P1450: autologous stem cell transplantation induces higher level of exhausted t-cells in dlbcl patients before leukapheresis for car-t cell therapy.

Background: Chimeric antigen receptor T-cells (CAR-T) are an highly effective therapy in relapse/refractory DLBCL, PMBCL and ALL, but almost half of treated patients (pts) still relapse. CAR-T cells generated from T-lymphocytes (T-Ly) enriched for early lineage T cells has shown durable persistence and replicative potential both in animal and in vivo models, while CAR-T cells from “exhausted” T-Ly have been related with poorer response. In the prospective BioCART BS study, we assessed the T-cell repertoire before the Ly-apheresis in DLBCL patients for evaluating the impact of previous treatments and efficacy of CAR-T therapy. Aims: To evaluate T-cells repertoire before the Ly-apheresis in high risk DLBCL pts. Methods: Between April and December 2021, a total of 11 DLBCL pts underwent Ly-apheresis for CAR-T manufacturing. Since cryopreservation of the apheresed Ly is allowed for Tisa-cel, DLBCL pts with poor prognostic risk factor (refractory to first line of treatment; PET positivity before Autologous Stem Cell Transplantation [ASCT]; first complete response less than 12 months) were enrolled in an early/“pre-emptive” Ly-apheresis program, scheduling leukapheresis as soon as possible, with the aim to collect more “fit” Ly. Combinations of monoclonal antibodies directed against CD45RA, CCR7, CD3, CD4, and CD8 were used by Flow Cytometry to evaluate the following CD4+/CD8+ T-ly subsets: T-naïve (CD45RA+CCR7+); central memory (CM, CD45RA-CCR7+); effector memory (EM, CD45RA-CCR7-); terminally differentiated (TD; CD45RA+CCR7-). For evaluating CAR-T cells expansion, pts’ cells were tested with a specific biotinilated CD19 CAR reagent followed by incubation with an anti-biotin PE conjugated MoAb. The time points were: at day +1, +3, +7, +10, +14, +21 after CAR-T cells infusion, and then monthly until 1 year or at any time for relapse onset. All stained samples were acquired on a Canto II (BD Bioscience) flow cytometer and analyzed using DIVA software version 8.0.2. Continuous and categorical variables were compared using an unpaired T-test, Mann-Whitney U test, or Chi-square test, as appropriate. Results: Table 1 shows the main patients’ characteristics. 5 out of the 11 (45%) pts performed Ly-apheresis before ASCT. At the time of Ly-apheresis, no significative differences were observed between pts who previously underwent ASCT and those who did not, except for age. ASCT induced a more “exhausted” T-cells repertoire. Indeed, pts who previously underwent ASCT had lower CD4+/CD8+ ratio (p 0.04) (Fig 1C) as well as lower levels of CD4+ Naïve T cells, both in terms of absolute count (p 0.05) and as percentage of CD4+ T cells (p 0, 01), and lower prevalence of CD8+ Naïve T cells (p 0.03) (Fig 1A). Moreover they also presented higher prevalence of CD4+ EM T cells (p 0.01) and of CD4+ TD T-cells (p 0.05), as well as higher level of both CD8+ CM (p 0.03) and TD (p 0.05) T cells (Fig 1B). 3 out of the 6 pts who underwent ASCT before Ly-apheresis and 2 of the 5 who did not, sent Ly for manufacturing. To date, no differences were apparently observed on CAR-T cells expansion comparing the two groups, but the number of pts analyzed is too small to make any consideration (Fig 1D for pts with longer follow up). - Ly-apheresis PRE ASCT Ly-apheresis POST ASCT p value Number 5 6 Age 51 (29-60) 67 (57-70) 0.02 Disease status @Ly-apheresis 1 2 0.08 Refractory 0 3 Relapse 4 1 Partial response Image:Summary/Conclusion: ASCT before Ly-apheresis may result in a higher prevalence of “exhausted” T-cells. In DLBCL pts primary refractory or at high risk of relapse after second line of treatment, timely pre-ASCT Ly-apheresis would be considered.