WO2022228539A1 - Procédé de préparation de cellules car-cik ayant une proportion élevée de cellules nkt, et son application - Google Patents

Procédé de préparation de cellules car-cik ayant une proportion élevée de cellules nkt, et son application Download PDF

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WO2022228539A1
WO2022228539A1 PCT/CN2022/090111 CN2022090111W WO2022228539A1 WO 2022228539 A1 WO2022228539 A1 WO 2022228539A1 CN 2022090111 W CN2022090111 W CN 2022090111W WO 2022228539 A1 WO2022228539 A1 WO 2022228539A1
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nac
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杨寒朔
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四川大学华西医院
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Definitions

  • the invention belongs to the field of tumor immunotherapy research, and in particular relates to a CAR-CIK cell optimized culture method for obtaining a high proportion of NKT cells.
  • CAR-T Immune cell therapy represented by CAR-T has become the most promising emerging therapy in the field of anti-tumor.
  • CAR-T cells have made breakthroughs in the treatment of hematological tumors, they still face many challenges when applied to solid tumors. Different from hematological tumors, solid tumors are inherently characterized by uneven expression of tumor antigens, while the killing effect of CAR-T on tumor cells is strictly target-dependent. CAR-triggered recognition and killing require tumor cells to express specific antigens. Antigens are used as targets, which is one of the important reasons for the poor efficacy of CAR-T cells on solid tumors.
  • tumor cells downregulate the expression of target antigens so that CAR-T cells cannot recognize them, even contact with CAR-T cells can evade immune killing. It has been reported that CAR-T cannot clear mixed tumor cell clusters containing only 10% target antigen-negative tumor cells (Ag-), because even if CAR-T cells eliminate 90% of target antigen-positive tumor cells, in this 10% of negative cells in the tumor environment become dominant growth cells, while CAR-T cells cannot effectively kill them, and Ag-tumor cells will rapidly clone and proliferate to cause drug resistance or disease recurrence. In fact, there are no fully positive tumors. Therefore, there is an urgent need to find an immunotherapeutic regimen that is effective against both target-antigen-positive and target-antigen-negative tumor cells.
  • Ag- target antigen-negative tumor cells
  • CIK cells are a mixed lymphocyte population with the activity of lysing target cells obtained by antibody activation and cultured with various cytokines in vitro.
  • Schmidt-Wolf et al. 2 and other cytokines were obtained by culturing PBMC.
  • the killing activity of CIK cells to tumor cells is not high, and the anti-tumor function of CIK cells alone is limited in vivo.
  • CAR-CIK cells are modified with CAR to obtain CAR-CIK cells, which have significantly enhanced killing activity against target antigen-positive tumor cells, but still cannot effectively kill target antigen-negative tumor cells. .
  • CIK cells include NKT cells, T cells and NK cells, among which NKT cells are the most important cells mediating the anti-tumor function of CIK.
  • NKT cells express the molecular markers of NK cells and T cells at the same time, and the killing of tumor cells has a broad spectrum and is not restricted by HLA.
  • the proportion of NKT in CIK cells cultured by conventional technology is limited. By optimizing the culture scheme to increase the proportion of NKT in CIK cells, the CAR-CIK cells prepared from this cell not only have a good effect on target antigen-positive tumor cells Killing activity, and still can effectively kill tumor cells negative for target antigen, can solve the problem of negative expression of target antigen on some or most tumor cells in solid tumors.
  • IL-2 is generally used to culture CAR-CIK cells at present, but some studies have shown that IL-2 can induce the exhaustion of immune cells, make immune cells survive for a short time in the body, and affect the anti-tumor effect. Therefore, further optimization of the preparation method is required.
  • the present invention proposes an optimized culture method for CAR-CIK cells, which can not only induce a high proportion of NKT cells, but also reduce the expression of depletion-related molecules on NKT, increase the lifespan of NKT cells, and be positive and negative for target antigens.
  • the tumor cells have good killing activity and show better anti-tumor activity.
  • One of the objectives of the present invention is to provide an optimized culturing method of CAR-CIK cells for obtaining a high proportion of NKT cells.
