Engineered immune effector cells with enhanced infiltration and immunomodulatory functions and uses thereof

Abstract

The application relates to the technical field of tumor immunotherapy, and discloses an engineered immune effector cell with enhanced infiltration and immune regulation functions and an application, wherein the immune effector cell simultaneously expresses a chimeric antigen receptor CAR, a transcription factor BCL6 and a chemokine receptor CXCR2; the preparation steps of the immune effector cell are as follows: S1, separating and activating T cells in human peripheral blood; S2, co-expressing a BCL6 gene, a CXCR2 gene and a CAR-Claudin18.2 in the T cells in a gene transfection mode to obtain BCL6+CXCR2+Claudin18.2 CAR-T cells. The immune effector cell can act on tumors, especially solid tumors such as pancreatic cancer, significantly enhances the infiltration capacity of CAR-T cells to tumor tissues, improves the tumor microenvironment, strengthens the anti-tumor function of T cells, and effectively improves the persistence and stability of the treatment effect.

Description

Technical Field

[0001] This application relates to the technical field of tumor immunotherapy, and in particular to an engineered immune effector cell with enhanced infiltration and immunomodulatory functions and its application. Background Technology

[0002] In recent years, tumor immunotherapy has become one of the important strategies for cancer treatment. CAR-T cells, which modify a patient's own T cells through in vitro genetic engineering technology to express chimeric antigen receptors that can specifically recognize tumor antigens, thereby precisely targeting and eliminating tumor cells, have shown significant efficacy in hematologic malignancies.

[0003] In existing technologies, the following technical approaches have been developed to improve the therapeutic effect of CAR-T cells: 1. Single-target CAR-T technology, such as CLDN18.2 CAR-T cells; Advantages: High targeting specificity, capable of accurately identifying CLDN18.2 positive tumor cells; 2. Exhaustion-resistant gene editing technologies, such as PD-1 knockout CAR-T cells; Advantages: By knocking out the PD-1 gene using CRISPR, T cell activity can be enhanced in the short term; 3. Microenvironment regulation technologies, such as IL-12 secretory CAR-T; Advantages: It reshapes the tumor microenvironment by locally secreting IL-12 and activates M1 macrophages to synergistically fight tumors.

[0004] However, despite its significant efficacy in some tumor types, this technology still faces numerous challenges in the treatment of solid tumors such as pancreatic cancer. For example, single-target CAR-T technology is prone to antigen-negative escape due to tumor antigen heterogeneity, with approximately 30-40% of patients experiencing tumor recurrence after treatment due to antigen escape; anti-exhaustion gene editing technology carries the risk of genome editing and cannot reverse terminal differentiation phenotypes; and microenvironment regulation technology has extremely high safety risks, such as the systemic IL-12 release caused by IL-12-secreting CAR-T, which may lead to severe toxicity (the incidence of grade 3 adverse events is >40% in clinical trials).

[0005] In summary, current technologies have not yet been able to address the two major bottlenecks in the treatment of solid tumors: on the one hand, the complex structure and high immunosuppressive nature of the solid tumor microenvironment severely limit the infiltration and function of CAR-T cells; on the other hand, existing CAR-T products often lack long-term survival and sustained response capabilities in vivo, are prone to functional exhaustion, and are difficult to maintain effective anti-tumor immune memory.

[0006] Therefore, how to enhance the infiltration ability of CAR-T cells in tumor tissues, especially solid tumors, and improve their sustained anti-tumor activity is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] This application addresses the shortcomings of existing CAR-T cell therapies in the treatment of solid tumors, particularly the poor infiltration and limited function of CAR-T cells in tumors with complex immunosuppressive microenvironments such as pancreatic cancer. This application provides an engineered immune effector cell with enhanced infiltration and immunomodulatory functions and its application. By synergistically promoting the overexpression of BCL6 and the high expression of CXCR2, it significantly enhances the infiltration ability of CAR-T cells into tumor tissues, improves the tumor microenvironment, and strengthens the anti-tumor function of T cells, thereby effectively overcoming the limitations of existing technologies and improving the sustainability and stability of treatment effects.

[0008] In a first aspect, this application provides an engineered immune effector cell with enhanced infiltration and immunomodulatory functions, employing the following technical solution: An engineered immune effector cell with enhanced infiltration and immunomodulatory functions, wherein the immune effector cell simultaneously expresses chimeric antigen receptor CAR, transcription factor BCL6, and chemokine receptor CXCR2.

[0009] Optionally, the immune effector cells are T cells.

[0010] Optionally, the CAR-targeted antigen is selected from Claudin18.2, mesothelin, Epha2, or EGFRvIII.

