Immune cells expressing non-secretory IL-10 for enhancing cellular immunotherapy, preparation method and uses

Abstract

The present invention discloses an immune cell expressing non-secretory IL-10 for enhancing cellular immunotherapy, a preparation method and a use thereof. The present invention unexpectedly discovered that non-secretory IL-10 can significantly reduce the mitochondrial dysfunction of CAR cells compared to secretory IL-10, and when facing target cell stimulation, compared with secretory IL-10, non-secretory IL-10 can effectively improve the viability of CAR cells and reduce the occurrence of CAR apoptosis. Based on this, the present invention expresses non-secretory IL-10 in CAR cells, while effectively reducing the main toxic and side effects of CAR cell therapy, by protecting the mitochondrial function of CAR cells and improving the viability of CAR cells, reducing the occurrence of CAR cell apoptosis and significantly improving the tumor killing ability of CAR cells, thereby achieving the attenuation and synergistic effect of CAR cells.

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 topic in the future development of human medicine due to its highly precise and personalized advantages. Immunotherapy refers to the use of autologous, allogeneic, or xenogeneic (non-human) cells, which are manipulated in vitro to enhance and modify the patient's immune system's ability to recognize and kill tumors, and then re-infused (or implanted) into the human body. Among them, chimeric antigen receptor T cell immunotherapy (CAR-T cell therapy) is an adoptive cellular immunotherapy that has emerged in recent years. It mainly combines the specificity of chimeric antigen receptors with the immune function of T cells, and then kills malignant tumor cells through specific recognition. In addition, due to the key role of NK cells in the innate immune response and their advantages such as wider access to sources, lower risk of graft-versus-host disease, and greater "off-the-shelf" supply potential, NK cell-based cellular immunotherapy has also received widespread attention and research. CAR-NK cell therapy not only utilizes the research and development ideas of CAR-T cell therapy, but also leverages the biological characteristics of NK cells.

[0003] Although CAR-T cell therapy has demonstrated excellent efficacy, the multiple toxic side effects and high relapse rates frequently reported in clinical trials remain significant challenges. These challenges present themselves as challenges for CAR-T cell therapy. The primary toxic side effects of CAR-T cell therapy are cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS). CRS is caused by the inherent characteristics of CAR-T cells. CRS is triggered by the release of inflammatory cytokines and chemokines, such as interferon (IFN-γ), tumor necrosis factor (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, IL-6, and IL-8, following CAR-T cell engagement with their target antigen. As a systemic inflammatory response, CRS affects multiple systems, including the circulatory, respiratory, urinary, and digestive systems. Patients may experience fever, rash, cardiac insufficiency, 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 existing technology and provide an immune cell expressing non-secretory IL-10 for enhancing cellular immunotherapy, as well as 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 alleviating 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 discovered that compared with secretory IL-10, non-secretory IL-10 can significantly reduce mitochondrial dysfunction of CAR-T / NK cells, and when faced with target cell stimulation, non-secretory IL-10 can effectively increase the activity 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, while effectively reducing the main toxic side effects of CAR-T / NK cell therapy, it protects the mitochondrial function of CAR-T / NK cells and improves the activity rate of CAR-T / NK, reduces the occurrence of CAR-T / NK apoptosis, and significantly improves the tumor killing ability of CAR-T / NK cells, 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 cells being restricted to the inside or around the cells, so that it cannot act on distant cells or other components through endocrine secretion, thereby weakening the cytokine release syndrome; the non-secretory IL-10 mainly includes: covalently bound IL-10, membrane protein fusion IL-10, etc., in particular, membrane protein fusion IL-10, i.e., membrane-bound IL-10, which is composed of IL-10, its fragments or variants, and hinge regions and transmembrane domains, and the produced IL-10 is bound to the cell membrane through the hinge region and transmembrane domain; the transmembrane domain can be, for example, 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 variant thereof.

[0008] In some embodiments, immune effector cells further 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 are capable of specifically binding 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 variants are one or more biologically active derivatives of membrane-bound IL-10, preferably having the amino acid sequence shown in SEQ ID No. 1. Generally, the term "variant" refers to a molecule having one or more additions, substitutions (usually conservative in nature), and / or deletions to the native sequence relative to the native molecule, provided that such modifications do not destroy its biological activity, and the variant is "substantially homologous" to the reference molecule. Typically, 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%, typically 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 of 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-secreted IL-10, a fragment or 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 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 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;

[0023] Figure 3 This is the result of chemiluminescence detection of in vitro killing function. Figure 3 A in the figure is the killing efficiency result of Raji cells at different effector-target ratios; Figure 3 Figure B shows the killing efficiency of Raji cells at 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 are shown 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 diagram showing the changes in mouse tumors after CAR-NK cell injection;

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

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

[0031] Figure 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 achieved by the present invention easy to understand, the present invention is further illustrated below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation manner, other embodiments obtained by those skilled in the art without making creative work all fall within the scope of protection of the present invention. In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the equipment and materials used in each embodiment are all the same. In the following examples, unless otherwise specified, % refers to volume percentage.

