 3, 27.0%).
  • the primary interleukin-2 did not increase the proportion of CD8+ cells compared to the control group (part 1 of Fig. 4, 15.5%) (part 2, 15.1% of Fig. 4), and fusion
  • the proportion of CD8+ T cells in peripheral blood of protein-treated mice (part 3 of Figure 4) was 22.8%.
  • In vivo experiments have shown that even with only a dose of native IL-210%, the immunological effects of the fusion proteins synthesized by the present invention can far exceed the native non-fusion proteins.
  • This example is the use of a bifunctional recombinant fusion protein for the preparation of a medicament for the treatment of a disease caused by immune cell failure.
  • the above dual-function recombinant fusion protein can be used alone or in combination with chemotherapy, targeted drugs, antibody drugs, and cell therapy to prepare drugs for treating diseases caused by immune cell failure, such as treating cancer drugs and treating chronic viral infections. Drugs, etc.
  • the fusion protein for restoring the function of the depleted immune cells of the present invention can recognize the depleted immune cells, activate the amplified immune cells, and restore the function of the immune cells to kill the antigen-positive cells. Since tumorigenesis and spread and chronic viral infection are the result of immune cell failure and immune system tolerance, the function of restoring depleted immune cells can enhance the body's ability to resist tumor and chronic viral infection. Therefore, the clinical status of the above fusion protein Application can enhance the function of inhibiting tumor growth and controlling viral infection, and has a good clinical prospect and a wide range of applications.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

一种恢复衰竭免疫细胞功能的融合蛋白，所述融合蛋白包含识别衰竭免疫细胞的功能区和激活扩增衰竭免疫细胞的功能区，两个功能区通过一定长度的非功能性氨基酸片段连接。识别功能区是利用免疫检查点特异性抗体识别衰竭性免疫细胞的表型受体；激活扩增功能区采用细胞因子或功能类似突变体、或者表面受体的配体或功能类似突变体、或者激活性抗体来激活衰竭性免疫细胞；其还涉及该融合蛋白的应用。试验表明，所述的融合蛋白既能识别衰竭性免疫细胞，又能激活扩增识别的免疫细胞，恢复免疫细胞杀伤抗原阳性细胞的功能，其可增强抑制肿瘤生长和控制病毒感染的功能，具有很好的临床前景和广泛的应用范围。

Description

一种恢复衰竭性免疫细胞功能的融合蛋白及其应用 技术领域
本发明涉及融合蛋白技术领域，尤其涉及一种恢复衰竭性免疫细胞功能的融合蛋白及其应用。
背景技术
癌症是常见且威胁人们生命和生活质量的最为严重疾病，由病毒感染而引起的慢性疾病更是困扰世界的难题。当前针对癌症的临床应用药物存在着各种不足，例如化疗药物副作用大，靶向药物容易产生耐药性(Curr Pharm Des.2010，16:3-10)，免疫检查点抑制剂有效率低(N Engl J Med 2012，366:2443-2454)，嵌合抗原受体T(Car-T)细胞疗法又有细胞因子风暴和复发率高的缺点(Curr Opin Pediatr.2017，29:27-33)。中国HBV(乙肝病毒)感染的人群达上亿人(APJCP,2011，12:1405-1408)，长期的病毒感染还会导致肝硬化、AIDS、子宫颈癌、肝癌等更为严重的疾病(PNAS 2012，109:1802-1829)，而市场上还缺乏治疗由HBV病毒引起疾病的有效药物(Clinical Science 2013，124:77-85)。因此，发现高效低毒新型治疗癌症与慢性病毒感染的药物，减少死亡率，提高患者的生活质量，是我国乃至世界医疗卫生领域最为迫切的课题和需求。
肿瘤的发生是由于机体细胞分裂过程中发生基因突变，突变细胞的生长失去调节控制的结果。如果肿瘤细胞不能在短期内被清除，会逐渐造成肿瘤抗原特异性T细胞的功能性衰竭(Nature Medicine 1999，5:677-685)，形成对肿瘤的耐受，免疫系统对肿瘤细胞不再敏感，造成肿瘤进一步的生长和扩散，形成癌症(PNAS.2002，99:12293-7)。由病毒感染导致的慢性疾病的发生也是抗原特异性细胞衰竭的结果(Trends Immunol.2014，35:51-60；Blood 2007，109:4671-4678；Cell Death and Disease 2015，6:e1694)。研究表明，大部分肿瘤侵润性淋巴细胞(TIL)和抗病毒特异性T细胞都表达免疫抑制性受体(PNAS 2010，107:7875-7880；JI 2007，178:2714-2720)，处于衰竭状态，丧失了特异性识别与杀伤抗原阳性靶细胞的功能。衰竭性T细胞丧失分泌细胞因子的能力，如伽马干扰 素(IFN-γ)等(Blood 2013，121:1367-1376；J.Biomed.Biotechnol.2011，451694)。即使机体内存在大量的特异性T细胞，衰竭的免疫细胞也不能清除抗原阳性靶细胞(J Immunol 2005；175:6169-6176)。在表型上，衰竭性T细胞普遍高表达免疫抑制检查点受体，例如程序性死亡受体(PD-1)，程序性死亡配体(PD-L)，T细胞免疫球蛋白3(Tim-3)，细胞毒T淋巴细胞抗原4(CTLA-4)和淋巴细胞活化基因3(Lag-3)等(Nat Immunol.2011；12:492-9)。这些信号通路的激活会抑制T细胞的扩增，降低效应T细胞的功能，促进T细胞的凋亡，引起免疫耐受(J Clin Invest.2011，121:2350-2360)。所以，激活这一类抗原特异性的免疫细胞群体，让他们重新恢复杀灭抗原阳性靶细胞的功能，才能清除肿瘤或病毒感染细胞(Trends Immunol.2015；36:265-276)，最终达到治愈的目的。
抗体可以特异性地识别靶细胞表面的蛋白抗原，检查点抑制性抗体可以阻抑衰竭性细胞表面的免疫抑制性信号，例如通过PD-1抗体Pembrolizumab和Nivolumab与PD-1的结合防止抑制性信号的传导与激活，恢复衰竭性免疫细胞的功能，已经在临床上产生治疗癌症的临床效果，尤其是在部分患者上获得了肿瘤完全消失(N Engl J Med 2012，366:2455-2465；N Engl J Med 2012，366:2443-2454)。但是，对缺乏效应细胞的患者临床效果不理想(NATURE 2014，515:568-571)，而且这种T细胞的恢复持续时间短(Science 2016，354:1160-1165)，患者体内的特异性免疫细胞又会很快回到衰竭状态(Science 2016，354:1165-1169)。同时，只是阻断T细胞抑制信号不会导致免疫细胞数量的增加，而缺乏T效应细胞的患者基本又不会产生临床效果(Nature 2014，515:568-571)，限制其临床的治疗效果。
