WO2022228538A1 - 一种表达免疫调节因子的car载体及其应用 - Google Patents

一种表达免疫调节因子的car载体及其应用 Download PDF

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WO2022228538A1
WO2022228538A1 PCT/CN2022/090106 CN2022090106W WO2022228538A1 WO 2022228538 A1 WO2022228538 A1 WO 2022228538A1 CN 2022090106 W CN2022090106 W CN 2022090106W WO 2022228538 A1 WO2022228538 A1 WO 2022228538A1
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car
cells
tumor
nucleic acid
application
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French (fr)
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杨寒朔
李琪琪
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West China Hospital of Sichuan University
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West China Hospital of Sichuan University
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Priority to AU2022263746A priority Critical patent/AU2022263746A1/en
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Priority to GB2318060.7A priority patent/GB2621770B/en
Priority to JP2023566450A priority patent/JP2024515319A/ja
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Definitions

  • the invention belongs to the field of bioengineering, and in particular relates to a CAR expression vector and its application.
  • Chimeric antigen receptor-modified T cells have shown good clinical effects in the treatment of hematological tumors such as acute B-cell lymphoma, but they are not effective in solid tumors.
  • the CAR structure consists of three parts, namely the extracellular region for recognizing tumor antigens, the transmembrane region for anchoring the CAR structure, and the intracellular T cell activation signaling domain.
  • the killing of tumor cells by CAR-T relies on the specific binding of the extracellular single-chain variable region to the corresponding tumor-associated antigen, and then initiates downstream signaling through the intracellular signaling domain, causing the activation and proliferation of CAR-T cells, and by releasing IFN - Cytokines such as gamma exert cytotoxicity.
  • IFN - Cytokines such as gamma exert cytotoxicity.
  • the current traditional CAR-T is not effective.
  • the intracellular region of the first-generation CAR structure has only 3 ITAM sequences of CD3 ⁇ or 2 ITAM sequences of FcR ⁇ , which can only provide the first signal of T cell activation, limited ability to secrete IL-2, and weak proliferation ability. Satisfactory results were not obtained.
  • the second-generation CAR structure adds a tandem costimulatory molecule signal domain after the intracellular signal sequence, and the proliferation and killing capacity are improved compared with the first-generation.
  • the intracellular signal of the third-generation CAR structure contains more costimulatory domains, however, studies have shown that its killing activity is not significantly improved, and the side effects are greater.
  • the fourth-generation CAR is based on the second-generation CAR to introduce new functional elements to enhance the anti-tumor activity of CAR-T cells, such as CAR-T that secretes cytokines IL-12, IL-15 or IL-18, which can enhance CAR-T cells.
  • CAR-T that secretes cytokines IL-12, IL-15 or IL-18, which can enhance CAR-T cells.
  • cytokines IL-12, IL-15 or IL-18 which can enhance CAR-T cells.
  • CAR-T cancer-associated fibroblasts
  • the tumor microenvironment can also help tumor cells evade immune surveillance, inhibit the body's anti-tumor response, and inhibit the infiltration, proliferation and survival of CAR-T cells, which is also the main reason for the ineffectiveness of CAR-T cells against solid tumors. one. Therefore, designing and constructing a CAR-T cell that can effectively infiltrate the tumor tissue and reverse the tumor immunosuppressive microenvironment can overcome the low response rate of the existing immunotherapy and have a more effective anti-tumor effect.
  • the present invention constructs a new type of CAR-T cell (CAR-GM-T) with high expression of GM-CSF on the basis of the improved second-generation CAR, which can alleviate the defects and deficiencies in the existing technology. .
  • the purpose of the present invention is to provide a novel CAR expression vector and its application.
  • the technical scheme of the present invention is:
  • a CAR expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) and a nucleic acid encoding an immunomodulatory factor, wherein the nucleic acid encoding the immunomodulatory factor is a granulocyte-macrophage colony stimulating factor encoding (GM-CSF) nucleic acid full length or fragment.
