WO2023197437A1 - 一种提升免疫细胞杀伤活性的嵌合受体及其应用 - Google Patents

一种提升免疫细胞杀伤活性的嵌合受体及其应用 Download PDF

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WO2023197437A1
WO2023197437A1 PCT/CN2022/098848 CN2022098848W WO2023197437A1 WO 2023197437 A1 WO2023197437 A1 WO 2023197437A1 CN 2022098848 W CN2022098848 W CN 2022098848W WO 2023197437 A1 WO2023197437 A1 WO 2023197437A1
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cancer
cells
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吴理达
顾雨春
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呈诺再生医学科技(北京)有限公司
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Priority to US18/516,502 priority Critical patent/US20240207316A1/en

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    • 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
    • C07K16/283—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 Fc-receptors, e.g. CD16, CD32, CD64
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    • A61K2239/17—Hinge-spacer domain
    • A—HUMAN NECESSITIES
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    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the invention belongs to the field of biomedicine, and specifically relates to a chimeric receptor that enhances the killing activity of immune cells and its application.
  • NK cells Natural killer cells
  • MHC MHC-derived cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic cytoplasmic factor, IL-2, antigen sensitization stimulation, antigen sensitization stimulation, and kill target cells.
  • the killing effect of NK cells occurs early after acting on target cells, and can be seen within 1 hour in vitro and 4 hours in vivo. Killing effect.
  • NK cells mainly include certain tumor cells, virus-infected cells, certain own tissue cells (such as blood cells), parasites, etc. Therefore, NK cells are an important immune factor for the body's anti-tumor and anti-infection.
  • the main mechanisms by which NK cells kill target cells 1. Cause cell lysis through the release of perforin and granzymes; 2. Cause apoptosis of target cells through the ligand-induced receptor-mediated apoptosis activation pathway; 3. Release of cytokines (Including: NK cell cytotoxic factors, NK cell tumor necrosis factor) to kill target cells; 4. Antibody-dependent cell-mediated cytotoxicity (ADCC, antibody-dependent cell-mediated cytotoxicity).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Fc receptors There are three main types of Fc receptors: Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), and Fc ⁇ RIII (CD16), of which the latter two can be divided into: Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIc, Fc ⁇ RIIIa, and Fc ⁇ RIIIb.
  • Different immune cells express specific Fc receptors.
  • neutrophils usually express Fc ⁇ RI, Fc ⁇ RII, and Fc ⁇ RIIIb, while NK cells only express Fc ⁇ RIIIa.
  • Fc ⁇ RIIIa is generally considered to be the key receptor that causes ADCC, so although NK cells, monocytes, macrophages, and neutrophils can all produce ADCC, NK cells are considered to be the most important cell population.
  • ADCC The strength of ADCC is related to many factors, such as the affinity of antibodies and antigens, the affinity of antibodies and Fc receptors, the density of tumor antigens, the characteristics of tumor target cells, and the characteristics of immune effector cells. Generally speaking, the closer the bridging binding between tumor target cells and immune effector cells is through antibodies, the stronger the ADCC effect. Therefore, antibodies with high affinity for antigens or Fc receptors mediate stronger ADCC effects. Tumor cells with high expression of target antigens are more sensitive to ADCC and are easily killed by ADCC.
  • NK cells into the tumor site also affects the effectiveness of immunotherapy.
  • the recruitment of NK cells into the tumor can effectively improve the anti-tumor immune response.
  • Chemokines and adhesion factors in the tumor microenvironment promote the increase of NK infiltration in tumor tissues by recruiting NK cells, which in turn causes NK cell surface-activated receptors to recognize corresponding ligands on the surface of tumor cells and release killing mediators such as perforin. Exert anti-tumor cytotoxic effects.
  • NK cell therapy is a promising field of clinical research. Studies have verified its good safety and preliminary efficacy in certain cancer patients. NK cell therapy will play an important role in future tumor immunotherapy. With the incidence of cancer increasing year by year, finding an efficient, non-toxic and side-effect treatment method is the common goal of doctors and patients. NK cell therapy can be used alone or in combination with other treatment modalities for the treatment of various cancers, and has extremely high application prospects.
  • this patent provides a genetically modified NK cell.
  • the genetically modified NK cells have a stronger killing effect than ordinary NK cells; further, when used in combination with antibodies, , the genetically modified NK cells of the present invention recognize the Fc end of the antibody, recognize the specific target through the antibody, and express a strong killing effect on tumor cells.
  • the first aspect of the present invention provides a fusion protein, which includes an extracellular part, an extracellular hinge region, a transmembrane region, and an intracellular region; the extracellular part includes the Ig-like C2-type of CD16A 1.
  • the extracellular part includes the Ig-like C2-type of CD16A 1.
  • CD16A Ig-like C2-type 2 CD64A Ig-like C2-type 1
  • CD64A Ig-like C2-type 2 CD64A Ig-like C2-type 3.
  • the extracellular portion is any of the following:
  • Ig-like C2-type 1 of CD16a, Ig-like C2-type 2 and Ig-like C2-type 3 of CD64 are connected in series;
  • Ig-like C2-type 1 of CD16A, Ig-like C2-type 2 of CD16A, Ig-like C2-type 1 of CD64A (CD64), Ig-like C2-type 2 of CD64A, Ig-like of CD64A C2-type 3 are connected in series;
  • CD64A Ig-like C2-type 1, CD64A Ig-like C2-type 2, CD64A Ig-like C2-type 3, CD16A Ig-like C2-type 1, CD16A Ig-like C2-type 2 are connected in series;
  • Ig-like C2-type 1 of CD64A, Ig-like C2-type 2 of CD64A, Ig-like C2-type 3 of CD64A, and Ig-like C2-type 1 of CD16A are connected in series;
  • Ig-like C2-type 1 of CD64A, Ig-like C2-type 2 of CD64A, Ig-like C2-type 3 of CD64A, and Ig-like C2-type 2 of CD16A are connected in series.
  • the extracellular hinge region (hinge region), transmembrane region, and intracellular region are respectively the extracellular hinge region of CD64, the transmembrane region of CD16a, and the intracellular region of CD16a.
  • amino acid sequence of the Ig-like C2-type 1 of CD16a is as shown in SEQ ID NO.: 1 or has 1, 2, 3, 4, 5 or more mutations with the sequence shown.
  • amino acid sequence of the Ig-like C2-type 2 of CD16a is as shown in SEQ ID NO.: 3 or has 1, 2, 3, 4, 5 or more mutations with the sequence shown.
  • amino acid sequence of the Ig-like C2-type 3 of CD64 is as shown in SEQ ID NO.: 5 or has 1, 2, 3, 4, 5 or more mutations with the sequence shown.
  • amino acid sequence of the extracellular hinge region of CD64 is as shown in SEQ ID NO.: 7 or has 1, 2, 3, 4, 5 or more mutations with the sequence shown.
  • amino acid sequence of the transmembrane region of CD16a is as shown in SEQ ID NO.: 9 or has 1, 2, 3, 4, 5 or more mutations from the sequence shown.
  • amino acid sequence of the intracellular region of CD16a is as shown in SEQ ID NO.: 11 or has 1, 2, 3, 4, 5 or more mutations with the sequence shown.
  • amino acid sequence of the intracellular region of CD64A Ig-like C2-type 1 is as shown in SEQ ID NO.: 15 or has 1, 2, 3, 4, 5 or more mutations with the sequence shown.
  • amino acid sequence of the intracellular region of CD64A Ig-like C2-type 2 is as shown in SEQ ID NO.: 17 or has 1, 2, 3, 4, 5 or more mutations with the sequence shown.
  • the fusion protein is the Ig-like C2-type 1 of CD16a, the Ig-like C2-type 2 of CD16a, the Ig-like C2-type 3 of CD64, the extracellular hinge region of CD64, and the transmembrane region of CD16a , the intracellular region of CD16a is connected sequentially.
  • amino acid sequence of the fusion protein of the present invention is the sequential connection of SEQ ID NO.: 1, 3, 5, 7, 9, and 11.
  • CD16a of the present invention also known as “Fc ⁇ RIIIA” is an activating Fc receptor, which is an Fc receptor that induces signal transduction events after being joined by the Fc region of an antibody. The event stimulates cells carrying the receptor. Perform effector functions.
  • the fusion protein is also referred to as Chimeric-FcyR in the present invention.
  • the present invention also provides coding nucleic acids encoding the fusion proteins of the present invention.
  • the present invention provides an isolated coding nucleic acid that sequentially codes for Ig-like C2-type 1 of CD16a, Ig-like C2-type 2 of CD16a, Ig-like C2-type 3 of CD64, and CD64
  • the coding nucleic acid sequence of Ig-like C2-type 1 of CD16a is shown in SEQ ID NO.: 2.
  • the coding nucleic acid sequence of Ig-like C2-type 2 of CD16a is shown in SEQ ID NO.: 4.
  • the coding nucleic acid sequence of Ig-like C2-type 3 of CD64 is shown in SEQ ID NO.: 6.
  • the coding nucleic acid sequence of the extracellular hinge region is shown in SEQ ID NO.: 8.
  • the coding nucleic acid sequence of the transmembrane region is shown in SEQ ID NO.: 10.
  • the coding nucleic acid sequence of the intracellular region is shown in SEQ ID NO.: 12.
  • the coding nucleic acid sequence of the CD64A Ig-like C2-type 1 is shown in SEQ ID NO.: 16.
  • the coding nucleic acid sequence of the CD64A Ig-like C2-type2 is shown in SEQ ID NO.: 18.
  • the nucleic acid encoding the Chimeric-Fc ⁇ R of the present invention is a DNA sequence composed of SEQ ID NO.: 2, 4, 6, 8, 10, and 12 connected in sequence.
