A car-cd8+ iTreg cell targeting cd38 and a preparation method and application thereof

Abstract

This invention relates to the field of biomedical technology, and more particularly to a CD38-targeting CAR-CD8+ iTreg cell, its preparation method, and its application. This invention utilizes CD8+ iTreg cells combined with the construction technology of a CD38-targeting chimeric antigen receptor (CAR) to generate novel CD38-targeting CAR-CD8+ iTreg cells. For the first time, these novel CD38-targeting CAR-CD8+ iTreg cells were applied to the treatment of systemic lupus erythematosus (SLE), demonstrating that they can effectively alleviate SLE and lupus nephritis, exhibiting good therapeutic effects. The mechanism may be that the CD38-targeting CAR-CD8+ iTreg cells exert a protective effect on lupus mice by depleting activated T cells, B cells, and NK cells that highly express CD38.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a CD38-targeting CAR-CD8+ iTreg cell, its preparation method, and its application. Background Technology

[0002] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease affecting multiple systems. Lupus nephritis (LN) is its most common organ manifestation and a leading cause of death. Approximately 50% of SLE patients present with clinical signs of kidney damage, and renal biopsy shows almost 100% kidney involvement. In my country, LN is the most common secondary immune glomerular disease and a significant cause of end-stage renal disease (ESRD). Currently, SLE and LN are incurable. Glucocorticoids and immunosuppressants remain the primary treatments for both SLE and LN, but their specificity is poor and they often cause severe toxic side effects, significantly impacting patients' quality of life and prognosis. Therefore, exploring effective and minimally invasive precision treatment options for lupus nephritis has become an urgent clinical challenge with significant implications.

[0003] Abnormal immune regulation plays a dominant role in the pathogenesis of SLE / LN. Patients experience a decrease in the number and dysfunction of regulatory T cells (Tregs), leading to persistent B cell activation and the production of autoantibodies. This weakens autoimmune tolerance, promoting disease development and progression. Our previous research found that under IL-2 / TGF-β in vitro culture conditions, naive CD8+ T cells (CD8+CD62L+T, naïve CD8+ T) in mice can be transformed into a novel CD8+ inducible regulatory T cell subset (CD8+ iTregs). This subset possesses the immunosuppressive function of regulatory T cells. Our previous results showed that CD8+ iTregs significantly reduced proteinuria in lupus mice, decreased the production and kidney deposition of autoantibodies such as anti-dsDNA antibodies, suppressed autoreactive B cells in vivo, and significantly prolonged the survival time of lupus mice. However, CD8+ iTregs also carry the risk of non-specific immunosuppression, limiting the further application of Tregs. We propose that this drawback can be overcome by constructing a specific chimeric antigen receptor (CAR) in CD8+iTreg.

[0004] Compared to antibody therapy, CAR-T cell therapy offers faster targeting and longer-lasting effects. CAR-T therapy is a chimeric antigen receptor T-cell immunotherapy. Through genetic engineering, autologous T cells express chimeric antigen receptors, specifically recognizing and efficiently killing target cells in the body. In August 2021, a team from the University of Erlangen-Nuremberg in Germany published a clinical research paper in the *New England Journal of Medicine* entitled "CD19-Targeted CAR T Cells in Refractory Systemic Lupus Erythematosus." The results of this trial showed that CD19-targeted CAR-T therapy has a rapid response, with symptoms resolving in patients with autoimmune diseases such as systemic lupus erythematosus, and temporarily eliminating the need for further treatment with hormones, immunosuppressants, or other drugs. However, current CAR-T therapy targeting CD19 also has the following drawbacks and limitations: 1. CAR-T therapy is restricted by HLA, and currently only autologous cell infusion can be performed, limiting its clinical application; 2. CAR-T cells recognize and kill target cells, releasing a large number of cytokines, including TNF-α, IL-1, IL-6, IFN-γ, MCP-1, and IL-8. The released cytokines further activate immune cells (such as T cells, B cells, monocytes, macrophages, etc.) or non-immune cells (such as endothelial cells), causing these cells to release even more cytokines, forming a positive feedback effect, further amplifying the level of cytokines and inflammation in the body, causing cytokine release syndrome (CRS), inducing systemic inflammatory response, and severe damage to tissues and organs; 3. Plasma blasts and long-lived plasma cells that produce autoantibodies do not express CD19, thus escaping the exhaustion caused by CD19 targeting, leading to relapse of lupus nephritis. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a CD38-targeting CAR-CD8+ iTreg cell and its application. This invention demonstrates that the novel CD38-targeting CAR-CD8+ iTreg cells can effectively alleviate systemic lupus erythematosus and lupus nephritis, exhibiting good therapeutic effects. To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a CAR-CD8+ iTreg cell that targets CD38, wherein the CAR-CD8+ iTreg cell expresses a chimeric antigen receptor that specifically binds to the CD38 antigen, CD39, and CD103.

