GPC3-targeted CAR-T cells expressing GLUT1 or AGK exhibit enhanced antitumor activity against hepatocellular carcinoma.

Chimeric antigen receptor-expressing T (CAR-T) cells induce robust antitumor responses in patients with hematologic malignancies. However, CAR-T cells exhibit only limited efficacy against solid tumors such as hepatocellular carcinoma (HCC), partially due to their limited expansion and persistence. CD8+ T cells, as key components of the adaptive immune response, play a central role in antitumor immunity. Aerobic glycolysis is the main metabolic feature of activated CD8+ T cells. In the tumor microenvironment, however, the uptake of large amounts of glucose by tumor cells and other immunosuppressive cells can impair the activation of T cells. Only when tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment have a glycolytic advantage might the effector function of T cells be activated. Glucose transporter type 1 (GLUT1) and acylglycerol kinase (AGK) can boost glycolytic metabolism and activate the effector function of CD8+ T cells, respectively. In this study, we generated GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK for the treatment of HCC. GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK specifically and effectively lysed GPC3-positive tumor cells in vitro in an antigen-dependent manner. Furthermore, GLUT1 or AGK overexpression protected CAR-T cells from apoptosis during repeated exposures to tumor cells. Compared with second-generation CAR-T cells, GPC3-targeted CAR-T cells overexpressing GLUT1 or AGK exhibited greater CD8+ T-cell persistence in vivo and better antitumor effects in HCC allograft mouse models. Finally, we revealed that GLUT1 or AGK maintained anti-apoptosis ability in CD8+ T cells via activation of the PI3K/Akt pathway. This finding might identify a therapeutic strategy for advanced HCC.

CXCR4-modified CAR-T cells suppresses MDSCs recruitment via STAT3/NF-κB/SDF-1α axis to enhance efficacy against pancreatic cancer.

Claudin18.2 (CLDN18.2)-specific chimeric antigen receptor (CAR-T) cells displayed limited efficacy in CLDN18.2-positive pancreatic ductal adenocarcinoma (PDAC). Strategies are needed to improve the trafficking capacity of CLDN18.2-specific CAR-T cells. PDAC has a unique microenvironment that consists of abundant cancer-associated fibroblasts (CAFs), which could secrete stromal cell-derived factor 1α (SDF-1α), the ligand of CXCR4. Then, we constructed and explored CLDN18.2-targeted CAR-T cells with CXCR4 co-expression in treating immunocompetent mouse models of PDAC. The results indicated that CXCR4 could promote the infiltration of CAR-T cells and enhance their efficacy in vivo. Mechanistically, the activation of signal transducer and activator of transcription 3 (STAT3) signaling was impaired in CXCR4 CAR-T cells, which reduced the release of inflammatory factors, such as tumor necrosis factor-α, IL-6, and IL-17A. Then, the lower release of inflammatory factors suppressed SDF-1α secretion in CAFs via the nuclear factor κB (NF-κB) pathway. Therefore, the decreased secretion of SDF-1α in feedback decreased the migration of myeloid-derived suppressor cells (MDSCs) in tumor sites. Overall, our study demonstrated that CXCR4 CAR-T cells could traffic more into tumor sites and also suppress MDSC migration via the STAT3/NF-κB/SDF-1α axis to obtain better efficacy in treating CLDN18.2-positive pancreatic cancer. Our findings provide a theoretical rationale for CXCR4 CAR-T cell therapy in PDAC.

FAP-targeted CAR-T suppresses MDSCs recruitment to improve the antitumor efficacy of claudin18.2-targeted CAR-T against pancreatic cancer.

