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Abstract Introduction: Dual targeted lentiviral anti-CD20/anti-CD19 (LV20.19) CAR T cell therapy has demonstrated high overall response rates and durable efficacy in patients (pts) with relapsed, refractory B-cell NHL (Shah NN. Nature Med 2020 and Shah NN JCO 2025). As part of a multi-cohort optimization study, we evaluated both length of manufacturing and impact of cryopreservation on clinical outcomes. We report final results of pts with DLBCL/FL treated on varying arms (8 vs 12 day and fresh vs cryopreserved/thawed product). Methods: We conducted a Phase 1/2 multi-cohort prospective trial (NCT04186520) evaluating LV20.19 CAR T-cells at a fixed dose of 2.5x10e6 cells/kg for pts with R/R B-cell non-Hodgkin Lymphoma. CAR T-cells were manufactured on site utilizing the CliniMACS Prodigy device with an 8–12-day manufacturing (MF) process depending on assigned cohort. This analysis is limited to CAR naïve pts with DLBCL and FL administered either fresh CAR T-cells on the day of harvest versus a CAR T-cell product after cryopreservation/thawing. To evaluate the impact of cryopreservation on the final product, we performed seahorse metabolic profiling on 24 patients comparing oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and ATP production across apheresed T cells, fresh CAR-T cells, and post-thaw CAR-T cells. Single Cell RNA sequencing was done on 6 patients to match fresh and cryopreserved CAR-T products to assess transcriptional changes. Results: In total, 42 pts with DLBCL or FL received LV20.19 CAR T-cells. DLBCL (including transformed DLBCL and Richters Transformation) was the diagnosis in 84% of pts. All pts achieved the target cell dose. Pts were assigned as per their manufacturing arm. There were 10 pts in the fresh 8-day arm, 9 pts in the fresh 12-day arm, and 23 pts in the frozen 8-day arm. There was no difference in mean age, sex, and lines of prior therapy between the fresh and cryopreserved/thawed cohorts. The ORR/CR for 8-day fresh vs 12-day fresh was 90%/70% vs 78%/22%, p=0.58 (ORR), and for 8-day fresh vs 8-day frozen was 90%/70% vs 74%/57%, p=0.40 (ORR). The 1-year PFS was 40% for 8-day fresh vs 22% for 12-day fresh vs 55% for 8-day frozen cohorts (p=0.08). Similarly, 1-year OS was 70% for 8-day fresh vs 56% for 12-day fresh vs 76% for 8-day frozen (p=0.18). Among treated pts any grade CRS occurred in 79%, with 1 pt experiencing Grade 3 CRS in the 8-day fresh cohort. There was a trend towards more frequent CRS in 8-day fresh products vs 8-day frozen (100% vs 74%, p=0.09). In total, 24% of pts experience any grade ICANS but only 2 pts (5%) had grade 3 ICANS with no difference among the cohorts. Correlative analyses performed on fresh and post-thaw CAR-T cells demonstrated significantly reduced OCR, ECAR, mitochondrial and glycolytic ATP production, maximal respiratory capacity, and spare respiratory capacity in the cryopreserved cohort (p<0.01). Transcriptomic analysis revealed downregulation of key cytotoxic and activation markers (IFNG, GZMB, GZMA, PRF1, NKG7), along with upregulation of genes associated with senescence and apoptosis in cryopreserved samples (p<0.05). Importantly, no major metabolic differences were observed between apheresed T cells and fresh CAR-T cells, indicating that cryopreservation—rather than manufacturing—drives the observed functional decline observed immediately post-thaw. Differentiation immunophenotyping of final CAR-T cell products showed that both CD4 and CD8 T CAR-T cell subsets were more terminally differentiated in 12-day products versus 8-day products (p<0.005). Conclusions: Through this multi-cohort optimization of LV20.19 CAR T cell study we did not see clinical differences among fresh versus cryopreserved treated pts; however, there were trends towards worse outcomes in pts with 12-day MF vs 8-day MF. ORR/CR were highest for 8-day fresh pts at 90%/70% although this did come with slightly higher rates of low-grade CRS. Correlative studies demonstrated diminished metabolism in LV20.19 CAR T cells post-cryopreservation with upregulation of genes associated with senescence and apoptosis. Immunophenotyping revealed improved differentiation profile of shorter manufactured cells. Taken together, these data suggest a short 8-day MF process with a fresh infusion is optimal for LV20.19 CAR T cell therapy, and this platform was subsequently utilized for all future arms on this clinical trial.