Production Of Chimeric Antigen Receptor Research Articles

Automated manufacturing and characterization of clinical grade autologous CD20 CAR T cells for the treatment of patients with stage III/IV melanoma.

Point-of-care (POC) manufacturing of chimeric antigen receptor (CAR) modified T cell has expanded rapidly over the last decade. In addition to the use of CD19 CAR T cells for hematological diseases, there is a growing interest in targeting a variety of tumor-associated epitopes. Here, we report the manufacturing and characterization of autologous anti-CD20 CAR T cells from melanoma patients within phase I clinical trial (NCT03893019). Using a second-generation lentiviral vector for the production of the CD20 CAR T cells on the CliniMACS Prodigy®. We demonstrated consistency in cell composition and functionality of the products manufactured at two different production sites. The T cell purity was >98.5%, a CD4/CD8 ratio between 2.5 and 5.5 and transduction rate between 34% and 61% on day 12 (harvest). Median expansion rate was 53-fold (range, 42-65-fold) with 1.7-3.8×109 CAR T cells at harvest, a sufficient number for the planned dose escalation steps (1×105/kg, 1×106/kg, 1×107/kg BW). Complementary research of some of the products pointed out that the CAR+ cells expressed mainly central memory T-cell phenotype. All tested CAR T cell products were capable to translate into T cell activation upon engagement of CAR target cells, indicated by the increase in pro-inflammatory cytokine release and by the increase in CAR T cell amplification. Notably, there were some interindividual, cell-intrinsic differences at the level of cytokine release and amplification. CAR-mediated T cell activation depended on the level of CAR cognate antigen. In conclusion, the CliniMACS Prodigy® platform is well suited for decentralized POC manufacturing of anti-CD20 CAR T cells and may be likewise applicable for the rapid and automated manufacturing of CAR T cells directed against other targets. https://clinicaltrials.gov/study/NCT03893019?cond=Melanoma&term=NCT03893019&rank=1, identifier NCT03893019.

Umbilical cord blood T cells can be isolated and enriched by CD62L selection for use in 'off the shelf' chimeric antigen receptor T-cell therapies to widen transplant options.

Umbilical cord blood (UCB) T cells exhibit distinct naive ontogenetic profiles and may be an attractive source of starting cells for the production of chimeric antigen receptor (CAR) T cells. Pre-selection of UCB-T cells on the basis of CD62L expression was investigated as part of a machine-based manufacturing process, incorporating lentiviral transduction, CRISPR-Cas9 editing, T-cell expansion and depletion of residual TCReeeT cells. This provided stringent mitigation against the risk of graft versus host disease (GVHD), and was combined with simultaneous knockout of CD52 to enable persistence of edited T cells in combination with preparative lymphodepletion using Alemtuzumab. Under compliant manufacturing conditions, two cell banks were generated with high levels of CAR19 expression and minimal carriage of TCReeeT cells. Sufficient cells were cryopreserved in dose-banded aliquots at the end of each campaign to treat dozens of potential recipients. Molecular characterisation captured vector integration sites and CRISPR editing signatures and functional studies, including in vivo potency studies in humanised mice, confirmed antileukaemic activity comparable to peripheral blood-derived universal CAR19 T cells. Machine manufactured UCB derived T cells banks offer an alternative to autologous cell therapies and could help widen access to CAR T cells.

Allogeneic chimeric antigen receptor T cells for children with relapsed/refractory B-cell precursor acute lymphoblastic leukemia.

Chimeric antigen receptor (CAR) T-cell therapy has emerged as a breakthrough cancer therapy over the past decade. Remarkable outcomes in B-cell lymphoproliferative disorders and multiple myeloma have been reported in both pivotal trials and real-word studies. Traditionally, the use of a patient's own (autologous) T cells to manufacture CAR products has been the standard practice. Nevertheless, this approach has some drawbacks, including manufacturing delays, dependence on the functional fitness of the patient's T cells, which can be compromised by both the disease and prior therapies, and contamination of the product with blasts. A promising alternative is offered by the development of allogeneic CAR-cell products. This approach has the potential to yield more efficient drug products and enables the use of effector cells with negligible alloreactive potential and a significant CAR-independent antitumor activity through their innate receptors (i.e., natural killer cells, γδ T cells and cytokine induced killer cells). In addition, recent advances in genome editing tools offer the potential to overcome the primary challenges associated with allogeneic CAR T-cell products, namely graft-versus-host disease and host allo-rejection, generating universal, off-the-shelf products. In this review, we summarize the current pre-clinical and clinical approaches based on allogeneic CAR T cells, as well as on alternative effector cells, which represent exciting opportunities for multivalent approaches and optimized antitumor activity.

