CAR Activation Research Articles

Engineered receptors for soluble cellular communication and disease sensing.

Despite recent advances in mammalian synthetic biology, there remains a lack of modular synthetic receptors that can robustly respond to soluble ligands and in turn activate bespoke cellular functions. Such receptors would have extensive clinical potential to regulate the activity of engineered therapeutic cells, but to date only receptors against cell surface targets have approached clinical translation1. To address this gap, we developed a receptor architecture called synthetic intramembrane proteolysis receptor (SNIPR), that has the added ability to be activated by soluble ligands, both natural and synthetic, with remarkably low baseline activity and high fold activation, through an endocytic, pH-dependent cleavage mechanism. We demonstrate the therapeutic capabilities of the receptor platform by localizing the activity of CAR T cells to solid tumors where soluble disease-associated factors are expressed, bypassing the major hurdle of on-target off-tumor toxicity in bystander organs. We further applied the SNIPR platform to engineer fully synthetic signaling networks between cells orthogonal to natural signaling pathways, expanding the scope of synthetic biology. Our design framework enables cellular communication and environmental interactions, extending the capabilities of synthetic cellular networking in clinical and research contexts.

Foresight Study of IoT Technology in Preventing Road Crash: A Case of Car Alcohol Detector

Alcohol detector in car is the application of IoT technology that senses alcohol level consumed by the driver’s breath and prevents them from driving. Road crash that caused by drunk driver recorded the number of deaths increase and it is uncontrollable and continuously happened. The aim of this study is to identify the key drivers and determine the futures implication wheel of road crash prevention by the implementation of alcohol detector installation in car. The STEEPV analysis is used to identify key drivers and for questionnaires development. A total of 223 respondents from Klang Valley participated in the survey with a response rate of 58.07%. Based on impact-uncertainty analysis, the top two drivers are “save human lives” and “safety feature increase car safety.” The implications of saving human lives are reducing the number of deaths, improving market needs, avoiding traffic congestion, and increasing the value of human life. Whereas the implications of safety feature that increasing car safety are that it increases production activity of new car, preventing road crash, increasing car cost as well as business capabilities. Overall, this study is important for future researchers and the automotive industry by contributing a significant idea for future needs technology in car.

In Vivo Monitoring and Magnetically‐Enhanced Delivery of CAR T‐Cells to Solid Tumor

AbstractChimeric antigen receptor (CAR) T‐cell therapy demonstrates efficacy for the treatment of hematologic cancers, but remains challenging in solid tumors because of the hostile tumor microenvironment and heterogeneity that restrict the infiltration and activity of CAR T‐cells. The lack of clinical imaging methods to monitor CAR T‐cell therapy in patients limits the potential to enhance the therapeutic approach and predict patient response. Here, a straightforward, non‐invasive method for Magnetic Resonance Imaging (MRI) tracking and magnetic targeting of CAR T‐cells to enhance T‐cell trafficking to solid tumors, in a setting that can be deployed in clinics, is reported. Iron oxide nanoparticles are loaded into T‐cells, allowing MRI detection and magnetic cell guidance, while preserving T‐cell viability, functions, and CAR specificity to target epidermal growth factor receptor (EGFR)‐expressing tumors. Longitudinal MRI monitoring of T‐cells over 14 days reveals differences in tumor infiltration rates and antitumor efficacy between EGFR‐targeted CAR T‐cells and non‐transduced T‐cell. Moreover, an external magnet placed over the tumor enhances the infiltration of CAR T‐cells and increases their antitumor effect. MRI monitoring and magnetic guidance represent a dual‐pronged clinically feasible therapeutic strategy to control and promote CAR T‐cells delivery into solid tumors, thereby improving the precision, efficacy, and safety of the treatment.

The Spectrum of CAR Cellular Effectors: Modes of Action in Anti-Tumor Immunity.

