Immune cell expressing non-secretory IL-10 for enhancing cellular immunotherapy, preparation method and application

Abstract

The invention discloses an immune cell for enhancing cell immunotherapy and expressing non-secreting IL-10, a preparation method and application thereof, and accidentally finds that compared with secreting IL-10, the non-secreting IL-10 can obviously relieve mitochondrial dysfunction of CAR cells, and compared with the secreting IL-10, the non-secreting IL-10 can obviously relieve the mitochondrial dysfunction of the CAR cells when the non-secreting IL-10 is stimulated by target cells, so that the non-secreting IL-10 can obviously relieve the mitochondrial dysfunction of the CAR cells. The non-secreting IL-10 can effectively improve the motility rate of CAR cells and reduce the occurrence of CAR apoptosis. On the basis, non-secreting IL-10 is expressed in the CAR cells, so that the main toxic and side effects of a CAR cell therapy are effectively relieved, meanwhile, the tumor killing ability of the CAR cells is remarkably improved by protecting the mitochondrial function of the CAR cells, improving the motility rate of the CAR cells and reducing the occurrence of CAR cell apoptosis, and the toxicity reducing and synergistic effects of the CAR cells are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to an immune cell expressing non-secretory IL-10 for enhancing cellular immunotherapy, a preparation method and use thereof. Background Art

[0002] The field of immunotherapy has become a hot direction for the development of human medicine in the future due to its advantages of high precision and personalization. Immunotherapy refers to the use of human autologous, allogeneic or xenogeneic (non-human) cells, which are enhanced and modified in vitro to improve the ability of the patient's immune system to recognize and kill tumors, and then returned (or implanted) into the human body. Among them, chimeric antigen receptor T cell immunotherapy (CAR-T cell therapy) is an adoptive cell immunotherapy that has emerged in recent years. It mainly combines the specificity of chimeric antigen receptors with the immune effects of T cells, and then kills malignant tumor cells through specific recognition. In addition, because NK cells play a key role in the innate immune response, and have the advantages of a wider source of acquisition, a lower risk of graft-versus-host disease, and more "spot-type" supply potential, NK cell-based cell immunotherapy has also received widespread attention and research. CAR-NK cell therapy not only uses the research and development ideas of CAR-T cell therapy, but also plays the biological characteristics of NK cells.

[0003] At present, although CAR-T cell therapy has a good therapeutic effect, the multiple toxic side effects and high recurrence rate frequently reported in clinical trials cannot be ignored, which has become the problem and challenge facing CAR-T cell therapy. The main toxic side effects of CAR-T cell therapy are cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). Among them, cytokine release syndrome (CRS) is caused by the characteristics of CAR-T cells themselves. CRS is triggered by inflammatory cytokines and chemokines released after the corresponding target antigen of CAR-T is engaged, such as interferon (IFN-γ), tumor necrosis factor (TNF-α), granulocyte-macrophage colony stimulating factor (GM-CSF), IL-2, IL-6 and IL-8. As a systemic inflammatory response, CRS involves multiple systems, such as circulatory, respiratory, urinary and digestive system diseases. Patients may experience fever, rash, heart failure, respiratory failure, renal failure, nausea, vomiting, liver dysfunction, disseminated intravascular coagulation disorder and neurotoxicity after receiving CAR-T cell therapy. Therefore, how to improve the efficacy of CAR-T cells while reducing the side effects of CRS is an urgent problem to be solved. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies of the prior art and provide an immune cell expressing non-secretory IL-10 for enhancing cellular immunotherapy, a preparation method and use thereof.

[0005] Non-secretory IL-10, especially membrane-bound IL-10, can effectively reduce the release of secretory IL-10, thereby reducing the related toxic side effects caused by IL-10 in CAR-T / NK cell therapy. Compared with CAR-T / NK cells that do not express IL-10, non-secretory IL-10 can effectively improve the tumor killing ability of CAR-T / NK cells. The present invention unexpectedly found that compared with secretory IL-10, non-secretory IL-10 can significantly reduce the mitochondrial dysfunction of CAR-T / NK cells, and when facing target cell stimulation, non-secretory IL-10 can effectively increase the activity rate of CAR-T / NK and reduce the occurrence of CAR-T / NK apoptosis. Based on this, the present invention proposes an immune cell expressing non-secretory IL-10 for enhancing cellular immunotherapy. By expressing non-secretory IL-10 in CAR-T / NK cells, the main toxic and side effects of CAR-T / NK cell therapy are effectively reduced. At the same time, by protecting the mitochondrial function of CAR-T / NK cells and improving the activity rate of CAR-T / NK, the occurrence of CAR-T / NK apoptosis is reduced, and the tumor killing ability of CAR-T / NK cells is significantly improved, thereby achieving the toxicity reduction and synergistic effect of CAR-T / NK cells.