[0006] This invention utilizes CD8+iTreg cells combined with the technology of constructing a specific chimeric antigen receptor (CAR) targeting CD38 to generate novel CAR-CD8+iTreg cells targeting CD38. The inventors found that CD8+iTregs exhibit increased expression levels of CD103 and CD39. CD8+iTregs with high expression of CD39 and CD103 are more effective in treating lupus nephritis mice than CD8+CD103+CD39-T cells or pretreated CD8+iTregs.

[0007] Compared with CAR-T cell therapy targeting CD19, novel CAR-CD8+ iTreg cells targeting CD38 have the following advantages: 1. Plasma blasts and long-lived plasma cells that produce autoantibodies do not express CD19, thus escaping the exhaustion caused by targeting CD19, leading to relapse of lupus nephritis; 2. CD38 is a typical marker of plasma cells. CAR-CD8+ iTreg cells targeting CD38 are more precise in treating lupus nephritis than the publicly disclosed CD19 treatment target in existing technologies. 3. Existing CAR-T cell therapies have side effects such as causing cytokine release syndrome (CRS) and inducing systemic inflammatory responses. The novel CAR-CD8+iTreg cells targeting CD38 prepared in this invention have regulatory T cell immunomodulatory properties, which significantly reduce the risk of CRS side effects. 4. It possesses regulatory T cell immunomodulatory properties, is not HLA-restricted, and enables allogeneic cell infusion.

[0008] Preferably, the chimeric antigen receptor includes a CD38 antigen-binding domain, a transmembrane domain, and an intracellular co-stimulatory domain.

[0009] Preferably, the amino acid sequence of the CD38 antigen-binding domain is shown in SEQ ID NO.1; the amino acid sequence of the transmembrane domain is shown in SEQ ID NO.2; and the amino acid sequence of the intracellular co-stimulatory domain is shown in SEQ ID NO.3.

[0010] Secondly, the present invention provides a method for preparing the above-mentioned CD38-targeting CAR-CD8+ iTreg cells, comprising the following steps: (1) Preparation of CD8+CD62L+ naive T cells; (2) HEK-293T cells were transfected with a plasmid containing a specific chimeric antigen receptor targeting CD38 to obtain a recombinant lentivirus; (3) The CD8+CD62L+ immature T cells from step (1) were infected with the recombinant lentivirus from step (2) to obtain CAR CD38 CD8+62L+ T cells; (4) CAR CD38 CD8+62L+ T cells were cultured in CD3 antibody, CD28 antibody, recombinant IL-2 and recombinant TGF-β, and CAR-CD8+ iTreg cells targeting CD38 were obtained by flow cytometry sorting.

[0011] This invention is the first to propose combining CD8+CD103+CD39+iTreg with CAR technology targeting CD38. CD8+ inducible regulatory T cells (CD8+CD103+CD39+ iTreg, hereinafter referred to as CD8+iTreg) are generated in vitro using IL-2 combined with TGF-β and sorted by flow cytometry. These CD8+iTregs are then combined with CAR technology to generate novel CAR-CD8+ iTreg cells targeting CD38.

[0012] Preferably, in step (4), the concentration ratio (μg / mL) of CD3 antibody, CD28 antibody and recombinant TGF-β is CD3 antibody:CD28 antibody:recombinant TGF-β = (1-3):(1-3):(0.002-0.005); the concentration of recombinant IL-2 is 50-100 IU / mL.

[0013] Preferably, in step (4), cells that highly express CD39 and CD103 are screened by flow cytometry.

[0014] Thirdly, the present invention provides the application of the above-mentioned CD38-targeting CAR-CD8+ iTreg cells in the preparation of drugs for treating systemic lupus erythematosus.

