DROP-CARs: Engineering Reversible, Drug-Controlled CAR T Cell Activity with a Clinically Approved Small Molecule.

Effective solid tumor cell therapy requires new strategies to improve T-cell activation, persistence, and durable function. We developed four complementary T-cell reprogramming technologies to enhance chimeric antigen receptor (CAR) T-cell therapy in solid tumors: 1) overexpression of the activator protein 1 (AP-1) family transcription factor c-Jun to delay T-cell exhaustion and improve antitumor activity; 2) nuclear receptor subfamily 4A member 3 (NR4A3) gene knockout (KO) to further delay exhaustion and enhance functionality; 3) Epi-RTM manufacturing protocols to preserve stem-like characteristics; and 4) Stim-RTM technology, a novel activating reagent to improve product potency compared with standard reagents. LYL119 is an investigational ROR1-targeted CAR T-cell product that combines these technologies to create potent and durable CAR T cells. Healthy or NSCLC patient donor T cells were manufactured with the Epi-R protocols, activated with Stim-R or a standard reagent, and transduced with a vector encoding a ROR1 CAR and c-Jun. The NR4A3 gene or a control gene was edited using SpyFiTM Cas9 nuclease (Aldevron®). Cytotoxicity, cytokine production, phenotype, and single-cell transcriptomic and epigenetic profiles were evaluated in vitro after antigen restimulation assays designed to promote exhaustion. CAR T cell activity was evaluated in vivo using a ROR1+ NSCLC xenograft mouse model. Research and clinical scale LYL119 products achieved ~90% genome editing efficiency at the NR4A3 target gene resulting in a 13-fold protein reduction compared to non-edited CAR T cells. LYL119 exhibited superior cytotoxicity and cytokine production upon antigen restimulation across 7 different ROR1+ solid tumor cell lines compared to CAR T cells that lacked one or more reprogramming technologies. After repeated rounds of tumor cell killing, LYL119 displayed reduced surface expression of exhaustion-related receptors (e.g. TIM-3) and higher expression of stemness-related markers (e.g. CD127) compared to non-edited CAR T cells. Furthermore, transcriptomic analysis revealed global downregulation of exhaustion-related gene signatures and retention of unique cell subsets characterized by upregulation of memory and effector-associated gene signatures. LYL119 exhibited robust antitumor efficacy in vivo across a 10-fold dose range, including a very low dose of 1 × 105 CAR T cells. Lastly, LYL119 derived from NSCLC patient donor T cells also demonstrated enhanced cytotoxicity in vitro compared to control CAR T cells. These nonclinical data suggest LYL119, which combines c-Jun overexpression, NR4A3 KO, Epi-R protocols, and Stim-R technology, can limit exhaustion, maintain stem-like features, and has the potential to provide effective and durable CAR T-cell antitumor activity in patients with ROR1+ solid tumors. Citation Format: Viola C. Lam, Aileen Li, Meritxell Galindo Casas, Jessica Barragan, Christina Cheung, Jessica Briones, Esha Afreen, Grant Vavra, Jia Lu, Purnima Sundar, Rowena Martinez, Candace Sims, Shobha Potluri, Omar Ali, Alexander S. Cheung, Rachel C. Lynn. LYL119, an investigational ROR1-targeted CAR T-cell product incorporating four novel reprogramming technologies designed for effective cell therapy for solid tumors [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 49.

<div>Abstract<p>Infiltration of tumor by T cells is a prerequisite for successful immunotherapy of solid tumors. In this study, we investigate the influence of tumor-targeted radiation on chimeric antigen receptor (CAR) T-cell therapy tumor infiltration, accumulation, and efficacy in clinically relevant models of pleural mesothelioma and non-small cell lung cancers. We use a non-ablative dose of tumor-targeted radiation prior to systemic administration of mesothelin-targeted CAR T cells to assess infiltration, proliferation, anti-tumor efficacy, and functional persistence of CAR T cells at primary and distant sites of tumor. A tumor-targeted, non-ablative dose of radiation promotes early and high infiltration, proliferation, and functional persistence of CAR T cells. Tumor-targeted radiation promotes tumor-chemokine expression and chemokine-receptor expression in infiltrating T cells, and results in a subpopulation of higher-intensity CAR-expressing T cells with high co-expression of chemokine receptors that further infiltrate distant sites of disease, enhancing CAR T-cell anti-tumor efficacy. Enhanced CAR T-cell efficacy is evident in models of both high-mesothelin-expressing mesothelioma and mixed-mesothelin-expressing lung cancer—two thoracic cancers for which radiation therapy is part of the standard of care. Our results strongly suggest that the use of tumor-targeted radiation prior to systemic administration of CAR T cells may substantially improve CAR T-cell therapy efficacy for solid tumors. Building on our observations, we describe a translational strategy of “sandwich” cell therapy for solid tumors that combines sequential metastatic site–targeted radiation and CAR T cells—a regional solution to overcome barriers to systemic delivery of CAR T cells.</p></div>