  • CAR chimeric antigen receptor
  • GM-CSF granulocyte-macrophage colony stimulating factor encoding
  • nucleic acid encoding the chimeric antigen receptor and the nucleic acid encoding the granulocyte-macrophage colony stimulating factor are linked by a sequence encoding the self-cleaving peptide P2A.
  • nucleic acid encoding the chimeric antigen receptor also includes an extracellular region targeting a specific tumor antigen, a transmembrane region anchoring the CAR structure, and an intracellular signaling domain containing multiple costimulatory molecules in series, Including CD3 ⁇ chain, 4-1BB and GM-CSF.
  • the targets targeting specific tumor antigens include one or more of Her2, B7-H3, Claudin18.2, CD70, MUC16, FSHR, FR or Meso.
  • Her2 mainly targets HER2-positive breast cancer, lung cancer, gastric cancer, ovarian cancer and sarcoma; Meso targets pancreatic cancer, ovarian cancer and lung cancer; B7-H3 and CD70 target melanoma, followed by MUC16, Both FSHR and FR target ovarian cancer.
  • Clasdin18.2 targets gastric cancer, esophagogastric junction cancer and pancreatic cancer.
  • a lentivirus comprising the above-mentioned CAR expression vector, the lentivirus includes pWPXLd, psPAX2 and/or pMD2.G.
  • a CAR-T cell expressing the above-mentioned chimeric antigen receptor A CAR-T cell expressing the above-mentioned chimeric antigen receptor.
  • the solid tumors mainly include breast cancer, ovarian cancer and lung cancer, but are not limited thereto.
  • the CAR-T cells can enhance the secretion of GM-CSF, IFN- ⁇ and IL-2.
  • the CAR-T cells directly enhance the killing activity of CAR-T cells by expressing granulocyte-macrophage colony-stimulating factor.
  • the CAR-T cells can enhance the infiltration ability, and more CAR-T cells can infiltrate into the solid tumor to exert the effect of specifically killing tumor cells.
  • the CAR-T cells after the CAR-T cells infiltrate into the solid tumor, they express granulocyte-macrophage colony-stimulating factor to play an immunoregulatory function to regulate the tumor microenvironment.
  • the CAR-T cells activate and recruit dendritic cells into the solid tumor by expressing granulocyte-macrophage colony-stimulating factor to activate the antigen-specific tumor immune response of endogenous T cells.
  • the CAR-T cells can inhibit the lymph node metastasis of tumor cells.
  • the "chemotaxis" mentioned in the present invention refers to the CAR-T prepared by the present invention, namely CAR-GM-T, which promotes the directional movement of DC cells to tumor cells.
  • DC cells are professional antigen-presenting cells in the body, express MHC II and costimulatory molecules, and can ingest, capture, and process tumor antigens, and then migrate to lymph nodes to activate the body's endogenous T cell anti-tumor immune response.
  • the present invention constructs a CAR-T cell (CAR-GM-T) with high expression of GM-CSF on the basis of the second-generation CAR-T.
  • CAR-GM-T CAR-T cell
  • GM-CSF is an important immunomodulator, GM-CSF can activate T cell immune response by activating and recruiting dendritic cells (DC), and can also activate other immune cells such as granulocytes, macrophages and NK cells play an important role in the regulation of tumor immune responses.
  • DC dendritic cells
  • the CAR-GM-T cells constructed in the present invention can highly express GM-CSF, which can not only directly enhance the killing activity and proliferation ability of the CAR-T cells themselves, but also make the CAR-T cells stronger than ordinary CAR-T cells. Cells have stronger immune regulation functions.
  • the CAR-GM-T cells prepared by the present invention have outstanding effects in the following aspects:
  • CAR-GM-T can significantly inhibit the growth of melanoma, and significantly prolong the survival time of experimental animals
  • CAR-GM-T has a stronger ability to infiltrate solid tumor tissue than conventional CAR-T cells, and exerts a stronger anti-tumor effect after infiltrating into solid tumors;
  • the present invention can reshape and reverse the tumor microenvironment, directly or assist in enhancing the activity of T cells, and provide a new strategy for the treatment of solid tumors.