[0015] This application is the first to apply CD38-targeting CAR-CD8+ iTreg cells to the treatment of systemic lupus erythematosus (SLE), demonstrating that CD38-targeting CAR-CD8+ iTreg cells can effectively alleviate SLE and lupus nephritis, exhibiting good therapeutic efficacy. The mechanism may be that CD38-targeting CAR-CD8+ iTreg cells exert a protective effect on lupus mice by depleting activated T cells, B cells, and NK cells that highly express CD38.

[0016] Fourthly, the present invention provides a pharmaceutical composition comprising the above-described CD38-targeting CAR-CD8+ iTreg cells.

[0017] Preferably, the dosage form of the drug includes granules or injections.

[0018] Preferably, the injection is administered via intravenous injection.

[0019] Preferably, the intravenous injection volume of cells is 1×10⁻⁶. 6 200 μL per cell.

[0020] Infusion of CD38-targeted CAR-CD8+ iTreg cells reduced serum autoantibody and urinary protein levels in lupus mice (MRL / Lpr mice), and alleviated renal immune complex deposition and pathological damage, indicating that CD38-targeted CAR-CD8+ iTregs have a protective effect on lupus mice. Furthermore, the expression levels of various cytokines in the treated mice were significantly downregulated, demonstrating that CAR-CD8 iTreg cell therapy has the advantage of reducing the side effects of cytokine release syndrome. In terms of mechanism, CD38-CAR CD8 iTregs were co-cultured with activated T cells, B cells, and NK cells from lupus mice. Proliferation assays, cytokine secretion assays, and cell killing and exhaustion assays preliminarily verified that CD38 CAR-CD8 iTregs have immunomodulatory functions on T cells, B cells, and NK cells in lupus mice.

[0021] Preferably, the drug further includes a pharmaceutically usable carrier.

[0022] The beneficial effects of this invention are as follows: This invention utilizes CD8+ iTreg cells combined with the construction of a CD38-targeting chimeric antigen receptor (CAR) to generate novel CD38-targeting CAR-CD8+ iTreg cells. For the first time, these novel CD38-targeting CAR-CD8+ iTreg cells were applied to the treatment of systemic lupus erythematosus (SLE), demonstrating that they can effectively alleviate SLE and lupus nephritis, exhibiting good therapeutic efficacy. The mechanism may be that the CD38-targeting CAR-CD8+ iTreg cells exert a protective effect on lupus mice by depleting activated T cells, B cells, and NK cells that highly express CD38. This invention provides a new strategy for the precision treatment of SLE and lupus nephritis. Attached Figure Description

[0023] Figure 1 Flow cytometry results for sorting CD8+CD62L+ naive T cells from the spleen of mice.

[0024] Figure 2 Flow cytometry image of CAR-CD8+ iTreg cells targeting CD38.

[0025] Figure 3 The figure shows the results of CAR-CD8+ iTreg cell therapy targeting CD38 in reducing kidney pathological damage and immune complex deposition in lupus mice.

[0026] Figure 4 Figure showing the effect of CD38-targeted CAR-CD8+ iTreg cell therapy on cytokine expression levels in lupus mice.

[0027] Figure 5 The figure shows the effect of CAR-CD8+ iTreg cells targeting CD38 on B cell proliferation. Detailed Implementation

[0028] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0029] SEQ ID NO.1: DIQMTQSPSSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPLTFGQGTKVEIKGGGGSGGGGSGGGG SQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYYMHWVRQAPGQGLEWMGGINPSNGGTNFQKFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGAGTTVTVSS; SEQ ID NO.2: FWVLVVVGGVLACYSLLVTVAFIIFWV; SEQ ID NO.3: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL.

