Activity Of Chimeric Antigen Receptor Research Articles

FOXO1 is a master regulator of memory programming in CAR T cells

A major limitation of chimeric antigen receptor (CAR) T cell therapies is the poor persistence of these cells in vivo1. The expression of memory-associated genes in CAR T cells is linked to their long-term persistence in patients and clinical efficacy2–6, suggesting that memory programs may underpin durable CAR T cell function. Here we show that the transcription factor FOXO1 is responsible for promoting memory and restraining exhaustion in human CAR T cells. Pharmacological inhibition or gene editing of endogenous FOXO1 diminished the expression of memory-associated genes, promoted an exhaustion-like phenotype and impaired the antitumour activity of CAR T cells. Overexpression of FOXO1 induced a gene-expression program consistent with T cell memory and increased chromatin accessibility at FOXO1-binding motifs. CAR T cells that overexpressed FOXO1 retained their function, memory potential and metabolic fitness in settings of chronic stimulation, and exhibited enhanced persistence and tumour control in vivo. By contrast, overexpression of TCF1 (encoded by TCF7) did not enforce canonical memory programs or enhance the potency of CAR T cells. Notably, FOXO1 activity correlated with positive clinical outcomes of patients treated with CAR T cells or tumour-infiltrating lymphocytes, underscoring the clinical relevance of FOXO1 in cancer immunotherapy. Our results show that overexpressing FOXO1 can increase the antitumour activity of human CAR T cells, and highlight memory reprogramming as a broadly applicable approach for optimizing therapeutic T cell states.

Abstract LB068: Repurposing chimeric antigen receptor regulatory T cells for treating cancer

Abstract Solid tumors are the deadliest form of cancer. Over the past decade, chimeric antigen receptor (CAR) T-cell therapy has led to remarkable regression rates in leukemia and lymphoma. However, solid tumors have remained refractory to CAR T-cell therapy, with CAR T cells either failing to penetrate the solid tumor microenvironment (TME) or becoming exhausted once in it. Mechanisms behind these properties of the TME include nutrient scarcity, aberrant vascularization, stromal cell deposition, expression of inhibitory immune receptors by cancer cells, and the presence of immunosuppressive cells. Interestingly, there is a subset of T cells found in the TME - regulatory T cells (Tregs). Dedicated to suppressing immune responses, Tregs constitute a barrier to anticancer immunity by migrating to and heavily accumulating in solid tumors, inhibiting local immunity. Tregs can be redirected towards any cell surface antigen using a CAR and have been shown to be cytotoxic towards target antigen presenting cells. Can Tregs be converted from tumor protectors to anti-tumor effector cells? To test the anti-tumor potential of CAR Tregs, we modified human Tregs with an anti-CD19 CD28-CD3z CAR. Tregs activated via the CAR or endogenous TCR/CD28 were evaluated at the functional and gene expression levels. CAR and endogenous TCR/CD28 activation resulted in similar cell expansion and Treg stability (FOXP3 and HELIOS expression). However, CAR-mediated activation markedly increased Treg proinflammatory cytokine production. Moreover, compared to their TCR/CD28-activated counterparts, CAR Tregs displayed increased cytotoxic activity towards tumor cells and a diminished capacity to modulate antigen-presenting cells and suppress T cell proliferation, suggesting a global shift from suppression to inflammation. Strikingly, CAR Tregs suppressed tumor growth in immunodeficient mice inoculated with antigen-expressing B-cell leukemia, myeloid leukemia, and lung epithelial carcinoma. Single-cell RNA sequencing identified a cell cluster enriched in CAR Tregs characterized by high expression of effector T cell genes, such as IFNG, GZMB, and CD40LG, revealing that CAR activation creates a highly active population of proinflammatory Tregs. Multiplex cytokine analysis, whole-transcriptome RNA-seq, and intracellular cytokine staining confirmed that IFNG production by FOXP3+ Tregs is induced specifically by CAR activation. Of note, murine CAR Tregs are also cytotoxic towards tumor cells. Current experiments are focused on pinpointing and validating transcription factors and surface markers characteristic of proinflammatory CAR Tregs and further assessing CAR Treg antitumor efficacy in vivo. In conclusion, CAR-mediated activation induces a novel proinflammatory Treg phenotype that can be exploited in the next generation of engineered immune cell therapies against solid tumors. Citation Format: Leonardo M. Ferreira, Russell W. Cochrane, Rob A. Robino, Bryan Granger, Eva Allen, Vishnu Gudivada, Aguirre A. de Cubas, Stefano Berto. Repurposing chimeric antigen receptor regulatory T cells for treating 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 LB068.

