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Antitumor treatments based on the infusion of T cells expressing chimeric antigen receptors (CAR T cells) are still relatively ineffective for solid tumors, due to the presence of immunosuppressive mediators [such as prostaglandin E2 (PGE2) and adenosine] and poor T-cell trafficking. PGE2 and adenosine activate protein kinase A (PKA), which then inhibits T-cell receptor (TCR) activation. This inhibition process requires PKA to localize to the immune synapse via binding to the membrane protein ezrin. We generated CAR T cells that expressed a small peptide called the “regulatory subunit I anchoring disruptor” (RIAD) that inhibits the association of PKA with ezrin, thus blunting the negative effects of PKA on TCR activation. After exposure to PGE2 or adenosine in vitro, CAR-RIAD T cells showed increased TCR signaling, released more cytokines, and showed enhanced killing of tumor cells compared with CAR T cells. When injected into tumor-bearing mice, the antitumor efficacy of murine and human CAR-RIAD T cells was enhanced compared with that of CAR T cells, due to resistance to tumor-induced hypofunction and increased T-cell infiltration of established tumors. Subsequent in vitro assays showed that both mouse and human CAR-RIAD cells migrated more efficiently than CAR cells did in response to the chemokine CXCL10 and also had better adhesion to various matrices. Thus, the intracellular addition of the RIAD peptide to adoptively transferred CAR T cells augments their efficacy by increasing their effector function and by improving trafficking into tumor sites. This treatment strategy, therefore, shows potential clinical application for treating solid tumors. Cancer Immunol Res; 4(6); 541–51. ©2016 AACR.

Supplementary Figure 3 - PDF file 97K, HIF1{alpha} and PLOD2 mediate sarcoma cell migration

Supplementary Figure 1 - PDF file 122K, Sarcoma cell lung colonization is dependent on HIF1{alpha} and PLOD2

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The majority of chimeric antigen receptor (CAR) T-cell research has focused on attacking cancer cells. Here, we show that targeting the tumor-promoting, nontransformed stromal cells using CAR T cells may offer several advantages. We developed a retroviral CAR construct specific for the mouse fibroblast activation protein (FAP), comprising a single-chain Fv FAP [monoclonal antibody (mAb) 73.3] with the CD8α hinge and transmembrane regions, and the human CD3ζ and 4-1BB activation domains. The transduced muFAP-CAR mouse T cells secreted IFN-γ and killed FAP-expressing 3T3 target cells specifically. Adoptively transferred 73.3-FAP-CAR mouse T cells selectively reduced FAPhi stromal cells and inhibited the growth of multiple types of subcutaneously transplanted tumors in wild-type, but not FAP-null immune-competent syngeneic mice. The antitumor effects could be augmented by multiple injections of the CAR T cells, by using CAR T cells with a deficiency in diacylglycerol kinase, or by combination with a vaccine. A major mechanism of action of the muFAP-CAR T cells was the augmentation of the endogenous CD8+ T-cell antitumor responses. Off-tumor toxicity in our models was minimal following muFAP-CAR T-cell therapy. In summary, inhibiting tumor growth by targeting tumor stroma with adoptively transferred CAR T cells directed to FAP can be safe and effective, suggesting that further clinical development of anti-human FAP-CAR is warranted. Cancer Immunol Res; 2(2); 154–66. ©2013 AACR.

CD19-specific KIR-CAR/Dap12 shows antigen-specific cytotoxicity in vitro.

Supplementary Figure 8 - PDF file 134K, Tumor cells are the major in vivo source of sarcoma collagen

PDF file - 1196K, Supplementary Figure 1. Phenotypic analysis of FAP+ stromal cells in untreated TC1 flank tumors in C57BL/6 mice. Supplementary Figure 2. Depletion of FAP+ cells. Supplementary Figure 3. Persistence and antitumor activities of FAP-CAR T cells employing either human 4-1BB (73.3-hBBz) or mouse CD28 (73.3-m28z) co-stimulatory domain in mice and their in vitro activity. Supplementary Figure 4. Deletion of DGKzeta enhanced effector functions and in vivo persistence of FAP-CAR T cells. Supplementary Figure 5. Body weight of FAP-CAR T cells-treated mice remained constant or increased. Supplementary Figure 6. Histology of bone marrows and pancreas following treatment with FAP-CAR T cells. Supplementary Figure 7. Activation-induced cell death of FAP-CAR T cells. Supplementary Figure 8. Differential FAP expression on tumor and pancreatic FAP+ stromal cells. Supplementary Figure 9. mAb 73.3 and mAb FAP5 are specific for distinct epitopes of murine FAP expressed by fibroblasts.

SS1-KIRS2 does not affect endogenous TCR expression.

Figure legends for supplemental data.

SS1-KIRS2/DAP12 T cells mediate robust anti-tumor activity in vivo.