[0033] Example 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 for NK cells (CD19 CAR) was constructed by using an anti-CD19 single-domain antibody sequence (SEQ ID NO.3) to bind to the extracellular, transmembrane, and cytoplasmic regions of the CD28 molecule (SEQ ID NO.4) and connect to the intracellular domain of CD3ζ (SEQ ID NO.5). Based on this CAR structure, a wild-type cytokine IL-10 coding sequence (amino acid sequence shown in SEQ ID NO.2) was connected to the CD3ζ intracellular domain using a self-cleaving peptide T2A to construct an IL-10 secreting CD19 targeting CAR structure (IL-10CD19 CAR). A wild-type cytokine IL-10 coding sequence, hinge region, and transmembrane domain (amino acid sequence shown in SEQ ID NO.1) were connected to the CD3ζ intracellular domain using a self-cleaving peptide T2A to construct an IL-10 membrane-bound CD19 targeting CAR structure (mIL-10CD19 CAR). The retroviral SFG vector was digested with enzymes and ligated with CD19CAR / IL-10CD19 CAR / mbIL-10CD19 CAR fragments, respectively. The cells were 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 recovered Phoenix-ampho cell line needs to be cultured for 2 generations before it can be used for virus packaging, the day before transfection, plating, at 1×10 6Cells were seeded into 6-well plates and cultured overnight. The next day, when the Phoenix-ampho cell confluency reached approximately 70%, plasmid transfection was performed. Opti-MEM / DNA mixture (calculated based on 0.25 mL per well) was prepared in a 1.5 mL EP tube: 125 μL of Opti-MEM + 2.5 μg of the retroviral vector (SFG core plasmid) with the correct sequence obtained in step 1.1 + 5 μL of P3000; and Opti-MEM / Lipo3000 mixture: 125 μL of Opti-MEM + Lipo3000 7.5μL; after mixing, let it stand at room temperature for 5 minutes. The Opti-MEM / Lipo3000 mixture was slowly added to the Opti-MEM / DNA mixture and allowed to stand at room temperature for 20 minutes. The cells to be transfected discarded 1mL of old culture medium and added dropwise to the mixed mixture. The cells were gently shaken 8 times in each direction of the "R" shape. After culturing for 6 hours in a cell culture incubator with 5vol% CO2, 37°C and saturated humidity, 2mL of fresh full DMEM medium preheated at 37°C was replaced and cultured for another 48 hours. After 48 hours, the supernatants were collected and filtered with a 0.45μm filter to obtain the corresponding three CAR gene γ-reverse transcription core plasmid packaging virus supernatants, which were stored at 4°C (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 medium to separate the separation medium and peripheral blood into two layers. The acceleration and deceleration rates were adjusted to "0", and the centrifugation was carried out at room temperature at 2200 rpm for 20 minutes. The PBMC layer was transferred to a new 50 mL centrifuge tube, and the acceleration and deceleration rates were adjusted back to "9". The tube was centrifuged at room temperature at 1500 rpm for 10 minutes, the supernatant was discarded, and the washing was repeated twice. After the final wash, the PBMC was resuspended in 40 mL of 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 separation (MACS) buffer containing 0.5 wt% BSA and 2 mM EDTA. 7PBMC was added with 80 μL MACS and 20 μL CD56 MicroBeads, 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 collect 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 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 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 medium RPMI 1640 (45 mL RPMI 1640 + 5 mL FBS + 1% P / S + 200 IU / mL IL-2), and resuspend the K562-2+ trophoblast cells in RPMI 1640 complete medium; add K562-2+ trophoblast cells (twice the number of NK cells) to the purified primary human NK cells to stimulate the proliferation of primary human NK cells. After mixing, add the cells to a culture flask of an appropriate volume and culture in a 5% CO2, 37°C, saturated humidity incubator.