Impact of Previous Allogeneic Hematopoietic Stem Cell Transplantation on Chimeric Antigen Receptor (CAR) T Cell Treatment for Relapsed/Refractory Multiple Myeloma

BackgroundAnti-BCMA-directed chimeric antigen receptor (CAR) T cells are effective treatment for patients with refractory/relapsed multiple myeloma (RRMM). However, little is known about the impact of previous allogeneic hematopoietic stem cell transplantation (allo-HSCT) on lymphocyte collection for production of CAR T cells and subsequent treatment with CAR T cells. Patients and MethodsWe performed a retrospective analysis of cellular composition of lymphocyte collections, CAR T cell expansion and treatment outcomes of RRMM patients undergoing therapy with idecabtagene vicleucel (ide-cel) with and without history of allo-HSCT.27 patients (11/27 female) with median age 63 (range 39-75) years were analyzed. Five patients (19%) had the history of allo-HSCT median of 5.5 years before ide-cel. ResultsPrior to apheresis, the white blood cell, absolute lymphocyte counts, CD3+ cells and monocytes did not differ in patients with and without prior allo-HSCT. We also noticed no differences in the collected CD3+ yields or cellular compositions of lymphocyte collections.One year after ide-cel infusion, the progression-free survival and overall survival of patients with and without previous allo-HSCT did not differ with 60% and 45% respectively (P = .58) and 66.7% and 74% respectively (P = .84).The highest expansion of CAR T was detected between day 7 after infusion and showed no difference regarding previous allo-HSCT (P = .71).No graft-versus-host disease during the follow-up was detected. ConclusionOur data confirm that the treatment with ide-cel is feasible for patients with prior allo-HSCT. Furthermore, allo-HSCT did not influence cellular composition of lymphocyte collections, clinical outcome or in vivo expansion of ide-cel.

CAR products from novel sources: a new avenue for the breakthrough in cancer immunotherapy.

Chimeric antigen receptor (CAR) T cell therapy has transformed cancer immunotherapy. However, significant challenges limit its application beyond B cell-driven malignancies, including limited clinical efficacy, high toxicity, and complex autologous cell product manufacturing. Despite efforts to improve CAR T cell therapy outcomes, there is a growing interest in utilizing alternative immune cells to develop CAR cells. These immune cells offer several advantages, such as major histocompatibility complex (MHC)-independent function, tumor microenvironment (TME) modulation, and increased tissue infiltration capabilities. Currently, CAR products from various T cell subtypes, innate immune cells, hematopoietic progenitor cells, and even exosomes are being explored. These CAR products often show enhanced antitumor efficacy, diminished toxicity, and superior tumor penetration. With these benefits in mind, numerous clinical trials are underway to access the potential of these innovative CAR cells. This review aims to thoroughly examine the advantages, challenges, and existing insights on these new CAR products in cancer treatment.

A novel lentiviral vector-based approach to generate chimeric antigen receptor T cells targeting Aspergillus fumigatus.

Invasive aspergillosis (IA) is a common and deadly mold infection in immunocompromised patients. As morbidity and mortality of IA are primarily driven by poor immune defense, adjunct immunotherapies, such as chimeric antigen receptor (CAR) T cells, are direly needed. Here, we propose a novel approach to generate Aspergillus fumigatus (AF)-CAR T cells using the single-chain variable fragment domain of monoclonal antibody AF-269-5 and a lentiviral vector system. These cells successfully targeted mature hyphal filaments of representative clinical and reference AF isolates and elicited a potent release of cytotoxic effectors and type 1 T cell cytokines. Furthermore, AF-CAR T cells generated from peripheral blood mononuclear cells of four healthy human donors and expanded with either of three cytokine stimulation regimens (IL-2, IL-2 + IL-21, or IL-7 + IL-15) significantly suppressed mycelial growth of AF-293 after 18 hours of co-culture and synergized with the immunomodulatory antifungal agent caspofungin to control hyphal growth for 36 hours. Moreover, cyclophosphamide-immunosuppressed NSG mice with invasive pulmonary aspergillosis that received two doses of 5 million AF-CAR T cells (6 and 48 hours after AF infection) showed significantly reduced morbidity on day 4 post-infection (P < 0.001) and significantly improved 7-day survival (P = 0.049) compared with mice receiving non-targeting control T cells, even without concomitant antifungal chemotherapy. In conclusion, we developed a novel lentiviral strategy to obtain AF-CAR T cells with high targeting efficacy, yielding significant anti-AF activity in vitro and short-term protection in vivo. Our approach could serve as an important steppingstone for future clinical translation of antifungal CAR T-cell therapy after further refinement and thorough preclinical evaluation.IMPORTANCEInvasive aspergillosis (IA) remains a formidable cause of morbidity and mortality in patients with hematologic malignancies and those undergoing hematopoietic stem cell transplantation. Despite the introduction of several new Aspergillus-active antifungals over the last 30 years, the persisting high mortality of IA in the setting of continuous and profound immunosuppression is a painful reminder of the major unmet need of effective antifungal immune enhancement therapies. The success of chimeric antigen receptor (CAR) T-cell therapy in cancer medicine has inspired researchers to translate this approach to opportunistic infections, including IA. Aiming to refine anti-Aspergillus CAR T-cell therapy and improve its feasibility for future clinical translation, we herein developed and validated a novel antibody-based CAR construct and lentiviral transduction method to accelerate the production of CAR T cells with high targeting efficacy against Aspergillus fumigatus. Our unique approach could provide a promising platform for future clinical translation of CAR T-cell-based antifungal immunotherapy.

