P1366: sar442257, a cd38/cd28/cd3 trispecific antibody, potentiates car t-cell activity against large b-cell lymphoma

Topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Research Background: CAR T cell therapy has significantly improved outcomes of patients with relapsed/refractory large B cell lymphoma (rrLBCL), but many do not experience long term benefit. The mechanisms of failure are multifaceted and includes CAR T-cell exhaustion. Understanding these mechanisms are likely to identify rational therapeutic avenues for improving outcomes. Aims: 1.To understand the impact of the rrLBCL immune microenvironment on clinical failure to CAR T therapy 2.Investigate targeting of CD38 with a novel CD38 T cell engaging trispecific antibody in combination with CD19 CAR T-cells Methods:Discovery analysis: 13 rrLBCL tumors, including 7 CAR T naïve and 6 CAR T refractory tumors, were subjected to scRNA-seq. Clustering was performed and frequency, clonal dominance, and expression profiles of major cell types and subclusters were compared between groups. Functional experiments: The effect of SAR442257 was assessed in cytotoxicity assays and compared to control antibodies lacking the CD38 or CD38/CD3/CD28 targeting regions. RL and HT lymphoma cell lines were CRISPR modified to knock-out CD19 and isogenic WT clones used as target cells. CD19 CAR T-cells were constructed from PBMCs of rrLBCL patients obtained at the time of apheresis using a construct like axicabtagene ciloleucel (axi-cel). For cytotoxicity assays, cell lines were co-cultured at E:T ratios of 1:1 for 24h or 48h. Results: scRNA-seq revealed that CAR T refractory rrLBCL tumors possessed significantly higher fractions of terminally exhausted LAG3+TIM3+CD38+ CD8 T-cells (CD8TEX) with high expression of T-cell dysfunction and TEX signatures compared to CAR T naïve tumors. CD8TEX were most frequent in CAR T refractory tumors and enriched within CAR+CD8 T-cells detected within all CAR T refractory tumors. We have previously identified similar cells enriched in axi-cel infusion products of rrLBCL patients failing to response (Deng et al., Nat. Med. 2020). TCR clonotype analysis revealed highly expanded T-cell clones within the CD8TEX cluster, significantly increased clonal dominance, and reduced clonal diversity of CD8TEX cells in CAR T refractory compared to CAR T naïve tumors. Single cell differential gene expression revealed significantly increased expression of LAG3, TIM3, and CD38 in CD8 T-cells from CAR T refractory compared to CAR T naïve tumors. We hypothesized that SAR442257, a CD38/CD28xCD3 trispecific antibody, could boost the activity of CD19 CAR T-cells from rrLBCL patients in part by allowing for dual antigen targeting of CD19 and CD38 on lymphoma cells and by inducing fratricide of CD38-expressing CD8TEX cells. Cytotoxicity assays showed that SAR442257 significantly increased the killing of CAR T-cells against HT and RL WT (CD19+/CD38+) cells (24-hr HT P-value=3e-4, 24-hr RL P-value=1e-2) which persisted at 48 hours. Addition of SAR442257 to CAR T-cells allowed killing of HT and RL CD19KO (CD19-/CD38+) (24-hr HT P-value=8e-7, 24-hr RL P-value=2e-8) at a level similar to the combination in WT cells. Addition of antibodies lacking CD38 or CD38/CD3/CD28-targeting regions did not boost CAR T cytotoxicity or rescue killing of CD19KO targets. We observed significant T-cell fratricide, which was beneficial or non-detrimental in light of significantly increased lymphoma cell killing. Summary/Conclusion: The tumor microenvironment of CAR T refractory rrLBCL is enriched in clonally expanded and terminally exhausted CD8 T-cells expressing CD38. The CD38/CD28xCD3 trispecific antibody SAR442257 boosted CAR T-cell activity through recognition of CD38 on the tumor, costimulation of CAR T-cells, and induced fratricide of CD38+ T-cells; resulting in superior tumor cell killing. In addition, SAR442257 allowed CD19 CAR T-cells to kill CD19-/CD38+ LBCL cells. Keywords: Diffuse large B cell lymphoma, Cellular therapy, Lymphoma therapy, CAR-T