WO2023169092A1 - 一种肿瘤微环境调控型car-单核/巨噬细胞及其制备方法和应用 - Google Patents

一种肿瘤微环境调控型car-单核/巨噬细胞及其制备方法和应用 Download PDF

Info

Publication number
WO2023169092A1
WO2023169092A1 PCT/CN2023/073798 CN2023073798W WO2023169092A1 WO 2023169092 A1 WO2023169092 A1 WO 2023169092A1 CN 2023073798 W CN2023073798 W CN 2023073798W WO 2023169092 A1 WO2023169092 A1 WO 2023169092A1
Authority
WO
WIPO (PCT)
Prior art keywords
car
macrophage
seq
monocyte
tmer
Prior art date
Application number
PCT/CN2023/073798
Other languages
English (en)
French (fr)
Inventor
宁蓬勃
Original Assignee
西安电子科技大学
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 西安电子科技大学 filed Critical 西安电子科技大学
Publication of WO2023169092A1 publication Critical patent/WO2023169092A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0645—Macrophages, e.g. Kuepfer cells in the liver; Monocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/243—Colony Stimulating Factors
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • 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
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • 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 relates to the technical fields of immunology and tumor therapy, and in particular to a tumor microenvironment-regulated CAR-monocyte/macrophage cell and its preparation method and application.
  • tumor cell immunotherapy has been widely used in experimental research and clinical treatment of various malignant tumors.
  • CAR-T cell immunotherapy has achieved good results in the field of hematological tumors.
  • the suppressive tumor microenvironment (Tumor Microenvironment, TME) usually forms an immune barrier to adoptive cellular immunotherapy through a variety of factors. Its dense cell and matrix barriers first form a physical barrier to the infiltration of engineered lymphocytes into solid tumors. .
  • the immunosuppressive microenvironment formed by the TME such as hypoxic and acidic environments, expression of immune checkpoint ligands, and especially a large number of immunosuppressive cells, such as tumor-associated macrophages (TAMs) ), myeloid-derived suppressor cells and regulatory T cells, the immunosuppressive barrier formed is one of the main difficulties in the current immunotherapy of solid tumors.
  • TME tumor-associated macrophages
  • TAMs tumor-associated macrophages
  • Tumors generate TAMs by recruiting circulating monocytes and tissue-resident macrophages to the TME through various soluble and mechanical factors and polarizing them into an M2 phenotype.
  • TAM's immune recognition and phagocytosis capabilities of tumor cells have significantly changed, and they have lost their basic immune function of recognizing and immune clearance of abnormal cells.
  • TAM also plays a key role in suppressing endogenous anti-tumor immunity by upregulating immunosuppressive surface proteins, secreting reactive oxygen species, producing cytokines that inhibit T cell function, and secreting chemokines that recruit Treg cells.
  • TAM promotes tumor angiogenesis and metastasis by secreting factors such as VEGF and matrix metalloproteinases, which can reshape the TME, increase blood vessel formation, and promote tumor cell migration. Since macrophage polarization is a gradual change process, with the increase of tumor suppressor signals in the tumor microenvironment, the TME may convert more macrophages that migrate into solid tumors from an M1 phenotype to an M2-like TAM phenotype. As the degree of tumor malignancy increases, TAM in solid tumors will reach 50% and continue to increase.
  • some current clinical immunotherapy methods include inhibiting the chemotaxis of macrophages to solid tumors, killing the number of macrophages, inducing the repolarization of M2 macrophages to M1, or promoting the reprogramming of TAMs to M1.
  • different strategies have shown that blocking monocyte differentiation can reduce the number of TAMs by blocking the CSF1 (also known as MCSF)/CSF1R axis, which is an important ligand-receptor pair for macrophage differentiation and survival. It also reduces the survival rate of existing TAMs.
  • CAR-Macrophage chimeric antigen receptor macrophage immunotherapy
  • CAR-M chimeric antigen receptor macrophage immunotherapy
  • monocytes/macrophages to perform CAR Construction
  • CAR-M cells can specifically accumulate into solid tumors and penetrate the dense stromal tissue surrounding tumors.
  • CAR-M combines direct tumor killing with the ability to enhance endogenous immunity through antigen presentation, making it a unique approach to the development of anti-tumor cell therapies.
  • the novel CAR-M technology has obvious advantages in solving solid tumors, as an adoptive immunotherapy of the macrophage system, the inhibitory TME inside the solid tumor usually hinders the therapeutic effect of CAR-M cells through a variety of factors, and CAR After -M enters the interior of solid tumors, there is also a risk of polarization into M2 macrophages.
  • How to avoid reinfused macrophages from developing and producing the cancer-promoting properties of TAMs is an important issue to be solved in the development of CAR-M technology.
  • the ideal design should not only maintain the M1-type characteristics of CAR-M itself, but also produce a TME reversal effect.
  • the design of specific elements for CAR-M signal transduction function provides the possibility to improve the anti-cancer potential of CAR-M.
  • the purpose of the present invention is to provide a tumor microenvironment-regulated CAR-monocyte/macrophage cell and its preparation method and application to solve the bottleneck problem of the above-mentioned existing technology in the development of the CAR-M field.
  • TMER CAR monouclear
  • CAR-M scFV targets and kills tumor cells, it can not only maintain the M1 phenotype in its own tumor microenvironment, but also be able to target TAMs in the TME. Achieve re-education, reverse the tumor microenvironment, and achieve better anti-tumor effects.
  • the present invention provides the following solutions:
  • the invention provides a tumor microenvironment-regulated CAR-monocyte/macrophage cell, which includes a chimeric antigen receptor
  • the composite structure can generate intracellular expression of GM-CSF and secrete it extracellularly, promote the differentiation of CAR-monocytes/macrophages into M1 macrophages, and further regulate the tumor microenvironment; wherein the GM-CSF
  • the nucleotide sequence is shown in SEQ ID NO:1 or SEQ ID NO:9.
  • the present invention also provides a chimeric antigen receptor, including GM-CSF, HER2 scFv, the transmembrane region of ⁇ 1 ⁇ 1 integrin, the intracellular ⁇ 1 ⁇ 1 integrin, and the intracellular Fc ⁇ RI transmembrane signaling domain.
  • a chimeric antigen receptor including GM-CSF, HER2 scFv, the transmembrane region of ⁇ 1 ⁇ 1 integrin, the intracellular ⁇ 1 ⁇ 1 integrin, and the intracellular Fc ⁇ RI transmembrane signaling domain.
  • the nucleotide sequences of the GM-CSF, HER2 scFv, the transmembrane region of ⁇ 1 ⁇ 1 integrin, the intracellular ⁇ 1 ⁇ 1 integrin and the intracellular Fc ⁇ RI transmembrane signaling domain are as follows: SEQ ID NO: 1/SEQ ID NO:9, SEQ ID NO:2, SEQ ID NO:3/SEQ ID NO:10, SEQ ID NO:4/SEQ ID NO:11, SEQ ID NO:5/SEQ ID NO:12.
  • the invention also provides a recombinant vector, including the chimeric antigen receptor.
  • the invention also provides a method for preparing CAR-monocyte/macrophage cells regulated by the tumor microenvironment, which includes constructing the recombinant vector and transfecting the CAR-monocyte/macrophage with the recombinant vector. Cellular steps.
  • the construction method of the recombinant vector includes:
  • the present invention also provides a pharmaceutical composition, including the CAR-monocyte/macrophage or the chimeric antigen receptor of claim 2 or the recombinant vector, together with pharmaceutically acceptable excipients or carriers .
  • the present invention also provides the application of the CAR-monocyte/macrophage in the preparation of drugs that promote the differentiation of M1 macrophages.
  • the present invention also provides the application of the CAR-monocyte/macrophage in the preparation of drugs for the treatment of solid tumors;
  • the solid tumors may be solid tumors with high HER2 expression, but are not limited thereto.
  • the present invention designs and constructs tumor microenvironment regulated TMER CAR (monocyte) macrophages (Tumor Microenvironment Regulated CAR-M, TMER CAR-M) based on the GM-CSF motif.
  • TMER CAR tumor microenvironment regulated TMER CAR
  • the GM-CSF motif and embedded Combined with the antigen receptor to form a complex structure, the constructed CAR-M can autosecret GM-CSF and transfer it to the outside of the cell, leading to the production of higher levels of inflammatory anti-cancer factors; it can precisely enable CAR-M scFV to target and kill tumors.
  • the cells maintain themselves to maintain the macrophage M1 phenotype and avoid reversal of the tumor microenvironment.
  • GM-CSF will enter the TME, allowing it to regulate the tumor microenvironment and enhance the anti-tumor effect.