Chimeric antigen receptor-T cells have spearheaded the field of adoptive cell therapy and have shown remarkable results in treating hematological neoplasia. Because of the different biology of solid tumors compared to hematological tumors, response rates of CAR-T cells could not be transferred to solid entities yet. CAR engineering has added co-stimulatory domains, transgenic cytokines and switch receptors to improve performance and persistence in a hostile tumor microenvironment, but because of the inherent cell type limitations of CAR-T cells, including HLA incompatibility, toxicities (cytokine release syndrome, neurotoxicity) and high costs due to the logistically challenging preparation process for autologous cells, the use of alternative immune cells is gaining traction. NK cells and γδ T cells that do not need HLA compatibility or macrophages and dendritic cells with additional properties such as phagocytosis or antigen presentation are increasingly seen as cellular vehicles with potential for application. As these cells possess distinct properties, clinicians and researchers need a thorough understanding of their peculiarities and commonalities. This review will compare these different cell types and their specific modes of action seen upon CAR activation.

An In Vitro Assay to Evaluate the Anti-Tumor Activity of Tumor-Specific CAR T Cells

An In Vitro Assay to Evaluate the Anti-Tumor Activity of Tumor-Specific CAR T Cells

An In Vitro Assay to Evaluate the Anti-Tumor Activity of Tumor-Specific CAR T Cells

An In Vitro Assay to Evaluate the Anti-Tumor Activity of Tumor-Specific CAR T Cells

CAR T cells outperform CAR NK cells in CAR-mediated effector functions in head-to-head comparison

BackgroundCAR NK cells as vehicles for engineered “off-the-shelf” cellular cancer immunotherapy have attracted significant interest. Nonetheless, a comprehensive comparative assessment of the anticancer activity of CAR T cells and CAR NK cells carrying approved benchmark anti-CD19 CAR constructs is missing. Here, we report a direct head-to-head comparison of CD19-directed human T and NK cells.MethodsWe generated CAR T and CAR NK cells derived from healthy donor PBMC by retroviral transduction with the same benchmark second-generation anti-CD19 CAR construct, FMC63.28z. We investigated IFN-γ secretion and direct cytotoxicity in vitro against various CD19+ cancer cell lines as well as in autologous versus allogeneic settings. Furthermore, we have assessed anticancer activity of CAR T and CAR NK cells in vivo using a xenograft lymphoma model in an autologous versus allogeneic setting and a leukemia model.ResultsOur main findings are a drastically reduced capacity for CAR-mediated IFN-γ production and lower CAR-mediated cytotoxicity of CAR NK cells relative to CAR T cells in vitro. Consistent with these in vitro findings, we report superior anticancer activity of autologous CAR T cells compared with allogeneic CAR NK cells in vivo.ConclusionsCAR T cells had significantly higher CAR-mediated effector functions than CAR NK cells in vitro against several cancer cell lines and autologous CAR T cells outperformed allogeneic CAR NK cells both in vitro and in vivo. CAR NK cells will likely benefit from further engineering to enhance anticancer activity to ultimately fulfill the promise of an effective off-the-shelf product.

Triclosan administration to humanized UDP-glucuronosyltransferase 1 neonatal mice induces UGT1A1 through a dependence on PPARα and ATF4

Triclosan (TCS) is an antimicrobial toxicant found in a myriad of consumer products and has been detected in human tissues, including breastmilk. We have evaluated the impact of lactational TCS on UDP-glucuronosyltransferase 1A1 (UGT1A1) expression and bilirubin metabolism in humanized UGT1 (hUGT1) neonatal mice. In hUGT1 mice, expression of the hepatic UGT1A1 gene is developmentally delayed resulting in elevated total serum bilirubin (TSB) levels. We found that newborn hUGT1 mice breastfed or orally treated with TCS presented lower TSB levels along with induction of hepatic UGT1A1. Lactational and oral treatment by gavage with TCS leads to the activation of hepatic nuclear receptors constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor alpha (PPARα), and stress sensor, activating transcription factor 4 (ATF4). When CAR-deficient hUGT1 mice (hUGT1/Car-/-) were treated with TCS, TSB levels were reduced with a robust induction of hepatic UGT1A1, leaving us to conclude that CAR is not tied to UGT1A1 induction. Alternatively, when PPARα-deficient hUGT1 mice (hUGT1/Pparα-/-) were treated with TCS, hepatic UGT1A1 was not induced. Additionally, we had previously demonstrated that TCS is a potent inducer of ATF4, a transcriptional factor linked to the integrated stress response. When ATF4 was deleted in liver of hUGT1 mice (hUGT1/Atf4ΔHep) and these mice treated with TCS, we observed superinduction of hepatic UGT1A1. Oxidative stress genes in livers of hUGT1/Atf4ΔHep treated with TCS were increased, suggesting that ATF4 protects liver from excessive oxidative stress. The increase oxidative stress may be associated with superinduction of UGT1A1. The expression of ATF4 in neonatal hUGT1 hepatic tissue may play a role in the developmental repression of UGT1A1.

