CAR-T Cell Products Research Articles

An adjusted digital droplets PCR assay for quantification of vector copy number in CAR-T cell and TCR-T cell products

Background and PurposeGenetically engineered T cell therapy holds immense promise in cancer immunotherapy. These T cell products are typically engineered by vectors that permanently integrate into the T cell genome, thus raising concerns about potential risks of insertional mutagenesis. Therefore, it becomes imperative to assess the integrated vector copy number (VCN) as a critical safety parameter for gene-engineered cell products. Materials and MethodsIn this study, we developed a robust assay for assessing the VCN of CAR-T cell and TCR T cell products, based on the digital droplet polymerase chain reaction (ddPCR) method. To provide accurate representation of the VCN in gene-engineered cells, we implemented a calculation that factors in the putative transduction efficiency based on Poisson distribution statistics. The adjusted VCN value (VCNadj) was also compared with VCN value from sorted transgene-positive cell populations, to validate its accuracy. ResultsThis assay consistently and accurately determines the average VCN for cell products. By comparing the VCN in sorted transgene-positive cell populations, we validated the refinement calculation provides a closer approximation to the actual VCN within transduced cells, offering a more realistic representation of the VCN for engineered cell products. ConclusionIn summary, we present a reliable and robust ddPCR-based assay for quantification of vector copy number in gene-engineered cell products.

Cellular Therapies for Multiple Myeloma: Engineering Hope.

Multiple myeloma (MM) treatment remains challenging due to its relapsed/refractory disease course as well as intra- and inter-patient heterogeneity. Cellular immunotherapies, especially chimeric antigen receptor (CAR)-T cells targeting B cell maturation antigen (BCMA), mark a major breakthrough, achieving long-lasting remissions and instilling hope for a potential cure. While ongoing clinical trials are increasingly driving approved cellular products towards earlier lines of therapy, novel targets as well as advanced approaches employing natural killer (NK) cells or dendritic cell (DC) vaccines are currently under investigation. Treatment resistance, driven by tumor-intrinsic factors such as antigen escape and the intricate dynamics of the tumor microenvironment (TME), along with emerging side effects such as movement and neurocognitive treatment-emergent adverse events (MNTs), are the major limitations of approved cellular therapies. To improve efficacy and overcome resistance, cutting-edge research is exploring strategies to target the microenvironment as well as synergistic combinatorial approaches. Recent advances in CAR-T cell production involve shortened manufacturing protocols and "off-the-shelf" CAR-T cells, aiming at decreasing socioeconomic barriers and thereby increasing patient access to this potential lifesaving therapy. In this review, we provide an extensive overview of the evolving field of cellular therapies for MM, underlining the potential to achieve long-lasting responses.

TMOD-02. PATIENT-DERIVED GLIOBLASTOMA ORGANOIDS AS REAL-TIME AVATARS FOR ASSESSING RESPONSES TO CLINICAL CAR-T CELL THERAPY

Abstract Patient-derived tumor organoids have been increasingly leveraged for disease modeling and preclinical studies, but rarely in real-time to aid with the interpretation of patient treatment responses in the clinical setting. Whether tumor organoids can be applied to mirror clinical activity in patients and even to predict responses for personalized medicine remains uncertain. We recently demonstrated promising early efficacy signals in a first-in-human, phase 1 study of dual-targeting chimeric antigen receptor T cells (EGFR-IL13Rα2 CAR-T cells) in patients with recurrent glioblastoma (clinical trial identifier: NCT05168423). Determination of meaningful clinical efficacy, in particular for CAR-T cell therapy, may take months to manifest. Given this challenge, real-time analysis of patient-derived glioblastoma organoids (GBOs) could help to provide early assessment of clinical efficacy and guide patient management. We analyzed six sets of GBOs generated on the same timeline as the production of patients’ CAR-T cell products. GBOs demonstrated the presence and retention of both targeted tumor-associated antigens EGFR and IL13Rα2 by immunohistochemistry. These GBOs were then treated with the same autologous CAR-T cell products received by patients in our phase 1 study in parallel with patient treatment. CAR-T cell treatment led to target antigen reduction assessed by immunohistochemistry and flow cytometry. Furthermore, we confirmed cytolysis of tumor cells in GBOs by cleaved caspase-3 immunostaining and by co-culture in the Axion Maestro Cellular Impedance platform. Importantly, the degree of cytolysis ex vivo significantly correlated with CAR-T cell engraftment detected in patients’ cerebrospinal fluid (CSF) for all six patients. Furthermore, cytotoxic activity of CAR-T cells as measured by TNFα, IL-2, and IFNγ release kinetics in GBOs mirrored cytokine levels in patient CSF samples over time. Our findings highlight a unique trial design and suggest GBOs as a valuable platform for real-time assessment of CAR-T cell bioactivity and for insights into immunotherapy efficacy.