[0011] Secondly, this application provides a method for preparing engineered immune effector cells with enhanced infiltration and immunomodulatory functions, employing the following technical solution: A method for preparing engineered immune effector cells with enhanced infiltration and immunomodulatory functions includes the following steps: Isolation and activation of S1 and T cells T cells were isolated from the mononuclear cell layer in human peripheral blood, and then activated T cells were obtained by stimulating the T cells with anti-CD3 / CD28. Preparation of S2, BCL6+CXCR2+Claudin18.2 CAR-T cells The CAR-Claudin18.2 sequence, BCL6 gene, and CXCR2 gene were cloned into the same or different expression vectors, then co-transfected into activated T cells, and cell viability and function were detected to obtain BCL6+CXCR2+Claudin18.2 CAR-T cells.

[0012] Optionally, the expression vector is a lentiviral vector.

[0013] Thirdly, this application provides a pharmaceutical composition, which adopts the following technical solution: A pharmaceutical composition comprising the aforementioned immune effector cells.

[0014] Optional, further including: pharmaceutically acceptable excipients.

[0015] Fourthly, this application provides the use of immune effector cells or a pharmaceutical composition in the preparation of a medicament for treating tumors.

[0016] Optionally, the tumor is a solid tumor.

[0017] Optionally, the tumor includes pancreatic cancer.

[0018] In summary, this application includes at least one of the following beneficial technical effects: 1. Synergistic enhancement of tumor invasion: Overexpression of CXCR2 enables CAR-T cells to respond efficiently to ligands such as CXCL5 highly expressed in the tumor microenvironment and actively chemotactically infiltrate to the core region of the tumor. 2. Remodeling the immune microenvironment and enhancing function: Overexpression of BCL6 induces some T cells (especially CD4 cells) to regenerate immune microenvironment and enhance function. + T cells differentiate into Tfh-like cells, which secrete CXCL13 and can recruit more endogenous CD8+ cells. + T cells and B cells secrete IL-21, which can directly enhance CD8. + CAR-T cells' ability to secrete IFN-γ (enhancing killing power) and promote B cells to produce antibodies can transform "cold" tumors into "hot" tumors. 3. Maintaining long-lasting anti-tumor immunity: BCL6 can effectively inhibit CD8. + Terminal differentiation of T cells maintains their stem cell-like memory or central memory phenotype, significantly prolongs the survival time of CAR-T cells in vivo, and provides long-term immune surveillance function to prevent tumor recurrence. 4. Significantly improved efficacy: In animal models of solid tumors such as pancreatic cancer, the Claudin18.2 CAR-T cells co-expressing BCL6 and CXCR2 in this application exhibited tumor clearance capacity and survival prolongation effects far exceeding those of traditional Claudin18.2 CAR-T cells.

[0019] In summary, this application, by co-expressing the BCL6 and CXCR2 genes in CAR-T cells, enables CAR-T cells to efficiently enter and persist in tumors with strong tumor clearance capabilities. This effectively solves the two technical problems of "insufficient tumor infiltration" and "exhaustion of immune effector cell function" in solid tumor immunotherapy, and has great clinical application and economic value. Attached Figure Description

[0020] Figure 1 This is an illustrative diagram of the cell construction in Example 1; wherein, Figure 1 A is a flowchart illustrating the construction steps of BCL6+CXCR2+Claudin18.2 CAR-T cells. Figure 1 Figure B shows the results of flow cytometry analysis of BCL6 (left) and CXCR2 (right) expression levels in T cells. Figure 1 Figure C shows the results of flow cytometry analysis of CAR (Claudin18.2) expression levels on the surface of T cells; Figure 2 This is an explanatory diagram of the in vitro functional experiment in Example 2; wherein, Figure 2 A is a schematic diagram of the Transwell experiment. Figure 2 B is a statistical graph showing the number of cells in the lower chamber of each group in the Transwell experiment. Figure 2 C is a statistical graph showing the secretion results of cytokine IFN-γ in the supernatant of each group; Figure 3 This is a diagram showing the results of the specific killing experiment of CAR-T cells against pancreatic cancer cells in Example 3; in which, Figure 3 A is a statistical graph showing the results of the cytotoxicity experiment. Figure 3 B is a statistical graph showing the results of flow cytometry detection of cell killing and apoptosis levels; Figure 4 This is a graph showing the results of the in vivo antitumor effect evaluation in mice in Example 4; where, Figure 4 A shows the tumor volume statistics of each group of mice from day 14 to 28. Figure 4 B is a statistical chart showing the survival time of mice in each group from 0 to 80 days. Figure 4 C is a statistical chart showing the number of CAR-T cells in each group. Figure 4 D is a statistical graph showing the IFN-γ secretion results of each group. Detailed Implementation

[0021] The following is in conjunction with the appendix Figure 1 - Appendix Figure 4This application provides a further detailed description; the specific embodiments described herein are intended to fully demonstrate the technical solution and effects of this application, but the scope of protection of this application is not limited thereto. Any equivalent changes or modifications made by those skilled in the art based on the substance of this application should be covered within the scope of protection of this application.