Abstract PO5-04-06: Enhancing Antitumor Activity of Her2 CAR T cells through TR2BB Co-Expression and Cytokine Signal 3 Incorporation

Abstract Chimeric antigen receptor T cells (CARTs) targeting Her2 represent a promising approach for the treatment of patients with Her2 expressing breast cancers (BC). Her2 CARTs confront major challenges within the immune-suppressive tumor microenvironment (TME) of breast cancer, which inhibits sustained T cell proliferation and persistent antitumor activity. To bolster Her2 CARTs' expansion and persistence, we engineered a TNF-related apoptosis-inducing ligand receptor 2 (TR2) specific scFv connected to the endodomain of 4-1BB (TR2BB). TR2BB co-expressing Her2 CARTs exhibited enhanced antitumor potential against BC tumors by triggering myelod derived suppressor cell (MDSC) apoptosis, thereby remodeling the TME and promoting T cell proliferation. Despite better tumor control in mice receiving CAR.TR2BB T cells, recent trials have demonstrated the importance of incorporating a cytokine signal for optimal tumor control in patients with solid tumors. Thus, we hypothesized that we could further augment anti-tumor activity of Her2 CAR.TR2BB T cells against BC by incorporating a cytokine signal 3 through IL-15 or C7R. We engineered the CARTs to (1) ectopically produce IL-15, a crucial T-cell homeostasis and survival cytokine, and (2) express a constitutively active IL-7 receptor (C7R) that triggers IL-7 receptor signaling independently of ligand or the common receptor gamma chain (γc). Our repeat weekly tumor challenge assay revealed that CAR.TR2BB.IL15 T cells outperformed CAR.TR2BB.C7R and CAR.TR2BB in T cell proliferation. Both CAR.TR2BB.IL15 and CAR.TR2BB.C7R exhibited improved cytotoxic potential after three weeks of antigen stimulation compared to CAR.TR2BB alone in comparison with CAR.TR2BB alone. Furthermore, in our in vivo xenograft NSG mouse models implanted with Her2 expressing breast cancer cell line and MDSCs, CAR.TR2BB.IL15 T cells showed superior tumor control and improved survival rates. In summary, the addition of IL15 transgene to our TME targeting Her2 CARTs provides an advantage in terms of T cell persistence, proliferation, and tumor control in in vitro and in vivo models. These promising preclinical results propel us to apply this optimized Her2 targeting CART therapy in a phase I clinical trial for patients with metastatic Her2-expressing breast cancers. Citation Format: Diego Chamorro, Lauren Somes, Emily Madaras, Saisha Nalawade, Paul Shafer, Andres Mosquera, Mary Kathryn McKenna, Valentina Hoyos. Enhancing Antitumor Activity of Her2 CAR T cells through TR2BB Co-Expression and Cytokine Signal 3 Incorporation [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-04-06.

Mitochondrial transcription is essential for the potent antitumor stem memory T cells

Abstract Adoptive T cell transfer (ACT) therapy is an effective approach to treat cancer patients, where expanding the patient’s T lymphocytes and redirecting them to recognize and regress solid tumors. Unfortunately, ACT can fail in some patients due to exhaustion or impaired metabolic fitness during T cell expansion. We discovered through transcriptomic analysis that the novel mitochondrial transcription regulator REXO2 induced by regulating PI3Kδ signaling in vitro - generated stem memory T cells (TSCM) - is responsible for the antitumor potency of TSCM cells. This finding is novel, as REXO2 has never been studied in immune cells and has only recently been appreciated in regulating heart muscles during embryonic development. We found that knocking out REXO2 via CRISPR in TSCM cells impaired their mitochondrial transcription and morphology in vitro. Moreover, they had reduced metabolic fitness marked by poor spare respiratory capacity, reduced mitochondrial potential and increased production of reactive oxygen species post tumor antigen recognition. Of clinical importance, REXO2 knockout in TSCM cells impaired the antitumor potency when infused into mice. This work also has translational potential, as REXO2 could be induced in human TIL or CAR T cell products by expanding them in the presence of PI3Kδ inhibitor. Our findings reveal that REXO2 is a promising target to bolster the antitumor activity of next generation TIL and CAR T cell products to treat cancer patients.