Identification and analysis of microplastic aggregation in CAR-T cells

Microplastics (MPs) are increasingly recognized as contaminants present in various environments and are widely acknowledged as potential hazards to the mammalian immune system. In our study of chimeric antigen receptor T cell (CAR-T) therapy, we observed the presence of MP in CAR-T cell products for the first time. It is worth exploring whether MP could enter CAR-T cells and how they might affect CAR-T cells’ functionality. Therefore, we analyzed how MP affected CD19 and BCMA-CAR-T cells. Based on flow cytometry, ELISA, and cytotoxicity analysis of in vitro and in vivo experiments, MP suppressed the activity of CAR-T cells. Subsequent investigation revealed that the exposure of CAR-T cells to varying concentrations of MP resulted in a notable increase in apoptosis, ferroptosis, and exhaustion levels. Furthermore, the hyperactivation of the mTOR signaling pathway in MP-treated CAR-T cells was verified. The partial restoration of CAR-T cell function in MP was achieved by inhibiting the mTOR pathway. MP present a threat to CAR-T cell function due to their role in inducing CAR-T cell apoptosis, ferroptosis, and T-cell exhaustion through the hyperactivation of mTOR signaling pathways.

Point-of-care manufacturing of anti-CD19 CAR-T cells using a closed production platform: Experiences of an academic in Thailand

Anti-CD19 chimeric antigen receptor (CAR)-T cell therapy has evolved as a standard of care for various forms of relapsed/refractory B cell malignancies in major developed countries. However, access to industry-driven CAR-T cell therapy is limited in developing countries, partly due to the centralized manufacturing system. Here, we demonstrated the feasibility of the point-of-care (POC) manufacturing of anti-CD19 CAR-T cells from heavily pretreated patients and healthy graft donors at an academic medical center in Thailand using a closed semi-automated production platform, CliniMACS Prodigy, and established in-process quality control and release testing to ensure their identity, purity, sterility, safety, and potency. Nine out of the nine products manufactured were used in a pilot study (ISRCTN17901467). However, we did observe that starting Tcells with CD4/CD8 ratios of less than one-third had a high chance of manufacturing failure, which could be minimized by serum supplementation. Further analysis of Tcell phenotypes in the infused versus circulating CAR-T cells revealed the differentiation from early memory subtypes toward effector cells invivo. The POC manufacturing and quality control settings herein could be applied to other CAR-T cell products and may benefit other academics, especially those in developing countries, making CAR-T cells more accessible.

Directed evolution-based discovery of ligands for in vivo restimulation of CAR-T cells.

Chimeric antigen receptor (CAR) T cell therapy targeting CD19 elicits remarkable clinical efficacy in B-cell malignancies, but many patients relapse due to failed expansion and/or progressive loss of CAR-T cells. We recently reported a strategy to potently restimulate CAR-T cells in vivo, enhancing their functionality by administration of a vaccine-like stimulus comprised of surrogate peptide ligands for a CAR linked to a lymph node-targeting amphiphilic PEG-lipid (termed CAR-T-vax). Here, we demonstrate a general strategy to generate and optimize peptide mimotopes enabling CAR-T-vax generation for any CAR. Using the clinical CD19 CAR FMC63 as a test case, we employed yeast surface display to identify peptide binders to soluble IgG versions of FMC63, which were subsequently affinity matured by directed evolution. CAR-T vaccines using these optimized mimotopes triggered marked expansion of both murine CD19 CAR-T cells in a syngeneic model and human CAR-T cells in a humanized mouse model of B cell acute lymphoblastic leukemia (B-ALL), and enhanced control of leukemia progression. This approach thus enables vaccine boosting to be applied to any clinically-relevant CAR-T cell product.

