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3184 Dual-Switch CAR-T Cells: Orthogonal Molecular Switches to Control Activation and Elimination of CAR-T Cells to Target CD123+ Cancer

Program: Oral and Poster Abstracts
Session: 703. Adoptive Immunotherapy: Poster II
Sunday, December 10, 2017, 6:00 PM-8:00 PM
Bldg A, Lvl 1, Hall A2 (Georgia World Congress Center)

J. Henri Bayle, PhD1*, MyLinh T Duong, PhD1*, An Lu1*, Eva Morschl, PhD2*, Matthew R. Collinson-Pautz, PhD2*, Kelly L Sharp2*, Slawomir Szymanski, PhD2*, Mary E. Brandt2*, Kevin M. Slawin, MD3*, Steven Toler, PhD2*, Eric Yvon, S.2*, Aaron Foster, PhD1 and David M. Spencer, PhD1

1Bellicum Pharmaceuticals, Houston, TX
2Bellicum Pharmaceuticals, Inc, Houston, TX
3Bellicum Pharmaceuticals, Inc., Houston, TX

Background

Improvement of the efficacy and safety of chimeric antigen receptor (CAR)-T immunotherapies requires controlled activation and termination of the T cells when transfused into patients. Here we present two independently regulated molecular switches that can elicit specific and rapid induction of cellular responses upon exposure to their cognate ligands. T cell costimulation is controlled by the homodimerizer rimiducid that triggers signaling cascades downstream of MyD88 and CD40 via an engineered protein termed iMC. A rapamycin-controlled pro-apoptotic switch (iRC9) that induces dimerization of caspase-9 mitigates possible CAR-T cell toxicity. iRC9 is a chimeric protein comprisingan FKBP-rapamycin binding (FRB) domain in tandem with FKBP12 and caspase-9. This design permits rapamycin, a heterodimerizing ligand, to function as a homodimerizer. When combined with a first generation CD123-specific CAR, these molecular switches allow for controlled, robust expansion of engineered T cells to control acute myelogenous leukemia (AML) in vitro and in vivo combined with a rapid and efficient safety mechanism to block excessive cytokine release.

Methods & Results

T cells were activated and co-transduced with pSFG-iMC.2A.CD123CAR.ζ and pSFG-iRC9.2A.ΔCD19 vectors to generate Dual-switch (DS) CAR-T cells. Combined transduction of iRC9 and iMC-CAR vectors produced CD123-directed CAR-T cells that eliminated CD123+ THP1 and MOLM13 AML cells, but not CD123- HPAC tumor cells, in a co-culture assay. Cytokine secretion and target cell killing were dependent on the dose of rimiducid (EC50 < 0.5 nM) to activate iMC costimulation. When challenged in a THP1-eGFPFluc tumor-bearing mouse xenograft model, activation of the on-switch by rimiducid (1 mg/kg) in DS CAR-T cells enhanced tumor killing measured by bioluminescence imaging and T-cell expansion determined by splenocyte flow cytometry and vector copy number analyses.

Deployment of the off-switch induced fast (½ Vmax ~ 8 hours) and efficient T cell elimination of in a caspase-3 activation assay with real-time monitoring by Incucyte microscopy as well as Annexin V detection by flow cytometry (DS CAR-T = 77.6% versus untransduced = 2.2% Annexin V+ when treated with 1 nM rapamycin). In vivo assessment of the suicide switch was performed with eGFPFluc-labeled CD123 DS CAR-T cells in NSG mice. Rapamycin, but not rimiducid, treatment efficiently eliminated DS CAR-T cells within 24 hours in NSG mice, which is similar to the clinically validated rimiducid-regulated iC9 switch. Importantly, the off-switch was insensitive to high rimiducid concentration, demonstrating that the on-switch regulator does not crosstalk with the safety switch.

Summary

Dual switch CAR-T, a novel platform comprising a first-generation CAR combined with regulated activation and apoptotic signaling elements, effectively controlled tumor growth and T cell expansion and elimination in vitro and in vivo. This dual switch technology provides a user-controlled system for managing persistence and safety of tumor antigen-specific CAR-T cells.

Disclosures: Bayle: Bellicum Pharmaceuticals: Employment, Equity Ownership. Duong: Bellicum Pharmaceuticals: Employment, Equity Ownership. Lu: Bellicum Pharmaceuticals: Employment. Morschl: Bellicum Pharmaceuticals: Employment, Equity Ownership. Collinson-Pautz: Bellicum Pharmaceuticals: Employment, Equity Ownership. Sharp: Bellicum Pharmaceuticals: Employment, Equity Ownership. Szymanski: Bellicum Pharmaceuticals: Employment, Equity Ownership. Brandt: Bellicum Pharmaceuticals: Employment, Equity Ownership. Slawin: Bellicum Pharmaceuticals: Consultancy, Equity Ownership. Toler: Bellicum Pharmaceuticals: Employment, Equity Ownership. Yvon: Bellicum Pharmaceuticals: Equity Ownership. Foster: Bellicum Pharmaceuticals: Employment, Other: stockholders . Spencer: Bellicum Pharmaceuticals: Employment, Equity Ownership, Other: stockholders .

*signifies non-member of ASH