[0006] The non-secretory IL-10 of the present invention refers to the IL-10 produced by the cell being restricted to the inside or around the cell, so that it cannot act on distant cells or other components through endocrine, thereby reducing cytokine release syndrome; the non-secretory IL-10 mainly includes: covalently bound IL-10, membrane protein fusion IL-10, etc., in particular, especially membrane protein fusion IL-10, i.e. membrane-bound IL-10, which is composed of IL-10, its fragments or variants, hinge regions and transmembrane domains, and the produced IL-10 is bound to the cell membrane through the hinge region and the transmembrane domain; the transmembrane domain can be CD8, CD28, 41BB, etc.

[0007] In some embodiments, an immune cell expressing non-secreted IL-10 for enhancing cellular immunotherapy comprises a recombinant construct encoding non-secreted IL-10, a fragment or a variant thereof.

[0008] In some embodiments, immune effector cells also include engineered immune receptors displayed on the cell surface. In some embodiments, engineered immune receptors are chimeric antigen receptors (CARs) comprising antigen binding domains, transmembrane domains, and intracellular signaling domains. In some embodiments, engineered immune receptors are engineered T cell receptors (TCRs). In some embodiments, engineered immune receptors can specifically bind to antigens expressed on the surface of target cells, wherein the antigen is a tumor-specific antigen or a tumor-associated antigen.

[0009] In some embodiments, the chimeric antigen receptor of the immune cell comprises a CD19 binding domain and a non-antigen binding domain, wherein the non-antigen binding domain includes the extracellular domain, transmembrane domain and intracellular co-stimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof.

[0010] In some embodiments, the immune cells include T cells, B cells, natural killer cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.

[0011] In some embodiments, the recombinant construct encoding the non-secreted IL-10, fragment or variant thereof is linked to a second recombinant construct encoding a CAR, TCR or any other synthetic tumor targeting motif.

[0012] In some embodiments, the recombinant construct encoding the non-secreted IL-10, fragment or variant thereof is linked to the second recombinant construct encoding CAR, TCR or any other synthetic tumor targeting motif via a sequence encoding a self-cleaving peptide.

[0013] The membrane-bound IL-10 variant is one or more biologically active derivatives of membrane-bound IL-10, preferably the amino acid sequence shown in SEQ ID No. 1. Generally, the term "variant" refers to a molecule with one or more additions, substitutions (usually conservative in nature) and / or deletions of a natural sequence relative to the natural molecule, as long as these modifications do not destroy its biological activity, the variant is "substantially homologous" to the reference molecule. Generally, the protein sequence of such variants has a high degree of sequence homology or identity with the reference sequence, for example, when the two sequences are aligned, the protein sequence homology or identity is greater than 25%, generally greater than 50% to 70%, even more particularly 80%, or 85% or higher, for example at least 90%, or 95% or higher.

[0014] In some embodiments, the immune cells are autologous, allogeneic, or xenogeneic.

[0015] In some embodiments, the method for preparing immune cells expressing non-secretory IL-10 for enhancing cellular immunotherapy of the present invention comprises:

[0016] obtaining immune cells from a subject;

[0017] Engineering an immune cell so that the immune cell comprises a recombinant construct encoding non-secretory IL-10, a fragment or a variant thereof; and

[0018] The immune cells are cultured to obtain immune cells expressing non-secretory IL-10 for enhancing cellular immunotherapy.

[0019] In some embodiments, a non-secretory IL-10 expressing immune cell for enhancing cellular immunotherapy is used in the preparation of a medicament for treating / preventing / diagnosing cancer or tumor, infection or autoimmune disease.

