Data from Tumor-targeted non-ablative radiation promotes solid tumor CAR T-cell therapy efficacy

<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>

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.

<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>

Efficacy of Chimeric Antigen Receptor T-Cell Therapy Is Not Impaired By Previous Bispecific Antibody Treatment in Patients with Large B-Cell Lymphoma

Mitochondrial Isocitrate Dehydrogenase Inhibition Enhances CAR T-Cell Function By Restraining Antioxidant Metabolism and Histone Acetylation

Acute Kidney Injury After the CAR-T Therapy Tisagenlecleucel

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

CAR T-cell therapy for solid tumours

Chimeric antigen receptor (CAR) T-cell therapy has produced promising response rates in patients with B cell malignancies. However, previous meta-analyses have demonstrated that CAR T-cell efficacy is unsatisfactory in patients with lymphoma unlike in patient with other hematological malignancies, but these studies included insufficient numbers of studies and patients with lymphoma. Furthermore, clinicians are interested in the effects of infusion dose, CAR structure, interleukin-2 (IL-2), and conditioning therapy regimen. All clinical trials administering autologous CAR T-cell therapy in lymphoma patients were searched in medical databases. A traditional meta-analysis was performed to assess the safety and efficacy of CAR T-cells in lymphoma treatment. Subgroup analysis was performed to determine the relationships between potential factors and efficacy. The best overall response rate (ORR), 6 month ORR (6m ORR), and severe cytokine release syndrome (sCRS) rate were calculated by Stata 14.0. A total of 411 patients across all the studies were included. Our analysis showed a best ORR of 0.71, a 6m ORR of 0.63, and an overall CRS (grade ≥ 3) rate of 0.18. The subgroup analysis showed that increased response rates and reduced CRS (grade ≥ 3) rates were associated with a low dose of CAR T-cells. No IL-2 administration and the use of a fludarabine-containing lymphodepletion regimen led to improved efficacy, while anti-CD19 CAR T cells led to a more successful outcome than anti-CD20 CAR T cells. In addition, 2nd- and 3rd-generation CAR T cells exhibited increased effectiveness in clinical studies, and no significant effect diversity was found between the 2nd- and 3rd-generation CAR T cells. sCRS was associated with a high dose of infused CAR T cells when IL-2 and fludarabine were excluded from the positive factors for sCRS. CAR T cells are promising in the treatment of relapsed or refractory lymphoma. Doses lower than 10/m, no IL-2 administration, fludarabine administration, and anti-CD19 CAR T cells were related to improved efficacy and safety.

Chimeric Antigen Receptor (CAR) T-cell therapy has been successful against hematological malignancies. However, it has not established efficacy in glioblastoma (GBM) patients. Significant challenges currently limiting the effectiveness of CAR T-cell therapy for patients with GBM include poor CAR-T cell trafficking to GBM due to the blood-brain barrier, and the presence of highly immunosuppressive and pro-tumoral tumor-associated macrophages (TAMs) in the tumor microenvironment. Furthermore, the heterogeneous expression of target antigens in GBM can lead to antigen escape and therapeutic resistance. Low-intensity pulsed ultrasound with microbubbles (LIPU/MB) has been shown to enhance CAR T-cell trafficking and improve survival in immunocompromised xenograft mice GBM models. In this study, we investigated whether multiple rounds of LIPU/MB can improve not only CAR T-cell but also endogenous T-cell infiltration into the tumors and induce myeloid cell trafficking into lymph nodes (LNs), thus enhancing CAR T-cell therapy against GBM in the immunocompetent syngeneic mice model. We recently developed a novel immunocompetent transgenic (Tg) mouse that expresses an anti-EGFRvIII CAR on T-cells and enables us to investigate the interaction between CAR T-cells and the endogenous immune environment. C57BL6/J mice bearing intracerebral EGFRvIII+ GBM received an intravenous administration of anti-EGFRvIII CAR T-cells derived from Tg-mice. The mice were stratified to receive single-LIPU (1 min before CAR T-cells administration) or triple-LIPU/MB (1 min before CAR T-cells administration, then two and four days later). Triple-LIPU/MB significantly suppressed tumor growth and improved survival and was associated with increased CAR T-cells and endogenous T-cells infiltration into the tumors compared with single-LIPU/MB or without LIPU. Furthermore, draining cervical LNs showed increased macrophages trafficking after triple-LIPU/MB. Moreover, TAMs isolated from triple-LIPU/MB mice brains showed higher levels of MHC-II expression. These results suggest that LIPU/MB may promote the tumor antigen presentation to endogenous T-cells and enhance the efficacy of CAR T-cell therapy for GBM.

