Novel cytokine delivery platform and application thereof

Abstract

The invention discloses a novel cytokine delivery platform and application thereof, and belongs to the technical field of biological medicine, the cytokine delivery platform is CD19 / IL-12EVs, that is, IL-12 and CD19 are simultaneously expressed on the surface of extracellular vesicles. The cytokine delivery platform can improve the function of CAR-T cells, improve the binding efficiency with the CAR-T cells, and improve the in-vivo proliferation and antitumor activity of the CAR-T cells. According to the invention, EVs are modified by using CD19, so that IL-12 is specifically delivered to anti-CD19 CAR-T cells, EVs (named as CD19 / IL-12 EVs) with CD19 and IL-12 displayed on envelopes are separated from parental cells expressing CD19 and IL-12 at the same time, and the influence of the EVs on the binding capacity and function of the anti-CD19 CAR-T cells is detected. In-vitro and in-vivo data show that the CD19 / IL-12EVs can effectively enhance the proliferation and cytotoxic functions of CAR-T cells and cannot cause systemic toxicity. The invention provides a principle evidence, and proves that the EVs modified by the CAR target spot is an effective and safe multi-effect cytokine IL-12 transmission system and is also a biological enhancer of CAR-T cells.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a novel cytokine delivery platform and application thereof. Background Art

[0002] Chimeric antigen receptor (CAR)-T cell therapy has achieved clinical success in the treatment of hematological tumors, especially B-cell malignancies. However, more than half of patients treated with CAR-T cell therapy fail to achieve long-term remission. In addition, the treatment of solid tumors with CAR-T cell therapy also faces major challenges. Several factors limit the clinical efficacy of CAR-T cells, such as insufficient T cell proliferation, weak anti-tumor activity, limited persistence, and immunosuppression by the tumor microenvironment (TME).

[0003] To overcome these obstacles, many preclinical studies and clinical trials have been conducted to enhance the function of CAR-T cells in vivo by combining cytokines, among which IL-12 is a promising candidate for combined immunotherapy. IL-12 is a heterodimeric protein composed of p40 and p35 subunits. It is a proinflammatory cytokine with strong anti-tumor activity. IL-12 is mainly produced by dendritic cells (DC), B lymphocytes and macrophages, and can bind to IL-12Rβ1 and IL-12Rβ2 on the surface of T cells. IL-12 promotes the differentiation of naive T cells into T helper type 1 (Th1) cells, and participates in the activation and proliferation of T cells, IFN-γ production and cytotoxicity. Many studies have shown that IL-12 can also enhance the cytokine release and anti-tumor activity of CAR-T cells, and significantly prolong their survival time in vivo.

[0004] In the rapidly developing field of nanomedicine, extracellular vesicles (EVs) have gradually become one of the safe and efficient nanovesicle delivery systems. EVs are membrane particles that can be secreted by almost all cells. Naturally released EVs can transport nucleic acids or proteins to nearby or distal cells and play a corresponding regulatory role. A large number of studies have shown that EVs can be used as carriers to deliver cytokines in vivo, affecting target cells in a stable and specific manner. In addition, studies have shown that surface molecules displayed by EVs can interact with receptors on T cells (including CAR-T cells), thereby regulating their function. Therefore, EVs modified with IL-12 on the surface may be an effective strategy to specifically deliver IL-12 to CAR-T cells and enhance their in vivo function. Summary of the invention

[0005] The efficacy of chimeric antigen receptor (CAR)-T cell therapy is limited by poor T cell persistence and low anti-tumor activity. In addition, interleukin-12 (IL-12) is an important drug in cancer immunotherapy, but its clinical application is limited by severe toxicity caused by systemic exposure. The present invention provides a novel cytokine delivery platform and its application, which is based on CAR target-modified extracellular vesicles (EVs) that can preferentially bind to CAR-T cells to improve the function of CAR-T cells. HEK-293 cells successfully produced EVs that simultaneously expressed CD19 and IL-12 on the surface. Compared with the same concentration of rhIL-12, in vitro experiments showed that IL-12EVs can significantly enhance the effector function of anti-CD19 CAR-T cells, including increased secretion of IFN-γ and TNF-α, cytolytic activity and T cell expansion. In addition, flow cytometry data showed that EVs co-expressing IL-12 and CD19 (CD19 / IL-12EVs) bound more efficiently to CAR-T cells than EVs expressing IL-12 alone, but not to T cells. In a xenograft mouse model bearing CD19+Raji tumors, intratumoral injection of CD19 / IL-12EVs produced a durable antitumor response and enhanced the in vivo expansion of CAR-T cells, which was superior to CD19 EVs, IL-12EVs, and control EVs, without causing systemic toxicity. RNA sequencing (RNA-seq) analysis of CAR-T cells stimulated with EVs showed that the improvement in CAR-T cell efficacy was driven by IL-12 signaling. These data suggest that CAR target antigen-modified EVs can serve as a targeted cytokine delivery system for CAR-T cells, providing a safe and effective strategy for enhancing CAR-T cell function.

[0006] In order to achieve the above purpose, this application adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a novel cytokine delivery platform, which is CD19 / IL-12EVs, i.e., IL-12 and CD19 are simultaneously expressed on the surface of extracellular vesicles.

[0008] In the above technical scheme, the method for constructing CD19 / IL-12EVs includes the following steps:

[0009] S1, HEK-293T cells were transfected with CD19 overexpression lentivirus and membrane-anchored IL-12 lentivirus. The transfection efficiency was detected 72-96 hours after transfection, and CD19 and IL-12 positive HEK-293T cells were sorted in single cell mode on Moflo XDP Flow Cytometer;

[0010] S2, cells were transferred to serum-free medium and cultured for 24 h, and the medium was obtained and centrifuged at 500 × g for 10 min to remove cell debris;

[0011] S3, the supernatant was filtered with a 0.45-μm sterile filter and centrifuged at 14,000 × g for 1 h at 4 °C to pellet EVs, and the purified EVs were resuspended in PBS and stored at −80 °C until use.

