MSLN-KIR CAR-T cells show robust cytotoxicity and antitumor efficacy in vitro and in vivo

Besides KIR-activation receptors, other activation receptors exist on the membranes of natural killer (NK) cells, including NKP44, NKP46, NKP30, and NKG2D. The functionality of these receptors in the CAR form of the NKR complex, instead of in the KIR, remains unknown. Here, we engineered new multiple-chain MSLN targeting CARs, each containing only one of the NKP44, NKP46, NKP30, or NKG2D intracellular, transmembrane, and short extracellular domains (Fig. 1A). As shown in Fig. 1B, flow cytometry revealed that all CARs were successfully expressed and presented to the T cell membrane, with the highest positivity for the KIR CAR. No significant differences were observed in the proliferative capacity of T cells transduced with different CAR structures (Fig. 1C). Furthermore, all CAR-T cells showed robust tumor cell-killing ability against MSLN high-expressing OVCAR3 cells; however, KIR CAR-T cells were significantly better at killing MSLN low-expressing SKOV3 cells (Fig. 1D). Additionally, KIR CAR-T cells secreted more interferon (IFN)-γ than other CAR-T cells under SKOV3 cell stimulation. In contrast, the secretion of IFN-γ by 30 CAR-T and two-dimensional (2D) CAR-T cells was relatively lower than that in other CAR-T cells when co-cultured with OVCAR3 cells (Fig. 1E). Moreover, KIR CAR-T cells secreted a higher level of IL-2 following stimulation with OVCAR3 cells than 30 CAR-T and 2D CAR-T cells (Fig. 1F). Moreover, the Tcm ratio of KIR CAR-T cells was higher than that of the other CAR-T cells (Fig. 1G). Since the cytotoxicity and cytokine secretion capacity of 2D CAR-T cells were poor, our subsequent in vivo efficacy assessments did not include 2D CAR-T cells. In the SKOV3 subcutaneous xenograft model, mice were infused with different CAR-T cells or control non-transduced (NTD) cells. As shown in Fig. 1H, only KIR CAR-T cells substantially inhibited or eradicated tumors, whereas the other CAR-T cells merely slowed tumor growth. In conclusion, KIR CAR confers T cells with stronger cytotoxic and antitumor effects than other activated NKR CARs, which could be a potential therapeutic strategy for inducing MSLN expression in patients with cancer.

Fig. 1

Killer cell immunoglobulin-like receptor (KIR) Chimeric antigen receptor (CAR) is the optimal CAR structure based on natural killer (NK) cell activation receptors. A CAR design based on various NK cell activation receptor combinations. B Flow cytometric analysis of CAR-positive T cell percentage 7 days after lentiviral transduction. C Expansion of different CAR-T and control non-transduced (NTD) cells. D xCELLigence RTCA system evaluation of in vitro cytotoxicity of different MSLN CAR-T and NTD cells against SKOV37 and OVCAR3 cells. E ELISA analysis of IFN-γ secretion by different MSLN CAR-T cells following co-culture with SKOV37 and OVCAR3 cells (E:T, 2:1). F ELISA analysis of IL-2 secretion by different MSLN CAR-T cells following co-culture with OVCAR3 cells (E:T, 2:1). G Flow cytometry-based gating strategy for identifying Tn, Tcm, Tem, and Tef subsets in NTD and different MSLN CAR-T cells. H Tumor growth curves demonstrating the antitumor efficacy of NTD or different MSLN CAR-T cells in a SKOV3-derived xenograft model. Statistical significance: **P < 0.01

Efficacy of MSLN-KIR CAR-T cells as a treatment for ovarian cancer and mesothelioma