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.

Pediatric brain tumors are the most common and deadliest childhood cancers, emphasizing the urgent need for improved treatments. Chimeric Antigen Receptor (CAR) T-cell therapy offers a promising, targeted approach but is limited by epigenetic challenges, particularly T-cell exhaustion. DNA methylation, a key regulatory mechanism, contributes to these challenges by suppressing CAR T-cell function. This study investigates enhancing CAR T-cell anti-tumor activity in solid and brain tumors through targeted manipulation of the DNA methylome using DNA methyltransferase inhibitors (DNMTis) during manufacturing. We hypothesize that altering the DNA methylome in CAR T-cells will enhance immune activation and anti-tumor efficacy. We optimized a transduction protocol that includes early treatment with DNMTis (including 5-Azacytidine and GSK-3484862) during the manufacturing process of murine and human CAR T-cells targeting B7-H3. Standard in vitro assays were conducted to assess whether DNMTi-treated B7-H3 CAR T-cells showed improved effector functions, including differentiation, cytokine response, persistence, and cytotoxicity. Results showed that incorporating DNMT inhibition during CAR T-cell manufacturing significantly enhanced proliferation and persistence, with improved sequential killing capacity in vitro. DNMTi-treated CAR T-cells expanded up to 55-fold more upon repeated stimulation with B7-H3-positive tumor cells and showed sustained cytokine secretion across stimulations. Treated CAR T-cells also maintained high cytotoxicity at lower effector-to-target ratios through 12 repeated stimulations, while untreated cells lost efficacy over time. Mechanistic insights indicated an enriched memory T-cell phenotype and epigenetic remodeling of exhaustion markers in DNMTi-treated CAR T-cells. In vivo studies in glioma and pediatric brain tumor models are ongoing, and preliminary data from ovarian cancer models show reduced disease burden and ascites in DNMTi-enhanced CAR T-treated groups. These findings highlight DNMT inhibition as a strategy to overcome epigenetic limitations in CAR T-cell therapy, providing a potential pathway to more effective treatments for glioma and other solid tumors through methylome-targeted reprogramming. Further research will validate these findings and assess their clinical potential. Citation Format: Elton L VanNoy, Zhongzhen Yi, Abigail Lee, Lynne El Ghorayeb, Katherine Chiappinelli, Dalia Haydar. Epigenetic reprogramming with DNA methylation inhibitors during CAR T cell manufacturing enhances anti-tumor activity, persistence, and cytotoxicity against solid and brain tumors [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr B021.

<p>Anti-CD19 Chimeric Antigen Receptor (CAR) T-cell therapy is shifting the treatment paradigm internationally for selected patients with relapsed and refractory B-cell Non- Hodgkin Lymphoma. Despite high response rates with durable responses achieved in a significant proportion of patients, over 50% of patients will have progressed at one year following treatment with the currently licensed anti-CD19 CAR T-cell therapies. This modality of therapy is also associated with acute and potentially life-threatening toxicities, requiring strict risk mitigation strategies. In this thesis, the design, preparation and implementation of a new third generation anti-CD19 CAR T-cell Phase 1 trial entitled ENABLE, for patients with relapsed and refractory B-cell Non-Hodgkin Lymphoma, is described in detail. Following a literature review of CAR T-cell therapy in patients with B-cell Non-Hodgkin Lymphoma, the rationale for the ENABLE trial design is discussed, along with regulatory and clinical requirements for setting up CAR T-cell therapy in New Zealand. The importance of international collaboration to inform aspects of study design, CAR T-cell product manufacturing and developing CAR T-cell toxicity management protocols, has been demonstrated. The early clinical experience on the ENABLE trial is presented along with provisional safety, pharmacokinetic and efficacy data from the first participant treated. This is the first time that CAR T-cell therapy has been administered in New Zealand, demonstrating CAR T-cell expansion in vivo; but also highlighting the complexities of the CAR T-cell product manufacturing process and the importance of evaluating feasibility of CAR T-cell manufacturing, as a key secondary endpoint of the study. Further clinical experience on the ENABLE trial is crucial to develop the potential for CAR T-Cell therapy to be a safe, feasible and effective option for selected New Zealand patients in the future.</p>