Targeting osteosarcoma with canine B7-H3 CAR T cells and impact of CXCR2 Co-expression on functional activity

The use of large animal spontaneous models of solid cancers, such as dogs with osteosarcoma (OS), can help develop new cancer immunotherapy approaches, including chimeric antigen receptor (CAR) T cells. The goal of the present study was to generate canine CAR T cells targeting the B7-H3 (CD276) co-stimulatory molecule overexpressed by several solid cancers, including OS in both humans and dogs, and to assess their ability to recognize B7-H3 expressed by canine OS cell lines or by canine tumors in xenograft models. A second objective was to determine whether a novel dual CAR that expressed a chemokine receptor together with the B7-H3 CAR improved the activity of the canine CAR T cells. Therefore, in the studies reported here we examined B7-H3 expression by canine OS tumors, evaluated target engagement by canine B7-H3 CAR T cells in vitro, and compared the relative effectiveness of B7-H3 CAR T cells versus B7-H3-CXCR2 dual CAR T cells in canine xenograft models. We found that most canine OS tumors expressed B7-H3; whereas, levels were undetectable on normal dog tissues. Both B7-H3 CAR T cells demonstrated activation and OS-specific target killing in vitro, but there was significantly greater cytokine production by B7-H3-CXCR2 CAR T cells. In canine OS xenograft models, little anti-tumor activity was generated by B7-H3 CAR T cells; whereas, B7-H3-CXCR2 CAR T cells significantly inhibited tumor growth, inducing complete tumor elimination in most treated mice. These findings indicated therefore that addition of a chemokine receptor could significantly improve the anti-tumor activity of canine B7-H3 CAR T cells, and that evaluation of this new dual CAR construct in dogs with primary or metastatic OS is warranted since such studies could provide a critical and realistic validation of the chemokine receptor concept.

Abstract 3993: A tunable safety switch for solid tumor CAR T-cell therapy

Abstract On-target off-tumor toxicity, cytokine release syndrome (CRS), and immune effector cell-associated neurotoxicity syndrome (ICANS) are severe immune-related adverse events (irAEs) that are frequently associated with Chimeric Antigen Receptor (CAR) T-cell therapy. Current efforts to manage such therapy-related toxicities involve incorporation of an inducible suicide agent within CAR constructs, such as iCaspase-9 or herpes simplex virus type 1 thymidine kinase that can be selectively activated to produce toxic effects within CAR T cells and attenuate their activity. However, while activation of these agents helps to mitigate or overcome such unwarranted toxicities, the therapeutic benefit of CAR T cells anti-tumor activity is also compromised. Therefore, to continue maintenance of CAR T cells’ therapeutic function while minimizing irAEs, an ideal safety switch should 1) rapidly inhibit the activation and proliferation of CAR T cells exposed to the target antigen, 2) reversibly inhibit activity without inducing CAR T cell elimination and 3) be clinically translatable for safe application in patients. Our laboratory investigated one such safety switch to inhibit CAR T-cell activity while maintaining their therapeutic function. We showed that incorporating a mutant variant of c-KIT D816V (KITv) in the intracellular domain of mesothelin-targeting second-generation CAR T cells (M28z-KITv) improved efficacy in solid tumors with low antigen, or an immunosuppressive environment. Herein, we evaluate the use of Dasatinib, a clinically available multitarget (BCR, SRC, c-KIT) tyrosine kinase inhibitor (TKI), as a tunable safety switch to reversibly inhibit M28z-KITv CAR T-cell functional activity. In cohorts of mice established with lung adenocarcinoma, daily administration of Dasatinib starting on day 1 or day 3 after CAR T-cell administration stabilized tumor growth, which otherwise continued to regress in untreated mice, indicating inhibition of CAR T-cell functional activity. Upon discontinuation of Dasatinib, tumors regressed, indicating reversal of CAR T-cell functional activity. In an experiment conducted to investigate functional persistence of CAR T cells upon long-term exposure to Dasatinib (1 month), we noted uninhibited activity of CAR T cells to rechallenged tumors. Dasatinib, thus may act as a tunable safety switch to regulate M28z-KITv CAR T-cell activity without compromising its therapeutic function. Citation Format: Kyohei Misawa, Meriem Taleb, Srijita Banerjee, William-Ray Vista, Navin K. Chintala, Prasad S. Adusumilli. A tunable safety switch for solid tumor CAR T-cell therapy [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 3993.