利用白细胞生长因子的临床应用治疗肿瘤具有一定的效果和不同程度的副作用(Semin Oncol.2015，42:539–548)。白介素2(IL-2)的临床应用是第一个癌症治疗的免疫疗法，能彻底清除肿瘤细胞，在部分癌症患者达到完全治愈的效果(Cancer 2008，113:293–301；JCO 2005，23:133-141)。但是，细胞因子对所有表达其受体的细胞都会产生激活作用，从而可能激活非目的性细胞，临床应用会产生一系列毒副作用，限制它的临床应用(JI 2014，192:5451-5458)，所以目前IL-2的临床应用范围和疗效有非常大的局限性(Immunity 2013，38:13-25)。利用激活性4-1BB抗体(Oncology 2010，37:508-516)和ox-40抗体 (Cancer Science 2008 99:361-367)激活患者的免疫系统同样也会产生副作用。同样，利用激活性配体4-1BBL和OX-40L也会激活T细胞，但这些作用都不能针对衰竭的特异性细胞(J.Leukoc.Biol.21011，89:989–999)
截止目前，还没有一种既能针对性地识别衰竭的特异性T细胞，又能恢复其功能和扩增其数量的蛋白药物。
发明内容
本发明的目的在于克服现有技术中的缺陷，提供一种恢复衰竭性免疫细胞功能的融合蛋白，其既可识别衰竭性免疫细胞，又能扩增免疫细胞的数量，恢复免疫细胞的功能。
为实现上述目的，本发明采用如下技术方案：
本发明提供一种恢复衰竭性免疫细胞功能的融合蛋白，包含识别衰竭性免疫细胞的功能区和激活扩增衰竭性免疫细胞的功能区，两个功能区通过一定长度的非功能性氨基酸片段连接，从而使两个功能区的蛋白折叠不存在相互干扰，保证融合蛋白的双功能特征。
为了进一步优化上述技术方案，本发明所采取的技术措施还包括：
优选地，所述识别衰竭性免疫细胞的功能区是利用免疫检查点特异性抗体识别衰竭性免疫细胞的表型受体，所述衰竭性免疫细胞的表型受体是具有共抑制性功能的免疫检查点。
优选地，所述衰竭性免疫细胞的表型受体包括(但不限于)PD-1、PD-L1、TIM-3、CTLA-4、LAG-3，此类受体基因在免疫细胞表面的过度表达，导致免疫细胞的衰竭和功能缺少。
优选地，所述PD-1的氨基酸序列为SEQ ID NO：1所示序列，所述PD-L1的氨基酸序列为SEQ ID NO：2所示序列，所述TIM-3的氨基酸序列为SEQ ID NO：3所示序列，所述CTLA-4的氨基酸序列为SEQ ID NO：4所示序列，所述LAG-3的氨基酸序列为SEQ ID NO：5所示序列。
优选地，所述表型受体的特异性抗体包括(但不限于)PD-1抗体、CTLA-4抗体、PD-L1抗体、TIM-3抗体、LAG-3抗体，通过抗体与受体的结合，一方面阻断受体与其配体的结合与信号传达，防止肿瘤组织或肿瘤微环境中检查点配体 对免疫系统产生抑制作用，同时将融合蛋白的活化功能部分携带到靶细胞表面，使激活功能区更容易发生效应，防止脱靶效应产生的副作用。
优选地，所述PD-1抗体的氨基酸序列包括SEQ ID NO：6～9所示序列，所述CTLA-4抗体的氨基酸序列包括SEQ ID NO：10～11所示序列，所述PD-L1抗体的氨基酸序列包括SEQ ID NO：12～13所示序列。
优选地，所述激活扩增衰竭性免疫细胞的功能区用于激活衰竭性免疫细胞，其采用细胞因子或功能类似突变体、或者表型受体的配体或功能类似突变体、或者激活性抗体。
优选地，所述细胞因子或功能类似突变体包括(但不限于)IL-2、IL-15、IL-21；其受体主要表达在T细胞和NK细胞表面。细胞因子功能类似突变体是指在不改变其基本功能的前提下，通过氨基酸序列突变或只是利用功能区多肽，增加其对目的细胞的激活反应，减少其对非目的细胞的作用。例如，通过IL-2上氨基酸序列的改变，突变体可能降低与T调节性细胞的激活，减少对非靶细胞及器官的作用，从而减少临床应用的副作用。
优选地，所述表型受体的配体或功能类似突变体包括(但不限于)4-1BBL、OX-40L。免疫细胞表面的激活性受体可以与相应的配体结合，通过激活与信号传递，达到激活与扩增靶细胞的目的；其功能类似突变体可能通过氨基酸序列突变或只是包含功能区部分，目的是改变与受体的亲和力或者部分功能。