  • the present invention also provides an expression vector expressing the aforementioned fusion protein or comprising the above encoding nucleic acid.
  • expression vector refers to bacterial plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenovirus, retrovirus, lentivirus or other vectors well known in the art.
  • any plasmid and vector can be used as long as it can replicate and be stable in the host body.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, a marker gene, and translation control elements.
  • the pLV-EF1a-IRES-Hygro lentiviral vector is used.
  • the present invention also provides host cells containing or expressing one or more of the aforementioned fusion proteins, encoding nucleic acids, and vectors.
  • the host cells are human immune cells or stem cells.
  • the host cells are NK cells, or iPSCs (induced pluripotent stem cells, full name: induced pluripotent stem cells) that can be induced into NK cells.
  • NK cells or iPSCs (induced pluripotent stem cells, full name: induced pluripotent stem cells) that can be induced into NK cells.
  • iPSCs induced pluripotent stem cells, full name: induced pluripotent stem cells
  • the host cells include autologous cells or allogeneic cells.
  • the host cell can be a mature commercial cell line product, or obtained by in vitro culture.
  • the host cell of the present invention can also be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; plant cells; CHO, COS, 293 cells, etc.
  • the present invention also provides a method for preparing immune cells with high killing activity, which method includes introducing one or more of the aforementioned fusion proteins, encoding nucleic acids, and vectors into immune cells, or the method includes introducing the aforementioned One or more of fusion proteins, coding nucleic acids, and vectors are introduced into stem cells and then induced to differentiate into immune cells.
  • the immune cells include one or more of T cells, B cells, K cells and NK cells.
  • the immune cells are NK cells.
  • killer activity when used to describe the activity of immune cells, such as NK cells, involves killing target cells by any of a variety of biological, biochemical or biophysical mechanisms.
  • the stem cells are induced pluripotent stem cells (iPSCs): cells with the potential to differentiate into a variety of cells obtained by transferring pluripotent factors into adult cells and then initiating genome expression profile reprogramming. Pluripotent stem cells.
  • iPSCs induced pluripotent stem cells
  • Pluripotent stem cells Pluripotent stem cells.
  • iNK iPSC-derived NK
  • iPSC-derived NK natural killer cells induced and differentiated from iPSCs.
  • the immune cells include autologous immune cells and allogeneic immune cells.
  • the stem cells include autologous stem cells and allogeneic stem cells.
  • fusion proteins encoding nucleic acids, and vectors into immune cells
  • techniques include, but are not limited to, electrophoresis and electroporation, protoplast fusion, calcium phosphate precipitation, cell fusion using enveloped DNA, microinjection, and transfection using intact viruses.
  • the viral vector is introduced into the host cell through lentiviral transfection technology, thereby obtaining a host cell that stably expresses the fusion protein, which means that the host cell contains the encoding nucleic acid, and The vector in which the encoding nucleic acid resides.
  • the present invention also provides a pharmaceutical composition comprising one or more of the aforementioned host cells, fusion proteins, encoding nucleic acids, and vectors.
  • the pharmaceutical composition also contains other drugs for treating cancer or structures recognized by Chimeric-Fc ⁇ R;
  • the drug is a monoclonal antibody drug.
  • the monoclonal antibody drugs include drugs that are already on the market, such as matuximab, trastuzumab, cetuximab, daclizumab, tanitizumab, abavo monoclonal antibody, adelimumab, afutuzumab, alemtuzumab, cultured aclizumab, amatuximab, avolizumab, baviliximab, betumolol monoclonal antibody, belimumab, bevacizumab, mo-bivalizumab, belemtuzumab-vedotin, mocantuzumab, lacantuzumab, carolizumab Pendetide, catuzumab, bocetalizumab, cetolumab, kanalimumab, daclizumab, daclizumab, detumomab, emeleximab anti-, eder
  • the monoclonal antibody drug can also be a commercial monoclonal antibody product that has not yet been clinically verified. As verified in the specific embodiments of the present invention, the antibody (FOLH1/3734) provided by abcam company with the product number ab268061.
  • the pharmaceutical composition further includes a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutically acceptable carrier, diluent or excipient includes, but is not limited to, any adjuvant, carrier, Excipients, glidants, sweeteners, diluents, preservatives, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, Surfactants or emulsifiers.
  • the pharmaceutical composition can be tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, suspensions, controlled release preparations, aerosols, films, injections, Intravenous drip preparations, transdermal absorption preparations, ointments, lotions, adhesive preparations, suppositories, small pills, nasal preparations, pulmonary preparations, eye drops, etc., oral or parenteral preparations.
  • the present invention is a method for killing target cells in vitro, which method includes contacting the target cells with one or more of the aforementioned fusion proteins, polynucleotides, vectors, host cells, and pharmaceutical compositions;
  • the target cells are cancer cells;
  • the cancer cells include cells from the following cancers: cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, skin cancer Squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, stomach cancer, esophageal cancer, thyroid cancer, bladder transitional epithelial cancer, leukemia, brain tumor, gastric cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, female reproductive tract Carcinoma, carcinoma in situ, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma;
  • the target cells are prostate cancer cells.
  • the present invention also provides a method for treating diseases, which method includes administering one or more of the aforementioned pharmaceutical compositions, host cells, fusion proteins, encoding nucleic acids, and vectors.
  • compositions described herein may be administered in a variety of methods well known in the art. Administration may include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotaxic), and the pharmaceutical composition may also be engineered into a cell-releasing biomaterial, such as a polymer matrix, Gels, permeable membranes, permeable systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g. up to 30 ⁇ m ), nanospheres (eg, less than 1 ⁇ m), microspheres (eg, 1-100 ⁇ m), or other suitable delivery vehicles to provide the desired release rate in varying proportions. Other methods of controlled release delivery of pharmaceutical compositions are known to the skilled artisan and are within the scope of this disclosure.
  • a cell-releasing biomaterial such as a polymer matrix, Gels, permeable membranes, permeable systems, multilayer coatings, microparticle
  • treatment means alleviation or alleviation of at least one symptom associated with such condition, or slowing or reversal of such condition. progression of the disease.
  • treatment also means inhibiting, delaying the onset of a condition (ie the period before clinical manifestation of the disease) and/or reducing the risk of development or progression of the disease.
  • treatment in relation to cancer may refer to eliminating or reducing a patient's tumor burden, or preventing, delaying or inhibiting metastasis, etc.
  • the present invention also provides the use of the aforementioned pharmaceutical compositions, host cells, fusion proteins, encoding nucleic acids, and vectors in the preparation of cancer immunotherapy drugs, autoimmune disease drugs, anti-aging drugs, medical beauty products, and metabolic disease drugs. the use of.
  • the cancer of the present invention may be a hematological cancer or a cancer with solid tumors.
  • the cancer includes cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, cutaneous squamous cell carcinoma, colon cancer, liver cancer, pancreatic cancer, gastric cancer, esophageal cancer Cancer, thyroid cancer, bladder transitional epithelial cancer, leukemia, brain tumor, gastric cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer, female reproductive tract cancer, carcinoma in situ, neurofibroma, bone cancer, skin cancer, Gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, glioma;
  • Exemplary autoimmune diseases of the present invention include achalasia; Addison's disease; adult Still's disease; agammaglobulinemia; alopecia areata; amyloidosis; ankylosing spondylitis; anti-GBM /Anti-TBM nephritis; antiphospholipid syndrome; autoimmune angioedema; autoimmune dysautonomia; autoimmune encephalomyelitis; autoimmune hepatitis; autoimmune inner ear disease (AIED); autoimmune myocarditis; Autoimmune oophoritis; autoimmune orchitis; autoimmune pancreatitis; autoimmune retinopathy; autoimmune urticaria.
  • Exemplary metabolic diseases of the present invention include diabetes, diabetic ketoacidosis, hyperglycemic hyperosmolar syndrome, hypoglycemia, gout, protein-energy malnutrition, vitamin A deficiency, scurvy, and vitamin D. Deficiency diseases, osteoporosis.
  • the metabolic diseases which are well known to those skilled in the art, are diseases caused by metabolic problems, including metabolic disorders, metabolic hyperactivity and other causes.
  • the present invention also provides the use of the pharmaceutical composition, host cells, fusion proteins, encoding nucleic acids, and vectors of the present invention in improving the therapeutic effect of monoclonal antibodies.
  • the application is to improve the use of PSMAmAb antibody (manufacturer abcam, product number ab268061) in killing LNCaP cells (human prostate cancer cells).
  • the present invention verified the killing activity of NK cells expressing the fusion protein of the present invention on cancer cells through prostate cancer cell LNCaP.
  • Figure 1 is a schematic structural diagram of Chimeric-FcyR according to the present invention.
  • Figure 2 is a schematic structural diagram of each variant of Chimeric-Fc ⁇ R.
  • Figure 3 is the verification of ADCC effect of Chimeric-Fc ⁇ R and its variants.
  • Figure 4 is a diagram showing the identification results of the expression levels of Chimeric-Fc ⁇ R or mutCD16A on the prepared iPSCs.
  • Figure 5 is a statistical result chart of the percentage of positive NK cells after activating NK cells.
  • Figure 6 is a statistical graph showing the killing activity of different groups of drugs against LNCaP cancer cell lines.
  • Figure 7 is a graph showing the statistical results of the effects of different groups of drugs on tumor weight in mice in the mouse experiment.
  • Example 1 Vector construction, lentiviral packaging, stably transfected NK cells and ADCC effect identification of Chimeric-Fc ⁇ R and its variants
  • pLV-EF1a-IRES-Hygro plasmid (addgene, Cat. No. Plasmid#85134) as the backbone vector of Chimeric-Fc ⁇ R and its variants, and the enzyme cutting sites are EcoRI and Hpa1;
  • Chimeric-Fc ⁇ R The structure of Chimeric-Fc ⁇ R is shown in Figure 1.