[0030] Example 1: Preparation of CD8+CD62L+ naive T cells CD8+CD62L+ immature T cells were obtained from the spleen or lymph nodes of mice using magnetic beads. The specific sorting steps are as follows: Mouse spleens were harvested and ground using a 1 mL syringe with a blunt tip through a 40 μm filter. The mixture was centrifuged at 1500 rpm at 4°C for 5 min. The supernatant was discarded, and the cells were resuspended in erythrocyte lysis buffer for 1 min to lyse the erythrocytes. The reaction was terminated by adding 2% FBS medium, and the mixture was centrifuged at 1500 rpm at 4°C for 5 min. After centrifugation, the supernatant was collected, and an appropriate amount of anti-mouse magnetic bead antibodies (including anti-CD45R / B220, CD11b, CD11c, CD4, and CD49b antibodies) was added according to the cell count. The mixture was incubated at 4°C for 20 min. 5 mL of 2% FBS medium was added, and the mixture was centrifuged at 1500 rpm at 4°C for 5 min, followed by one wash. The supernatant was discarded, and the cells were resuspended in a small amount of 2% FBS medium. An appropriate amount of anti-Biotin magnetic bead antibody was added, and the mixture was incubated at 4°C for 15 min. 5 mL of 2% FBS medium was added, and the mixture was centrifuged at 1500 rpm at 4°C for 5 min, followed by one wash. After discarding the supernatant, resuspend the cells in 2 mL of PBS. Take a negative tube (excluding magnetic beads and antibodies) to obtain CD8+ T cells, and centrifuge at 1500 rpm, 4°C for 5 min. After discarding the supernatant, resuspend the cells in a small amount of 2% FBS medium, add an appropriate amount of anti-mouse CD62L microsphere antibody, and incubate at 4°C for 15 min. Add 5 mL of 2% FBS medium, centrifuge at 1500 rpm, 4°C for 5 min, and wash once. After discarding the supernatant, resuspend the cells in 1 mL of PBS. Add 10 μL / CD62L-bead and incubate at 4℃ for 15 min; wash once with 10 mL of 2% FBS 1640 medium, centrifuge, resuspend cells in MACS Buffer to 500 μL, add to MS column (pre-washed with 1-2 mL MACS Buffer), then rinse the centrifuge tube with 1-2 mL MACS Buffer, and add to MS column again. At this point, the positive tube contains CD8+CD62L+ naive T cells.

[0031] Flow cytometry was used to detect the proportion of CD8+CD62L+ immature T cells. Figure 1 The purity reaches over 95%, thus obtaining the CD8+CD62L+ naive T cells described in this invention.

[0032] Example 2: Preparation of CAR-CD8+ iTreg cells targeting CD38 Constructing a specific chimeric antigen receptor (CAR) technology targeting CD38: (1) Design a chimeric antigen receptor construct targeting CD38: containing an extracellular CD38 antigen-binding domain, a transmembrane domain, and an intracellular co-stimulatory domain composed of CD28 and 4-1BB; transiently transfect HEK-293T cells to obtain recombinant lentivirus; (2) CAR-CD8+ T cells were obtained by infecting CD8+62L+ naive T cells with recombinant lentivirus through lentiviral transfection technology; (3) CAR-CD8+ T cells were cultured in vitro in the presence of CD3 antibody (1 μg / mL), CD28 antibody (1 μg / mL), recombinant IL-2 (50 IU / mL), and recombinant TGF-β (2 ng / mL). After induction at 37℃ for 72 h, the cells were sorted by flow cytometry. Figure 2 Cells that highly express CD39 and CD103 were selected to ultimately obtain CAR-CD8+ iTreg cells that target CD38.

[0033] Example 3: CAR-CD8+ iTreg cells targeting CD38 for the treatment of MRL / Lpr lupus mice CD8+ T control cells (2×10) 6 / mouse), CAR-CD8+ iTreg cells targeting CD38 (2×10 6 PBS (disease model group) and PBS (mouse) were intravenously injected into lupus mice (MRL / Lpr mice) at approximately 10 weeks of age.

[0034] Following cell injection, urinary protein levels in mice were measured weekly using proteinuria test strips. Peripheral blood was collected from lupus mice every two weeks via tail vein sampling, and serum was obtained after high-speed centrifugation. Serum anti-dsDNA antibodies in lupus mouse serum were detected using a mouse anti-double-stranded DNA (dsDNA) antibody ELISA kit. Twenty weeks after cell injection, lupus mice were sacrificed, and kidneys from each group were collected. These kidneys were fixed in 10% formalin solution for 72 h and then transferred to 75% ethanol. Routine paraffin embedding was performed, sections were cut to 2 μm, dewaxed using a gradient method, and stained with HE, PAS, PASM, and Masson staining. Results are as follows: Figure 3 As shown.

[0035] Compared with the control group, infusion of CD38-targeting CAR-CD8+ iTreg cells reduced serum autoantibody and urinary protein levels (reflected in IgG antibody and complement C3 levels) in lupus mice (MRL / Lpr mice), and alleviated renal immune complex deposition and pathological damage, indicating that CD38-targeting CAR-CD8+ iTreg cells have a protective effect on lupus mice.