Co-expression of IL-21-Enhanced NKG2D CAR-NK cell therapy for lung cancer

BackgroundAdoptive cell therapy has achieved great success in treating hematological malignancies. However, the production of chimeric antigen receptor T (CAR-T) cell therapy still faces various difficulties. Natural killer (NK)-92 is a continuously expandable cell line and provides a promising alternative for patient’s own immune cells.MethodsWe established CAR-NK cells by co-expressing natural killer group 2 member D (NKG2D) and IL-21, and evaluated the efficacy of NKG2D-IL-21 CAR-NK cells in treating lung cancer in vitro and in vivo.ResultsOur data suggested that the expression of IL-21 effectively increased the cytotoxicity of NKG2D CAR-NK cells against lung cancer cells in a dose-dependent manner and suppressed tumor growth in vitro and in vivo. In addition, the proliferation of NKG2D-IL-21 CAR-NK cells were enhanced while the apoptosis and exhaustion of these cells were suppressed. Mechanistically, IL-21-mediated NKG2D CAR-NK cells function by activating AKT signaling pathway.ConclusionOur findings provide a novel option for treating lung cancer using NKG2D-IL-21 CAR-NK cell therapy.

Expanding CAR-T cell immunotherapy horizons through microfluidics.

Chimeric antigen receptor (CAR)-T cell therapies have revolutionized cancer treatment, particularly in hematological malignancies. However, their application to solid tumors is limited, and they face challenges in safety, scalability, and cost. To enhance current CAR-T cell therapies, the integration of microfluidic technologies, harnessing their inherent advantages, such as reduced sample consumption, simplicity in operation, cost-effectiveness, automation, and high scalability, has emerged as a powerful solution. This review provides a comprehensive overview of the step-by-step manufacturing process of CAR-T cells, identifies existing difficulties at each production stage, and discusses the successful implementation of microfluidics and related technologies in addressing these challenges. Furthermore, this review investigates the potential of microfluidics-based methodologies in advancing cell-based therapy across various applications, including solid tumors, next-generation CAR constructs, T-cell receptors, and the development of allogeneic "off-the-shelf" CAR products.

Reinforcement Learning Based Resource Management for CAR T-Cell Therapies

This paper focuses on optimizing resource management strategies in chimeric antigen receptor (CAR) T-cell therapies using reinforcement learning (RL). CAR T-cell therapy is an innovative and promising treatment within the field of immunotherapy, which is based on the isolation of patients’ own T-cells, genetically modifying these cells to express a CAR for tumor recognition, cultivating and expanding these T cells and infusing them back to the patient. These therapies require several reusable but scarce resources, including special equipment, such as the bioreactor that is used to expand CAR T cells, and medical resources, such as the hospital staff, e.g., doctors and nurses. Considering the stochastic nature of medical procedures and the production of CAR T cells, the scheduling of the therapies and the efficient allocation of the required resources pose a significant challenge inside the hospital environment. Here, we propose a derivative-free policy gradient algorithm that utilizes a simulation model of the therapy, built using real-world data, to obtain efficient control policies for the resource management problem. The proposed method is designed to minimize the expected number of deviations from the therapy protocol as well as the expected overall completion time of the therapies for every patient. The effectiveness of the proposed resource management approach is demonstrated via simulation experiments.

A production scheduling and control system for CAR T cell manufacturing

This paper addresses some of the new challenges posed by scheduling and controlling the production of chimeric antigen receptor (CAR) T cells for personalized therapies. In this therapy, the patient’s T cells are genetically modified by adding a gene for a receptor to help T cells attach to the cancer cell antigens. Then, the modified cells are grown and multiplied (called cell expansion) and finally returned to the patient. The production takes place in a manufacturing system that comprises several devices, which are capable of processing cells from multiple patients at the same time. The efficient operation of such a complex system requires a sophisticated control system accompanied by a scheduler to optimize the production of CAR T cells for several patients simultaneously. We propose a scheduling algorithm, which can handle the uncertain duration of cell expansion, and the maximum time lags between consecutive processes simultaneously, a combination hardly studied in the literature. We also describe the main features of an adaptive control system including service-oriented device connections as well as centralized control and monitoring. Finally, we describe how the overall system works, including a JSON based communication between scheduler and control system.

CAR-modified Cellular Therapies in Chronic Lymphocytic Leukemia: Is the Uphill Road Getting Less Steep?