  • the extracellular part includes Ig-like C2-type 1 of CD16a, Ig-like C2-type 2 and Ig-like C2-type 3 of CD64.
  • Chimeric-Fc ⁇ R also has Including the hinge region of CD64A, the transmembrane region of CD16a, and the intracellular region of CD16a.
  • Cell inoculation Inoculate 1.5 ⁇ 10 7 293T cells in a 10cm dish. Add 10 ml of DMEM medium containing 10% FBS, culture overnight in a 37°C, 5% CO2 incubator, and transfect after 16-24 hours.
  • Transfection medium replacement After 16-18 hours, remove the culture medium containing the transfection reagent, add 10 ml of DMEM containing 10% FBS, and continue culturing under 5% CO 2 and 37°C.
  • First virus harvest 48 hours after the start of transfection, harvest the cell supernatant, transfer to a 50ml centrifuge tube, centrifuge at 3,000 rpm for 10 minutes, filter the supernatant with a 0.45 ⁇ m filter, and store at 4°C. Add 10 ml of DMEM containing 10% FBS to the cells, and continue culturing under 5% CO 2 and 37°C.
  • Second harvest of viruses Harvest the cell supernatant, transfer to a 50ml centrifuge tube, centrifuge at 3,000 rpm for 10 minutes, filter the supernatant with a 0.45 ⁇ m filter, and store at 4°C. The cells were treated with 10% disinfectant (84 disinfectant) and discarded.
  • Virus concentration Filter the collected lentivirus components with a 0.45 ⁇ m filter to remove bacterial contamination. Mix the filtered components with Lenti-XTM Concentrator at a volume ratio of 3:1, and mix gently by inverting.
  • Lentivirus lenti-A, lenti-B, lenti-C, lenti-D, lenti-E, lenti-F, lentiG and lenti-Chimeric-Fc ⁇ R expressing the above structures were obtained through lentiviral packaging, and the above viruses were infected into NK92 cells respectively. Lines were screened using Hexadimethrine bromide for 7-14 days to obtain positive cell lines, which were named NK92-A, NK92-B, NK92-C, NK92-D, NK92-E, NK92-F, NK92-G and NK-Chimeric. -Fc ⁇ R
  • target NK92-A, NK92-B, NK92-C, NK92-D, NK92-E, NK92-F, NK92-G, NK-Chimeric-Fc ⁇ R cells and tumor cells at a ratio of 1:1.
  • the cells were inoculated into a 96-well plate, 1 ⁇ g/mL of PSMAmAb was added to the culture medium, and cultured in a 37-degree cell culture incubator.
  • NK92-A and NK92-E are better than those of other variants, indicating that the three Ig-like C2 structures of CD64A are complete, which is more conducive to NK cells to exert the ADCC effect.
  • the effect of NK92-E is better than that of NK92-A, indicating that the IgG Fc binding domain of CD64A is farther away from the cell membrane structure, which is conducive to its ADCC effect.
  • NK92-A, NK92-B, NK92-C, NK92-D and NK92-Chimeric-Fc ⁇ R have better ADCC effects, indicating that CD64A is an Ig-like C2-type 3 Compared with the other two structures, this structure is more conducive to NK's ADCC effect.
  • NK92-E Comparing NK92-E, NK92-F, and NK92-G, the ADCC effect of NK92-E is better, indicating that the Ig-like C2-type 3 structure of CD64A followed by a structure in series will affect the ADCC effect of NK92, but the ADCC effect of CD16A in series is intact. The two structures will not affect its ADCC effect.
  • mutCD16A amino acid sequence such as SEQ ID NO.: 14, nucleotide sequence such as SEQ ID NO.: 13
  • mutCD16A amino acid sequence such as SEQ ID NO.: 14, nucleotide sequence such as SEQ ID NO.: 13
  • the stably transfected cell lines are named NK92-Chimeric-Fc ⁇ R-iPSC (Chimeric-Fc ⁇ R-iPSC) and mutCD16A-iPSC respectively.
  • the reaction system is as follows:
  • the detection primer sequence is as follows
  • MutCD16A structure detection primers :
  • iNK Use STEMdiff TM NK Cell Kit to differentiate iPSC, Chimeric-Fc ⁇ R-iPSC and mutCD16A-iPSC into iNK cells.
  • the obtained iNK cells are named iNK, Chimeric-Fc ⁇ R-iNK and mutCD16A-iNK respectively.
  • K562 mitomycin C treated
  • PMA/lonomycin were used to activate NK cells, and the percentage of activated NK cells was detected.
  • NK cells When NK cells are activated, unmodified CD16A will be cleaved by metalloenzyme (ADAM17).
  • ADAM17 metalloenzyme
  • the experimental results measured the percentage of NK cells and found that the percentage of unmodified iNK cells decreased significantly.
  • mutCD16A-iNK cells and Chimeric-Fc ⁇ R-iNK mutCD16A and Chimeric-Fc ⁇ R proteins will not be cleaved by metalloenzymes, so the proportion of iNK-positive cells is still at a high level (the results are shown in Figure 5).
  • the Chimeric-Fc ⁇ R-iNK of the present invention will have better killing activity than unmodified iNK cells, so the following continues to compare the killing effect of Chimeric-Fc ⁇ R-iNK and mutCD16A on tumor cells at the cell level and animal experiment level.
  • the mutCD16A-iNK and Chimeric-Fc ⁇ R-iNK groups are much more lethal to LNCaP cells, indicating that the mutCD16A, Chimeric -Fc ⁇ R can enhance the killing ability of NK.
  • mutCD16A-iNK and mutCD16A-iNK+PSMAmAb groups showed that the mutCD16A-iNK+PSMAmAb group had stronger killing effect on LNCaP cells, and mutCD16A-iNK+PSMAmAb had a stronger ADCC effect.
  • NK cells expressing the fusion protein of the present invention have better killing activity than unmodified NK cells.
  • NOD SCID mice were injected subcutaneously with 1 ⁇ 10 6 fluorescently labeled C42 cells, and visible tumors formed after 3 weeks.
  • the tumor model is formed, it is first analyzed through the small animal imaging system, and then different groups are injected through the tail vein, respectively, 5 ⁇ 10 6 iNK, iNK+100 ⁇ g PSMAmAb mutCD16A-iNK+100 ⁇ g PSMAmAb, Chimeric-Fc ⁇ R-iNK+ 100 ⁇ g PSMAmAb. At the same time, set a control without processing
  • the tumor status will be analyzed weekly through the small animal imaging system.
  • mutCD16A-iNK+PSMAmAb Compared with iNK+PSMAmAb, mutCD16A-iNK+PSMAmAb, and Chimeric-Fc ⁇ R-iNK+PSMAmAb groups, mutCD16A-iNK+PSMAmAb and Chimeric-Fc ⁇ R-iNK+PSMAmAb groups have stronger ADCC effects and stronger tumor killing effects.
  • the Chimeric-Fc ⁇ R-iNK+PSMAmAb group has a stronger ADCC effect and a stronger tumor killing effect.