Efficient combinatorial adaptor-mediated targeting of acute myeloid leukemia with CAR T-cells

CAR T-cell products targeting lineage-specific cell-of-origin antigens, thereby eliminating both tumor and healthy counterpart cells, are currently clinically approved therapeutics in B- and plasma-cell malignancies. While they represent a major clinical improvement, they are still limited in terms of efficacy by e.g. single, sometimes low-expressed antigen targeting, and in terms of safety by e.g., lack of on-off activity. Successful cell-of-origin non-discriminative targeting of heterogeneous hematopoietic stem and progenitor cell malignancies, such as acute myeloid leukemia (AML), will require antigen-versatile targeting and off-switching of effectors in order to then allow rescue by hematopoietic stem cell transplantation (HSCT), preventing permanent myeloablation. To address this, we developed adaptor-CAR (AdFITC-CAR) T-cells targeting fluoresceinated AML antigen-binding diabody adaptors. This platform enables the use of adaptors matching the AML-antigen-expression profile and conditional activity modulation. Combining adaptors significantly improved lysis of AML cells in vitro. In therapeutic xenogeneic mouse models, AdFITC-CAR T-cells co-administered with single diabody adaptors were as efficient as direct CAR T-cells, and combinatorial use of adaptors further enhanced therapeutic efficacy against both, cell lines and primary AML. Collectively, this study provides proof-of-concept that AdFITC-CAR T-cells and combinations of adaptors can efficiently enhance immune-targeting of AML.

Next-generation BCMA-targeted chimeric antigen receptor CARTemis-1: the impact of manufacturing procedure on CAR T-cell features.

CAR therapy targeting BCMA is under investigation as treatment for multiple myeloma. However, given the lack of plateau in most studies, pursuing more effective alternatives is imperative. We present the preclinical and clinical validation of a new optimized anti-BCMA CAR (CARTemis-1). In addition, we explored how the manufacturing process could impact CAR-T cell product quality and fitness. CARTemis-1 optimizations were evaluated at the preclinical level both, in vitro and in vivo. CARTemis-1 generation was validated under GMP conditions, studying the dynamics of the immunophenotype from leukapheresis to final product. Here, we studied the impact of the manufacturing process on CAR-T cells to define optimal cell culture protocol and expansion time to increase product fitness. Two different versions of CARTemis-1 with different spacers were compared. The longer version showed increased cytotoxicity. The incorporation of the safety-gene EGFRt into the CARTemis-1 structure can be used as a monitoring marker. CARTemis-1 showed no inhibition by soluble BCMA and presents potent antitumor effects both in vitro and in vivo. Expansion with IL-2 or IL-7/IL-15 was compared, revealing greater proliferation, less differentiation, and less exhaustion with IL-7/IL-15. Three consecutive batches of CARTemis-1 were produced under GMP guidelines meeting all the required specifications. CARTemis-1 cells manufactured under GMP conditions showed increased memory subpopulations, reduced exhaustion markers and selective antitumor efficacy against MM cell lines and primary myeloma cells. The optimal release time points for obtaining the best fit product were > 6 and < 10 days (days 8-10). CARTemis-1 has been rationally designed to increase antitumor efficacy, overcome sBCMA inhibition, and incorporate the expression of a safety-gene. The generation of CARTemis-1 was successfully validated under GMP standards. A phase I/II clinical trial for patients with multiple myeloma will be conducted (EuCT number 2022-503063-15-00).

Good manufacturing practice-grade generation of CD19 and CD123-specific CAR-T cells using piggyBac transposon and allogeneic feeder cells in patients diagnosed with B-cell non-Hodgkin lymphoma and acute myeloid leukemia.

The non-viral production of CAR-T cells through electroporation of transposon DNA plasmids is an alternative approach to lentiviral/retroviral methods. This method is particularly suitable for early-phase clinical trials involving novel types of CAR-T cells. The primary disadvantage of non-viral methods is the lower production efficiency compared to viral-based methods, which becomes a limiting factor for CAR-T production, especially in chemotherapy-pretreated lymphopenic patients. We describe a good manufacturing practice (GMP)-compliant protocol for producing CD19 and CD123-specific CAR-T cells based on the electroporation of transposon vectors. The lymphocytes were purified from the blood of patients undergoing chemotherapy for B-NHL or AML and were electroporated with piggyBac transposon encoding CAR19 or CAR123, respectively. Electroporated cells were then polyclonally activated by anti-CD3/CD28 antibodies and a combination of cytokines (IL-4, IL-7, IL-21). The expansion was carried out in the presence of irradiated allogeneic blood-derived mononuclear cells (i.e., the feeder) for up to 21 days. Expansion in the presence of the feeder enhanced CAR-T production yield (4.5-fold in CAR19 and 9.3-fold in CAR123). Detailed flow-cytometric analysis revealed the persistence of early-memory CAR-T cells and a low vector-copy number after production in the presence of the feeder, with no negative impact on the cytotoxicity of feeder-produced CAR19 and CAR123 T cells. Furthermore, large-scale manufacturing of CAR19 carried out under GMP conditions using PBMCs obtained from B-NHL patients (starting number=200x10e6 cells) enabled the production of >50x10e6 CAR19 in 7 out of 8 cases in the presence of the feeder while only in 2 out of 8 cases without the feeder. The described approach enables GMP-compatible production of sufficient numbers of CAR19 and CAR123 T cells for clinical application and provides the basis for non-viral manufacturing of novel experimental CAR-T cells that can be tested in early-phase clinical trials. This manufacturing approach can complement and advance novel experimental immunotherapeutic strategies against human hematologic malignancies.

