Mesothelin CAR T Cells Secreting Anti-FAP/Anti-CD3 Molecules Efficiently Target Pancreatic Adenocarcinoma and its Stroma.

Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.

Wehrli M ，Guinn S ，Birocchi F ，Kuo A ，Sun Y ，Larson RC ，Almazan AJ ，Scarfò I ，Bouffard AA ，Bailey SR ，Anekal PV ，Montero Llopis P ，Nieman LT ，Song Y ，Xu KH ，Berger TR ，Kann MC ，Leick MB ，Silva H ，Salas-Benito D ，Kienka T ，Grauwet K ，Armstrong TD ，Zhang R ，Zhu Q ，Fu J ，Schmidts A ，Korell F ，Jan M ，Choi BD ，Liss AS ，Boland GM ，Ting DT ，Burkhart RA ，Jenkins RW ，Zheng L ，Jaffee EM ，Zimmerman JW ，Maus MV ... -  《-》

Chimeric antigen receptor macrophages targeting c-MET(CAR-M-c-MET) inhibit pancreatic cancer progression and improve cytotoxic chemotherapeutic efficacy.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors. Macrophages are abundant in the tumor microenvironment, making them an attractive target for therapeutic intervention. While current immunotherapies, including immune checkpoint inhibition (ICI) and chimeric antigen receptor T (CAR-T) cells, have shown limited efficacy in pancreatic cancer, a novel approach involving chimeric antigen receptor macrophages (CAR-M) has, although promising, not been explored in pancreatic cancer. In this study, we first investigated the role of CAR-M cells targeting c-MET in pancreatic cancer. The effectiveness and rationality of c-MET as a target for CAR-M in pancreatic cancer were validated through bioinformatic analyses and immunohistochemical staining of samples from pancreatic cancer patients. We utilized flow cytometry and bioluminescence detection methods to demonstrate the specific binding and phagocytic killing effect of CAR-M on pancreatic cancer cells. Additionally, we observed the process of CAR-M engulfing pancreatic cancer cells using confocal microscopy and a long-term fluorescence live cell imaging system. In an in situ tumor model transplanted into NOD/SCID mice, we administered intraperitoneal injections of CAR-M to confirm its inhibitory function on pancreatic cancer. Furthermore, we validated these findings in human monocyte-derived macrophages (hMDM). Bioinformatics and tumor tissue microarray analyses revealed significantly higher expression levels of c-MET in tumor tissues, compared to the paired peritumoral tissues, and higher c-MET expression correlated with worse patient survival. CAR-M cells were engineered using human monocytic THP-1 cell line and hMDM targeting c-MET (CAR-M-c-MET). The CAR-M-c-MET cells demonstrated highly specific binding to pancreatic cancer cells and exhibited more phagocytosis and killing abilities than the pro-inflammatory polarized control macrophages. In addition, CAR-M-c-MET cells synergized with various cytotoxic chemotherapeutic drugs. In a NOD/SCID murine model, intraperitoneally injected CAR-M-c-MET cells rapidly migrated to tumor tissue and substantially inhibited tumor growth, which did not lead to obvious side effects. Cytokine arrays and mRNA sequencing showed that CAR-M-c-MET produced higher levels of immune activators than control macrophages. This study provides compelling evidence for the safety and efficacy of CAR-M therapy in treating pancreatic cancer. The results demonstrate that CAR-M-c-MET significantly suppresses pancreatic cancer progression and enhances the effectiveness of cytotoxic chemotherapy. Remarkably, no discernible side effects occur. Further clinical trials are warranted in human pancreatic cancer patients.

Zheng H ，Yang X ，Huang N ，Yuan S ，Li J ，Liu X ，Jiang Q ，Wu S ，Ju Y ，Kleeff J ，Yin X ，Liao Q ，Liu Q ，Zhao Y ... -  《Molecular Cancer》

Novel banana lectin CAR-T cells to target pancreatic tumors and tumor-associated stroma.