[0012] In a second aspect, the present invention provides the application of the above-mentioned cytokine delivery platform in improving the function of CAR-T cells.

[0013] In the above technical scheme, CD19 / IL-12EVs can improve the binding efficiency with CAR-T cells and enhance the proliferation and anti-tumor activity of CAR-T cells in vivo.

[0014] In a third aspect, the present invention provides the use of the above-mentioned cytokine delivery platform in the preparation of a drug for treating acute B lymphocytic leukemia.

[0015] In the above technical scheme, CD19 / IL-12EVs can be used to specifically promote CAR-T cell expansion and functional persistence in vitro and in vivo during CAR-T treatment of acute B-lymphocytic leukemia.

[0016] In a fourth aspect, the present invention provides the use of the above-mentioned cytokine delivery platform in the preparation of a drug for treating B-cell lymphoma.

[0017] In a fifth aspect, the present invention provides the use of the above-mentioned cytokine delivery platform in the preparation of drugs for treating solid tumors.

[0018] In the above technical scheme, CD19 / IL-12EVs utilize different target modifications to improve the function of CAR-T cells targeting solid tumors.

[0019] The beneficial effects of the present invention are:

[0020] The present invention aims to modify EVs with CD19 (the most commonly used target in CAR-T cell therapy) to specifically deliver IL-12 to anti-CD19CAR-T cells, and to separate EVs (named CD19 / IL-12EVs) showing CD19 and IL-12 on the envelope from parental cells expressing both CD19 and IL-12, and to detect their binding ability and effects on anti-CD19 CAR-T cells. In vitro and in vivo data show that CD19 / IL-12EVs can effectively enhance the proliferation and cytotoxic function of CAR-T cells without causing systemic toxicity. The present invention provides proof of principle that EVs modified with CAR targets are an effective and safe multi-functional cytokine IL-12 delivery system and a bioenhancer for CAR-T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 EVs with functional IL-12 displayed on their surface were designed and manufactured. (A) IL-12EVs display single-chain IL-12 on their surface through the membrane anchor structure GPI. (B) Imaging analysis flow cytometry was used to detect IL-12 on the surface of IL-12EVs and control EVs. (C) The expression of Annexin A1 and IL-12 in EVs was detected by immunoblotting. (D) The concentration of IL-12 in EVs was determined by ELISA. (E) IL-12EVs were stored at -80℃ and their concentration was determined by ELISA analysis at different time points. (F) Dose-response curve of IFN-γ secretion by T cells after treatment with rhIL-12 or IL-12EVs. The EC50 values ​​obtained from the figure were 0.0549 ng / mL and 0.0094 ng / mL, respectively. ****P<0.0001.

[0022] Figure 2It is IL-12EVs that enhance the function of CAR-T cells. (A) CAR-T cells were treated with control EVs or IL-12EVs for 30 minutes, and then the phosphorylation of STAT4 was analyzed by immunoblotting. (BG) CAR-T cells and Raji cells were treated with PBS, control EVs, rhIL-12, and IL-12EVs at a ratio of 1:1. (B, C) After 24 hours of co-culture, the cytokines secreted by CAR-T cells were detected by ELISA. (D) After 72 hours of co-culture, the proportion of CAR-T cells was analyzed by flow cytometry. (E) The expression of CD107a in CD8+CAR-T cells was detected by flow cytometry. (F) The proportion of Raji cell death was determined by PI after 24 hours and analyzed by flow cytometry. (G) The subpopulations in CAR-T cells were detected by flow cytometry 7 days after treatment. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0023] Figure 3 CD19 / IL-12EVs specifically bind to anti-CD19 CAR-T cells and enhance their anti-tumor activity. (A) Schematic diagram of CD19 / IL-12EVs. (B) Imaging analysis flow cytometry was used to detect CD19 and IL-12 on the surface of IL-12EVs and control EVs. (C) IL-12EVs or CD19 / IL-12EVs were added to CAR-T cells for 45 minutes, and CAR-T cells modified by EVs were detected by imaging analysis flow cytometry. (D) CAR-T cells were incubated with IL-12EVs or CD19 / IL-12EVs for 45 minutes, and imaging analysis flow cytometry showed the efficiency of EVs binding to CAR-T cells and non-CAR-T cells. (E) CAR-T cells were treated with DiO-labeled IL-12EVs or CD19 / IL-12EVs for 4 hours, and the cellular internalization of DiO-labeled EVs by CAR-T cells was analyzed by flow cytometry. (F, G) CAR-T cells were mixed with Raji cells at a 1:1 effector to target ratio and treated with control EVs, CD19EVs, IL-12EVs, or CD19 / IL-12EVs for 24 h. (G) The death ratio of Raji cells was determined using PI and analyzed using flow cytometry. (H) Schematic diagram of the ΔCAR plasmid, which removes CD3ζ from the normal CAR structure. (I) ΔCAR-T cells were treated with control EVs, CD19 EVs, IL-12EVs, or CD19 / IL-12EVs for 8 h, and the level of IFN-γ in CD8+CAR-T cells was measured by flow cytometry. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0024] Figure 4 Intratumoral administration of CD19 / IL-12EVs increased the proliferation and in vivo antitumor activity of CAR-T cells. (A) Experimental scheme of mice (5 mice per group). (B) Monitoring tumor growth by bioluminescence imaging. (C) Tumor growth curve of mice, and the tumor volume was calculated as follows: volume = (length × width × width) / 2. (D) The proportion of CAR-T cells in peripheral blood was analyzed by flow cytometry on day 9 after T cell infusion. (E) Quantification of CAR lentiviral copy number in peripheral blood by droplet digital PCR was used to determine the cell dynamics of CAR-T cells in mice. (F) Liver function of mice on day 14 after T cell infusion was detected by chemical test. (G) The levels of cytokines (IFN-γ and IL-6) in mouse serum were quantified using cytokine detection kit on day 9 after T cell infusion. *P<0.05, **P<0.01, ***P<0.001.