To evaluate the safety and efficacy of MSLN-KIR CAR-T cells (KT032) in ovarian cancer and mesothelioma therapy, we conducted a single-arm, open-label clinical trial at the First Affiliated Hospital of Nanjing Medical University, China, between August 2021 and February 2024. Nine MSLN-positive patients with advanced recurrent refractory ovarian cancer (eight patients) or mesothelioma (one patient) were enrolled in the trial after MSLN screening, and signed informed consent forms were obtained from all participants from August 2021 to September 2022. All nine patients underwent leukapheresis, which failed in one patient with ovarian cancer (Fig. 2A). All eight patients with successful leukapheresis had previously received at least two prior standard lines of therapy, which are shown in Table 1. MSLN KIR CAR-T cells were manufactured via lentiviral transduction in the GMP facility at Nanjing CART Medical Technology Co., Ltd., then cryopreserved and transferred to the First Affiliated Hospital of Nanjing Medical University. Among the eight patients, three received a low dose (0.5 × 106 cells/kg), two received a medium dose (1.0 × 106 cells/kg), and three received a high dose (1.5 × 106 cells/kg) of CAR-T cells, which were infusion following fludarabine- and cyclophosphamide-mediated lymphodepletion. We adjusted the doses of fludarabine (50–93 mg∙m−2 day−1, 1 day) and cyclophosphamide (500–930 mg∙m−2 day−1, 1 day) according to the physical condition of each patient (Table 1). As of the evaluation cutoff date (28 days after infusion), two of eight patients discontinued this study after their CAR-T cell infusion due to rapid disease progression (Fig. 2A).

Fig. 2

A–I Evaluation of the efficacy and safety of KT032 in patients with ovarian cancer and mesothelioma. A The study schema is shown. Subjects were screened for enrolment, underwent leukapheresis for CAR-T cell manufacturing, and infused with varying doses of CAR-T cells. B A swimmer plot was drawn to show patients’ response and time to disease progression in months. C A waterfall plot was drawn to show the determined percent change in tumor burden. D CT images showing the longest dimension of an initially responding tumor lesion at baseline, and at different time points after receiving KT032 cell infusion

Table 1 Patient characteristics

Of the six evaluable patients, two achieved partial response (PR), and four achieved stable disease (SD) status. However, all the six patients progressed within 9 months, and two patients were still alive until the latest follow-up. The median progression-free survival (PFS) was 5.5 months, and the median overall survival (OS) was 10.5 months (Fig. 2B and C). An enhanced computed tomography (CT) scan indicated that liver metastases were reduced by 31% and peritoneal effusion disappeared in Patient 1. Patient 2 exhibited a 42% reduction in the total maximum diameter of the measurable lesion. Meanwhile, the changes in tumor lesions or metastases were not significant in other patients (− 18.7% to + 4.56%) (Fig. 2D–I). Notably, the level of cancer antigen 125 (CA125) in Patient 1 was significantly decreased from 1687 U/mL to 150 U/mL 32 d after CAR-T therapy, while Patients 2 and 3 also showed a decrease in CA125 after CAR-T treatment, albeit with a rebound increase observed at day 20. For Patients 4 and 5, CAR-T therapy failed to reduce the CA125 level (Additional file2: Fig S1A-B).

Pharmacokinetics and safety of MSLN-KIR CAR-T cells

Flow cytometry and qPCR analysis showed that MSLN-KIR CAR-T cells in the peripheral blood peaked at approximately 7 days (6–10 days) after infusion (Fig. 3A). However, the peak of CAR-T cell expansion represented by flow cytometry was not very consistent with data from the qPCR analysis, which may be due to the discrepancy in total cell count in peripheral blood. Activation and expansion of MSLN-KIR CAR-T cells induced the secretion of high levels of cytokines IL6 and IFNγ in five of six patients (besides Patient 6, who showed slight changes in IL6 and IFNγ levels), while levels of TNFα, IL2, IL4, and IL10, etc. were slightly affected by CAR-T cell treatment. As a result, all five patients besides Patient 6, developed cytokine release syndrome (CRS) (one patient with grade 3, four patients with grade 1) accompanied by high levels of IL-6 and IFNγ (Fig. 3B–G); accordingly, corticosteroids, indomethacin, or tocilizumab (8 mg/kg) were used to treat CRS. Among the five patients with CRS, Patient 1 experienced immune pneumonia and received mechanical ventilation and high-dose corticosteroids prednisone (1 g/day) combined with tocilizumab (8 mg/kg). The patient’s condition improved significantly after treatment. Moreover, CT evaluation indicated that immune-related pneumonia was relieved (Additional file2: Fig S1C). Notably, the proliferation of CAR-T cells was limited due to the high-dose corticosteroids in this patient, while the number of CAR-T cells increased again (constituting 6.65% of the total peripheral blood T cells) on the 25th day after infusion, indicating that CAR-T cell activity may be restored by the withdrawal of corticosteroids (Fig. 3A and B). The other most common adverse effects during the MSLN-KIR CAR-T cell therapy were nausea, which was observed in 83% (5/6 patients), and no immune effector cell-associated neurotoxicity syndrome was observed in any patients. Other adverse events related to the MSLN-KIR CAR-T cell treatments were summarized in Table 2.