优选地，所述激活性抗体包括针对4-1BB的抗体、XO-40L的抗体、CD28的抗体、CD3的抗体、CD27的抗体。通过与激活性受体上激活位点的结合，激活性抗体可以达到类似于配体的功能，刺激靶细胞上的激活信号，达到扩增靶细胞的目的。
优选地，所述IL-2的氨基酸序列为SEQ ID NO：14所示序列，所述IL-15的氨基酸序列为SEQ ID NO：15所示序列，所述IL-21的氨基酸序列为SEQ ID NO：16所示序列。
优选地，所述4-1BBL的氨基酸序列为SEQ ID NO：17所示序列，所述OX-40L的氨基酸序列为SEQ ID NO：18所示序列。
优选地，所述4-1BB的抗体的氨基酸序列包括SEQ ID NO：19～20所示序列，所述XO-40L的抗体的氨基酸序列包括SEQ ID NO：21～22所示序列。
优选地，识别衰竭性免疫细胞的功能区是利用PD-1抗体识别衰竭性免疫细胞的表型受体PD-1，激活扩增衰竭性免疫细胞的功能区采用IL-2来激活衰竭性免疫细胞。
优选地，一种恢复衰竭性免疫细胞功能的融合蛋白的制备步骤如下：
步骤1)由人PD-1抗体重链的C端和白介素2的N端氨基酸序列通过非功能氨基酸连接，组成融合蛋白重链构造基因；
步骤2)将步骤1)所述的融合蛋白构造基因和合成的PD-1抗体的轻链基因共转入表达载体，并转染至仓鼠卵巢细胞；
步骤3)将步骤2)所述的仓鼠卵巢细胞放置在孵箱中培养一段时间，取上清液，纯化后制得重组融合蛋白。
优选地，所述人PD-1抗体重链为SEQ ID NO：6所示序列，所述合成的PD-1抗体的轻链为SEQ ID NO：7所示序列，所述白介素2为SEQ ID NO：14所示序列，所述非功能氨基酸的氨基酸序列为SGGGGSGGGGSGGGGSG，所述融合蛋白重链构造基因为SEQ ID NO：23所示序列，所述表达载体为真核动物表达载体pcDNA3.1。
优选地，所述衰竭性免疫细胞为衰竭的特异性T细胞、衰竭的NK细胞中的至少一种。
本发明还提供一种上述恢复衰竭性免疫细胞功能的融合蛋白的的应用，该融合蛋白的医药用途用于非诊断或非治疗目的。
优选地，所述应用为融合蛋白的单独应用或为融合蛋白与化疗、靶向药物、抗体药物、细胞治疗组成的联合应用，以用于制备治疗因免疫细胞衰竭而引起的疾病的药物。
优选地，所述疾病包括癌症和慢性病毒感染疾病。
优选地，所述癌症包括肾细胞癌、黑色素瘤、淋巴瘤、结直肠癌，肝癌、软巢癌、头颈部鳞癌、膀胱癌、肺癌；所述慢性病毒感染疾病中的病毒包括HIV、HBV、HCV、EBV，HPV、CMV。
与现有技术相比，本发明具有以下有益效果：
本发明所述的融合蛋白能够满足患者免疫恢复的需要，该重组融合蛋白既能识别衰竭的免疫细胞，又能扩增其数量，恢复其功能；且其临床应用可以增强抑 制肿瘤生长和控制病毒感染的功能，具有很好的临床前景和广泛的应用范围。
附图说明
图1为本发明一实施例中制备的重组融合蛋白的凝胶电泳分析图：A带为还原状态下的蛋白，B带为原始状态下的蛋白，MK为蛋白标准分子量标记。
图2为本发明一实施例中制备的重组融合蛋白人PBMC体外试验CD8+T细胞的比例的示意图：其中融合蛋白组为图2的第1部分，PD-1抗体组为图2的第2部分，白介素2组为图2的第3部分。
图3为本发明一实施例中制备的重组融合蛋白人PBMC体外试验PD-1+细胞在CD8+T细胞中的比例的示意图：其中融合蛋白组为图3的第1部分，PD-1抗体组为图3的第2部分，白介素2组为图3的第3部分。
图4为本发明一实施例中的重组融合蛋白小鼠体内试验CD8+T细胞和NK细胞在外周血淋巴细胞中的比例的示意图：其中融合蛋白组为图4的第3部分，白介素2组为图4的第2部分，PBS对照组为图4的第1部分。
具体实施方式
本发明提供了一种恢复衰竭性免疫细胞功能的融合蛋白，包含识别衰竭性免疫细胞的功能区和激活扩增衰竭性免疫细胞的功能区，两个功能区通过一定长度的非功能性氨基酸片段连接；本发明还提供了上述融合蛋白在制备治疗因免疫细胞衰竭而引起的疾病的药物中的应用。
下面结合附图和实施例，对本发明的具体实施方式作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。
实施例1
本实施例为重组融合蛋白的基因构建和生产纯化。
