Abstract 3745: Dual-switch TCR: A two-ligand system to control PRAME TCR-modified T cell proliferation and death using inducible MyD88/CD40 and caspase-9
Authors: Pentcheva-Hoang Tsvetelina , Rodriguez Tania , Torres David , Korngold Ana , Crisostomo Jeannette , Duong MyLinh T. , Collinson-Pautz Matthew , Bayle J. Henri , Heemskerk Mirjam HM , Falkenburg J.H. Frederik , Moseley Annemarie , Slawin Kevin M. , Spencer David , Foster Aaron E. 
Published: July 2017

T cells engineered to express the α and β chains of antigen-specific T cell receptors (TCRs) have shown promise as a cancer immunotherapy treatment; however, durable responses have been limited by poor persistence of gene-modified T cells. Additionally, severe toxicities, including patient deaths, have occurred upon infusion of large numbers of TCR-modified T cells. To enhance T cell persistence while providing a safeguard against life-threatening toxicity, we developed a dual-switch αβ TCR platform that uses a rapamycin (Rap)-induced caspase-9 (iRC9) together with a rimiducid (Rim)-controlled activation switch, inducible MyD88/CD40 (iMC).

The αβ TCR sequence derived from an HLA-A2-restricted, PRAME-specific T cell clone was synthesized and placed in-frame with iMC, comprising signaling domains from MyD88 and CD40 fused to tandem Rim-binding mutant FKBP12v36 domains to generate the iMC-PRAME TCR. Caspase-9 was fused to FRB and wild-type FKBP12 domains and cloned in-frame with a selectable marker, truncated CD19 (ΔCD19), to generate iRC9-ΔCD19 retrovirus. All modules were separated by 2A polypeptide, bond-skipping sequences. Activated human T cells were dual-transduced with iMC-PRAME TCR and iRC9-ΔCD19 viruses and subsequently enriched for CD19 expression using magnetic selection. iMC and iRC9 were activated by exposing transduced T cells to 10 nM Rim or Rap, respectively. Proliferation, cytokine production and cytotoxicity of TCR-modified T cells were assessed in co-culture assays with U266 (myeloma) and THP-1 (AML) cells in the presence or absence of inducible ligands.

T cells transduced with iMC-PRAME TCR and iRC9-ΔCD19 showed efficient and stable expression for TCR and ΔCD19 post-CD19 selection (82±9% CD3+Vβ1+, 96±2% CD3+CD19+). In coculture assays, dual-switch PRAME TCR-modified T cells demonstrated specific lysis of HLA-A2+PRAME+ THP-1 and U266 tumor cells compared to an irrelevant TCR (CMVpp65-specific)-modified T cells with or without iMC activation. However, Rim exposure induced a 42-fold induction of IL-2 (9±0.3 versus 385±180 pg/ml IL-2) and resulted in 13-fold greater expansion of TCR-modified T cells. The expression of iRC9 did not interfere with TCR function, nor with the synergy between TCR and iMC activation. Furthermore, exposure to Rap triggered rapid apoptosis of dual-switch TCR-modified T cells (72±5% Annexin-V+ with Rap versus 14±4% without drug), indicating that the suicide switch is also functional.

iMC is a potent “Go” switch that utilizes rimiducid to provide costimulation to TCR-engineered T cells. In addition, iRC9 is a novel rapamycin-inducible suicide switch that can eliminate T cells in case of severe toxicity. This iMC-enhanced iRC9-incorporating TCR is the first reported prototype of a dual switch TCR-engineered T cell therapy to increase efficacy, durability and safety of adoptive T cell therapies.

Citation Format: Tsvetelina Pentcheva-Hoang, Tania Rodriguez, David Torres, Ana Korngold, Jeannette Crisostomo, MyLinh T. Duong, Matthew Collinson-Pautz, J. Henri Bayle, Mirjam HM Heemskerk, J.H. Frederik Falkenburg, Annemarie Moseley, Kevin M. Slawin, David Spencer, Aaron E. Foster. Dual-switch TCR: A two-ligand system to control PRAME TCR-modified T cell proliferation and death using inducible MyD88/CD40 and caspase-9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3745. doi:10.1158/1538-7445.AM2017-3745