PURPOSE: The claudin 18.2 (CLDN18.2) antigen is frequently expressed in malignant tumors, including pancreatic ductal adenocarcinoma (PDAC). Although CLDN18.2-targeted CAR-T cells demonstrated some therapeutic efficacy in PDAC patients, further improvement is needed. One of the major obstacles might be the abundant cancer-associated fibroblasts (CAFs) in the PDAC tumor microenvironment (TME). Targeting fibroblast activation protein (FAP), a vital characteristic of CAFs provides a potential way to overcome this obstacle. In this study, we explored the combined antitumor activity of FAP-targeted and CLDN18.2-targeted CAR-T cells against PDAC. METHODS: Novel FAP-targeted CAR-T cells were developed. Sequential treatment of FAP-targeted and CLDN18.2-targeted CAR-T cells as well as the corresponding mechanism were explored in immunocompetent mouse models of PDAC. RESULTS: The results indicated that the priorly FAP-targeted CAR-T cells infusion could significantly eliminate CAFs and enhance the anti-PDAC efficacy of subsequently CLDN18.2-targeted CAR-T cells in vivo. Interestingly, we observed that FAP-targeted CAR-T cells could suppress the recruitment of myeloid-derived suppressor cells (MDSCs) and promote the survival of CD8+ T cells and CAR-T cells in tumor tissue. CONCLUSION: In summary, our finding demonstrated that FAP-targeted CAR-T cells could increase the antitumor activities of sequential CAR-T therapy via remodeling TME, at least partially through inhibiting MDSCs recruitment. Sequential infusion of FAP-targeted and CLDN18.2-targeted CAR-T cells might be a feasible approach to enhance the clinical outcome of PDAC.

Enforced expression of Runx3 improved CAR-T cell potency in solid tumor via enhancing resistance to activation-induced cell death.

Limited T cell persistence restrains chimeric antigen receptor (CAR)-T cell therapy in solid tumors. To improve persistence, T cells have been engineered to secrete proinflammatory cytokines, but other possible methods have been understudied. Runx3 has been considered a master regulator of T cell development, cytotoxic T lymphocyte differentiation, and tissue-resident memory T (Trm)-cell formation. A study using a transgenic mouse model revealed that overexpression of Runx3 promoted T cell persistence in solid tumors. Here, we generated CAR-T cells overexpressing Runx3 (Run-CAR-T cells) and found that Run-CAR-T cells had long-lasting antitumor activities and achieved better tumor control than conventional CAR-T cells. We observed that more Run-CAR-T cells circulated in the peripheral blood and accumulated in tumor tissue, indicating that Runx3 coexpression improved CAR-T cell persistence in vivo. Tumor-infiltrating Run-CAR-T cells showed less cell death with enhanced proliferative and effector activities. Consistently, in vitro studies indicated that AICD was also decreased in Run-CAR-T cells via downregulation of tumor necrosis factor (TNF) secretion. Further studies revealed that Runx3 could bind to the TNF promoter and suppress its gene transcription after T cell activation. In conclusion, Runx3-armored CAR-T cells showed increased antitumor activities and could be a new modality for the treatment of solid tumors.

Radiation enhances the efficacy of EGFR-targeted CAR-T cells against triple-negative breast cancer by activating NF-κB/Icam1 signaling.

Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype, with limited treatment options. Epidermal growth factor receptor (EGFR) is reported to be expressed in 50%-75% of TNBC patients, making it a promising target for cancer treatment. Here we show that EGFR-targeted chimeric antigen receptor (CAR) T cell therapy combined with radiotherapy provides enhanced antitumor efficacy in immunocompetent and immunodeficient orthotopic TNBC mice. Intriguingly, this combination therapy resulted in a substantial increase in the number of tumor-infiltrating CAR-T cells. The efficacy of this combination was independent of tumor radiosensitivity and lymphodepleting preconditioning. Cytokine profiling showed that this combination did not increase the risk of cytokine release syndrome (CRS). RNA sequencing (RNA-seq) analysis revealed that EGFR-targeting CAR-T therapy combined with radiotherapy increased the infiltration of CD8+ T and natural killer (NK) cells into tumors. Mechanistically, radiation significantly increased Icam1 expression on TNBC cells via activating nuclear factor κB (NF-κB) signaling, thereby promoting CAR-T cell infiltration and killing. These results suggest that CAR-T therapy combined with radiotherapy may be a promising strategy for TNBC treatment.

Chimeric anti-GPC3 sFv-CD3ε receptor-modified T cells with IL7 co-expression for the treatment of solid tumors.