Abstract 6331: A novel automated platform for rapid manufacturing of CAR T cells

Abstract Autologous cell therapy using genetically modified T cells to express chimeric antigen receptor (CAR) has yielded durable responses in cancers patients. However, currently only a small portion of patients are privileged to receive the treatment. High cost and long manufacturing time contribute to the key limitations prohibiting cell therapy broad adoption. Researchers discovered that reducing ex vivo culture improves anti-tumor activity of CAR T cells1 and recently demonstrated the process of generating functional CAR T cells within 24 hours2. This rapid process provides great promise in cost reduction of CAR T therapy and opportunity to have cells manufactured in close-to-patient settings. CART cell manufacturing process involves: (1) collection of peripheral blood; (2) isolation of T cells; (3) performing transduction via viral vector; (4) washing CAR T cells to remove viral vector, and (5) harvesting CAR T cells in IV fluid. Conventional process uses magnetic separation and serial centrifugation technologies on multiple platforms with manual operations. Here we demonstrate the feasibility of rapid manufacturing CAR T cells on an automated platform with closed fluidics without centrifugation steps. The platform, MARS® Atlas, is built with innovative technologies in immune-magnetic cell isolation and acoustic cell washing and implemented as a modular system. In this feasibility test, T cells were isolated from peripheral blood from a healthy donoron MARS® magnetic module. eGFP expressing lentivirus were added to the enriched T cells. After short incubation, cells and virus were washed with fresh medium on the MARS® acoustic module and cells were incubated with virus overnight without activation. Infected cells were washed on the acoustic module to remove free virus. Product CAR T cells were harvested in saline solution and ready for QC analysis. Starting from 20mL of peripheral blood, magnetic T cell separation was done 2mL/min achieving 90% T cell purity, 90% recovery. In the first acoustic wash, medium exchange of the mixture of cells and virus took place at 0.9mL/min flow rate. As a result, 90% removal of contaminating molecule and 99.6% recovery of T cells were achieved. The second acoustic wash completed medium exchange of transfected cells at 0.9mL/min with 96.7% removal of virus and 90% CAR T cell recovery. As a result, 18 million GFP expressing T cells were produced. In this study we have demonstrated the feasibility of rapidly manufacturing CAR-T cells on an automated platform MARS® Atlas. This result indicates the MARS® Atlas being a potential CAR T manufacturing platform to be used in close-to-patient settings to enable low-cost cell therapy. 1 Saba Ghassemi, Reducing Ex Vivo Culture Improves the Antileukemic Activity of Chimeric Antigen Receptor (CAR) T Cells, Cancer Immunol Res; 6(9) September 2018 2 Saba Ghassemi, Rapid manufacturing of non-activated potent CAR T cells, Nature Biomedical Engineering | VOL 6 | February 2022 | 118-128 Citation Format: Silin Sa, Ethan Nguyen, Jie Pan, Liping Yu. A novel automated platform for rapid manufacturing 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 6331.

Abstract 3612: Preclinical & clinical activity of autologous mRNA engineered chimeric antigen receptor monocytes for targeted cancer immunotherapy

Abstract Introduction: The tumor microenvironment (TME) is a dynamic and multifaceted system that comprises largely myeloid lineage immune cells including neutrophils, macrophages, dendritic cells and monocytes. When activated, these cells are can effectively infiltrate TME and orchestrate anti-tumor immune response. Reprogramming myeloid cells by arming them with a tumor specific chimeric antigen receptor (CAR) presents an attractive strategy to overcome current limitations in solid tumor treatment. In this study, we present the isolation and mRNA-based engineering of monocytes with chimeric antigen receptors (CARs), followed by their in vivo application for cancer treatment in rodent models and in human T cell lymphoma patients. Results: Engineered monocytes demonstrated the abilities to infiltrate the TME, undergo activation, and differentiate into macrophages and dendritic cells. Upon CAR activation, these reprogrammed myeloid cells utilized both direct and indirect mechanisms to eliminate tumor cells. This included direct tumor cell killing through phagocytosis and TNF-associated mechanisms. Furthermore, engineered monocytes exhibited the capacity to recruit natural killer (NK) cells and T cells to the TME. In the case of T cells, these engineered myeloid cells facilitated the presentation of neoantigens to T cells and stimulate an adaptive immune response. In patients with T cell lymphoma, treatment with engineered monocytes expressing a CAR targeting CD5 exhibited a favorable safety profile and was associated with TME remodeling, expansion of novel T cell clones, and promising survival outcomes. Conclusions: These findings underscore the potential of reprogrammed myeloid cells as a novel avenue for cancer immunotherapy, offering hope for improved treatments and outcomes in the fight against cancer. Citation Format: Yuxiao Wang, Michele Gerber, Michael Gorgievski, Josephine D'Alessandro, Neha Diwanji, Ronald Vale, Siddhartha Mukherjee, Daniel Getts. Preclinical & clinical activity of autologous mRNA engineered chimeric antigen receptor monocytes for targeted cancer immunotherapy [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 3612.