Comparing the Differences in Adverse Events among Chimeric Antigen Receptor T-Cell Therapies: A Real-World Pharmacovigilance Study.

In this study, we compared the similarities and differences in adverse events (AEs) among CAR T-cell products through signal mining via the FDA Adverse Event Reporting System (FAERS) and identified unknown AEs to provide a reference for safe clinical medication. Data from the FAERS database spanning from the fourth quarter of 2017 to the first quarter of 2024 were extracted. Signals were identified using the reporting odds ratio (ROR) method and the Medicines and Healthcare Products Regulatory Agency (MHRA) method. A total of 11,386 AE reports related to six CAR T-cell products were selected. The top three categories of AEs reported were nervous system disorders, immune system disorders, and general disorders and administration site conditions. However, there were variations in the AE spectra among the different CAR T-cell products. The BCMA-targeting drugs idecabtagene vicleucel (Ide-cel) and ciltacabtagene autoleucel (Cilta-cel) were found to be associated with parkinsonism, which were not observed in CD19-targeting drugs. Tisagenlecleucel (Tisa-cel) and axicabtagene ciloleucel (Axi-cel) exhibited cerebrovascular accident-related AEs, graft versus host disease, and abnormal coagulation indices. Cilta-cel was associated with cerebral hemorrhage, intracranial hemorrhage, cranial nerve disorder, and facial nerve disorder. Cardiopulmonary toxicity, including hypoxia, tachypnoea, cardiorenal syndrome, and hypotension, exhibited strong signal intensities and considerable overlap with CRS. The number of positive signals for cardiopulmonary toxicity associated with drugs targeting CD-19 is greater. Clinicians should assess patients prior to medication and closely monitor their vital signs, mental status, and laboratory parameters during treatment.

CAR-T cell therapy in AML: recent progress and future perspectives.

Despite several small-molecule drugs that have revolutionized the current treatment strategy for acute myeloid leukemia (AML), hematopoietic stem cell transplantation remains the only curative treatment in most cases to date. Chimeric antigen receptor (CAR)-T cell therapy is one of the most promising next-generation cancer therapies for hematological malignancies and is clinically available for treatment of AML. However, developing AML-targeted CAR-T therapy is challenging because of the heterogeneity of target antigen expression across leukemic cells and patients, the difficulty in excluding on-/off-target tumor effects, and the immunosuppressive tumor microenvironment. To date, various targets, including CD33, NKG2D, CD123, CLL-1, and CD7, have been actively studied for CAR-T cells. Although no CAR-T cell products are close to practical use, several clinical trials have shown promising results, particularly for CAR-T cells targeting CLL-1 or CD123. Meanwhile, research exploring the ideal target for AML-targeted CAR-T therapy continues. Furthermore, as collecting autologous lymphocytes from patients with AML is difficult, development of off-the-shelf CAR-T products is being actively pursued. This review discusses the challenges in AML-targeted CAR-T cell therapy development from the perspectives of target antigen characteristics and AML-specific on-target/off-tumor toxicity. Moreover, it discusses the clinical development and prospects of AML-targeting CAR-T cells.

Single center, real-world retrospective study of CAR-T cell therapy for relapsed/refractory large B-cell lymphoma beyond second line: five-year results at the University Hospitals Leuven