[0020] In some embodiments, the cancer or tumor comprises one or more combinations of glioblastoma, head and neck cancer, lung cancer, thyroid cancer, bronchial cancer, breast cancer, gastric cancer, liver cancer, pancreatic cancer, colon cancer, colorectal cancer, kidney cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, prostate cancer, skin cancer, melanoma, lymphoma, and leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of using target protein combined with flow cytometry to detect the CAR positivity rate of three types of CAR-NK cells (first row) and the expression of IL-10 on the membrane (second row);

[0022] Figure 2 The results of using Annexin V and PI to detect the early apoptosis, late apoptosis and live cell ratio of CAR-NK cells without target cell stimulation and after target cell stimulation;

[0023] Figure 3 This is the result of chemiluminescence detection of in vitro killing function. Figure 3 A in the figure is the result diagram of killing efficiency of Raji cells at different effector-target ratios; Figure 3 B in the figure is the killing efficiency result of Raji cells with effector-target ratios of 2:1 and 1:1;

[0024] Figure 4 This is the result of flow cytometry detection of in vitro killing function, in which the effector-target ratio is 1:4;

[0025] Figure 5 The mitochondrial function results of the three types of CAR-NK cells without target cell stimulation and after target cell stimulation;

[0026] Figure 6This is the result of using CBA kit to detect the cytokine secretion levels of three types of CAR-NK, including IL-10, Ganzyme B, TNF-α, and IFN-γ;

[0027] Figure 7 This is a schematic diagram of the in vivo functional evaluation of CAR-NK cells in mice;

[0028] Figure 8 This is a graph showing the changes in mouse tumors after CAR-NK cell injection;

[0029] Fig. 9 This is a graph showing the weight changes of mice after CAR-NK cell injection;

[0030] Fig.10 This is a graph showing the changes in mouse tumors after CAR-T cell injection;

[0031] Fig.11 This is a graph showing the weight changes of mice after CAR-T cell injection. DETAILED DESCRIPTION

[0032] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific examples below, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same. In the following embodiments, unless otherwise specified, % refers to volume percentage.

[0033] Embodiment 1:

[0034] 1 Construction of chimeric antigen receptor retroviral vector and virus preparation

[0035] 1.1 Construction of chimeric antigen receptor vector

[0036] A CAR structure targeting CD19 (CD19 CAR) that can be used for NK cells was constructed by using the anti-CD19 single-domain antibody sequence (SEQ ID NO.3) to bind to the extracellular region, transmembrane region and cytoplasmic region of the CD28 molecule (SEQ ID NO.4) and connect to the intracellular domain of CD3ζ (SEQ ID NO.5); on the basis of this CAR structure, the wild-type cytokine IL-10 coding sequence (amino acid sequence as shown in SEQ ID NO.2) was connected to the self-cleaving peptide T2A after the CD3ζ intracellular domain to construct an IL-10 secreting CD19 targeting CAR structure (IL-10 CD19 CAR); the wild-type cytokine IL-10 coding sequence and the hinge region and transmembrane domain (amino acid sequence as shown in SEQ ID NO.1) were connected to the self-cleaving peptide T2A after the CD3ζ intracellular domain to construct an IL-10 membrane-bound CD19 targeting CAR structure (mIL-10 CD19 CAR). The retroviral SFG vector was digested with enzymes, and the CD19CAR / IL-10 CD19 CAR / mbIL-10 CD19 CAR fragments were connected respectively, transformed, cloned, plasmid extracted, and sequenced to obtain a retroviral vector with the correct sequence.