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Abstract

本发明属于生物医药领域，具体涉及一种提升免疫细胞杀伤活性的嵌合受体及其应用。具体地，本发明提供了一种融合蛋白，所述融合蛋白包括胞外部分、胞外铰链区、跨膜区、胞内区胞内部分。最优选地，本发明所述融合蛋白的氨基酸序列是SEQ ID NO.：1、3、5、7、9、11的依次连接，表达所述融合蛋白的免疫细胞具有强杀伤活性。

Description

一种提升免疫细胞杀伤活性的嵌合受体及其应用 技术领域
本发明属于生物医药领域，具体涉及一种提升免疫细胞杀伤活性的嵌合受体及其应用。
背景技术
由于NK细胞(natural killer cell，自然杀伤细胞)的杀伤活性无MHC限制，因此称为自然杀伤活性。与T细胞和B细胞不同，NK细胞不需要特异性的抗原致敏刺激就可识别并杀伤靶细胞，NK细胞作用于靶细胞后杀伤作用出现早，在体外1小时、体内4小时即可见到杀伤效应。
NK细胞的靶细胞主要包括某些肿瘤细胞、病毒感染细胞、某些自身组织细胞(如血细胞)、寄生虫等，因此NK细胞是机体抗肿瘤、抗感染的重要免疫因素。NK细胞杀伤靶细胞的主要机制：1、通过释放穿孔素和颗粒酶引起细胞溶解；2、通过配体诱导的受体介导的凋亡激活途经引起靶细胞的凋亡；3、释放细胞因子(包括：NK细胞细胞毒因子、NK细胞肿瘤坏死因子)杀伤靶细胞；4、抗体依赖细胞介导的细胞毒性作用(ADCC，antibody-dependent cell-mediated cytotoxicity)。
当抗体通过抗原结合部位结合肿瘤细胞表面抗原以及Fc部位结合免疫效应细胞表面Fc受体时，免疫效应细胞得到激活，杀死肿瘤细胞，这个过程称为抗体依赖的细胞介导的细胞毒性作用(ADCC)。Fc受体主要有三种：FcγRI(CD64)、FcγRII(CD32)、FcγRIII(CD16)，其中后两种又可分为：FcγRIIa、FcγRIIb、FcγRIIc、FcγRIIIa、FcγRIIIb。不同的免疫细胞表达特定的Fc受体，如中性粒细胞通常表达FcγRI、FcγRII、FcγRIIIb，而NK细胞只表达FcγRIIIa。FcγRIIIa通常认为是引起ADCC的关键受体，所以虽然NK细胞、单核巨噬细胞和中性粒细胞都可以产生ADCC作用，但是NK细胞被认为是最重要的细胞种群。
ADCC强弱跟许多因素有关，比如抗体与抗原的亲和力，抗体与Fc受体的亲和力、肿瘤抗原的密度、肿瘤靶细胞的特性以及免疫效应细胞的特性等。一般情况下，肿瘤靶细胞与免疫效应细胞通过抗体的桥联结合越紧密，ADCC作用越强。因此，对抗原或Fc受体亲和力高的抗体介导的ADCC作用更强。表达靶抗原高的肿瘤细胞对ADCC更为敏感，容易被ADCC作用所杀死。
NK细胞在肿瘤部位的浸润程度也对免疫治疗的效果有影响，NK细胞向肿瘤内部募集可有效改善抗肿瘤免疫应答。肿瘤微环境中的趋化因子和黏附因子等，通过募集NK细胞，促使肿瘤组织中NK浸润程度增加，继而使得NK细胞表面活化受体识别肿瘤细胞表面相应配体，释放穿孔素等杀伤介质，发挥抗肿瘤的细胞毒性作用。
NK细胞疗法是一个很有前途的临床研究领域，已有研究验证了其对某些癌症患者具有良好的安全性和初步疗效，NK细胞疗法将在未来肿瘤免疫治疗中扮演重要的角色。 随着肿瘤发病率的逐年提高，寻找一种高效、无毒副作用的治疗方法是是医生和患者共同的努力方向。NK细胞疗法既可单独用于治疗，亦可联合其他治疗方式用于各种癌症的治疗，具有极高的应用前景。
发明内容
为进一步提高NK细胞的杀伤作用，本专利提供了一种基因修饰的NK细胞，所述基因修饰的NK细胞相较于普通的NK细胞有更强的杀伤作用；进一步地，与抗体联合使用时，本发明所述的基因修饰的NK细胞识别抗体的Fc端，通过抗体识别特异性靶点，表达对肿瘤细胞的强杀伤效果。
本发明的第一方面，提供了一种融合蛋白，所述融合蛋白包括胞外部分、胞外铰链区、跨膜区、胞内区；所述胞外部分包括CD16A的Ig-like C2-type 1、CD16A的Ig-like C2-type 2、CD64A的Ig-like C2-type 1、CD64A的Ig-like C2-type 2、CD64A的Ig-like C2-type 3中的一种或多种。
优选地，所述胞外部分是以下任意一种：
1)CD16a的Ig-like C2-type 1，Ig-like C2-type 2和CD64的Ig-like C2-type 3依次串联；
2)CD16A的Ig-like C2-type 1、CD16A的Ig-like C2-type 2、CD64A(CD64)的Ig-like C2-type 1、CD64A的Ig-like C2-type 2、CD64A的Ig-like C2-type 3依次串联；
3)CD64A的Ig-like C2-type 1、CD64A的Ig-like C2-type 2、CD64A的Ig-like C2-type 3、CD16A的Ig-like C2-type 1、CD16A的Ig-like C2-type 2依次串联；
4)CD64A的Ig-like C2-type 1、CD64A的Ig-like C2-type 2、CD64A的Ig-like C2-type 3、CD16A的Ig-like C2-type 1依次串联；
5)CD64A的Ig-like C2-type 1、CD64A的Ig-like C2-type 2、CD64A的Ig-like C2-type 3、CD16A的Ig-like C2-type 2依次串联。
优选地，所述胞外铰链区(铰链区)、跨膜区、胞内区分别为CD64的胞外铰链区、CD16a的跨膜区、CD16a的胞内区。
优选地，所述CD16a的Ig-like C2-type 1的氨基酸序列如SEQ ID NO.:1所示或与所示序列有1、2、3、4、5或更多个突变。
优选地，所述CD16a的Ig-like C2-type 2的氨基酸序列如SEQ ID NO.:3所示或与所示序列有1、2、3、4、5或更多个突变。
优选地，所述CD64的Ig-like C2-type 3的氨基酸序列如SEQ ID NO.:5所示或与所示序列有1、2、3、4、5或更多个突变。
优选地，所述CD64的胞外铰链区的氨基酸序列如SEQ ID NO.:7所示或与所示序列有1、2、3、4、5或更多个突变。
优选地，所述CD16a的跨膜区的氨基酸序列如SEQ ID NO.:9所示或与所示序列有1、2、3、4、5或更多个突变。
优选地，所述CD16a的胞内区的氨基酸序列如SEQ ID NO.:11所示或与所示序列有1、2、3、4、5或更多个突变。
优选地，所述CD64A Ig-like C2-type 1的胞内区的氨基酸序列如SEQ ID NO.:15所示或与所示序列有1、2、3、4、5或更多个突变。
优选地，所述CD64A Ig-like C2-type 2的胞内区的氨基酸序列如SEQ ID NO.:17所示或与所示序列有1、2、3、4、5或更多个突变。
优选地，所述融合蛋白即CD16a的Ig-like C2-type 1、CD16a的Ig-like C2-type 2、CD64的Ig-like C2-type 3、CD64的胞外铰链区、CD16a的跨膜区、CD16a的胞内区的依次连接。
也即本发明所述融合蛋白的氨基酸序列是SEQ ID NO.:1、3、5、7、9、11的依次连接。
本发明所述“CD16a”也就是“FcγRIIIA”，是一种激活性Fc受体，其被抗体Fc区接合之后引出信号转导事件的Fc受体，所述事件刺激带有该受体的细胞履行效应器功能。
所述融合蛋白在本发明中也称为Chimeric-FcγR。
另一方面，本发明还提供编码本发明所述融合蛋白的编码核酸。
也即，本发明提供一种分离的编码核酸，所述编码核酸依次编码CD16a的Ig-like C2-type 1、CD16a的Ig-like C2-type 2、CD64的Ig-like C2-type 3、CD64的胞外铰链区、CD16a的跨膜区、CD16a的胞内区。
优选地，所述CD16a的Ig-like C2-type 1的编码核酸序列如SEQ ID NO.:2所示。
优选地，所述CD16a的Ig-like C2-type 2的编码核酸序列如SEQ ID NO.:4所示。
优选地，所述CD64的Ig-like C2-type 3的编码核酸序列如SEQ ID NO.:6所示。
优选地，所述胞外铰链区的编码核酸序列如SEQ ID NO.:8所示。
优选地，所述跨膜区的编码核酸序列如SEQ ID NO.:10所示。
优选地，所述胞内区的编码核酸序列如SEQ ID NO.:12所示。
优选地，所述CD64A Ig-like C2-type 1的编码核酸序列如SEQ ID NO.:16所示。
优选地，所述CD64A Ig-like C2-type2的编码核酸序列如SEQ ID NO.:18所示。
优选地，所述编码本发明所述Chimeric-FcγR的核酸即SEQ ID NO.:2、4、6、8、10、12依次连接所构成的DNA序列。
另一方面，本发明还提供了表达前述融合蛋白或包含以上编码核酸的表达载体。
术语“表达载体”指本领域熟知的细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、慢病毒或其他载体。
总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一 个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
示例性的本发明具体实施例中使用了pLV-EF1a-IRES-Hygro慢病毒载体。
另一方面，本发明还提供了包含或表达前述融合蛋白、编码核酸、载体中的一种或多种的宿主细胞。
优选地，所述宿主细胞是人的免疫细胞、干细胞。
更优选地，所述宿主细胞是NK细胞，或是可以诱导为NK细胞的iPSC(诱导性多能干细胞，全称为induced pluripotent stem cells)。
优选地，所述宿主细胞包括自体细胞或异体细胞。
优选地，所述宿主细胞可以是成熟的商品化细胞系产品，或由体外培养获得。