  • the specific technical solutions are as follows:
  • IL-15 can accelerate the induction of the generation of memory T cells, increase the persistence of T cells and thereby improve anti-tumor activity, and the role of IL-15 has been verified in NK cells and NKT cell subtypes. Therefore, the present invention hopes to increase the killing activity of CAR-CIK cells on tumor cells and reduce the apoptosis of CAR-CIK cells by adding IL-15.
  • NAC can promote the synthesis of intracellular reduced glutathione (GSH), which is ROS. scavenger.
  • the invention reduces the ROS level in the CAR-CIK by adding NAC, and enhances the continuous killing ability of the CAR-CIK.
  • the method includes the following steps:
  • the CIK cells are derived from peripheral blood or umbilical cord blood;
  • the prepared CAR-CIK cells were cultured with 50ng/mL IL-15 and NAC greater than or equal to 2mM to obtain CAR-CIK cells with a high NKT ratio.
  • the CAR-CIK cells prepared in step 2) also contain co-stimulatory intracellular signaling domains including CD3 and/or 4-1BB.
  • Another aspect of the present invention also provides a combination scheme, which is specifically as follows:
  • NAC in combination with CAR-CIK cells co-cultured with IL-15 and NAC in the preparation of anti-solid tumor drugs.
  • the solid tumor includes target antigen positive, target antigen negative and/or mixed target antigen positive and negative tumor cell populations.
  • the solid tumors include solid tumors targeting Mesothelin (MSLN) and HER2.
  • the IL-15 and NAC can stimulate the CAR-CIK to differentiate into more NKT cells for enhancing the broad-spectrum anti-cancer ability of the drug.
  • the IL-15 and NAC can reduce the apoptosis and depletion of the CAR-CIK, so as to improve the lethality of the drug to the tumor.
  • the drug is administered by injection, firstly injecting CAR-CIK cells co-cultured with IL-15 and NAC, and then boosting the injection of NAC.
  • One aspect of the present invention provides a new method for culturing CAR-CIK cells, which uses IL-15 and NAC to replace IL-2 to culture CAR-CIK cells, so that the proportion of NKT cells increases significantly, from 28% to 40% , so that the CAR-CIK cells prepared by the present invention have better broad-spectrum anti-cancer ability.
  • the CAR-CIK cells obtained by the culture method of the present invention have a killing effect on solid tumors of target antigen-positive, target antigen-negative and/or mixed target antigen-positive and negative tumor cell groups, so it is useful for controlling immune cell therapy.
  • the recurrence of the disease caused by the dominant proliferation of target antigen-negative cells has important clinical significance.
  • the in vitro proliferation ability of the CAR-CIK cultured in the present invention is enhanced, and the apoptosis and depletion after continuous killing are reduced, so the ability and efficiency of the CAR-CIK cells to kill tumors are improved as a whole.
  • the CAR-CIK cells prepared in the present invention are of great significance for overcoming the limitations faced by immune cell therapy in solid tumors and controlling drug resistance or recurrence after CAR-T therapy.
  • Another aspect of the present invention also provides a technical solution for the combined use of IL-15, NAC and CAR-CIK. It has been observed in in vivo animal models that the CAR-CIK/IL-15+NAC regimen has a good inhibitory effect on tumors in vivo, further prolonging the survival period of experimental animals, and has far-reaching clinical application value.
  • Figure 1 shows the in vitro proliferation ability and induced NKT cell differentiation of the optimized cultured CAR-CIK cells
  • Figure 2 shows the phenotyping of optimized cultured CAR-CIK cells
  • Figure 3 shows the killing activity of CAR-CIK cells cultured with different concentrations of NAC on target antigen-positive tumor cells
  • Figure 4 is a graph of flow cytometry results showing a reduction in apoptotic and depleted CAR-CIK cells (optimized culture) after multiple rounds of sequential killing;
  • Figure 5 is a graph showing the killing effect of CAR-CIK cells targeting MSLN (optimized culture) on target antigen-positive tumor cells (SK-OV-3-MSLN) and target antigen-negative tumor cells (SK-OV-3);
  • Figure 6 shows that target antigen-positive (Ag+) and target antigen-negative (Ag-) tumor cells were labeled with green and red fluorescent proteins, respectively, mixed 1:1 as target cells and incubated with CAR-CIK cells (optimized culture) , observe and count the surviving tumor cells by fluorescence microscope;
  • Figure 7 is a graph showing the in vivo antitumor activity and mouse survival time of optimized cultured CAR-CIK cells in the peritoneal tumor NSG mouse model.