  • Fig. 1 is the construction diagram of CAR-hGM expression vector
  • Figure 2 shows that GM-CSF secreted by CAR-GM-T cells is significantly higher than that of conventional CAR-T cells
  • Figure 3 is a graph of the killing activity of CAR-GM-T cells
  • Figure 4 shows that the killing efficiency of Meso-CAR-GM-T cells on SK-OV3-Meso is significantly higher than that of Meso-CAR-T, and the killing efficiency increases with the increase of the effector-target ratio;
  • Figure 5 shows the GM-CSF secreted when CAR-GM-T kills tumor cells, which is significantly higher than that of conventional CAR-T cells;
  • Figure 6 shows that when the effector-target ratio is 5:1, the IFN- ⁇ secreted by CAR-GM-T cells is 4.5 times that of conventional CAR-T cells, and when CAR-GM-T cells contact tumor cells at different effector-target ratios The IFN- ⁇ was significantly higher than that of conventional CAR-T cells;
  • Figure 7 shows that IL-2 secreted by CAR-GM-T cells in killing tumor cells is also significantly enhanced under different effector-target ratios
  • Figure 8 is a graph showing the effect of CAR-GM-T cells in the treatment of abdominal tumors in mice
  • Figure 9 shows that conventional CAR-T has no obvious inhibitory effect on the growth of B16F10-Her2 subcutaneous melanoma xenografts, nor can it prolong the survival of mice, while CAR-GM-T treatment can significantly inhibit the growth of melanoma xenografts in mice;
  • FIG. 10 shows that CAR-GM-T treatment can significantly prolong the survival time of mice
  • Figure 11 is a graph showing the proportion of CAR-T cells in the tumor and lymph node of the mouse on the 24th day after the mice were treated, isolated and prepared as a single cell suspension;
  • Figure 12 shows the further detection of CAR-GM-T infiltration in human tumor tissue. 28 days after the treatment of abdominal tumor in NSG mice, the residual tumor in the abdominal cavity was taken for immunofluorescence staining to detect the infiltration of CD3 + T cells;
  • Figure 13 shows that the CD45.2 + immune cells in the conventional CAR-T treatment group accounted for only 5%, and the infiltration of immune cells in the tumor of the mice in the CAR-GM-T treatment group was significantly better than that in the conventional CAR-T treatment group;
  • Figure 14 shows that CD3 + T lymphocytes in the tumors of mice in the CAR-GM-T treatment group accounted for 11% of the CD45.2 + cells, while the CD3 + T lymphocytes in the conventional CAR-T treatment group accounted for only 5%, indicating that high Expression of GM-CSF significantly increased the proportion of endogenous CD3 + T cells in mouse tumors after CAR-T cell therapy;
  • Figure 15 shows that the proportion of CD11c + MHCII hi DC cells in the tumor in the CAR-GM-T treatment group was significantly higher than that in the conventional CAR-T treatment group;
  • Figure 16 shows the results of HE staining of the draining lymph nodes, showing that CAR-GM-T can inhibit the lymph node metastasis of tumor cells.
  • the term "about” is typically expressed as +/- 5% of the stated value, more typically +/- 4% of the stated value, and more typically + /-3%, more typically +/-2% of said value, even more typically +/-1% of said value, even more typically +/-0.5% of said value.
  • Human ovarian cancer cell line (SK-OV3-Luc) expressing luciferase and mouse melanoma cell line (B16F10-Her2) overexpressing Her2 protein were preserved by the State Key Laboratory of Biotherapy, Sichuan University. 10% calf serum in DMEM medium, 37°C, 5% CO 2 , and cultured under normoxia.
  • Her2-CAR and hGM-CSF were linked with the self-cleaving polypeptide P2A to construct a Her2-CAR-hGM expression vector (pWPXLd-Her2-CAR-hGM-CSF) with high expression of hGM-CSF ( Figure 1).
  • the Her2-CAR-P2A fragment was amplified with the pWPXLd-Her2-CAR-EGFP vector as the template, and the hGM-CSF fragment was amplified with the pWPXLd-hGM-CSF vector as the template.