  • the present invention designs polarization elements suitable for macrophage function, and designs and implements a tumor microenvironment-regulated TMER CAR-M (TMER CAR-M) cell technology platform.
  • TMER CAR-M tumor microenvironment-regulated TMER CAR-M
  • This technology platform has significant Improving the anti-tumor properties of CAR-M plays a role in maintaining the M1 phenotype and regulating the tumor microenvironment, and is suitable for the application and extension design of various types of CAR-M in the treatment of solid tumors in the future.
  • Figure 1 shows the construction and characterization of macrophage-specific activated TMER CAR-M of the present invention: A: Confocal microscopy to observe the transmembrane expression of TMER CAR; B: qPCR to detect the expression of TMER CAR; C: Confocal detection of TMER CAR -M's HER2 protein binding ability, scale bar size is 30 ⁇ m;
  • FIG. 2 shows the activity detection of TMER CAR-M of the present invention
  • Figure 3 shows the in vitro M1 phenotype polarization detection of TMER CAR-M of the present invention
  • A qPCR detection of CD80 expression
  • B qPCR detection of IFN- ⁇ expression
  • C qPCR detection of iNOS expression
  • D qPCR detection The expression of CD206
  • E qPCR detects the expression of Arg-1
  • F qPCR detects the expression of IL-10
  • * indicates significant differences between different groups, *** means P ⁇ 0.001, ** means P ⁇ 0.01, * means P ⁇ 0.05;
  • Figure 4 shows the in vitro anti-M2 repolarization detection of TMER CAR-M of the present invention
  • A qPCR detection of CD80 expression
  • B qPCR detection of IFN- ⁇ expression
  • C qPCR detection of iNOS expression
  • D qPCR detection The expression of CD206
  • E qPCR detects the expression of Arg-1
  • F qPCR detects the expression of IL-10
  • * indicates significant differences between different groups, *** means P ⁇ 0.001, ** means P ⁇ 0.01, * means P ⁇ 0.05;
  • Figure 5 is an analysis of the reversal status of TMER CAR-M of the present invention in regulating M2 macrophages in vitro;
  • A. qPCR detects the expression of CD206;
  • B. qPCR detects the expression of Arg-1;
  • C. qPCR detects the expression of IL-10 ;
  • qPCR detects the expression of CD80;
  • E. qPCR detects the expression of IFN- ⁇ ;
  • Figure 6 shows the mouse HER2+-4T1 tumor model of the present invention to verify the anti-tumor results of TMER CAR-M;
  • A TMER CAR-M treated mouse HER2+-4T1 tumor model diagram;
  • B TMER CAR-M treated mouse tumor growth curve ;
  • C TMER CAR-M treated mouse tumor survival curve;
  • D TMER CAR-M treated mouse weight; * indicates significant differences between different groups, *** means P ⁇ 0.001, ** means P ⁇ 0.01 , * means P ⁇ 0.05;
  • Figure 7 is the imaging system IVIS Lumina II monitoring the anti-tumor results of the TMER CAR-M of the present invention inhibiting the nude mouse SKOV3 (human ovarian cancer cell) abdominal tumor model.
  • HER2 scFv the transmembrane region of ⁇ 1 ⁇ 1 integrin, ⁇ 1 ⁇ 1 integrin intracellularly, Fc ⁇ RI transmembrane signaling domain intracellularly and GFP were connected using a linker, and then P2A was used to connect the GM-CSF motif to synthesize Chimeric antigen receptor HER2 scFv-GM-CSF (sequence synthesis by Sangon Bioengineering (Shanghai) Co., Ltd.) is transfected into macrophages to construct macrophage-specific CAR.
  • the scFv-GM-CSF-CAR lentiviral plasmid is co-transfected with Rev, Gag, and VSV helper plasmids to package it into a complete HER2 scFv-GM-CSF-CAR lentivirus.
  • virus titer (TU/mL) average number of green fluorescent cells ⁇ virus dilution factor/volume of inoculated virus solution (mL), the unit is TU/mL.
  • HER2 scFv sequence (SEQ ID NO:2):
  • GFP sequence (SEQ ID NO:6):
  • P2A sequence (SEQ ID NO:8):
  • HER2 scFv-GM-CSF-CAR lentivirus was used to infect macrophages to obtain TMER CAR-M stably transduced cell line, and macrophages were inoculated into 6-well plates.
  • HER2 scFv-GM-CSF-CAR lentiviral virus particles to the macrophage culture medium at a ratio of 9:1, and replace with fresh DMEM before and after 24 hours.
  • Complete culture medium, lentiviral infection of macrophages will be completed after 48 hours; observe the cell growth and morphological changes, and pass them into new culture bottles or dishes for amplification or cryopreservation.
  • TMER CAR-293T and TMER CAR-M represents cell membrane localization
  • GFP represents the CAR structure expressed across the membrane
  • Merged represents the overlay.
  • the confocal results show that the CAR structure realized by the technology of the present invention is successfully expressed on the cell membrane.
  • Figure 1B further qPCR quantitative analysis verified the significant high expression of the CAR sequence in the corresponding design group, supporting the successful construction of TMER CAR-M.
  • TMER CAR-M represents the results of field photography
  • HER2 scFv-GFP represents TMER CAR-M
  • HER2-PE represents PE-labeled HER2 tumor antigen protein
  • Merge represents the overlay, showing TMER CAR -M. Effective binding to HER2 is achieved through the expression of transmembrane HER2 scFv.
  • Figure 1 The results verify that the TMER CAR-M fusion protein was successfully expressed. Not only was the TMER CAR-M successfully constructed, but it also had significant HER2 Tumor antigen binding activity; this provides a cell therapy model for later stage studies.
  • the TMER CAR-M constructed above was cultured and detected in 6-well plates at a density of 2 ⁇ 10 5 cells/mL.
  • the specific operations are as follows:
  • the tumor cell culture supernatant was used to simulate the tumor suppressive microenvironment system in vitro, and TMER CAR-M, CAR-Like and control group ordinary macrophages were respectively acted on to successfully construct TMER CAR-M cells, CAR-Like and First generation
  • the CAR-M group was subjected to control analysis.
  • the tumor culture supernatant was induced by TMER CAR-M for 5 days, and total cellular RNA was extracted. qPCR detection further verified the anti-polarization ability of TMER CAR-M. Specific steps include:
  • TMER CAR-M regulates the reversion of M2 macrophages to M1 macrophages in vitro
  • the tumor cell culture supernatant was used to simulate the tumor suppressive microenvironment system in vitro, and TMER CAR-M, CAR-Like and control group ordinary macrophages were respectively acted on to successfully construct TMER CAR-M cells, CAR-Like and First generation
  • the CAR-M group was subjected to control analysis.
  • the tumor culture supernatant was used to induce ordinary macrophages, and then TMER CAR-M supernatant was used for induction, and total cellular RNA was extracted. qPCR detection further verified the anti-polarization ability of TMER CAR-M. Specific steps include:
  • the mouse HER2 + -4T1 tumor model was used to verify the anti-tumor effect of TMER CAR-M.
  • the establishment of the HER2 + -4T1 tumor model includes the treatment process as follows:
  • the mouse breast cancer cell line HER2 + -4T1 was cultured in 1640 medium (hyclone) in a 37°C, 5% CO 2 cell incubator;
  • HER2 + -4T1 cells form tumors, set up parallel experiments with 4 mice in each group. Inject 100 ⁇ L of PBS or cell resuspension through the tail vein. UTD-M, Empty, CAR-like, The First generation CAR-M and TMER CAR-M groups were injected with 3 ⁇ 10 6 cells respectively. On the 5th day after the first injection, the second injection was performed. The injection volume of each group was 3 ⁇ 10 6 cells. The total injection twice.
  • mice tumor volume (length* width2 )/2. Measure once every two days, and finally calculate Mouse survival curve.
  • Figure 6A is a schematic diagram of the treatment plan.
  • Figure 6B shows the volume growth of mice in different treatment groups and control groups during treatment.
  • Figure 6C represents the survival conditions of mice in different treatment groups and control groups.
  • Figure 6D represents The weight monitoring of mice in different groups showed that compared with the CAR-like group and the First generation CAR-M group, the tumors in the TMER CAR-M group were significantly suppressed, the survival period was prolonged, and significant therapeutic effects were achieved.
  • the body weight of the TMER CAR-M group did not change significantly, suggesting that it has better safety.
  • the mouse HER2+-4T1 tumor model verified the anti-tumor effect of TMER CAR-M, and then the abdominal tumor model of human ovarian cancer SK-OV-3 (HER2 high expression) was used to further verify the TMER CAR-M anti-tumor design based on this patent.
  • Tumor effect the establishment of SK-OV-3 abdominal tumor model includes the treatment process as follows:
  • Human ovarian cancer cell line SK-OV-3 was cultured in DMEM medium (hyclone) in a 37°C, 5% CO 2 cell incubator;
  • nude mice aged 4-6 weeks need to be raised in a constant-temperature sterile ventilated mouse breeding room for more than one week before conducting mouse experiments;