  • 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.
  • CAR-CIK cells targeting MSLN have strong killing on target antigen-positive tumor cells (SK-OV-3-MSLN), and can also effectively kill target antigen-negative cells tumor cells (SK-OV-3); target antigen-positive (Ag+) and target antigen-negative (Ag-) tumor cells were labeled with green and red fluorescent proteins, respectively, and mixed 1:1 as target cells with CAR- CIK cells were co-incubated, and the surviving tumor cells were observed and counted by fluorescence microscope. It was found that CAR-CIK cells also had strong killing ability to Ag-cells in mixed tumor cells, suggesting that CAR-CIK cells can effectively avoid the target of tumor cells. Immune escape due to loss of antigen expression ( Figures 1 and 3).
  • the CAR-CIK cells of the new culture protocol provided by the present invention have stronger in vitro proliferation ability, and can induce a higher proportion of NKT cell differentiation, increasing from 28% to 40% (Fig. 5).
  • HER2ScFv is a mouse-derived single-chain variable region fragment FRP5.
  • the MSLN ScFv in the MSLN-CAR molecular sequence involved in the present invention is the human single-chain variable region fragment m912.
  • CAR molecules include: signal peptide, ScFv, hinge region, CD8 transmembrane region, 4-1BB costimulatory signal region and CD3 ⁇ signaling domain.
  • Lentiviral packaging was performed using calcium phosphate precipitation.
  • the prepared mixed solution was gently added dropwise to a HEK 293T cell culture dish of suitable density (80%-90%) in an amount of 1 mL per dish. Shake the petri dish slightly, mix the medium and the mixture, and place it in a 37°C cell incubator to continue culturing.
  • peripheral blood of healthy volunteers was drawn into a heparin sodium anticoagulant tube, and PBMC were separated by Ficoll separation medium. Take a clean six-well plate, add 2mL X-VIVO15 complete medium to resuspend PBMC, add 1000IU/mL IFN- ⁇ to activate it, and add 100ng/mL CD3 antibody and 500IU/mL to the medium after 24 hours. mL of IL-2, and thereafter continue to culture CIK cells with IL-2-containing medium.
  • RetroNectin Add 1 mL of 25 ⁇ g/mL RetroNectin to a non-adherent 12-well plate and place it at 4°C overnight.
  • the RetroNectin in the well plate was sucked off, washed with DPBS, the liquid was sucked off, and 1 mL of filtered 2% BSA solution was added again to block for 30 min.
  • the concentrated virus was added to a 12-well plate, centrifuged at 32°C, 1000 g for 2 h, and the acceleration and deceleration were both set to 2.
  • CAR expression was detected by flow cytometry after culturing for 2-3 days in an incubator at 37°C.
  • CAR-CIK cells can be cultured as long as needed.
  • step 1 in a new plate after 24 hours of co-incubation to plate the desired target cells for Round 2.
  • the tumor cells positive or negative for the target antigen were counted respectively, and the tumor cells were plated in advance in a 48-well plate. Count the CAR-CIK cells and resuspend them with an appropriate amount of X-VIVO15 double-free medium, adjust the concentration to the appropriate effect-target ratio, mix well and add to the tumor cell plate for co-incubation, and observe under a fluorescence microscope after 24 hours of killing. Photographs were taken, and the viable fluorescent cells in the field were counted using Image J software.
  • the CAR-CIK cells were divided into four different phenotypes by the expression of CD45RO and CD62L. Optimized cultured CAR-CIK cells have a higher proportion of CD45RO+ memory cell phenotype, Tcm with memory phenotype is considered to have stronger viability, and a higher proportion of Tcm tends to predict better antitumor response (Figure 2 ).
