Introduction: Conventional CD19-directed CAR-T manufacturing requires 7–14 days, potentially delaying treatment and promoting T-cell exhaustion. In contrast, next-day CAR-T platforms enable rapid production and preserve a less-differentiated phenotype, yet their clinical impact relative to conventional CAR-T remains undefined.

Methods: We developed a next-day manufacturing platform (InstanCAR-T) and compared it with conventional CAR-T (ConvenCAR-T) across three domains: (1) In vitro assays assessing T-cell differentiation and exhaustion following stimulation; (2) B-ALL murine xenograft models evaluating antitumor efficacy, CAR-T expansion, and persistence; (3) Clinical comparison of a Phase I trial of InstanCAR-T in R/R B-ALL (NCT06209671), administered at two fixed total doses: 1×10⁷ (INS DL-1) and 2×10⁷ (INS DL-2) T cells. Safety, pharmacokinetics, preliminary efficacy, and high-dimensional immune profiling (single-cell RNA sequencing and 40-marker CyTOF mass cytometry) were analyzed and compared to a matched cohort receiving ConvenCAR-T in a separate Phase I trial (NCT05309213), dosed at 5×10⁴ (IM DL-1), 1×10⁵ (IM DL-2), and 3×10⁵ (IM DL-3) CAR-T cells/kg.

Results: InstanCAR-T was successfully manufactured within 2 days, in contrast to the 7–12 days required for ConvenCAR-T. Phenotypic analyses demonstrated that InstanCAR-T retained a significantly higher proportion of naïve and stem cell memory T cells (CCR7⁺CD45RA⁺) compared to ConvenCAR-T (57.3% vs. 18.2%). Functionally, InstanCAR-T exhibited enhanced proliferation kinetics upon repeated tumor antigen stimulation and demonstrated greater CAR expression density, which was associated with improved persistence.

In B-ALL murine xenograft models, InstanCAR-T achieved durable tumor control, with superior in vivo expansion and persistence. Between Day 15 and 24, tumor recurrence occurred in 50% (3/6) of mice treated with ConvenCAR-T, whereas no recurrence was observed in the InstanCAR-T group. The area under the curve (AUC) for CAR-T cell expansion over time was higher in the InstanCAR-T group.

In two parallel Phase I trials, 10 patients received InstanCAR-T and 9 received ConvenCAR-T. By Day 28 post-infusion, the incidence of cytokine release syndrome (CRS) was comparable between groups (p = 1.0): Grade 2 CRS occurred in 40.0% (InstanCAR-T) vs. 44.4% (ConvenCAR-T); Grade 3 CRS occurred in 10.0% vs. 11.1%, respectively. ICANS incidence was also similar (p = 0.582), with no Grade ≥3 events reported.Efficacy at Day 28 was comparable: objective response rate (ORR) was 90.0% for InstanCAR-T and 88.8% for ConvenCAR-T.

High-dimensional immune profiling via CyTOF revealed a sustained increase in T naive and TCM subsets from Day 14 to Day 60 in both peripheral blood (PB) and bone marrow (BM), alongside reductions in exhaustion markers PD-1, TIGIT, and TIM-3. CD127⁺CAR-T cells, indicative of enhanced self-renewal and differentiation capacity, increased over time. CD27 expression initially rose but declined significantly by Day 60, suggesting transient in vivo activation with retained memory features.

Single-cell RNA sequencing of CD8⁺ and CD4⁺ T cells from InstanCAR-T (Day 0) and PBMCs (Day 14) revealed that CD8⁺ T cells followed a continuous differentiation trajectory bifurcating into two terminal states, characterized by differential expression of memory-associated genes including CCR7, IL7R, LEF1, SELL, and TCF7, supporting preserved stem-like features.

Conclusion: InstanCAR-T is a next-day, CD19-targeted CAR-T product that demonstrates potent anti-leukemic activity with favorable safety and efficacy profiles. Integrated phenotypic, functional, and high-dimensional immune profiling highlights the enrichment of stem-like T-cell subsets, reduced exhaustion, and sustained in vivo persistence. These findings support the potential of InstanCAR-T to improve clinical outcomes for patients with relapsed/refractory B-ALL by delivering rapid, robust, and durable CAR-T cell therapy.

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2025
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