ABSTRACT Introduction Large B-cell lymphomas (LBCL) are the most frequently aggressive B-cell non-Hodgkin lymphomas. Anti-CD19 chimeric antigen receptor (CAR)-T cell therapy has emerged as a new, powerful treatment for relapsed or refractory (R/R) disease. Two CAR-T cell products, tisagenlecleucel (tisa-cel,) and axicabtagene ciloleucel (axi-cel), are reimbursed in Belgium for R/R LBCL beyond second line. Objectives and methods We conducted a retrospective cohort study to report the outcome with tisa-cel and axi-cel for R/R LBCL beyond second line in the years 2019–2023 at the University Hospitals Leuven for 79 patients selected for apheresis and CAR-T infusion. Results Eleven patients (14%) did not proceed to CAR-T cell infusion. For infused patients (n = 68), the best overall response rate (ORR)/complete response (CR) rate was 64%/49% for tisa-cel and 88%/66% for axi-cel (p = 0.04 for ORR). After a median follow-up of 13.8 months, progression-free survival (PFS) and overall survival (OS) at 1 year were 30% and 43% for tisa-cel and 48% and 62% for axi-cel. Cytokine release syndrome (CRS) (all grades/grade ≥3) occurred in 82%/9% after tisa-cel and in 97%/0% after axi-cel. Immune effector cell-associated neurotoxicity syndrome (ICANS) (all grades/grade ≥3) occurred in 24%/18% after tisa-cel and in 54%/40% after axi-cel. The non-relapse mortality in the infusion cohort was 13%. Conclusion Our real-world data show high and durable response rates, with a non-significant trend towards a higher efficacy and higher toxicity for axi-cel compared to tisa-cel. Our results are in line with other real-world registries except for a shorter median OS and more high-grade ICANS.

A Multicenter Real-life Prospective Study of Axicabtagene Ciloleucel versus Tisagenlecleucel Toxicity and Outcomes in Large B-cell Lymphomas.

This real-world prospective observational study across 21 Italian centers (CART-SIE) compares axicabtagene ciloleucel (axi-cel) and tisagenlecleucel (tisa-cel) outcomes in 485 patients with relapsed/refractory large B-cell lymphoma with baseline characteristics matched by stabilized inverse propensity score weighting. Axi-cel versus tisa-cel had higher all-grade cytokine release syndrome (78.6% vs. 89.3%, P = 0.0017) and neurotoxicity (9.9% vs. 32.2%, P < 0.0001) but also superior progression-free survival (PFS) at 1 year (46.5% vs. 34.1%, P = 0.0009). Even among patients who failed bridging therapy, axi-cel PFS was superior to tisa-cel (37.5% vs. 22.7%, P = 0.0059). Differences in overall survival and high-grade immune toxicities were not significant. The CAR-HEMATOTOX score not only predicted hematologic toxicity but also 1-year survival outcomes (51.5% in CAR-HEMATOTOX high vs. 77.2% in CAR-HEMATOTOX low, P < 0.0001). Twenty patients developed second primary malignancies, including two cases of T-cell neoplasms. These findings enable more informed selection of anti-CD19 CAR T-cell therapy, balancing bridging, safety, and efficacy considerations for individual patients. Significance: The findings of this study on 485 patients with relapsed/refractory large B-cell lymphoma treated with commercial axi-cel and tisa-cel indicate axi-cel's superior PFS after propensity score weighting. The predictive utility of CAR-HEMATOTOX in assessing not only toxicity but also outcomes across both CAR T-cell products may guide future risk-stratified management strategies.

Analysis of polyfunctionality for enhanced BAFF-R CAR T-cell therapy for hematologic malignancies

AbstractChimeric antigen receptor (CAR) T-cell therapy has emerged as a promising immunotherapeutic strategy for eradicating human cancers. Their therapeutic success and durability of clinical responses hinges, in large part, on their functional capacity, including the ability of these engineered cells to simultaneously expand and persist after infusion into patients. CD19 CAR T-cell polyfunctionality, assessing the simultaneous functions of cytokine production, proliferation, and cytotoxicity has been reported to correlate with clinical outcomes. Assay optimization is potentially limited by the heterogeneous nature of CAR T-cell infusion products and target specificity. We optimized a single-cell platform for polyfunctionality using CAR T-cell products manufactured from healthy donors, engineered against a novel target, B-cell–activating factor receptor (BAFF-R) and validated the protocol using CD19 CAR T cells. We observed distinct qualitative differences between BAFF-R and CD19 CAR T cells relative to the proportions of stimulatory vs effector cytokines, based on target antigen density, and, generally, CD19 CAR T cells exhibited lower indices of polyfunctionality. Finally, we applied our assay to the autologous BAFF-R CAR T-cell product generated from the first patient with non-Hodgkin lymphoma treated in an ongoing clinical trial who had progressed after prior CD19 CAR T-cell therapy. We observed robust indicators of polyfunctionality, which correlated with successful CAR T-cell expansion after infusion and achievement of durable complete remission ongoing after 18 months. The precise identification of factors determining the role of BAFF-R CAR T-cell fitness in toxicity and clinical outcome will require the application of this robust assay in the analysis of additional treated patients. This trial was registered at www.ClinicalTrials.gov as #NCT05370430.