[0045] 3. Preparation of CAR-NK cells

[0046] After 120 hours of stimulation of K562-4-1BBL-mIL21 trophoblast cells after irradiation (100Gy) of human primary NK cells, the virus supernatant containing the γ-reverse transcription core plasmid packaging virus of 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 1mL of the corresponding γ-reverse transcription core plasmid packaging virus supernatant and 0.5mL of RPMI 1640 medium containing only FBS and P / S, mix gently by pipetting, and add to the coated plate, with 5×10 per well. 5cells; after sealing with sealing film, centrifuge at 32°C and 3000rpm for 90min; remove the sealing film and place in a cell culture incubator for 24h; replace with fresh RPMI 1640 complete medium containing IL-2 (200IU / mL) and continue to culture for 48h to obtain three types of NK cells: CD19 CAR-NK cells (not expressing IL-10), IL-10CD19CAR-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 rate 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 has successfully constructed three types of CAR-NK cells. The obtained CAR-NK cells were observed every 2 to 3 days and replenished with RPMI 1640 complete 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-10CD19CAR-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 and co-cultured for 24 hours at a ratio of CAR-NK:Raji=1:4 for target cell stimulation. 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 2 As can be seen, without target cell stimulation, none of the three CAR-NK cells showed significant differences in apoptosis. After target cell stimulation, CD19 CAR-NK cells had the lowest proportion of early and late apoptotic cells in the total cells and the highest proportion of live cells; IL-10CD19 CAR-NK cells had the highest proportion of early and late apoptotic cells in the total cells and the lowest proportion of live cells; mbIL-10CD19 CAR-NK cells had a lower proportion of early and late apoptotic cells in the total cells and a higher proportion of live cells. This indicates that when faced with target cell stimulation, membrane-bound IL-10 can, compared to secreted IL-10, improve the activity of CAR-NK cells and reduce the occurrence of CAR-NK apoptosis to a certain extent.

[0050] 4.2 Verification of killing function in vitro

[0051] Chemiluminescence assay to detect killing function in vitro:

[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 (the 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. At the same time, a blank group with no effector cells and only target cells was set; after co-culturing CAR-T cells and target cells in a 37°C cell incubator for 16 to 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 a microplate reader, and the killing efficiency of CAR-T cells was calculated, killing efficiency = (1-experimental group / blank group) × 100. The results are shown in Figure 2. 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 detection of in vitro killing function:

[0054] After detecting the expression level of CAR on the surface of NK cells, CD19-expressing Raji cells 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 proportion of Raji cells, and the number of remaining Raji cells was calculated. The more Raji cells remained, the worse the killing effect of CAR-NK, and the fewer Raji cells remained, the better the killing effect of CAR-NK.

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

[0056] 4.3 Mitochondrial function assay

[0057] After detecting the expression level of CAR on the surface of NK cells, CD19-expressing Raji cells 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 5As shown in the results, without target cell stimulation, the three CAR-NK cells had similar mitochondrial functions; after target cell stimulation, the proportion of dysfunctional mitochondria in IL-10CD19 CAR-NK cells increased, while CD19 CAR-NK cells and mbIL-10CD19 CAR-NK cells had similar mitochondrial functions, indicating that membrane-bound IL-10 is more effective than secreted IL-10 in protecting the mitochondrial function of CAR-NK cells, thereby improving the killing ability of mbIL-10CD19 CAR-NK cells. 4.4 In vitro cytokine release capacity detection

[0058] To detect the different cytokine secretion levels of the three CAR-NK cells, target cells were stimulated for 24 hours using a CAR-NK:Raji (E:T) ratio of 4:1; the supernatant of the stimulated or unstimulated CAR-NK cell culture medium was collected and the cytokine secretion levels of CAR-NK were detected using a CBA kit. Figure 6 As shown, only IL-10CD19 CAR-NK cells can effectively secrete IL-10, while no significant IL-10 was detected in the supernatant of the other two CAR-NK cells, indicating that membrane-bound IL-10 was successfully constructed. Compared with cells without target cell stimulation, the various cytokines produced by mbIL-10CD19 CAR-NK cells stimulated with target cells were significantly lower than those of IL-10CD19 CAR-NK cells, thereby effectively alleviating cytokine release syndrome.

[0059] 5. In vivo functional evaluation of membrane-bound CD19 CAR-NK cells in mice

[0060] like Figure 7 As shown, the right armpit of NSG mice was wiped and disinfected with 75% alcohol cotton balls, and 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. According to the tumor area, the mice were randomly divided into 4 groups: Blank NK, CD19 CAR-NK, IL-10CD19 CAR-NK, and mbIL-10CD19 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); 4 groups of mice were injected with 5×10 6The mice were treated with NK cells, CD19 CAR-NK cells, IL-10CD19 CAR-NK cells, and mbIL-10CD19CAR-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 Figure 9 As shown in the figure, the tumor area of the mbIL-10CD19CAR-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-10CD19 CAR-NK groups, the body weight of the mice in the mbIL-10CD19 CAR-NK group remained constant, indicating that it had less toxic side effects and Figure 6 The results are consistent.

[0061] The above results show that the present invention can effectively reduce the main toxic 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.

[0062] Example 2:

[0063] 1 Construction of chimeric antigen receptor retroviral vector and virus preparation

[0064] The chimeric antigen receptor retroviral vector was constructed and the virus was prepared using the same method as in Example 1.