[0025] Figure 5 Preparation and characterization of control EVs and IL-12 EVs. (A) Schematic diagram of the lentiviral plasmid structure overexpressing membrane chimeric IL-12. (B) Flow cytometry detection of IL-12 expression on the surface of HEK293T monoclonal cell line carrying IL-12 on the membrane surface. (C) Brownian motion of EVs under NTA monitoring. (D) Measured particle size distribution. (E) Morphology of EVs under electron microscopy.

[0026] Figure 6 The effect of IL-12EVs on the expression of immune checkpoints in CAR-T cells. (A, B) CAR-T cells were mixed with Raji cells at a 1:1 effector-target ratio and treated with PBS, control EVs, rhIL-12, and IL-12EVs, respectively. After 7 days of treatment, the expression of PD1 and LAG3 in CAR-T cells was analyzed by flow cytometry.

[0027] Figure 7 Preparation and characterization of CD19 EVs and CD19 / IL-12 EVs. (A) Flow cytometry was used to detect the expression of CD19 and IL-12 in the EV-derived cell line HEK293T. (B, C) NTA was used to detect the Brownian motion and size distribution of CD19 EVs and CD19 / IL-12 EVs.

[0028] Figure 8The safety of intratumoral injection of CD19 / IL-12EVs. (A) Body weight of mice during various treatments. (B) Renal function of mice was measured by chemical test on day 14 after ACT. (C) Cytokine levels (TNF-α, IL-2, IL-12, and IL-6) in mouse serum were quantified by CBA kit on day 9 after ACT. (D) Representative images of H&E staining of important organs on day 30 after ACT. Scale bar is 50 μm. DETAILED DESCRIPTION

[0029] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as being limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete, and the concept of the present invention will be fully conveyed to those skilled in the art, and the present invention will only be limited by the claims.

[0030] 1. Experimental Materials

[0031] 1. Preparation of EVs

[0032] HEK293T cells were transferred to serum-free medium and cultured for 24 hours. The medium was then collected and centrifuged at 500×g for 10 minutes to remove cell debris. The supernatant was then filtered through a 0.45 μm sterile filter (ThermoScientific) and further centrifuged at 14,000×g for 1 hour at 4°C (Beckman Coulter, Optima XPN-100) to obtain EVs. EVs were resuspended in PBS and stored at -80°C until use.

[0033] To quantify EVs, they were lysed with RIPA lysis buffer (Beyotime, China) containing protease inhibitors (Roche, Switzerland), and their protein content was determined using a BCA assay kit (Beyotime, China).

[0034] Nanoparticle tracking analysis (NTA): After EVs were diluted with PBS, nanoparticle tracking analysis was performed on a ZetaView multi-parameter particle tracking analyzer to observe the Brownian motion of EVs and determine the particle size.

[0035] 2. Preparation of CD19 EVs

[0036] HEK-293T cells were transfected with CD19 overexpressing lentivirus, and the transfection efficiency was detected 72-96 hours after transfection. CD19-positive HEK-293T cells were sorted in single cell mode using MofloXDP Flow Cytometer (Beckman Coulter). CD19 EVs were obtained by the above EVs preparation method.

[0037] 3. Preparation of IL-12EVs

[0038] The present invention connects the two subunits p35 and p40 of IL-12 through a flexible connection structure, and then adds a membrane anchor structure GPI ( Figure 1 A, Figure 5 A), HEK293T cells were overexpressed with membrane-anchored IL-12 by lentiviral transfection, and a monoclonal cell line (HEK293T-mIL-12) with high IL-12 expression on the cell membrane was obtained by flow cytometry, with an IL-12 expression rate of more than 99% ( Figure 5 B). HEK293T-mIL-12 monoclonal cell line will be used as the source cell line for preparing IL-12EVs, while HEK293T transfected with empty lentiviral plasmid will be used as the source cell line for preparing control EVs (ctrlEVs). By observing the Brownian motion of ctrl EVs and IL-12EVs ( Figure 5 C) and compare their particle size ( Figure 5 D) found that the movement process and size of EVs (distributed between 50 and 500 nm) did not change significantly after the membrane surface was modified by IL-12. At the same time, the two types of EVs were observed using electron microscopy ( Figure 5 E). Imaging analysis flow cytometry and Western Blot both detected the expression of IL-12 in IL-12EVs, and the former further proved that IL-12 can be detected on the surface of EVs ( Figure 1 B, Figure 1 C).

[0039] The ELISA kit can be used to quantitatively analyze the IL-12 carried on the surface of IL-12EVs ( Figure 1 D). The IL-12 concentration of IL-12EVs did not change significantly after one week at -80°C, and was the same as the IL-12 concentration after separation and purification on day 0 ( Figure 1E). These data indicate that the IL-12EVs designed and prepared by us can carry IL-12 and can be stored at -80°C. The IL-12 concentration remains stable for at least one week and can be used for subsequent experiments. In addition, we found that at the same IL-12 concentration, IL-12EVs promoted T cells to secrete more IFN-γ than soluble human recombinant IL-12 (rhIL-12). Figure 1 F), the half effective concentration (EC50) of IL-12EVs is much lower than that of rhIL-12, which indicates that the concentration of IL-12EVs required to achieve the same T cell function activation effect is much lower than that of secretory IL-12, which improves the safety of IL-12EVs application to a certain extent.