  • effector cells were taken into the flow tube and washed with 1 mL of DPBS. After centrifugation, aspirate the supernatant, add 100 ⁇ L of DPBS, and add 1 ⁇ L of the following antibodies to the flow tube: PE anti-human PD-1, PE anti-human TIM-3, and APC anti-human LAG-3 antibodies, beat and mix well . After incubating at 4°C for 30 min in the dark, wash with DPBS, add 400 ⁇ L of DPBS to resuspend, mix well and put on the machine.
  • the experimental animals are 4-6 weeks old NSG mice weighing 18-20g, which need to be adapted to the environment in the SPF laminar flow room of the animal room for one week in advance.
  • the experimental animals met the feeding standards, and the bedding, feed, drinking water and cages were changed twice a week to ensure the cleanliness of the living environment, and the physiological state of the mice was closely observed.
  • mice were digested to make single cell suspension, and the cell concentration was counted.
  • the mice were intraperitoneally injected with 2 ⁇ 10 5 cells/200 ⁇ L of tumor cells. Mice were imaged in vivo 2 days after tumor inoculation to observe tumor formation. The mice were evenly grouped according to the fluorescence value. On day 3, mice in the corresponding group were intraperitoneally injected with 1 ⁇ 10 6 cells/200 ⁇ L of effector cells (ie, optimized cultured CAR-CIK cells), and the PBS group was intraperitoneally injected with an equal volume of PBS solution.
  • effector cells ie, optimized cultured CAR-CIK cells
  • mice were injected with NAC solution in the intraperitoneal cavity, the injection dose of NAC was 1 g/kg, twice a day.
  • biotin fluorescence imaging was performed on the mice in each group, and the tumor size of the mice was observed by taking pictures, and the scale was adjusted to an appropriate size. Tumor growth in mice was observed with intravital imaging at one-week intervals. The survival status of the mice was observed, and body weight changes and survival time were recorded on time.
  • the present invention respectively constructs second-generation CAR recombinant plasmids targeting human epidermal growth factor receptor (HER2) and mesothelin (MSLN) containing costimulatory molecule 4-1BB, and uses HEK293T cells to package CAR lentivirus.
  • CIK cells derived from peripheral blood mononuclear cells (PBMC) were infected with lentivirus to prepare CAR-CIK cells.
  • the anti-tumor activity of the prepared CAR-CIK cells was verified by in vitro killing experiments to detect the killing efficiency and the release of cytokines during the killing process.
  • the CD4/CD8 cell grouping and CD3/CD56 expression of CAR-CIK were detected by flow cytometry.
  • SK-OV-3-MSLN tumor cells overexpressing MSLN were constructed by lentivirus system, and HER2 and MSLN-positive human ovarian cancer SK-OV-3 and SK-OV-3-MSLN were selected as target cells, respectively, and HER2-CAR -CIK and MSLN-CAR-CIK cells were co-incubated, and different effector-target ratios were set, and the killing efficiency was detected using a lactate dehydrogenase (LDH) kit.
  • LDH lactate dehydrogenase
  • CD3 and CD28 antibodies were used to activate T cells in PBMC, and then lentivirus infection was used to prepare CAR-T cells to compare the expansion capabilities of CAR-T and CAR-CIK cells. , and used flow cytometry to detect cell populations and functional phenotypic changes of effector cells.
  • the present invention uses target antigen-positive or negative tumor cells as target cells, which are incubated with CAR-T or CAR-CIK cells respectively for in vitro killing experiments.
  • the lentivirus system was used to construct SK-OV-3-MSLN-GFP stably expressing green fluorescent protein and SK-OV-3-mcherry stably expressing red fluorescence, and the above tumor cells were mixed in 1:1 as target cells.
  • In vitro killing experiments were performed by co-incubating with MSLN-CAR-T and MSLN-CAR-CIK, respectively. The killing situation and pictures were observed under a fluorescence microscope, and the fluorescent cells in the field of view were counted by Image J, and the killing efficiency of each group was compared.
  • the invention also simulates the killing environment in vivo through multiple rounds of continuous killing experiments, and detects the apoptosis of effector cells and the depletion molecules PD-1, TIM-3 and LAG on the surface of CAR-CIK cells after repeated antigen stimulation by flow cytometry -3 expression level.
  • the present invention compares the proliferation ability and NKT cell differentiation changes of CAR-CIK cultured by the new culture protocol (IL-15 and NAC) and traditional IL-2.
  • the effect of the new culture protocol on the killing process of CAR-CIK cells was explored through a multi-round killing system.
  • Ovarian cancer peritoneal tumor model was established in NSG mice using tumor cells with Luciferase gene, and the tumor growth process of PBS group, CIK group, CAR-CIK group and CAR-CIK/IL-15+NAC group was monitored by in vivo imaging system. To study the effect of the new culture protocol on the antitumor effect of CAR-CIK cells in vivo.
  • the CAR-CIK cells constructed in the present invention have strong in vitro proliferation ability, and can be activated and exert an anti-tumor effect after contacting the target antigen. Since CAR-CIK cells contain CD3+CD56+NKT cells, the killing of target antigen-negative tumor cells is significantly stronger than that of CAR-T cells, and for a mixture of target antigen-positive (Ag + ) and negative (Ag - ) tumor cells In the mixed cell population, the killing effects of CAR-CIK cells on Ag + , Ag - tumor cells and the overall mixed tumor cell population were significantly stronger than CAR-T cells.
  • CAR-CIK cells optimized by the new culture protocol have stronger in vitro expansion ability, can differentiate more NKT cells, and increase the ability of CAR-CIK to kill target cells in vitro s efficiency.
  • the new culture protocol can reduce the expression levels of apoptosis and exhaustion molecules in CAR-CIK cells after multiple rounds of serial killing.
  • the new culture protocol can improve the killing effect of CAR-CIK cells in vivo, further prolong the survival period of mice, and has a good anti-tumor effect in vivo.
  • the CAR-CIK cells prepared in the present invention can effectively kill target cells.
  • the killing ability of CAR-CIK cells was higher than that of CAR-T cells.
  • the CAR-CIK cells optimized by the new culture protocol have further improved proliferation ability, and can induce a higher proportion of NKT cells earlier, thereby enhancing the killing effect on target antigen-positive, negative and Ag + /Ag - mixed tumor cells.
  • apoptosis and depletion of CAR-CIK cells after serial killing were reduced.
  • good in vivo antitumor activity of CAR-CIK/IL-15+NAC cells was also observed in in vivo animal models.
  • CAR-CIK cells help to overcome the "antigen dilemma" faced by CAR-T therapy in solid tumors with large heterogeneity, and it is used to control the proliferation of target antigen-negative cells after immune cell therapy.
  • the resulting disease recurrence has important clinical significance.
  • the new culture protocol can induce a higher proportion of NKT cells and enhance the effect of CAR-CIK cells, thereby improving the feasibility of CAR-CIK cell therapy in clinical applications.
  • Oxidative stress mediates a reduced expression of the activating receptor NKG2D in NK cells from end-stage real disease patients[J].J Immunol,2009,182(3):1696 -705.

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Abstract

La présente invention concerne le domaine de la recherche d'immunothérapie antitumorale, et en particulier un procédé de culture optimisé pour des cellules CAR-CIK pour obtenir une proportion élevée de cellules NKT. Le procédé consiste à ajouter de l'IL-15 et de la N-acétylcystéine (NAC) pour la co-culture dans un processus de culture CAR-CIK, au lieu de l'IL-2 traditionnelle. La présente invention peut améliorer de manière significative la proportion de NKT, et renforcer l'activité anti-tumorale des cellules CAR-CIK afin que lesdites cellules aient toujours une activité de destruction sur les cellules tumorales cibles négatives, et donc, l'effet anti-tumoral est considérable.
PCT/CN2022/090111 2021-04-30 2022-04-29 Procédé de préparation de cellules car-cik ayant une proportion élevée de cellules nkt, et son application WO2022228539A1 (fr)

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