Abstract 2194: Inducible MyD88/CD40 (iMC) enhances CAR-T cell expansion and persistence by overcoming T cell exhaustion
Authors: Duong MyLinh T. , Mahendravada Aruna , Brandt Mary E. , Sharp Kelly L. , Foster Aaron E. , Bayle J. Henri 
Published: September 2020

Abstract
Background: Chimeric antigen receptor (CAR) T cell therapy targeting solid tumors is challenged by inhibitory signals in the tumor microenvironment that may lead to CAR-T exhaustion. Previously, we described a GoCAR platform that uses a rimiducid (Rim)-inducible cytoplasmic costimulatory switch, inducible MyD88/CD40 (iMC), to drive the proliferation, survival, cytokine release and anti-tumor efficacy of T cells expressing a 1st generation CAR. To further understand how iMC signaling improves CAR-T potency, we directly compared iMC-based costimulation to conventional costimulatory domains (i.e., CD28 and 4-1BB) against hematological and solid tumor models.
Methods: PBMCs from four donors were activated with anti-CD3/CD28 and transduced with retrovirus encoding 1st generation (CAR.ζ), 2nd generation (CAR.CD28.ζ CAR.4-1BB.ζ or GoCAR (iMC-2A-CAR.ζ) components targeting HER2, CD19 or GD2. Cocultures were established at a 1:1 ratio with antigen-expressing targets and cultures serially passaged weekly against fresh targets for up to six weeks. For in vivo efficacy evaluations NSG mice were engrafted s.c. with 1 × 106 HER2+ OE19-GFPffLuc esophageal carcinoma cells and challenged on day 4 with 5 × 106 CAR-T cells marked with RLuc each monitored by IVIS imaging.
Results: 1st generation CAR-T cells produced low levels of IL-2 and IFNγ after the first antigen stimulation and quickly expressed markers of exhaustion including PD-1. 2nd generation CAR-T cells proliferated in early passage but became phenotypically and functionally exhausted following a second stimulation. Conversely, GoCAR-T cells were capable of expanding and sustaining IL-2 production beyond five passages, but only when stimulated with Rim. Lower levels of PD-1, TIM-3, and LAG-3 expression were observed in Rim-treated GoCAR-T cells when compared to 1st gen, 2nd gen and GoCAR-T cells without iMC activation, and PD1 expression remained repressed for six weeks in Rim-treated GoCAR-T cells.
In NSG mice engrafted with HER2+ OE19 tumors, Rim treatment drove robust iMC-HER2.ζ CAR-T cell expansion compared to HER2.ζ, HER2.BB.ζ and HER2.28.ζ CAR-T cells over 5 weeks (p &lt; 0.05). iMC-HER2.ζ CAR-T cells significantly enhanced tumor killing with Rim activation relative to vehicle-control and HER2.ζ-expressing T cells days post T cell infusion (tumor radiance = 1.63 × 107 ± 1.7E7 vs 1.40 × 108 ± 5.27E7 vs 1.70 × 108 ± 8.65E7, respectively, p&lt;0.05). When rechallenged with tumor cells at day 35, persistent GoCAR-T cells continued to control tumor growth while 2nd generation CAR-T cells failed to control tumor growth. Furthermore, mice treated with iMC-HER2.ζ CAR-T cells and Rim produced high levels of proinflammatory cytokines including IFN-γ, GM-CSF, and IP-10 consistent with supporting T cell activation and proliferation.
Conclusions: iMC activation via Rim infusion provides on-demand control of CAR-T cell signaling to resist CAR-T cell exhaustion, enhance persistence, produce cytokines, and induce antitumor toxicity against solid tumors.
Citation Format: MyLinh T. Duong, Aruna Mahendravada, Mary E. Brandt, Kelly L. Sharp, Aaron E. Foster, J. Henri Bayle. Inducible MyD88/CD40 (iMC) enhances CAR-T cell expansion and persistence by overcoming T cell exhaustion [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 2194.