Abstract 6330: A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors

Abstract Introduction: Chimeric Antigen Receptor (CAR)-T cell therapy is a highly innovative form of immunotherapy that has proven to be successful in the treatment of B cell malignancies and multiple myeloma. As this treatment modality continues to evolve toward the targeting of novel tumor antigens and engineering of CAR constructs that enhance cell persistence, there is a need for reagents that can be leveraged to selectively interrogate multiple aspects of CAR-T cell biology. Here, we report on a novel and highly versatile recombinant rabbit monoclonal antibody raised against the Gly4Ser peptide linker, which is commonly integrated into single-chain variable fragment (scFv)-based CARs. This antibody can be leveraged for flow cytometry-based CAR detection, bead-based CAR-T cell enrichment, and selective activation of CAR transduced cells. Methods: The recombinant monoclonal antibody, E7O2V, was generated by immunizing rabbits with a synthetic peptide containing multiple repeats of the Gly4Ser core pentapeptide. E7O2V was directly conjugated to a panel of fluorophores and validated for specificity in a live cell flow cytometry assay using non-transduced and CAR-transduced primary human T cells. To assess its utility in cell enrichment using bead-based sorting, a biotinylated conjugate of E7O2V was used in combination with streptavidin releasable magnetic meads to immunoaffinity purify CAR transduced cells from a mixed population. Finally, a plate-bound antibody stimulation assay was leveraged to assess the ability of E7O2V to selectively activate CAR transduced cells. Results: Live cell flow cytometric analysis of CAR-transduced primary human T cells revealed that E7O2V could detect surface expressed CARs, independent of scFv specificity. No specific staining was observed on non-transduced cells. Furthermore, flow cytometric analysis of bead-free, purified cells revealed that biotinylated E7O2V could enrich CAR transduced cells to a high degree of purity and viability. E7O2V was found to selectively activate CAR transduced cells, but not non-transduced cells, as demonstrated by upregulation of cell surface activation markers, including CD69 and CD25. Conclusions: Exploiting the commonly used Gly4Ser linker of scFv-based CARs for antibody discovery, we identified a novel and highly versatile monoclonal antibody, E7O2V. This antibody binds to CARs of varying antigen specificity and can be leveraged in multiple assay formats to interrogate various attributes of CAR transduced cells, including CAR expression, CAR signaling, and transcriptome profiling enabled by bead-based enrichment. Citation Format: Amrik Singh, Sarah L'Heureux, Jeremy Fisher. A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors [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 6330.

Abstract 5228: Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile

Abstract Acute myeloid leukemia (AML) is an aggressive hematological malignancy that, despite advances in treatment, has an overall 5-year survival rate of less than 30%. There is an urgent unmet need for new and effective therapies for AML. Chimeric antigen receptor (CAR)-modified cell therapies are emerging as promising options. However, the selection of suitable antigens is challenging, due to the heterogeneous expression of potential antigen targets in AML. Most antigens, such as CD123, are expressed on both leukemic cells and normal hematopoietic stem and progenitor cells. In addition, conventional autologous or allogeneic T cell therapies present many limitations, such as cytokine release syndrome (CRS) and graft-vs-host disease (GvHD). We therefore evaluated CD123-targeting CAR designs in vδ1-enriched γδT cells, an innate immune cell platform that is inherently non-MHC restricted, has low risks of CRS and GvHD, and is capable of discriminating between malignant and healthy cells in preclinical models. We engineered vδ1-enriched γδT cells from peripheral blood of healthy individuals to express various CD123-targeting CAR constructs. The CD123-targeting CAR-engineered vδ1-enriched γδT cells expressed high levels of natural cytotoxicity receptors, suggesting capability to target innate receptor ligands on AML cell surface. CD123-targeting CAR-expressing vδ1-enriched γδT cells demonstrated enhanced cytotoxicity in vitro against multiple AML cell lines, in both short-term co-culture and repeated antigen stimulation assays. CD123-targeting CAR-expressing vδ1-enriched γδT cells also showed enhanced efficacy and survival in vivo against mouse models of human AML. Interestingly, attenuation of CAR activity via novel designs preserved the inherent selectivity of vδ1-enriched γδT cells against AML cells relative to normal hematopoietic stem or progenitor cells, which also express CD123. Together, our results demonstrated through rational CAR designing and engineering in vδ1-enriched γδT cells, an allogeneic cell therapy targeting CD123 can be generated with selective cytotoxicity against AML cells while sparing normal hematopoietic stem cells. Such product designs have the potential to be a transformative therapeutic option for a broad population of AML patients. Citation Format: Mei Rosa Ng, Sarah L. Hayward, Di Zhang, Gary Gu, Bri Paulhus, Donjeta Gjuka, Collin Crowley, Sophie McLaughlin, Anya Lublinsky, Angel Mathew, Evans Berreondo Giron, Doanh Mai, Madisen Lee, Megha Wal, Rebecca Alade, Andre Simoes, Robin Lytle, James Gavin, Shira Cramer, Zhong-hua Yan, Taylor Hickman, Rashmi Choudhary, Cori Abikoff, Yana Wang, Istvan Kovacs, Nandhu M. Sobhana, Lan Cao. Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile [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 5228.

Aptamer-Based Nongenetic Reprogramming of CARs Enables Flexible Modulation of T Cell-Mediated Tumor Immunotherapy.

Innovating the design of chimeric antigen receptors (CARs) beyond conventional structures would be necessary to address the challenges of efficacy, safety, and applicability in T cell-based cancer therapy, whereas excessive genetic modification might complicate CAR design and manufacturing, and increase gene editing risks. In this work, we used aptamers as the antigen-recognition unit to develop a nongenetic CAR engineering strategy for programming the antitumor activity and specificity of CAR T cells. Our results demonstrated that aptamer-functionalized CAR (Apt-CAR) T cells could be directly activated by recognizing target antigens on cancer cells, and then impart a cytotoxic effect for cancer elimination in vitro and in vivo. The designable antigen recognition capability of Apt-CAR T cells allows for easy modulation of their efficacy and specificity. Additionally, multiple features, e.g., tunable antigen-binding avidity and the tumor microenvironment responsiveness, could be readily integrated into Apt-CAR design without T cell re-engineering, offering a new paradigm for developing adaptable immunotherapeutics.