Fig. 3

KT032 cell pharmacokinetics and cytokine levels in peripheral blood of patients. A The pharmacokinetics of KT032 cell copy number in patients were evaluated using qPCR. The pharmacokinetics of KT032 cells in patients’ peripheral blood were assessed using qPCR and flow cytometry. B ELISA analysis of IFN-γ levels in patients’ peripheral blood. C ELISA analysis of TNFα levels in patients’ peripheral blood. D ELISA analysis of IL-2 levels in patients’ peripheral blood. E ELISA analysis of IL-4 levels in patients’ peripheral blood. F ELISA analysis of IL-6 levels in patients’ peripheral blood. G ELISA analysis of IL-10 levels in patients’ peripheral blood

Table 2 Summary of reported adverse events related to KT032 cells by grade reported in more than one subject (unless ≥ grade 3)Associations between MSLN CAR-T cell subtypes and clinical response

The proportion of CAR-positive T cells in the manufactured CAR-T products and the CD4/CD8 ratio of CAR-T cells were not related to patient responses. We hypothesized that subpopulations and functional gene signatures of individual CAR-T products may influence patient outcomes; hence, we conducted scRNA-seq on MSLN CAR-T cells produced from four patients. Cryopreserved CAR-T cells from four patients with ovarian cancer undergoing treatment were thawed and prepared for scRNA-seq. The number of CAR-T cells that passed the initial quality control ranged from 8093 to 9498 per patient. Further dimensionality reduction and unsupervised clustering analysis showed that 12 clusters (0–11) were identified, and CAR-T cells belonging to each cluster were detectable in all four patients. Next, we evaluated the differences in the clusters and transcriptomic signatures of the KT032 products from patients who had a good response (R) compared with those who did not. Patients 1 (HUME) and 4 (YUQI) were defined as having a good response, as Patient 1 achieved PR, while Patient 4 demonstrated signs of tumor lysis, as evaluated using CT during the two weeks after KT032 treatment. Meanwhile, Patients 5 (YLFE) and 6 (SXME) were defined as not responding, since both were determined to have progressed using fluorodeoxyglucose positron emission tomography. No significant differences in CD4 + and CD8 + T cell subsets were observed between the two patient groups (Fig. 4A–C). Differential gene expression analysis showed significant upregulation of GSTM1 and NOTCH2NLB gene expression in CAR-T cells from the responding patients (Fig. 4D). GSTM11 is essential for controlling the intracellular redox state of immune cells, while NOTCH2NLB is functionally similar to the intracellular structural domain (NICD) of Notch1 and activates the Notch signaling pathway. These findings indicate that the control of the intracellular redox state and the Notch signaling pathway may be important for the long-term antitumor activity of MSLN-KIR CAR-T cells.

Fig. 4

Single-cell sequencing analysis of the properties of KT032 from different patients. A The bar chart shows the proportional distribution (left) and the absolute distribution (right) of various T cell types in four cases with KT032 cell transfusion. B The bubble plot displays the expression levels of key marker genes in various types of T cells with KT032 cell transfusion. The size of the bubbles represents the percentage of gene expression in the cells, and the color of the bubbles represents the average level of gene expression. C The distribution of various types of T cells in the t-distributed stochastic neighbor embedding (tSNE) plot for four cases with KT032 cell transfusion. D The volcano plot of the differential analysis shows the upregulated and downregulated genes in various T cell clusters with KT032 cell transfusion products compared with PR samples. Upregulated genes are shown in red, downregulated genes in blue, and the symbols of the top five genes with the largest differences in upregulation and downregulation are labeled. The differential threshold is set as fold change > 2 and P < 0.05

Gene expression signatures of immune and cancer cells in the ovarian cancer tumor microenvironment (TME) after MSLN-CAR-T cells infusion determined using scRNA-seq

To further explore whether immune cell components and their gene signatures in the immune microenvironment of patients with ovarian cancer correlated with clinical responses, we first compared the representation of immune cell types and functional states between one patient with PR and one with SD. As shown in Fig. 5A, T cells, fibroblasts, and pericytes were significantly enriched within the TME of the patient who achieved PR, while epithelial cells were enriched within the TME of the patient with SD. Moreover, dimensionality reduction and unsupervised clustering analysis identified 11 CD4 + and CD8 + T cell clusters in both patients, but patients with PR had relatively higher proportions of CD4 naive, CD4 Teff, CD4 Tcm, and CD8 naive cells, while patients with SD had much more CD8 Teff cells, including CD8 Teff, CD8 Teff CD74, and CD8 Teff mitotic cells (Fig. 5A–D). This suggests that more T cells, especially CD4 clusters, within the TME may be associated with a good response to CAR-T cell infusion.