The clinical development of chimeric antigen receptor (CAR) T-cell therapy has been more challenging for chronic lymphocytic leukemia (CLL) compared to other settings. One of the main reasons is the CLL-associated state of immune dysfunction that specifically involves patient-derived T cells. Here, we provide an overview of the clinical results obtained with CAR T-cell therapy in CLL, describing the identified immunologic reasons for the inferior efficacy. Novel CAR T-cell formulations, such as lisocabtagene maraleucel, administered alone or in combination with the Bruton tyrosine kinase inhibitor ibrutinib, are currently under investigation. These approaches are based on the rationale that improving the quality of the T-cell source and of the CAR T-cell product may deliver a more functional therapeutic weapon. Further strategies to boost the efficacy of CAR T cells should rely not only on the production of CAR T cells with an improved cellular composition but also on additional changes. Such alterations could include (1) the coadministration of immunomodulatory agents capable of counteracting CLL-related immunological alterations, (2) the design of improved CAR constructs (such as third- and fourth-generation CARs), (3) the incorporation into the manufacturing process of immunomodulatory compounds overcoming the T-cell defects, and (4) the use of allogeneic CAR T cells or alternative CAR-modified cellular vectors. These strategies may allow to develop more effective CAR-modified cellular therapies capable of counteracting the more aggressive and still incurable forms of CLL.

CAR T Cell Exhaustion but Not Ex Vivo Cytotoxicity Is Predictive of Patient Clinical Response: An Interim Analysis of ACIT001/EXC002, a Phase Ib/II Trial of Decentralized Production of CAR T Cells for Treatment of Relapsed/Refractory Aggressive NHL and ALL

Chimeric antigen receptor (CAR) T cell therapy is an increasingly prominent form of next-generation immunotherapy in both hematologic and solid tumor malignancies. Multiple CAR T products have received regulatory approval for use as treatment in relapsed and refractory leukemias and lymphomas. However, approximately half of these patients do not respond to therapy and production of CAR T cells is a difficult, expensive, and time-consuming process. Although much has been learned about the fundamental biology of CAR T cells over the past decade, we are still unable to accurately predict which patients will respond to therapy. In particular, few studies have specifically examined biological characteristics of manufactured CAR T cells and correlated these to relevant clinical outcomes. In this study, we analyzed the lymphocyte populations and CAR T cells from patients enrolled in ACIT001/EXC002, a Canadian multi-centre phase Ib/II single-arm clinical trial of decentralized production of second-generation CD19/41BB/CD3z CAR T cells for treatment of multiply relapsed/refractory non-Hodgkin lymphoma (NHL) and acute lymphoblastic leukemia (ALL). CD3-positive T cells and CAR T products were comprehensively immunophenotyped by flow cytometry. Thecapacity for manufactured CAR T cells to kill tumor cells was assessed using an ex vivo cytotoxicity assay. CAR T cell immunophenotype and cytotoxic potential were correlated with clinical outcomes including response to therapy, progress-free survival (PFS) and incidence of serious adverse events. To date, 23 patients have been enrolled, of which 16 had an aggressive NHL while the remaining 7 had been diagnosed with B cell ALL. Mean age of participants at time of enrollment was 57 (range: 26-77) and 65.2% were male. CAR T product was manufactured using lentiviral transduction with a median transduction efficiency of 32.1% (range: 9.1% to 55.5%). A majority of patients achieved a complete response as evaluated by PET scan on day 28 post-CAR T cell infusion with only 3 patients showing disease progression, 1 patient achieving only partial response, and 3 patients being unevaluable. We were able to evaluate PFS at 6 months in 15 out of 23 patients, with 46.7% of patients experiencing progression and/or mortality within this period. Surprisingly, while CAR T cell cytotoxicity ex vivo trended higher in patients that achieved a complete metabolic response compared to those with partial response or disease progression, this did not reach statistical significance (p = 0.280). However, expression of T cell exhaustion markers such as PD-1 CAR T products was found to be significantly increased in non-responders (p = 0.010). Higher expression of exhaustion markers was also found to be associated with decreased CAR T persistence in vivo as measured by circulating CAR T cell levels at day 14 post-infusion. In our study cohort, CAR T products had either similar or significantly greater proportions of CD4+ as compared to CD8+ T cells. CAR T cells were found to express high levels of HLA-DR and CD127, however this did not correlate with cytotoxicity, response to therapy or PFS. Patient CAR T cells, regardless of the level of exhaustion marker expression, tended to be highly skewed towards the T effector memory subset, with almost no populations of naïve or memory T cells identified. In this analysis of the impact of biological characteristics of CAR T cell products on patient outcomes, we found that CAR T cell exhaustion rather than cytotoxic activity is correlated with response to treatment and higher PFS at 6 months. Interestingly, the degree of patient T cell exhaustion had an impact on transduction efficiency but not the cytotoxic potential of the manufactured CAR T cell product. Taken together, our data indicate that CAR T cell exhaustion and persistence following infusion rather than cytotoxic potential is a key predictor for patient response to treatment. While our conclusions are currently limited by small sample size, more patients are presently being accrued to allow for more robust analysis in the future.

