Abstract
Introduction: Use of T cells engineered to express antigen-specific T cell receptors (TCRs) has shown promise as a cancer immunotherapy treatment; however, durable responses have been limited by poor T cell persistence and expansion in vivo. Additionally, MHC class I downregulation on tumor cells further reduces therapeutic efficacy. Therefore, we co-expressed in human T cells a novel, small molecule dimerizer (rimiducid)-dependent T cell "activation switch", called inducible MyD88/CD40 (iMC), along with tumor antigen-specific TCRs to regulate T cell activation and expansion, while upregulating MHC class I expression on tumor cells.
Methods: Human T cells were activated with anti-CD3/CD28 and transduced with g-retroviruses encoding TCR α and β chains recognizing either the cancer-testes antigen PRAME (HLA-A*201-restricted SLLQHLIGL) or the B cell-specific transcriptional co-activator, Bob1/OBF-1 (HLA-B*702-restricted APAPTAVVL). Parallel "GoTCR" vectors co-expressed the αβ TCR and iMC, comprising signaling domains from MyD88 and CD40 fused in-frame with tandem rimiducid-binding FKBP12v36 domains. Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed EGFPluc-expressing T2 cells (PRAME only) or PRAME+/Bob1+, HLA-A2+ HLA-B7+ EGFPluc-expressing U266 myeloma cells ± rimiducid (10 nM). MHC class I upregulation on tumor cells was measured using transwell assays and flow cytometry. In vitro tumor killing and T cell proliferation were analyzed using T cell and tumor coculture assays by either measuring loss of luciferase activity overnight or by flow cytometry over a period of 4-7 days. Finally, in vivo efficacy was determined using immune-deficient NSG mice engrafted i.v. with U266 cells and treated i.v. with 5x106-1x107 transduced T cells. iMC was activated in vivo by weekly or biweekly i.p. rimiducid injections (1-5 mg/kg). Tumor size and T cell expansion was measured using in vivo bioluminescence imaging and flow cytometry, respectively.
Results: All vectors efficiently (~85%) transduced activated T cells and showed antigen-specific IFN-g production and cytolytic function against peptide-pulsed T2 cells and/or PRAME+Bob1+ U266 myeloma cells. However, both TCR ligation and rimiducid-dependent iMC costimulation were required for IL-2 production against PRAME peptide-pulsed T2 cells. Coculture assays against U266 cells showed that tumor elimination was optimized with concurrent rimiducid-driven iMC activation in both "GoPRAME" and "GoBob1" constructs, and this was accompanied by greatly increased IL-2 secretion and robust T cell proliferation (~ 50-fold vs PRAME or Bob1-specific TCRs alone). Further, iMC activation produced IFN-g independently of TCR ligation, which significantly increased MHC class I expression on tumor cells (~ 7-fold) relative to PRAME TCR-transduced T cells. In NSG mice engrafted with PRAME+ U266 myeloma tumors, GoPRAME TCR-modified T cells persisted for at least 81 days post-injection and prevented tumor growth, unlike any of the other T cell groups. Importantly, weekly rimiducid injection dramatically expanded iMC-PRAME TCR-expressing T cell numbers by ~1000-fold on day 81 post-injection compared to T cells expressing only the PRAME TCR (p < 0.001).
Summary: iMC is a novel "Go" switch that utilizes rimiducid, a small molecule dimerizer, to provide costimulation to PRAME and Bob1-specific TCR-engineered T cells while sensitizing tumors to TCR-mediated recognition via cytokine-induced MHC I upregulation. These iMC-enhanced TCRs are prototypes of novel "GoTCR" engineered T cell therapies that may increase efficacy, safety and durability of adoptive T cell therapies.
Disclosures
Pentcheva-Hoang: Bellicum Pharmaceuticals: Employment. Torres:Bellicum Pharmaceuticals: Employment. Rodriguez:Bellicum Pharmaceuticals: Employment. Korngold:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Crisostomo:Bellicum Pharmaceuticals: Employment. Moseley:Bellicum Pharmaceuticals: Employment, Membership on an entity's Board of Directors or advisory committees. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership. Foster:Bellicum Pharmaceuticals: Employment.
Unconventional Peptide Presentation by Classical MHC Class I and Implications for T and NK Cell Activation