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Public Health (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Oncology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Hematology (AREA)
  • Epidemiology (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

提供一种肿瘤微环境调控型CAR-单核/巨噬细胞及其制备方法和应用，属于免疫学和肿瘤治疗学技术领域。所述CAR-单核/巨噬细胞形成嵌合抗原受体复合结构的同时，可胞内表达GM-CSF并自分泌至胞外，促进CAR-单核/巨噬细胞形成M1型巨噬细胞分化，保持自身抗肿瘤微环境M2型逆转的同时，可进一步调控肿瘤微环境，进而增敏CAR-单核/巨噬细胞的抗肿瘤效应。基于GM-CSF构建了肿瘤微环境调控型的CAR巨噬细胞技术平台，能够维持CAR-M自身的M1型特性，又能产生TME逆转效应，进而实现CAR-单核/巨噬细胞的高效抗肿瘤效应。

Description

一种肿瘤微环境调控型CAR-单核/巨噬细胞及其制备方法和应用 技术领域
本发明涉及免疫学和肿瘤治疗学技术领域，特别是涉及一种肿瘤微环境调控型CAR-单核/巨噬细胞及其制备方法和应用。
背景技术
近年来，肿瘤细胞免疫疗法功能被广泛应用于各种恶性肿瘤的实验研究和临床治疗，尤其是CAR-T细胞免疫疗法在血液系统肿瘤领域取得很好的疗效。但在实体肿瘤中，抑制性肿瘤微环境(Tumor Microenvironment，TME)通常会通过多种因素对过继细胞免疫治疗形成免疫壁垒，其致密的细胞和基质屏障首先对工程淋巴细胞浸润实体肿瘤形成物理屏障。即便是进入实体肿瘤，TME形成的免疫抑制微环境，如缺氧和酸性环境、免疫检查点配体的表达、特别是大量的免疫抑制细胞，如肿瘤相关巨噬细胞(tumor-associated macrophage，TAM)、髓系来源的抑制细胞和调节性T细胞，形成的免疫抑制屏障是当前实体肿瘤免疫治疗的主要难点之一。
TME形成的免疫抑制机制十分复杂，这其中重要的因素，即为肿瘤相关巨噬细胞(tumor-associated macrophage，TAM)形成的免疫抑制机制。肿瘤通过各种可溶性和机械因素将循环中的单核细胞和组织驻留的巨噬细胞招募到TME，并将它们极化成M2表型，从而产生TAM。TAM相对于生理条件下的巨噬细胞，对肿瘤细胞的免疫识别和吞噬能力发生显著性改变，失去了对异常细胞识别并免疫清除的基本免疫功能。同时，TAM还通过上调免疫抑制表面蛋白，分泌活性氧，产生抑制T细胞功能的细胞因子，以及分泌招募Treg细胞的趋化因子，在抑制内源性抗肿瘤免疫中发挥关键作用。此外，TAM通过分泌VEGF和基质金属蛋白酶等因子来促进肿瘤血管生成和转移，这些因子可以重塑TME，增加血管形成，促进肿瘤细胞迁移。由于巨噬细胞极化是一个渐进变化过程，随着肿瘤微环境抑癌信号的增加，TME可能将更多趋化至实体瘤内部的巨噬细胞从M1表型转变为M2样TAM表型。随着肿瘤恶性程度的增加，实体瘤内TAM会达到50％，并且继续增多。
针对TAM的免疫抑制特性，当前一部分临床免疫治疗，采取抑制巨噬细胞向实体肿瘤趋化、杀灭巨噬细胞数量、诱导M2型巨噬细胞向M1型复极化或者促进TAM重编程为M1型等不同策略。有研究通过阻断CSF1(也称为MCSF)/CSF1R轴，这是巨噬细胞分化和存活的重要配体-受体对，从而阻断单核细胞分化来减少TAM的数量，同 时也降低了现有TAM的存活率。此外，有研究使用CSF1R的小分子抑制剂阻断诱导TAM从M2向M1表型的复极化，使TAM具有抗肿瘤作用。这些策略仍着眼于对TAM的数量改变的治疗水平。
针对实体肿瘤的免疫治疗难点，嵌合抗原受体巨噬细胞免疫治疗(CAR-Macrophage，CAR-M)是作为近年来发展的新颖的过继细胞免疫治疗技术，采用单核/巨噬细胞进行CAR构建，为攻克实体肿瘤提供了一种可能的解决方案。CAR-M细胞可特异地向实体瘤集聚，并穿透肿瘤周围致密的间质组织。此外，CAR-M将直接杀瘤手段与通过抗原提呈增强内源性免疫的能力结合在一起，使其成为抗肿瘤细胞治疗发展的独特途径。研究发现，CAR-M能够在不添加可溶性调理因子的情况下驱动对靶肿瘤细胞的吞噬和杀伤。Morrissey等发现，利用抗CD19和抗CD22 CAR构建小鼠巨噬细胞系和小鼠骨髓来源的巨噬细胞，介导了对抗原珠和肿瘤细胞的吞噬作用，证实了CAR诱导吞噬途径的能力。Zhang等利用诱导性多能干细胞(Induced pluripotent stem cells，iPSCs)表达CAR，并将CAR-iPSCs诱导为CAR-iMac。在没有抗原刺激的情况下，CAR-iMac偏向M2样表型。在抗原刺激后，CAR介导的信号促进CAR-iMac的吞噬作用。
虽然新颖的CAR-M技术在解决实体瘤方面优势明显，但是作为巨噬细胞体系的过继免疫治疗，实体瘤内部的抑制性TME通常会通过多种因素阻碍CAR-M细胞的治疗效果，并且CAR-M在进入实体瘤内部之后，也存在极化成为M2型巨噬细胞的风险。如何避免回输的巨噬细胞发展并产生TAM的促癌特性，是CAR-M技术发展中要解决的重要问题。对此，理想的设计应既能维持CAR-M自身的M1型特性，又能产生TME逆转效应。而CAR-M信号转导功能的特异性元件的设计，为提高CAR-M的抗癌潜能提供了可能性。
发明内容
本发明的目的是提供一种肿瘤微环境调控型CAR-单核/巨噬细胞及其制备方法和应用，以解决上述现有技术在CAR-M领域发展中的瓶颈问题，基于GM-CSF基序设计构建的肿瘤微环境调控型TMER CAR(单核)巨噬细胞，CAR-M scFV靶向杀伤肿瘤细胞后，不但能够维持自身肿瘤微环境中维持M1表型，并能够对TME中的TAM实现再教育，逆转肿瘤微环境，实现更优抗肿瘤效应。
为实现上述目的，本发明提供了如下方案：
本发明提供一种肿瘤微环境调控型CAR-单核/巨噬细胞，其为包括嵌合抗原受体 的复合结构，可产生GM-CSF胞内表达并分泌至胞外，促进CAR-单核/巨噬细胞形成M1型巨噬细胞分化的同时，进一步调控肿瘤微环境；其中所述GM-CSF的核苷酸序列如SEQ ID NO:1或SEQ ID NO:9所示。
本发明还提供一种嵌合抗原受体，包括GM-CSF、HER2 scFv、α1β1整合素的跨膜区域、α1β1整合素胞内以及FcγRI跨膜信号传导域胞内。
优选的是，所述GM-CSF、HER2 scFv、α1β1整合素的跨膜区域、α1β1整合素胞内以及FcγRI跨膜信号传导域胞内的核苷酸序列分别如SEQ ID NO:1/SEQ ID NO:9、SEQ ID NO:2、SEQ ID NO:3/SEQ ID NO:10、SEQ ID NO:4/SEQ ID NO:11、SEQ ID NO:5/SEQ ID NO:12所示。
本发明还提供一种重组载体，包括所述的嵌合抗原受体。
本发明还提供根据所述的肿瘤微环境调控型CAR-单核/巨噬细胞的制备方法，包括构建所述的重组载体，并将所述重组载体转染所述CAR-单核/巨噬细胞的步骤。
优选的是，所述重组载体的构建方法包括：
(1)构建嵌合抗原受体：将HER2 scFv、α1β1整合素的跨膜区域、α1β1整合素胞内、FcγRI跨膜信号传导域胞内使用linker进行连接后，再用P2A连接GM-CSF，合成嵌合抗原受体；