Chimeric antigen receptor (CAR) T cells targeting glypican-3 (GPC3) demonstrated early signs of therapeutic efficacy to hepatocellular carcinoma patients with a risk of cytokine release syndrome (CRS). Several adoptive cell therapies (ACTs) with T cells using the natural T cell receptor (TCR) signaling induced more efficient antitumor function and reduced cytokine production relative to CARs in solid tumors. To improve the efficacy and safety of GPC3-targeted ACTs, T cells were modified with anti-GPC3 single-chain fragment variable(sFv) linked to CD3ε, which could be incorporated into the entire TCR/CD3 complex to form chimeric sFv-CD3ε receptor (sFv-ε). sFv-ε T cells showed competitive antitumor activity and lower cytokine release compared to 28ζ or BBζ CAR T cells, which may be ascribed to moderately less activated Ca2+-calcineurin-NFAT signaling pathway. We further generated murine sFv-ε T cells with interleukin-7 co-expression (7sFv-ε) to promote T cell survival and to mobilize the endogenous immune system. In immunocompetent mouse models, 7sFv-ε T cells showed superior persistence, antitumor efficacy, and immunological memory while preserving the low production of cytokines associated with CRS compared to conventional sFv-ε T cells. These results indicate that GPC3-specific 7sFv-ε T cells could serve as a promising therapeutic strategy for solid tumors.

Olaparib Suppresses MDSC Recruitment via SDF1α/CXCR4 Axis to Improve the Anti-tumor Efficacy of CAR-T Cells on Breast Cancer in Mice.

A hostile tumor microenvironment is one of the major obstacles for the efficacy of chimeric antigen receptor modified T (CAR-T) cells, and combination treatment might be a potential way to overcome this obstacle. Poly(ADP-ribose) polymerase inhibitor (PARPi) has demonstrated tremendous potential in breast cancer. In this study, we explored the possible combination of the PAPRi olaparib with EGFRvIII-targeted CAR (806-28Z CAR) T cells in immunocompetent mouse models of breast cancer. The results indicated that the administration of olaparib could significantly enhance the efficacy of 806-28Z CAR-T cells in vivo. Interestingly, we observed that olaparib could suppress myeloid-derived suppressor cell (MDSC) migration and promote the survival of CD8+ T cells in tumor tissue. Mechanistically, olaparib was shown to reduce the expression of SDF1α released from cancer-associated fibroblasts (CAFs) and thereby decreased MDSC migration through CXCR4. Taken together, this study demonstrated that olaparib could increase the antitumor activities of CAR-T cell therapy at least partially through inhibiting MDSC migration via the SDF1α/CXCR4 axis. These findings uncover a novel mechanism of PARPi function and provide additional mechanistic rationale for combining PARPi with CAR-T cells for the treatment of breast cancer.

Coexpression of IL7 and CCL21 Increases Efficacy of CAR-T Cells in Solid Tumors without Requiring Preconditioned Lymphodepletion.

PURPOSE: T-cell recruitment, survival, and proliferation are the important limitations to chimeric antigen receptor (CAR) T cells therapy in the treatment of solid tumors. In this study, we engineered CAR-T cells to coexpress cytokines IL7 and CCL21 (7 × 21 CAR-T), a cytokine combination in order to improve proliferation and chemotaxis of CAR-T cells. EXPERIMENTAL DESIGN: CLDN18.2-specific second-generation CAR-T cells coexpressing cytokines were prepared using retroviral vector transduction. The proliferation and migration of genetically engineered CAR-T cells were evaluated in vitro. The antitumor activities of genetically engineered CAR-T cells were evaluated against multiple solid tumors in C57BL/6 mice in vivo. RESULTS: In vitro, the proliferation and chemotaxis of 7 × 21 CAR-T cells are significantly improved when compared with those of the conventional CAR-T cells. In vivo, 7 × 21 CAR-T cells revealed superior therapeutic effects to either conventional CAR-T cells or 7 × 19 CAR-T cells which coexpress IL7 and CCL19 as previously reported in three different solid tumors without cyclophosphamide precondition. Interestingly, 7 × 21 CAR-T cells could also suppress the tumor growth with heterogeneous antigen expression and even induce tumor complete remission. Mechanistically, IL7 and CCL21 significantly improved survival and infiltration of CAR-T cells and dendritic cells in tumor. In addition, CCL21 also inhibited the tumor angiogenesis as proved by IHC. CONCLUSIONS: Coexpression of IL7 and CCL21 could boost CAR-T cells' antitumor activity, and 7 × 21 CAR-T cells may be served as a promising therapy strategy for solid tumors.