  • the vector pWPXLd was linearized with restriction enzymes BamHI and EcoRI, and the fragments Her2-CAR and hGM-CSF were ligated into the linearized vector pWPXLd by homologous recombination.
  • Lentiviral packaging was performed using a lentiviral three-plasmid system.
  • the plasmid transduction method was calcium phosphate-DNA co-precipitation method
  • the helper plasmids were psPAX2 and pMD2.G
  • the packaging cells were 293T cells.
  • 48 hours and 72 hours after transfection the virus supernatant was collected respectively, and the virus supernatant was filtered with a 0.22 ⁇ m disposable syringe filter (PES membrane). °C refrigerator.
  • peripheral blood mononuclear lymphocytes According to the number of peripheral blood mononuclear lymphocytes, take out an appropriate amount of Human T-Expander CD3/CD28 Dynabeads magnetic beads, wash the magnetic beads once with 5 mL of DPBS, and resuspend in T cell complete medium. The peripheral blood mononuclear lymphocyte suspension was mixed with the washed magnetic beads and placed in a T75 culture at 37 °C, 5% CO 2 .
  • peripheral blood mononuclear lymphocytes According to the number of peripheral blood mononuclear lymphocytes, take out an appropriate amount of Human T-Expander CD3/CD28 Dynabeads magnetic beads, wash the magnetic beads once with 5 mL of DPBS, and resuspend in T cell complete medium. The peripheral blood mononuclear lymphocyte suspension was mixed with the washed magnetic beads and placed in a T75 culture at 37 °C, 5% CO 2 .
  • the secretion of cytokines was determined by ELISA, including IL-2, IFN ⁇ , and GM-CSF.
  • the samples were derived from the culture supernatant of CAR-T cells or the supernatant after CAR-T cells killed tumor cells.
  • LDH lactate dehydrogenase
  • each effector-target ratio is set to the following 6 groups MOCK T (effect Spontaneous cell group), MOCK T+Tumor (experimental group), CAR-T, CAR-T+Tumor, CAR-GM-CSF-T, CAR-GM-CSF-T+Tumor, 3 sub-wells in each group, simultaneously The blank medium group, the spontaneous tumor cell group, and the tumor cell maximal release group were set, and the final volume of each well was 200 ⁇ L.
  • MOCK T effect Spontaneous cell group
  • MOCK T+Tumor experimental group
  • the blank medium group, the spontaneous tumor cell group, and the tumor cell maximal release group were set, and the final volume of each well was 200 ⁇ L.
  • mice Regularly monitor the body weight of mice.
  • mice On the 6th day after inoculation, each mouse was imaged in vivo, and the mice were divided into 3 groups according to the fluorescence value of the image, with 6 mice in each group, namely the PBS group, the Her2-CAR group, and the Her2-CAR group. -GM-CSF group.
  • mice Regularly monitor the mice's mental state, activity, hair gloss, and eating conditions, observe whether there are adverse reactions, and detect the weight of the mice.
  • mice were anesthetized and sacrificed by de-neck, and the residual tumor in the abdominal cavity was taken for immunofluorescence staining to detect the infiltration of CAR-T cells.
  • mice can be used for the construction of subcutaneous xenograft models after acclimating to the environment for 1 week.
  • each tumor-bearing mouse was injected with cyclophosphamide at a dose of 200 mg/kg, intraperitoneally.
  • mice On the 12th day after inoculation, the volume of the transplanted tumor was measured, and the mice were divided into 3 groups according to the tumor volume, with 15 mice in each group, namely the PBS group, the Her2-CAR group, and the Her2-CAR-GM-CSF group.
  • Her2-CAR-T and Her2-CAR-GM-CSF-T cells prepared from lymphocytes of C57BL/6 CD45.1 mice with good growth status, and resuspend them in 1640 medium to The final concentration is 3*10 ⁇ 7/mL.
  • mice were sacrificed by decapitation after anesthesia, and the spleen, draining lymph nodes, lymph nodes, and tumors of the mice were isolated, and HE staining and flow cytometry were performed respectively.