[0065] 2. Isolation and purification of activated T cells

[0066] PBMCs were isolated by the same method as in Example 1, and then a magnetic bead separation (MACS) buffer containing 0.5% BSA and 2 mM EDTA was prepared. 7 PBMCs were added with 80 μL MACS and 20 μL CD3 MicroBeads, mixed by pipetting, and incubated at 4°C in the dark for 20 min. MACS was added to the cell / Beads mixture at the end of the reaction to bring the final volume to 500 μL or 1 mL. The mixture was added to an MS or LS separation column. A 15 mL centrifuge tube was used to collect the effluent. The MS / LS separation column was rinsed twice with 500 μL MACS. All cells collected in the centrifuge tube were CD3-negative cells. The MS or LS separation column was placed at the top of a new 15 mL centrifuge tube, 1 mL MACS was added, and the plunger included in the column package was used to slowly but firmly push the liquid to the bottom. This was repeated twice. The cells pushed out of the column were the isolated and purified primary human T cells, which were counted and the yield was calculated.

[0067] Prepare T cell complete culture medium X-VIVO15 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°C, saturated humidity incubator.

[0068] 3. Preparation of CAR-T cells

[0069] Transfection was performed using the supernatant of the γ-retroviral core plasmid containing the three corresponding CAR genes. The specific steps 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 added to the T cells. The mixture was gently pipetted and mixed. The cells were then added to the coated plate, with 5 × 10 cells per well. 5 cells; after sealing with sealing film, centrifuge at 32°C and 3000rpm for 90 minutes; remove the sealing film and place in a cell culture incubator for 24 hours; replace with fresh X-VIVO15 complete medium containing IL-7 and IL-15 and continue to culture for 48 hours; use IgG to recognize CD19 CAR molecule expression positive rate and use IL-10 antibody to recognize T cell surface IL-10 molecule expression level 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 complete medium containing cytokines, and adjust the cell density to 1×10 6 / mL.

[0070] 4. In vivo functional evaluation of membrane-bound CD19 CAR-T cells in mice

[0071] Use 75% alcohol cotton balls to wipe and disinfect the right armpit 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. According to the tumor area, the mice were randomly divided into 4 groups: Blank T, CD19CAR-T, IL-10CD19 CAR-T, and mbIL-10CD19 CAR-T, with 5 mice in each group. The tumor area was calculated as follows: tumor area (mm 2) = major axis (mm) × minor axis (mm); 4 groups of mice were injected with 5 × 10 6 The mice were treated with T cells, CD19 CAR-T cells, IL-10CD19 CAR-T cells, and mbIL-10CD19 CAR-T cells for 4 weeks. The mice were then observed twice a week, and the long and short axes of the tumors were measured. The data were collected and the tumor area was calculated. Figure 10 and Figure 11 As shown, the tumor area of the mbIL-10CD19 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. Compared with the CD19CAR-T and IL-10CD19CAR-T groups, the body weight of the mice in the mbIL-10CD19 CAR-T group was not significantly different from that of 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 activity 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.

[0072] 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 principles of the present invention should be included in the scope of protection 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-secretory IL-10; the non-secretory IL-10 is membrane-bound IL-10, which is composed of IL-10, a hinge region, and a transmembrane domain; the amino acid sequence of IL-10 is shown in SEQ ID No. 1; The immune cells are T cells and natural killer cells; the immune cells further comprise a second recombinant construct encoding CAR, wherein the recombinant construct encoding non-secretory IL-10 is connected to the second recombinant construct encoding CAR; the CAR comprises a CD19 binding domain and a non-antigen binding domain, and the non-antigen binding domain includes the extracellular domain, transmembrane domain and intracellular co-stimulatory domain of CD28, and the signaling domain of CD3ζ.

2. The non-secretory IL-10 expressing immune cell for enhancing cellular immunotherapy according to claim 1, characterized in that The recombinant construct encoding the non-secretory IL-10 is connected to the second recombinant construct encoding CAR through a sequence encoding a self-cleavage peptide.

3. The non-secretory IL-10 expressing immune cell for enhancing cellular immunotherapy according to any one of claims 1 to 2, characterized in that: The immune cells are autologous, allogeneic or xenogeneic.

4. A method for preparing immune cells expressing non-secretory IL-10 for enhancing cellular immunotherapy according to any one of claims 1-2, characterized in that: include: obtaining immune cells from a subject; engineering an immune cell so that the immune cell contains a recombinant construct encoding non-secretory IL-10; as well as The immune cells are cultured to obtain immune cells expressing non-secretory IL-10 for enhancing cellular immunotherapy.

5. Use of the non-secretory IL-10-expressing immune cells for enhancing cellular immunotherapy according to any one of claims 1 to 2 in the preparation of a medicament for treating / preventing cancer or autoimmune diseases; the cancer is lymphoma or leukemia.

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