Genetic and epigenetic mechanisms of GPRC5D loss after anti-GPRC5D CAR T-cell therapy in multiple myeloma.

Development and Evaluation of an Optimal Human Single-Chain Variable Fragment-Derived BCMA-Targeted CAR T Cell Vector.

γ-Secretase inhibition increases efficacy of BCMA-specific chimeric antigen receptor T cells in multiple myeloma

Advancing CAR T-Cell Care in Relapsed/Refractory Multiple Myeloma: Insights from a Collaborative Quality Improvement Initiative

EZH1/2 Inhibition Improves the Anti-Tumor Efficacy of CAR and TCR T-Cell Based Therapies Against Multiple Liquid and Solid Tumors

Universal SLAMF7-Specific CAR T-Cells As Treatment for Multiple Myeloma

217 Background: Neuroendocrine prostate cancers (NEPC) currently have no effective therapies. While CAR (Chimeric Antigen Receptor) T cell therapies targeting PSMA and PSCA receptors are currently in phase 1/2 studies in metastatic castrate-resistant prostate cancer (mCRPC), antigen targets for NEPC remain limited. Lewis Y antigen (LeY) is an oncofetal antigen expressed only in adult tumour cells and is considered a target for CAR T cell therapy in haematological, lung, ovarian and colon cancer. We previously found that LeY is a promising target for CAR T cell therapy in prostate cancer and that preconditioning LeY-directed CAR T cells with carboplatin further enhances tumour responses in mCRPC. We investigated if LeY can be used as a novel CAR T cell target for NEPC. Methods: The expression of the Lewis Y antigen was assessed in our patient-derived prostate cancer xenograft collection using immunohistochemistry (IHC). To assess the efficacy of LeY-CAR T cells, we used specimens established from a patient (Patient 508) with NEPC who had incurable metastatic disease and failed standard treatments. We generated CAR T cells from his peripheral blood mononuclear cells (PBMCs) to test their specificity and efficacy in vitro against established cell lines. Results: In our MURAL PDX collection of 59 prostate tumours, 81% (9/11) NEPCs expressed LeY antigen on IHC. Next, we successfully produced anti-LeY CAR T cells from the patient’s PBMCs using retroviral transduction. Flow cytometry verified efficient transduction of the CAR constructs. Previous studies found that a 1:1 ratio of CD4 + :CD8 + T cells improved outcomes following CAR T cell therapy 5,6 , so we further phenotyped patient T cells by flow cytometry. The CD4 + :CD8 + T cell ratio was nearly 1:1 (51.7% and 41.4% of total T cells, respectively). Next, we used an immunobead assay to investigate CAR-T specificity in releasing proinflammatory cytokines. Significant cytokine production was observed after a 4-hour co-culture with LeY+ human ovarian cancer cell line, OVCAR3, vs co-culture with control LeY- MDA-MB435 cells (p=0.0004), demonstrating the specificity of the CAR T cells.Lastly, we showed that patient CAR T cells effectively killed LeY+ cells (OVCAR3), but not control LeY- cells (MDA-MB435; p<0.0001). Tumour cell death was measured by the amount of released chromium following 16-hour co-culture. CAR T cells killed LeY+ target cells more effectively than their non-transduced equivalents at a range of effector-to-target ratios, with up to 15-fold increase in cell death. Conclusions: Our preclinical studies show that most NEPC tumors express the LeY antigen and that patient-derived CAR T cells specifically kill those cells. These findings identified a potential CAR T treatment target for NEPC for a phase 1/2 study.

A Novel and Highly Potent CAR T Cell Drug Product for Treatment of BCMA-Expressing Hematological Malignances

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.

Incidence of Secondary Malignancy after Treatment with Anti-CD19 and Anti-BCMA Chimeric Antigen Receptor T-Cell Therapies