[0037] 1.2 Retrovirus preparation

[0038] Phoenix-ampho cell plating. The revived Phoenix-ampho cell line needs to be cultured for two generations before it can be used for virus packaging. The day before transfection, plating was performed at 1×10 6Cells were seeded into plates (6-well plates) and cultured overnight. The next day, when the confluency of Phoenix-ampho cells reached about 70%, plasmid transfection was performed. Opti-MEM / DNA mixture was prepared in a 1.5 mL EP tube (calculated based on 0.25 mL system per well): Opti-MEM 125 μL + 2.5 μg of the retroviral vector (SFG core plasmid) with the correct corresponding sequence obtained in step 1.1 + 5 μL of P3000 and Opti-MEM / Lipo3000 mixture: Opti-MEM 125 μL + Lipo3000 7.5 μL; after mixing, let stand at room temperature for 5 min, and then slowly add the Opti-MEM / Lipo3000 mixture to the Opti-MEM / DNA mixture and let stand at room temperature for 20 min; 1 mL of the old culture medium was discarded from the transfected cells, and the mixture was added dropwise, and the mixture was gently shaken 8 times in each direction in a "rice" shape, and placed in a cell culture incubator with 5 vol% CO2, 37 ℃, and saturated humidity for 6 hours, and then 2 mL of 37 ℃ preheated fresh full DMEM medium, and continue to culture for 48 hours. After 48 hours, the supernatants were collected and filtered with a 0.45μm filter to obtain the corresponding γ-reverse transcription core plasmid packaging virus supernatants of the three CAR genes, which were stored at 4 ℃ (no longer than 1 week) for subsequent infection.

[0039] 2 Trophoblast cells expand and activate NK cells

[0040] 2.1 PBMC Isolation

[0041] 30-50 mL of peripheral blood was collected from healthy volunteers (Donor) (ethics approval number: NO. 2020-003); the peripheral blood was diluted with PBS (PBS: peripheral blood = 1:1), and 20 mL of the diluted peripheral blood was slowly added along the wall of a 50 mL centrifuge tube into an equal volume of Ficoll lymphocyte separation solution to separate the separation solution and peripheral blood into two layers, and the acceleration and deceleration speeds were adjusted to "0", and the centrifugation was performed at room temperature at 2200 rpm for 20 min; the PBMC layer was transferred to a new 50 mL centrifuge tube, and the acceleration and deceleration speeds were adjusted back to "9", and the tube was centrifuged at room temperature at 1500 rpm for 10 min, the supernatant was discarded, and the washing was repeated twice; after the last wash, the PBMC was resuspended in 40 mL PBS, and the cell viability and density were detected using a cell counter, and the number of PBMC was calculated.

[0042] 2.2 NK cell purification, culture and activation

[0043] Prepare magnetic bead sorting (MACS) buffer containing 0.5 wt% BSA and 2 mM EDTA for 1 × 10 780 μL MACS and 20 μL CD56 MicroBeads were added to PBMC, mixed by pipetting, and incubated at 4°C in the dark for 20 min; MACS was added to the cell / Beads mixture after the reaction to make the final volume reach 500 μL or 1 mL, and the mixture was added to the MS or LS sorting column. A 15 mL centrifuge tube was used to receive the effluent, and the MS / LS sorting column was rinsed twice with 500 μL MACS. All cells collected in the centrifuge tube were CD56 negative cells; the MS or LS sorting column was placed in the mouth of a new 15 mL centrifuge tube, 1 mL MACS was added, and the piston included in the column package was used to slowly but firmly push the liquid to the bottom, and repeated twice; the cells pushed down from the column were the isolated and purified human primary NK cells, and the cells were counted and the yield was calculated. The purified human primary NK cells were resuspended in RPMI 1640 complete medium and the density was adjusted to 1×10 6 cells / mL.

[0044] Resuscitate K562-4-1BBL-mIL21 (K562-2+) trophoblast cells; prepare cytokine-containing NK cell complete culture medium RPMI 1640 (45 mL RPMI 1640 + 5 mL FBS + 1% P / S + 200IU / mL IL-2), and resuspend K562-2+ trophoblast cells with RPMI1640 complete culture medium; add K562-2+ trophoblast cells (twice the number of NK cells) to the purified human primary NK cells to stimulate the proliferation of human primary NK cells, mix well, add to a culture flask of appropriate volume, and culture in a 5% CO2, 37 ℃, saturated humidity incubator.