[0040] 4. Preparation of CD19 / IL-12 EVs

[0041] HEK-293T cells were transfected with CD19 overexpression lentivirus and membrane-anchored IL-12 lentivirus, and the transfection efficiency was detected 72-96 hours after transfection. CD19 and IL-12 positive HEK-293T cells were sorted in single cell mode on Moflo XDP Flow Cytometer (Beckman Coulter). CD19 / IL-12EVs were obtained by the above EVs preparation method.

[0042] 5. CAR-T cell generation

[0043] Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors and patients using lymphocyte separation medium (Axis-Shield), and human T cells were isolated from human PBMCs using a CD3 sorting kit (Miltenyi, 130-097-043). They were activated for 24 h in culture medium (Gibco, A3021002) containing 200 IU mL-1 recombinant IL-2 (PeproTech) using CD3 / CD28 dybeads (Thermo Science, 11131D). Activated T cells were transfected with anti-CD19 lentivirus at an infection multiplicity of 3 and cultured for 10 to 14 days. The culture medium was renewed every 2 to 3 days, and the cell concentration was adjusted to 0.5 × 10 6 / mL~2.5×10 6 Pieces / mL.

[0044] 6. Cell lines and cell culture

[0045] HEK293T (human embryonic kidney cells), Raji (Burkitt's lymphoma cells) and K562 (chronic myeloid leukemia cells) cell lines were from the American Type Culture Collection (ATCC, USA). For in vivo studies, Raji cells were transduced with luciferase lentivirus and sorted to obtain 100% purity.

[0046] Raji and K562 cells were cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA) and 1% glutamine (Gibco, USA); HEK293T cells were cultured in DMEM (Gibco, USA) supplemented with 10% FBS and 1% glutamine (Gibco, USA). All cell cultures were cultured in a humidified incubator at 37°C and 5% CO2.

[0047] 2. Experimental Methods

[0048] 1. Plasmid preparation

[0049] The CD19-encoding lentiviral vector pCDH-CMV-CD19 was obtained from GeneCopoeia. To express IL-12, a plasmid encoding membrane-anchored IL-12 was constructed by linking the human IL-12 subunit p35 and p40 sequences via a flexible linker linked to a GPI anchor signal sequence. This DNA fragment was synthesized, cloned, and inserted into a lentiviral expression vector provided by GenScript. The anti-CD19 CAR lentiviral plasmid was constructed as described previously and consists of an anti-CD19 scFv (FMC63), a CD8a hinge / transmembrane region, CD28 and 4-1BB costimulatory domains, and a CD3ζ signaling domain. For laboratory validation, the anti-CD19CAR gene was linked to a truncated sequence of the epidermal growth factor receptor (EGFRt) via a T2A sequence. The CD3ζ-deficient anti-CD19CAR lentiviral plasmid was generated by removing the CD3ζ signaling domain from the above construct.

[0050] 2. Preparation and transfection of lentivirus

[0051] HEK293T cells were transiently transfected with PAX2 and pMD2.G packaging vectors and lentiviral expression vectors containing the target plasmid at a ratio of 1:1 using PEI reagent to prepare lentiviral particles. 48 hours after transfection, the viral supernatant was collected and centrifuged at 4°C for 30 minutes to remove debris. The supernatant was passed through a 0.45μm Thermo Scientific filter and then centrifuged at 4°C and 30,000×g for 150 minutes to concentrate the viral particles (Avanti J-26S XPI high-performance centrifuge, Beckman Coulter). HEK293T cells were infected with lentivirus containing IL-12 at an MOI of 20 and then sorted using a Moflo XDP flow cytometer (Beckman Coulter) to obtain stably expressed human IL-12. HEK293T cells expressing CD19 and cells expressing both CD19 and IL-12 were prepared using the same procedure.

[0052] 3. Preparation of EVs

[0053] EVs were isolated from HEK293T cells, HEK293T-CD19 cells, and HEK293T-IL12 and CD19-IL12 co-expressing cell lines by previously described methods. Briefly, cells were transferred to serum-free medium for 24 h, and then the culture medium was obtained and centrifuged at 500 × g for 10 min to remove cell debris. The supernatant was then filtered with a 0.45-μm sterile filter (Thermo Scientific) and centrifuged at 14,000 × g for 1 h at 4°C (Beckman Coulter, Optima XPN-100) to precipitate EVs. Purified EVs were resuspended in PBS and stored at -80°C until use.

[0054] 4. Characterization of EVs

[0055] The particle size distribution and Brownian motion of purified EVs were determined by nanoparticle tracking analysis (NTA) using a ZetaView multiparameter particle tracking analyzer. The morphology of EVs was determined by transmission electron microscopy (TEM), and images were obtained by a Tecnai G2 20TWIN microscope operating at 80 kV. The protein concentration of EVs was quantified using a BCA protein assay kit (Bio-University, China). In addition, the concentration of human IL-12 in EVs was determined using a human IL-12p40 quantitative ELISA kit (Xinbaosheng, China).

[0056] 5. EVs labeling and data collection

[0057] Purified EVs were stained with PE-conjugated anti-human IL-12p70 (clone: ​​20C2; BD Biosciences) and APC-conjugated anti-human CD19 (clone: ​​HIB19; BioLegend), diluted in PBS and filtered through a 0.22 μm pore size membrane, incubated at 4°C in the dark for 30 min, washed with filtered PBS, and then resuspended in filtered PBS. Samples were immediately subjected to flow cytometry (ImageStreamxMkII cytometer, Amnis), and data were analyzed using IDEAS 6.2 software (Luminex) as described previously. The fluid dynamics setting was “low speed / high sensitivity” when all sample data were acquired at 60× magnification.

