Background Solid tumors present unique barriers to treatment with CAR T cells, including poor tumor infiltration into a highly immunosuppressive and metabolically challenging tumor microenvironment (TME). Objectives To enhance both CAR T cell efficacy and the overall immune response against solid tumors, this study explored the therapeutic potential of combining chimeric antigen receptor (CAR) T cells with CD40 stimulation via an agonistic CD40 antibody (αCD40). We hypothesized that CAR T cells could serve as targeted vaccines, promoting antigen release and cooperating with αCD40 to activate and mobilize the endogenous immune cells, thus "heating up" the TME and potentially rendering it more receptive to subsequent therapies. Methods We used a syngeneic mouse model of pancreatic ductal adenocarcinoma (PDAC) and further validated our findings in a triple-negative breast cancer (TNBC) mouse model Results This combined strategy was associated with enhanced anti-tumor activity over CAR T cells alone. This included rapid and sustained tumor necrosis, increased immune cell activation both systemically and within the TME, as well as an overall improvement in survival rates. Comprehensive immune profiling at early timepoints revealed mechanistic insights into the enhanced anti-tumor effects of CAR T cell therapy and αCD40 treatment. Conclusions These findings set the stage for future clinical applications of CAR T cells in combination with CD40 agonists for the treatment of challenging solid tumors.

Mesothelin is an attractive target for CAR T therapy on a number of cancer types; however, the efficacy of this therapy is diminished because the bulk of the cell surface-expressed mesothelin is shed through naturally occurring proteolysis leaving behind a short juxtamembrane peptide "stump". The two problems this creates are one, the bulk of the target protein is no longer on the tumor cell and two, soluble, shed mesothelin persists in the tumor microenvironment and circulates in blood and other body fluids, where it can bind mesothelin-targeted CAR T cells and act as a decoy that reduces engagement with tumor cell-surface mesothelin. These issues have contributed at least in part to the lack of desired efficacy in human clinical trials utilizing CAR T cells that target membrane distal regions of mesothelin (i.e., the shed domain) such as those utilizing the variable domains of anti-mesothelin monoclonal antibodies SS1 and M5. In addition, there have been safety concerns regarding the targeting of mesothelin on normal tissues. Here we describe CAR T cells that utilize novel phage display-derived antibodies specific for the mesothelin stump domain, thus being unaffected by the natural process of mesothelin shedding. Mesothelin "stump-specific" CAR T cells (CAR 422) had cytotoxicity and in vivo activity that were comparable to previously studied anti-mesothelin CAR T cells. Importantly, CAR 422 T cells were effective against tumor cells that were resistant to conventional anti-mesothelin CAR T cells and showed reduced on-target/off tumor toxicity in a human mesothelin knock-in mouse model. Thus, CAR 422 holds potential as a next-generation therapy for challenging solid tumors.

Patients can develop human anti-mouse immune responses against CD19-specific chimeric antigen receptor (CAR) T cells due to the use of a murine CD19-specific single-chain variable fragment to redirect T cells. We screened a yeast display library to identify an array of fully human CD19 single-chain variable fragment binders and performed a series of studies to select the most promising fully human CAR. We observed significant differences in the ability of CARs employing these CD19 binders to be expressed on the cell surface, induce tonic signaling, redirect T-cell function, mediate tumor killing, recognize lower levels of CD19 antigen, and maintain function upon continuous antigen exposure. From this initial analysis, CAR T cells using two binders (42 and 52) were selected for additional studies. Although CAR T cells using both binders controlled tumor growth well in vivo, we advanced a CAR construct using binder 42 for more advanced preclinical testing because of its greater similarity to binders based on the antibody FMC63, which is the murine antibody underlying four FDA-approved CD19-specific CAR T-cell therapies, and ability to robustly respond to tumors expressing lower levels of CD19. We found that this binder uniquely bound CD19 using distinct contact residues than FMC63 and with ∼40-fold lower affinity. CARs using binder 42 were non-inferior to those using the FMC63 binder in a mouse model of acute lymphoblastic leukemia, indicating that CAR T cells using binder 42 should be considered for clinical use.

