Estefanía García-Guerrero

BCMA-directed CAR-T cell therapy has demonstrated remarkable clinical efficacy in multiple myeloma, but robust methods to evaluate product quality and predict long-term functionality remain underdeveloped. While immunophenotypic features of leukapheresis products correlate with clinical outcomes, the characterization of infused CAR-T cell products remains challenging and inconsistent across studies. We compared two versions of our academic BCMA-CAR product, CARTemis-1, differing only in their costimulatory domains (4-1BB vs. CD28), and performed a detailed functional and molecular characterization before and after antigen stimulation to identify which analytical approaches best capture functionally relevant differences between CAR-T cell products. Analyses included conventional flow cytometry, short-term cytotoxicity assays, repetitive antigen stimulation, metabolic profiling, and transcriptomic evaluation. Finally, metabolic profile of patient derived CAR-T cells products (n = 15) was also analyzed. Although previous studies have reported functional differences between CAR-T cell products incorporating distinct costimulatory domains, conventional assays in our system revealed no significant differences in viability, expansion dynamics, immunophenotype, or activation/exhaustion marker expression. In contrast, more functionally demanding assays revealed clear differences between constructs. Repetitive antigen stimulation revealed superior long-term cytotoxicity in CARTemis-1-BB, while metabolic profiling demonstrated enhanced spare respiratory capacity and maximal respiration after antigen exposure. Transcriptomic analysis further showed distinct pathway regulation, with enrichment of T cell activation signatures in CARTemis-1-BB and Wnt/TGFβ-related responses in CARTemis-1-28. Importantly, metabolic profiling of infused CAR-T cell products also identified distinct signatures associated with clinical outcome, distinguishing products administered to long-term responders from those infused into short-term or non-responding patients. Persistence and metabolic analyses represent key tools for optimizing CAR-T cell construct selection during preclinical development, providing a more accurate assessment of CAR-T cell product quality than standard immunophenotyping and enabling the identification of constructs with superior therapeutic potential.

The manufacturing of lentiviral vectors (LV) under Good Manufacturing Practices (GMP) remains a critical bottleneck limiting the clinical translation of ac

Graft-versus-host disease (GVHD) is a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. BCL2 overexpression has been related to a higher risk of GVHD and glucocorticoid resistance. In this study, we aimed to evaluate the role of BCL2 in GVHD pathophysiology. Remarkably, we observed that the pharmacological inhibition of BCL2 increased the risk of GVHD in murine models. In addition, we used a GVHD model with C57BL/6 donor mice that constitutively overexpress human BCL2 gene in hematopoietic cells (Vav-BCL2). Interestingly, the overexpression of BCL2 in donor lymphocytes was related to a lower risk and severity of GVHD. In vitro, the BCL2 inhibitor venetoclax significantly hampered resting T-cell viability, but it did not affect activated lymphocytes, at least in part because of reduced BCL2 interacting mediator of cell death–BCL2 binding after activation. Moreover, BCL2 overexpressing T cells had compromised functionality. Overexpression of BCL2 had no deleterious effect on graft-versus-leukemia activity. Finally, in vitro studies showed that exposure to steroids enriched the populations of T cells displaying higher BCL2 expression levels. Therefore, our study shows that high expression of BCL2 decreases the risk and severity of GVHD.

Chronic graft-versus-host disease (cGVHD) remains a major hurdle to the success of hematopoietic stem cell transplantation, directly affecting patient morbidity and mortality. Impaired regulatory T-cell (Treg) recovery in patients with cGVHD has led to clinical studies aiming to increase peripheral Treg numbers. We conducted phase 1 dose-escalation clinical trials testing the feasibility and safety of using freshly isolated donor-derived Treg infusions in steroid-refractory/dependent cGVHD. The phase 1 was extended to a preliminary phase 2 trial, resulting in a total of 33 treated patients. We report that Treg purification from donor leukapheresis using CliniMACS were feasible and that Treg infusions were safe. Importantly, Treg infusions resulted in improved symptoms, particularly at higher Treg doses. Global responses were observed in 71% of patients, and 52% of patients had at least a 2-point improvement in the cGVHD severity scale. Furthermore, improvement in cGVHD symptoms resulted in reductions in corticosteroids, ruxolitinib, and mycophenolate in 58%, 83%, and 33% of patients, respectively, whereas calcineurin inhibitors were discontinued in 75% of patients. Exploratory analyses revealed the detection of infused Treg clonotypes up to 12 months after infusion and suggest increased Treg numbers in circulation. We observed increases in serum levels of interleukin-7 and interferon gamma and decreases in soluble CD13 and suppression of tumorigenicity 2 over time, which were not statistically significant after adjustment for multiple comparisons. Although these studies were not powered to assess efficacy, they suggest potential therapeutic benefits of donor-derived Treg in cGVHD treatment and highlight the need for larger phase 2 clinical trials. The trials were registered at www.clinicaltrials.gov as #NCT02385019 and #NCT03683498.