[0058] The same gating strategy was used for the analysis of different samples. Controls included unstained samples, pure buffer controls, and buffer plus reagent controls, and single-stained samples were used for fluorescence compensation. Low SSC areas were circled by side scatter (Ch06) and then used for further analysis of fluorescent events (more than 10,000 events were acquired). Based on these single-positive fluorescent populations, single-positive gates were established, and double-positive gates were set based on the boundaries of the single-positive gates. The lower end of the various gates was determined by the unstained sample.

[0059] 6. Protein Blot

[0060] To identify the characteristics of molecules present in EVs and cells, Western blot detection was performed. Briefly, purified EVs or cells were lysed with RIPA lysis buffer supplemented with 1mM PMSF and protease inhibitor cocktail. The protein lysate (40μg per sample) was then separated by 4-12% SDS-PAGE. The resulting proteins were transferred to a PVDF membrane (Millipore, USA) and blocked with 5% skim milk for 1 hour at 25°C. To identify EVs molecules, the membrane was incubated with the following antibodies overnight on a shaker at 4°C: anti-annexin A1 antibody (Abcam, ab214486, 1:2000), anti-CD19 antibody (Abcam, ab245235, 1:1000) and anti-IL-12p70 antibody (Abcam, ab245235, 1:1000). Total STAT4 and phosphorylated STAT4 in T cells were detected using anti-STAT4 (C46B10) antibody (CST, 2653S, 1:1000) and anti-phospho-STAT4 (Tyr693) antibody (CST, 5267S, 1:1000). After washing the membrane three times with TBST, it was incubated with HRP-conjugated secondary antibody (1:1000) at room temperature for 1 hour. The membrane was then developed using Pierce ECL reagent (Thermo Fisher, USA), and all images were collected using Image Lab software (Bio-Rad).

[0061] 7. Generate CAR-T cells

[0062] Peripheral blood mononuclear cells (PBMCs) were collected from whole blood using Ficoll-Paque Plus (GE Healthcare, USA), and CD3+ T cells were isolated from human PBMCs using Miltenyi Biotec CD3 microbeads (Germany). Primary T cells were stimulated with T-Activator CD3 / CD28 magnetic beads (Thermo Fisher Scientific, 11131D) at a bead / cell ratio of 1:1 and cultured in CTS medium (Gibco, USA) supplemented with 2% human FBS, 2mM l-glutamine (Gibco, USA), and 200IU / mL human recombinant IL-2 (PeproTech, USA). After 24 hours, activated T cells were transfected with anti-CD19 CAR virus, and untransfected T cells were used as negative controls. Throughout the culture process, the culture density of T cells and CAR-T cells was 0.5-2.0×10 6 / mL, and the culture medium was replaced every 2-3 days.

[0063] 8. Flow cytometer

[0064] For cell surface staining, cells were resuspended in PBS supplemented with antibodies and placed at room temperature for 15 minutes. Flow cytometric analysis was performed using a NovoCyte flow cytometer (ACEA Biosciences) or a MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec), and data were analyzed using NovoExpress software (ACEA Biosciences) or FlowJo software (Tree Star).

[0065] PE-conjugated anti-human IL-12P70 (clone: ​​20C2; BD Biosciences) and APC-conjugated anti-human CD19 antibody (clone: ​​HIB19; BioLegend) were used to assess the expression of CD19 and IL12 on 293T cells. The following antibodies were used to detect the phenotype and function of CAR-T cells: PE / Cyanine7 conjugate anti-human CD8 (clone: ​​RPA-T8, BioLegend), BV421 conjugate anti-human CD4 (clone: ​​RPA-T4; BD Biosciences), FITC conjugate anti-human CD3 (clone: ​​HIT3a; BD Biosciences), APC conjugate anti-human CD45RO (clone: ​​UCHL1, BioLegend), BV605 conjugate anti-human CD62L (clone: ​​DREG-56; BD Biosciences), BV785 conjugate anti-human PD-1 (clone: ​​EH12.2H7, BioLegend), BV650 conjugate anti-human LAG3 (clone: ​​HC3C65, BioLegend), and PerCPCy5.5 conjugate anti-human CD4 (clone: ​​RPA-T4; BioLegend) antibodies.

[0066] For CAR expression detection, cells were stained with PE-labeled or FITC-labeled human CD19 protein (Acro Biosystems, Cat. CD9-HP2H3) or APC-conjugated anti-human epidermal growth factor receptor (clone: ​​AY13; BioLegend).

[0067] In the degranulation assay, CAR-T cells were treated with EVs or PBS and incubated with Raji cells for 4 h in RPMI 1640 medium containing CD107a antibody (clone H4A3; BioLegend) at a 1:1 E:T ratio. In the CFSE dilution assay, 2 μM CellTrace TM Violet (Thermo Fisher Scientific) was used to label T cells.

[0068] 9. Functional analysis of CAR-T cells

[0069] Anti-CD19 CAR-T cells were cultured at 3×10 5 Cells were seeded into 48-well plates at a density of 1:1 and co-cultured with Raji cells or K562 cells at a ratio of 1:1 with different EVs (167 μg / mL) or recombinant IL-12. For cytokine release assays, after 24 h of culture, the culture supernatants were harvested by centrifugation at 300 × g for 10 min and stored at −80 °C. The concentrations of cytokines IFN-γ, TNF-α, and IL-2 released by CAR-T cells in the supernatants were quantified using cytokine-specific ELISA kits (New Biotech, China). For activation analysis, cells were collected after 24 h and analyzed for the expression of cell activation markers by flow cytometry. For phenotypic analysis, cells cultured with EVs for 7 days were analyzed for in vitro CAR-T cell differentiation and exhaustion markers. For CFSE proliferation assays, CFSE-labeled CAR-T cells were co-cultured with mitomycin-treated Raji cells or K562 cells treated with different types of EVs at a ratio of 1:1 for 72 h. After 72 h of culture, cell proliferation was assessed by flow cytometry.