Cardiotoxic profiles of CAR-T therapy and bispecific T-cell engagers in hematological cancers

BackgroundChimeric antigen receptor (CAR) T-cell therapy and bispecific T-cell engagers, which redirect T-cells to tumor antigens, have immensely benefitted patients with relapsed/refractory B-cell cancers. How these therapies differ in cardiotoxicity is underexplored. We used the World Health Organization pharmacovigilance database, VigiBase, to compare cardiotoxicity profiles between CD19-targeted CAR-T therapy and blinatumomab (a CD19/CD3-targeted bispecific T-cell engager).MethodsSafety reports in VigiBase were filtered for diffuse large B-cell lymphoma (DLBCL, n = 17,479) and acute lymphocytic leukemia (ALL, n = 28,803) for all adverse reactions. Data were further filtered for patients taking CAR-T therapy or blinatumomab. Reporting odds ratios (ROR) and fatality rates were compared between CAR-T cell products (e.g. tisagenlecleucel and axicabtagene ciloleucel), and between CAR-T therapy and blinatumomab.ResultsTisagenlecleucel is associated with cardiac failure (IC025 = 0.366) with fatality rates of 85.7% and 80.0% in DLBCL and pediatric ALL patients respectively. For DLBCL patients, axicabtagene ciloleucel has greater reporting for hypotension than tisagenlecleucel (ROR: 2.54; 95% CI: 1.28–5.03; p = 0.012), but tisagenlecleucel has higher fatality rates for hypotension than axicabtagene ciloleucel [50.0% (tisagenlecleucel) vs 5.6% (axicabtagene ciloleucel); p < 0.001]. Blinatumomab and tisagenlecleucel have similar fatality rates for hypotension in pediatric ALL patients [34.7% (tisagenlecleucel) vs 20.0% (blinatumomab); p = 0.66].ConclusionsTisagenlecleucel is associated with severe and fatal adverse cardiac events, with higher fatality rates for hypotension compared to axicabtagene ciloleucel in DLBCL patients, but similar hypotension fatality rates compared to blinatumomab in pediatric ALL patients. Effective management necessitates experienced physicians, including cardio-oncologists, skilled in interdisciplinary approaches to manage these toxicities.

Predictors of severe hematotoxicity after CAR T-cell therapy.

7024 Background: The EHA/EBMT immune effector cell-associated hematotoxicity (ICAHT) grading system characterizes hematotoxicity after CAR T-cell therapy based on depth and duration of neutropenia (Rejeski et al, Blood, 2023). We evaluated pre- and post-infusion factors associated with grade 3-4 ICAHT and developed a predictive model in 602 patients with hematologic malignancies undergoing CAR T-cell therapy at Fred Hutch Cancer Center. Methods: Grading of early hematotoxicity (day-0-30 after CAR T-cell cell infusion) was automated using the heatwaveR package. Associations with 50 patient, disease-related, and laboratory factors, and grade 3-4 ICAHT were modeled using univariate and multivariable logistic regression. Results: The most common disease types were aggressive non-Hodgkin lymphoma (NHL; n = 293; 49%), indolent NHL (n = 150; 25%), and acute lymphoblastic leukemia (ALL; n = 94; 16%). The most common CAR T-cell products were the investigational CD19 CAR T-cell product JCAR014 (n = 197; 33%), axicabtagene ciloleucel (n = 129; 21%), and lisocabtagene maraleucel (n = 73; 12%). Incidences of early ICAHT grades 1, 2, 3, and 4 were 319 (53%), 96 (16%), 60 (10%), and 35 (6%) patients, respectively. Baseline patient factors associated with grade 3-4 ICAHT included ALL (reference: aggressive NHL, OR = 4.18, 95% CI, 2.41-7.26, p &lt; 0.001) , Hispanic or Latino ethnicity (OR = 2.17, 95% CI, 1.07-4.20, p = 0.025), and lower age (OR = 1.03, 95% CI, 1.02-1.05, p &lt; 0.001). Pre-lymphodepletion (LD) laboratory factors associated with grade 3-4 ICAHT in univariate analyses included lower ANC (OR = 6.67 per log10 cells/μL, 95% CI, 4.0-11.1, p &lt; 0.001), LDH (OR = 8.70per log10U/L, 95% CI, 4.03-19.4, p &lt; 0.001), CRP (OR = 3.18 per log10mg/L, 95% CI, 2.06-5.07, p &lt; 0.001), and ferritin (OR = 6.07 per log10mg/L, 95% CI, 3.65-10.6, p &lt; 0.001). Peak CRP (OR = 15.6, 95% CI, 7.27-36.4, p &lt; 0.001), peak ferritin (OR = 7.41, 95% CI, 5.02-11.3, p &lt; 0.001), peak CRS grade (OR = 2.01, 95% CI, 1.59-2.56, p &lt; 0.001), and peak ICANS grade (OR = 1.51, 95% CI, 1.29-1.77, p &lt; 0.001) after CAR T-cell infusion were also strongly associated with grade 3-4 ICAHT. A multivariable model using restricted cubic splines and including disease type, pre-LD ANC, pre-LD LDH, peak CRP, peak ferritin, and CRS grade demonstrated high discrimination to predict grade 3-4 ICAHT (C-index = 0.89). Internal validation using bootstrapping showed near-perfect calibration without overfitting. Sensitivity and specificity based on the Youden criteria was 74% and 90%, respectively. The sensitivity and specificity of the CAR-HEMATOTOX score in predicting grade 3-4 ICAHT was 93% and 30%, respectively. Conclusions: We identified pre- and post-infusion predictors of grade 3-4 ICAHT and internally validated a multivariable logistic regression model including disease-type, pre-LD ANC, pre-LD LDH, peak CRP, peak ferritin, and CRS grade. We plan to further evaluate our model in an external cohort.