Supplementary Figure 7 - PDF file 786K, Minoxidil treatment does not affect primary tumor volume or overall animal weight

Supplementary Figure 4 - PDF file 151K,HIF1{alpha} and PLOD2 mediate migration via a cell extrinsic mechanism in KP cells

Chimeric antigen receptors (CAR) bearing an antigen-binding domain linked in cis to the cytoplasmic domains of CD3ζ and costimulatory receptors have provided a potent method for engineering T-cell cytotoxicity toward B-cell leukemia and lymphoma. However, resistance to immunotherapy due to loss of T-cell effector function remains a significant barrier, especially in solid malignancies. We describe an alternative chimeric immunoreceptor design in which we have fused a single-chain variable fragment for antigen recognition to the transmembrane and cytoplasmic domains of KIR2DS2, a stimulatory killer immunoglobulin-like receptor (KIR). We show that this simple, KIR-based CAR (KIR-CAR) triggers robust antigen-specific proliferation and effector function in vitro when introduced into human T cells with DAP12, an immunotyrosine-based activation motifs-containing adaptor. T cells modified to express a KIR-CAR and DAP12 exhibit superior antitumor activity compared with standard first- and second-generation CD3ζ-based CARs in a xenograft model of mesothelioma highly resistant to immunotherapy. The enhanced antitumor activity is associated with improved retention of chimeric immunoreceptor expression and improved effector function of isolated tumor-infiltrating lymphocytes. These results support the exploration of KIR-CARs for adoptive T-cell immunotherapy, particularly in immunotherapy-resistant solid tumors. Cancer Immunol Res; 3(7); 815–26. ©2015 AACR.

Supplementary Figure 5 - PDF file 173K, HIF1{alpha} and PLOD2 mediate migration via a cell extrinsic mechanism in HT-1080 cells

Cytotoxicity induced by SS1-KIRS2, but not receptor expression at the cell surface, depends upon DAP12 co-expression.

Supplementary Figure 2 - PDF file 124K, HIF1{alpha} is not required for primary sarcoma formation

Supplementary Figure 6 - PDF file 110K, Expression of WT and mutant PLOD2 in sarcoma cells

Mesothelin-specific CD3ζ CARs and SS1-KIRS2/Dap12 show comparable antigen-specific in vitro cytotoxicity toward EM-meso cells.

Supplementary Figure Legends

Supplementary Figure Legends - PDF file 104K, Supplementary Figure Legends

FAP-specific KIR-CAR/Dap12 T cells induce bone marrow hypocellularity and loss of body weight.

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Supplemental Figure 1. Schematic representations of viral-engineered constructs for transductions of T cells. Supplemental Figure 2. Successful transduction of primary T cells and detection of transgenes. Supplemental Figure 3. Phenotypic characterization of human CAR T cells at baseline. Supplemental Figure 4. Phenotypic characterization of murine CAR T cells at baseline. Supplemental Figure 5. Heightened cytokine production by CAR-RIAD T cells upon in vitro restimulation with mesothelin-coated beads. Supplemental Figure 6. Densitometry analyses of signaling proteins. Supplemental Figure 7. Expression of PGE2 in our tumor cells and the effect of mesoCAR-RIAD T cells on tumors that do not express the antigen mesothelin. Supplemental Figure 8. Greater influx of CD4+ CAR T cells in human and murine tumors.

Supplementary Data from Monitoring Therapeutic Response to Anti-FAP CAR T Cells Using [18F]AlF-FAPI-74

Purpose:Despite the success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies, successful targeting of solid tumors with CAR T cells has been limited by a lack of durable responses and reports of toxicities. Our understanding of the limited therapeutic efficacy in solid tumors could be improved with quantitative tools that allow characterization of CAR T–targeted antigens in tumors and accurate monitoring of response.Experimental Design:We used a radiolabeled FAP inhibitor (FAPI) [18F]AlF-FAPI-74 probe to complement ongoing efforts to develop and optimize FAP CAR T cells. The selectivity of the radiotracer for FAP was characterized in vitro, and its ability to monitor changes in FAP expression was evaluated using rodent models of lung cancer.Results:[18F]AlF-FAPI-74 showed selective retention in FAP+ cells in vitro, with effective blocking of the uptake in presence of unlabeled FAPI. In vivo, [18F]AlF-FAPI-74 was able to detect FAP expression on tumor cells as well as FAP+ stromal cells in the tumor microenvironment with a high target-to-background ratio. We further demonstrated the utility of the tracer to monitor changes in FAP expression following FAP CAR T-cell therapy, and the PET imaging findings showed a robust correlation with ex vivo analyses.Conclusions:This noninvasive imaging approach to interrogate the tumor microenvironment represents an innovative pairing of a diagnostic PET probe with solid tumor CAR T-cell therapy and has the potential to serve as a predictive and pharmacodynamic response biomarker for FAP as well as other stroma-targeted therapies. A PET imaging approach targeting FAP expressed on activated fibroblasts of the tumor stroma has the potential to predict and monitor therapeutic response to FAP-targeted CAR T-cell therapy.See related commentary by Weber et al., p. 5241

Supplementary Figure from Monitoring Therapeutic Response to Anti-FAP CAR T Cells Using [18F]AlF-FAPI-74

Making CAR T cells in vivo Cardiac fibrosis is the stiffening and scarring of heart tissue and can be fatal. Rurik et al . designed an immunotherapy strategy to generate transient chimeric antigen receptor (CAR) T cells that can recognize the fibrotic cells in the heart (see the Perspective by Gao and Chen). By injecting CD5-targeted lipid nanoparticles containing the messenger RNA (mRNA) instructions needed to reprogram T lymphocytes, the researchers were able to generate therapeutic CAR T cells entirely inside the body. Analysis of a mouse model of heart disease revealed that the approach was successful in reducing fibrosis and restoring cardiac function. The ability to produce CAR T cells in vivo using modified mRNA may have a number of therapeutic applications. —PNK

PDF file - 37K, CAR spleen infiltrating lymphocytes have lower levels of inhibitory receptors than CAR tumor infiltrating lymphocytes.