An IL-4/21 Inverted Cytokine Receptor Improving CAR-T Cell Potency in Immunosuppressive Solid-Tumor Microenvironment.

Incorporation of inverted cytokine receptor (ICR) such as interleukin (IL)-4 vs. IL-7 (4/7) ICR is one strategy to improve the antitumor activities of chimeric antigen receptor (CAR) modified T (CAR-T) cells facing immunosuppressive cytokines. Here we report a novel interleukin (IL)-4 vs. IL-21 ICR (4/21 ICR) that enhanced CAR-T cell potency in IL-4+ tumor milieu via a different working-mechanism from 4/7 ICR. Upon IL-4 stimulation, 4/21 ICR activated the STAT3 pathway and promoted Th17-like polarization and tumor-targeted cytotoxicity in CAR-T cells in vitro. Furthermore, 4/21 ICR-CAR T cells persisted and eradicated established IL-4+ tumors in vivo. Thus, 4/21 ICR is a promising clinical CAR-T cell therapeutics for solid tumors rich in IL-4.

Combined Antitumor Effects of Sorafenib and GPC3-CAR T Cells in Mouse Models of Hepatocellular Carcinoma.

Our previous study indicated that GPC3-targeted chimeric antigen receptor (CAR) T cell therapy has a high safety profile in patients with hepatocellular carcinoma (HCC). However, the response rate requires further improvement. Here, we analyzed the combined effect of GPC3-CAR T cells and sorafenib in both immunocompetent and immunodeficient mouse models of hepatocellular carcinoma. In immunocompetent mouse model, mouse CAR (mCAR) T cells induced regression of small tumors (approximately 130 mm3 tumor volume) but had no effect on large, established tumors (approximately 400 mm3 tumor volume). Sorafenib, at a subpharmacologic but not a pharmacologic dose, augmented the antitumor effects of mCAR T cells, in part by promoting IL12 secretion in tumor-associated macrophages (TAMs) and cancer cell apoptosis. In an immunodeficient mouse model, both subpharmacologic and pharmacologic doses of sorafenib had limited impacts on the function of human CAR (huCAR) T cells in vitro and showed synergistic effects with huCAR T cells in vivo, which can at least partially be ascribed to the upregulated tumor cell apoptosis induced by the combined treatment. Thus, this study applied two of the most commonly used mouse models for CAR T cell research and demonstrated the clinical potential of combining sorafenib with GPC3-targeted CAR T cells against HCC.

Combined Adjuvant of Poly I:C Improves Antitumor Effects of CAR-T Cells.

Chimeric antigen receptor modified T cells (CAR-T) therapy is an emerging immunotherapy against malignancies. However, only limited success was obtained in solid tumors. Polyinosinic-polycytidylic acid (poly I:C), ligand of TLR3, mediates innate immune and adaptive immune and shows broad antitumor effect on many types of cancer. In the present study, we combined EGFRvIII-targeted CAR-T cells with poly I:C treatment and evaluated the synergic antitumor effect in vitro and in immunocompetent mice bearing subcutaneous colon or orthotopic breast cancer xenografts. Poly I:C significantly promoted more IL-2 and IFN γ production as well as higher lytic activity of CAR-T cells. Upon systemic administration in vivo, CAR-T cells obviously suppressed tumor growth, and poly I:C significantly enhanced the suppression. Further study showed that poly I:C exerted antitumor effect dependent on type I IFNs. In addition, poly I:C decreased myeloid-derived suppressor cells (MDSC) number in peripheral blood and spleen, and attenuated the immunosuppressive activity of MDSC on proliferation and cytolytic function of CAR-T. Depletion of MDSC with anti-Gr1 Ab further increased the antitumor effect of CAR-T cells plus poly I:C treatment. In conclusion, CAR-T treatment combined with intratumoral delivery of poly I:C resulted in synergistic antitumor activity. We thus provide a rationale to translate this immunotherapeutic strategy to solid tumors.