Preclinical Development of CAR T Cells with Antigen-Inducible IL18 Enforcement to Treat GD2-Positive Solid Cancers.

Cytokine-engineering of chimeric antigen receptor-redirected T cells (CAR T cells) is a promising principle to overcome the limited activity of canonical CAR T cells against solid cancers. We developed an investigational medicinal product, GD2IL18CART, consisting of CAR T cells directed against ganglioside GD2 with CAR-inducible IL18 to enhance their activation response and cytolytic effector functions in the tumor microenvironment. To allow stratification of patients according to tumor GD2 expression, we established and validated immunofluorescence detection of GD2 on paraffin-embedded tumor tissues. Lentiviral all-in-one vector engineering of human T cells with the GD2-specific CAR with and without inducible IL18 resulted in cell products with comparable proportions of CAR-expressing central memory T cells. Production of IL18 strictly depends on GD2 antigen engagement. GD2IL18CART respond to interaction with GD2-positive tumor cells with higher IFNγ and TNFα cytokine release and more effective target cytolysis compared with CAR T cells without inducible IL18. GD2IL18CART further have superior in vivo antitumor activity, with eradication of GD2-positive tumor xenografts. Finally, we established GMP-compliant manufacturing of GD2IL18CART and found it to be feasible and efficient at clinical scale. These results pave the way for clinical investigation of GD2IL18CART in pediatric and adult patients with neuroblastoma and other GD2-positive cancers (EU CT 2022- 501725-21-00). See related commentary by Locatelli and Quintarelli, p. 3361.

Abstract CT129: A phase 1 dose escalation study of a novel coupled CAR T cell therapy, GCC19CART, for patients with metastatic colorectal cancer

Abstract Background: GCC19CART, the first clinical candidate from the CoupledCAR solid tumor platform, is designed to overcome the limitations of conventional CAR T-cells in solid tumor malignancies by pairing solid tumor CAR T-cells with CD19-targeting CAR T-cells to amplify the proliferation and activation of the solid tumor CAR T component. GCC19CART targets guanylate cyclase-C (GCC), which is reported to be expressed in the metastatic lesions of 70%-80% of subjects with colorectal cancers and largely restricted to the intestinal tract. A Phase 1 investigator-initiated clinical trial conducted in China showed preliminary efficacy with an overall response rate (ORR) at dose level 1 (1x106 CAR T-cells/kg) of 15.4% ORR (2/13) and 50% ORR (4/8) in dose level 2 (2x106 CAR T-cells/kg). Common side effects included cytokine release syndrome (CRS) and diarrhea/colitis. Immune effector cell-associated neurotoxicity syndrome (ICANS) was seen in a smaller proportion of patients. A phase 1 study was initiated in the United States in August 2022 for patients with metastatic colorectal cancer refractory to standard of care chemotherapy. As of a data cutoff on ​November 1, 2023, 4 subjects have been treated in the initial dose level. Methods: Subjects were initially screened for GCC expression; this requirement was later discontinued when immunohistochemistry studies revealed that the frequency of positive GCC expression exceeded 95%. Eligible subjects underwent leukapheresis, a single dose of lymphodepleting chemotherapy (fludarabine 30mg/m2 and cyclophosphamide 300mg/m2) 3 days prior to infusion, and then administration of a single infusion of GCC19CART. Responses were assessed by RECIST v1.1 by site investigator review and an independent third-party imaging contract research organization. Results: 4 subjects have been dosed at level 1 (1x106 cells/kg) and completed the DLT period at the time of data cutoff. The most common adverse events were CRS in 4/4 subjects (Grade 1: 2/4 (50%) or Grade 2: 2/4 (50%)) and diarrhea in 3/4 subjects (Grade 1: 1/4 (25%), Grade 2: 1/4 (25%), or Grade 3: 1/4 (25%)). Three out of four patients had a mild tremor (Grade 1) that resolved with corticosteroids and no other evidence of neurotoxicity. The combined overall response rate (ORR) was 50% (2/4) by independent review. Two subjects demonstrated a partial response (PR) while 1 additional subject had partial metabolic response (PMR) on PET/CT with stable disease (SD) and 1 patient had progressive disease per RECIST 1.1. Per site investigator review, the ORR was 25%, with 1 confirmed PR and 3 SD assessed as best response. The median PFS was 3.8 months in the dose level 1 group. Conclusions: Preliminary results demonstrate that GCC19CART has meaningful clinical activity and an acceptable safety profile in refractory metastatic colorectal cancer. This trial is ongoing and updated data will be presented, including from patients treated at dose level 2 (2x106 CAR T-cells/kg). Clinical trial information: NCT05319314. Citation Format: Bridget P. Keenan, Christopher H. Lieu, Marwan Fakih, Dongqi Chen, Lucy Lu, Eric Rowinsky, Victor Lu, Lei Xiao, Alan P. Venook, Benjamin Schlechter. A phase 1 dose escalation study of a novel coupled CAR T cell therapy, GCC19CART, for patients with metastatic colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT129.