根据人PD-1抗体重链(SEQ ID NO：6)C端和白介素2(SEQ ID NO：14)的N端氨基酸序列，通过基因合成，酶切和进一步克隆，将两部分通过17个非功能氨基酸(SGGGGSGGGGSGGGGSG)连接，组成融合蛋白重链构造基因(SEQ ID NO：23)，然后转入真核动物表达载体pcDNA3.1。通过基因合成，酶切和 进一步克隆，将PD-1抗体的轻链(SEQ ID NO：7)基因转入真核动物表达载体pcDNA3.1。最后将融合蛋白的重链表达载体和轻链表达载体同时转染到中国仓鼠卵巢细胞(CHO)中。转染的细胞置于37℃、5％CO2孵箱中培养，72小时后取上清液，进一步通过ProteinA亲和层析纯化，最后纯化的蛋白为双功能重组融合蛋白。同时，根据PD-1抗体序列(SEQ ID NO：6、SEQ ID NO：7)和白介素2序列(SEQ ID NO：14)用上述方法制作做非融合蛋白，作为试验的对照。纯化的蛋白经过电泳检测确认分子量(图1)，证明依据本发明设计的重组融合蛋白可以通过CHO细胞生产。最后用分光光度计测试蛋白浓度，稀释在PBS中，用作进一步体内外的活性测试及功能研究。
实施例2
本实施例为双功能重组融合蛋白对人PBMC细胞体外培养的活性及功能测定。
人外周血经利用淋巴细胞密度梯度离心法(Ficoll)分离纯化，在24孔板中用X-Vivo15培养基中稀释细胞密度到5×106/ml，加入试验蛋白至最终浓度为2μg/ml。37℃孵育30分钟，离心后更换X-Vivo15培养基，至最终细胞密度为5x105/ml。然后置于37℃、5％CO2孵箱中培养72小时后取出，细胞收集后用CD8和PD-1抗体染色，用流式细胞仪进行表型测定和数据分析。图2中显示，与未融合的PD-1抗体(图2的第2部分)和白介素2(图2的第3部分)相比，融合蛋白处理(图2的第1部分)能显著增加培养细胞中CD8+细胞的比例(58.8％，52.9％，51.4％)。同时，如图3显示，培养后融合蛋白处理组中PD-1+的细胞占CD8+T细胞的比例为3.16％(图3的第1部分)，而PD-1抗体处理组为1.53％(图3的第2部分)，白介素2处理组为1.31％(图3的第3部分)，所以，融合蛋白能显著加强CD8+T细胞中衰竭性细胞(PD-1+)的扩增。试验证明，依据本发明设计的重组融合蛋白不但能选择性地扩增CD8+的T细胞，还能定向性扩增衰竭性的CD8+PD-1+的细胞，达到融合蛋白的预期目的。
实施例3
本实施例为双功能重组融合蛋白对体内效应细胞的影响。
小黑鼠分别通过腹腔连续3天注射融合蛋白(4μg/只/天)或白介素2(40μg/只/天)或PBS(对照组)，第4天从尾部取外周血，用抗小鼠CD8和NK1.1流 式抗体染色后检测，数据分析如图4。与对照组(图4的第1部分，4.0％)和白介素2组(图4的第2部分，15.3％)相比，重组融合蛋白显著增加了NK细胞在淋巴细胞中的比例(图4的第3部分，27.0％)。同时，在CD8+T细胞部分，与对照组(图4的第1部分，15.5％)相比，原生性白介素2没有增加CD8+细胞的比例(图4的第2部分，15.1％)，而融合蛋白处理的小鼠外周血中CD8+T细胞比例(图4的第3部分)达22.8％。体内试验证明，即使只有原生性IL-210％的剂量，本发明合成的融合蛋白免疫学效应可以远远超过原生性的非融合蛋白。
实施例4
本实施例为双功能重组融合蛋白在制备治疗因免疫细胞衰竭而引起的疾病的药物中的应用。上述双功能重组融合蛋白可单独应用或与化疗，靶向药物，抗体药物，细胞治疗组成联合应用，用于制备治疗因免疫细胞衰竭而引起的疾病的药物，例如治疗癌症药物和治疗慢性病毒感染的药物等。
由上述实施例可知，本发明所述的恢复衰竭性免疫细胞功能的融合蛋白，既能识别衰竭性免疫细胞，又能激活扩增识别的免疫细胞，恢复免疫细胞杀伤抗原阳性细胞的功能。由于肿瘤的发生与扩散和慢性病毒感染是免疫细胞衰竭与免疫系统耐受的结果，而恢复衰竭性免疫细胞的功能可以增强机体抗肿瘤和抗慢性病毒感染的能力，因此，上述融合蛋白的临床应用可以增强抑制肿瘤生长和控制病毒感染的功能，具有很好的临床前景和广泛的应用范围。
以上对本发明的具体实施例进行了详细描述，但其只作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对该实用进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。