(2)将所述嵌合抗原受体与慢病毒载体重组，构建所述重组载体。
本发明还提供一种药物组合物，包括所述的CAR-单核/巨噬细胞或权利要求2所述的嵌合抗原受体或所述的重组载体，连同药学上可接受的辅料或载体。
本发明还提供所述的CAR-单核/巨噬细胞在制备促进M1型巨噬细胞分化的药物中的应用。
本发明还提供所述的CAR-单核/巨噬细胞在制备治疗实体瘤药物中的应用；所述实体瘤可选择HER2高表达的实体肿瘤，但不限于此。
本发明公开了以下技术效果：
本发明基于GM-CSF基序设计构建了肿瘤微环境调控型TMER CAR(单核)巨噬细胞(Tumor Microenvironment Regulated CAR-M，TMER CAR-M)，基于本技术平台GM-CSF基序与嵌合抗原受体形成复合结构，其构建的CAR-M能够自分泌GM-CSF并至胞外，并导致产生更高水平的炎性抗癌细胞因子；恰能够使CAR-M scFV靶向杀伤肿瘤细胞的同时，维持自身保持巨噬细胞M1表型，免遭肿瘤微环境逆转，同时， GM-CSF会进入TME中，恰能够使其起到调控肿瘤微环境的作用，增强抗肿瘤效应。
本发明基于GM-CSF基序，设计适用于巨噬细胞功能的极化元件，设计并实施了一种肿瘤微环境调控型TMER CAR-M(TMER CAR-M)细胞技术平台，该技术平台显著提高CAR-M抗肿瘤特性，起到了维持M1表型以及调控肿瘤微环境的作用，适用于今后各类型CAR-M治疗实体肿瘤的应用及延伸设计。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明巨噬细胞特异活化型TMER CAR-M的构建及其表征：A：共聚焦显微镜观察TMER CAR的跨膜表达；B：qPCR检测TMER CAR的表达；C：共聚焦检测TMER CAR-M的HER2蛋白结合能力，标尺大小为30μm；
图2为本发明TMER CAR-M的活力检测；A：CCK-8检测TMER CAR-M的增殖情况；B：qPCR检测p53的表达情况；C：qPCR检测CCND-1的表达情况；D：qPCR检测Ki67的表达情况；*表示不同组之间的显著性差异，***为P<0.001，**为P<0.01，*为P<0.05；
图3为本发明TMER CAR-M体外M1表型极化检测；A.qPCR检测CD80的表达情况；B.qPCR检测IFN-γ的表达情况；C.qPCR检测iNOS的表达情况；D.qPCR检测CD206的表达情况；E.qPCR检测Arg-1的表达情况；F.qPCR检测IL-10的表达情况；*表示不同组之间的显著性差异，***为P<0.001，**为P<0.01，*为P<0.05；
图4为本发明TMER CAR-M体外抗M2复极化检测；A.qPCR检测CD80的表达情况；B.qPCR检测IFN-γ的表达情况；C.qPCR检测iNOS的表达情况；D.qPCR检测CD206的表达情况；E.qPCR检测Arg-1的表达情况；F.qPCR检测IL-10的表达情况；*表示不同组之间的显著性差异，***为P<0.001，**为P<0.01，*为P<0.05；
图5为本发明TMER CAR-M体外调控M2型巨噬细胞逆转情况分析；A.qPCR检测CD206的表达情况；B.qPCR检测Arg-1的表达情况；C.qPCR检测IL-10的表达情况；D.qPCR检测CD80的表达情况；E.qPCR检测IFN-γ的表达情况；F.qPCR检测iNOS的表达情况；*表示不同组之间的显著性差异，***为P<0.001，**为P<0.01，* 为P<0.05；
图6为本发明小鼠HER2+-4T1肿瘤模型验证TMER CAR-M的抗肿瘤结果；A.TMER CAR-M治疗小鼠HER2+-4T1肿瘤模式图；B.TMER CAR-M治疗小鼠肿瘤生长曲线；C.TMER CAR-M治疗小鼠肿瘤生存曲线；D.TMER CAR-M治疗小鼠体重；*表示不同组之间的显著性差异，***为P<0.001，**为P<0.01，*为P<0.05；
图7为成像系统IVIS Lumina II监测本发明TMER CAR-M抑制裸鼠SKOV3(人卵巢癌细胞)腹腔肿瘤模型的抗肿瘤结果。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。
实施例1一种肿瘤微环境调控型CAR(单核)巨噬细胞的制备方法及功能验证
1、肿瘤微环境调控型TMER CAR-M的构建及表征
本研究前期参考CAR-T的体胞外结合理念，受益于包膜病毒的侵染出芽表达机制，采用慢病毒载体构建巨噬细胞的特异性CAR以及肿瘤微环境调控型TMER CAR-M， 以HER2为模式分子成功建立肿瘤微环境调控型TMER CAR-M平台，于细胞膜表面表达跨膜HER2 scFv胞外段，筛选获得具有高亲和HER2结合活性的TMER CAR-M稳转细胞系。
1.1慢病毒的构建和包装
基于慢病毒载体，通过将HER2 scFv、α1β1整合素的跨膜区域、α1β1整合素胞内、FcγRI跨膜信号传导域胞内和GFP使用linker进行连接，随后使用P2A连接GM-CSF基序，合成嵌合抗原受体HER2 scFv-GM-CSF(由生工生物工程(上海)股份有限公司进行的序列合成)，将其转染巨噬细胞，构建巨噬细胞特异性CAR，通过将合成的HER2 scFv-GM-CSF-CAR慢病毒质粒与Rev、Gag、VSV三种辅助质粒进行共转染，使其包装成为完整的HER2 scFv-GM-CSF-CAR慢病毒。
具体步骤如下：
(1)将4×105个处于对数生长期的HEK293T细胞均匀接种于6孔板中，在37℃、5％CO2细胞培养箱中静置培养至细胞汇合度达到60％-70％，按3:1:1:1比例分别取质粒pCDH-HER2 scFv-GM-CSF、Rev、Gag和VSV加入到装有Opti-MEM的离心管中混匀室温孵育，再加入TurboFect Transfection Reagent转染试剂，枪头轻柔吹打混匀，室温孵育；
(2)将上述孵育好的混合液缓慢滴加到接种了293T细胞的6孔板中，边滴加边轻轻摇动培养板，置于37℃、5％CO2培养16-24h后，弃掉6孔板各孔中的转染混合液，添加2-3mL包含10％胎牛血清(FBS)的Advanced DMEM完全培养基，将细胞培养板重置于37℃、5％CO2培养48h，待合成为HER2 scFv-GM-CSF-CAR慢病毒；
(3)为去除细胞碎片，将含有慢病毒的培养基于4℃、4000g下离心10min。用0.22μm的过滤器过滤上清液并收集，离心机4℃、15000g离心2h。弃上清，利用病毒保存液重悬病毒颗粒，再10000g离心5min后取上清于-80℃中保存备用；
(4)随后，检测包装出来的慢病毒滴度，向9个无菌的1.5mL离心管中加入90μL DMEM完全培养基，再将10μL已获得的重组慢病毒颗粒HER2 scFv-GM-CSF-CAR加入第一个离心管中吹打均匀，吸出10μL混合液加入到第二个离心管中，如此连续直至最后一管，每组设置三个重复。将5×104个HEK293T细胞均匀接种于96孔板中，细胞汇合度达到60％-70％时依次加入上述稀释好的各梯度慢病毒稀释液，同时设置不加入病毒稀释液的293T细胞作为阴性对照；培养24h后，更换新鲜的DMEM完全培养 基，48h后于倒置荧光显微镜下观察各孔应该情况。慢病毒滴度计算公式为：病毒滴度(TU/mL)＝平均发绿色荧光细胞数×病毒稀释倍数/接种病毒液的体积(mL)，单位为TU/mL。
上述构建嵌合抗原受体的各元件序列如下：
鼠源GM-CSF序列(SEQ ID NO:1)：
HER2 scFv序列(SEQ ID NO:2)：