Stereotypes and Stereotyping: Measuring the Accuracy of Lifestyle-Based Judgments on Political Affiliation

Abstract People often draw inferences about others’ underlying characteristics from single and static samples of their appearance, such as facial features, or attractiveness. Evidence also suggests that these judgments occur spontaneously and rapidly. Are humans also able to detect political preferences based on appearance? This article examines to what extent observable lifestyle characteristics influence people’s judgments about one’s political affiliation and, more importantly, to what degree these judgments are accurate. A conjoint analysis allows for the identification of the specific lifestyle cues that people use to infer one’s political affiliation. These results are contrasted with a large and unique dataset (n = 64,745), enabling the assessment of how accurate these cues are. Results suggest that certain lifestyle characteristics, such as type of car or leisure activities, are clearly associated with different political parties, at least in people’s minds. Results also suggest that, despite the potential detrimental effects of appearance-based judgments, people are generally pretty good at guessing others’ political preferences. This study contributes to a growing body of research on the relationship between lifestyle and political preferences. More generally, it sheds light on the diagnostic value of appearances in everyday social judgments.

Abstract 3995: A novel chimeric Fas signal redirect receptor enhances the durability of anti-tumor activity and serial killing potential of CAR T cells

Abstract Chimeric antigen receptor (CAR) T-cell therapies have demonstrated remarkable success in hematological malignancies but only modest efficacy in solid tumors. Apoptosis induced by Fas receptor signaling can be a substantial barrier to CAR T-cell antitumor activity, as many solid tumors express high levels of Fas ligand (FasL). Here we describe a novel synthetic construct where the ectodomain of Fas receptor is fused to the signaling endodomain of a stimulatory receptor, creating a Fas signal redirect receptor (Fas-SRR). This unique design not only prevents Fas-dependent cell death, but also augments the long-term killing potential of induced pluripotent stem cell (iPSC)-derived CAR-T (CAR-iT) cells. Fas-SRR constructs with unique T-cell activation signaling endodomains were designed and compared to two control arms: a dominant negative receptor (Fas-DNR), which blocks Fas signal but does not promote activation; and a Fas-41BB fusion receptor, where the 41BB endodomain is fused to FasR ectodomain to direct activation upon FasL:Fas engagement. Fas-SRR designs were validated in Jurkat reporter cells treated with Fas agonist to measure signal redirection via Fas-SRR. To conduct a head-to-head comparison, CAR-iT cells were engineered to express a CAR under the regulation of the TRAC locus and to express either a Fas-DNR, a Fas-41BB, or FAS-SRR constructs at high purity (>90% transgene expression). No major differences were seen in maturation phenotype (>95% TRAC-CAR expression) of these cells, indicating compatibility of the FAS-SRR constructs with CAR-iT cell generation. Functional testing of the Fas-SRR+ CAR-iT cells was performed in a long-term restimulation assay where effector cells were repeatedly exposed to target cells over multiple rounds of co-culture. Complete loss of target cell lysis was observed by round six in the co-cultures with FAS-DNR cells, where loss of CAR-iT cell persistence led to accumulation of target cells. After eight rounds of co-culture, the Fas-41BB cells and the majority of Fas-SSR cell constructs also began to exhibit minimal activity toward target cells. However, the top Fas-SRR cell constructs continued to show durable antitumor activity (>97% cytolysis) through the end of the 9-round rechallenge assay. Of note, GSEA analysis identified significant upregulation of NFkB and RELA target genes in cells having the top FAS-SRR construct versus the less durable designs. These results reinforce the advantage of attenuating Fas-FasL interactions to prevent apoptosis and, more importantly, demonstrate the unique therapeutic value in redirecting Fas signaling to improve T-cell potency over an extended duration. Additional studies are underway to further investigate the implications of Fas signal redirection in CAR T and CAR-iT cells and to further understand the module of genes involved in supporting serial stimulation performance. Citation Format: Christopher Ecker, Eigen Peralta, Hui-Yi Chu, Lorraine Loter, Masanao Tsuda, Earl Avramis, Matthew Denholtz, Alec Witty, Tom Lee, Bahram Valamehr. A novel chimeric Fas signal redirect receptor enhances the durability of anti-tumor activity and serial killing potential of CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3995.