[0022] Unless otherwise specified, the materials and equipment used in the various embodiments of this application are all commercially available products in the art.

[0023] Example 1: Construction of Claudin18.2 CAR-T cells co-expressing BCL6 and CXCR2 The flowchart of the construction steps of BCL6+CXCR2+Claudin18.2 CAR-T cells is as follows: Figure 1 As shown in Figure A.

[0024] 1. Isolation and activation of T cells (1) The mononuclear cell layer was separated from the patient’s peripheral blood by density gradient centrifugation using Ficoll-Paque (catalog number: 17544602-1, GE Healthcare) separation medium; (2) T cells were isolated from the mononuclear cell layer using a T cell sorting kit (catalog number: 17851, Stemcell), and their proliferation and activation were promoted by stimulation with anti-CD3 / CD28 (catalog number: 10970, Stemcell). The cells were then cultured in RPMI-1640 medium containing IL-2 (catalog number: 200-02-100UG, PeproTech) at a concentration of 50 U / mL for 72 hours to obtain activated T cells.

[0025] 2. Transfection and amplification of BCL6 and CXCR2 genes (1) Using a lentiviral vector system, the BCL6 gene and CXCR2 gene were cloned into lentiviral vectors to construct overexpression lentiviruses of BCL6 and CXCR2; The vector ID of the above lentiviral vector is VB010000-9492agg, produced by Yunzhou Biotechnology (Guangzhou) Co., Ltd. The RefSeq ID of the BCL6 gene mentioned above is: NM_001706.5; The RefSeq ID of the CXCR2 gene mentioned above is: NM_001557.4; (2) The constructed BCL6 and CXCR2 overexpression lentiviruses were co-cultured with activated T cells, and the transfection efficiency was enhanced to over 90% using lentiviral transduction enhancer (LVTE). (3) After transfection, the transfected T cells were cultured in RPMI-1640 medium containing IL-2 at a concentration of 50 U / mL, and the proliferation and survival of T cells were observed. IL-2 was added to maintain cell viability, and the culture time was 7-14 days, during which the medium was changed regularly. (4) 72 hours after transduction, T cell surface markers were detected by flow cytometry to confirm the expression levels of BCL6 (nuclear staining, catalog number: 358503, Biolegend) and CXCR2 (catalog number: 320704, Biolegend). The results are as follows: Figure 1 As shown in Figure B, T cells co-expressing BCL6 and CXCR2 were obtained.

[0026] In other embodiments, the BCL6 and CXCR2 genes can be co-transduced in two different vectors. Furthermore, in addition to using viral vectors, gene editing technologies such as CRISPR / Cas9 and TALEN can be used to precisely integrate BCL6 and / or CXCR2 into specific safe sites (such as AAVS1) in the T cell genome.

[0027] 3. Preparation of BCL6+CXCR2+Claudin18.2 CAR-T cells (1) CAR-Claudin18.2 was transfected into T cells co-expressing BCL6 and CXCR2 using the same lentiviral vector system as described above; The aforementioned CAR-Claudin18.2 sequence can be found in patent WO2018006882A1; (2) After transfection, CAR-T cells were further expanded to ensure cell viability and function; 72 hours after transduction, T cell surface markers were detected by flow cytometry to confirm the expression level of CAR (detected by specific antibodies or target proteins). The results are as follows: Figure 1 As shown in Figure C; the results indicate that BCL6+CXCR2+Claudin18.2 CAR-T cells were successfully constructed.

[0028] In other embodiments, the lentiviral vector containing the BCL6 gene, CXCR2 gene, and CAR-Claudin18.2 can be co-transfected into activated T cells. Cells that express all three genes positively are selected as BCL6+CXCR2+Claudin18.2 CAR-T cells.