Apheresis for Production of Chimeric Antigen Receptor (CAR) T Cells in Patients with Multiple Myeloma - Clinical Factors, Cell Composition and T Cell Senescence

Chimeric antigen receptor (CAR) T cells have been proven as a highly promising immunotherapy for patients with multiple myeloma (MM). Apheresis of autologous CD3+ lymphocytes is the first pivotal step in the production process of chimeric antigen receptor (CAR) T cells. Repetitive activation of T lymphocytes can affect the qualitative properties, i.e. the fitness of CD3 cells leading to progressive loss of expression of the costimulatory receptors CD27 and CD28. CD3+27-28- cells are non-proliferating memory, senescent cells. In that regard, the factors influencing the manufacturing process of CAR T cells in patients with MM have not been sufficiently explored. We performed a retrospective analysis of clinical parameters influencing both collection of autologous lymphocytes and the subsequent manufacturing of idecabtagene-vicleucel with a special focus on T cell senescence, defined as loss of CD27 and CD28. Furthermore, we focused on cell-compositions of collections resulting in out of specification (OOS) events during CAR T cell manufacturing. Between January 2022 and January 2023, 36 lymphocyte collections of 32 patients with relapsed/refractory (r/r) MM were performed for subsequent CAR T cell therapy with idecabtagene-vicleucel. All collections were forwarded for further manufacturing without prior cryokonservation. 9 (25%) collections resulted in OOS; 7/9 resulting in insufficient proliferation or expansion of T cells, while 2/9 showed a microbial contamination during the manufacturing process after viral transduction for CAR generation. At apheresis, the patients were a median 63 (range 39-75) years old, 12 (38%) were female with median body weight 79 kg (49-103). The patients had a median of 7 previous treatment lines (range 3-13) and 15/36 collections were performed at partial remission or better. 8/36 collection had prior exposition to bispecific antibodies whereas in 13/36 collections the unspecific positivity for cytomegalovirus (CMV) in polymerase chain reaction (PCR) was detected. The collections were performed on a Spectra Optia cell-separator by processing median 3x total blood volume. CD3+ cells in peripheral blood (pB) were median 513.9 /µl (range 70.6-3616.0). The collections showed a median CD3+ yield with 29.8 x10 8 (range 4.9-171.6), with CD3+4+ and CD3+8+ yields 10.6 x10 8 (range 1-663.4) and 16.3 x10 8 (range 3.2-121.2) respectively, resulting in median CD4:CD8 ratio of 0.6 (range 0.1-2.6). The median yields for CD3+27+28+ and CD3+27-28- cells were 14.8 (range 2.3-88.8) x10 8 and 2.1 (range 0.1-59.8) x10 8 respectively. Noteworthy, OOS events were more common in patients with detectable cytomegalovirus replication at apheresis ( P=0.037) with a trend for prior exposition to bispecific antibodies ( P=0.086). In contrast, the collections with OOS did not differ regarding patients' age ( P=0.498), sex ( P=0.442), body weight ( P=0.364), number of previous treatment lines ( P=0.185) or remission status at apheresis ( P=0.443). In OOS collections, CD3+ cells in pB were significantly higher with median 991 /µl (range 558-2968) compared to 448.9 /µl (70.6-3616.0) ( P=0.002) and showed a trend for higher CD3+ yields with 38.9 x10 8 (range 23.9-62.2) and 28.2 x10 8 (range 4.9-171.6) respectively ( P=0.096). Interestingly, OOS products also had higher median yields of CD3+CD8+ cells with 28.1 x10 8 (range 12.2-52.3) compared to 15.3 x10 8 (range 3.2-121.2), ( P=0.046) and showed a trend for lower median CD3+CD4+ yields with 9.1 x10 8 (range 3.8-11.9) compared to 11.4 x10 8 (range 1.6-63.4), ( P=0.071). Median CD3+27+28+ yields in OOS and successful manufacturings were 9.7 x10 8 (4.7-26.0) and 17.0 x10 8(range 2.4-88.8) respectively ( P=0.030). OOS events yielded with higher median CD3+27-28- with 17.8 x10 8(range 0.2-46.1) compared to 1.4 x10 8 (0.1-59.8), ( P=0.031). We observed a negative prognostic role of CMV replication as well as of senescence of T lymphocytes on the manufacturing process. In contrast, a successful manufacturing was not influenced by patient age, sex, remission status at apheresis or number of previous treatment lines. Further analyses are required to determine measures appropriate for further optimization of autologous lymphocyte collections and improvement of collection outcomes in patients with MM.