Obecabtagene autoleucel (obe-cel, AUTO1) in adults with relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL): Overall survival (OS), event-free survival (EFS) and the potential impact of chimeric antigen receptor (CAR)-T cell persistency and consolidative stem cell transplantation (SCT) in the open-label, single-arm FELIX phase Ib/II study.

6504 Background: Obe-cel is an autologous CAR-T cell product with a fast off-rate CD19 binder designed to mitigate immunotoxicity and improve expansion/persistence. Pooled results from the pivotal FELIX phase Ib/II study (NCT04404660) of obe-cel in adults with R/R B-ALL were recently presented (Roddie C et al. Blood 2023;142[Suppl 1]:222). Here, OS and EFS in all patients (pts) treated with obe-cel are reported, alongside the impact of CAR-T cell persistency and consolidative SCT for pts in remission. Methods: Pts aged ≥18 yrs with R/R B-ALL were enrolled. CAR-T products were generated via an automated process (Roddie C et al. Blood 2023;142[Suppl 1]:222). Pts received bridging therapy as appropriate and underwent lymphodepletion (fludarabine, 4×30mg/m2; cyclophosphamide, 2×500mg/m2), followed by obe-cel split dose infusions on Days 1 and 10 based on pre-lymphodepletion leukemic burden at a target dose of 410×106 CAR-T cells. Results: A total of 127/153 (83%) enrolled pts were infused. At screening, pts’ median age was 47 yrs; 42%/31%/44% had received prior blinatumomab/inotuzumab ozogamicin/allogeneic SCT; median bone marrow blast burden was 36% (range: 0−100). At data cut-off (13 September 2023), median follow-up was 16.6 mos (range 3.7−36.6 mos). The overall complete remission or complete remission with incomplete count recovery rate among infused pts was 78%. Among responding pts, 17/99 (17%) proceeded to consolidative SCT while in remission; all 17 (100%) were in measurable residual disease (MRD)-negative remission (≤10–4 leukemic blasts) and 10/17 (59%) showed CAR-T cell persistency prior to SCT. Loss of CAR-T cell persistency was associated with a hazard risk of relapse or death 2.9 times compared with pts who had ongoing CAR-T cell persistency. Pts who experienced B-cell recovery had a hazard risk of relapse or death 1.7 times compared with pts without B-cell recovery. At 12 mos, the EFS rate was 50% and 43% with or without censoring for consolidative SCT or new therapies, respectively; the OS rate was 61% and 59% with or without censoring for SCT, respectively. Conclusions: Ongoing CAR-T cell persistency and B-cell aplasia were associated with improved EFS without further consolidation post-obe-cel. At the current follow-up, consolidative SCT for pts in MRD-negative remission post-obe-cel did not improve EFS or OS. Clinical trial information: NCT04404660 .