<div>Abstract<p>Infiltration of tumor by T cells is a prerequisite for successful immunotherapy of solid tumors. In this study, we investigate the influence of tumor-targeted radiation on chimeric antigen receptor (CAR) T-cell therapy tumor infiltration, accumulation, and efficacy in clinically relevant models of pleural mesothelioma and non-small cell lung cancers. We use a non-ablative dose of tumor-targeted radiation prior to systemic administration of mesothelin-targeted CAR T cells to assess infiltration, proliferation, anti-tumor efficacy, and functional persistence of CAR T cells at primary and distant sites of tumor. A tumor-targeted, non-ablative dose of radiation promotes early and high infiltration, proliferation, and functional persistence of CAR T cells. Tumor-targeted radiation promotes tumor-chemokine expression and chemokine-receptor expression in infiltrating T cells, and results in a subpopulation of higher-intensity CAR-expressing T cells with high co-expression of chemokine receptors that further infiltrate distant sites of disease, enhancing CAR T-cell anti-tumor efficacy. Enhanced CAR T-cell efficacy is evident in models of both high-mesothelin-expressing mesothelioma and mixed-mesothelin-expressing lung cancer—two thoracic cancers for which radiation therapy is part of the standard of care. Our results strongly suggest that the use of tumor-targeted radiation prior to systemic administration of CAR T cells may substantially improve CAR T-cell therapy efficacy for solid tumors. Building on our observations, we describe a translational strategy of “sandwich” cell therapy for solid tumors that combines sequential metastatic site–targeted radiation and CAR T cells—a regional solution to overcome barriers to systemic delivery of CAR T cells.</p></div>

<div>Abstract<p>Infiltration of tumor by T cells is a prerequisite for successful immunotherapy of solid tumors. In this study, we investigate the influence of tumor-targeted radiation on chimeric antigen receptor (CAR) T-cell therapy tumor infiltration, accumulation, and efficacy in clinically relevant models of pleural mesothelioma and non–small cell lung cancers. We use a nonablative dose of tumor-targeted radiation prior to systemic administration of mesothelin-targeted CAR T cells to assess infiltration, proliferation, antitumor efficacy, and functional persistence of CAR T cells at primary and distant sites of tumor. A tumor-targeted, nonablative dose of radiation promotes early and high infiltration, proliferation, and functional persistence of CAR T cells. Tumor-targeted radiation promotes tumor-chemokine expression and chemokine-receptor expression in infiltrating T cells and results in a subpopulation of higher-intensity CAR-expressing T cells with high coexpression of chemokine receptors that further infiltrate distant sites of disease, enhancing CAR T-cell antitumor efficacy. Enhanced CAR T-cell efficacy is evident in models of both high-mesothelin-expressing mesothelioma and mixed-mesothelin-expressing lung cancer—two thoracic cancers for which radiotherapy is part of the standard of care. Our results strongly suggest that the use of tumor-targeted radiation prior to systemic administration of CAR T cells may substantially improve CAR T-cell therapy efficacy for solid tumors. Building on our observations, we describe a translational strategy of “sandwich” cell therapy for solid tumors that combines sequential metastatic site–targeted radiation and CAR T cells—a regional solution to overcome barriers to systemic delivery of CAR T cells.</p></div>