[0070] 10. In vitro cytotoxicity test

[0071] 1 μM CellTrace TM Violet (Thermo Fisher Scientific) was used to label Raji or K562 cells, and then washed with PBS to remove excess dye. Subsequently, CAR-T cells (3 × 10 5 Cells / well, 48-well plate) were co-cultured with CFSE-labeled Raji or K562 cells at an effector: tumor (E:T) ratio of 1:1. Cells were harvested 24 hours later and stained with propidium iodide (PI) (BD Pharmingen). Flow cytometry was used to analyze the cytotoxicity of CAR-T cells against tumor cells by quantifying the percentage of dead tumor cells in CFSE-labeled tumor cells.

[0072] 11. Intracellular cytokine staining

[0073] To evaluate the effects of different EVs on IFN-γ production by CAR-T cells, 2×10 6CAR-T cells were co-cultured with different EVs for 8 h, and protein transport blockers (BD Bioscience, 54655) were added. Cell surface staining was performed, followed by cell fixation and permeabilization using the Cytofix / Cytoperm Kit (BD Biosciences, 558050). Cells were then stained with FITC-conjugated anti-human IFN-γ (clone 4S.B3; BioLegend) intracellular antibodies at 4°C for 30 min. After a washing step, flow cytometry analysis was performed to determine the level of IFN-γ within CAR-T cells.

[0074] 12. EVs Binding and Cellular Uptake

[0075] EVs were labeled with 10 μM DIO (Invitrogen, USA) in PBS at 4°C for 2 h, washed twice with PBS to remove free dye, and then ultracentrifuged at 14,000 × g for 1 h at 4°C. To analyze the efficiency of EV uptake by CAR-T cells, 3x10 5 CAR-T cells were incubated with different DIO-labeled EVs at 37°C for 4 h and then analyzed by flow cytometry (NovaExpress).

[0076] 13. Imaging of the interaction between CAR-T cells and EVs

[0077] To image and evaluate the interaction between CAR-T cells and EVs, 1 × 10 6 CAR-T cells were treated with different EVs for 45 min at 37 ° C. The cells were then collected and washed with PBS to remove unbound EVs, followed by staining with anti-EGFR-APC, anti-CD3-FITC, and anti-IL-12p70-PE antibodies for 30 min at room temperature.

[0078] Samples were immediately evaluated by flow cytometry (ImageStreamxMkII cytometer, Amnis). At least 5000–15000 cell events were acquired for each sample with low acquisition rate and high sensitivity at 60× magnification. At least 2000 cell events were acquired for compensation of a single stained tube. The acquired images and data were analyzed by IDEAS 6.2 software (Luminex) as described previously. The gating strategy used the following steps: events were focused based on the gradient RMS value; single cells were identified by evaluating the ratio of the area of ​​the bright field (Ch01) to the aspect ratio of the bright field (Ch01), defined as aspect ratio greater than 0.8; single cells were further selected using CD3-FITC (Ch02) to identify T cells; within the T cell gate, EGFR-APC (Ch05) was used to distinguish between CAR-positive and CAR-negative T cells; within the CAR-positive T cell population, cells bound to EVs were detected by IL-12p70-PE (Ch03) positivity, as EVs are labeled with IL-12p70. The same gating strategy was used for the analysis of different samples.

[0079] 14. In vivo experiments in mice

[0080] All animal experiments were performed in accordance with the regulations of the Experimental Animal Care and Use Committee of Tongji Medical College, Wuhan, China.

[0081] Female NCG mice aged 5-6 weeks (obtained from GemPharmatech, China) were injected subcutaneously on the right side of the abdomen with Raji-luc lymphoma cells (5 × 10 6 cells, 50 μl Matrigel matrix and 50 μl PBS). After tumor implantation, the length and width of the tumor were measured every 3-5 days with a digital caliper, and the tumor volume was calculated by the formula (width ^ 2 × length) / 2. In addition, the Raji-luc cells in mice were imaged live every week by IVIS imaging system, and the acquired data were analyzed using LivingImage software. When the subcutaneous tumors of mice reached 50 to 100 mm 3 When 1×10 6 CAR-T cells or T cells. Mice were randomly assigned to different treatment groups and received intratumoral injections of control EVs, CD19 EVs, IL-12EVs, CD19 / IL-12EVs, or CD19+IL-12EVs (200 μg) on ​​days 1, 3, and 5 after CAR-T cell treatment. The body weight and overall condition of the mice were monitored every three days. If the mice showed symptoms such as a loss of more than 20% of the initial body weight, obvious lethargy, hunchback, severe diarrhea, severe dermatitis, or a tumor volume exceeding 1500mm 3 , the mice were euthanized.

[0082] To evaluate cytokine secretion levels and monitor CAR-T cell expansion, 100 μl of peripheral blood was collected from each mouse by orbital bleeding for subsequent flow cytometric analysis and cytokine detection.

[0083] 15. Droplet Digital PCR

[0084] As previously described, the expansion and persistence of CAR-T cells in mice were detected by droplet digital PCR. The following primers were used in this study: CAR forward primer 5'-CAGCAAAAA TACGACCTCCTCACT-3', reverse primer 5'-TGGTGCTGCCTTTGATCTCA-3', and probe 5'-FAM-TTGGGCGGAGGGACC-3'. Samples were detected by the QuantalifeQX200 droplet digital PCR system (Bio-Rad). Data were analyzed using QuantaSoft software version 1.7.4 (Bio-Rad).