  • the killing efficiency of Her2-CAR-GM-T cells to SK-OV3 cells naturally expressing Her2 protein was significantly higher than that of Her2-CAR-T, and the killing efficiency was enhanced with the increase of the effective-target ratio.
  • the killing efficiency of Her2-CAR-GM-T cells to SK-OV3 reached 30% when the effector-target ratio was 1:1, which was significantly higher than that of the Her2-CAR-T group (P ⁇ 0.001), while Her2-CAR-T
  • Meso CAR-T is a meso-targeted CAR-T therapy developed by the University of Pennsylvania and Novartis, and Meso CAR-T is also Novartis' first CAR-T product for solid tumors, with indications including ovarian cancer, lung cancer and Pancreatic cancer.
  • CAR-T had no obvious inhibitory effect on the growth of B16F10-Her2 subcutaneous melanoma xenografts, and could not prolong the survival of mice, while CAR-GM-T treatment could significantly inhibit the growth of melanoma xenografts in mice (P ⁇ 0.05).
  • Fig. 9 the survival time of mice was significantly prolonged (Fig. 10).
  • the tumors and lymph nodes of the mice were isolated and prepared as single-cell suspensions, and the proportion of CAR-T cells in the tumors and lymph nodes of the mice was detected by flow cytometry.
  • the results showed that the CAR + T cells in the conventional CAR-T treatment group accounted for 1.2% of the CD3 + T cells, while the intratumor CAR + T cells in the CAR-GM-T treatment group accounted for 2.4% of the CD3 + T cells . -2 times of T treatment group, the difference was statistically significant (P ⁇ 0.01).
  • CAR + T cells in the tumor-draining lymph nodes of the conventional CAR-T treatment group accounted for only 0.04% of CD3 + T cells, while the CAR-GM-T treatment group of mice
  • the CAR + T cells in the tumor-draining lymph nodes accounted for 0.4% of the CD3 + T cells, which was 10 times that of the conventional CAR-T treatment group and significantly higher than the conventional CAR-T treatment group (P ⁇ 0.01) ( Figure 11).
  • Endogenous immune cells of CD45.2 + were analyzed in mouse tumor cells, and the infiltration of endogenous immune cells was analyzed.
  • the results showed that CD45.2 + immune cells accounted for 8% in the tumors of the mice in the CAR-GM-T treatment group, while the CD45.2 + immune cells in the CAR-T treatment group accounted for only 5%.
  • the infiltration of immune cells in the tumor of the mice was significantly better than that of the CAR-T treatment group (P ⁇ 0.05) ( Figure 13).
  • CD3 + T lymphocytes were analyzed among CD45.2 + endogenous immune cells. The results showed that CD3 + T lymphocytes in the tumors of the mice in the CAR-GM-T treatment group accounted for 11% of the CD45.2 + cells, while the CD3 + T lymphocytes in the CAR-T treatment group accounted for only 5%, indicating a high expression of CD3+ T lymphocytes.
  • GM-CSF significantly increased the proportion of endogenous CD3 + T cells in mouse tumors after CAR-T cell treatment (P ⁇ 0.01) (Fig. 14).
  • CD11b + CD11c + cells were analyzed among CD45.2 + cells, and CD11c + MHCII hi DC cells were analyzed from this fraction of cells.
  • the results showed that the proportion of CD11c + MHCII hi DC cells in the tumor in the CAR-GM-T treatment group was significantly higher than that in the conventional CAR-T treatment group (P ⁇ 0.01) ( Figure 15), indicating that the tumor in the CAR-GM-T treatment group more mature DC cells.
  • CAR-GM-T cells inhibit tumor cell lymph node metastasis
  • B16F10 is a high-metastatic mouse melanoma cell line.
  • the team of the present invention used CAR-T cells and CAR-GM-T cells to treat the B16F10-Her2 melanoma subcutaneous transplanted tumor model.
  • the results of HE staining of the draining lymph nodes showed that there were a large number of melanin plaques in the PBS treatment group, a small amount of melanin plaques in the CAR-T treatment group, and a large number of lymphocytes and no melanin plaques in the CAR-GM-T treatment group.

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