[0045] 3 Preparation of CAR-NK cells

[0046] After 120 hours of stimulation of irradiated (100 Gy) human primary NK cells with K562-4-1BBL-mIL21 trophoblast cells, the γ-retroviral core plasmid packaging virus supernatant containing the three CAR genes obtained in step 1 can be used for virus infection; the details are as follows: 1 day in advance, use RetroNectin to coat the plate; take the NK cells after 120 hours of stimulation, add 1 mL of the corresponding γ-retroviral core plasmid packaging virus supernatant and 0.5 mL of RPMI 1640 medium containing only FBS and P / S, mix gently by pipetting, and add to the coated plate, 5×10 per well. 5cells; after sealing with sealing film, centrifuge at 32 ℃ and 3000 rpm for 90 min; remove the sealing film and place in a cell culture incubator for 24 hours; replace with fresh RPMI1640 complete medium containing IL-2 (200IU / mL) and continue to culture for 48 hours to obtain three types of NK cells: CD19 CAR-NK cells (not expressing IL-10), IL-10CD19 CAR-NK cells (expressing secretory IL-10) and mbIL-10CD19 CAR-NK cells (expressing non-secretory IL-10); use antibodies that recognize the IgG fragment of scFv in CAR combined with flow cytometry to detect the CAR positivity of the three types of CAR-NK cells. The results are as follows Figure 1 As shown, Figure 1 The results of the positive expression rate of CAR molecules using IgG to recognize CD19 and the expression level of IL-10 molecules on the surface of NK cells using IL-10 antibodies show that the present invention successfully constructed three types of CAR-NK cells. The obtained CAR-NK cells were observed every 2 to 3 days, and the cells were replenished with RPMI 1640 full culture medium containing IL-2 (200 IU / mL) to adjust the cell density to 1×10 6 / mL.

[0047] 4 In vitro functional evaluation of membrane-bound IL-10 CAR-NK cells

[0048] 4.1 Apoptosis detection

[0049] CD19 CAR-NK cells, IL-10CD19 CAR-NK cells and mIL-10CD19 CAR-NK cells were cultured in RPMI 1640, a complete NK cell culture medium without cytokines. Raji cells expressing CD19 were used as target cells. The target cells were stimulated by co-culture at a ratio of CAR-NK: Raji = 1:4 for 24 hours. Annexin V and PI were used to detect the early apoptosis, late apoptosis and live cell ratio of CAR-NK cells without target cell stimulation and after target cell stimulation. Figure 2It can be seen that without target cell stimulation, the three CAR-NK cells did not show obvious differences in apoptosis. After target cell stimulation, the early and late apoptotic cells of CD19 CAR-NK cells accounted for the lowest proportion of total cells, and the highest proportion of live cells; the early and late apoptotic cells of IL-10 CD19 CAR-NK cells accounted for the highest proportion of total cells, and the lowest proportion of live cells; the early and late apoptotic cells of mbIL-10CD19 CAR-NK cells accounted for a lower proportion of total cells, and the proportion of live cells was higher. This shows that when facing target cell stimulation, membrane-bound IL-10 can improve the activity rate of CAR-NK to a certain extent and reduce the occurrence of CAR-NK apoptosis compared with secretory IL-10.

[0050] 4.2 In vitro killing function verification

[0051] Chemiluminescence detection of in vitro killing function:

[0052] After detecting the expression level of CAR on the surface of NK cells, the number of CAR-NK cells required for different effector-target ratios (ratio of the number of effector cells (E) to target cells (T) (E:T), 2:1, 1:1, 1:2, 1:4, 1:8, 1:16, 1:32) was calculated according to the positive rate, and the blank group with no effector cells but only target cells was set; after co-culturing CAR-T cells and target cells in a 37 ℃ cell culture incubator for 16-18 hours, 100 μL of D-luciferin substrate (1.5 mg / mL) was added to each well, and the chemiluminescence of each well was detected using an enzyme-labeled instrument, and the killing efficiency of CAR-T cells was calculated, killing efficiency = (1-experimental group / blank group) × 100. The results are shown in Figure 3 As shown in the figure, the killing efficiency of IL-10CD19 CAR-NK cells and mbIL-10CD19 CAR-NK cells was higher than that of CD19 CAR-NK cells.

[0053] Flow cytometry to detect in vitro killing function:

[0054] After detecting the expression level of CAR on the surface of NK cells, Raji cells expressing CD19 were used as target cells. After co-culture for 24 hours at a ratio of CAR-NK: Raji = 1:4, an antibody recognizing CD19 was used to detect the cell ratio of Raji, and the number of remaining Raji cells was calculated. The more remaining Raji cells, the worse the killing effect of CAR-NK, and the fewer remaining Raji cells, the better the killing effect of CAR-NK.

[0055] The results are as follows Figure 4As shown in the figure, CD19 CAR-NK cells had the worst killing effect, while IL-10 CD19 CAR-NK cells and mIL-10 CD19 CAR-NK cells killed Raji cells more effectively, which was consistent with the results of in vitro killing function detection by chemiluminescence.

[0056] 4.3 Mitochondrial function assay

[0057] After detecting the expression level of CAR on the surface of NK cells, Raji cells expressing CD19 were used as target cells and co-cultured for 24 hours at a ratio of CAR-NK: Raji = 1:4 for target cell stimulation; MitoTracker DeepRed and MitoTracker Green dyes were then used to stain the mitochondria of stimulated or unstimulated CAR-NK cells. Figure 5 As shown in the data, without target cell stimulation, the three CAR-NK cells had similar mitochondrial functions; after target cell stimulation, the proportion of dysfunctional mitochondria in IL-10CD19CAR-NK cells increased, while CD19CAR-NK cells and mbIL-10CD19CAR-NK cells had similar mitochondrial functions, indicating that membrane-bound IL-10 can more effectively protect the mitochondrial function of CAR-NK cells than secreted IL-10 to enhance the killing ability of CAR-NK cells.

[0058] 4.4 In vitro cytokine release capacity detection

[0059] To detect the different cytokine secretion levels of the three CAR-NK cells, target cells were stimulated for 24 hours using a co-culture ratio of CAR-NK: Raji (E:T) = 4:1; the supernatant of stimulated or unstimulated CAR-NK cell culture medium was then collected, and the cytokine secretion level of CAR-NK was detected using a CBA kit. Figure 6 As shown in the figure, only IL-10 CD19 CAR-NK can effectively secrete IL-10, and no significant IL-10 was detected in the supernatant of the other two CAR-NKs, indicating that the membrane-bound IL-10 was successfully constructed. Compared with cells without target cell stimulation, the various cytokines of mbIL-10 CD19 CAR-NK cells stimulated with target cells were significantly lower than those of IL-10 CD19 CAR-NK, which can effectively alleviate cytokine release syndrome.

[0060] 5. Evaluation of the function of membrane-bound CD19 CAR-NK cells in mice

[0061] like Figure 7As shown, the right armpit of NSG mice was wiped and disinfected with 75% alcohol cotton balls, and 200 μL of 1×10 resuspended in PBS was drawn using a 1 mL syringe. 6 Raji tumor cells were injected subcutaneously; 7 days after tumor cell inoculation, the long and short axes of the tumor of each mouse were measured using a vernier caliper, and the mice were randomly divided into 4 groups according to the tumor area, namely Blank NK, CD19 CAR-NK, IL-10 CD19 CAR-NK, and mbIL-10 CD19 CAR-NK, with 5 mice in each group. The tumor area was calculated as follows: tumor area (mm 2 ) = long axis (mm) × short axis (mm); the four groups of mice were injected with 5×10 6 The mice were treated with NK cells, CD19 CAR-NK cells, IL-10 CD19 CAR-NK cells, and mbIL-10CD19 CAR-NK cells for 4 weeks. The mice were weighed and the long and short axes of the tumors were measured twice a week. The data were collected and the tumor area was calculated. Figure 8 and Fig. 9 As shown in the figure, the tumor area of ​​the mbIL-10 CD19 CAR-NK cell group was significantly lower than that of the other groups, indicating that its tumor control effect was enhanced to a certain extent. Compared with the CD19 CAR-NK and IL-10 CD19 CAR-NK groups, the body weight of the mice in the mbIL-10 CD19 CAR-NK group remained constant, indicating that it had less toxic side effects and Figure 6 The results are consistent.

[0062] The above results indicate that the present invention can effectively reduce the main toxic and side effects of CAR-NK cell therapy by expressing non-secretory IL-10 in CAR-NK cells, while protecting the mitochondrial function of CAR-NK cells and improving the activity of CAR-NK, reducing the occurrence of CAR-NK apoptosis, and significantly improving the tumor killing ability of CAR-NK cells, thereby achieving the toxicity-reducing and synergistic effect of CAR-NK cells.