Infiltration of tumor by T cells is a prerequisite for successful immunotherapy of solid tumors. In this study, we investigate the influence of tumor-targeted radiation on chimeric antigen receptor (CAR) T-cell therapy tumor infiltration, accumulation, and efficacy in clinically relevant models of pleural mesothelioma and non-small cell lung cancers. We use a nonablative dose of tumor-targeted radiation prior to systemic administration of mesothelin-targeted CAR T cells to assess infiltration, proliferation, antitumor efficacy, and functional persistence of CAR T cells at primary and distant sites of tumor. A tumor-targeted, nonablative dose of radiation promotes early and high infiltration, proliferation, and functional persistence of CAR T cells. Tumor-targeted radiation promotes tumor-chemokine expression and chemokine-receptor expression in infiltrating T cells and results in a subpopulation of higher-intensity CAR-expressing T cells with high coexpression of chemokine receptors that further infiltrate distant sites of disease, enhancing CAR T-cell antitumor efficacy. Enhanced CAR T-cell efficacy is evident in models of both high-mesothelin-expressing mesothelioma and mixed-mesothelin-expressing lung cancer-two thoracic cancers for which radiotherapy is part of the standard of care. Our results strongly suggest that the use of tumor-targeted radiation prior to systemic administration of CAR T cells may substantially improve CAR T-cell therapy efficacy for solid tumors. Building on our observations, we describe a translational strategy of "sandwich" cell therapy for solid tumors that combines sequential metastatic site-targeted radiation and CAR T cells-a regional solution to overcome barriers to systemic delivery of CAR T cells.

CAR T-cell therapy for solid tumours

Promoter usage regulating the surface density of CAR molecules may modulate the kinetics of CAR-T cells in vivo

Genetic deletion of vasoactive intestinal peptide (VIP) and PI3K signaling pathways impair CAR T cell function

Acute Kidney Injury After the CAR-T Therapy Tisagenlecleucel

BackgroundGlucose deprivation inhibits T-cell metabolism and function. Glucose levels are low in the tumor microenvironment of solid tumors and insufficient glucose uptake limits the antitumor response of T cells. Furthermore,...

Rationale: The recent success of chimeric antigen receptor (CAR) T cell therapy in treating leukemia has brought new enthusiasm to this strategy. While the majority of CAR research has been primarily focused on targeting tumor cells, our group is developing a new CAR construct to target Fibroblast Activation Protein (FAP), a molecule that is highly and selectively expressed on cancer-associated fibroblasts (CAFs). It is well known that these CAFs play an important role in tumor development and tumor progression. Thus, we hypothesized that the immune-mediated destruction of CAFs by adoptively transferred FAP-CAR T cells would inhibit tumor growth in pre-clinical tumor models. Experimental procedures: The 73.3 anti-mouse FAP hybridoma was sequenced to obtain the single chain Fv to insert into our existing CAR construct with the 4-1BB and CD3ζ intracellular signaling domains. Different combinations of FAP-CAR constructs were synthesized and transduced into human T cells first to screen for optimal structure-activity response. The best two FAP-CAR constructs were then selected to transduce into mouse T cells for in vitro cytotoxicity and cytokine assays, as well as in vivo studies. To evaluate the in vivo efficacy, 2 tumor cell lines, AE17 (mouse mesothelioma) and TC1 (mouse lung cancer), were inoculated subcutaneously into flanks of syngeneic C57Bl6 mice. When the flank tumors reached 150 mm3, 10 million CAR T cells were injected through tail vein and tumors were then monitored. Tumor tissues were collected at the end of the study to evaluate the effect of CAR T cells on tumor microenvironment by flow cytometry and/or by immunohistochemistry. Results: All 8 different combinations of anti-mouse FAP CAR constructs expressed on human T cells showed target-specific activities against 3T3parental (FAP-null) versus 3T3FAP cells, in terms of cytotoxicity and cytokine production. The two best FAP-CAR constructs were then transduced into mouse T cells and re-tested for cytotoxicity and cytokine production against 3T3parental and 3T3-FAP fibroblasts, and again showed target-specific killing and cytokine production. When FAP-CAR T cells were adoptively transferred into tumor bearing mice, tumor growth inhibition (~50%) was observed in both flank tumor models while control T cells did not show any efficacy. No toxicity was detected in the animal studies. We found that FAP-CAR T cell-treated tumors had significantly more apoptotic and less proliferating tumor cells, less matrix (collagen, fibronectin), less M2 macrophages and fewer blood vessels, in comparison with the untreated and control T cell-treated tumors. To assure specificity, similar in vivo studies were also carried out in FAP knockout mice. In the KO mice, all the anti-tumor activity of FAP-CAR T cells was lost. Conclusion: Here, we demonstrate the feasibility of inhibiting tumor growth by targeting tumor stroma, instead of tumor cells, with adoptively transferred CAR T cells. We detected no safety issues with these stroma-targeted CAR T cells. We also showed that the anti-tumor effects induced by our T cells were specific to FAP. We are currently evaluating different CAR constructs against human FAP with a goal of conducting a clinical trial. This abstract is also presented as Poster A5. Citation Format: Liang-Chuan S. Wang, Albert Lo, John Scholler, Carl H. June, Ellen Pure, Steven M. Albelda. Fibroblast activation protein as a universal target for chimeric antigen receptor T cell therapy in solid tumors. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr PR14.