BackgroundMultiplex gene-edited chimeric antigen receptor (CAR) T-cell therapies face significant challenges, including potential oncogenic risks associated with double-strand DNA breaks. Targeted microRNAs (miRNAs) may provide a safer, functional, and tunable alternative for gene silencing without the need for DNA editing.MethodsAs a proof of concept for multiplex gene silencing, we employed an optimized miRNA backbone and gene architecture to silence T-cell receptor (TCR) and major histocompatibility complex class I (MHC-I) in mesothelin-directed CAR (M5CAR) T cells. The efficacy of this approach was compared to CD3ζ and β2-microglobulin (β2M) CRISPR/Cas9 knockout (KO) cells. miRNA-expressing cassettes were incorporated into M5CAR lentiviral vectors, enabling combined gene silencing and CAR expression. Antitumor activity was evaluated using in vitro assays and in vivo pancreatic ductal adenocarcinoma models.ResultsSilenced (S) M5CAR T cells retained antitumor functionality comparable to, and in some cases exceeding, that of KO cells. In vivo, S M5CAR T cells achieved tumor control with higher persistence and superior metastasis prevention. In vitro assays demonstrated enhanced resistance to alloreactive natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs).ConclusionsTitratable multiplex gene silencing via targeted miRNAs offers an alternative to gene editing for CAR T cells, with potential advantages in potency, persistence, metastasis prevention, and immune evasion for allogeneic products. This strategy may overcome tumor-induced immunosuppression while avoiding the risks associated with DNA double-strand breaks.

Mesothelin is an attractive target for CAR-T therapy on a number of cancer types; however, the efficacy of this therapy is diminished because the bulk of the cell surface-expressed mesothelin is shed through naturally occurring proteolysis leaving behind a short juxtamembrane peptide 'stump'. The two problems this creates are (1) the bulk of the target protein is no longer on the tumor cell and (2), the free soluble shed mesothelin remains in the tumor microenvironment and becomes present in blood/other body fluids binding to the mesothelin-targeted CAR-T and interfering with their ability to target the mesothelin that remains on the surface of the tumor. These issues have likely contributed at least in part to the lack of desired efficacy of CAR-T cells that target membrane distal regions of mesothelin (i.e., the shed domain) such as those utilizing anti-mesothelin scFvs SS1 and M5. Here we describe CAR T cells that utilize novel antibodies specific for the mesothelin stump domain thus being unaffected by the natural process of mesothelin shedding. Mesothelin 'stump' specific CAR T cells (ST4-22) had cytotoxicity and in vivo activity that was comparable to standard anti-mesothelin CAR T cells. Importantly, CAR T cells expressing ST4-22 were effective against tumor cells that were resistant to standard anti-mesothelin CAR T cells.

Background This study explores the synergistic potential of combining mouse mesothelin-specific chimeric antigen receptor (mmeso-CAR) T cells and a CD40 agonist (αCD40) to enhance CAR T cell and overall immune response against pancreatic ductal adenocarcinoma (PDAC) in syngeneic mouse models. Methods The subcutaneous syngeneic PDAC model was established using a KPC-derived cell line. Mice were treated with mmeso-CAR T cells, αCD40, or combinations of both. In vivo therapeutic efficacy was evaluated in endpoint and time course models, monitoring tumor volume and histological changes over time, and assessing CAR T cell and immune cell distribution and activation in secondary lymphoid organs (SLOs): tumor draining lymph node (TdLN) and spleen, and the tumor microenvironment (TME). Our methodology encompassed real-time live cell assays, in vivo imaging, multiplex cytokine assays, multi-parametric flow cytometry, histology, RNAscope, digital spatial profiling, and scRNAseq. Additionally, the combination treatment is currently being evaluated in an orthotopic syngeneic model of triple-negative breast cancer (TNBC). Results Combining mmeso-CAR T cells with αCD40 yielded improved tumor control and long-term survival outcomes. αCD40 treatment induced significant tumor necrosis within 24 hours (39.5 ± 29.1% and 33.8 ± 20.5% of tumor area, with or without mmeso-CAR T cells, respectively). The necrotic effect persisted after seven days when combined with mmeso-CAR T cells (25.9 ± 20.7% versus 2.1 ± 3.3% with αCD40 alone), associated with a greater reduction in tumor weights and PanCK/mesothelin+ tumor areas (figure 1). αCD40 treatment promoted the expansion of mmeso-CAR T cells and modulated their activation in SLOs and within the TME. The combination therapy engaged APCs, host T and NK cells, increasing recruitment and activation in SLOs (figure 2) and in the TME, associated with elevated blood levels of pro-inflammatory cytokines and chemokines. ScRNAseq analysis on immune cells from tumor and TdLN confirmed the involvement of both CAR-dependent and independent antitumor responses. Conclusions This study unveils the synergistic effect of combining mmeso-CAR T cells and αCD40 in delaying tumor growth in a syngeneic PDAC model. The combination therapy led to rapid and sustained tumor necrosis with increased infiltration and activation of immune cells in the SLOs and the TME. Our comprehensive characterization provided valuable mechanistic insights into the underlying synergistic mechanisms. Ongoing evaluation of the mmeso-CAR T cells/αCD40 therapy in a syngeneic model of TNBC aims to assess the effectiveness in various solid tumors expressing mesothelin. These findings may open potential applications of meso-CAR T cells/αCD40 combination therapy in the clinic. Acknowledgements We express gratitude to the following facilities at the University of Pennsylvania: the Stem Cell and Xenograft Core (SCXC) for providing equipment for in vivo procedures; the Pathology Core at the Children's Hospital of Philadelphia for tissue processing, immunohistochemistry and image acquisition; the Comparative Pathology Core at the School of Veterinary Medicine for immunohistochemistry, image acquisition, and pathological assessment; and the Translational and Correlatives Studies Laboratory at the Center for Cellular Immunotherapies for support and access to the nanoString® GeoMx platform. We thank the Beatty, Stanger and Tchou Laboratories at the Center for Cellular Immunotherapies for providing PDAC and TNBC cell lines. Ethics Approval The University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) approved animal experiments (protocol n°804226). Animal procedures were performed in the animal facility at the University of Pennsylvania in accordance with Federal and Institutional IACUC requirements.

