Neoantigens in the Application of Adoptive Cell Therapy for Solid Tumors

Chimeric Antigen Receptor (CAR)-T cell therapy has demonstrated remarkable success in treating hematological malignancies, yet its efficacy in solid tumors remains limited, with response rates as low as ∼9% and limited by high relapse rates (∼50% within one year). Furthermore, CAR-T therapies have demonstrated challenges for reduced efficacy, largely attributed to poor penetration and poor infiltration of CAR-T cells into the tumor microenvironment (TME), combined with immune suppressive factors such as T cell exhaustion, metabolic fatigue, and anti-inflammatory cytokines. To address these barriers, we investigate the potential of photobiomodulation (PBM), a non-invasive light-based treatment to enhance CAR-T cell function in both solid tumors and hematological malignancies. PBM, which stimulates cellular activity, could improve CAR-T cell survival, metabolic fitness, and persistence, and thus, therapeutic efficacy. Using optimized wavelengths, we evaluated CAR-T cell function in vitro, focusing on cellular metabolism and expansion. Metabolic analysis using the Seahorse XF platform revealed a marked upregulation in oxygen consumption rates (OCR) indicative of enhanced glycolytic activity (p<0.001) in CAR-T cells exposed to PBM. Furthermore, we observed a significant increase in T cell counts over a 14-day period, validated by CFSE labeling and flow cytometry with AO/PI and hemacytometer counting, in addition to evaluation of cytotoxicity and differentiation of PBM-treated CAR-T cells compared to control CAR-T cells. These findings suggest PBM preconditioning significantly enhances CAR-T cell energy production, proliferation, metabolic activity, and persistence, potentially overcoming challenges related to TME penetration, immune suppression, and exhaustion in solid tumors. Additionally, the PBM treatment could help address issues of persistence and proliferation of CAR-T cells in both solid tumors and hematological malignancies, improving their overall therapeutic potential and optimizing treatment protocols for clinical application. This combinational approach of CAR-T cell therapy and PBM represents a promising strategy to refine and optimize CAR-T functional longevity in both hematological and solid malignancies for clinical translation. As my lab experience focuses on immunology and the development of CAR-T therapies for pre-clinical use, this project will be in collaboration with the Jenkins/Moffitt Lab for their expertise in PBM. An invention disclosure was made to CWRU for the use of PBM for adoptive cell therapy. It is expected that a provisional application will be filed in the future prior to public disclosure. CWRU is the full owner of this intellectual property. Citation Format: Jude S. Franklin. Enhancing chimeric antigen receptor (CAR)-T cell therapy for solid tumors through photobiomodulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3190.

Pediatric cancer poses a major health challenge globally, especially in low-middle-income countries like Indonesia. The survival rate of pediatric cancer in many high-income countries (HICs) reaches 90%, while it only ranges from 5 to 60% in LMICs. Over 80% of children with cancer live in low-middle-income countries, indicating the urgency to improve the survival rate of pediatric cancer in LMICs [1]. In Indonesia, the prevalence of pediatric cancer was 43.5% in 2020, making it the highest among Southeast Asian countries [2]. According to Dharmais Cancer Hospital (2024), the national cancer referral center for all of Indonesia, the 5-year survival rate of high-risk pediatric acute lymphoblastic leukemia is only 48.8% (unpublished data).One key factor contributing to the low survival rate of pediatric cancer in Indonesia is the lack of effective therapy options, especially for high-risk and relapsed or refractory patients. Several therapeutic approaches, such as immunotherapy, have been widely used in HICs but are still not very popular in Indonesia. CAR (Chimeric Antigen Receptor) T-cell therapy is one of the most promising immunotherapeutic approaches to treat pediatric cancer. Implementing CAR T Cell therapy in Indonesia offers promising prospects for improving the survival rates of pediatric cancer patients.CAR T cell therapy utilizes the body's immune system to specifically target and eliminate cancer cells. This innovative therapy entails extracting a patient's T cells, genetically modifying them to express chimeric antigen receptors specific to tumor-associated antigens, and then reinfusing them into the patient. Once infused, these engineered T cells recognize and eliminate cancer cells bearing the targeted antigen, thereby offering a highly targeted and potentially curative treatment option [3]. This innovative therapy has demonstrated remarkable success in treating certain hematologic malignancies, including pediatric leukemia. The most extensively studied case in childhood patients involves CAR T cells that target CD19, a B cell surface receptor [4].CAR T cell therapy holds great promise for improving survival rates among pediatric cancer patients in Indonesia. Children with refractory or relapsed leukemia, such as B-cell acute lymphoblastic leukemia (B-ALL), who have exhausted standard treatment options, can benefit from CAR T cell therapy. Most relapsed or refractory pediatric cancer patients in Indonesia do not have effective therapy options to treat the disease. CAR T cell therapy emerges as a novel therapy that can significantly improve the survival of this subset of patients. Numerous studies have documented high remission rates (ranging from 70% to 90%) in adults and children diagnosed with refractory B-ALL [4]. A study by Maude et al. [5] reported high remission rates and durable responses in young adults and children with refractory or relapsed B-ALL treated with CAR T cells. Similarly, Park et al. [6] demonstrated long-term remissions and improved survival in pediatric leukemia patients receiving CAR T cell therapy. Several groups also have observed the persistence of CAR T cells and sustained remission lasting over six months in the majority of patients examined [4]. Efforts have been made to implement CAR T cells in Indonesia. Dharmais Cancer Hospital, as a National Cancer Center in Indonesia, has initiated this effort by collaborating with iCarTAB Biomed Inc., a China-based CAR T cell manufacturer with one of its manufacturing sites located in Malaysia. However, this approach involves sending patients' blood samples that have been processed through leukapheresis to Malaysia for CAR T cell manufacturing, followed by the shipment of the manufactured cells back to Indonesia for administration to patients. This process is impractical and incurs intangible costs such as transportation and cryopreservation, ultimately making it more expensive for patients. Regulatory issues related to the shipment of cells across borders in the region and early preparation of patients for CAR T cell therapy soon after relapse before they succumb to treatment-related mortality or relapse-related complications are also challenges that need to be addressed [7]. Reflecting on the abovementioned issue, CAR T cell therapy adoption in Indonesia faces significant challenges. Limited healthcare infrastructure, including specialized facilities for cell therapy manufacturing and administration, poses logistical hurdles. Moreover, cost remains a major barrier, as CAR T cell therapy is often expensive and inaccessible to many patients in Indonesia. Furthermore, the lack of local expertise in cellular immunotherapy may impede the successful implementation of CAR T cell therapy programs.Efforts to address these challenges and maximize the potential of CAR T cell therapy in Indonesia are essential. This requires a multi-faceted approach involving investment in healthcare infrastructure, including establishing specialized centers equipped for CAR T cell therapy manufacturing and administration. Two alternative models have been proposed for manufacturing CAR-T cell therapy: centralized and de-centralized models [8]. In the centralized manufacturing model, point of manufacturing and point of care are located in different geographical areas, while decentralized manufacturing focuses on establishing point of care and manufacturing in close proximity. A decentralized manufacturing model might be the best approach to be implemented in LMICs like Indonesia. Building hospital-based cellular therapy manufacturing reduces the need for transportation and cryopreservation. The decentralized system's geographic proximity improves communication between manufacturing and treatment teams, facilitating the creation of customized products based on a patient's phenotype. This setup also reduces administration time and the risk of delays and mix-ups compared to centralized manufacturing, making hospital-based cellular therapy manufacturing a potentially more cost-effective option [8].In addition, initiatives to reduce the cost of therapy through partnerships with pharmaceutical companies, government subsidies, or philanthropic endeavors can improve affordability and access. Furthermore, capacity-building initiatives aimed at training local healthcare professionals in cellular immunotherapy techniques are essential for ensuring the successful implementation and sustainability of CAR T cell therapy programs in Indonesia. Collaboration between local institutions, international organizations, and industry stakeholders can facilitate knowledge transfer and technology transfer, fostering indigenous expertise in this cutting-edge treatment modality.CAR T cell therapy represents a transformative approach to improving survival rates among pediatric cancer patients in Indonesia. By harnessing the power of immunotherapy, specifically tailored to target cancer cells, CAR T cell therapy offers hope for children with refractory or relapsed leukemia who have limited treatment options. Through continued research, collaboration, and investment in healthcare infrastructure, CAR T cell therapy potentially could greatly improve the prognosis and quality of life for pediatric cancer patients in Indonesia.

CAR T-cell therapy for solid tumours

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.

Purpose: The aim is to review the current advances in designing safer and more efficient CAR-T cells and discuss the future research possibilities for the treatment of both hematological malignancies and solid tumors. Study Design: An extensive review was carried out on the basic structure of CARS, current advances to design safer and more efficient CAR-T cells, and future research possibilities for the treatment of both hematological malignancies and solid tumors. Results: Encouragement of chimeric antigen receptor-T (CAR-T) cell therapy as one of adoptive immunotherapy is increasingly important in recent years. Its preparation is based on the genetic modification of individual T cells. The innovation of the functional intracellular signaling domain is a critical part of the genetically modified T cells and requires a long journey of development that has resulted in several improvements in the safety and effectiveness of CAR-T cells. CAR-T cell therapy can be modified rapidly and has great and strong application potential according to a large number of global clinical trials. This article briefly describes the basic structure and design of CARs and discusses current trends in the development of safer and more efficient CAR-T cells for the treatment of both hematological and solid malignancies and looks forward to future research possibilities. Conclusion: It is concluded that conclude that the prospect of this technology lies in CAR-T cell engineering which can overcome aggressive TMEs and recruiting an endogenous tumor response. The final task for researchers in this field is to carry out clinical trials and secure the funding needed to complete their clinical trials. This immunotherapy continues to progress and more records of successful malignancy eradication occur.

