Expression of MyD88/CD40 Drives In Vivo Activation and Proliferation of Chimeric Antigen Receptor-Modified T Cells That Can be Effectively Regulated By Inducible Caspase-9
Authors: Foster Aaron , Chang Peter , Lin Pei-Yi , Crisostomo Jeannette , Mahendravada Aruna , Lu An , Khalil Mariam , Saha Sunandan , Shaw Joanne , Morschl Eva , Slawin Kevin M , Spencer David M 
Published: October 2019

Abstract
Introduction: Efficacy of chimeric antigen receptor (CAR)-modified T cells is dependent on their in vivo survival and expansion following infusion. The addition of accessory molecules (e.g., costimulatory and cytokine genes) may improve CAR-T proliferation and potency, but may also increase toxicity of these next generation CAR-T cell therapies, suggesting that the incorporation of a built in "safety switch" would balance safety and efficacy in a single, controllable therapy. Here, we demonstrate that cytosolic coexpression of a MyD88/CD40-derived fusion protein dramatically enhances CAR-T activation, cytokine production, and proliferation in vivo, resulting in improved antitumor efficacy. Importantly, CAR-T cell numbers, elevated cytokine levels, and observed CAR-T-related toxicity could be controlled by titratable rimiducid administration to reduce or eliminate CAR-T cells by activating the inducible caspase-9 (iC9) suicide gene.
Methods: Human T cells were activated with anti-CD3/CD28 and transduced with retrovirus encoding, iC9, a first generation CAR (with CD3ζ) targeting CD19, Her2 or PSCA, and a detached, fusion protein comprising signaling domains from MyD88 and CD40 (MC). For comparison, additional CARs were constructed without MC, with MyD88 or CD40 elements only, or with conventional CARs coexpressing CD28 within the CAR molecule (CAR.28.ζ). Transduced T cells were assessed in vitro for cytotoxicity, cytokine production and proliferation against tumor cell lines (CD19+: Daudi, Raji; Her2+: SK-BR-3; PSCA+: Capan-1, HPAC). In vivo antitumor efficacy of CAR-modified T cells was assessed using immunodeficient NSG mice engrafted with antigen-matched tumor cell lines (5x105 Raji, i.v.; 1x106 SK-BR-3, s.c; 2x106 HPAC, s.c.) followed by i.t. or i.v. injection of variable doses of T cells. Reduction or elimination of CAR-T cells was performed by i.p. injection of rimiducid (0 - 5 mg/kg). Tumor cell lines expressing luciferase or T cells co-transduced with luciferase-encoding vectors were used for bioluminescence imaging (BLI) to measure tumor growth or T cell expansion/elimination, respectively. Serum cytokine levels were assessed by blood draws and CAR-T cell frequency was measured by flow cytometry.
Results: All CAR constructs were stably expressed in T cells (30-90%). CAR vectors coexpressing MC induced high IL-2 levels in vitro when exposed to target antigen+ tumor cells (CD19 = 4246 ± 52, Her2 = 2613 ± 1298, and PSCA = 3263 ± 1393 pg/ml per 1x105 T cells over 48 hrs) and corresponded to improved CAR-T cell proliferation and tumor elimination compared to control vectors. In NSG mice, MC costimulation resulted in >2,000-fold expansion of CD19-targeted CAR-T cells and complete tumor control for >100 days in 100% of mice engrafted with CD19+ Raji cells (p = 0.0002) following injection of 5x106 CAR-T cells, followed on day 7 with a single i.p. dose of rimiducid (5 mg/kg) to control toxicity. MC-enabled CAR-T cells were eliminated or partially reduced by rimiducid titrations, which corresponded to decreased cytokine (IL-6, IFN-γ, TNF-α) levels and restoration of health in animals showing signs of toxicity (e.g., ≥15% weight loss). For solid tumors, Her2-targeted, MC-enabled CAR-T cells showed a 150-fold in vivo expansion and compared favorably to first (Her2.ζ; p = 0.01) and second generation (Her2.28.ζ; p = 0.01) CARs, causing 100% elimination of SK-BR-3 tumors and enhanced survival for >60 days following i.t. injection (p = 0.0015). PSCA-targeted CARs expressing MC also drove complete and durable (>42 days) elimination of large (200 mm3) HPAC tumors in 100% of mice, after a single i.v. injection of 1x107 CAR-T cells followed on day 14 with a single 5 mg/kg i.p. rimiducid dose to reverse toxicity.
Summary: Coexpression of MC, and the cell therapy safety switch "CaspaCIDe", in combination with a first generation CAR, together comprising the novel "CIDeCAR" platform technology, dramatically increases efficacy against a number of tumor targets by enhancing T cell engraftment and proliferation following infusion, while incorporating an effective, built-in safety mechanism. In three distinct tumor models, rimiducid administration promptly eliminated signs and symptoms of CAR toxicity without subsequent loss of tumor control. CIDeCAR technology may allow the development of safer and more effective CAR-T cell therapies for a range of difficult-to-treat liquid and solid tumors.

Disclosures
Foster: Bellicum Pharmaceuticals: Employment. Chang:Bellicum Pharmaceuticals: Employment. Lin:Bellicum Pharmaceuticals: Employment. Crisostomo:Bellicum Pharmaceuticals: Employment. Mahendravada:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Khalil:Bellicum Pharmaceuticals: Employment. Saha:Bellicum Pharmaceuticals: Employment. Shaw:Bellicum Pharmaceuticals: Employment. Morschl:Bellicum Pharmaceuticals: Employment. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership.