Compared to the standard 7-day method, the 3-day approach resulted in expression of genes associated with a more stem-like phenotype while reducing manufacturing time and cost, and may provide a practical alternative for decentralized CAR T-cell manufacturing, particularly in resource-limited settings.
Abstract
Chimeric antigen receptor (CAR) T-cell therapy has transformed the treatment landscape for many hematological malignancies. However, high relapse rates and limited accessibility remain significant challenges. We developed a 3-day streamlined process to address these limitations. T-cells were isolated from whole blood collected from healthy donors via an automated density gradient separation that included a T-isopure™ antibody cocktail, which isolates T-cells through negative selection. T-cells were activated then transduced with a lentiviral vector encoding a trispecific CAR. T-cells were cultured in G-Rex vessels and harvested at day 3 or day 7 for analysis. CAR expression and T-cell phenotype were assessed by flow cytometry and gene expression analysis. Functional activity was evaluated by measuring cytotoxicity and cytokine secretion following co-culture with target cell lines. The T-isopure isolation enriched CD3+ T-cells in whole blood from 18.9% to 88.5% of CD45+ cells, with a mean recovery of 40.6%. RBCs were depleted with ≥ 99% efficiency, with monocytes and NK cells comprising the bulk of remaining CD45+ cells. The 3-day manufacturing process produced T-cells with > 95% viability, 53% transduction efficiency, and vector copy number < 3 copies/cell. Phenotypic analysis revealed a high proportion of stem/central memory T-cells at both timepoints, with no significant differences observed. Cytotoxicity assays demonstrated strong and sustained killing of NALM6 tumor cells, comparable between both products. Gene expression profiling indicated that day 3 products were less differentiated, exhibiting a memory-like phenotype and reduced inflammatory signaling, further supported by protein analysis of culture supernatants. This study establishes a rapid, GMP-compliant method for manufacturing polyfunctional, CAR T-cells directly from whole blood. The workflow outlined here achieved a potent, phenotypically favorable CAR T-cell product without compromising viability or cytotoxic function. Compared to the standard 7-day method, the 3-day approach resulted in expression of genes associated with a more stem-like phenotype while reducing manufacturing time and cost. This method may provide a practical alternative for decentralized CAR T-cell manufacturing, particularly in resource-limited settings.
Backgrounds: Chimeric antigen receptor (CAR) T cell therapy has recently exhibited promise as cancer treatment, especiallyfor hematologic malignancies. Currently, all of the FDA-approved CAR T therapies are autologous in nature. This presentsas a limitation since generating CAR T cell using the patient’s own blood leads to higher costs and a 3–4 week productiontime. To overcome this, allogeneic CAR T cell-therapy is being explored.
Objective: In this study, CD45RA depletion was used to generate allogeneic anti-BCMA CAR TT cells. This approach aimsto utilize the CD45RA-negative fraction containing memory T cells which has lower alloreactivity and reduced risk to causegraft-versus-host disease (GVHD).
Methods: The CD45RA-negative T cells were obtained through magnetic separation of CD45RA-labeled PBMCs. Thisprocess yielded the flow-through containing the unlabeled CD45RA-negative fraction which was then used to produceCAR T cells through lentiviral transduction. The generated CD45RA-negative anti-BCMA CAR T cells were compared tonondepleted anti-BCMA CAR T cells according to transduction efficiency, cell expansion, memory phenotype and cellsubset. Cytotoxic function was also assessed through co-culture assays.
Results: Results show higher transduction efficiency and MFI of CD45RA-negative CAR T cells on days 4 and 11,respectively with no difference in expansion. They also feature more CD4+ cell subset and Tem memory phenotype.
Results show no statistically significant difference between non-depleted and depleted anti-BCMA CAR T cells in in vitroco-culture assay.
Conclusion: The data demonstrate CD45RA depletion as a feasible approach to generating allogeneic anti-BCMA CART cells.
Arianwen Rollan, Koramit Suppipa, S. Tawinwung et al.· Journal of Medical Bioscienc...· 0 citations
These findings suggest CAR spatial organization correlates with functional states and demonstrate the power of integrating advanced flow and imaging cytometry to resolve CAR T cell phenotypes and spatial dynamics with high precision.
G. Baracho, Aruna Ayer, Joseph Cantor et al.· Journal of Immunology· 0 citations
Multiple myeloma (MM) is an aggressive blood cancer arising from plasma cells. B cell maturation antigen (BCMA)-targeted chimeric antigen receptor T cell (α-BCMA-CAR-T) immunotherapies currently provide life-saving treatment for MM patients. Unfortunately, severe toxicities along with the high cost and complexity of autologous CAR-T manufacturing remain important limitations. Novel research is underway to use CAR-expressing natural killer (NK) cells as an allogeneic CAR-T alternative, but studies have yet to evaluate long-term CAR-NK efficacy against MM.
NK cells were isolated, expanded via feeder-cell stimulation, and engineered to express α-BCMA-CAR and IL-15 co-expression. The functional characteristics of α-BCMA-CAR-IL15-expressing NK cells were initially assessed in vitro, followed by long-term testing in a luciferase-expressing MM-xenograft mouse model to examine the persistence and therapeutic effect of α-BCMA-CAR-IL15 NK.
α-BCMA-CAR NK cells have enhanced cytokine production and cytotoxicity against BCMA-high MM cells compared to untransduced NK cells, with IL-15 co-expression required for CAR-NK persistence. When injected into NSG mice, both α-BCMA-CAR and IL-15 expression were required for persistent restriction of MM growth. Despite near complete and sustained elimination of MM in hematopoietic tissues, long-term assessment of mice treated with α-BCMA-CAR-IL15 NK cells revealed the emergence of extramedullary disease (EMD) in the form of BCMA-positive MM plasmacytomas.