本发明所述宿主细胞还可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；植物细胞；CHO、COS、293细胞等。
另一方面，本发明还提供了制备高杀伤活性的免疫细胞的方法，所述方法包括将前述融合蛋白、编码核酸、载体中的一种或多种引入免疫细胞，或者所述方法包括将前述融合蛋白、编码核酸、载体中的一种或多种引入干细胞，然后诱导分化为免疫细胞。
优选地，所述免疫细胞包括T细胞、B细胞、K细胞和NK细胞中的一种或多种。
优选地，所述免疫细胞是NK细胞。
如本文所用，术语“杀伤活性”当用于描述免疫细胞诸如NK细胞的活性时，涉及通过多种生物学、生物化学或生物物理机制中的任一种杀灭靶细胞。
优选地，所述干细胞是诱导多能干细胞(induced pluripotent stem cell，iPSC)：是通过在成体细胞中转入多能性因子继而起始基因组表达谱重新编程得到的具有分化为多种细胞潜能的多能性干细胞。而iNK(iPSC-derived NK)则是由iPSC诱导分化而来的自然杀伤细胞。
优选地，所述免疫细胞包括自体免疫细胞、异体免疫细胞。
优选地，所述干细胞包括自体干细胞、异体干细胞。
所述将前述融合蛋白、编码核酸、载体中的一种或多种引入免疫细胞可以通过本领域的技术人员公知的多种技术来完成。这些技术包括但不限于，电泳和电穿孔、原生质体融合、磷酸钙沉淀、使用有包膜DNA的细胞融合、显微注射以及使用完整病毒转染。
如本发明具体实施例所使用的，通过慢病毒转染技术将病毒载体导入宿主细胞，从而获得了稳定表达所述融合蛋白的宿主细胞，也就代表了所述宿主细胞中含有编码核酸、以及编码核酸所在的载体。
另一方面，本发明还提供了包含前述宿主细胞、融合蛋白、编码核酸、载体中的 一种或多种的药物组合物。
所述药物组合物中还含有其他治疗癌症的药物或被Chimeric-FcγR识别的结构；
更优选地，所述药物是单克隆抗体药物。
示例性的，所述单克隆抗体药物包括已经上市的药物，例如马妥昔单抗、曲妥珠单抗、西妥昔单抗、达利珠单抗、他尼珠单抗、阿巴伏单抗、阿德木单抗、阿夫土珠单抗、阿仑单抗、培化阿珠单抗、阿麦妥昔单抗、阿泊珠单抗、巴维昔单抗、贝妥莫单抗、贝利木单抗、贝伐单抗、莫-比伐珠单抗、贝伦妥单抗-维多汀、莫坎妥珠单抗、拉坎妥珠单抗、卡罗单抗喷地肽、卡妥索单抗、泊西他珠单抗、西妥木单抗、可那木单抗、达西珠单抗、达洛珠单抗、地莫单抗、依美昔单抗、依决洛单抗、依洛珠单抗、恩司昔单抗、依帕珠单抗、厄马索单抗、埃达珠单抗、法勒珠单抗、芬妥木单抗、加利昔单抗、吉妥珠单抗、吉妥珠单抗、吉瑞昔单抗、格莱木单抗-维多汀、替伊莫单抗、伊戈伏单抗、拉英达西单抗、英妥木单抗、伊珠单抗奥佐米星、伊匹木单抗、伊妥木单抗、拉贝珠单抗、来沙木单抗、林妥珠单抗、莫洛伏珠单抗，包括其抗原结合片段；
所述单克隆抗体药物也可以是商品化的、尚未经临床验证的单抗产品，如本发明具体实施例所验证的，abcam公司所提供的货号为ab268061的抗体(FOLH1/3734)。
优选地，所述药物组合物还包括药学上可接受的载体、稀释剂或赋形剂。
优选地，所述药学上可接受的载体、稀释剂或赋形剂包括但不限于已经由美国食品和药品管理局或中国食品药品监督管理局批准可用于人或家畜的任何佐剂、载体、赋形剂、助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、增味剂、表面活性剂、润湿剂、分散剂、悬浮剂、稳定剂、等渗剂、溶剂、表面活性剂或乳化剂。
优选地，所述药物组合物可以为片剂，丸剂，粉剂，颗粒剂，胶囊剂，锭剂，糖浆剂，液体，乳剂，混悬剂，控制释放制剂，气雾剂，膜剂，注射剂，静脉滴注剂，透皮吸收制剂，软膏剂，洗剂，粘附制剂，栓剂，小药丸，鼻制剂，肺制剂，眼睛滴剂等等，口服或胃肠外制剂。
另一方面，本发明一种在体外杀伤靶细胞的方法，所述方法包括将靶细胞与前述融合蛋白、多核苷酸、载体、宿主细胞、药物组合物中的一种或多种相接触；
优选地，所述靶细胞是癌细胞；所述癌细胞包括来着以下癌症的细胞：宫颈癌、精原细胞瘤、睾丸淋巴瘤、前列腺癌、卵巢癌、肺癌、直肠癌、乳腺癌、皮肤鳞状细胞癌、结肠癌、肝癌、胰腺癌、胃癌、食管癌、甲状腺癌、膀胱移行上皮癌、白血病、脑瘤、胃癌、腹膜癌、头颈癌、子宫内膜癌、肾癌、雌性生殖道癌、原位癌、神经纤维瘤、骨癌、皮肤癌、胃肠道间质瘤、肥大细胞肿瘤、多发性骨髓瘤、黑色素瘤、胶质瘤；
优选地，所述靶细胞是前列腺癌细胞。
另一方面，本发明还提供了一种治疗疾病的方法，所述方法包括施用前述药物组合物、宿主细胞、融合蛋白、编码核酸、载体中的一种或多种。
本文描述的药物组合物可以以本领域众所周知的各种方法进行施用。施用可以包 括例如涉及以下的方法:经口摄取、直接注射(例如全身或立体定向)，所述药物组合物还可以被改造为可释放细胞的生物材料，所述生物材料例如:聚合物基质、凝胶、渗透膜、渗透性系统、多层包衣、微粒、可植入基质装置、微型渗透泵、植入泵、可注射凝胶和水凝胶、脂质体、胶束(例如高达30μm)、纳米球(例如小于1μm)、微球体(例如1-100μm)、或其它合适的递送媒介物，以提供以不同比例的所需释放速率。药物组合物的控制释放递送的其它方法是技术人员已知的，并且在本公开内容的范围内。
在本发明的上下文中，在涉及任一本文所引用病症的范围内，术语“治疗”、“疗法”等意味着缓解或减轻与此类病症相关的至少一种症状，或减缓或逆转这种病症的进展。在本发明的含义中，术语“治疗”还表示抑制、延缓病症发作(即，疾病临床表现之前的时期)和/或降低疾病发展或恶化的风险。例如，与癌症相关的术语“治疗”可以指消除或减少病人的肿瘤负荷，或预防、延迟或抑制转移等。
另一方面，本发明还提供了前述药物组合物、宿主细胞、融合蛋白、编码核酸、载体在制备癌症免疫治疗药物、自身免疫性疾病药物、抗衰老药物、医美产品、代谢性疾病药物中的用途。
本发明所述癌症可以是血液癌症或具有实体瘤的癌症。优选地，所述癌症包括宫颈癌、精原细胞瘤、睾丸淋巴瘤、前列腺癌、卵巢癌、肺癌、直肠癌、乳腺癌、皮肤鳞状细胞癌、结肠癌、肝癌、胰腺癌、胃癌、食管癌、甲状腺癌、膀胱移行上皮癌、白血病、脑瘤、胃癌、腹膜癌、头颈癌、子宫内膜癌、肾癌、雌性生殖道癌、原位癌、神经纤维瘤、骨癌、皮肤癌、胃肠道间质瘤、肥大细胞肿瘤、多发性骨髓瘤、黑色素瘤、胶质瘤；
本发明所述自身免疫性疾病示例性的包括贲门失弛缓症；阿狄森氏病；成人斯蒂尔氏病；无丙种球蛋白血症；斑秃；淀粉样变性；强直性脊柱炎；抗GBM/抗TBM肾炎；抗磷脂综合征；自身免疫性血管性水肿；自身免疫性自主神经异常；自身免疫性脑脊髓炎；自身免疫性肝炎；自身免疫性内耳病(AIED)；自身免疫性心肌炎；自身免疫性卵巢炎；自身免疫性睾丸炎；自身免疫性胰腺炎；自身免疫性视网膜病变；自身免疫性荨麻疹。
本发明所述代谢性疾病示例性的包括糖尿病、糖尿病酮症酸中毒、高血糖高渗综合征、低血糖症、痛风、蛋白质-能量营养不良症、维生素A缺乏病、坏血病、维生素D缺乏病、骨质疏松症。本领域技术人员所熟知的所述代谢性疾病即因代谢问题引起的疾病，包括代谢障碍和代谢旺盛等原因。
另一方面，本发明还提供了本发明所述药物组合物、宿主细胞、融合蛋白、编码核酸、载体在提高单抗治疗效果中的应用。
更优选地，所述应用是提高PSMAmAb抗体(厂商abcam，货号ab268061)在杀伤LNCaP细胞(人前列腺癌细胞)中的应用。
如本发明具体实施例，本发明通过前列腺癌细胞LNCaP验证了表达本发明所述融合蛋白的NK细胞对癌细胞的杀伤活性。
附图说明
图1是本发明所述Chimeric-FcγR的结构示意图。
图2是Chimeric-FcγR各个变体的结构示意图。
图3是Chimeric-FcγR及其变体的ADCC效应验证。
图4是制备得到的iPSC上Chimeric-FcγR或mutCD16A表达量进行的鉴定结果图。
图5是激活NK细胞后对阳性NK细胞百分比进行的统计结果图。
图6是不同组别药物对LNCaP癌细胞系的杀伤活性的统计结果图。
图7是小鼠实验中，不同组别药物对小鼠体内肿瘤重量的影响的统计结果图。
具体实施方式
下面结合实施例对本发明做进一步的说明，以下所述，仅是对本发明的较佳实施例而已，并非对本发明做其他形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更为同等变化的等效实施例。凡是未脱离本发明方案内容，依据本发明的技术实质对以下实施例所做的任何简单修改或等同变化，均落在本发明的保护范围内。
实施例1、Chimeric-FcγR及其变体的载体构建、慢病毒包装、稳转NK细胞及ADCC效应鉴定
载体构建
1、实验材料
骨架载体：pLV-EF1a-IRES-Hygro质粒(addgene，货号Plasmid#85134)
2、实验方法
1、以pLV-EF1a-IRES-Hygro质粒(addgene，货号Plasmid#85134)为Chimeric-FcγR及其变体的骨架载体，酶切位点是EcoRI和Hpa1；
2、Chimeric-FcγR的结构如图1所示，胞外部分包括CD16a的Ig-like C2-type 1，Ig-like C2-type 2和CD64的Ig-like C2-type 3，Chimeric-FcγR上还包括CD64A的铰链区、CD16a的跨膜区、CD16a的胞内区。
3、Chimeric-FcγR各变体的结构如图2所示，Chimeric-FcγR及其变体的胞外部分总结如下表，铰链区、跨膜区、胞内区与Chimeric-FcγR一致。
Figure PCTCN2022098848-appb-000001
Figure PCTCN2022098848-appb-000002
4、基因合成以上表中的各序列，合成公司为安徽通用生物科技有限公司。
5、将Chimeric-FcγR及其变体分别插入pLV-EF1a-IRES-Hygro质粒的EcoRI和Hpa1之间，获得的载体。
慢病毒包装
1、实验材料
名称 公司 货号
FBS hyclone SH30084.03
DMEM 赛默飞世尔科技 11965084
PEI POLYSCIENCES 23966
pMD2.G addgene #12259
pCMV-VSVG addgene #8454
pRSV-Rev addgene #12253
Lenti-XTM Concentrator clonetech 631232
2、实验方法