Despite the promising results of CAR-T cell therapy, still a significant proportion of patients experience treatment failure due to absence of response, progression or treatment-related toxicities. Our research aims to investigate how the CAR-T cell product characteristics and particularly the percentage of CAR-negative cells present in the infusion product affects its efficacy and safety. CAR-positive (CAR-pos) and CAR-negative (CAR-neg) cells were analyzed during in vitro expansion with IL-2 or IL-7/IL-15. FACS-sorted populations were assessed for cytotoxic effect and secretion of cytokines. Transcriptomic analyses were conducted for CAR-pos and CAR-neg populations. A significant reduction in the percentage of transduced (CAR-pos) T cells was observed throughout the in vitro cell culture, where CAR-neg T cells displayed greater expansion along the procedure compared to CAR-pos T cells, using either IL-2 or IL-7/IL-15. CAR-pos T cells exhibited increased expression of activation and exhaustion markers. Comparative analyses of CAR-pos and CAR-neg showed an increased cytotoxic effect in purified samples with 100% CAR-pos T cells compared to non-purified product. Quantification of cytokine levels revealed a significant decrease in IL-6 in purified compared to non-purified products. RNA sequencing analysis revealed significant differential gene expression profile between CAR-pos and CAR-neg cells, the prior exhibiting an upregulation of genes associated with immune checkpoint regulation, cytokine-cytokine receptor interaction and Th1/Th2 and Th17 cell differentiation pathways. CAR-neg T cells exhibit a significantly greater expansion compared to CAR-pos T cells alongside CAR-T cell production. The percentage of CAR-neg cells influences the specific cytotoxicity of the product and on the cytokine pattern, which might influence both efficacy and toxicity and, therefore, the number of CAR-neg T cells within the infusion product must be considered in the clinical setting. Purification of CAR-pos T cells could potentially optimize product characteristics.

Purpose 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. Methods 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. Results 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). Conclusions 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).

Disclosures. Reguera:AMGEN: Speakers Bureau; KITE: Speakers Bureau; BMS: Speakers Bureau; Janssen: Consultancy, Speakers Bureau. Einsele:Takeda: Honoraria, Othe

Purpose: CAR-T cell therapy has proven to be a disruptive treatment in the hematology field, however, less than 50% of patients maintain long-term response and early predictors of outcome are still inconsistently defined. Here, we aimed to optimize the detection of CD19 CAR-T cells in blood and to identify phenotypic features as early biomarkers associated with toxicity and outcomes.Experimental design: In this study, monitoring by flow cytometry and digital PCR (dPCR), and immunophenotypic characterization of circulating CAR-T cells from 48 patients treated with Tisa-cel or Axi-cel was performed.Results: Validation of the flow cytometry reagent for the detection of CAR-T cells in blood revealed CD19 protein conjugated with streptavidin as the optimal detection method. Kinetics of CAR-T cell expansion in blood confirmed median day of peak expansion at seven days post-infusion by both flow cytometry and digital PCR. Circulating CAR-T cells showed an activated, proliferative, and exhausted phenotype at the time of peak expansion. Patients with increased expansion showed more severe CRS and ICANs. Immunophenotypic characterization of CAR-T cells at the peak expansion identified the increased expression of co-inhibitory molecules PD1 and LAG3 and reduced levels of the cytotoxicity marker CD107a as predictors of a better long-term disease control. Conclusions: These data show the importance of CAR-T cells in vivo monitoring and identify the expression of PD1LAG3 and CD107a as early biomarkers of long-term disease control after CAR-T cell therapy.

Current data on CAR-T cell-based therapy is really promising in multiple myeloma, especially in terms of response. In heavily pretreated patients, who have already received proteasome inhibitors, immunomodulatory drugs and monoclonal antibodies, current trials report an overall response rate ranging from 81 to 97% and 45 to 67% of complete remission rates. Data are less encouraging in terms of duration of response, although most recent trials have shown significant improvements in terms of event-free survival, with medians ranging from 8 to 14 months and up to 77% progression-free survival at 12 months with an acceptable toxicity profile. These data will be consolidated in future years and will provide new evidence on the best timing for CAR-T cell therapy. Moreover, new CAR-T designs are underway and will challenge the current results.

Multiple myeloma (MM) remains an incurable disease regardless of recent advances in the field. Therefore, a substantial unmet need exists to treat patients with relapsed/refractory myeloma. The use of novel agents such as daratumumab, elotuzumab, carfilzomib, or pomalidomide, among others, usually cannot completely eradicate myeloma cells. Although these new drugs have had a significant impact on the prognosis of MM patients, the vast majority ultimately become refractory or can no longer be treated due to toxicity of prior treatment, and thus succumb to the disease. Cellular therapies represent a novel approach with a unique mechanism of action against myeloma with the potential to defeat drug resistance and achieve long-term remissions. Genetic modification of cells to express a novel receptor with tumor antigen specificity is currently being explored in myeloma. Chimeric antigen receptor gene-modified T-cells (CAR T-cells) have shown to be the most promising approach so far. CAR T-cells have shown to induce durable complete remissions in other advanced hematologic malignancies like acute lymphocytic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). With this background, significant efforts are underway to develop CAR-based therapies for MM. Currently, several antigen targets, including CD138, CD19, immunoglobulin kappa (Ig-Kappa) and B-cell maturation antigen (BCMA), are being used in clinical trials to treat myeloma patients. Some of these trials have shown promising results, especially in terms of response rates. However, the absence of a plateau is observed in most studies which correlates with the absence of durable remissions. Therefore, several potential limitations such as lack of effectiveness, off-tumor toxicities, and antigen loss or interference with soluble proteins could hamper the efficacy of CAR T-cells in myeloma. In this review, we will focus on clinical outcomes reported with CAR T-cells in myeloma, as well as on CAR T-cell limitations and how to overcome them with next generation of CAR T-cells.