PROTEOMIC PROFILING IDENTIFIES GRANZYME B INHIBITOR SERPIN B9 AS MEDIATOR OF RESISTANCE TO CAR T‐CELL AND BISPECIFIC ANTIBODY TREATMENT IN NODAL B‐CELL LYMPHOMA

Abstract

Introduction: Treatment options for relapsed or refractory (r/r) B-cell non-Hodgkin lymphomas (B-NHL) have broadened towards T-cell engaging therapies, including CD19-targeting chimeric antigen receptor T-cells (CD19-CAR) and bispecific antibodies (CD19-BsAb). Although CD19-CAR and CD19-BsAb induce durable responses in some r/r B-NHL patients, response remains heterogenous and a significant proportion of patients experience insufficient responses or relapse. A thorough understanding of tumor-intrinsic mechanisms of resistance is crucial for the clinical improvement of T-cell immunotherapy. Methods: Aiming to identify lymphoma cell-inherent mechanisms that impair response to T-cell engaging therapy, we quantified the in-vitro response of 46 B-NHL cell lines to 3rd generation CD19-CAR and CD19-BsAb. Response was measured using a high-throughput flow cytometry-based assay. In parallel, cell lines were subjected to proteomic profiling and protein abundance was regressed on in-vitro response in order to identify proteomic determinants of response. We further combined CD19-CAR treatment with clinically relevant drugs and assessed how killing of lymphoma cells and expansion of T-cells was altered. Results: Response to CD19-CAR and CD19-BsAb was highly heterogeneous across cell lines and B-NHL entities, but not significantly linked to their proliferation rate or CD19 expression. Integration of the proteomic profiles revealed that abundance of Serpin B9, endogenous inhibitor of T-cell effector molecule granzyme B, was associated with poor response to CD19-CAR and CD19-BsAb. This finding was validated in overexpression and knock-out models. Overexpression of SERPINB9 indeed attenuated response to CD19-CAR and CD19-BsAb, while its knock-out rendered cell lines more sensitive. The observed changes in susceptibility to T-cell-mediated cytotoxicity upon modulation of SERPINB9 expression were independent of T-cell donor and expression of T-cell activation and proliferation markers. We sought to improve T-cell-mediated cytotoxicity specifically in samples with high SERPINB9 expression and found that immune checkpoint inhibitors and lenalidomide enhanced expansion of CAR T-cells and increased killing of lymphoma cells. In addition, vorinostat and antibody-drug-conjugate polatuzumab vedotin specifically killed lymphoma cells without affecting T-cell expansion. However, the observed improvement in T-cell-mediated cytotoxicity was independent of SERPINB9 expression. Keywords: aggressive B-cell non-Hodgkin lymphoma, immunotherapy, indolent non-Hodgkin lymphoma No conflicts of interests pertinent to the abstract.