[0063] Embodiment 2:

[0064] 1 Construction of chimeric antigen receptor retroviral vector and virus preparation

[0065] The chimeric antigen receptor retroviral vector was constructed and the virus was prepared in the same manner as in Example 1.

[0066] 2. Isolation and purification of activated T cells

[0067] PBMCs were isolated by the same method as in Example 1, and then a magnetic bead sorting (MACS) buffer containing 0.5% BSA and 2 mM EDTA was prepared. 7 80 μL MACS and 20 μL CD3 MicroBeads were added to PBMC, mixed by pipetting, and incubated at 4°C in the dark for 20 min; MACS was added to the cell / Beads mixture after the reaction to make the final volume reach 500 μL or 1 mL, and the mixture was added to the MS or LS sorting column. A 15 mL centrifuge tube was used to receive the effluent, and the MS / LS sorting column was rinsed twice with 500 μL MACS. All cells collected in the centrifuge tube were CD3-negative cells; the MS or LS sorting column was placed at the mouth of a new 15 mL centrifuge tube, 1 mL MACS was added, and the piston included in the column package was used to slowly but firmly push the liquid to the bottom, and repeated twice; the cells pushed down from the column were the isolated and purified human primary T cells, and the cells were counted and the yield was calculated.

[0068] Prepare X-VIVO15 complete T cell culture medium containing cytokines (45 mL X-VIVO15 + 5 mL FBS + 1% P / S + 5 ng / μL IL-7 + 5 ng / mL IL-15). Resuspend the purified human primary T cells in X-VIVO15 complete culture medium and adjust the density to 1×10 6 cells / mL, add CD3 / CD28 dynabeads with an equal number of T cells to stimulate the proliferation of human primary T cells, mix well, add to a culture flask of appropriate volume, and culture in a 5% CO2, 37 ℃, saturated humidity incubator.

[0069] 3 Preparation of CAR-T cells

[0070] Transfection was performed using the γ-retroviral core plasmid packaging virus supernatant containing the three corresponding CAR genes; the details were as follows: 1 day in advance, the plate was coated with RetroNectin; after stimulation, 1 mL of the corresponding γ-retroviral supernatant and 0.5 mL of X-VIVO15 medium containing only FBS and P / S were taken from the stimulated T cells, the mixture was gently pipetted and mixed, and the cells were added to the coated plate, with 5×10 cells per well. 5cells; after sealing with sealing film, centrifuge at 32 °C and 3000 rpm for 90 min; remove the sealing film and place in a cell culture incubator for 24 h; replace with fresh X-VIVO15 full culture medium containing IL-7 and IL-15 and continue to culture for 48 h; use IgG to recognize the positive expression rate of CAR molecules of CD19 and use IL-10 antibody to recognize the expression level of IL-10 molecules on the surface of T cells to verify the successful preparation of three types of CAR-T cells. After that, observe the cells every 2 to 3 days, replenish with X-VIVO15 full culture medium containing cytokines, and adjust the cell density to 1×10 6 / mL.

[0071] 4 Evaluation of the function of membrane-bound CD19 CAR-T cells in mice

[0072] Use 75% alcohol cotton balls to wipe and disinfect the right axilla of NSG mice, and use a 1 mL syringe to draw 200 ml of 1×10 6 Raji tumor cells were injected subcutaneously; 7 days after tumor cell inoculation, the long and short axes of the tumor of each mouse were measured using a vernier caliper, and the mice were randomly divided into 4 groups according to the tumor area: Blank T, CD19CAR-T, IL-10 CD19 CAR-T, and mbIL-10 CD19 CAR-T, with 5 mice in each group. The tumor area was calculated as follows: tumor area (mm 2 ) = long axis (mm) × short axis (mm); the four groups of mice were injected with 5×10 6 The mice were treated with T cells, CD19 CAR-T cells, IL-10 CD19 CAR-T cells, and mbIL-10 CD19 CAR-T cells for 4 weeks. The mice were then weighed and the long and short axes of the tumors were measured twice a week. The data were collected and the tumor area was calculated. Fig.10 and Fig.11 As shown, the tumor area of ​​the mbIL-10 CD19 CAR-T cell group was significantly lower than that of the other groups, indicating that its tumor control effect was enhanced to a certain extent, and compared with the CD19 CAR-T and IL-10 CD19 CAR-T groups, the body weight of mice in the mbIL-10 CD19 CAR-T group was not significantly different from that in the Blank T group, indicating that it had less toxic side effects. The above results show that the present invention can effectively reduce the main toxic side effects of CAR-T cell therapy by expressing non-secretory IL-10 in CAR-T cells, while protecting the mitochondrial function of CAR-T cells and improving the viability of CAR-T, reducing the occurrence of CAR-T apoptosis, and significantly improving the tumor killing ability of CAR-T cells, thereby achieving the attenuation and synergistic effect of CAR-T cells.

[0073] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An immune cell expressing non-secretory IL-10 for enhancing cellular immunotherapy, characterized in that: The immune cell comprises a recombinant construct encoding non-secreted IL-10, a fragment or a variant thereof.

2. The immune cell expressing non-secretory IL-10 for enhancing cellular immunotherapy according to claim 1, characterized in that: The immune cells include T cells, B cells, natural killer cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and / or peripheral blood mononuclear cells.

3. The immune cell expressing non-secretory IL-10 for enhancing cellular immunotherapy according to claim 1, characterized in that: The immune cell further comprises a second recombinant construct encoding CAR, TCR or any other synthetic tumor targeting motif, wherein the recombinant construct encoding non-secreted IL-10, a fragment or variant thereof is linked to the second recombinant construct encoding CAR, TCR or any other synthetic tumor targeting motif.

4. The immune cell expressing non-secretory IL-10 for enhancing cellular immunotherapy according to claim 3, characterized in that: The recombinant construct encoding the non-secretory IL-10, its fragment or variant is connected to the second recombinant construct encoding CAR, TCR or any other synthetic tumor targeting motif via a sequence encoding a self-cleaving peptide.

5. The immune cell expressing non-secretory IL-10 for enhancing cellular immunotherapy according to claim 3, characterized in that: The CAR comprises a CD19 binding domain and a non-antigen binding domain, wherein the non-antigen binding domain includes the extracellular domain, transmembrane domain and intracellular co-stimulatory domain of CD28 or a functional variant thereof, and the signaling domain of CD3ζ or a functional variant thereof.

6. The immune cell expressing non-secretory IL-10 for enhancing cellular immunotherapy according to claim 1, characterized in that: The non-secretory IL-10, fragment or variant thereof is membrane-bound IL-10, fragment or variant thereof.

7. The immune cell expressing non-secretory IL-10 for enhancing cellular immunotherapy according to claim 6, characterized in that: The membrane-bound IL-10 is composed of IL-10, a fragment or variant thereof, a hinge region and a transmembrane domain.

8. The immune cell expressing non-secretory IL-10 for enhancing cellular immunotherapy according to any one of claims 1 to 7, characterized in that: The immune cells are autologous, allogeneic or xenogeneic.

9. A method for preparing immune cells expressing non-secretory IL-10 for enhancing cellular immunotherapy according to any one of claims 1 to 8, characterized in that: include: obtaining immune cells from a subject; Modifying an immune cell so that the immune cell comprises a recombinant construct encoding non-secretory IL-10, a fragment or a variant thereof; as well as The immune cells are cultured to obtain immune cells expressing non-secretory IL-10 for enhancing cellular immunotherapy.

10. Use of the immune cells expressing non-secretory IL-10 for enhancing cellular immunotherapy according to any one of claims 1 to 8 in the preparation of a medicament for treating / preventing / diagnosing cancer or tumor, infection or autoimmune disease.

11. The use according to claim 10, characterized in that The cancer or tumor includes: glioblastoma, head and neck cancer, lung cancer, thyroid cancer, bronchial cancer, breast cancer, gastric cancer, liver cancer, pancreatic cancer, colon cancer, colorectal cancer, kidney cancer, bladder cancer, cervical cancer, uterine cancer, ovarian cancer, prostate cancer, skin cancer, melanoma, lymphoma, leukemia, or one or more combinations thereof.

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