CAR-T therapy targeting B-cell antigens is effective for relapsed hematologic malignancies, but antigen escape remains a challenge. We previously showed dual CD19/CD20-4-1BB CAR-T therapy was highly effective (Nat Med, 2020). We further developed trispecific CAR-T cells (CD19/CD20/CD22) incorporating ICOS signaling (Sci Transl Med, 2021). Despite promising preclinical activity, trispecific-ICOS CAR-T cells failed to expand and showed no efficacy in a Phase I trial (NCT05094206; ASH 2024). Here, we investigated mechanisms underlying this failed response.
Based on our IRB approved protocol and using patient-derived PBMCs and leftover samples from treated patients, we generated tested bispecific-4-1BB, bispecific-ICOS and trispecific-ICOS CAR-T cells.
NSG mice receiving bispecific- or trispecific-ICOS CAR-T cells had worse survival than those with 4-1BB CAR-T cells. In vivo, ICOS CAR-T cells failed to expand and showed an exhaustion phenotype. When co-culture with Raji cells, ICOS CAR-T cells significantly enhanced surface-FMC63 loss compared to 4-1BB counterparts. Surface-FMC63 expression was mostly recovered at 40 hours on 4-1BB CAR-T cells, but not on ICOS CAR-T cells. Since trogocytosis can mediate CAR internalization and antigen loss on tumor, we examined CD19 transfer. ICOS CAR-T cells displayed higher and more sustained CD19 uptake and fratricide reflected by apoptosis than 4-1BB cells. Consistently, when co-culture with patient autologous B cells, ICOS CAR-T cells again showed greater CAR loss and apoptosis. Extended co-culture mimicking chronic antigen stimulation revealed expansion of 4-1BB but not ICOS CAR-T cells, the latter showing reduced proliferation (Ki67) and elevated exhaustion markers.
In conclusion, the ICOS domain promotes trogocytosis and fratricide, explaining the poor persistence and function of ICOS CAR-T cells observed in our trial. Thus, our findings raise significant caution for using CAR constructs that rely on ICOS for CAR activation.
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Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)
Yongxia Wu, Allison Pugel, Katie A. Palen et al.· Journal of Immunology· 0 citations
Disease recurrence is the main cause of treatment failure after CD19-directed CAR T cells, often due to CD19 antigen loss, stability and/or coverage. To overcome single-antigen escape, we evaluated a trispecific CAR targeting CD19, CD20, and CD22 with OX40 co-stimulatory domain. Preclinical studies demonstrated potent, antigen-specific cytotoxicity in in vitro and in vivo lymphoma models. We then conducted a first-in-human phase I trial in patients with relapsed/refractory B-cell malignancies. Sixteen patients received infusions at a median vein-to-vein time of 7 days, at doses of 0.5-2×10⁶ cells/kg. No severe cytokine release syndrome nor neurotoxicity occurred. Overall response rate was 50%, including complete responses in 83% of lymphoma patients. One-year overall survival rate was 61%, with durable remissions observed in lymphoma. CAR T expansion did not correlate with dose or response. T-cell exhaustion in apheresis cells correlated with progressive disease. Trispecific CAR T cells are safe and potentially active in lymphoma.
S. Vasu, N. Denlinger, No-Joon Song et al.· Blood Cancer Discovery· 0 citations
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.
Isabella Vignola, M. Procházková, L. Shao et al.· Journal of Translational Med...· 1 citation