Enhancing chimeric antigen receptor T cell therapy by modulating the p53 signaling network with Δ133p53α

Chimeric antigen receptor (CAR) T cell dysfunction is a major barrier to achieving lasting remission in hematologic cancers, especially in chronic lymphocytic leukemia (CLL). We have shown previously that Δ133p53α, an endogenous isoform of the human TP53 gene, decreases in expression with age in human T cells, and that reconstitution of Δ133p53α in poorly functional T cells can rescue proliferation [A. M. Mondal et al., J. Clin. Invest. 123, 5247-5257 (2013)]. Although Δ133p53α lacks a transactivation domain, it can form heterooligomers with full-length p53 and modulate the p53-mediated stress response [I. Horikawa et al., Cell Death Differ. 24, 1017-1028 (2017)]. Here, we show that constitutive expression of Δ133p53α potentiates the anti-tumor activity of CD19-directed CAR T cells and limits dysfunction under conditions of high tumor burden and metabolic stress. We demonstrate that Δ133p53α-expressing CAR T cells exhibit a robust metabolic phenotype, maintaining the ability to execute effector functions and continue proliferating under nutrient-limiting conditions, in part due to upregulation of critical biosynthetic processes and improved mitochondrial function. Importantly, we show that our strategy to constitutively express Δ133p53α improves the anti-tumor efficacy of CAR T cells generated from CLL patients that previously failed CAR T cell therapy. More broadly, our results point to the potential role of the p53-mediated stress response in limiting the prolonged antitumor functions required for complete tumor clearance in patients with high disease burden, suggesting that modulation of the p53 signaling network with Δ133p53α may represent a translationally viable strategy for improving CAR T cell therapy.

Optical sensing and control of T cell signaling pathways.

T cells regulate adaptive immune responses through complex signaling pathways mediated by T cell receptor (TCR). The functional domains of the TCR are combined with specific antibodies for the development of chimeric antigen receptor (CAR) T cell therapy. In this review, we first overview current understanding on the T cell signaling pathways as well as traditional methods that have been widely used for the T cell study. These methods, however, are still limited to investigating dynamic molecular events with spatiotemporal resolutions. Therefore, genetically encoded biosensors and optogenetic tools have been developed to study dynamic T cell signaling pathways in live cells. We review these cutting-edge technologies that revealed dynamic and complex molecular mechanisms at each stage of T cell signaling pathways. They have been primarily applied to the study of dynamic molecular events in TCR signaling, and they will further aid in understanding the mechanisms of CAR activation and function. Therefore, genetically encoded biosensors and optogenetic tools offer powerful tools for enhancing our understanding of signaling mechanisms in T cells and CAR-T cells.

Abstract A044: Screening and characterization of CD38 chimeric antigen receptors for the development of natural killer cell-based therapies

Abstract Background: CD38 is a transmembrane protein that is often overexpressed in multiple myeloma (MM) and other hematologic cancers. The success of anti-CD38 monoclonal antibodies, like daratumumab and isatuximab, in the treatment of MM supports the potential of CD38-targeting therapy candidates in development, including chimeric antigen receptor (CAR) engineered natural killer (NK) cells. Because CD38 is expressed on normal hematopoietic cells and other tissues, it is important to assess the affinity and epitope-specificity of the CD38-targeting moiety to limit potential on-target/off-tumor toxicities. To determine how different biophysical characteristics impact CAR activity, we identified a diverse panel of CD38-specific antibodies, which were shown to display a wide range of affinities and epitopes towards CD38 and converted these clones into CARs to evaluate their expression and target-specific and non-specific activation in a high-throughput (HTP) CD38-knockout (CD38 KO) Jurkat screen. Methods: Antibodies against CD38 were identified through hybridoma screening. Humanized mice were immunized with the extracellular domain of CD38, and lymphocytes from lymph nodes were fused with SP2/0 cells to generate hybridoma cells. Supernatants from single cell sorted hybridoma clones were screened against CD38+ and CD38 KO Jurkat cells and were assessed for affinity and epitope via surface plasmon resonance detection with the Carterra LSA. 96 unique clones were converted into CARs and transduced into CD38 KO Jurkat cells. CAR-expressing Jurkat cells were assayed for CAR expression and activation (via CD69 expression) when cultured overnight in the presence or absence of MM.1R target cells. Seven CARs were selected for additional characterization in primary NK cells. NK cells from healthy donors were expanded on our proprietary NKSTIM cell line, knocked out for CD38 using a CRISPR-Cas system, and transduced with CD38 CAR constructs. CD38 CAR NK-mediated cytotoxicity against MM.1S target cells was assessed by IncuCyte® S3 live cell analysis system. Results: Hybridoma screening identified many unique CD38-specific antibodies, displaying a wide range of affinities and epitopes towards CD38. Despite these biophysical differences, many of these antibodies, when converted to CARs, confer target-specific activity. Multiple epitopes on CD38 may be targeted and even low affinity antibodies were sufficient for target-specific CAR activation. Furthermore, the seven selected CAR candidates that were expressed in CD38 KO donor NK cells displayed potent in vitro target cell killing against MM.1S target cells. Conclusions: In summary, we have identified and characterized a range of CD38-specific antibodies. When converted into CARs, these targeting moieties mediate target-specific activation, despite differences in CD38 epitope and in affinity, demonstrating the utility of HTP CAR screening. Moreover, expression of the top seven CAR candidates in CD38 KO NK cells resulted in potent target cell killing, supporting further pre-clinical evaluation in relevant models. Citation Format: Emily N Kang, Jacinda T Chen, Bing Li, Kate Jamboretz, Nitin Patel, Daniel Bedinger, Kyle T Pratt, Jessica Hsieh, Luxuan TE Buren, Chao Guo, Ivan Chan, James B Trager, Sasha Lazetic. Screening and characterization of CD38 chimeric antigen receptors for the development of natural killer cell-based therapies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A044.