Disclosure: W.J. Kulakowska: None. R.A. Taylor: None. W. Ranasinghe: None. P.K. Darcy: None. J.A. Trapani: None. G.P. Risbridger: None. Background and aims While CAR (Chimeric Antigen Receptor) T cell therapies targeting PSMA and PSCA receptors are currently in phase 1/2 studies in metastatic castrate-resistant prostate cancer (mCRPC), antigen targets for neuroendocrine prostate cancer (NEPC) remain limited. Lewis Y antigen (LeY) is an oncofetal antigen expressed only in adult tumour cells and is considered a target for CAR T cell therapy in haematological, lung, ovarian and colon cancer. We previously found that LeY is a promising target for CAR T cell therapy in prostate cancer and that preconditioning LeY-directed CAR T cells with carboplatin further enhances tumour responses in mCRPC. We investigated if LeY can be used as a novel CAR T cell target for NEPC. Methods: The expression of the Lewis Y antigen was assessed in our patient-derived prostate cancer xenograft (PDX) collection using immunohistochemistry (IHC). To assess the efficacy of LeY-CAR T cells, we used specimens established from a patient (Patient 508) with NEPC who had incurable metastatic disease and failed standard treatments. We generated CAR T cells from his peripheral blood mononuclear cells (PBMCs) to test their specificity and efficacy in vitro. Results: In our MURAL PDX collection of 59 prostate tumours, 81% (9/11) NEPCs expressed LeY antigen on IHC, including Patient 508. We successfully produced anti-LeY CAR T cells from the patient’s PBMCs using retroviral transduction. Generated CAR T cells presented a nearly 1:1 CD4+:CD8+ T cell ratio (51.7% and 41.4% of total T cells, respectively), which according to existing literature yields better treatment outcomes. Next, we used an immunobead assay to investigate CAR-T specificity in releasing proinflammatory cytokines. Significant cytokine production was observed after a co-culture with LeY+ human ovarian cancer cell line, OVCAR3, vs co-culture with control LeY- MDA-MB435 cells (p=0.0004), demonstrating the specificity of the CAR T cells. Lastly, we showed that patient CAR T cells effectively killed LeY+ cells (OVCAR3), but not control LeY- cells (MDA-MB435; p<0.0001) in a 16-hour chromium release assay. CAR T cells killed LeY+ target cells more effectively than their non-transduced equivalents at a range of effector-to-target ratios, with up to 15-fold increase in cell death. Conclusion: Our preclinical studies show that most NEPC tumors express the LeY antigen and that patient-derived CAR T cells can specifically kill those cells. These findings identified a potential CAR T treatment target for NEPC for a phase 1/2 study. Presentation: Monday, July 14, 2025

Advancing Collaboration across the Multiple Myeloma Treatment Journey from Oncology Clinic to CAR T-Cell Center: A Paired Center Transformative Quality Improvement Initiative

<div>Abstract<p>Chimeric antigen receptor (CAR) T-cell therapy for multiple myeloma targeting B-cell maturation antigen (BCMA) induces high overall response rates. However, relapse still occurs and novel strategies for targeting multiple myeloma cells using CAR T-cell therapy are needed. SLAMF7 (also known as CS1) and CD38 on tumor plasma cells represent potential alternative targets for CAR T-cell therapy in multiple myeloma, but their expression on activated T cells and other hematopoietic cells raises concerns about the efficacy and safety of such treatments. Here, we used CRISPR/Cas9 deletion of the <i>CD38</i> gene in T cells and developed DCAR, a double CAR system targeting CD38 and CS1 through activation and costimulation receptors, respectively. Inactivation of <i>CD38</i> enhanced the anti–multiple myeloma activity of DCAR T <i>in vitro</i>. Edited DCAR T cells showed strong <i>in vitro</i> and <i>in vivo</i> responses specifically against target cells expressing both CD38 and CS1. Furthermore, we provide evidence that, unlike anti-CD38 CAR T-cell therapy, which elicited a rapid immune reaction against hematopoietic cells in a humanized mouse model, DCAR T cells showed no signs of toxicity. Thus, DCAR T cells could provide a safe and efficient alternative to anti-BCMA CAR T-cell therapy to treat patients with multiple myeloma.</p></div>

<div>Abstract<p>Chimeric antigen receptor (CAR) T-cell therapy for multiple myeloma targeting B-cell maturation antigen (BCMA) induces high overall response rates. However, relapse still occurs and novel strategies for targeting multiple myeloma cells using CAR T-cell therapy are needed. SLAMF7 (also known as CS1) and CD38 on tumor plasma cells represent potential alternative targets for CAR T-cell therapy in multiple myeloma, but their expression on activated T cells and other hematopoietic cells raises concerns about the efficacy and safety of such treatments. Here, we used CRISPR/Cas9 deletion of the <i>CD38</i> gene in T cells and developed DCAR, a double CAR system targeting CD38 and CS1 through activation and costimulation receptors, respectively. Inactivation of <i>CD38</i> enhanced the anti–multiple myeloma activity of DCAR T <i>in vitro</i>. Edited DCAR T cells showed strong <i>in vitro</i> and <i>in vivo</i> responses specifically against target cells expressing both CD38 and CS1. Furthermore, we provide evidence that, unlike anti-CD38 CAR T-cell therapy, which elicited a rapid immune reaction against hematopoietic cells in a humanized mouse model, DCAR T cells showed no signs of toxicity. Thus, DCAR T cells could provide a safe and efficient alternative to anti-BCMA CAR T-cell therapy to treat patients with multiple myeloma.</p></div>