[0029] Example 2: In vitro functional experiment The Transwell chamber was used, with pancreatic cancer organoids (secreting CXCL5) added to the lower chamber and equal amounts of different groups of CAR-T cells added to the upper chamber. The groups were: a) BCL6 monoexpression group; b) CXCR2 monoexpression group; c) dual expression group; d) dual expression group + anti-CXCL5 (catalog number: MAB254, R&D). Figure 2 As shown in A; The above-mentioned methods for constructing pancreatic cancer organoids are referenced in the article Antigen / HLA-agnostic strategies for Characterizing Tumor-responsive T cell receptors in PDAC patients via single-cell sequencing and autologous organoid application (PMID: 38395378). The above-mentioned methods for preparing cells expressing BCL6 and CXCR2 are as follows: the BCL6 gene or CXCR2 gene is cloned into a lentiviral vector and then transfected into activated T cells. The concentration of anti-CXCL5 added was 10 μg / mL; After culturing for 24 hours, the cells in the lower chamber were counted, and the results were as follows: Figure 2 As shown in B; the results showed that both the single expression group of BCL6 and the single expression group of CXCR2 had migration ability, but the dual expression group showed the strongest migration ability, and the migration ability depended on CXCL5.

[0030] Furthermore, the cytokine IFN-γ in the supernatant was detected by ELISA (catalog number: 97024ES96, Yisheng Biotechnology (Shanghai) Co., Ltd.), and the results are as follows. Figure 2 As shown in Figure C. The results indicate that the BCL6 monoexpression group had limited IFN-γ secretion, the CXCR2 monoexpression group activated IFN-γ secretion due to effective migration, and the dual expression group had the highest IFN-γ secretion level.

[0031] In summary, CAR-T cells simultaneously overexpress both the BCL6 and CXCR2 genes. CXCR2 focuses on enhancing infiltration, providing the spatial basis for BCL6-mediated immunomodulation and functional persistence (i.e., allowing cells to enter the tumor). BCL6 focuses on enhancing function and persistence, providing the temporal extension guarantee for CXCR2-mediated infiltration (i.e., allowing cells to function persistently within the tumor). The two genes work synergistically; overexpression of BCL6 induces the growth of some T cells (especially CD4+). + T cells differentiate into Tfh-like cells, which secrete CXCL13 and can recruit more endogenous CD8+ cells.+ T cells and B cells secrete IL-21, which can directly enhance CD8. + CAR-T cells' ability to secrete IFN-γ (enhancing killing power) and promote B cells to produce antibodies can transform "cold" tumors into "hot" tumors. This, in turn, enables BCL6+CXCR2+Claudin18.2 CAR-T cells to have navigational infiltration capabilities and cellular functionality. BCL6+CXCR2+Claudin18.2 CAR-T cells can efficiently respond to ligands such as CXCL5, which are highly expressed in the tumor microenvironment, and actively chemotactically infiltrate to the core region of the tumor.

[0032] Example 3: Specific killing experiment of CAR-T cells against pancreatic cancer cells The killing efficiency of BCL6+CXCR2+Claudin18.2 CAR-T cells against pancreatic cancer cells was evaluated using a cytotoxicity assay (catalog number: BC0680, Solarbio). T cells were used as the control. Results are as follows: Figure 3 As shown in A; The pancreatic cancer cells mentioned above were PANC-1 and CRL-1469, purchased from ATCC.

[0033] The results showed that the cell lysis rate of CAR-T cells was significantly higher than that of ordinary T cells at different effector-to-target ratios (E:T ratio = 2, 4, 8, 16); and with the increase of effector-to-target ratio, the efficient and specific killing ability of CAR-T cells against pancreatic cancer cells also gradually improved.

[0034] Furthermore, flow cytometry was used to detect the apoptosis level of pancreatic cancer cells (7-AAD, catalog number: 640922, Biolegend) after treatment with CAR-T cells or T cells. The results are as follows: Figure 3 As shown in Figure B, the results indicate that CAR-T cells can significantly induce apoptosis or death in pancreatic cancer cells.

[0035] Example 4: Evaluation of in vivo antitumor effect A mouse model of pancreatic cancer (subcutaneous xenograft) was established, and the tumor grew to approximately 60 mm. 3 At that time, the mice were randomly divided into 3 groups of 5 mice each. The groups were as follows: c. BCL6+CXCR2+Claudin18.2 CAR-T cell therapy group: 1×10⁻⁶ cells per injection 6 BCL6+CXCR2+Claudin18.2 CAR-T cells were used in PBS. b. Traditional Claudin18.2 CAR-T cell therapy group: 1×10⁻⁶ injections per dose. 6One set of traditional Claudin18.2 CAR-T cells were used in PBS. a. Control group: Infused with an equal volume of PBS buffer; The above-mentioned pancreatic cancer mouse model and the construction method of traditional Claudin18.2 CAR-T cells are described in the article "Ectopic CXCR2 expression cells improve the anti-tumor efficiency of CAR-T cells and remodel the immune microenvironment of pancreatic ductaladenocarcinoma" (PMID: 38430267).