Phase 2 open label, multicenter study evaluating CRG-022, a CD22-directed autologous CAR T-cell therapy, in patients (pts) with relapsed/refractory (R/R) large B-cell lymphoma (LBCL) after CD19-directed CAR T-cell therapy.

TPS7085 Background: Autologous (auto) CD19-directed CAR T-cell therapy can induce long-term remissions for pts with R/R LBCL but approximately 60% will experience disease progression resulting in poor outcomes (Neelapu, 2023; Zurko, 2023). CRG-022 is a novel CAR T-cell product targeting CD22, a common B-cell antigen widely expressed in LBCL. Key features of CRG-022 include a fully human scFv derived from the m971 monoclonal antibody and 4-1BB/CD3z intracellular signaling domains (Singh, 2021). This CD22 CAR construct was developed at the National Cancer Institute and was investigated in phase (ph) 1 studies of pediatric/young adult pts with R/R CD22+ malignancies (Shah, 2020; NCT02315612) and at Stanford University in adults with R/R LBCL (Baird, 2021; NCT04088890) primarily in pts who received prior CD19-directed CAR-T therapy. The Stanford ph 1b study reported an overall response rate (ORR) and complete response rates of 66% and 52%, respectively in 29 pts treated at the recommended Phase 2 dose (RP2D) of 1x106 CAR+ T cells/kg (Frank, 2023). After a median follow-up of 27.3 months, the median overall survival had not been reached at the RP2D (Su, 2023). At the RP2D, no grade &gt;3 cytokine release syndrome (CRS), immune-effector cell associated neurotoxicity syndrome (ICANS), or immune effector cell associated hemophagocytic lymphohistiocytosis-like syndrome (IEC-HS) occurred; two pts (7%) developed grade 2 IEC-HS (Su, 2023; Srinagesh, 2023). Based on these notable results, this potentially pivotal ph 2 trial has been initiated. Methods: This Phase 2 study will evaluate the safety/efficacy of CRG-022 at 1x106 CAR+ T cells/kg in pts with R/R LBCL whose disease has progressed after CD19-directed CAR T-cell therapy. The primary endpoint is ORR according to Lugano response criteria (Cheson, 2014) by blinded independent review. Adverse events (AEs) are graded per CTCAE v5 except with CRS, ICANS, and IEC-HS graded per ASTCT guidelines. Pts with diffuse large B-cell lymphoma (DLBCL), high grade B-cell lymphoma, DLBCL transformed from follicular or marginal zone lymphoma, follicular large B-cell lymphoma (FLBL)/FL Grade 3B, and primary mediastinal B-cell lymphoma are eligible. Other eligibility criteria include adequate hematologic and organ function, and no prior allogeneic stem cell transplant. Pts with prior treatment to both a CD19-directed auto CAR-T and a bispecific T-cell engaging antibody are also eligible within a separate cohort. Lymphodepletion with fludarabine 30 mg/m2/day and cyclophosphamide 500 mg/m2/day for 3 days will be administered ahead of a single infusion of CRG-022. The study is designed to enroll approximately 123 pts. Study enrollment commenced in the US in August 2023, with 9 pt infused as of 08 JAN 2024. Clinical trial information: NCT05972720 .

SPENT MEDIA ANALYSIS AIDS IN DETERMINING METABOLIC MECHANISMS OF SUPERIOR CAR T-CELL PRODUCTS

SPENT MEDIA ANALYSIS AIDS IN DETERMINING METABOLIC MECHANISMS OF SUPERIOR CAR T-CELL PRODUCTS

Nanomaterials Boost CAR-T Therapy for Solid Tumors.

T cell engineering, particularly via chimeric antigen receptor (CAR) modifications for enhancing tumor specificity, has shown efficacy in treating hematologic malignancies. The extension of CAR-T cell therapy to solid tumors, however, is impeded by several challenges: The absence of tumor-specific antigens, antigen heterogeneity, a complex immunosuppressive tumor microenvironment, and physical barriers to cell infiltration. Additionally, limitations in CAR-T cell manufacturing capacity and the high costs associated with these therapies restrict their widespread application. The integration of nanomaterials into CAR-T cell production and application offers a promising avenue to mitigate these challenges. Utilizing nanomaterials in the production of CAR-T cells can decrease product variability and lower production expenses, positively impacting the targeting and persistence of CAR-T cells in treatment and minimizing adverse effects. This review comprehensively evaluates the use of various nanomaterials in the production of CAR-T cells, genetic modification, and in vivo delivery. It discusses their underlying mechanisms and potential for clinical application, with a focus on improving specificity and safety in CAR-T cell therapy.