Comparison of Healthcare Utilization in Patients Undergoing CAR T-Cell Therapy for Multiple Myeloma and Lymphoma

Background: Recent studies with autologous chimeric antigen receptor (CAR)-redirected T-cells against CD19 have demonstrated long-term durable remissions in patients with B-cell leukemia and lymphoma, indicating that CAR T-cell therapy is a promising approach for refractory malignancies. Signaling lymphocytic activation molecule F7 (SLAMF7, also called CS1) is highly expressed on multiple myeloma (MM) tumor cells and is present in only a subset of hematopoietic cells among normal tissues. Because of its high expression on MM tumor cells and restricted expression in normal cells, SLAMF7 is a potential target for CAR T-cell therapy approach in MM. We had previously demonstrated that allogeneic “off-the-shelf” CAR T-cells lacking the ability to induce graft versus host disease could be generated for universal use by inactivating the TCRα constant (TRAC) gene using TALEN® gene editing technology. We also demonstrated that to minimize the risk of fratricide of SLAMF7-specific CAR+ T-cells, SLAMF7 could be inactivated by TALEN® in the T-cells prior to introduction of the CAR construct (Galetto et al., ASH 2015, Abstract 116). Here, we report the efficacy of these double KO (TRAC and SLAMF7) SLAMF7-specific universal CAR T cells (UCARTCS1) against MM in in vitro and in vivo studies. Methods: We tested the efficacy of UCARTCS1 cells against MM cell lines and primary tumor cells from MM patients for their capacity to specifically a) degranulate when co-cultured with MM tumor cells as determined by CD107a assay, b) lyse MM cell lines and primary MM cells in in vitro cytotoxicity assay, c) produce cytokines in culture supernatants, d) proliferate in presence of MM cells as determined by CFSE proliferation assay, and e) eradicate primary MM tumors in a patient-derived xenograft model as determined by serum tumor immunoglobulin (M-protein) levels and survival analysis. Results: UCARTCS1 but not control double KO T-cells (lacking SLAMF7 CAR) specifically lysed the MM cell line, MM.1S (median, 93% lysis; range, 78-98% with UCARTCS1 vs. median, 17%; range, 15-47% with control T-cells; n=10). UCARTCS1 cells similarly induced significant lysis of tumor cells from primary MM samples (n=10) (median 59%; range, 20-90%) compared to control T cells (median 9%; range, 0-36%). In agreement with this, we observed specific degranulation in both CD4+ and CD8+ UCARTCS1 cells but not control T-cells in presence of MM.1S cells and primary MM tumor cells. In addition, significant and specific proliferation of both CD4+ and CD8+ UCARTCS1 cells but not control T-cells was observed when they were co-cultured with MM.1S or primary MM tumor samples (n=8). Analysis of culture supernatants for ten cytokines (IFN-γ, GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, and TNF-α) showed that UCARTCS1 cells primarily produced IFN-γ and GM-CSF in presence of primary MM tumor cells (n=6), indicating a Th1/Tc1 response. To test the efficacy of UCARTCS1 cells in vivo, we injected 1x106 primary MM tumor cells into human fetal bone implanted under the skin of NSG mice. After the tumor has been established for 6 to 8 weeks and the serum M-protein was sufficiently elevated, mice were treated intravenously with either 10x106 total cells/mouse of UCARTCS1 or control T-cells. Mice treated with control T-cells developed gradual increase in M-protein levels (median, 339 µg/ml; range, 100-700 µg/mL) whereas the M-protein levels rapidly became undetectable in the mice treated with UCARTCS1 cells and remained undetectable until they were euthanized at approximately 50 days after adoptive transfer. Conclusion: Our results demonstrated that UCARTCS1 cells were highly cytotoxic against primary MM tumor cells in both in vitro and in vivo studies. In addition, UCARTCS1 cells specifically degranulated, produced Th1/Tc1 cytokines, and proliferated in response to primary MM tumor cells. These data provide a rationale for evaluating UCARTCS1 cells as a universal “off-the-shelf” allogeneic CART product in patients with MM. This abstract is also being presented as Poster A46. Citation Format: Rohit Mathur, Zheng Zhang, Jin He, Roman Galetto, Agnes Gouble, Isabelle Chion-Sotinel, Stephanie Filipe, Annabelle Gariboldi, Tanooha Veeramachaneni, Elisabet Manasanch, Sheeba Thomas, Hans C. Lee, Krina Patel, Donna Weber, Richard Eric Davis, Robert Orlowski, Julianne Smith, Jing Yang, Sattva S. Neelapu. Targeting multiple myeloma with universal SLAMF7-specific CAR T-cells [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr PR01.