Claims (17)

  1. 一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在于，包含识别衰竭性免疫细胞的功能区和激活扩增衰竭性免疫细胞的功能区，两个功能区通过一定长度的非功能性氨基酸片段连接。
  2. 根据权利要求1所述的一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在于，所述识别衰竭性免疫细胞的功能区是利用免疫检查点特异性抗体识别衰竭性免疫细胞的表型受体，所述衰竭性免疫细胞的表型受体是具有共抑制性功能的免疫检查点。
  3. 根据权利要求2所述的一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在于，所述衰竭性免疫细胞的表型受体包括PD-1、PD-L1、TIM-3、CTLA-4、LAG-3，所述表型受体的特异性抗体包括PD-1抗体、CTLA-4抗体、PD-L1抗体、TIM-3抗体、LAG-3抗体。
  4. 根据权利要求3所述的一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在于，所述PD-1为SEQ ID NO：1所示序列，所述PD-L1为SEQ ID NO：2所示序列，所述TIM-3为SEQ ID NO：3所示序列，所述CTLA-4为SEQ ID NO：4所示序列，所述LAG-3为SEQ ID NO：5所示序列。
  5. 根据权利要求3所述的一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在于，
    所述PD-1抗体包括SEQ ID NO：6～9所示序列，所述CTLA-4抗体包括SEQ ID NO：10～11所示抗体，所述PD-L1抗体包括SEQ ID NO：12～13所示序列。
  6. 根据权利要求1所述的一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在于，所述激活扩增衰竭性免疫细胞的功能区是采用细胞因子或功能类似突变体、或者表型受体的配体或功能类似突变体、或者激活性抗体来激活衰竭性免疫细胞。
  7. 根据权利要求6所述的一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在于，所述细胞因子或功能类似突变体包括IL-2、IL-15、IL-21。
  8. 根据权利要求7所述的一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在于，所述IL-2为SEQ ID NO：14所示序列，所述IL-15为SEQ ID NO：15所示序列；所述IL-21为SEQ ID NO：16所示序列。
  9. 根据权利要求6所述的一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在于，所述表型受体的配体或功能类似突变体包括4-1BBL、OX-40L。
  10. 根据权利要求9所述的一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在于，所述4-1BBL为SEQ ID NO：17所示序列；所述OX-40L为SEQ ID NO：18所示序列。
  11. 根据权利要求6所述的一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在于，所述激活性抗体包括针对4-1BB的抗体、XO-40L的抗体、CD28的抗体、CD3的抗体、CD27的抗体。
  12. 根据权利要求11所述的一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在于，所述4-1BB的抗体包括SEQ ID NO：19～20所示序列，所述XO-40L的抗体包括SEQ ID NO：21～22所示序列。
  13. 根据权利要求1所述的一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在于，识别衰竭性免疫细胞的功能区利用PD-1抗体识别衰竭性免疫细胞的表型受体PD-1，激活扩增衰竭性免疫细胞的功能区采用IL-2来激活衰竭性免疫细胞。
  14. 根据权利要求13所述的一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在于，根据如下步骤制备所述融合蛋白：
    步骤1)由人PD-1抗体重链的C端和IL-2的N端氨基酸序列通过非功能氨基酸连接，组成融合蛋白重链构造基因；
    步骤2)将步骤1)所述的融合蛋白重链构造基因和合成的PD-1抗体的轻链基因共转入表达载体，并转染至仓鼠卵巢细胞；
    步骤3)将步骤2)所述的仓鼠卵巢细胞放置在孵箱中培养一段时间，取上清液，纯化后制得重组融合蛋白。
  15. 根据权利要求14所述的一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在于，所述人PD-1抗体重链为SEQ ID NO：6所示序列；所述IL-2为SEQ ID NO：14所示序列；所述合成的PD-1抗体的轻链为SEQ ID NO：7所示序列；所述融合蛋白重链构造基因为SEQ ID NO：23所示序列；所述非功能氨基酸的氨基酸序列为SGGGGSGGGGSGGGGSG；所述表达载体为真核动物表达载体pcDNA3.1。
  16. 根据权利要求1所述的一种恢复衰竭性免疫细胞功能的融合蛋白，其特征在 于，所述衰竭性免疫细胞为衰竭的特异性T细胞、衰竭的NK细胞中的至少一种。
  17. 一种如权利要求1～12中任一项所述的恢复衰竭性免疫细胞功能的融合蛋白的应用。
PCT/CN2017/079679 2017-03-29 2017-04-07 一种恢复衰竭性免疫细胞功能的融合蛋白及其应用 WO2018176505A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US16/496,348 US10815303B2 (en) 2017-03-29 2017-04-07 Fusion protein for restoring the functions of failing immune cells and application thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201710197757.8A CN107082812B (zh) 2017-03-29 2017-03-29 一种恢复衰竭性免疫细胞功能的融合蛋白及其应用
CN201710197757.8 2017-03-29