Armored Inducible Expression of IL-12 Enhances Antitumor Activity of Glypican-3-Targeted Chimeric Antigen Receptor-Engineered T Cells in Hepatocellular Carcinoma.

Adoptive immunotherapy based on chimeric antigen receptor-modified T (CAR-T) cells has been demonstrated as one of the most promising therapeutic strategies in the treatment of malignancies. However, CAR-T cell therapy has shown limited efficacy for the treatment of solid tumors. This is, in part, because of tumor heterogeneity and a hostile tumor microenvironment, which could suppress adoptively transferred T cell activity. In this study, we, respectively, engineered human- or murine-derived-armored glypican-3 (GPC3)-specific CAR-T cells capable of inducibly expressing IL-12 (GPC3-28Z-NFAT-IL-12) T cells. The results showed that GPC3-28Z-NFAT-IL-12 T cells could lyse GPC3+ tumor cells specifically and increase cytokine secretion compared with GPC3-28Z T cells in vitro. In vivo, GPC3-28Z-NFAT-IL-12 T cells augmented the antitumor effect when encountering GPC3+ large tumor burdens, which could be attributed to IL-12 increasing IFN-γ production, favoring T cells infiltration and persistence. Furthermore, in immunocompetent hosts, low doses of GPC3-m28Z-mNFAT-mIL-12 T cells exerted superior antitumor efficacy without prior conditioning in comparison with GPC3-m28Z T cells. Also, mIL-12 secretion decreased regulatory T cell infiltration in established tumors. In conclusion, these findings demonstrated that the inducible expression of IL-12 could boost CAR-T function with less potential side effects, both in immunodeficient and immunocompetent hosts. The inducibly expressed IL-12-armored GPC3-CAR-T cells could broaden the application of CAR-T-based immunotherapy to patients intolerant of lymphodepletion chemotherapy and might provide an alternative therapeutic strategy for patients with GPC3+ cancers.

Antitumor efficacy of chimeric antigen receptor T cells against EGFRvIII-expressing glioblastoma in C57BL/6 mice.

Chimeric antigen receptor (CAR) T cell therapy has shown remarkable success in hematological tumors. However, many challenges remain in improving the efficacy of CAR T cells in solid tumors. The epidermal growth factor receptor variant III (EGFRvIII) is only expressed in tumors but barely found in normal tissues, making it a good target for CAR T therapy. It is reported that 31-64% of glioblastoma (GBM) patients are EGFRvIII positive. Here we report the robust antitumor activities of CAR T cells targeting EGFRvIII-expressing mouse GBM cells. In vitro and in vivo, 806-28Z CAR T cells were able to lyse GL261/EGFRvIII cellsin a dose-dependent manner. A low dose of 806-28Z CAR T cells suppressed GL261/EGFRvIII tumor growth, whereas a high dose of 806-28Z CAR T cells completely eradicated xenograft tumors. Higher concentrations of granzyme B in mice plasma were correlated with increased CAR T cells infusion. Enhanced CD8+ T cells infiltration within the tumors were detected by immunohistochemistry in sections from the mice treated by CAR T cells. The 806-28Z CAR T cells can also inhibit the growth of antigenic heterogeneous GBM tumors. More importantly, additional rechallenge experiments indicated that GL261/EGFRvIII cells or parental GL261 cells could not grow in the cured mice. Therefore, the cell dose is a crucial determinant for CAR T efficacy against EGFRvIII-expressing GBM and granzyme B release is a predictive marker for the antitumor efficacy of CAR T cells.

Growth suppression of colorectal cancer expressing S492R EGFR by monoclonal antibody CH12.