<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>

Combinational CAR T-cell therapy for solid tumors: Requisites, rationales, and trials

Advancements in adoptive cell therapy over the last four decades have revealed various new therapeutic strategies, such as chimeric antigen receptors (CARs), which are dedicated immune cells that are engineered and administered to eliminate cancer cells. In this context, CAR T-cells have shown significant promise in the treatment of hematological malignancies. However, many obstacles limit the efficacy of CAR T-cell therapy in both solid tumors and hematological malignancies. Consequently, CAR-NK and CAR-M cell therapies have recently emerged as novel therapeutic options for addressing the challenges associated with CAR T-cell therapies. Currently, many CAR immune cell trials are underway in various human malignancies around the world to improve antitumor activity and reduce the toxicity of CAR immune cell therapy. This review will describe the comprehensive literature of recent findings on CAR immune cell therapy in a wide range of human malignancies, as well as the challenges that have emerged in recent years.

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.

Altered cancer metabolism and implications for next-generation CAR T-cell therapies

<div>AbstractPurpose:<p>Despite the success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies, successful targeting of solid tumors with CAR T cells has been limited by a lack of durable responses and reports of toxicities. Our understanding of the limited therapeutic efficacy in solid tumors could be improved with quantitative tools that allow characterization of CAR T–targeted antigens in tumors and accurate monitoring of response.</p>Experimental Design:<p>We used a radiolabeled FAP inhibitor (FAPI) [<sup>18</sup>F]AlF-FAPI-74 probe to complement ongoing efforts to develop and optimize FAP CAR T cells. The selectivity of the radiotracer for FAP was characterized <i>in vitro</i>, and its ability to monitor changes in FAP expression was evaluated using rodent models of lung cancer.</p>Results:<p>[<sup>18</sup>F]AlF-FAPI-74 showed selective retention in FAP<sup>+</sup> cells <i>in vitro</i>, with effective blocking of the uptake in presence of unlabeled FAPI. <i>In vivo</i>, [<sup>18</sup>F]AlF-FAPI-74 was able to detect FAP expression on tumor cells as well as FAP<sup>+</sup> stromal cells in the tumor microenvironment with a high target-to-background ratio. We further demonstrated the utility of the tracer to monitor changes in FAP expression following FAP CAR T-cell therapy, and the PET imaging findings showed a robust correlation with <i>ex vivo</i> analyses.</p>Conclusions:<p>This noninvasive imaging approach to interrogate the tumor microenvironment represents an innovative pairing of a diagnostic PET probe with solid tumor CAR T-cell therapy and has the potential to serve as a predictive and pharmacodynamic response biomarker for FAP as well as other stroma-targeted therapies. A PET imaging approach targeting FAP expressed on activated fibroblasts of the tumor stroma has the potential to predict and monitor therapeutic response to FAP-targeted CAR T-cell therapy.</p><p><i><a href="https://aacrjournals.org/clincancerres/article/doi/10.1158/1078-0432.CCR-22-2565" target="_blank">See related commentary by Weber et al., p. 5241</a></i></p></div>

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

<div>AbstractPurpose:<p>Despite the success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies, successful targeting of solid tumors with CAR T cells has been limited by a lack of durable responses and reports of toxicities. Our understanding of the limited therapeutic efficacy in solid tumors could be improved with quantitative tools that allow characterization of CAR T–targeted antigens in tumors and accurate monitoring of response.</p>Experimental Design:<p>We used a radiolabeled FAP inhibitor (FAPI) [<sup>18</sup>F]AlF-FAPI-74 probe to complement ongoing efforts to develop and optimize FAP CAR T cells. The selectivity of the radiotracer for FAP was characterized <i>in vitro</i>, and its ability to monitor changes in FAP expression was evaluated using rodent models of lung cancer.</p>Results:<p>[<sup>18</sup>F]AlF-FAPI-74 showed selective retention in FAP<sup>+</sup> cells <i>in vitro</i>, with effective blocking of the uptake in presence of unlabeled FAPI. <i>In vivo</i>, [<sup>18</sup>F]AlF-FAPI-74 was able to detect FAP expression on tumor cells as well as FAP<sup>+</sup> stromal cells in the tumor microenvironment with a high target-to-background ratio. We further demonstrated the utility of the tracer to monitor changes in FAP expression following FAP CAR T-cell therapy, and the PET imaging findings showed a robust correlation with <i>ex vivo</i> analyses.</p>Conclusions:<p>This noninvasive imaging approach to interrogate the tumor microenvironment represents an innovative pairing of a diagnostic PET probe with solid tumor CAR T-cell therapy and has the potential to serve as a predictive and pharmacodynamic response biomarker for FAP as well as other stroma-targeted therapies. A PET imaging approach targeting FAP expressed on activated fibroblasts of the tumor stroma has the potential to predict and monitor therapeutic response to FAP-targeted CAR T-cell therapy.</p><p><i><a href="https://aacrjournals.org/clincancerres/article/doi/10.1158/1078-0432.CCR-22-2565" target="_blank">See related commentary by Weber et al., p. 5241</a></i></p></div>