Cell therapies for solid tumors are thwarted by the hostile tumor microenvironment (TME) and by heterogeneous expression of tumor target antigens. We address both limitations with a novel class of chimeric antigen receptors based on plant lectins, which recognize the aberrant sugar residues that are a 'hallmark' of both malignant and associated stromal cells. We have expressed in T cells a modified lectin from banana, H84T BanLec, attached to a chimeric antigen receptor (H84T-CAR) that recognizes high-mannose (asparagine residue with five to nine mannoses). Here, we tested the efficacy of our novel H84T CAR in models of pancreatic ductal adenocarcinoma (PDAC), intractable tumors with aberrant glycosylation and characterized by desmoplastic stroma largely contributed by pancreatic stellate cells (PSCs). We transduced human T cells with a second-generation retroviral construct expressing the H84T BanLec chimeric receptor, measured T-cell expansion, characterized T-cell phenotype, and tested their efficacy against PDAC tumor cells lines by flow cytometry quantification. In three-dimensional (3D) spheroid models, we measured H84T CAR T-cell disruption of PSC architecture, and T-cell infiltration by live imaging. We tested the activity of H84T CAR T cells against tumor xenografts derived from three PDAC cell lines. Antitumor activity was quantified by caliper measurement and bioluminescence signal and used anti-human vimentin to measure residual PSCs. H84T BanLec CAR was successfully transduced and expressed by T cells which had robust expansion and retained central memory phenotype in both CD4 and CD8 compartments. H84T CAR T cells targeted and eliminated PDAC tumor cell lines. They also disrupted PSC architecture in 3D models in vitro and reduced total tumor and stroma cells in mixed co-cultures. H84T CAR T cells exhibited improved T-cell infiltration in multicellular spheroids and had potent antitumor effects in the xenograft models. We observed no adverse effects against normal tissues. T cells expressing H84T CAR target malignant cells and their stroma in PDAC tumor models. The incorporation of glycan-targeting lectins within CARs thus extends their activity to include both malignant cells and their supporting stromal cells, disrupting the TME that otherwise diminishes the activity of cellular therapies against solid tumors.

McKenna MK ，Ozcan A ，Brenner D ，Watanabe N ，Legendre M ，Thomas DG ，Ashwood C ，Cummings RD ，Bonifant C ，Markovitz DM ，Brenner MK ... -  《Journal for ImmunoTherapy of Cancer》

Therapeutic Efficacy of IL7/CCL19-Expressing CAR-T Cells in Intractable Solid Tumor Models of Glioblastoma and Pancreatic Cancer.

Cancer immunotherapy using immune checkpoint inhibitors and its combination with other anticancer therapies has emerged as a new standard of care because of the encouraging therapeutic effects in various solid cancers. Nonetheless, glioblastoma and pancreatic cancer remain resistant to immunotherapy and represent intractable cancers with the poorest prognosis. We investigated the therapeutic effects of next-generation chimeric antigen receptor (CAR) T cells producing IL7 and chemokine (C-C motif) ligand 19 (CCL19; referred to as 7 × 19 CAR-T) in these intractable cancers. Cytotoxic activities and therapeutic effects of 7 × 19 CAR-T were evaluated in vitro and in vivo, in a model using EGFR variant III (EGFRvIII)-positive glioblastoma and anti-EGFRvIII CAR-T generated from healthy donor peripheral blood mononuclear cells (PBMC), or a model using HER2-positive pancreatic cancer organoids and anti-HER2 CAR-T generated from the same patient's PBMC. Anti-EGFRvIII 7 × 19 CAR-T exhibited cytotoxic activity specific to EGFRvIII-positive tumor, induced complete rejection of glioblastoma with massive T-cell infiltration and tumor cell death in the tumor tissues, and consequently prolonged mouse survival. Anti-HER2 7 × 19 CAR-T demonstrated a potent cytotoxic activity against autologous HER2-positive pancreatic cancer organoids and induced complete rejection of autologous tumor along with prolonged mouse survival. Our results suggest that 7 × 19 CAR-T could become a therapeutic option for glioblastoma and pancreatic cancer. To the best of our knowledge, this is the first study to demonstrate the therapeutic efficacy of next-generation CAR-T in an autologous model using patient-derived tumor organoids and CAR-T generated from the same patient's PBMC, in which unwanted allogeneic immune responses are fully excluded. Despite the clinical development of CAR T-cell therapy, its efficacy in solid cancers has yet to be established. This study explored the therapeutic potential and immunologic mechanisms of IL7/CCL19-producing CAR-T therapy in preclinical solid cancer models of glioblastoma and pancreatic cancer. We found that IL7/CCL19-producing CAR-T cells generated from the patient's PBMC showed potent therapeutic effects against the solid cancer model established by inoculating organoids from the autologous tumor tissue.