Fig. 5

Single-cell sequencing analysis of the properties of the immune and tumor cells after infusion of KT032 in patients with ovarian cancer. A The bar chart shows the proportional distribution of various cell types in tumor samples from patients with ovarian cancer before and after KT032 treatment. B The bar chart shows the proportional distribution of various T cells in tumor samples from patients with ovarian cancer before and after KT032 treatment. C The tSNE plot illustrates the distribution of different types of T cells in tumor samples from patients with ovarian cancer before and after KT032 treatment. D The bubble plot displays the expression levels of key marker genes for different types of T cells in tumors from patients with ovarian cancer. The size of the bubbles represents the percentage of gene expression in the cells, while the color represents the average level of gene expression. E The volcano plot of the differential analysis shows the upregulated and downregulated genes in various T cell clusters of tumors from patients with ovarian cancer after treatment compared with before treatment. Upregulated genes are shown in red, downregulated genes in blue, and the symbols of the top five genes with the largest differential expression are labeled. The differential threshold is fold change (FC) > 2 and P <0.05. F The bar chart illustrates the proportional distribution of different myeloid cells in tumor samples from patients with ovarian cancer before and after CAR-T treatment. G The bar chart displays the proportional distribution of different malignant cells in tumor samples from patients with ovarian cancer before and after CAR-T treatment. H The tSNE plot shows the distribution of different malignant cells in tumor samples of patients with ovarian cancer before and after CAR-T treatment. I The bubble plot shows the expression levels of the top five upregulated genes in various malignant cells in ovarian cancer tumor samples. The size of the bubbles represents the percentage of gene expression in the cells, while the color represents the average level of gene expression. J The volcano plot of differential analysis shows the upregulated and downregulated genes in tumor samples from patients with ovarian cancer after treatment compared with before treatment. Upregulated genes are shown in red, downregulated genes in blue, and the symbols of the top five genes with the largest differential expression are labeled. The differential threshold is FC > 2 and P < 0.05

The proportions of myeloid cells, T cells, and NK_NKT cells increased while that of epithelial cells decreased (Fig. 5A). For T cell clusters, only the proportions of CD4 + Tregs and CD8 + Tact cells increased slightly, while other clusters were not significantly influenced by CAR-T cell infusion (Fig. 5B). Differentially expressed gene (DEG) enrichment analysis showed that these DEGs in CD4 + Tregs post-CAR-T cell infusion were enriched in response to cytokine, cytokine-mediated signaling pathways, immune response, etc., indicating that CAR-T cells and their secreted cytokines may influence Treg cell phenotypes (Additional file2: Fig S2A). Moreover, T cells from patients with PR showed a higher expression of IL12RB2, GZMB, and IL10, indicating greater cytotoxicity and immunomodulatory properties (Fig. 5E). Furthermore, patients with PR had much more MDSC-like macrophages but less CD24-TAM abundance compared with patients with SD. In contrast, the proportions of FN1-TAM and MDSC-like macrophages increased after CAR-T cell therapy (Fig. 5F and Additional file2: Fig S2B-E). Further analysis demonstrated that dysregulated genes in these CD24-TAM cells were enriched in antigen processing and presentation of peptide antigen via MHC class I, cellular response to interferon-gamma, myeloid leukocyte mediated immunity, etc., which indicated that CAR-T cell therapy might induce the transformation of the TAM (Additional file2: Fig S2F). Moreover, the DEGs in MDSC-like macrophages post CAR-T cell treatment were enriched in immune response, cellular response to cytokine stimulus, response to cytokines, etc. as well as CD4 + Tregs (Additional file2: Fig S2G), which indicated that CAR-T cells would alter the pre-existing suppressive tumor microenvironment. These findings indicate that CAR-T cell infusion may recruit more immune cells and temporarily remodel the TME.