1、细胞接种：10cm dish接种1.5×10 7个293T细胞。加10ml含10％FBS的DMEM培养基，37℃，5％CO 2培养箱过夜培养，16-24h后转染。
2、细胞转染:细胞生长的交汇度达到80-90％，准备转染。转染体系如下:
Figure PCTCN2022098848-appb-000003
B液逐滴加入A液中，边加边摇匀，室温22-26℃静置15min。逐滴加到培养皿中，轻轻摇匀，5％CO 2，37℃过夜培养。
3、转染换液:16-18h后，去掉含转染试剂的培养基，加入10ml含10％FBS的DMEM，5％CO 2、37℃条件下继续培养。
4、病毒第一次收获:从转染开始算48h后，收获细胞上清，转移到50ml离心管中，3,000rpm离心10min，上清用0.45μm滤膜过滤，4℃保存。细胞加入10ml含10％FBS的DMEM，5％CO 2、37℃条件下继续培养。
5、病毒第二次收获：收获细胞上清，转移到50ml离心管中，3,000rpm离心10min，上清用0.45μm滤膜过滤，4℃保存。细胞用10％消毒液(84消毒液)处理后丢弃。
6、病毒浓缩：将收集到的慢病毒组分用0.45μm滤器过滤去除细菌污染，将过滤后组分与Lenti-XTM Concentrator按照体积比3:1混合，轻轻颠倒混匀。
7、4℃孵育30min或过夜。
8、4℃1,500g离心45min，离心后会在管底看到白色沉淀。
9、小心吸去上清液，不能破坏白色沉淀。
10、用适当体积慢病毒保存液重悬沉淀，并将获得的慢病毒分装、保存于-80℃。
细胞杀伤试验
1、实验材料
名称 公司 货号
LNCaP细胞(人前列腺癌) 武汉普诺赛生命科技有限公司 CL-0143
Caspase-3/7 Green Apoptosis Assay Reagent Essen Bioscience 4440
PSMAmAb抗体 abcam ab268061
2、实验方法
通过慢病毒包装获得表达上述结构的慢病毒lenti-A、lenti-B、lenti-C、lenti-D、lenti-E、lenti-F、lentiG以及lenti-Chimeric-FcγR，将上述病毒分别感染NK92细胞系，使用Hexadimethrine bromide筛选7-14天，获得阳性细胞系，分别命名为NK92-A、NK92-B、NK92-C、NK92-D、NK92-E、NK92-F、NK92-G以及NK-Chimeric-FcγR
1、以每孔4000个细胞将LNCaP细胞铺种到96孔板，孵育24小时。
2、24小时后，收集3个孔的LNCaP细胞，计数，并求平均值。
3、计数后，分别将96孔板的LNCaP细胞与Caspase-3/7 Green Apoptosis Assay Reagent孵育30分钟。
4、根据计数，分别将NK92-A、NK92-B、NK92-C、NK92-D、NK92-E、NK92-F、NK92-G以及NK-Chimeric-FcγR细胞和肿瘤细胞按照1:1的靶标比接种到96孔板中，培养基中均添加1μg/mL的PSMAmAb，放入37度细胞培养箱中培养。
5、每隔3小时分析一次。结果如图3所示。
3、实验结果说明
1、NK92-A，NK92-E的ADCC相对于其他变体效果更好，说明CD64A的3个Ig-like C2结构完整，更加有利于NK细胞发挥ADCC效应。NK92-E的效果要优于NK92-A，表明CD64A的IgG Fc结合域离细胞膜结构远一些，有利于其发挥ADCC效应。
2、NK92-A，NK92-B，NK92-C，NK92-D以及NK92-Chimeric-FcγR比较，NK92-A和NK92-Chimeric-FcγR的ADCC效果更好，表明CD64A的Ig-like C2-type 3结构相对于另外2个结构，更加有利于NK发挥ADCC效果。
3、NK92-E，NK92-F，NK92-G比较，NK92-E的ADCC效果更好，表明CD64A的Ig-like C2-type 3结构后面串联一个结构会影响NK92的ADCC效应，但是串联CD16A完整的2个结构，则不会影响其ADCC效应。
4、NK92-E和NK92-Chimeric-FcγR的ADCC效应基本无差异，表明CD16A的Ig-like C2-type1，Ig-like C2-type2两个结构和CD64A的Ig-like C2-type1，Ig-like C2-type2两个结构相似，CD64A的Ig-like C2-type3能够增强NK92的ADCC效应。
根据图3的结果分析，Chimeric-FcγR的结构使NK细胞具有最强的ADCC效应，进一步对表达Chimeric-FcγR的NK细胞进行研究。
实施例2、iPSC来源的Chimeric-FcγR-iNK的制备、及其在激活条件下的稳定性检测
构建实施例1所述Chimeric-FcγR，并且构建mutCD16A(氨基酸序列如SEQ ID NO.:14，核苷酸序列如SEQ ID NO.:13)作为对照，进一步验证本发明Chimeric-FcγR的特性。
Chimeric-FcγR-iPSC细胞稳转及筛选
1、实验材料
名称 公司 货号
嘌呤霉素 Merck P9620
mTeSR1 Stem cell technologies #85850
Dispase II Merck D4693
Hexadimethrine bromide Merck H9268
2、实验方法
1、第0天，在初始转导前约24小时，当细胞密度达到80％左右，使用1mg/mL Dispase II进行细胞传代。
2、以1:3的比例将iPSC细胞接种到24孔培养板中，注意将细胞团保持在直径约为50-60μm。
3、第一天，用500μL的预热(37℃)mTeSR1孵育细胞，培养基中添加6μg/mL Hexadimethrine bromide，将细胞放入培养箱中孵育15分钟。
4、按每孔10μL的1x10 6TU/mL病毒颗粒感染iPSC细胞。在37℃，5％CO 2和95％湿度下孵育18-20小时。
5、第2天，去除培养基，加500μL的预热(37℃)mTeSR1孵育细胞，培养基中添加6μg/mL Hexadimethrine bromide，将细胞放入培养箱中孵育15分钟。
6、在培养基中加入三倍于初始量(从第1天开始)的病毒颗粒。即30μL的1x10 6TU/mL病毒颗粒进行二次感染。在37℃，5％CO 2和95％湿度下孵育18-20小时。
7、第3天和第4天，每天去除培养基，并更换为500μL不含Hexadimethrine bromide的预热培养基
8、第5-8天，每天使用500μL的预热培养基换液，培养基中添加1μg/mL嘌呤霉素。
9、持续使用1μg/mL嘌呤霉素筛选阳性细胞，直到细胞稳定为止。
10、稳转细胞系分别命名为NK92-Chimeric-FcγR-iPSC(Chimeric-FcγR-iPSC)、mutCD16A-iPSC。
Chimeric-FcγR-iPSC稳转细胞鉴定
1、实验材料
Figure PCTCN2022098848-appb-000004
2、实验方法
1.收集细胞200W Chimeric-FcγR-iPSC、mutCD16A-iPSC细胞加1ml TRIZOL，提取RNA并测定RNA浓度，取1μgRNA反转为cDNA，按如下表体系进行预混，
Figure PCTCN2022098848-appb-000005
2.然后将上述体系放入Light cycler仪器中按照3步法进行反应，循环数为45，反应体系如下：
Figure PCTCN2022098848-appb-000006
Figure PCTCN2022098848-appb-000007
3.检测引物序列如下
Chimeric-FcγR结构检测引物：
Forwordprimer:ACTCAAAGACAGCGGCTCCTA
Reverse primer:ACAGCTCAGGGTGACCAGATT
MutCD16A结构检测引物：
Forwordprimer:CCTCCTGTCTAGTCGGTTTGG
Reverse primer:TCGAGCACCCTGTACCATTGA
3、实验结果
如图4所示，检测Chimeric-FcγR或MutCD16A的表达量，可以确定pLV-EF1a-Chimeric-FcγR-IRES-Hygro或者pLV-EF1a-mutCD16A-IRES-Hygro载体都已经成功表达。
Chimeric-FcγR稳定性检测
1、实验材料
名称 公司 货号
STEMdiff TM NK Cell Kit Stem cell technologies #100-0170
PMA/Ionomycin mixture(250X) MultiSciences 70-CS1001
DPBS 赛默飞世尔科技 14190144
Anti-Human CD16 BD Biosciences 560995
K-562 武汉普诺赛生命科技有限公司 CL-0130
丝裂霉素C Sigma M5353
2、实验方法
1、使用STEMdiff TM NK Cell Kit将iPSC，Chimeric-FcγR-iPSC和mutCD16A-iPSC分化为iNK细胞，得到的iNK细胞分别命名为iNK，Chimeric-FcγR-iNK和mutCD16A-iNK。
2、取200万iNK、Chimeric-FcγR-iNK和mutCD16A-iNK，使用1乘PMA/Ionomycin mixture(250X)刺激细胞4小时。或者使用等比例的丝裂霉素C处理过后的K562细胞，分别与iNK、Chimeric-FcγR-iNK和mutCD16A-iNK孵育4小时。同时设置空白对照，不对NK细胞进行上述任何刺激。
3、细胞激活后，使用DPBS清洗细胞2遍，将细胞重悬在100μl 2％FBS的DPBS中，按照厂商说明，将细胞分别与Anti-Human CD16孵育1h。
4、孵育结束后，使用DPBS清洗细胞2遍，将细胞重悬在100μl 2％FBS的DPBS中，然后对上述细胞进行流式分析。
3、实验结果