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

A challenge when targeting T-cell lymphoma with chimeric antigen receptor (CAR) T-cell therapy is that target antigens are often shared between T cells and tumor cells, resulting in fratricide between CAR T cells and on-target cytotoxicity on normal T cells. CC chemokine receptor 4 (CCR4) is highly expressed in many mature T-cell malignancies, such as adult T-cell leukemia/lymphoma (ATLL) and cutaneous T-cell lymphoma (CTCL), and has a unique expression profile in normal T cells. CCR4 is predominantly expressed by type-2 and type-17 helper T cells (Th2 and Th17) and regulatory T cells (Treg), but it is rarely expressed by other T helper (Th) subsets and CD8+ cells. Although fratricide in CAR T cells is generally thought to be detrimental to anticancer functions, in this study, we demonstrated that anti-CCR4 CAR T cells specifically depleted Th2 and Tregs, while sparing CD8+ and Th1 T cells. Moreover, fratricide increased the percentage of CAR+ T cells in the final product. CCR4-CAR T cells were characterized by high transduction efficiency, robust T-cell expansion, and rapid fratricidal depletion of CCR4-positive T cells during CAR transduction and expansion. Furthermore, mogamulizumab-based CCR4-CAR T cells induced superior antitumor efficacy and long-term remission in mice engrafted with human T-cell lymphoma cells. In summary, CCR4-depleted anti-CCR4 CAR T cells are enriched in Th1 and CD8+ T cells and exhibit high antitumor efficacy against CCR4-expressing T-cell malignancies.

PURPOSE Despite the success of chimeric antigen receptor (CAR) T cell therapy against hematological 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. DESIGN We used a radiolabeled fibroblast activation protein (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 both 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. CONCLUSION This non-invasive 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 stromal cell-targeted therapies.

AbstractPurpose: 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

Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy 1 , 2 . Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common γ-chain (γ c ) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γ c cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rβ-ECD–IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells, o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours.

Background For cellular immunotherapies, clinical outcomes depend on the proliferative potency and metabolic fitness of the therapeutic product. For their most successful indications, CAR T cells are effective 'serial killers,' each T cell recognizing and eliminating many target cells. What fuels CAR T cell serial killing is unknown. Two critical events define the efficiency of T cell serial killing: migration and immune synapse formation/cytolysis. Importantly, each event is influenced by the local metabolic milieu. Methods Using a specialized CAR T cell conditioning regimen, the goal of this research to is determine the relative energy cost of migration versus cytolysis using innovative eSIGHT RTCA technology. Our project will reveal how the spare respiratory capacity (SRC), supports CAR T cell migration and/or cytolysis, founded on the hypothesis that cells that can replenish their SRC in repetitive antigen stimulation models, are more efficient serial killers. Results Our data sheds light on critical metabolic states that impair CAR T cell cytolytic activity. We also use multi-omic approaches to identify arginosuccinate synthase 1 (ASS1), a gene distinguishable at the metabolic (Seahorse), transcriptional (RNAseq), epigenetic level (ATAC seq), and functional (tumor clearance in vivo) in CAR T cells. Supporting the premise of our work, we show that ASS1 supports high SRC levels despite frequent antigen encounter in repetitive stimulation models in vitro. In parallel work, we provide data that reductive glutamine metabolism is enhanced in 28zCARTs, suggesting mechanisms for why 28zCARTs outperform BBz CARTs in some hypoxic tumor models (figure1B). We also show that expressing isoforms of the GOT family of amino transaminases enhance CAR T cell anti-tumor function (figure 1A&C). Given the prior link connecting reductive glutamine metabolism and GOT1 in Jurkat cells we hypothesize a fundamental link, involving reductive glutamine metabolism, GOT1-mediated aspartate replenishment, and fumarate production via ASS1, to support mitochondrial function and serial killing in CAR T cells (figure 1D). Conclusions Our findings reveal unique conditioning and genetic strategies to arm CAR T cells with unique metabolic attributes against solid tumors.