鼠源α1β1整合素的跨膜区域序列(SEQ ID NO:3)：
鼠源α1β1整合素胞内序列(SEQ ID NO:4)：
鼠源FcγR I跨膜信号传导域胞内序列(SEQ ID NO:5)：
GFP序列(SEQ ID NO:6)：
Linker序列(SEQ ID NO:7)：
P2A序列(SEQ ID NO:8)：
人源GM-CSF序列(SEQ ID NO:9)：
人源α1β1整合素的跨膜区域序列(SEQ ID NO10)：
人源α1β1整合素胞内序列(SEQ ID NO11)：
人源FcγR I跨膜信号传导域胞内序列(SEQ ID NO12：
1.2制备TMER CAR-M细胞系
在包装为完整的慢病毒并检测了其滴度后，使用HER2 scFv-GM-CSF-CAR慢病毒感染巨噬细胞，获得TMER CAR-M稳转细胞系，取巨噬细胞接种于6孔板内，待其生长到细胞汇合度至60-70％时，以每9：1的比例向巨噬细胞的培养基中加入HER2 scFv-GM-CSF-CAR慢病毒病毒颗粒，24h前后更换新鲜DMEM完全培养基，48h后即完成巨噬细胞的慢病毒感染；观察细胞生长情况及形态变化，传代至新的培养瓶或培养皿中进行扩增或者冻存。
1.3 TMER CAR-M构建的qPCR和共聚焦显微技术检测
提取上述稳转细胞株的总RNA，利用SuperReal PreMix Plus(SYBR Green)试剂盒检测上述的稳转细胞株中融合蛋白的表达情况，按照试剂盒说明书进行实验，qPCR实验步骤如下：
(1)正常溶解2×SuperReal Premix Plus、50×ROX Reference Dye、模板、引物和RNase-free ddH2O，并将所有试剂在室温下平衡并彻底混匀；
(2)在冰上将反转得到的cDNA模板以一定体系与正向引物、反向引物、Mix、ROX、ddH2O混合，利用移液枪将混合液添加入八连管中，20μL体系包括：10μL2×SuperReal Premix Plus、0.6μL正向引物(10μM)、0.6μL反向引物(10μM)、0.1-2μL cDNA模板、0.4μL 50×ROX Reference Dye，使用RNase-free ddH2O补充至总体积20μL；
(3)盖上管盖，吹打混匀，用微量离心机离心5-10s，确保所有组分均在管底；
(4)将反应体系放置于RT-qPCR仪中，设置仪器参数，95℃预变性15min、95℃变性10s、60℃±1℃退火20s，72℃延伸31s，40个循环，运行程序，待程序结束后导出Ct值，并利用2-ΔΔCt(Livak)法计算最终结果。
如图1A，TMER CAR-293T及TMER CAR-M中DiI代表细胞膜定位，GFP代表跨膜表达的CAR结构，Merged代表叠加图，共聚焦结果显示本发明技术实现的CAR结构成功地表达在细胞膜上；如图1B，进一步的qPCR定量分析验证CAR序列在对应设计组的显著性高表达，支持了TMER CAR-M的构建成功。如图1C共聚焦显微镜结果，TMER CAR-M中BF代表明场拍照结果，HER2 scFv-GFP代表TMER CAR-M，HER2-PE代表PE标记的HER2肿瘤抗原蛋白，Merge代表叠加图，显示TMER CAR-M.通过跨膜HER2 scFv的表达与HER2实现了有效结合。图1结果验证了TMER CAR-M融合蛋白成功表达，不但实现了的TMER CAR-M成功构建，并且具有显著性的HER2 肿瘤抗原结合活性；这为后期研究提供细胞治疗模型。
2、TMER CAR-M的活力检测
(1)CCK细胞增殖试验
①于96孔板中每孔加入100μL的不同组的巨噬细胞悬液，分别为J774A.1(UTD-M)、Empty-pCDH J774A.1(Empty)、HER2 scFv J774A.1(CAR-like)、HER2 scFv CAR J774A.1(First generation CAR-M)、HER2 scFv-GM-CSF CAR J774A.1(TMER CAR-M)密度为1×104个细胞/mL，置于37℃、5％CO2的培养箱中静置培养；
②细胞培养24-48h之后，吸去96孔板中的培养基，用PBS清洗细胞2-3次，再向96孔板中加入10μL的CCK8检测溶液，在37℃、5％CO2的培养箱中培养3-4h；
③用酶标仪测定各孔在450nm处的吸光度，靶细胞细胞活性大小＝(效靶比作用组吸光度－效应细胞对照组吸光度)/(靶细胞阳性对照组吸光度－空白孔吸光度)。
(2)同时，分别提取各组细胞总RNA，采用qPCR检测TMER CAR-M活性，检测方法同上述的qPCR的检测方法。
结果如图2所示，TMER CAR-M组细胞增殖显著增强于CAR-Like组和First generation CAR-M组(图2A)。qPCR检测结果显示，TMER CAR-M组细胞的增殖因子CCND1和Ki67的mRNA表达水平均显著强于CAR-Like组和First generation CAR-M组，且p53表达水平低于CAR-Like组和First generation CAR-M组，见图2B-E。研究结果提示，TMER CAR-M的基础活力显著增强，这意味着GM-CSF的表达增强了TMER CAR-M的活力。
3、TMER CAR-M的体外M1表型极化检测
将上述构建的TMER CAR-M，分别基于2×105个细胞/mL的密度，分别于6孔板中进行培养并检测，具体操作为：
于6孔板中分别接种4×105个UTD-M、Empty、CAR-like、First generation CAR-M、TMER CAR-M，以10％FBS的DMEM高糖培养基于37℃、5％CO2培养箱中静置培养48h，随后分别提取各组细胞总RNA，采用qPCR检测TMER CAR-M极化，检测方法同上述的qPCR的检测方法。
结果如图3所示，与CAR-like组和和First generation CAR-M相比，TMER CAR-M组M1型因子CD80、IFN-γ和iNOS表达水平均显著上升，但其M2型因子CD206、Arg-1和IL-10表达水平均显著下降，说明TMER CAR-M组相对于CAR-like组和First  generation CAR-M组均呈现显著性的M1型抗肿瘤巨噬细胞特性，这意味着GM-CSF在TMER CAR-M的极化方面发挥了作用，提示TMER CAR-M可能抗M2复极化。
4、TMER CAR-M体外抗M2复极化验证
采用肿瘤细胞培养上清液体外模拟肿瘤抑制性微环境体系，分别作用TMER CAR-M、CAR-Like及对照组普通巨噬细胞，将构建成功的TMER CAR-M细胞与CAR-Like和First generation CAR-M组进行对照分析，肿瘤培养上清对TMER CAR-M诱导5d，提取其提取细胞总RNA，qPCR检测进一步验证TMER CAR-M抗极化能力。具体步骤包括：
(1)于6孔板中每孔加入2×105个的UTD-M、CAR-like、First generation CAR-M和TMER CAR-M细胞，置于37℃、5％CO2的培养箱中静置培养；
(2)待UTD-M、CAR-like、First generation CAR-M和TMER CAR-M组细胞贴壁后，吸去孔板中的培养基，用PBS清洗细胞2-3次，分别向6孔板中每组加入肿瘤细胞培养上清，每组实验设置3个平行孔。
(3)细胞诱导5d后，吸去6孔板中的培养基，用PBS清洗细胞2-3次，再向6孔板中每孔加入1mL的Trizol，提取RNA，利用SuperReal PreMix Plus(SYBR Green)试剂盒，并按照试剂盒说明书进行实验。