Despite the success of HER2 targeted therapies in HER2+ breast and gastric cancer, additional therapies are needed to address treatment-resistant metastatic disease. Adoptive immune cell therapy is a promising therapeutic modality given the remarkable clinical responses seen with autologous chimeric antigen receptor (CAR) T cells in hematological malignancies. However, success of cell therapy in solid tumors has been more limited. Three major impediments to the success of adoptive cell therapies in solid tumors are the heterogeneity of antigen expression, the immunosuppressive tumor microenvironment (TME), and the inherent challenges of manufacturing autologous cells and consequent variability of these cell products. Engineered, off-the-shelf, allogeneic Natural Killer (NK) cells provide a solution to these challenges. We describe here CAT-179, a novel engineered CAR-NK cell therapeutic for HER2+ solid tumors. CAT-179 cells express three transgenes: a HER2-directed CAR to effectively eliminate tumor cells, a Transforming Growth Factor (TGF) β dominant negative receptor (DNR) for resistance to TGFβ -mediated immunosuppression in the TME, and Interleukin 15 (IL15) cytokine to enhance NK cell persistence and activity for durable response. High efficiency engineering of the large (~3.7Kb) cargo containing CAR, IL15, and DNR in CAT-179 is enabled by the non-viral TC Buster™ Transposon System. Transposon engineering of CAT-179 results in high and stable expression of CAR (45% CAR at day 7 post gene delivery) without the need for post-engineering selection. CAT-179 demonstrates both CAR-dependent and innate NK receptor-dependent tumor cell killing in vitro, reducing the likelihood of tumor escape through antigen loss. CAT-179 effectively kills in vitro both high HER2-expressing SKOV3 cells as well as lower HER2-expressing HT-29 cells. CAT-179 also demonstrates resistance to TGFβ mediated immunosuppression, as evidenced by 75% reduction in TGFβ -induced phosphorylation of SMAD2 as well as prevention of TGFβ induced downregulation of NK cell activating receptors and restoration of NK cell cytotoxic activity. These data suggest CAT-179 cells will be protected from TGFβ -mediated immune suppression in the TME. Finally, the addition of IL15 in CAT-179 significantly enhances persistence for at least fourteen days in vitro without the need for exogenous cytokines. Moreover, CAT-179 administration to NSG mice showed expansion and persistence of the transferred cell product. CAT-179 addresses key hurdles to allogeneic cell therapy for solid tumors and is a promising new therapeutic approach for HER2 expressing breast, gastric and other tumors. Citation Format: Celeste Richardson, Finola Moore, Andres Alvarez, Alexia Barandiaran, Luke Barron, Eugene Choi, Tucker Ezell, Charlotte Franco, Bashar Hamza, Jennifer Johnson, Annie Khamhoung, Taeyoon Kyung, Marilyn Marques, Dominic Picarella, Jared Sewell, Alex Storer, Meghan Walsh, Vipin Suri. Allogeneic Natural Killer cells engineered to express HER2 CAR, Interleukin 15 and TGF beta dominant negative receptor effectively control HER2+ tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 555.