This study showcases α-BCMA-CAR-IL15 NK cell therapy as a potent anti-MM therapeutic, achieving sustained MM elimination from the bone marrow and greatly extending survival in a MM-xenograft model. However, α-BCMA-CAR-IL15 NK cells appeared ineffective at eliminating extramedullary disease. By demonstrating the strengths and weaknesses of α-BCMA-CAR-IL15 cells, our study provides a valuable pre-clinical MM model for studying and developing interventions for aggressive MM-EMD.
Canadian Institutes of Health Research
Translational and Interventional Immunology (TI)
Seung-Hwan Lee, Shelby Kaczmarek, Safa Ghaziasgar et al.· Journal of Immunology· 0 citations
Ex vivo-manufactured chimeric antigen receptor (CAR) T cells targeting CD19 have transformed treatment strategies for patients with B cell malignancies and shown promising results in autoimmune disease. Yet despite their success, significant barriers remain that impede patient access to these therapies, including their complex manufacturing and the need for lymphodepleting chemotherapy. Moreover, many patients who receive ex vivo-manufactured CD19 CAR T cells do not achieve durable responses, suggesting that the expansion and persistence of CAR T cells may need improvement, and/or that strategies targeting alternative antigens may be needed for patients who are refractory to or relapse following treatment with CD19-directed therapies. Umoja is developing a surface-engineered lentiviral vector (LVV) platform capable of generating CAR T cells in vivo without lymphodepletion. UB-VV400 delivers a payload comprised of a fully human anti-CD22 CAR and the rapamycin-activated cytokine receptor (RACR), which enable preferential expansion and enrichment of CAR T cells in response to rapamycin. Here, we describe preclinical studies demonstrating that UB-VV400 generates anti-CD22 CAR T cells that kill CD22-expressing B cells and tumor cells in vitro and in vivo, and that engagement of RACR results in the selective expansion and enrichment of CAR T cells in vivo leading to complete tumor clearance and B cell depletion.
Travis J. Friesen, Alyssa Sheih, Nikole Perdue et al.· Molecular Therapy· 0 citations
Chimeric antigen receptor (CAR)-T cell therapy is effective for hematologic malignancies; however, the response of solid tumors is limited because of the immunosuppressive tumor microenvironment, antigen heterogeneity, and lack of persistence of transferred T cells. To overcome these challenges, CAR-T cells expressing interleukin-7 and CC chemokine ligand 19 (7 × 19 CAR-T) were generated to achieve potent antitumor efficacy through the recruitment and proliferation of CAR-T cells and endogenous immune cells. To elucidate the underlying mechanism of 7 × 19 CAR-T cells against solid tumors, we analyzed the cellular composition and gene expression profiles of host immune cells following CAR-T cell infusion in a murine solid tumor model. Antihuman CD20 7 × 19 CAR-T cells were prepared using Thy 1.1 congenic mice and administered to C57BL/6N mice bearing subcutaneous MC38 tumors expressing human CD20. The tumors were harvested 4 days postinfusion to capture early immune responses before overt tumor regression. CD90.1- recipient immune cells were subjected to flow cytometry analysis, and transcriptomic changes were determined using AmpliSeq and single-cell RNA seq. An increase in recipient CD8 + T cells and macrophages was observed in the tumor of mice treated with 7 × 19 CAR-T cells, but not with conventional CAR-T. The expression of chemokines and genes associated with the inflammatory pathway was upregulated only in recipient immune cells of the 7 × 19 CAR-T-treated mice. Single-cell RNA-seq analysis revealed upregulation of pro-inflammatory genes and chemokines in the dendritic cell and monocyte/macrophage populations. These results indicate that 7 × 19 CAR-T cells initiate the early recruitment and activation of host immune cells, which contributes to their superior antitumor activity compared with conventional CAR-T cells.
Yo Muraki, Yuumi Okuzono, Rina Kurisu et al.· PLoS ONE· 0 citations
On the basis that T-cell acute lymphoblastic leukemia (T-ALL) cells overexpress IL7 receptor (IL7R), which promotes resistance to chemotherapy and disease relapse, here we develop IL7Rα (CD127)–targeted chimeric antigen receptor (CAR) T cells with low- and high-affinity single-chain variable fragments. We establish the antitumor efficacy of CD127 CAR against T-ALL cells in vitro, in female mouse models of T-ALL, and against blasts from patients with T-ALL using the patients’ own T cells transduced with CD127 CAR. Antitumor efficacy is higher with low-affinity CAR T cells than high-affinity CAR T cells, albeit with fratricide of CAR T cells following eradication of CD127-overexpressing blasts. CRISPR-Cas9 knockout of CD127 eliminates fratricide at the risk of prolonged lymphopenia in vivo. To overcome fratricide, we investigate short-term ( < 7 days) co-culture with or without dasatinib, a tyrosine kinase inhibitor, versus a natural selection method (10 days) and demonstrate that co-culturing with dasatinib facilitates higher CAR T-cell yield, improved fitness, and preserved functionality. In vivo, dasatinib can be used to temporarily and reversibly suppress CAR T-cell activity. With this supporting translational data, we are initiating a trial with low-affinity CD127 CAR T cells for adult and pediatric patients with relapsed or refractory T-ALL. Chimeric antigen receptor (CAR) T cell therapies for T-cell acute lymphoblastic leukemia are explored in the clinic. Here the authors propose IL7-Receptor (IL7R/CD127) as an alternative target for T-ALL, showing the antitumor efficacy of IL7Rα/CD127 CAR-T cells against T-ALL in preclinical models.
H. Hocine, Undrakh Ganbaatar, Alfredo Amador-Molina et al.· Nature Communications· 0 citations