(4)正常溶解2×SuperReal Premix Plus、50×ROX Reference Dye、模板、引物和RNase-free ddH2O，并将所有试剂在室温下平衡并彻底混匀；
(5)在冰上将反转得到的cDNA模板以一定体系与正向引物、反向引物、Mix、ROX、ddH2O混合，利用移液枪将混合液添加入八连管中，20μL体系包括：10μL 2×SuperReal Premix Plus、0.6μL正向引物(10μM)、0.6μL反向引物(10μM)、0.1-2μL cDNA模板、0.4μL 50×ROX Reference Dye，使用RNase-free ddH2O补充至总体积20μL；
(6)盖上管盖，吹打混匀，用微量离心机离心5-10s，确保所有组分均在管底；
(7)将反应体系放置于RT-qPCR仪ABI 7300中，设置仪器参数，95℃预变性15min、95℃变性10s、60±1℃退火20s，72℃延伸31s，40个循环，运行程序，待程序结束后导出Ct值，并利用2-ΔΔCt(Livak)法计算最终结果。
结果如图4所示，在肿瘤上清液的作用下，与TMER CAR-M组相比，CAR-like组、First generation CAR-M组及普通巨噬细胞组M2型因子CD206、Arg-1和IL-10表达水平均显著上升(如图4E-F)，提示CAR-like组、和First generation CAR-M组及普 通巨噬细胞组在肿瘤微环境诱导性出现M2型极化，而TMER CAR-M组仍然保持显著性的M1型巨噬细胞特征。
5、TMER CAR-M体外调控M2型巨噬细胞逆转为M1型
采用肿瘤细胞培养上清液体外模拟肿瘤抑制性微环境体系，分别作用TMER CAR-M、CAR-Like及对照组普通巨噬细胞，将构建成功的TMER CAR-M细胞与CAR-Like和First generation CAR-M组进行对照分析，肿瘤培养上清对普通巨噬细胞诱导，随后使用TMER CAR-M上清进行诱导，提取其提取细胞总RNA，qPCR检测进一步验证TMER CAR-M抗极化能力。具体步骤包括：
(1)于6孔板中每孔加入2×105个普通巨噬细胞，使用肿瘤细胞培养上清诱导5d，置于37℃、5％CO2的培养箱中静置培养，每组实验设置3个平行孔；
(2)细胞诱导5d后，吸去6孔板中的培养基，用PBS清洗细胞2-3次，使用UTD-M、CAR-like、First generation CAR-M和TMER CAR-M细胞培养液上清进行诱导；
(3)细胞诱导5d后，吸去6孔板中的培养基，用PBS清洗细胞2-3次，再向6孔板中每孔加入1mL的Trizol，提取RNA，检测方法同上述的qPCR的检测方法。
结果如图5A-C所示，与CAR-like组、First generation CAR-M组及普通巨噬细胞组相比，在TMER CAR-M组上清液的作用下，CAR-like组、First generation CAR-M组及普通巨噬细胞组M2型因子CD206、Arg-1和IL-10表达水平均显著上升，提示CAR-like组、First generation CAR-M组及普通巨噬细胞组在肿瘤微环境中不能诱导M2型巨噬细胞重编程为M1表型，而TMER CAR-M组上清诱导的巨噬细胞显示出显著性的M1型巨噬细胞特征，说明TMER CAR-M上清能够调控M2型巨噬细胞逆转为M1型，初步评价其体外肿瘤微环境调控能力。
6、TMER CAR-M体内治疗肿瘤效果验证
本研究前期初探TMER CAR-M在体内靶向肿瘤能力的验证，使用小鼠HER2+-4T1肿瘤模型验证TMER CAR-M的抗肿瘤效果，HER2+-4T1肿瘤模型的建立包括治疗过程如下：
(1)小鼠乳腺癌细胞系HER2+-4T1利用1640培养基(hyclone)于37℃、5％CO2细胞培养箱中培养；
(2)4-6周龄的磁性BALB/c小白鼠购买后需于恒温无菌通风小鼠饲养间饲养一周以上后再进行小鼠实验；
(3)将HER2+-4T1细胞扩大培养，待小鼠饲养约一周后，消化HER2+-4T1细胞，用PBS清洗2-3次，并利用PBS重悬，稀释密度为107个细胞/mL，将100μL细胞重悬液以皮下注射的方式注射到小鼠右肢上方皮下；
(4)将UTD-M、Empty、CAR-like、First generation CAR-M和TMER CAR-M细胞均利用DMEM高糖培养基于37℃、5％CO2细胞培养箱中培养；
(5)待HER2+-4T1细胞成瘤后，每组设置4只小鼠的平行实验，分别将100μL PBS或细胞重悬液通过尾静脉方式进行注射，UTD-M、Empty、CAR-like、First generation CAR-M和TMER CAR-M组分别注射3×106个细胞，在注射第一次后的第5d，进行第二次注射，每组注射量为3×106个细胞，总共注射了两次。
(6)用电子游标卡尺对小鼠肿瘤的长和宽分别进行测量，利用电子天平对小鼠体重进行测量，小鼠肿瘤体积＝(长*宽2)/2，每两天测量一次，最后计算小鼠生存曲线。
结果见图6，图6A是治疗方案的模式图，图6B展示不同治疗组与对照组治疗期间的小鼠体积增长情况，图6C代表不同治疗组与对照组的小鼠生存情，图6D代表不同分组的小鼠体重监测情况，研究结果说明相对于CAR-like组和First generation CAR-M组，TMER CAR-M组肿瘤得到了明显抑制，生存期延长，取得了显著的治疗效果。另外，相对于CAR-like组和First generation CAR-M组，TMER CAR-M组体重无明显变化，提示其具有较好安全性。
7、TMER CAR-M体内治疗腹腔肿瘤效果验证
小鼠HER2+-4T1肿瘤模型验证了TMER CAR-M的抗肿瘤效果，随后使用人卵巢癌SK-OV-3(HER2高表达)的腹腔瘤模型，进一步验证基于本专利设计的TMER CAR-M抗肿瘤效果，SK-OV-3的腹腔瘤模型建立包括治疗过程如下：
(1)人卵巢癌细胞系SK-OV-3利用DMEM培养基(hyclone)于37℃、5％CO2细胞培养箱中培养；
(2)4-6周龄的裸鼠购买后需于恒温无菌通风小鼠饲养间饲养一周以上后再进行小鼠实验；
(3)将SK-OV-3细胞扩大培养，待小鼠饲养约一周后，消化SK-OV-3细胞，用PBS清洗2-3次，并利用PBS重悬，稀释密度为5×106个细胞/mL，将100μL细胞重悬液以腹腔注射的方式注射到小鼠腹部；
(4)将来源于人单核细胞THP-1的UTD-M和TMER CAR-M细胞均利用1640 培养基于37℃、5％CO2细胞培养箱中培养；
(5)待SK-OV-3细胞注射进入腹腔2-4h后，每组设置5只小鼠的平行实验，分别将100μL PBS或细胞重悬液通过腹腔方式进行注射，UTD-M和TMER CAR-M组分别注射1.5×106个细胞，仅注射一次。
(6)使用小动物活体成像系统IVIS Lumina II实时监测小鼠腹腔肿瘤的生长情况。
结果见图7，与PBS组、UTD-M组相比较，TMER CAR-M组代表肿瘤细胞数量的荧光强度逐渐降低、治疗组小鼠实验期内全部存活，而PBS组、UTD-M组荧光强度逐渐增加、并且出现部分小鼠的死亡，说明相对于PBS组、UTD-M组，TMER CAR-M组的SK-OV-3肿瘤得到了显著地抑制，TMER CAR-M取得了显著的治疗效果。TMER CAR-M可有效抑制SK-OV-3肿瘤的生长。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。