Abstract 58: Immunocytokines with specificity for invariant region of CAR molecules enhance effector function of multiple CAR T cell therapies

Abstract The effector functions of CAR T cells can be enhanced through delivery of immunostimulatory cytokines, such as IL-12, IL-18, or IL-23. However, systemic delivery of recombinant cytokines or paracrine secretion by CAR T cells can elicit toxicities due to non-specific targeting of the cytokines. Here, we designed immunocytokines with antibodies specific for the invariant linker peptides that join variable domains in the CAR scFv and immunostimulatory cytokines. The immunocytokines were capable of targeting CAR molecules agnostic of target antigen, demonstrating broad applicability without the need for further genetic engineering of CAR therapies, such as co-expression of a surrogate molecule (tEGFR, etc.). The addition of immunocytokines enhanced CAR T cell cytolytic activity and secretion of type I cytokines, even at low concentrations of immunocytokine. In a stringent Nalm6 xenograft model, the addition of immunocytokines delivering IL-12 enhanced the anti-tumor activity of anti-CD19 CAR T cells and overall survival compared to controls of antibody alone or PBS. This technological development presents the opportunity to specifically enhance the activity of multiple CAR T cell therapies in the absence of non-specific toxicities. Citation Format: J T. Keane, Oula K. Dagher, Fang Liu, Avery D. Posey. Immunocytokines with specificity for invariant region of CAR molecules enhance effector function of multiple CAR T cell therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 58.

Abstract 6326: Targeting PRDM1 to enhance CAR T cell efficacy against diffuse large B cell lymphoma (DLBCL)