Enhancing brain entry and therapeutic activity of chimeric antigen receptor T cells with intra-arterial NEO100 in a mouse model of CNS lymphoma.

Malignancies of the CNS are difficult to treat because the blood-brain barrier (BBB) prevents most therapeutics from reaching the intracranial lesions at sufficiently high concentrations. This also applies to chimeric antigen receptor (CAR) T cells, for which systemic delivery is inferior to direct intratumoral or intraventricular injection of the cells. The authors previously reported on a novel approach to safely and reversibly open the BBB of mice by applying intra-arterial (IA) injections of NEO100, a pharmaceutical-grade version of the natural monoterpene perillyl alcohol. The authors hypothesized that this method would enable enhanced brain entry and therapeutic activity of intravenously delivered CAR T cells, which the authors tested in a mouse model of CNS lymphoma. Human Raji lymphoma cells were implanted into the brains of immune-deficient mice. After tumor uptake was confirmed with bioluminescent imaging, 0.3% NEO100 was injected intra-arterially, which was followed by intravenous (IV) delivery of CD19-targeted CAR T cells. After this single intervention, tumor growth was monitored with imaging, long-term survival of mice was recorded, and select mice were euthanized to analyze the distribution of CAR T cells in brain tissue. Intravenously injected CAR T cells could be readily detected in brain tumor areas after IA injection of NEO100 but not after IA injection of the vehicle (without NEO100). Although all untreated control animals died within 3 weeks, all mice that received IA NEO100 followed by IV CAR T cells survived and thrived for 200 days, when the experiment was terminated. Of the mice that received IV CAR T cells without prior IA NEO100, 3 died within 3 weeks and 2 survived long-term. BBB opening by IA NEO100 facilitates brain entry of intravenously delivered CD19 CAR T cells. The long-term survival of all mice with CNS lymphoma, along with the disappearance of the tumor as determined with imaging, suggests that this one-time therapeutic intervention was curative. BBB opening by IA NEO100 may offer a novel option to increase brain access by CAR T cells.

Modular chimeric cytokine receptors with leucine zippers enhance the antitumour activity of CAR T cells via JAK/STAT signalling.

The limited availability of cytokines in solid tumours hinders maintenance of the antitumour activity of chimeric antigen receptor (CAR) T cells. Cytokine receptor signalling pathways in CAR T cells can be activated by transgenic expression or injection of cytokines in the tumour, or by engineering the activation of cognate cytokine receptors. However, these strategies are constrained by toxicity arising from the activation of bystander cells, by the suboptimal biodistribution of the cytokines and by downregulation of the cognate receptor. Here we show that replacement of the extracellular domains of heterodimeric cytokine receptors in T cells with two leucine zipper motifs provides optimal Janus kinase/signal transducer and activator of transcription signalling. Such chimeric cytokine receptors, which can be generated for common γ-chain receptors, interleukin-10 and -12 receptors, enabled T cells to survive cytokine starvation without induction of autonomous cell growth, and augmented the effector function of CAR T cells in vitro in the setting of chronic antigen exposure and in human tumour xenografts in mice. As a modular design, leucine zippers can be used to generate constitutively active cytokine receptors in effector immune cells.

Development of a Regulated, Optimized Mesothelin Chimeric Antigen Receptor (CAR) for the Treatment of Mesothelin Positive Cancers