Claims (9)

  1. 一种肿瘤微环境调控型CAR-单核/巨噬细胞，其特征在于，其为包括嵌合抗原受体的复合结构，可产生GM-CSF胞内表达并分泌至胞外，促进CAR-单核/巨噬细胞形成M1型巨噬细胞分化的同时，进一步调控肿瘤微环境；其中所述GM-CSF的核苷酸序列如SEQ ID NO:1或SEQ ID NO:9所示。
  2. 一种嵌合抗原受体，其特征在于，包括GM-CSF、HER2 scFv、α1β1整合素的跨膜区域、α1β1整合素胞内以及FcγRI跨膜信号传导域胞内。
  3. 根据权利要求2所述的嵌合抗原受体，其特征在于，所述GM-CSF、HER2 scFv、α1β1整合素的跨膜区域、α1β1整合素胞内以及FcγRI跨膜信号传导域胞内的核苷酸序列分别如SEQ ID NO:1/SEQ ID NO:9、SEQ ID NO:2、SEQ ID NO:3/SEQ ID NO:10、SEQ ID NO:4/SEQ ID NO:11、SEQ ID NO:5/SEQ ID NO:12所示。
  4. 一种重组载体，其特征在于，包括权利要求2或3所述的嵌合抗原受体。
  5. 根据权利要求1所述的肿瘤微环境调控型CAR-单核/巨噬细胞的制备方法，其特征在于，包括构建权利要求4所述的重组载体，并将所述重组载体转染所述CAR-单核/巨噬细胞的步骤。
  6. 根据权利要求5所述的制备方法，其特征在于，所述重组载体的构建方法包括：
    (1)构建嵌合抗原受体：将HER2 scFv、α1β1整合素的跨膜区域、α1β1整合素胞内、FcγRI跨膜信号传导域胞内使用linker进行连接后，再用P2A连接GM-CSF，合成嵌合抗原受体；
    (2)将所述嵌合抗原受体与慢病毒载体重组，构建所述重组载体。
  7. 一种药物组合物，其特征在于，包括权利要求1所述的CAR-单核/巨噬细胞或权利要求2所述的嵌合抗原受体或权利要求4所述的重组载体，连同药学上可接受的辅料或载体。
  8. 根据权利要求1所述的CAR-单核/巨噬细胞在制备促进M1型巨噬细胞分化的药物中的应用。
  9. 根据权利要求1所述的CAR-单核/巨噬细胞在制备治疗实体瘤药物中的应用。
PCT/CN2023/073798 2022-03-11 2023-01-30 一种肿瘤微环境调控型car-单核/巨噬细胞及其制备方法和应用 WO2023169092A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202210237581.5A CN114657143B (zh) 2022-03-11 2022-03-11 一种肿瘤微环境调控型car-单核/巨噬细胞及其制备方法和应用
CN202210237581.5 2022-03-11

Publications (1)

Publication Number Publication Date
WO2023169092A1 true WO2023169092A1 (zh) 2023-09-14

Family

ID=82029181

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/073798 WO2023169092A1 (zh) 2022-03-11 2023-01-30 一种肿瘤微环境调控型car-单核/巨噬细胞及其制备方法和应用

Country Status (2)

Country Link
CN (1) CN114657143B (zh)
WO (1) WO2023169092A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116240173A (zh) * 2023-02-02 2023-06-09 西安电子科技大学 一种冷热肿瘤调控型car-单核/巨噬细胞及其制备方法和应用

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114657143B (zh) * 2022-03-11 2022-10-25 西安电子科技大学 一种肿瘤微环境调控型car-单核/巨噬细胞及其制备方法和应用