Publications (1)

Publication Number Publication Date
WO2018176505A1 true WO2018176505A1 (zh) 2018-10-04

Family

ID=59614186

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/079679 WO2018176505A1 (zh) 2017-03-29 2017-04-07 一种恢复衰竭性免疫细胞功能的融合蛋白及其应用

Country Status (3)

Country Link
US (1) US10815303B2 (zh)
CN (1) CN107082812B (zh)
WO (1) WO2018176505A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11518808B2 (en) 2018-01-12 2022-12-06 Amgen Inc. Anti-PD-1 antibodies and methods of treatment
US11541103B2 (en) 2017-08-03 2023-01-03 Amgen Inc. Interleukin-21 mutein/ anti-PD-1 antibody conjugates
US11725034B2 (en) 2019-12-20 2023-08-15 Regeneron Pharmaceuticals, Inc. IL2 agonists and methods of use thereof
US12024559B2 (en) 2020-10-23 2024-07-02 Asher Biotherapeutics, Inc. Fusions with CD8 antigen binding molecules for modulating immune cell function

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11421029B2 (en) 2017-09-01 2022-08-23 Sichuan Kelun-Biotech Biopharmaceutical Co., Ltd. Recombinant bispecific antibodies to PD-L1 and CTLA-4
CN109575140B (zh) * 2017-09-29 2021-02-23 北京比洋生物技术有限公司 靶向pd-1或pd-l1且靶向vegf家族的双靶向融合蛋白及其用途
CN109503718B (zh) * 2017-11-14 2020-02-21 拜西欧斯（北京）生物技术有限公司 包含免疫检查点抑制剂的融合物及其制备方法和应用
CN108285493B (zh) * 2018-02-02 2020-09-15 上海科医联创生物科技有限公司 一种恢复衰竭性免疫细胞功能的融合蛋白及其应用
CN108610422B (zh) * 2018-03-29 2019-05-28 中国人民解放军军事科学院军事医学研究院 抑制pd-1/pd-l1信号通路的结合分子
SG11202011349PA (en) * 2018-05-14 2020-12-30 Werewolf Therapeutics Inc Activatable interleukin-2 polypeptides and methods of use thereof
KR20210040103A (ko) * 2018-07-31 2021-04-12 피어이스 파마슈티컬즈 게엠베하 Cd137 및 pd-l1에 특이적인 신규한 융합 단백질
CN110894241B (zh) * 2018-09-13 2022-01-18 中国科学院生物物理研究所 一种靶向抗原特异性t细胞诱导其向记忆干性细胞分化的融合蛋白
US20220048966A1 (en) * 2018-09-28 2022-02-17 Pierre Fabre Medicament New immunocytokines for the treatment of cancer
US11845801B2 (en) 2019-06-12 2023-12-19 AskGene Pharma, Inc. IL-15 prodrugs and methods of use thereof
WO2021006604A1 (ko) * 2019-07-08 2021-01-14 주식회사 프로젠 신규 융합단백질 및 그의 용도
TWI815194B (zh) 2020-10-22 2023-09-11 美商基利科學股份有限公司 介白素2-Fc融合蛋白及使用方法
EP4376870A1 (en) * 2021-07-28 2024-06-05 Anwita Biosciences, Inc. Interleukin-2 muteins, fusion proteins, pharmaceutical compositions, and therapeutic applications
CN114426585B (zh) * 2022-02-15 2023-10-03 广东香雪干细胞再生医学科技有限公司 融合蛋白及其表达细胞株与应用

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101365799A (zh) * 2005-12-16 2009-02-11 Lfb生物技术公司 制备激活性Fc受体的选择性抗体的方法
CN101426532A (zh) * 2005-12-08 2009-05-06 路易斯维尔大学研究基金会有限公司 体内细胞表面工程化
CN103768596A (zh) * 2012-10-17 2014-05-07 傅阳心 用于肿瘤治疗的组合产品、其用途及相关方法
CN103965361A (zh) * 2013-02-06 2014-08-06 上海细胞治疗工程技术研究中心有限公司 一种t细胞信号的嵌合分子转换器及其用途
CN104024276A (zh) * 2012-06-21 2014-09-03 卡姆普根有限公司 Lsr抗体及其用于癌症治疗的用途
CN104185681A (zh) * 2012-02-01 2014-12-03 卡姆普根有限公司 C1orf32抗体及其用于治疗癌症的用途
CN104334573A (zh) * 2012-04-30 2015-02-04 比奥孔有限公司 靶向/免疫调节性融合蛋白及其制造方法
CN105263521A (zh) * 2014-01-15 2016-01-20 卡德门企业有限公司 免疫调节剂
WO2016027764A1 (ja) * 2014-08-19 2016-02-25 国立大学法人 岡山大学 免疫細胞の機能増強方法及び免疫細胞の多機能性評価方法
CN105531288A (zh) * 2013-09-13 2016-04-27 百济神州有限公司 抗pd1抗体及其作为治疗剂与诊断剂的用途
CN105828834A (zh) * 2013-11-05 2016-08-03 同源生物服务股份有限公司 检查点抑制剂和治疗剂的组合以治疗癌症
CN105874061A (zh) * 2013-02-26 2016-08-17 纪念斯隆-凯特琳癌症中心 用于免疫疗法的组合物和方法
CN106132436A (zh) * 2014-02-21 2016-11-16 Ibc药品公司 通过诱导对trop‑2表达细胞的免疫应答的疾病疗法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2791383C (en) * 2010-03-05 2022-09-20 The Johns Hopkins University Compositions and methods for targeted immunomodulatory antibodies and fusion proteins
WO2016141387A1 (en) * 2015-03-05 2016-09-09 Xencor, Inc. Modulation of t cells with bispecific antibodies and fc fusions
US9567399B1 (en) * 2016-06-20 2017-02-14 Kymab Limited Antibodies and immunocytokines
BR112019017329A2 (pt) * 2017-04-03 2020-04-14 Hoffmann La Roche imunoconjugados, um ou mais polinucleotídeos e vetores, métodos para a produção de um imunoconjugado, tratamento de uma doença e para a estimulação do sistema imunológico, composição, uso do imunoconjugado, invenção e usos da composição

