SAR442257, a CD38/CD28/CD3 trispecific antibody, potentiates CAR T‐cell activity against large B‐cell lymphoma

CART 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 including CAR T exhaustion. Understanding these mechanisms are likely to identify therapeutic avenues for improving outcomes. 13 rrLBCL tumors (7 CART naïve, 6 CART refractory), were subjected to scRNA-seq. Clustering was performed and frequency, clonal dominance, and expression of major cells and subclusters were compared. The effect of SAR442257, a CD38/CD28xCD3 trispecific antibody, was assessed in cytotoxicity assays and compared to control antibodies without CD38 or CD38/CD3/CD28 targeting regions. RL and HT cell lines were CRISPR modified to knock-out CD19 and isogenic WT used as target cells. CD19 CAR T were constructed from PBMCs of rrLBCL patients obtained at time of apheresis using a construct like axicabtagene ciloleucel. For cytotoxicity assays, cell lines were co-cultured at E:T ratios of 1:1 for 24 h or 48 h. scRNA-seq revealed that CART refractory rrLBCL tumors possessed 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 CART refractory tumors and enriched within CAR+CD8 T-cells detected. 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 CART naïve tumors. Single cell differential gene expression revealed significantly increased expression of LAG3, TIM3, and CD38 in CD8 T-cells from CART refractory compared to CART naïve tumors. We hypothesized that SAR442257 could boost the activity of CD19 CART-cells from rrLBCL patients through dual antigen targeting of CD19 and CD38 on lymphoma cells and by 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-h HT P-value = 3e-4, 24-h RL P-value = 1e-2) that persisted at 48 h. Addition of SAR442257 to CART-cells allowed killing of HT and RL CD19KO (CD19-/CD38+) (24-h HT P-value = 8e-7, 24-h RL P-value = 2e-8) at a level similar to the combination in WT. Addition of antibodies lacking CD38 or CD38/CD3/CD28-targeting regions did not boost cytotoxicity or rescue killing of CD19KO targets. We observed significant T-cell fratricide, which was beneficial or non-detrimental in light of increased lymphoma cell killing. The tumor microenvironment of CART refractory rrLBCL is enriched in clonally expanded and terminally exhausted CD8 T-cells expressing CD38. The CD38/CD28xCD3 trispecific antibody SAR442257 boosted CART-cell activity through recognition of CD38 on the tumor, costimulation of CART-cells, and induced fratricide of CD38+ T-cells. In addition, SAR442257 allowed CD19 CART-cells to kill CD19-/CD38+ LBCL cells. Encore Abstract - previously submitted to EHA 2023 The research was funded by: Sanofi Keywords: Aggressive B-cell non-Hodgkin lymphoma, Bioinformatics, Cellular therapies, Computational and Systems Biology Conflicts of interests pertinent to the abstract. P. Kim Employment or leadership position: Sanofi K. Bisht Employment or leadership position: Sanofi H. Van De Velde Employment or leadership position: Sanofi H. Wang Employment or leadership position: Sanofi