K562(丝裂霉素C处理过的)和PMA/lonomycin用于激活NK细胞，检测NK细胞受激活后的百分比。NK细胞在激活的状态下，未经改造的CD16A会被金属酶(ADAM17)切除，实验结果检测NK细胞百分比后发现，未经改造的iNK细胞百分比明显下降。而mutCD16A-iNK细胞和Chimeric-FcγR-iNK在细胞激活后，mutCD16A和Chimeric-FcγR蛋白不会被金属酶切除，因此iNK的阳性细胞比例依旧处于较高水平(结果如图5所示)。
因此推断本发明Chimeric-FcγR-iNK会有优于未经改造的iNK细胞的杀伤活性，所以以下继续细胞水平和动物实验水平比较Chimeric-FcγR-iNK和mutCD16A对肿瘤细胞的杀伤效果。
细胞杀伤试验
1、实验方法
1、以每孔4000个细胞将LNCaP细胞铺种到96孔板，孵育24小时。
2、24小时后，收集3个孔的LNCaP细胞，计数，并求平均值。
3、计数后，分别将96孔板的LNCaP细胞与Caspase-3/7Green Apoptosis Assay Reagent孵育30分钟。
4、根据计数，分别将iNK，Chimeric-FcγR-iNK和mutCD16A-iNK细胞和肿瘤细胞按照1:1的靶标比接种到96孔板中，分别设置下表所示组别，放于IncuCyt培养箱中培养。
Figure PCTCN2022098848-appb-000008
5、之后每隔3小时拍照记录，分析，统计结果如图6所示。
2、实验结果
1、mutCD16A-iNK，Chimeric-FcγR-iNK和iNK+PSMAmAb组比较，mutCD16A-iNK和Chimeric-FcγR-iNK组对LNCaP细胞杀伤强很多，说明mutCD16A-iNK和Chimeric-FcγR-iNK组的mutCD16A、Chimeric-FcγR能够增强NK的杀伤能力。
2、iNK组和iNK+PSMAmAb组比较显示，iNK+PSMAmAb组对LNCaP细胞杀 伤较强，iNK有一定ADCC效应。
3、mutCD16A-iNK和mutCD16A-iNK+PSMAmAb组比较显示，mutCD16A-iNK+PSMAmAb组对LNCaP细胞杀伤较强，mutCD16A-iNK+PSMAmAb有更强的ADCC效应。
4、Chimeric-FcγR-iNK和Chimeric-FcγR-iNK+PSMAmAb组比较显示，Chimeric-FcγR-iNK+PSMAmAb组对LNCaP细胞杀伤较强，Chimeric-FcγR-iNK+PSMAmAb有更强的ADCC效应。
5、mutCD16A-iNK+PSMAmAb和Chimeric-FcγR-iNK+PSMAmAb组比较显示，Chimeric-FcγR-iNK+PSMAmAb组对LNCaP细胞杀伤稍强，Chimeric-FcγR-iNK+PSMAmAb有更强的ADCC效应。
综上，对LNCaP细胞杀伤的实验结果表明，无论有无抗体存在，表达本发明所述融合蛋白的NK细胞皆具有优于未经改造的NK细胞的杀伤活性。
实施例3、体内杀伤实验
1、实验材料
名称 公司 货号
C42细胞(人前列腺癌细胞) ATCC CRL-3314
NOD SCID小鼠 维通利华 406
2、实验方法
1、NOD SCID小鼠皮下注射1×10 6个荧光标记的C42细胞，3周后形成肉眼可见的肿瘤。
2、肿瘤模型形成后，先通过小动物成像系统分析，然后通过尾静脉注射不同组别，分别注射5×10 6个iNK、iNK+100μg PSMAmAb mutCD16A-iNK+100μg PSMAmAb、Chimeric-FcγR-iNK+100μg PSMAmAb。同时设置不做处理的对照
3、注射NK后，每周通过小动物成像系统分析肿瘤情况。
4、实验结束后，解剖动物，分离肿瘤组织称重。
3、实验结果
对实验结束后肿瘤重量进行统计如图7所示。实验结果说明：
1、iNK组和iNK+PSMAmAb组比较显示，iNK+PSMAmAb组有更强的ADCC效应，肿瘤杀伤效果更强。
2、iNK+PSMAmAb，mutCD16A-iNK+PSMAmAb，Chimeric-FcγR-iNK+PSMAmAb组比较，mutCD16A-iNK+PSMAmAb，Chimeric-FcγR-iNK+PSMAmAb组有更强的ADCC效应，肿瘤杀伤效果更强。
3、mutCD16A-iNK+PSMAmAb和Chimeric-FcγR-iNK+PSMAmAb组比较，Chimeric-FcγR-iNK+PSMAmAb组有更强的ADCC效应，肿瘤杀伤效果更强。

Claims (22)

  1. 一种融合蛋白，所述融合蛋白包括胞外部分、胞外铰链区、跨膜区、胞内区胞内部分，所述胞外部分是以下任意一种：
    1)CD16a的Ig-like C2-type 1，Ig-like C2-type 2和CD64的Ig-like C2-type 3依次串联；
    2)CD16A的Ig-like C2-type 1、CD16A的Ig-like C2-type 2、CD64A的Ig-like C2-type 1、CD64A的Ig-like C2-type 2、CD64A的Ig-like C2-type 3依次串联；
    3)CD64A的Ig-like C2-type 1、CD64A的Ig-like C2-type 2、CD64A的Ig-like C2-type 3、CD16A的Ig-like C2-type 1、CD16A的Ig-like C2-type 2依次串联；
    4)CD64A的Ig-like C2-type 1、CD64A的Ig-like C2-type 2、CD64A的Ig-like C2-type 3、CD16A的Ig-like C2-type 1依次串联；
    5)CD64A的Ig-like C2-type 1、CD64A的Ig-like C2-type 2、CD64A的Ig-like C2-type 3、CD16A的Ig-like C2-type 2依次串联；
    所述胞外铰链区、跨膜区、胞内区分别为CD64的胞外铰链区、CD16a的跨膜区、CD16a的胞内区。
  2. 如权利要求1所述的融合蛋白，其特征在于，所述CD16a的Ig-like C2-type 1的氨基酸序列如SEQ ID NO.：1所示或与所示序列相比有1、2、3、4、5或更多个突变，
    所述CD16a的Ig-like C2-type 2的氨基酸序列如SEQ ID NO.：3所示或与所示序列相比有1、2、3、4、5或更多个突变，
    所述CD64的Ig-like C2-type 3的氨基酸序列如SEQ ID NO.：5所示或与所示序列相比有1、2、3、4、5或更多个突变，
    所述CD64的胞外铰链区的氨基酸序列如SEQ ID NO.：7所示或与所示序列相比有1、2、3、4、5或更多个突变，
    所述CD16a的跨膜区的氨基酸序列如SEQ ID NO.：9所示或与所示序列相比有1、2、3、4、5或更多个突变，
    所述CD16a的胞内区的氨基酸序列如SEQ ID NO.：11所示或与所示序列相比有1、2、3、4、5或更多个突变，
    所述CD64A Ig-like C2-type 1的胞内区的氨基酸序列如SEQ ID NO.：15所示或与所示序列有1、2、3、4、5或更多个突变，
    所述CD64A Ig-like C2-type 2的胞内区的氨基酸序列如SEQ ID NO.：17所示或与所示序列有1、2、3、4、5或更多个突变。
  3. 一种分离的编码核酸，所述编码核酸编码权利要求1所述的融合蛋白。
  4. 如权利要求3所述的编码核酸，其特征在于所述CD16a的Ig-like C2-type 1的编码核酸序列与SEQ ID NO.：2所示序列有85％及以上同源性、或与SEQ ID NO.：2所示序列部分互补或完全互补、或如SEQ ID NO.：2所示，
    所述CD16a的Ig-like C2-type 2的编码核酸序列与SEQ ID NO.：4所示序列有85％及以上同源性、或与SEQ ID NO.：4所示序列部分互补或完全互补、或如SEQ ID NO.：4所示，
    所述CD64的Ig-like C2-type 3的编码核酸序列与SEQ ID NO.：6所示序列有85％及以上同源性、或与SEQ ID NO.：6所示序列部分互补或完全互补、或如SEQ ID NO.：6所示，
    所述胞外铰链区的编码核酸序列与SEQ ID NO.：8所示序列有85％及以上同源性、或与 SEQ ID NO.：8所示序列部分互补或完全互补、或如SEQ ID NO.：8所示，
    所述跨膜区的编码核酸序列与SEQ ID NO.：10所示序列有85％及以上同源性、或与SEQID NO.：10所示序列部分互补或完全互补、或如SEQ ID NO.：10所示，
    所述胞内区的编码核酸序列与SEQ ID NO.：12所示序列有85％及以上同源性、或与SEQID NO.：12所示序列部分互补或完全互补、或如SEQ ID NO.：12所示，
    所述CD64AIg-like C2-type 1的编码核酸序列与SEQ ID NO.：16所示序列有85％及以上同源性、或与SEQ ID NO.：16所示序列部分互补或完全互补、或如SEQ ID NO.：16所示，
    所述CD64AIg-like C2-type2的编码核酸序列与SEQ ID NO.：18所示序列有85％及以上同源性、或与SEQ ID NO.：18所示序列部分互补或完全互补、或如SEQ ID NO.：18所示。
  5. 表达权利要求1所述融合蛋白、或包含权利要求3所述编码核酸的表达载体。
  6. 如权利要求5所述的载体，所述表达载体包括但不限于细菌质粒载体、噬菌体载体、酵母质粒载体、腺病毒载体、逆转录病毒载体、慢病毒载体。
  7. 含有或表达权利要求1所述融合蛋白、权利要求3所述多核苷酸、权利要求5所述载体中的一种或多种的宿主细胞。
  8. 如权利要求7所述的宿主细胞，其特征在于，所述宿主细胞包括人的免疫细胞或干细胞。
  9. 如权利要求8所述的宿主细胞，其特征在于，所述免疫细胞包括T细胞、B细胞、K细胞和NK细胞中的一种或多种。
  10. 如权利要求8所述的宿主细胞，其特征在于，所述免疫细胞是NK细胞，所述干细胞是iPSC。
  11. 制备高杀伤活性的免疫细胞的方法，所述方法包括将权利要求1所述融合蛋白、权利要求3所述多核苷酸、权利要求5所述载体中的一种或多种引入免疫细胞；