Introduction: Mesothelin (MSLN) is an attractive target for chimeric antigen receptor T cell (CAR-T) immunotherapy as it is highly expressed on mesothelioma, ovarian, lung cancer, and some forms of leukemia, including approximately 30% of acute myeloid leukemia (AML); however, MSLN directed CAR-T cells are prone to exhaustion, and soluble proteins, such as shed MSLN and MUC16 can inhibit anti-tumor efficacy. To address these deficiencies, we developed a novel, fully-human MSLN-specific CAR using our OutSpacer™ technology to optimize the immune synapse geometry. Further, this MSLN CAR is expressed using a novel regulatory response element to dynamically express the CAR molecule to enhance CAR-T potency and durability. Methods: A set of fully-human single domain binders (VHH) were produced and screened against a spacer library to identify high-performing CARs. The spacer library is composed of human extracellular polypeptides selected for optimal biophysical properties (e.g., flexibility), enabling systematic sampling to identify CAR constructs with optimal immune synapse geometry. More than 300 MSLN-directed CAR candidates were evaluated in vitro using 2D or 3D (spheroid) culture assays to measure tumor killing and cytokine production against several MSLN + cell lines that express different levels of the target protein. These functional assays were also performed in the presence of recombinant MSLN (10ug/mL) or MUC16 (1ug/mL) to assess potential inhibition by shed target or ligand. Top performing MSLN CARs were subsequently tested in NSG mice (2e6, 4e6 and 8e6 cells/animal; 2 donors) subcutaneously engrafted with MSLN + H1650 tumor cells to identify the best CAR candidate. To further improve the durability of T cell response, the MSLN CAR was expressed under the control of an engineered promoter (OP1) to dynamically regulate expression, and compared to CARs expressed constitutively using the MND promoter. CAR-T cells were repetitively stimulated (4x) by plate-bound antigen in combination with CD58 and ICAM1, and subsequently tested in a tumor spheroid assay for killing, cytokine production, CAR expression and T cell activation/ exhaustion markers (i.e., PD-1, TIGIT, CD39 and TIM3). Results: Anti-MSLN VHH domains targeting region 1 or 3 (RI and RIII) of the MLSN extracellular domain were paired with a defined spacer library to define the optimal immune synapse. Contrary to the conclusions of prior studies, RI (membrane-distal) binders outperformed RIII (membrane-proximal) binders when paired with their optimal spacer. Based on tumor killing and cytokine production, RI targeted CARs preferred a spacer length of approximately 75 Å, consistent with our previously-reported optimal CAR synapse of ~200 Å. All spacer optimized MSLN-specific CARs resisted inhibition by soluble MSLN and MUC16. In vivo studies with two donors in NSG mice engrafted with MSLN + tumors showed that one CAR (B8S8) eliminated tumors at a dose of 2e6 and 4e6 CAR-T cells per animal, where other CAR constructs, including a clinically-used MSLN-specific CAR, failed to control tumors even at 8e6 cells per animal. Secondly, we assessed whether a novel “stim-on” promoter (OP1) could enhance CAR-T activity by preserving T cell function. Here, B8S8 CAR was expressed via OP1 or the constitutive MND promoter (n=3 donors). Following chronic antigen exposure by plate-bound antigen, OP1 regulated MSLN CAR T cells retained the capacity to eliminate MSLN + H226 tumor spheroids compared to MND-driven CAR T constructs. OP1-regulated CAR T cells also continued to produce high levels of cytokines (IFN-γ and IL-2) whereas MND CAR-T cells lost cytokine production ability. Further, phenotypic analyses showed that MND CAR-T cells expressed elevated levels of PD-1 and TIGIT compared to OP1 CAR-T cells. Conclusions: Contrary to the conclusions of prior studies with MSLN-targeted CARs, membrane distal epitopes were superior to membrane proximal epitopes when binders were paired with the correct spacer, and shed MSLN and MUC16 were not inhibitory for the best-performing CARs, regardless of target epitope. Furthermore, regulated expression enhanced the potency and durability of response compared to constitutive expression. This approach may improve CAR activity in patients with MSLN + solid tumors or leukemia.

Fine-tuning the antigen sensitivity of CAR T cells: emerging strategies and current challenges.

Chimeric antigen receptor (CAR) T cells are "living drugs" that specifically recognize their target antigen through an antibody-derived binding domain resulting in T cell activation, expansion, and destruction of cognate target cells. The FDA/EMA approval of CAR T cells for the treatment of B cell malignancies established CAR T cell therapy as an emerging pillar of modern immunotherapy. However, nearly every second patient undergoing CAR T cell therapy is suffering from disease relapse within the first two years which is thought to be due to downregulation or loss of the CAR target antigen on cancer cells, along with decreased functional capacities known as T cell exhaustion. Antigen downregulation below CAR activation threshold leaves the T cell silent, rendering CAR T cell therapy ineffective. With the application of CAR T cells for the treatment of a growing number of malignant diseases, particularly solid tumors, there is a need for augmenting CAR sensitivity to target antigen present at low densities on cancer cells. Here, we discuss upcoming strategies and current challenges in designing CARs for recognition of antigen low cancer cells, aiming at augmenting sensitivity and finally therapeutic efficacy while reducing the risk of tumor relapse.

Noninvasive longitudinal PET/CT imaging of CAR T cells using PSMA reporter gene.