Two of the major obstacles that adoptive cell transfer immunotherapy needs to overcome to be successful in solid tumors are 1) the immunosuppressive tumor microenvironment (TME) and 2) the lack of robust biomarkers that allow for the identification of patients that would benefit from the therapy and for monitoring of the treatment response. Here we paired a CAR T cell therapy with a companion Positron Emission Tomography (PET) imaging approach that allows for serial, non-invasive, whole-body visualization of a biological target of interest -in this specific case, Fibroblast Activating Protein (FAP). FAP is a cell surface serine protease that is highly expressed by cancer associated fibroblasts in the TME that participate in the generation of the immunosuppressive stromagenic response in solid tumors. We designed a novel FAP CAR construct based on the scFv of the 4G5 antibody developed, in house, against canine FAP that cross-reacts against mouse and human FAP. In this study, we utilized the 18F-radiolabeled FAP inhibitor (FAPI), [18F]AlF-FAPI-74, to image FAP in two different mouse tumor models. First, the probe specificity was evaluated in the I45 human mesothelioma tumor model (a line which does not induce FAP+ fibroblasts). I45 WT and I45 cells transduced with human FAP were injected s.c. into the opposite flanks of a mouse and imaged following 2 weeks of tumor growth. We observed a 7.5-fold higher uptake of [18F]AlF-FAPI-74 in the I45 huFAP tumor compared to the WT, demonstrating the high specificity of the probe for FAP. Next, we used a more clinically-relevant A549 model where the tumor cells do not express FAP but induce a stromagenic response and drive the recruitment of FAP+ stromal cells in the TME. [18F]AlF-FAPI-74 PET/CT following 3 weeks of tumor growth showed a 6.5-fold increased radiotracer uptake in the tumor relative to the muscle at the baseline scan. Using this model, we evaluated the potential of our new CAR T cells to reduce tumor burden, as well as evaluating the potential of the [18F]AlF-FAPI-74 tracer as a tool to monitor the clearance of FAP-expressing cells in response to FAP CAR T cell therapy. Immediately after the baseline scan, we injected 5x106 FAP CAR T cells iv. At day 14 post-T cell injection, mice treated with FAP CAR T cells had significantly smaller tumors relative to the control group, which were treated with T cells that do not express the CAR, highlighting the therapeutic efficacy of the FAP CAR T cell therapy. Moreover, [18F]AlF-FAPI-74 PET/CT imaging showed no detectable tracer uptake in the tumors treated with FAP targeted CAR T. These findings were confirmed by immunofluorescence, indicating successful clearance of the FAP+ stroma by the injected FAP CAR T cells. In conclusion, the new 4G5 FAP CAR shows specific targeting toward mouse stroma infiltrating lung adenocarcinoma xenografts. PET imaging of FAP could be a highly useful approach to stratify patients prior to FAP CAR T therapy, as well as to monitor the pharmacodynamic response for FAP-targeted therapies. Citation Format: Estela Noguera-Ortega, Iris K Lee, Zebin Xiao, Leslie Todd, John Scholler, Decheng Song, Maria Liousia, Katheryn Lohith, Kexiang Xu, Kimberly J Eduards, Michael D Farwell, Carl H June, Steven M Albelda, Ellen Puré, Mark A Sellmyer. FAP CAR T cell therapy for solid tumors with PET imaging [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B19.

Successful tumor eradication by chimeric antigen receptor-expressing (CAR-expressing) T lymphocytes depends on CAR T cell persistence and effector function. We hypothesized that CD4+ and CD8+ T cells may exhibit distinct persistence and effector phenotypes, depending on the identity of specific intracellular signaling domains (ICDs) used to generate the CAR. First, we demonstrate that the ICOS ICD dramatically enhanced the in vivo persistence of CAR-expressing CD4+ T cells that, in turn, increased the persistence of CD8+ T cells expressing either CD28- or 4-1BB-based CARs. These data indicate that persistence of CD8+ T cells was highly dependent on a helper effect provided by the ICD used to redirect CD4+ T cells. Second, we discovered that combining ICOS and 4-1BB ICDs in a third-generation CAR displayed superior antitumor effects and increased persistence in vivo. Interestingly, we found that the membrane-proximal ICD displayed a dominant effect over the distal domain in third-generation CARs. The optimal antitumor and persistence benefits observed in third-generation ICOSBBz CAR T cells required the ICOS ICD to be positioned proximal to the cell membrane and linked to the ICOS transmembrane domain. Thus, CARs with ICOS and 4-1BB ICD demonstrate increased efficacy in solid tumor models over our current 4-1BB-based CAR and are promising therapeutics for clinical testing.