Infections Drive Non-Relapse Mortality Following CAR-T Therapy across Disease Entities and CAR Products - a Meta-Analysis of Clinical Trials and Real-World Studies

Introduction: Chimeric antigen receptor (CAR) T cell therapy has markedly improved treatment outcomes for patients with relapsed/refractory B-cell malignancies. However, CAR-T cells are also associated with a distinct toxicity profile including cytokine release syndrome (CRS) and neurotoxicity (ICANS). Due to their nature as a ‘living drug’, CAR-T cells can also exert profound and long-lasting immune deficits - predisposing for a broad range of infectious complications. While there is a growing body of evidence concerning treatment response and relapse mechanisms, data on non-relapse mortality (NRM) remains scarce. To gain further insights into drivers of non-relapse related deaths after CAR-T therapy, we performed a meta-analysis of published clinical trials and real-world studies (RWS). Methods: For our literature search of clinical trials, we included all studies which led to approval of the six commercially available CAR-T products (axi-cel, tisa-cel, ide-cel, cilta-cel, liso-cel, brexu-cel) and the corresponding phase I-II (Ph I/II) trials. For RWS, articles had to be published until June 2023 and had to fulfill the following criteria: (1) use of commercially available CAR products in a real-world setting, (2) inclusion of &amp;gt; 100 patients, (3) reports on adult patients. Studies which did not differentiate between relapse and non-relapse related deaths were excluded. Data extraction included basic study characteristics (patient numbers, follow-up time, CAR product), NRM, causes of death, and treatment-association. Results: From a total number of 511 screened studies, 15 clinical trials (Ph I/II: 9, Ph III: 6) and 9 RWS compromising 5,313 patients fulfilled the inclusion criteria. As expected, median follow-up was longer for Ph III trials compared to RWS (Ph III: 18.0 (16.3-34.9) months, RWS: 12.9 (9.7-14.2) months, p=0.04), and comparable to Ph I/II trials (17.5 (15.9-28) months, p&amp;gt;0.9). Among all studies, the most common entity was large B-cell lymphoma (LBCL, 3,770 patients), followed by multiple myeloma (MM, 959), mantle cell lymphoma (MCL, 339) and indolent lymphomas (IL, 245). The distribution of encountered CAR products in the study cohort was: 10 axi-cel, 6 tisa-cel, 4 ide-cel, 4 liso-cel, 3 brexu-cel, and 2 cilta-cel. Of note, over half of the studies did not report any measure of NRM (58%), highlighting deficits in toxicity reporting among CAR-T cell studies. We observed considerable heterogeneity in reporting of NRM with follow-up times ranging from 3 to 12 months and NRM rates ranging between 3.0-9.1%. To integrate studies without a reported NRM, we calculated a point estimate of NRM by dividing non-relapse related deaths by the total number of respective patients ( Fig. 1A). Importantly, the comparison of reported NRMs and NRM point estimates was similar (p=0.89). We noted a trend towards higher NRM estimates in patients with MM (9.4% IQR 6.5-12%) followed by MCL (8.7% IQR 8.1-9.7%), LBCL (6.2% IQR 4.4-8.4%) and IL (4.1% IQR 3.1-5.1%, Kruskal-Wallis p=0.12). NRM was lowest with tisa-cel (3.4% IQR 2.8-4.3%) and highest with cilta-cel (14.3% IQR 13.1-15.4%, Kruskal-Wallis p=0.014). Numerically increased NRM was noted for CD28z- vs. 4-1BB harboring CAR products (8.2 vs. 6.2%, p=0.13) and for phase III studies (Ph I/II vs. III vs. RWS: 6.6 vs. 11.9 vs. 6.2%, Kruskal-Wallis p=0.16). To further elucidate the etiology of non-relapse related deaths, we categorized all reported deaths into one of the following categories: CRS, neurotoxicity, infection, hemorrhage, secondary malignancy, HLH, organ failure not otherwise specified (NOS), others/unknown. In total, 441 deaths were reported of which nearly half were caused by infections (n=210, 47.6%). The CAR-T specific adverse events CRS, ICANS, or HLH led to death in 11.1% of cases. Of interest, secondary malignancies contributed to death in 6.1% of cases and hemorrhages in 3% of cases. We observed a similar distribution of CAR-specific toxicities across entities ( Fig. 1B chi 2 p=0.78). Conclusions: Infections were by far the main determinant of non-relapse mortality across all disease entities, underlining the profound immune deficits conferred during CAR-T therapy. The high incidence of secondary malignancies of 6% is concerning and warrants further systematic study. High-quality and long-term reporting of NRM is critical to recognize emerging side effects and to identify potential toxicity signals of novel CAR products.

Uncovering the Role of CD4+ CAR T Cells in Cancer Immunotherapy.