Ohta K ，Sakoda Y ，Adachi K ，Shinozaki T ，Nakajima M ，Yasuda H ，Nagano H ，Tamada K ... -  《-》

Pancreatic cancer cell-intrinsic transglutaminase-2 promotes T cell suppression through microtubule-dependent secretion of immunosuppressive cytokines.

Pancreatic ductal adenocarcinoma (PDAC) is mostly refractory to immunotherapy due to immunosuppression in the tumor microenvironment and cancer cell-intrinsic T cell tolerance mechanisms. PDAC is described as a "cold" tumor type with poor infiltration by T cells and factors leading to intratumoral T cell suppression have thus received less attention. Here, we identify a cancer cell-intrinsic mechanism that contributes to a T cell-resistant phenotype and describes potential combinatorial therapy. We used an unbiased screening approach of T cell resistant and sensitive murine KPC (KrasLSL-G12D/+; Trp53fl/fl; Ptf1aCre/+ ) PDAC cells in a three-dimensional co-culture platform with syngeneic antigen-educated T cells to identify potential cell-intrinsic drivers of T cell suppression in PDAC. Comparative transcriptomic analysis was performed to reveal promising candidates that mediate resistance to T cells. We investigated their contribution by shRNA-mediated knockdown and pharmacological inhibition in murine in vitro and in vivo studies, as well as in patient-derived organoids (PDOs). A combination of transcriptomic analyses, cytometric and immunohistochemistry techniques allowed us to validate the underlying T cell response phenotypes of PDAC cells. The action of TGM2 via interaction with tubulin and the impact of microtubule dynamics and vesicle trafficking were evaluated by protein analyses and live-cell imaging. Correlation analyses via TCGA data complemented the functional studies. We identified transglutaminase 2 (TGM2) as a mediator of T cell suppression in PDAC. We report that high levels of TGM2 expression in patients' tumors correlate with immunosuppressive signatures and poor overall survival. We found that TGM2 regulates vesicle trafficking by modulating microtubule network density and dynamics in pancreatic cancer cells, thus facilitating the secretion of immunosuppressive cytokines, which impair effector T cell functionality. In TGM2-expressing PDOs, pharmacological TGM2 inhibition or treatment with nocodazole increased T cell-mediated apoptosis. Also, pretreatment of TGM2high PDOs with sublethal doses of the spindle poisons paclitaxel or vincristine increased CD8+T cell activation and sensitized PDOs toward T cell-mediated cytotoxicity. These findings indicate that targeting microtubular function therapeutically may enhance antitumor T cell responses by impacting activity of immunosuppressive cytokines in the PDAC microenvironment.

Lahusen A ，Minhöfer N ，Lohse KA ，Blechner C ，Lindenmayer J ，Eiseler T ，Wellstein A ，Kleger A ，Seufferlein T ，Windhorst S ，Lin YN ... -  《Journal for ImmunoTherapy of Cancer》