In addition, to investigate which genes in tumor cells may influence ovarian cancer cell resistance to MSLN-DAP CAR-T cell-mediated killing, we compared the changes in tumor cell subpopulations and gene expression profiles after CAR-T cell infusion. Seven clusters were identified by dimensionality reduction and unsupervised clustering analysis (Fig. 5G–I). As shown in Fig. 5J, patients with PR had fewer M0 tumor cells that highly expressed FLOR3, PAGE2B, PAGE2, and C19orf33, and more M1 cells that had low expression of FLOR3, PAGE2B, PAGE2, and C19orf33 compared with patients with SD. The number of M2 cluster cells with high expression of NFKB1, MIR222HG, and LINC00472 was significantly decreased in both patients, while there was an increase in the number of M3 cluster cells that expressed CCL4, NKG7, and CD53. These findings suggest that tumor cells expressing NFKB1, MIR222HG, and LINC00472 may be more sensitive to MSLN-DAP CAR-T cell killing, whereas tumor cells expressing CCL4, NKG7, and CD53 may be resistant to MSLN-DAP CAR-T cell-mediated eradication.

Gene expression signatures of immune and cancer cells in mesothelioma after MSLN-CAR-T cell infusion

We further explored the representation of immune cell types and functional states in patients with mesothelioma who had the longest survival times of 15 and 45 days after CAR-T cell infusion. As shown in Fig. 6A–B, dimensionality reduction and unsupervised clustering analysis showed significant enrichment of NK cells, endothelial cells, and pericytes within the TME 45 days after CAR-T cell therapy compared with 15 days. Conversely, the ratio of mesothelial to myeloid cells decreased, whereas the proportion of T cells did not. Compared with day 15, the increased endothelial cells highly expressed KLF4, BMP4, and FCN3 and had low expression of CHI3L1, HLA-DRA, and PRG4, while the NK cells had upregulated ZNF420 expression, and the pericytes showed increased levels of SNAI1, EFNA1, and BRD9 (Fig. 6C). Moreover, a total of seven clusters of CD4 + and CD8 + T cells were identified, and relatively higher proportions of CD8 + Tact and a lower ratio of CD8 Mitotic characterized in the TME 45 days after CAR-T cell therapy compared with 15 days (Fig. 6D). These altered genes were enriched for protein binding, NADH dehydrogenase (ubiquinone) activity, and catalytic activity (Fig. 6E). Additionally, seven clusters of mesothelioma cells were identified using dimensionality reduction and unsupervised clustering analyses. As shown in Fig. 6F and G, the ratio of M1 clusters with highly expressed SPATC1L, DDX52, BMPR2, and TMEM126B increased 45 days after CAR-T cell infusion, while the ratio of M3 clusters with highly expressed EPGN, NRK, WFDC2, and PRR9 decreased. Moreover, the DEGs in the specimen 45 days after CAR-T cell therapy were enriched in C–C motif chemokine receptor 1 chemokine receptor binding, chemokine receptor binding, and T cell receptor binding (Fig. 6H), indicating that CAR-T cell treatment may recruit and reactivate immune cells in the TME, which in turn induces an antitumor immune response.

Fig. 6

Single-cell sequencing analysis of the properties of the immune and tumor cells after infusion of KT032 in a patient with mesothelioma. A The bar chart shows the proportional distribution of different cell types in tumor samples from patients with mesothelioma after KT032 therapy in two stages. B The tSNE plot illustrates the distribution of different cell types in tumor samples from patients with mesothelioma after KT032 therapy in two stages. C The volcano plot of differential analysis displays the upregulated and downregulated genes in tumor samples from patients with mesothelioma after KT032 therapy in stage two compared with stage one. Upregulated genes are shown in red, downregulated genes in blue, and the top five genes with the largest differences in upregulation or downregulation are labeled with their symbols. The differential threshold is FC > 2 and P < 0.05. D The bar chart presents the proportional distribution of different T cell types in tumor samples from patients with mesothelioma after KT032 therapy in two stages. E The bubble plot shows the top 20 enriched pathways (q-value) for differentially expressed genes between CD8+ mitotic T cells (left) and CD8+ Tact cells (right) in tumor samples from patients with mesothelioma after KT032 therapy in two stages. F The bar chart displays the proportional distribution of different malignant cell types in tumor samples from patients with mesothelioma after KT032 therapy in two stages. G The heatmap illustrates the expression levels of the top five upregulated genes in different malignant cell types in tumor samples from patients with mesothelioma. The color represents the average gene expression level. H The bubble plot shows the top 20 enriched pathways (q-value) for upregulated genes in the M3 subgroup (left) and M4 subgroup (right) of tumor samples from patients with mesothelioma after KT032 therapy