Chimeric antigen receptor (CAR) T-cell therapy has transformed clinical care against blood malignancies and is seeing encouraging progress against solid tumors. While scientific advancement has been rapid, our mechanistic understanding of intrinsic features of CAR-engineered T cells is still evolving. CAR products typically consist of CD4+ and CD8+ T-cell subsets at variable ratios, yet a clear understanding of how each subset contributes together and independently to therapeutic response is lacking. CD8+ CAR T cells are well characterized for their perforin-dependent killing effects; however, the role of CD4+ CAR T cells as "helpers" versus "killers" has been variable across models and warrants more in-depth investigation. A recent study by Boulch and colleagues published in Nature Cancer demonstrates that CD4+ CAR T cells, alone, can exert potent antitumor activity through a mechanism involving IFNγ. CD4+ CAR T-cell production of IFNγ creates a cytokine field that can act at a distance to kill both antigen-positive and -negative tumor cells that are sensitive to the proapoptotic effects of IFNγ. These new findings reveal important insights for the antitumor effects mediated by CD4+ CAR T cells, which could have significant clinical implications.

Single center experience of using Anakinra prophylactically at Day 0 to reduce CD19 CART toxicities.

e19503 Background: Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 is an effective line of the treatment for relapsed/refractory CD19 B cell cancers, however navigating Cytokine Release Syndrome (CRS) and Immune effector Cell-Associated Neurotoxicity (ICANS) still remains a challenging task. Corticosteroids and Tocilizumab are currently the mainstay treatment for toxicities but Anakinra, an IL-1 receptor antagonist has also been successfully used to treat patients receiving CART who develop CRS/ICANS. In this study we set out to describe the utility of prophylactic use of Anakinra in patients receiving CART. Methods: We conducted a single center retrospective analysis of patients who received Anakinra for CRS and ICANS prophylaxis per institutional protocol. Patient data including demographics, cancer history, CRS &amp; ICANS American Society for Transplantation and Cellular Therapy Grades, toxicity treatment history and outcomes during their hospitalization were extracted from our medical records. Institutional review board approval was obtained for this review. Results: A total of 19 patients {Diffuse Large B Cell Lymphoma = 10, Follicular = 2,Mantle cell = 2 and Acute Lymphoblastic Leukemia (ALL) = 5} were enrolled in our cohort. The median age of the patients at the time of T cell infusion was 63 years (range, 26-75). CD19 CAR products included Axicabtagene ciloleucel (8 pts; 42.1%), Brexucabtagene autoleucel (6 pts; 31.5%) and Tisagenlecleucel (5 pts; 26.3%). CRS of all grades was observed in 13 patients (68.4%) with severe CRS (grade 3-4) observed in 1 patient (5.2%) (grade 4;12 days). Median CRS duration was 3 days (range, 1-18). Median max CRS grade was observed for 2 days (range, 1-12). Median time to CRS was 5 days (range; 1-8). ICANS of all grades was observed in 12 patients (63.1%), with severe ICANS (grade3-4) in 5 patients (26.3%) {1 patient with grade 4 for 12 days; 4 patients with grade 3 for a median of 2 days (range;1-4)}. Median time to ICANS was 6 days (range, 1-11). Tocilizumab and corticosteroids were used in 9 patients (47.3%) and 11 patients (57.8%), respectively. Median starting day of Tocilizumab and steroid was on day 4(range, 1-6) and day4 (range, 1-21) respectively. Median dose of Tocilizumab was 2 (range, 1-3). Median dose of total Dexamethasone and Methylprednisolone use was 125mg (range, 10-276) and 3.92 g (range, 3-7.4) respectively for a median of 6 cumulative days (range, 1-22). Three patients (15.7%) required ICU transfer with a median stay of 5 days (range, 4-16). 1 patient died of Neurotoxicity on Day 27(Mantle cell) and 2 ALL patients died due to progression after discharge on Day61 and 256. Conclusions: We observed that prophylactically starting Anakinra at Day 0 appeared to improve CRS and ICANS toxicities in patients receiving CART. Further prospective investigations with larger population pools would be required to better understand its role in CAR T toxicity suppression.

Impact of serum‑free media on the expansion and functionality of CD19.CAR T‑cells.