[0085] 16. Measurement of serum cytokine levels

[0086] Mouse serum was separated from whole blood supernatant for cytokine analysis. The levels of human IFN-γ, TNF-α, IL-12p40, IL-2, IL-10, and IL-4 in serum were assessed by the Human Cytokine Cytometry Bead Array Kit (BD Biosciences) according to the manufacturer's instructions. Data were collected using a NovoCyte flow cytometer (ACEA Biosciences) and analyzed using FCAP.GUI software.

[0087] 17. Histopathological analysis

[0088] After treatment, the main organs of the mice were removed, fixed with 4% paraformaldehyde, and embedded in paraffin. The sections were 3 microns in diameter and pathologically examined by hematoxylin and eosin (H&E) staining.

[0089] 18. Statistical analysis

[0090] All experiments were performed three or more times, and all statistical analyses were performed using GraphPad Prism version 9.0. The statistical significance of differences between two groups was determined by two-tailed paired or unpaired t-test, while comparisons between multiple groups were performed by one-way or two-way analysis of variance. Animal survival was assessed by the log-rank (Mantel-Cox) test. Error bars in this study represent mean ± standard error of the mean (SEM). P values ​​less than 0.05 were considered statistically significant, and the significance level was indicated by an asterisk (ns indicates not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001).

[0091] 3. Experimental Conclusion

[0092] 1. IL-12EVs can enhance the function of CAR-T cells

[0093] Since IL-12 can activate STAT phosphorylation in T cells, we studied the effect of IL-12EVs on STAT4 phosphorylation in anti-CD19CAR-T cells in vitro. Western blot experiments confirmed that the level of STAT4 phosphorylation in CAR-T cells treated with IL-12EVs was higher than that in control cells ( Figure 2 A). To clarify the effect of IL-12EVs on CAR-T cell function, we co-cultured anti-CD19 CAR-T cells with Raji cells at a ratio of 1:1 between effector cells and target cells, and treated them with PBS, control EVs, rhIL-12, or IL-12EVs. After 24 hours, CAR-T cells in the IL-12EVs group secreted significantly more IFN-γ and TNF-α than those in the control EVs group, and at the same IL-12 concentration (667 pg / mL), CAR-T cells in the IL-12EV group secreted more IFN-γ and TNF-α than those in the rhIL-12 group ( Figure 2 B. Figure 2 C). Similarly, the proportion of CAR-T cells in the IL-12EV group was the highest among the above four groups ( Figure 2 D). In addition, IL-12EVs significantly increased the degranulation level and tumor killing ability of CAR-T cells ( Figure 2 E, Figure 2 F). Next, we analyzed the effects of IL-12EVs on the cell subsets and immune checkpoint expression of CAR-T cells and found that IL-12EVs induced an increase in CAR-T cells with a central memory phenotype, while a decrease in CAR-T cells with an effector memory phenotype, that is, IL-12EVs promoted the stemness differentiation of CAR-T cells ( Figure 2 G). Interestingly, IL-12EVs induced higher levels of LAG3 expression than other EVs, but there was no significant difference in PD1 expression ( Figure 6 A. Figure 6 B). In summary, IL-12EVs can promote the function of CAR-T cells, mainly by promoting their proliferation ability and key cytokine secretion, reducing terminal differentiation, and obtaining sustained and potent tumor cell killing ability.

[0094] 2. Verification of the targeting of anti-CD19 CAR-T cells by CD19 / IL-12EVs

[0095] Although IL-12 has been shown to enhance the function of CAR-T cells, systemic administration of IL-12 at therapeutic doses induces severe inflammatory responses and uncontrollable toxicity, thus limiting its clinical use in combination with CAR-T cell immunotherapy. Previously, we demonstrated that EVs presenting CAR-specific antigens can be preferentially bound by CAR-T cells and modulate their function. Therefore, we utilized IL-12 EVs with surface antigen expression as a strategy for targeted delivery of IL-12 to CAR-T cells, thereby specifically enhancing the function of CAR-T cells in vivo.

[0096] First, we constructed a HEK293T cell line that co-expressed IL-12 and CD19 on the cell surface ( Figure 7 A) to obtain CD19 / IL-12 EVs ( Figure 3 A). Notably, CD19 EVs showed no difference in Brownian motion or size distribution from EVs co-expressing IL-12 and CD19 (hereafter referred to as CD19 / IL-12EVs). Figure 7 B. Figure 7 C). Imaging flow cytometry confirmed that both IL-12 and CD19 were expressed on the surface of CD19 / IL-12 EVs ( Figure 3 B). Next, to test the delivery efficiency of CD19 / IL-12EVs in vitro, we cultured the same dose of IL-12EVs or CD19 / IL-12EVs with anti-CD19 CAR-T cells for 45 minutes. Flow cytometric analysis showed that CAR-T cells in the CD19 / IL-12EVs group bound more IL-12 molecules than those in the IL-12EVs group ( Figure 3 C). In addition, IL-12 signaling was only observed in CAR-T cells but not in T cells ( Figure 3D), indicating that the increase in IL-12 expression is due to the specific interaction between CD19 and CAR. In addition, to verify whether the increase in IL-12 expression in CAR-T cells is the result of exogenous delivery of EVs or the result of CAR-induced downstream cell signaling activation, we treated CAR-T cells with DiO-labeled IL-12EVs or CD19 / IL-12EVs for 4 hours and evaluated the cellular internalization of DiO-labeled EVs by flow cytometry. As a result, the number of DiO-labeled CAR-T cells in the CD19 / IL-12EV group was significantly more than that in the IL-12EV group ( Figure 3 E), indicating that CAR-dependent antigen / receptor binding facilitates the targeted delivery of EVs membrane-bound molecules, including IL-12.