[0036] Example 4: CAR-CD8+ iTreg cell therapy targeting CD38 led to a significant downregulation of cytokine levels in lupus mice. As described in Example 3, peripheral blood was collected from the tail vein of lupus mice every two weeks, and serum was obtained after high-speed centrifugation. Various cytokines (such as IL-1, IL-6, TNF-α, IFN-γ, etc.) in the serum of lupus mice were detected using a mouse ELISA kit. The results are as follows: Figure 4 As shown.

[0037] The results showed that after treating lupus mice with CD38-targeted CAR-CD8+ iTreg cells, the expression levels of various cytokines (such as IL-1, IL-6, TNF-α, IFN-γ, etc.) in the mice were significantly downregulated. CAR-CD8+ iTreg cells targeting CD38 have the advantage of reducing the side effects of cytokine release syndrome.

[0038] Example 5: CAR-CD8+ iTreg cells targeting CD38 inhibit B cells in vitro CD8+ T control cells (0.3×10⁻⁶) 6 / well), CAR-CD8+ iTreg cells targeting CD38 (0.3×10 6 / well) under in vitro inflammatory stimulation with LPS (1.0 μg / mL) + IL-6 (10 ng / mL) and CFSE-labeled (1 μmol / L) B cells (0.3 × 10⁻⁶) 6 / well) co-culture. After 72 hours of culture, cells were collected, and the fluorescence intensity of CFSE was detected by flow cytometry to evaluate the proliferation of B cells labeled with CFSE. The results are as follows: Figure 5 As shown.

[0039] The results showed that the novel CAR-CD8 iTreg cells targeting CD38 inhibited B cells in vitro.

[0040] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A CAR-CD8+ iTreg cell targeting CD38, characterized in that, The CAR-CD8+ iTreg cells express a chimeric antigen receptor that specifically binds to the CD38 antigen, as well as CD39 and CD103.

2. The CAR-CD8+ iTreg cells targeting CD38 according to claim 1, characterized in that, The chimeric antigen receptor includes a CD38 antigen-binding domain, a transmembrane domain, and an intracellular co-stimulatory domain.

3. The CAR-CD8+ iTreg cells targeting CD38 according to claim 2, characterized in that, The amino acid sequence of the CD38 antigen-binding domain is shown in SEQ ID NO.1; the amino acid sequence of the transmembrane domain is shown in SEQ ID NO.2; and the amino acid sequence of the intracellular co-stimulatory domain is shown in SEQ ID NO.

3.

4. The method for preparing CD38-targeting CAR-CD8+ iTreg cells according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Preparation of CD8+CD62L+ naive T cells; (2) HEK-293T cells were transfected with a plasmid containing a specific chimeric antigen receptor targeting CD38 to obtain a recombinant lentivirus; (3) The CD8+CD62L+ immature T cells from step (1) were infected with the recombinant lentivirus from step (2) to obtain CAR CD38 CD8+62L+ T cells; (4) CAR CD38 CD8+62L+ T cells were cultured in CD3 antibody, CD28 antibody, recombinant IL-2 and recombinant TGF-β, and CAR-CD8+ iTreg cells targeting CD38 were obtained by flow cytometry sorting.

5. The method for preparing CD38-targeting CAR-CD8+ iTreg cells according to claim 4, characterized in that, In step (4), the concentration ratio (μg / mL) of CD3 antibody, CD28 antibody and recombinant TGF-β is CD3 antibody:CD28 antibody:recombinant TGF-β = (1-3):(1-3):(0.002-0.005); the concentration of recombinant IL-2 is 50-100 IU / mL.

6. The use of the CD38-targeting CAR-CD8+ iTreg cells according to any one of claims 1-3 in the preparation of a medicament for treating systemic lupus erythematosus.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises CAR-CD8+ iTreg cells targeting CD38 as described in any one of claims 1-3.

8. The pharmaceutical composition according to claim 7, characterized in that, The dosage form of the drug includes granules or injections.

9. The pharmaceutical composition according to claim 8, characterized in that, The injection is administered via intravenous injection.

10. The pharmaceutical composition according to claim 9, characterized in that, The intravenous cell injection volume was 1×10⁻⁶. 6 200 μL per cell.

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