Fetal bovine serum (FBS) or human serum is widely used in the production of chimeric antigen receptor (CAR) T‑cells. In order to overcome a lot‑to‑lot inconsistency, the use of chemically defined medium that is free of animal-components would be highly desirable. The present study compared three serum‑free media [Prime‑XV™ T Cell CDM, Fujifilm™ (FF), LymphoONE™ T‑Cell Expansion Xeno‑Free Medium, Takara Bio™ (TB) and TCM GMP‑Prototype, CellGenix™ (CG)] to the standard CAR T‑cell medium containing FBS (RCF). After 12days of CD19.CAR T‑cell culture, the expansion, viability, transduction efficiency and phenotype were assessed using flow cytometry. The functionality of CAR T‑cells was evaluated using intracellular staining, a chromium release assay and a long‑term co‑culture assay. Expansion and viability did not differ between the CAR T‑cells generated in serum‑free media compared to the standard FBS‑containing medium. The CG CAR T‑cells had a statistically significant higher frequency of IFNγ+ and IFNγ+TNF‑α+ CAR T‑cells than the CAR T‑cells cultured with FBS (22.5 vs. 7.6%, P=0.0194; 15.3 vs. 6.2%, P=0.0399, respectively) as detected by intracellular cytokine staining. The CAR T‑cells generated with serum‑free media exhibited a higher cytotoxicity than the CAR T‑cells cultured with FBS in the evaluation by chromium release assay [CG vs. RCF (P=0.0182), FF vs. RCF (P=0.0482) and TB vs. RCF (P=0.0482)]. Phenotyping on day12 of CAR T‑cell production did not reveal a significant difference in the expression of the exhaustion markers, programmed cell death protein 1, lymphocyte‑activation gene3 and T‑cell immunoglobulin and mucin‑domain containing‑3. The CAR T‑cells cultured in FF had a higher percentage of central memory CAR T‑cells (40.0 vs. 14.3%, P=0.0470) than the CAR T‑cells cultured with FBS, whereas the CAR T‑cells in FF (6.2 vs. 24.2%, P=0.0029) and CG (11.0% vs. 24.2%, P=0.0468) had a lower frequency of naïve CAR T‑cells. On the whole, the present study demonstrates that in general, the functionality and expansion of CAR T cells are maintained in serum‑free media. Given the advantages of freedom from bovine material and consistent quality, serum‑free media hold promise for the future development of the field of GMP manufacturing of CAR T‑cells.

Clinical applications of gene therapy for rare diseases: A review.

Rare diseases collectively exact a high toll on society due to their sheer number and overall prevalence. Their heterogeneity, diversity, and nature pose daunting clinical challenges for both management and treatment. In this review, we discuss recent advances in clinical applications of gene therapy for rare diseases, focusing on a variety of viral and non-viral strategies. The use of adeno-associated virus (AAV) vectors is discussed in the context of Luxturna, licenced for the treatment of RPE65 deficiency in the retinal epithelium. Imlygic, a herpes virus vector licenced for the treatment of refractory metastatic melanoma, will be an example of oncolytic vectors developed against rare cancers. Yescarta and Kymriah will showcase the use of retrovirus and lentivirus vectors in the autologous ex vivo production of chimeric antigen receptor T cells (CAR-T), licenced for the treatment of refractory leukaemias and lymphomas. Similar retroviral and lentiviral technology can be applied to autologous haematopoietic stem cells, exemplified by Strimvelis and Zynteglo, licenced treatments for adenosine deaminase-severe combined immunodeficiency (ADA-SCID) and β-thalassaemia respectively. Antisense oligonucleotide technologies will be highlighted through Onpattro and Tegsedi, RNA interference drugs licenced for familial transthyretin (TTR) amyloidosis, and Spinraza, a splice-switching treatment for spinal muscular atrophy (SMA). An initial comparison of the effectiveness of AAV and oligonucleotide therapies in SMA is possible with Zolgensma, an AAV serotype 9 vector, and Spinraza. Through these examples of marketed gene therapies and gene cell therapies, we will discuss the expanding applications of such novel technologies to previously intractable rare diseases.

Hypoimmune anti-CD19 chimeric antigen receptor T cells provide lasting tumor control in fully immunocompetent allogeneic humanized mice

Manufacturing autologous chimeric antigen receptor (CAR) T cell therapeutics is complex, and many patients experience treatment delays or cannot be treated at all. Although current allogeneic CAR products have the potential to overcome manufacturing bottlenecks, they are subject to immune rejection and failure to persist in the host, and thus do not provide the same level of efficacy as their autologous counterparts. Here, we aimed to develop universal allogeneic CAR T cells that evade the immune system and produce a durable response. We generated human hypoimmune (HIP) T cells with disrupted B2M, CIITA, and TRAC genes using CRISPR-Cas9 editing. In addition, CD47 and anti-CD19 CAR were expressed using lentiviral transduction. These allogeneic HIP CD19 CAR T cells were compared to allogeneic CD19 CAR T cells that only expressed the anti-CD19 CAR (allo CAR T). In vitro assays for cancer killing and exhaustion revealed no differences between allo CAR T and HIP CAR T cells, confirming that the HIP edits did not negatively affect T cell performance. Clearance of CD19+ tumors by HIP CAR T cells in immunodeficient NSG mice was comparable to that of allo CAR T cells. In fully immunocompetent humanized mice, HIP CAR T cells significantly outperformed allo CAR T cells, showed improved persistence and expansion, and provided lasting cancer clearance. Furthermore, CD47-targeting safety strategies reliably and specifically eliminated HIP CAR T cells. These findings suggest that universal allogeneic HIP CAR T cell-based therapeutics might overcome the limitations associated with poor persistence of allogeneic CAR T cells and exert durable anti-tumor responses.

Automated, scaled, transposon-based production of CAR T cells

BackgroundThere is an increasing demand for chimeric antigen receptor (CAR) T cell products from patients and care givers. Here, we established an automated manufacturing process for CAR T cells on...