[0097] We then mixed anti-CAR-T cells with Raji cells at a 1:1 ratio of effector cells to target cells and treated them with control EVs, CD19 EVs, IL-12EVs, or CD19 / IL-12EVs for 24 h to determine whether IL-12 delivered by EVs could exert a bioactive effect. Both IL-12EVs and CD19 / IL-12EVs induced more IFN-γ secretion and cytotoxicity compared with the control group ( Figure 3 F. Figure 3 G). In addition, to exclude the effect of CD19 / IL-12EVs on T cell activation via anti-CD19 CAR, we generated CAR-T cells using a CAR construct lacking the CD3ζ signal sequence (hereafter referred to as ΔCAR) ( Figure 3 H), so the effect of targeted delivery of IL-12 by CD19 / IL-12EVs is unrelated to CAR-induced T cell signaling.

[0098] The experimental results showed that the IFN-γ secreted by CD8+ΔCAR-T cells in the CD19 / IL-12EV group was significantly higher than that in the control group, CD19 EVs or IL-12EVs group ( Figure 3 I). This indicates that CD19 / IL-12EVs specifically enhance the cytokine secretion level of cytotoxic CAR-T cells, and functionally validates the targeting of CD19 / IL-12EVs to CAR-T cells. In summary, these research results indicate that CD19 / IL-12EVs can preferentially bind to anti-CD19 CAR-T cells and enhance their anti-tumor effects in vitro.

[0099] 3. CD19 / IL-12EVs can enhance the proliferation and anti-tumor activity of CAR-T cells in vivo

[0100] To explore the effects of CD19 / IL-12EVs on CAR-T cells in vivo, we injected CAR-T cells or control T cells into xenograft mice bearing CD19+Raji tumors ( Figure 4 A). We injected control EVs, CD19 EVs, IL-12EVs, or CD19 / IL-12EVs into tumor-bearing xenograft mice by intratumoral injection to reduce potential systemic toxicity. Compared with T cells, CAR-T cells can significantly control the tumor burden of xenograft model mice within 30 days after infusion. Among mice treated with CAR-T cells and intratumoral injection of EVs, the tumor burden in the CD19 / IL-12EVs group was the lowest among all groups, indicating that the combination of CAR-T cells and CD19 / IL-12EVs achieved the best tumor control effect ( Figure 4 B. Figure 4 C). In addition, compared with other types of EVs, CD19 / IL-12EVs induced a more prominent expansion of CAR-T cells, as demonstrated by both flow cytometry and digital PCR techniques ( Figure 4 D. Figure 4 E). The above results prove that CD19 / IL-12EVs can promote the proliferation of CAR-T cells in vivo and enhance their anti-tumor effects.

[0101] The application of IL-12 in vivo may have side effects, such as liver dysfunction and severe cytokine release syndrome (CRS); therefore, we monitored the liver and kidney functions of mice and found that there was no significant difference in liver and kidney functions among the groups of mice ( Figure 4 F. Figure 8 B). We also detected the concentrations of cytokines in mouse serum, including IFN-γ, IL-6, TNF-α, IL-2, IL-12, and IL-4. The level of IL-6 in the CD19 / IL-12EV group was slightly increased, but the difference was not statistically significant. There were no significant differences in other cytokines among the groups ( Figure 4 G, Figure 8 C). Mice treated with different EVs appeared normal and gained weight over time ( Figure 8 A). We also collected important organs of mice in each group for HE staining and found that the physiological structure of each tissue was normal ( Figure 8 D).

[0102] In summary, intratumoral administration of CD19 / IL-12EVs increased the proliferation and antitumor activity of CAR-T cells in vivo without systemic toxicity.

[0103] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A novel cytokine delivery platform, characterized in that: The cytokine delivery platform is CD19 / IL-12EVs, that is, IL-12 and CD19 are simultaneously expressed on the surface of extracellular vesicles.

2. The cytokine delivery platform according to claim 1, characterized in that: The method for constructing CD19 / IL-12 EVs includes the following steps: S1, HEK-293T cells were transfected with CD19 overexpression lentivirus and membrane-anchored IL-12 lentivirus. The transfection efficiency was detected 72-96 hours after transfection, and CD19 and IL-12 positive HEK-293T cells were sorted in single cell mode on Moflo XDP Flow Cytometer; S2, cells were transferred to serum-free medium and cultured for 24 h, and the medium was obtained and centrifuged at 500 × g for 10 min to remove cell debris; S3, the supernatant was filtered with a 0.45-μm sterile filter and centrifuged at 14,000 × g for 1 h at 4 °C to pellet EVs, and the purified EVs were resuspended in PBS and stored at −80 °C until use.

3. Application of the cytokine delivery platform of claim 1 in improving the function of CAR-T cells.

4. The application according to claim 3, characterized in that: CD19 / IL-12EVs can improve the binding efficiency with CAR-T cells and enhance the proliferation and anti-tumor activity of CAR-T cells in vivo.

5. Use of the cytokine delivery platform according to claim 1 in the preparation of a drug for treating acute B-lymphocytic leukemia.

6. The use according to claim 5, characterized in that: CD19 / IL-12EVs can be used to specifically promote CAR-T cell expansion and functional persistence in vitro and in vivo during CAR-T therapy for acute B-lymphocytic leukemia.

7. Use of the cytokine delivery platform according to claim 1 in the preparation of a drug for treating B-cell lymphoma.

8. Use of the cytokine delivery platform according to claim 1 in the preparation of drugs for treating solid tumors.

9. The use according to claim 8, characterized in that: CD19 / IL-12EVs utilize different target modifications to improve the function of CAR-T cells targeting solid tumors.

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