Colorectal cancer (CRC) is a common malignant tumor in the digestive tract, and 30%-85% of CRCs express epidermal growth factor receptors (EGFRs). Recently, treatments using cetuximab, also named C225, an anti-EGFR monoclonal antibody, for CRC have been demonstrated to cause an S492R mutation in EGFR. However, little is known about the biological function of S492R EGFR. Therefore, we attempted to elucidate its biological function in CRC cells and explore new treatment strategies for this mutant form. Our study indicated that EGFR and S492R EGFR accelerate the growth of CRC cells in vitro and in vivo and monoclonal antibody CH12, which specifically recognizes an EGFR tumor-specific epitope, can bind efficiently to S492R EGFR. Furthermore, mAb CH12 showed significantly stronger growth suppression activities and induced a more potent antibody-dependent cellular cytotoxicity effect on CRC cells bearing S492R EGFR than mAb C225. mAb CH12 obviously suppressed the growth of CRC xenografts with S492R EGFR mutations in vivo. Thus, mAb CH12 may be a promising therapeutic agent in treating patients with CRC bearing an S492R EGFR mutation.

Target-Dependent Expression of IL12 by synNotch Receptor-Engineered NK92 Cells Increases the Antitumor Activities of CAR-T Cells.

IL12 is an immune-stimulatory cytokine for key immune cells including T cells and NK cells. However, systemic administration of IL12 has serious side effects that limit its clinical application in patients. Recently, synthetic Notch (synNotch) receptors have been developed that induce transcriptional activation and deliver therapeutic payloads in response to the reorganization of specific antigens. NK92 cell is a human natural killer (NK) cell line which has been developed as tools for adjuvant immunotherapy of cancer. Here, we explored the possibility of using synNotch receptor-engineered NK92 cells to selectively secrete IL12 at the tumor site and increase the antitumor activities of chimeric antigen receptor (CAR)-modified T cells. Compared with the nuclear factor of activated T-cells (NFATs) responsive promoter, which is another regulatory element, the synNotch receptor was better at controlling the expression of cytokines. NK92 cells transduced with the GPC3-specific synNotch receptor could produce the proinflammatory cytokine IL12 (GPC3-Syn-IL12-NK92) in response to GPC3 antigen expressed in cancer cells. In vivo GPC3-Syn-IL12-NK92 cells controlling IL12 production could enhance the antitumor ability of GPC3-redirected CAR T cells and increase the infiltration of T cells without inducing toxicity. Taken together, our results demonstrated that IL12 supplementation by synNotch-engineered NK92 cells could secrete IL12 in a target-dependent manner, and promote the antitumor efficiency of CAR-T cells. Local expression of IL12 by synNotch-engineered NK92 cells might be a safe approach to enhance the clinical outcome of CAR-T cell therapy.

Claudin18.2-Specific Chimeric Antigen Receptor Engineered T Cells for the Treatment of Gastric Cancer.

BACKGROUND: Claudin18.2 (CLDN18.2), a gastric-specific membrane protein, has been regarded as a potential therapeutic target for gastric cancer and other cancer types. The aim of our study was to elucidate whether chimeric antigen receptor T (CAR T) cells redirected to CLDN18.2 have the potential to be used in the treatment of this deadly disease. METHODS: CLDN18.2-specific humanized antibodies were developed using hybridoma and humanization technology. CLDN18.2-specific CAR T cells were prepared by lentiviral vector transduction. In vitro antitumor activities and cytokine production of the CAR T cells to gastric cancer cell lines were examined by cytotoxicity and ELISA assay. In vivo antitumor activities of CAR T cells were evaluated in mice bearing gastric cancer cell line and patient-derived tumor xenograft (PDX) models (n ≥ 6 mice per group). All statistical tests were two-sided. RESULTS: Humanized CLDN18.2-specific hu8E5 and hu8E5-2I single-chain fragment variables (scFv) were successfully developed. CLDN18.2-specific CAR T cells were developed using hu8E5 or hu8E5-2I scFv as targeting moieties. Both hu8E5-28Z and hu8E5-2I-28Z CAR T cells comprising the CD28 costimulatory domain potently suppressed tumor growth in a cancer cell line xenograft mouse model (mean [SD] tumor volume: hu8E5-28Z = 118.0 [108.6] mm3 and hu8E5-2I-28Z group = 75.5 [118.7] mm3 vs untransduced T cell group = 731.8 [206.3] mm3 at day 29 after tumor inoculation, P < .001). Partial or complete tumor elimination was observed in CLDN18.2-positive gastric cancer PDX models treated with the hu8E5-2I-28Z CAR T cells (P < .001), which persist well in vivo and infiltrate efficiently into the tumor tissues. Although the CLDN18.2 CAR T cells could lyse target cells expressing murine CLDN18.2 (mCLDN18.2), no obvious deleterious effect on the normal organs including the gastric tissues was observed in the mice. CONCLUSIONS: CLDN18.2-specific CAR T cells could be a promising treatment strategy for gastric cancer and potentially other CLDN18.2-positive tumors.