[0036] The mice were injected via tail vein every 7 days for a total of two injections; tumor volume was measured regularly and mouse survival was recorded. Results are as follows: Figure 4 A and Figure 4 As shown in B.

[0037] The results showed that tumors in mice treated with the conventional Claudin18.2 CAR-T cell therapy (b) were somewhat inhibited, but tumor growth in mice treated with the BCL6+CXCR2+Claudin18.2 CAR-T cell therapy (c) was significantly inhibited or delayed, and the survival rate of these mice was still over 50% at 80 days, indicating a significant extension of survival. This may be because BCL6 effectively inhibits terminal differentiation of T cells, maintains their stem cell-like memory or central memory phenotype, significantly prolongs the survival time of CAR-T cells in vivo, provides long-term immune surveillance, exhibits sustained anti-tumor activity, and prevents tumor recurrence.

[0038] Tumor tissue was collected 28 days after tumor formation for flow cytometry analysis, and the results were as follows: Figure 4 C and Figure 4 As shown in D, the BCL6+CXCR2+Claudin18.2 CAR-T cell therapy group (c) exhibited the highest cell infiltration rate, with a cell count reaching 1.26 × 10⁻⁶. 6 / cm 3 Furthermore, its IFN-γ secretion level can reach up to 850 pg / mL. This indicates that overexpression of BCL6 and CXCR2 can synergistically enhance the anti-tumor effect of Claudin18.2 CAR-T cells.

[0039] In summary, this application obtained BCL6+CXCR2+Claudin18.2 CAR-T cells by co-expressing BCL6, CXCR2, and CAR-Claudin18.2 in T cells. The highly efficient chemotaxis mediated by CXCR2 and the functional enhancement mediated by BCL6 in these cells, with the two genes working synergistically, solve the technical problems of "insufficient tumor invasion" and "exhaustion of immune effector cell function" in solid tumor treatment, enhancing the tumor invasion and killing capabilities of Claudin18.2 CAR-T cells. Furthermore, in animal models of solid tumors such as pancreatic cancer, it exhibits tumor clearance capacity and prolonged survival effects exceeding those of traditional Claudin18.2 CAR-T cells.

[0040] Preferably, this dual-gene modification strategy can be applied to CAR-T cells or TCR-T cells that target other solid tumor antigens (such as mesothelin MSLN, Epha2, EGFRvIII, etc.), and is not limited to Claudin18.2 CAR.

[0041] The specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Those skilled in the art can make modifications to these embodiments without contributing any inventive step after reading this specification, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. An engineered immune effector cell with enhanced infiltration and immunomodulatory functions, characterized in that, The immune effector cells simultaneously express chimeric antigen receptor CAR, transcription factor BCL6, and chemokine receptor CXCR2.

2. The immune effector cells as described in claim 1, characterized in that, The immune effector cells are T cells.

3. The immune effector cells as described in claim 1, characterized in that, The CAR-targeted antigen is selected from Claudin18.2, mesothelin, Epha2, or EGFRvIII.

4. A method for preparing engineered immune effector cells with enhanced infiltration and immunomodulatory functions, characterized in that, Includes the following steps: Isolation and activation of S1 and T cells T cells were isolated from the mononuclear cell layer in human peripheral blood, and then activated T cells were obtained by stimulating the T cells with anti-CD3 / CD28. Preparation of S2, BCL6+CXCR2+Claudin18.2 CAR-T cells The CAR-Claudin18.2 sequence, BCL6 gene, and CXCR2 gene were cloned into the same or different expression vectors, then co-transfected into activated T cells, and cell viability and function were detected to obtain BCL6+CXCR2+Claudin18.2 CAR-T cells.

5. The preparation method according to claim 4, characterized in that, The expression vector is a lentiviral vector.

6. A pharmaceutical composition, characterized in that, Includes the immune effector cells as described in any one of claims 1-3.

7. The pharmaceutical composition according to claim 6, characterized in that, Further includes: Pharmaceutically acceptable excipients.

8. The use of the immune effector cells as described in any one of claims 1-3 or the pharmaceutical composition as described in any one of claims 6-7 in the preparation of a medicament for treating tumors.

9. The application as described in claim 8, characterized in that, The tumor is a solid tumor.

10. The application as described in claim 9, characterized in that, The tumors include pancreatic cancer.

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