T cells modified by chimeric antigen receptor (CAR) have the advantage of major histocompatibility complex-independent recognition of tumor-associated antigens, so can achieve efficient response to tumor targets. Chimeric antigen receptor (CAR) T cell therapy has shown a good therapeutic effect in hematological malignancies; however, its efficacy is generally not satisfactory for solid tumors. The reasons include the lack of tumor specific antigen target on solid tumors, the uncertainty of homing ability of engineered T cells and the inhibitory immune microenvironment of tumors. In clinical trials, the targets of CAR-T cell therapy for solid tumors are mainly disialoganglioside (GD2), claudin-18 isoform 2 (CLDN18.2), mesenchymal, B7 homolog 3 (B7H3), glypican (GPC) 3 and epidermal growth factor receptor variant Ш (EGFRvШ)Ⅲ. Combination of CAR-T cells with oncolytic viruses, tyrosine kinase inhibitors, and programmed death ligand-1 monoclonal antibodies may increase its efficacy. The CAR-T cell therapy for solid tumors can be optimized through gene editing to enhance the activity of CAR-T cells, adding corresponding regulatory components to make the activation of CAR-T cells safer and more controllable, and enhancing the persistence of CAR-T cells. In this article, we review the latest advances of CAR-T cell therapy in solid tumors to provide new insights for clinical application.

Recently (July 2024), a CAR-T therapy (lisocabtagene maraleucel, or Breyanzi.) was approved by the U.S. FDA for resistant mantle cell lymphoma (MCL). This extends the list of approvals for this revolutionary cancer treatment to six, with all approvals being restricted to hematological malignancies. In select leukemias and lymphomas, CAR-T cells have produced remarkable responses, showing objective response rates of 80% or more. However, significant challenges limit the efficacy of CAR-T cells for solid tumors. Physical and environmental barriers to creating effective CAR-T cell therapy for solid tumor indications are broadly grouped into mechanistic categories of: 1) cell trafficking; 2) tumor metabolism; and 3) immunological regulation (1). Thus, a multi-pronged effort is needed to develop and implement innovative CAR-T cells that improve anti-solid tumor activity and decrease toxicity. Furthermore, with the exciting new approach of using CAR-T cells as a treatment for select auto-immune disorders (2), the need for significant improvements in efficacy is heightened. In this issue, we present five articles that summarize the state of the art of CAR-T cell therapy for solid tumors or present experimental strategies to overcome some of the challenges that limit efficacy. In their review article, Zhou et al summarize novel engineering and pharmaceutical interventions designed to address many barriers, and they discuss the latest studies that are expected to reach the clinic in the next few years. They close by presenting future potential directions, including CRISPR-based CAR genetic modifications and the generation of CAR T cells from progenitor-like T cells. Smirnov et al focus on improvements in our understanding of CAR-T cell signaling. For example, they discuss how the field is now using fifth generation CAR-T cells in preclinical trials, whereas second generation CAR-T cells introduced a costimulatory domain for better long-term CAR-T cell engraftment and efficacy. They highlight how the processes that orchestrate the activation and differentiation of CAR-T cells into a specific phenotype required for long-term persistence are not fully understood, and they discuss research aimed at elucidating how CAR domains and T-cell signaling molecules are involved in these processes.Satapathy et al, in their systematic review, analyze combination therapy involving CAR-T cells and anti PD-1 agents, an approach that is aimed at overcoming barriers of tumor inhibition, T-cell exhaustion, heightened T-cell activation, and unwanted toxicities. Topics discussed include the ability to achieve ORR and progression-free survival in both pre-clinical and clinical models and the implications and feasibility of combination immunotherapies. Finally, two papers present new experimental data on models that use innovative approaches; for example, Nagy et al are working to improve the penetration of universal CAR-T cells into the extracellular matrix of ADCC-resistant tumors, using molecular tags to target a variety of different tumor antigens. In contrast, Yang et al hypothesize that the delivery of radiation to tumors can synergize with CAR-T therapy, resulting in enhanced antitumor immunity and tumor response. They studied the feasibility of this approach by delivering b-emitting 177Lu-DOTATATE to subcutaneous tumors along with tumor-infiltrating CAR T cells expressing somatostatin receptor 2.

<h3>Background</h3> Next-generation strategies to improve T-cell functional activity, persistence, and durability are needed for effective cellular immunotherapy against solid tumors. Overexpression of the activator protein 1 (AP-1) family transcription factor...