Disruption of PD-1 Enhanced the Anti-tumor Activity of Chimeric Antigen Receptor T Cells Against Hepatocellular Carcinoma.

Cancer immunotherapy has made unprecedented breakthrough in the fields of chimeric antigen receptor-redirected T (CAR T) cell therapy and immune modulation. Combination of CAR modification and the disruption of endogenous inhibitory immune checkpoints on T cells represent a promising immunotherapeutic modality for cancer treatment. However, the potential for the treatment of hepatocellular carcinoma (HCC) has not been explored. In this study, the gene expressing the programmed death 1 receptor (PD-1) on the Glypican-3 (GPC3)-targeted second-generation CAR T cells employing CD28 as the co-stimulatory domain was disrupted using the CRISPR/Cas9 gene-editing system. It was found that, in vitro, the CAR T cells with the deficient PD-1 showed the stronger CAR-dependent anti-tumor activity against native programmed death 1 ligand 1-expressing HCC cell PLC/PRF/5 compared with the wild-type CAR T cells, and meanwhile, the CD4 and CD8 subsets, and activation status of CAR T cells were stable with the disruption of endogenous PD-1. Additionally, the disruption of PD-1 could protect the GPC3-CAR T cells from exhaustion when combating with native PD-L1-expressing HCC, as the levels of Akt phosphorylation and anti-apoptotic protein Bcl-xL expression in PD-1 deficient GPC3-CAR T cells were significantly higher than those in wild-type GPC3-CAR T cells after coculturing with PLC/PRF/5. Furthermore, the in vivo anti-tumor activity of the CAR T cells with the deficient PD-1 was investigated using the subcutaneous xenograft tumor model established by the injection of PLC/PRF/5 into NOD-scid-IL-2Rγ-/- (NSG) mice. The results indicated that the disruption of PD-1 enhanced the in vivo anti-tumor activity of CAR T cells against HCC, improved the persistence and infiltration of CAR T cells in the NSG mice bearing the tumor, and strengthened the inhibition of tumor-related genes expression in the xenograft tumors caused by the GPC3-CAR T cells. This study indicates the enhanced anti-tumor efficacy of PD-1-deficient CAR T cells against HCC and suggests the potential of precision gene editing on the immune checkpoints to enhance the CAR T cell therapies against HCC.

Selective Targeting of Glioblastoma with EGFRvIII/EGFR Bitargeted Chimeric Antigen Receptor T Cell.

The heterogeneous expression of EGFRvIII [variant III mutant of epidermal growth factor receptor (EGFR)] in glioblastoma has significant impact on the clinical response to the treatment of EGFRvIII-specific chimeric antigen receptor-engineered T (CAR T) cells. We hypothesized that CAR T cells that could target both EGFRvIII and the form of EGFR expressed on tumor cells, but not EGFR on normal cells, would greatly improve efficacy without inducing on-target, off-tumor toxicity. Therefore, we developed a humanized single-chain antibody, M27, with a single specificity that binds to an epitope found both on wild-type EGFR- and EGFRvIII-overexpressing tumor cells, but not EGFR-expressing normal cells, including primary keratinocytes, on which wild-type EGFR is highly expressed. M27-derived CAR T cells effectively lysed EGFRvIII- or EGFR-overexpressing tumor cells, but showed no observable toxicity on normal cells. Inclusion of the CD137 (4-1BB) costimulatory intracellular domain in the M27-28BBZ CAR provided CAR T cells with higher tumor lysis activity than when not included (as in the M27-28Z CAR). The growth of established EGFR- or EGFRvIII-overexpressing glioma xenografts was suppressed by M27-28BBZ CAR T cells as well. The growth of heterogeneic xenograft tumors, created by mixing EGFR- and EGFR-overexpressing glioblastoma cells, was also effectively inhibited by M27-28BBZ CAR T cells. The survival of mice in the orthotopic models was significantly prolonged after M27-28BBZ CAR T-cell infusion. These results suggested that tumor-selective, bitargeted anti-EGFR/EGFRvIII CAR T cells may be a promising modality for the treatment of patients with EGFR/EGFRvIII-overexpressing glioblastoma. Cancer Immunol Res; 6(11); 1314-26. ©2018 AACR.