Effects of caNFATc1 expression in CN-/- fibroblasts on collagen remodeling.

PDF file - 38K, CAR TILs upregulate inhibitory receptors.

Purpose: Immunotherapy using vaccines or adoptively transferred tumor-infiltrating lymphocytes (TIL) is limited by T-cell functional inactivation within the solid tumor microenvironment. The purpose of this study was to determine whether a similar tumor-induced inhibition occurred with genetically modified cytotoxic T cells expressing chimeric antigen receptors (CAR) targeting tumor-associated antigens.Experimental Design: Human T cells expressing CAR targeting mesothelin or fibroblast activation protein and containing CD3ζ and 4–1BB cytoplasmic domains were intravenously injected into immunodeficient mice bearing large, established human mesothelin-expressing flank tumors. CAR TILs were isolated from tumors at various time points and evaluated for effector functions and status of inhibitory pathways.Results: CAR T cells were able to traffic into tumors with varying efficiency and proliferate. They were able to slow tumor growth, but did not cause regressions or cures. The CAR TILs underwent rapid loss of functional activity that limited their therapeutic efficacy. This hypofunction was reversible when the T cells were isolated away from the tumor. The cause of the hypofunction seemed to be multifactorial and was associated with upregulation of intrinsic T-cell inhibitory enzymes (diacylglycerol kinase and SHP-1) and the expression of surface inhibitory receptors (PD1, LAG3, TIM3, and 2B4).Conclusions: Advanced-generation human CAR T cells are reversibly inactivated within the solid tumor microenvironment of some tumors by multiple mechanisms. The model described here will be an important tool for testing T cell–based strategies or systemic approaches to overcome this tumor-induced inhibition. Our results suggest that PD1 pathway antagonism may augment human CAR T-cell function. Clin Cancer Res; 20(16); 4262–73. ©2014 AACR.

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Live cell imaging of PDACs on FDMs.

PDF file - 756K, Supplemental Fig 1. EMP and EMMESO mesothelin expression. Supplemental Fig 2. EMMESO flank tumors retain antigen expression in vivo. Supplemental Fig 3. MesoCAR TILs are unable to secrete IL-2 in response to antigen. Supplemental Fig 4. Supplemental IL-2 helps but is not necessary for CAR TIL recovery. Supplemental Fig 5. IFNγ and TNFα secretion by CAR TILs recovers after resting away from tumor.

Fibroblast activation is a crucial step in tumor growth and metastatic progression. Activated fibroblasts remodel the extracellular matrix (ECM) in primary tumor and metastatic microenvironments, exerting both pro- and antitumorigenic effects. However, the intrinsic mechanisms that regulate the activation of fibroblasts are not well-defined. The signaling axis comprising the calcium-activated Ser/Thr phosphatase calcineurin (CN), and its downstream target nuclear factor of activated T cells, has been implicated in endothelial (EC) and immune cell activation, but its role in fibroblasts is not known. Here, we demonstrate that deletion of CN in fibroblasts in vitro altered fibroblast morphology and function consistent with an activated phenotype relative to wild-type fibroblasts. CN-null fibroblasts had a greater migratory capacity, increased collagen secretion and remodeling, and promoted more robust EC activation in vitro. ECM generated by CN-null fibroblasts contained more collagen with greater alignment of fibrillar collagen compared with wild-type fibroblast-derived matrix. These differences in matrix composition and organization imposed distinct changes in morphology and cytoskeletal architecture of both fibroblasts and tumor cells. Consistent with this in vitro phenotype, mice with stromal CN deletion had a greater incidence and larger lung metastases. Our data suggest that CN signaling contributes to the maintenance of fibroblast homeostasis and that loss of CN is sufficient to promote fibroblast activation.Significance:Calcineurin signaling is a key pathway underlying fibroblast homeostasis that could be targeted to potentially prevent fibroblast activation in distant metastatic sites.

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FAP-CAR T cells restrict AE17.OVA tumor growth through reduced proliferation, increased apoptosis and altered tumor stroma.

Alteration of tumor structure by FAP-CAR T cells in A549 tumors.

PDF file, 8785K, Notch1 ablation does not induce DNA damage in vitro.

Combination of FAP-CAR T cells and gemcitabine treatment augments anti-tumor response in 4662 tumors.

Reagents for immunohistochemistry studies.

PDF file, 346K, Notch1 ablation does not affect p53/MDM2 binding.