Chimeric antigen receptor (CAR) T cell therapy has achieved great success in treating hematologic malignancies. However, it is yet to prove effective in the treatment of solid tumors. Thus, it is necessary to develop appropriate methodology for the long-term, accurate, and quantitative evaluation of the distribution and activities of CAR T cells in solid tumors. In the present study, we engineered TfR ΔPSMA CAR (CAR-ΔPSMA) T cells, which targeted the transferrin receptor (TfR) expressed by tumor cells and could be tracked in vivo via a reporter gene encoding the truncated prostate specific membrane antigen (ΔPSMA). We then quantitatively monitored these CAR T cells in vitro and in vivo using [68Ga]Ga-PSMA-617 positron emission tomography (PET)/computed tomography (CT). The CAR-ΔPSMA T cells were genetically engineered by transducing T cells with a lentiviral vector encoding TfR41BBζ-T2A-ΔPSMA. Firstly, the target expression, activation, and cytotoxicity of CAR-ΔPSMA T cells were validated in vitro. Secondly, the minimum thresholds of CAR-ΔPSMA T cells detection for [68Ga]Ga-PSMA-617 PET/CT were also determined in vitro and in vivo respectively. Lastly, the feasibility of monitoring the biodistribution and infiltration of CAR-ΔPSMA T cells after systematic administration was evaluated in the breast cancer subcutaneous xenograft model. The CAR-ΔPSMA T cells retained activation and tumor killing capacity after transduction of the ΔPSMA-encoding reporter gene. Next, the CAR-ΔPSMA T cells could be reliably tracked by [68Ga]Ga-PSMA-617 PET/CT, the detection sensitivity of which was 250 cells/mm3 in vitro and 100 cells/mm3 in vivo. Next, the sequential imaging assays revealed that [68Ga]Ga-PSMA-617 PET/CT could be used to specifically visualize ΔPSMA+ CAR T cells at the tumor site. The increase in the [68Ga]Ga-PSMA-617 signal intensity over time allowed us to effectively detect CAR T cells in vivo. Our findings preliminarily confirmed that [68Ga]Ga-PSMA-617 PET/CT could reliably detect CAR-ΔPSMA T cells in vitro and in vivo in solid tumors, laying the foundation for the monitoring CAR T cell therapy in the future.

Poliovirus receptor-based chimeric antigen receptor T cells combined with NK-92 cells exert potent activity against glioblastoma.

Poliovirus receptor interacts with 3 receptors: T-cell immunoglobulin immunoreceptor tyrosine-based inhibitory motif, CD96, and DNAX accessory molecule 1, which are predominantly expressed on T cells and natural killer (NK) cells. Many solid tumors, including IDH wild-type glioblastoma, have been reported to overexpress poliovirus receptor, and this overexpression is associated with poor prognosis. However, there are no preclinical or clinical trials investigating the use of cell-based immunotherapies targeting poliovirus receptor in IDH wild-type glioblastoma. We analyzed poliovirus receptor expression in transcriptome sequencing databases and specimens from IDH wild-type glioblastoma patients. We developed poliovirus receptor targeting chimeric antigen receptor T cells using lentivirus. The antitumor activity of chimeric antigen receptor T cells was demonstrated in patient-derived glioma stem cells, intracranial and subcutaneous mouse xenograft models. We verified poliovirus receptor expression in primary glioma stem cells, surgical specimens from IDH wild-type glioblastoma patients, and organoids. Accordingly, we developed poliovirus receptor-based second-generation chimeric antigen receptor T cells. The antitumor activity of chimeric antigen receptor T cells was demonstrated in glioma stem cells and xenograft models. Tumor recurrence occurred in intracranial xenograft models because of antigen loss. The combinational therapy of tyrosine-based inhibitory motif extracellular domain-based chimeric antigen receptor T cells and NK-92 cells markedly suppressed tumor recurrence and prolonged survival. Poliovirus receptor-based chimeric antigen receptor T cells were capable of killing glioma stem cells and suppressing tumor recurrence when combined with NK-92 cells.

A genetically encoded protein tag for control and quantitative imaging of CAR T cell therapy

Chimeric antigen receptor (CAR) Tcell therapy has been successful for hematological malignancies. Still, a lack of efficacy and potential toxicities have slowed its application for other indications. Furthermore, CAR Tcells undergo dynamic expansion and contraction invivo that cannot be easily predicted or controlled. Therefore, the safety and utility of such therapies could be enhanced by engineered mechanisms that engender reversible control and quantitative monitoring. Here, we use a genetic tag based on the enzyme Escherichia coli dihydrofolate reductase (eDHFR), and derivatives of trimethoprim (TMP) to modulate and monitor CAR expression and Tcell activity. We fused eDHFR to the CAR C terminus, allowing regulation with TMP-based proteolysis-targeting chimeric small molecules (PROTACs). Fusion of eDHFR to the CAR does not interfere with cell signaling or its cytotoxic function, and the addition of TMP-based PROTACs results in a reversible and dose-dependent inhibition of CAR activity via the proteosome. We show the regulation of CAR expression invivo and demonstrate imaging of the cells with TMP radiotracers. Invitro immunogenicity assays using primary human immune cells and overlapping peptide fragments of eDHFR showed no memory immune repertoire for eDHFR. Overall, this translationally-orientied approach allows for temporal monitoring and image-guided control of cell-based therapies.

CD8+ chimeric antigen receptor T cells manufactured in absence of CD4+ cells exhibit hypofunctional phenotype

BackgroundCell culture conditions during manufacturing can impact the clinical efficacy of chimeric antigen receptor (CAR) T cell products. Production methods have not been standardized because the optimal approach remains unknown....