Increased antitumor activities of glypican-3-specific chimeric antigen receptor-modified T cells by coexpression of a soluble PD1-CH3 fusion protein.

Our recent clinical study demonstrated that glypican-3 (GPC3)-specific chimeric antigen receptor-modified T (CAR-T) cells are a promising treatment for hepatocellular carcinoma (HCC). However, the interaction of programmed cell death 1 (PD-1) and PD-L1-mediated T-cell inhibition is involved in immune evasion in a wide range of solid tumors, including HCC. To overcome this problem, we introduced a fusion protein composed of a PD-1 extracellular domain and CH3 from IgG4 into GPC3-specific CAR-T cells (GPC3-28Z) to block the PD-1/PD-L1 pathway. GPC3-specific CAR-T cells carrying the PD-1-CH3 fusion protein (sPD1) specifically recognized and lysed GPC3-positive HCC cells. The proliferation capacity of GPC3-28Z-sPD1 T cells after weekly stimulation with target cells was much higher than that of control GPC3-28Z T cells. Additionally, the coexpression of sPD1 could protect CAR-T cells from exhaustion when incubated with target cells, as phosphorylated AKT and Bcl-xL expression levels were higher in GPC3-28Z-sPD1 T cells than in GPC3-28Z cells. Importantly, in two HCC tumor xenograft models, GPC3-28Z-sPD1 T cells displayed a significantly higher tumor suppression capacity than GPC3-28Z T cells. In addition, an increased number of CD3+ T cells in the circulation and tumors and increased granzyme B levels and decreased Ki67 expression levels in the tumors were observed in the mice treated with GPC3-28Z-sPD1 T cells. Together, these data indicated that GPC3-specific CAR-T cells carrying sPD1 show promise as a treatment for patients with HCC.

The activation of Toll-like receptor 4 reverses tumor differentiation in human glioma U251 cells via Notch pathway.

Toll-like receptors (TLRs) are closely related to cancer. However, the mechanism for TLR regulation of cancer is not fully understood. Our previous studies demonstrated that toll-like receptor (TLR) 4 functions to maintain the un-differential stem cell phenotypes of human endothelial progenitor cells. In this study, we found that human glioma cells expressed several TLRs. The activation of TLR4 by LPS in glioma U251 cells induced the expression of cytokines, including IL-1ß, IL-6, IL-8, and TNFα, suggesting the functional expression of TLR4. Nude mouse in vivo studies showed that LPS treatment promoted tumor growth, and decreased mouse survival. But LPS treatment did not promote tumor cell proliferation in vitro. Meanwhile, we found that LPS treatment down-regulated the expression of glial fibrillary acidic protein (GFAP), an important differentiation maker of glioma, at both mRNA and protein levels. TLR4 activation also down-regulated GFAP in glioma Hs683 cells. LPS did not induce the activation of MAPKs, but induced the activation of NF-κB. However, pharmacological inhibition of NF-κB signaling did not reverse the down-regulation of GFAP. Furthermore, we found that LPS induced the activation of Notch pathway, which was MyD88-dependent, and Notch inhibition reversed the down-regulation of GFAP. In addition, LPS treatment up-regulated stem cell makers, including CD34 and CD133. Taken together, these results suggested that in human glioma U251 cells, TLR4 functions to reverse tumor differentiation, and it may be a target for glioma prevention and therapy.