Abstract CAR T cell therapy has shown remarkable success in treating patients with relapsed or refractory (r/r) DLBCL, but a majority (>60%) will experience disease progression. A key failure mechanism is the rapid terminal differentiation and exhaustion of CAR T cells, driven by persistent antigen exposure in vitro during manufacturing and in vivo in the DLBCL environment, which culminates in poor persistence and short-term effector functions. Recent studies have identified the transcriptional repressor PR/SET domain 1 (PRDM1) as a key regulator that drives differentiation of T cells toward these unwanted fates. Further, single cell transcriptomic profiling of r/r DLBCL biopsies has revealed PRDM1 enrichment in a cluster of exhausted-like CD8+ CAR T cells. These observations indicate that targeting PRDM1 could prevent or delay terminal differentiation/exhaustion in CAR T cells, thereby enhancing their persistence for lasting anti-tumor activity. Using CRISPR/Cas9 targeting, we have demonstrated that PRDM1-deficient CAR T cells exhibit enhanced proliferative capacity, memory characteristics, and resilience against exhaustion upon repeated stimulation. Moreover, genetic ablation of PRDM1 also promoted the in vivo expansion and persistence of CAR T cells, which led to significantly improved survival of lymphoma-bearing mice. Mechanistically, loss of PRDM1 leads to de-repression of PPARGC1A (PGC1α), a master regulator of mitochondrial biogenesis and antioxidant activity. This confers elevated tolerance to oxidative damage in CAR T cells lacking PRDM1, which showed increased capacity to buffer oxidative stress induced by mitochondrial uncouplers and constant stimulation. These findings suggest that PRDM1 loss may improve CAR T cell fitness through PGC1α upregulation and mitochondrial activation. From a translational perspective, we assessed the potential synergistic effects of PGC1α agonist, bezafibrate, with CAR T cells. Dual therapy with bezafibrate and CAR T cells mediated more robust expansion and anti-tumor immunity of CAR T cells without causing discernible adverse events. Importantly, bezafibrate alone had no direct tumoricidal effects, which indicates that bezafibrate suppressed tumor growth through modulating CAR T cell functions. Moving forward, we will uncover the previously uncharted role of PRDM1 in orchestrating the mitochondrial and metabolic activity of CAR T cells. Building upon a correlation between PRDM1 expression and CAR T cell dysfunction, we aim to pioneer the use of PRDM1 and/or its target genes as novel biomarkers for predicting clinical responses of r/r DLBCL patients to CAR T cell therapy. Our endeavors will shed light on the long-standing conundrum of CAR T cell dysfunction and offer solutions that may be applicable for improving T cell-based therapy against cancer in general. Citation Format: Sidney Wang, Lishi Xie, Xiufen Chen, Alan Cooper, Joanna Chorazeczewski, Justin Kline. Targeting PRDM1 to enhance CAR T cell efficacy against diffuse large B cell lymphoma (DLBCL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6326.

Abstract 6332: Evaluating the relationship of affinity, functional avidity, and in vivo potency in KIR-CAR T cells

Abstract Chimeric antigen receptors (CARs) targeting CD19 demonstrate remarkable clinical outcomes in patients (pts) with B-cell malignancies. Importantly, all four FDA-approved CD3ζ-based CAR T cell therapies use the same FMC63-derived single-chain variable fragment (scFv) to target CD19+ tumors. Although response rates are high in diffuse large B cell lymphoma, only ~40% of pts achieve durable, long-term remissions. Improved CAR T cell therapies are therefore needed. We have previously shown that a novel killer immunoglobulin receptor (KIR)-based CAR expressed in T cells as a multichain receptor with DAP12 (KIR-CAR T) has enhanced anti-tumor activity in several preclinical solid tumor models suggesting that this platform may show better activity in lymphoma. Some recent studies report that lower-affinity CD19 binders compared to the benchmark FMC63-scFv yield equivalent or increased anti-tumor activity of CAR T in pts, raising the question of optimal affinity for a KIR-based CAR. In this study, we aimed to evaluate the relationship of affinity, functional avidity, and in vivo potency of CD19 binders in a KIR-based CAR T cell. Using a highly-diverse 40-billion member canine scFv phage display library, we generated 19 unique human CD19-specific binders that compete with FMC63 binder supporting a similar binding epitope. Different KIR-CAR T cells were generated, and relative affinities were compared via binding to a recombinant (rec) CD19 protein. We found that although CAR receptor expression was similarly expressed, the concentration of soluble rec CD19 protein required to saturate the surface CAR varied more than 2-log among the different scFv binders suggesting a range of affinities relative to FMC63. We selected several scFvs with lower, and equivalent CD19 binding MFI compared to the FMC63-scFv and found that all scFvs could mediate similarly robust in vitro potency. In vivo anti-tumor efficacy was next assessed using the NSG xenograft mouse model of B-cell leukemia NALM6 cells. Interestingly, despite their comparable in vitro functions, some binders with relatively similar binding MFI failed to control the tumors. In contrast, two binders with an ~30-fold apparent affinity relative to FMC63 exhibited robust anti-tumor activity comparable to the benchmark scFv. These preliminary data suggest that the in vivo functional activity of chimeric KIR-based immunoreceptors is insensitive to binder affinity over a wide range of affinities. The failure of some scFvs with comparable affinity and binding epitope suggest that other, yet to be characterized attributes of the binder may be critical for optimal function. Further characterization of these binders is being conducted to understand the relationship between affinity, functional avidity, antigen density, and in vivo potency. This study paves the way for developing an improved CD19-specific KIR-CAR T cell therapy for treating pts with B-cell malignancies. Citation Format: Jun Xu, Selene Nunez-Cruz, John M. Leferovich, Gayathri Gulendran, Chune Zhang, Nora D. Yucel, Megan C. Blair, William S. Stanley, Laura A. Johnson, Don L. Siegel, Michael C. Milone. Evaluating the relationship of affinity, functional avidity, and in vivo potency in KIR-CAR T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6332.