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101426532A (zh) * 2005-12-08 2009-05-06 路易斯维尔大学研究基金会有限公司 体内细胞表面工程化
CN101365799A (zh) * 2005-12-16 2009-02-11 Lfb生物技术公司 制备激活性Fc受体的选择性抗体的方法
CN104185681A (zh) * 2012-02-01 2014-12-03 卡姆普根有限公司 C1orf32抗体及其用于治疗癌症的用途
CN104334573A (zh) * 2012-04-30 2015-02-04 比奥孔有限公司 靶向/免疫调节性融合蛋白及其制造方法
CN104024276A (zh) * 2012-06-21 2014-09-03 卡姆普根有限公司 Lsr抗体及其用于癌症治疗的用途
CN103768596A (zh) * 2012-10-17 2014-05-07 傅阳心 用于肿瘤治疗的组合产品、其用途及相关方法
CN103965361A (zh) * 2013-02-06 2014-08-06 上海细胞治疗工程技术研究中心有限公司 一种t细胞信号的嵌合分子转换器及其用途
CN105874061A (zh) * 2013-02-26 2016-08-17 纪念斯隆-凯特琳癌症中心 用于免疫疗法的组合物和方法
CN105531288A (zh) * 2013-09-13 2016-04-27 百济神州有限公司 抗pd1抗体及其作为治疗剂与诊断剂的用途
CN105828834A (zh) * 2013-11-05 2016-08-03 同源生物服务股份有限公司 检查点抑制剂和治疗剂的组合以治疗癌症
CN105263521A (zh) * 2014-01-15 2016-01-20 卡德门企业有限公司 免疫调节剂
CN106132436A (zh) * 2014-02-21 2016-11-16 Ibc药品公司 通过诱导对trop‑2表达细胞的免疫应答的疾病疗法
WO2016027764A1 (ja) * 2014-08-19 2016-02-25 国立大学法人 岡山大学 免疫細胞の機能増強方法及び免疫細胞の多機能性評価方法

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11541103B2 (en) 2017-08-03 2023-01-03 Amgen Inc. Interleukin-21 mutein/ anti-PD-1 antibody conjugates
US11518808B2 (en) 2018-01-12 2022-12-06 Amgen Inc. Anti-PD-1 antibodies and methods of treatment
US11725034B2 (en) 2019-12-20 2023-08-15 Regeneron Pharmaceuticals, Inc. IL2 agonists and methods of use thereof
US12024559B2 (en) 2020-10-23 2024-07-02 Asher Biotherapeutics, Inc. Fusions with CD8 antigen binding molecules for modulating immune cell function

Also Published As

Publication number Publication date
US10815303B2 (en) 2020-10-27
CN107082812A (zh) 2017-08-22
CN107082812B (zh) 2018-11-13
US20200040080A1 (en) 2020-02-06

Similar Documents

Publication Publication Date Title
WO2018176505A1 (zh) 一种恢复衰竭性免疫细胞功能的融合蛋白及其应用
Musella et al. Type-I-interferons in infection and cancer: Unanticipated dynamics with therapeutic implications
US20210147548A1 (en) Fusion protein of interferon (ifn) and anti-pd-l1 antibody and use thereof
JP6754532B2 (ja) 二重特異性抗体をコードする腫瘍溶解性アデノウイルスおよびそれに関連する方法と使用
US20200087377A1 (en) Multifunctional Fusion Protein and Applications Thereof
Alexandroff et al. Role for CD40–CD40 ligand interactions in the immune response to solid tumours
Horiuchi et al. Transmembrane TNF-α: structure, function and interaction with anti-TNF agents
Casey et al. IL-21 promotes differentiation of naive CD8 T cells to a unique effector phenotype
Xu et al. The role of CD40-CD154 interaction in cell immunoregulation
WO2016180034A1 (zh) 抗ctla-4和pd-1的双重可变结构域免疫球蛋白
Cao et al. Next generation of tumor-activating type I IFN enhances anti-tumor immune responses to overcome therapy resistance
Zhang et al. Biological effects of IL-15 on immune cells and its potential for the treatment of cancer
US11926654B2 (en) Fusion proteins composed of an interleukin-2 mutein and type I interferon
AU2014255733A1 (en) Enhanced adoptive cell therapy
CN107557336B (zh) 一种抗muc16安全型嵌合抗原受体修饰的免疫细胞及其应用
CA2179196A1 (en) Method of preventing or treating disease characterized by neoplastic cells expressing cd40
JPH06508821A (ja) ウイルス病治療のための細胞表面受容体を標的とする分子の使用
CN106554416B (zh) 一种抗pd-l1人源化单克隆抗体联合干扰素基因刺激蛋白（sting）激动剂在抗肿瘤中的应用
CN104231068A (zh) 人白细胞介素ii突变体及其应用
US20230310548A1 (en) APPLICATION OF IFN-y IN PREPARING ANTI-TUMOR ADJUVANT DRUG
CN110396133A (zh) 一种以白介素12为活性成分的融合蛋白型药物前体
CN108300699A (zh) 修饰的nk细胞及其用途
CN110075275A (zh) IL-36β的应用
CN109666074B (zh) 一种趋化因子受体cxcr5的用途
Krautwald et al. Ectopic expression of CCL19 impairs alloimmune response in mice

Legal Events

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

Ref document number: 17903170

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17903170

Country of ref document: EP

Kind code of ref document: A1