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6210963B1 (en) * 1993-08-25 2001-04-03 Institut National De La Sante Et De La Recherche Medicale Recombinant cells from the monocyte-macrophage cell line for gene therapy
US20170087185A1 (en) * 2015-09-09 2017-03-30 Seattle Children's Hospital (dba Seattle Children' s Research Institute) Genetic engineering of macrophages for immunotherapy
US20180028631A1 (en) * 2016-07-29 2018-02-01 Lan Bo Chen Anti-ssea4 chimeric antigen receptors and their use for treating cancer
US20180244748A1 (en) * 2015-07-28 2018-08-30 The Trustees Of The University Of Pennsylvania Modified Monocytes/Macrophage Expressing Chimeric Antigen Receptors and Uses Thereof
CN111733139A (zh) * 2020-01-13 2020-10-02 西安电子科技大学 一种基于功能化巨噬细胞/单核细胞的靶向递送系统及其构建与应用
CN113106067A (zh) * 2020-01-10 2021-07-13 南京大学 一种嵌合抗原受体-单核/巨噬细胞(car-m)的构建及其应用
CN113402616A (zh) * 2021-06-18 2021-09-17 浙江大学 巨噬细胞专属嵌合抗原受体、表达该受体的可控极化单核/巨噬细胞及其制备方法和应用
WO2021223376A1 (zh) * 2020-05-08 2021-11-11 浙江大学 嵌合性抗原受体及表达其的巨噬细胞、调节巨噬细胞极化的方法和应用
CN114410588A (zh) * 2022-01-29 2022-04-29 西安电子科技大学 一种α1β1整合素依赖增强型CAR巨噬细胞及其制备方法和应用
CN114657143A (zh) * 2022-03-11 2022-06-24 西安电子科技大学 一种肿瘤微环境调控型car-单核/巨噬细胞及其制备方法和应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109266618B (zh) * 2018-10-18 2021-04-23 赛元生物科技(杭州)有限公司 能够靶向肿瘤细胞的巨噬细胞及其制备方法
CN110684121B (zh) * 2019-10-12 2021-08-10 华夏源（上海）细胞基因工程股份有限公司 一种靶向her2联合表达pd1-mica融合蛋白的嵌合抗原受体及其表达载体和应用
CN111925448B (zh) * 2020-08-03 2022-06-21 山东大学 在体生成car-巨噬细胞的制备方法及肿瘤免疫治疗中的应用
CN113398258B (zh) * 2021-06-23 2022-06-28 河北大学 一种m1型巨噬细胞外泌体疫苗及其制备方法和应用

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6210963B1 (en) * 1993-08-25 2001-04-03 Institut National De La Sante Et De La Recherche Medicale Recombinant cells from the monocyte-macrophage cell line for gene therapy
US20180244748A1 (en) * 2015-07-28 2018-08-30 The Trustees Of The University Of Pennsylvania Modified Monocytes/Macrophage Expressing Chimeric Antigen Receptors and Uses Thereof
US20170087185A1 (en) * 2015-09-09 2017-03-30 Seattle Children's Hospital (dba Seattle Children' s Research Institute) Genetic engineering of macrophages for immunotherapy
US20180028631A1 (en) * 2016-07-29 2018-02-01 Lan Bo Chen Anti-ssea4 chimeric antigen receptors and their use for treating cancer
CN113106067A (zh) * 2020-01-10 2021-07-13 南京大学 一种嵌合抗原受体-单核/巨噬细胞(car-m)的构建及其应用
CN111733139A (zh) * 2020-01-13 2020-10-02 西安电子科技大学 一种基于功能化巨噬细胞/单核细胞的靶向递送系统及其构建与应用
WO2021223376A1 (zh) * 2020-05-08 2021-11-11 浙江大学 嵌合性抗原受体及表达其的巨噬细胞、调节巨噬细胞极化的方法和应用
CN113402616A (zh) * 2021-06-18 2021-09-17 浙江大学 巨噬细胞专属嵌合抗原受体、表达该受体的可控极化单核/巨噬细胞及其制备方法和应用
CN114410588A (zh) * 2022-01-29 2022-04-29 西安电子科技大学 一种α1β1整合素依赖增强型CAR巨噬细胞及其制备方法和应用
CN114657143A (zh) * 2022-03-11 2022-06-24 西安电子科技大学 一种肿瘤微环境调控型car-单核/巨噬细胞及其制备方法和应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KLICHINSKY, M.ET AL.: "Human chimeric antigen receptor macrophages for cancer immunotherapy", NATURE BIOTECHNOLOGY, vol. 38, 31 August 2020 (2020-08-31), XP055926928, DOI: 10.1038/s41587-020-0462-y *
SLOAS, C. ET AL.: "Engineered CAR-Macrophages as Adoptive Immunotherapies for Solid Tumors", FRONTIERS IN IMMUNOLOGY, vol. 12, 24 November 2021 (2021-11-24), XP055964792, DOI: 10.3389/fimmu.2021.783305 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116240173A (zh) * 2023-02-02 2023-06-09 西安电子科技大学 一种冷热肿瘤调控型car-单核/巨噬细胞及其制备方法和应用

Also Published As

Publication number Publication date
CN114657143A (zh) 2022-06-24
CN114657143B (zh) 2022-10-25

Similar Documents

Publication Publication Date Title
Mathewson et al. Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis
WO2023169092A1 (zh) 一种肿瘤微环境调控型car-单核/巨噬细胞及其制备方法和应用
CN108504668A (zh) 靶向cd19和cd22嵌合抗原受体及其用途
CN114410588B (zh) 一种α1β1整合素依赖增强型CAR巨噬细胞及其制备方法和应用
US20220347217A1 (en) Anti CD70 CAR-T Cell, and Preparation Method Therefor and Use Thereof
KR101702629B1 (ko) 체세포로부터 혈관 전구 세포로의 직접교차분화 유도용 조성물 및 이의 용도
CN111344398A (zh) 分离的重组溶瘤腺病毒、药物组合物及其在治疗肿瘤和/或癌症的药物中的用途
WO2022007804A1 (zh) T淋巴细胞及其应用
WO2017120998A1 (zh) 治疗脑胶质母细胞瘤的治疗组合物
CN110964697A (zh) 一种抗肿瘤nk细胞及其制备方法和应用
CN105209617B (zh) 微囊泡及其制造方法
CN113214408B (zh) 一种嵌合抗原受体巨噬细胞及其制备方法和用途
CN113896801B (zh) 靶向人Claudin18.2和NKG2DL的嵌合抗原受体细胞及其制备方法和应用
CN109913422A (zh) 一种包含肿瘤抗原识别受体的免疫细胞及其应用
CN110055269A (zh) 人间皮素嵌合抗原受体、其t细胞及其制备方法和用途
CN108456247A (zh) 靶向ny-eso-1的t细胞受体及其用途
CN109288855B (zh) 试剂在制备药物中的用途、干涉片段、抑制肝癌肿瘤干细胞自我更新方法和治疗肝癌药物
US20190008897A1 (en) Compositions and methods for producing pro-inflammatory macrophages
CN116003622A (zh) 一种用于卵巢癌治疗的嵌合抗原受体及其应用
US20140275219A1 (en) Uses of the human zfx gene and drugs associated with same
CN103800919A (zh) Tuft1在制备肝癌诊断和治疗制剂中的应用
CN108728458A (zh) 靶向mesothelin的嵌合抗原受体并联合表达IL-15的方法和用途
CN103882057B (zh) 携带p21ras单链抗体基因肿瘤特异性腺病毒载体的构建及其应用
CN114053294A (zh) miR-150及负载miR-150模拟物的外泌体在制备治疗结直肠癌药物上的应用
CN108424458A (zh) 靶向ny-eso-1的嵌合抗原受体及其用途

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: 23765672

Country of ref document: EP

Kind code of ref document: A1