Abstract 4021: The development of CAR T cell therapeutics targeting tumor specific CD146

Abstract Chimeric antigen receptor (CAR) T cells represent a promising class of "living drugs" for cancer treatment. These primary T cells are genetically engineered to express a synthetic antigen receptor, incorporating various domains to enhance their effector functions. While CAR T cells have shown success against liquid cancers, their application to solid cancers is hindered by significant "on-target/off-tumor" toxicities due to the absence of specific target proteins. In this study, we aim to enhance the tumor specificity of CAR T cells by targeting a tumor-specific version of CD146. CD146 is a glycoprotein widely expressed in several solid tumors, particularly melanoma and ovarian cancer. However, it is also expressed on benign cells, such as endothelial cells, pericytes, and smooth muscle cells. Nevertheless, a CD146 isoform exists that is enriched in cancer cells. We assessed CAR activity using flow cytometry to measure surface CD69 expression (an early activation marker) on CAR-expressing Jurkat cells co-cultured with various CD146-positive and CD146-negative human melanoma and ovarian cancer cell lines. The killing ability of primary CAR-T cells was evaluated by measuring the relative luciferase activity of target cells, and their effector functions were further analyzed by ELISA for the secretion of IFN-g and TNF-a. Through multiple rounds of protein engineering, we have developed a CAR construct capable of reprogramming T cells to respond to a tumour-enriched CD146 isoform. Our CAR demonstrated antigen-dependent activation of Jurkat cells when co-cultured with CD146-positive human melanoma and ovarian cancer cell lines, comparable to a CAR targeting pan-CD146. Moreover, primary CAR T cells exhibited proportional killing activity and other effector functions. We have successfully engineered a CAR with specificity for a tumour-enriched isoform of CD146. Our future work will involve rigorously evaluating the tumor selectivity of our CAR, as well as assessing the ability of primary CAR T cells expressing this CAR to control tumor growth in vivo. Citation Format: Erez Uzuner, Holly Mole, Chun W. Wong, Macarena L. Fernandez Carro, Rotem Leshem, Kieran Sefton, Helen Tovey, Andrew Picken, Marcel Blot-Chabaud, Adam F. Hurlstone. The development of CAR T cell therapeutics targeting tumor specific CD146 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4021.

A versatile CRISPR-Cas13d platform for multiplexed transcriptomic regulation and metabolic engineering in primary human T cells

CRISPR technologies have begun to revolutionize Tcell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the Tcell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human Tcells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR Tcell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of Tcell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathwaysto enhance CAR Tcell fitness and anti-tumor activity invitro and invivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.

Miniaturized CAR knocked onto CD3ε extends TCR function with CAR specificity under control of endogenous TCR signaling cascade

Immunotherapy using TCR and especially CAR transgenic T cells is a rapidly advancing field with the potential to become standard of care for the treatment of multiple diseases. While all current FDA approved CAR T cell products are generated using lentiviral gene transfer, extensive work is put into CRISPR/Cas mediated gene delivery to develop the next generation of safer and more potent cell products. One limitation of all editing systems is the size restriction of the knock-in cargo. Targeted integration under control of an endogenous promotor and/or signaling cascades opens the possibility to reduce CAR gene size to absolute minimum. Here we demonstrate that a first-generation CAR payload can be reduced to its minimum component - the antigen-binding domain - by targeted integration under control of the CD3ε promoter generating a CAR-CD3ε fusion protein that exploits the endogenous TCR signaling cascade. Miniaturizing CAR payload in this way results in potent CAR activity while simultaneously retaining the primary antigen recognition function of the TCR. Introducing CAR-specificity using a CAR binder only while maintaining endogenous TCR function may be an appealing design for future autologous CAR T cell therapies.