Abstract 5641: CD28 expression on multiple myeloma cells enhances the cytotoxic activity of CD38/CD28xCD3 trispecific T cell engager

SAR442257 (CD38/CD28xCD3) is a trispecific T cell engager designed for the treatment of multiple myeloma (MM). Its cytotoxic activity and anti-tumor efficacy is dependent on the expression of CD38 by malignant plasma cells and the recruitment and activation of T cells via CD3 and CD28. Here we show that MM cells also express CD28 on their membrane and investigate how this expression impacts the cytotoxic activity of the trispecific antibody. Using CRISPR, CD28 knock-out (KO) models were generated from two MM cell lines that have different receptor densities of CD38 and CD28: KMS-11 (CD38 ~3 000/cell; CD28 ~35 000/cell) and RPMI8226 (CD38 ~129 000/cell; CD28 ~84 000/cell). T-cell-mediated cytotoxicity was evaluated in vitro using either effector CD8+ T cells or total PBMC isolated from healthy donors. Binding of SAR442257 to tumor cells in the presence of saturating concentrations of anti-CD38 antibodies was evaluated by flow cytometry using APC-labeled SAR442257 and FITC-labeled anti-CD38 antibodies. In vitro killing assays showed that SAR442257 is active on both CD38 high and CD38 low MM cell lines (EC50 in the pM range for WT cell lines; Table 1). Following CD28 KO, SAR442257 remained active but displayed reduced cytotoxic activity as compared to WT CD38+ CD28+ cells. The reduced cytotoxic activity was noticeable in both CD38 low and CD38 high MM models suggesting that the expression of CD28 by MM cells contributes to the cytotoxic activity of the trispecific antibody. We further show that when other anti-CD38 antibodies are bound to CD38, binding of SAR442257 to tumor cells and T-cell mediated cytotoxicity are rescued by CD28 as evidenced by loss of binding and cytotoxic activity in CD28KO cells. Overall, these data show that SAR442257 is active on both CD38 high and low MM models and that CD28 expressed on MM tumor cells can be directly targeted by SAR442257, enhancing its cytotoxicity and allowing it to bind MM cells when CD38 is occupied by other anti-CD38 antibodies Table 1.T cell mediated killing assays. EC50rel, Relative EC50; CI, Confidence interval.EC50rel (pM) [±CI95%]KMS-11RPMI8226WTCD28KOWTCD28KOEffector CD8+ T cells4.1 [2.9 ; 5.9]30.2 [16.9 ; 54.0]0.2 [0.1 ; 0.3]0.5 [0.3 ; 0.8]Total PBMC26.0 [7.8 ; 87]>250032.0 [14.8 ; 69.2]304.0 [118.0 ; 787.0] Citation Format: Nizar El-Murr, Christophe Henry, Elisa Francesconi, Florence Attenot, Angela Virone-Oddos, Laurent Vidard, Lan Wu, Zhi-Yong Yang, Marielle Chiron. CD28 expression on multiple myeloma cells enhances the cytotoxic activity of CD38/CD28xCD3 trispecific T cell engager [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5641.