    或者，所述方法包括将权利要求1所述融合蛋白、权利要求3所述多核苷酸、权利要求5所述载体中的一种或多种引入干细胞，然后诱导干细胞分化为免疫细胞。
  12. 如权利要求11所述的方法，其特征在于，所述引入的方法包括电穿孔、原生质体融合、磷酸钙沉淀、使用有包膜DNA的细胞融合、显微注射以及使用完整病毒转染。
  13. 包含权利要求1所述融合蛋白、权利要求3所述多核苷酸、权利要求5所述载体、权利要求7所述宿主细胞中的一种或多种的药物组合物。
  14. 如权利要求13所述药物组合物，其特征在于，所述药物组合物中还含有其他治疗癌症的药物和/或被权利要求1所述融合蛋白所识别的结构。
  15. 如权利要求14所述的药物组合物，其特征在于，所述药物是单克隆抗体药物，所述单克隆抗体药物包括abcam公司所提供的货号为ab268061的抗体或已经上市的单克隆抗体药物，所述已经上市的单克隆抗体药物包括马妥昔单抗、曲妥珠单抗、西妥昔单抗、达利珠单抗、他尼珠单抗、阿巴伏单抗、阿德木单抗、阿夫土珠单抗、阿仑单抗、培化阿珠单抗、阿麦妥昔单抗、阿泊珠单抗、巴维昔单抗、贝妥莫单抗、贝利木单抗、贝伐单抗、莫-比伐珠单抗、贝伦妥单抗-维多汀、莫坎妥珠单抗、拉坎妥珠单抗、卡罗单抗喷地肽、卡妥索单抗、泊西他珠单抗、西妥木单抗、可那木单抗、达西珠单抗、达洛珠单抗、地莫单抗、依美昔单抗、依决洛单抗、依洛珠单抗、恩司昔单抗、依帕珠单抗、厄马索单抗、埃达珠单抗、法勒珠单抗、芬妥木单抗、加利昔单抗、吉妥珠单抗、吉妥珠单抗、吉瑞昔单抗、格莱木单抗-维多汀、替伊莫单 抗、伊戈伏单抗、拉英达西单抗、英妥木单抗、伊珠单抗奥佐米星、伊匹木单抗、伊妥木单抗、拉贝珠单抗、来沙木单抗、林妥珠单抗、莫洛伏珠单抗。
  16. 如权利要求14所述的药物组合物，其特征在于，所述药物组合物还包括药学上可接受的载体、稀释剂或赋形剂，
    所述药学上可接受的载体、稀释剂或赋形剂包括但不限于已经由美国食品和药品管理局或中国食品药品监督管理局批准可用于人或家畜的任何佐剂、载体、赋形剂、助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、增味剂、表面活性剂、润湿剂、分散剂、悬浮剂、稳定剂、等渗剂、溶剂、表面活性剂或乳化剂，
    所述药物组合物可以为片剂、丸剂、粉剂、颗粒剂、胶囊剂、锭剂、糖浆剂、液体、乳剂、混悬剂、控制释放制剂、气雾剂、膜剂、注射剂、静脉滴注剂、透皮吸收制剂、软膏剂、洗剂、粘附制剂、栓剂。
  17. 一种在体外杀伤癌细胞的方法，所述方法包括将靶细胞与权利要求1或2所述融合蛋白、权利要求3或4所述多核苷酸、权利要求5或6所述载体、权利要求7-10任意一种宿主细胞、权利要求14-16任意一种药物组合物中的一种或多种相接触。
  18. 如权利要求17所述的方法，其特征在于，所述癌细胞是前列腺癌细胞。
  19. 权利要求1或2所述融合蛋白、权利要求3或4所述多核苷酸、权利要求5或6所述载体、权利要求7-10任意一种宿主细胞、权利要求14-16任意一种药物组合物在制备癌症免疫治疗药物、自身免疫性疾病药物、抗衰老药物、医美产品、代谢性疾病药物中的用途。
  20. 如权利要求19所述的用途，其特征在于，所述癌症包括宫颈癌、精原细胞瘤、睾丸淋巴瘤、前列腺癌、卵巢癌、肺癌、直肠癌、乳腺癌、皮肤鳞状细胞癌、结肠癌、肝癌、胰腺癌、胃癌、食管癌、甲状腺癌、膀胱移行上皮癌、白血病、脑瘤、胃癌、腹膜癌、头颈癌、子宫内膜癌、肾癌、雌性生殖道癌、原位癌、神经纤维瘤、骨癌、皮肤癌、胃肠道间质瘤、肥大细胞肿瘤、多发性骨髓瘤、黑色素瘤、胶质瘤，
    所述自身免疫性疾病包括贲门失弛缓症、阿狄森氏病、成人斯蒂尔氏病、无丙种球蛋白血症、斑秃、淀粉样变性、强直性脊柱炎、抗GBM/抗TBM肾炎、抗磷脂综合征、自身免疫性血管性水肿、自身免疫性自主神经异常、自身免疫性脑脊髓炎、自身免疫性肝炎、自身免疫性内耳病、自身免疫性心肌炎、自身免疫性卵巢炎、自身免疫性睾丸炎、自身免疫性胰腺炎、自身免疫性视网膜病变、自身免疫性荨麻疹，
    所述代谢性疾病包括糖尿病、糖尿病酮症酸中毒、高血糖高渗综合征、低血糖症、痛风、蛋白质-能量营养不良症、维生素A缺乏病、坏血病、维生素D缺乏病、骨质疏松症。
  21. 权利要求1或2所述融合蛋白、权利要求3或4所述多核苷酸、权利要求5或6所述载体、权利要求7-10任意一种所述宿主细胞、权利要求14-16任意一种药物组合物在提高单抗治疗效果中的应用。
  22. 如权利要求21所述的应用，其特征在于，所述治疗效果是针对前列腺癌症的治疗效果。
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Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114478806B (zh) * 2022-04-14 2022-07-01 呈诺再生医学科技(北京)有限公司 一种提升免疫细胞杀伤活性的嵌合受体及其应用
WO2024067682A1 (zh) * 2022-09-29 2024-04-04 上海先博生物科技有限公司 工程改造的免疫效应细胞及其组合物和应用
CN116732039B (zh) * 2023-08-03 2023-11-14 呈诺再生医学科技(北京)有限公司 一种促进rna序列在细胞内直接环化翻译的序列组合及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111556892A (zh) * 2017-12-08 2020-08-18 菲特治疗公司 使用增强的iPSC衍生的效应细胞的免疫疗法
CN111556756A (zh) * 2017-10-26 2020-08-18 明尼苏达大学董事会 重组免疫细胞、制备方法和使用方法
CN113583139A (zh) * 2020-07-06 2021-11-02 上海鑫湾生物科技有限公司 一种嵌合受体及其应用
WO2021260696A1 (en) * 2020-06-22 2021-12-30 Ramot At Tel-Aviv University Ltd. Multi subunit protein modules, cells expressing same and uses thereof
WO2022032864A1 (zh) * 2019-12-09 2022-02-17 深圳市体内生物医药科技有限公司 一种识别Fc片段的嵌合抗原受体及其应用
CN114478806A (zh) * 2022-04-14 2022-05-13 呈诺再生医学科技(北京)有限公司 一种提升免疫细胞杀伤活性的嵌合受体及其应用

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2688490C (en) * 2007-06-01 2022-06-21 Scott E. Strome Immunoglobulin constant region fc receptor binding agents
WO2009158696A1 (en) * 2008-06-27 2009-12-30 Zymogenetics, Inc. SOLUBLE HYBRID Fcγ RECEPTORS AND RELATED METHODS
CN104087607B (zh) * 2013-04-01 2017-06-20 科济生物医药（上海）有限公司 编码嵌合抗原受体蛋白的核酸及表达嵌合抗原受体蛋白的t淋巴细胞
CN105601746B (zh) * 2014-11-21 2021-02-19 三生国健药业（上海）股份有限公司 一种嵌合Fc受体的融合蛋白及其制备方法和应用
CN108250301A (zh) * 2016-12-29 2018-07-06 天津天锐生物科技有限公司 一种多靶点嵌合抗原受体
CN110494162A (zh) * 2017-01-30 2019-11-22 优努姆治疗公司 改良的抗体-偶联t细胞受体构建体及其治疗用途
US20210340219A1 (en) * 2018-09-07 2021-11-04 Sotio, LLC Chimeric receptor polypeptides in combination with trans metabolism molecules modulating intracellular lactate concentrations and therapeutic uses thereof
CN113913379B (zh) * 2020-07-07 2024-02-06 深圳市菲鹏生物治疗股份有限公司 T淋巴细胞及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111556756A (zh) * 2017-10-26 2020-08-18 明尼苏达大学董事会 重组免疫细胞、制备方法和使用方法
CN111556892A (zh) * 2017-12-08 2020-08-18 菲特治疗公司 使用增强的iPSC衍生的效应细胞的免疫疗法
WO2022032864A1 (zh) * 2019-12-09 2022-02-17 深圳市体内生物医药科技有限公司 一种识别Fc片段的嵌合抗原受体及其应用
WO2021260696A1 (en) * 2020-06-22 2021-12-30 Ramot At Tel-Aviv University Ltd. Multi subunit protein modules, cells expressing same and uses thereof
CN113583139A (zh) * 2020-07-06 2021-11-02 上海鑫湾生物科技有限公司 一种嵌合受体及其应用
CN114478806A (zh) * 2022-04-14 2022-05-13 呈诺再生医学科技(北京)有限公司 一种提升免疫细胞杀伤活性的嵌合受体及其应用

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