<p>Anti-CD19 Chimeric Antigen Receptor (CAR) T-cell therapy is shifting the treatment paradigm internationally for selected patients with relapsed and refractory B-cell Non- Hodgkin Lymphoma. Despite high response rates with durable responses achieved in a significant proportion of patients, over 50% of patients will have progressed at one year following treatment with the currently licensed anti-CD19 CAR T-cell therapies. This modality of therapy is also associated with acute and potentially life-threatening toxicities, requiring strict risk mitigation strategies. In this thesis, the design, preparation and implementation of a new third generation anti-CD19 CAR T-cell Phase 1 trial entitled ENABLE, for patients with relapsed and refractory B-cell Non-Hodgkin Lymphoma, is described in detail. Following a literature review of CAR T-cell therapy in patients with B-cell Non-Hodgkin Lymphoma, the rationale for the ENABLE trial design is discussed, along with regulatory and clinical requirements for setting up CAR T-cell therapy in New Zealand. The importance of international collaboration to inform aspects of study design, CAR T-cell product manufacturing and developing CAR T-cell toxicity management protocols, has been demonstrated. The early clinical experience on the ENABLE trial is presented along with provisional safety, pharmacokinetic and efficacy data from the first participant treated. This is the first time that CAR T-cell therapy has been administered in New Zealand, demonstrating CAR T-cell expansion in vivo; but also highlighting the complexities of the CAR T-cell product manufacturing process and the importance of evaluating feasibility of CAR T-cell manufacturing, as a key secondary endpoint of the study. Further clinical experience on the ENABLE trial is crucial to develop the potential for CAR T-Cell therapy to be a safe, feasible and effective option for selected New Zealand patients in the future.</p>

<div><p>The success of chimeric antigen receptor (CAR) T-cell therapy against hematologic malignancies has altered the treatment paradigm for patients with these diseases. Nevertheless, the occurrence of relapse due to antigen escape or heterogeneous antigen expression on tumors remains a challenge for first-generation CAR T-cell therapies as only a single tumor antigen can be targeted. To address this limitation and to add a further level of tunability and control to CAR T-cell therapies, adapter or universal CAR T-cell approaches use a soluble mediator to bridge CAR T cells with tumor cells. Adapter CARs allow simultaneous or sequential targeting of multiple tumor antigens, control of immune synapse geometry, dose control, and the potential for improved safety. Herein, we described a novel CAR T-cell adapter platform that relies on a bispecific antibody (BsAb) targeting both a tumor antigen and the GGGGS (G<sub>4</sub>S) linker commonly used in single-chain Fv (ScFv) domains expressed on CAR T-cell surfaces. We demonstrated that the BsAb can bridge CAR T cells to tumor cells and potentiate CAR T-cell activation, proliferation, and tumor cell cytolysis. The cytolytic activity of CAR T-cells was redirected to different tumor antigens by changing the BsAb in a dose-dependent manner. This study highlights the potential of G<sub>4</sub>S-displaying CAR T cells to be redirected to engage alternative tumor-associated antigens (TAA).</p>Significance:<p>New approaches are needed to address relapsed/refractory disease and manage potential toxicities associated with CAR T-cell therapy. We describe an adapter CAR approach to redirect CAR T cells to engage novel TAA-expressing cells via a BsAb targeting a linker present on many clinical CAR T-cell therapeutics. We anticipate the use of such adapters could increase CAR T-cell efficacy and reduce potential CAR-associated toxicities.</p></div>