Abstract B59: Enhancing T-cell therapy through TCR signaling-responsive nanogel drug delivery
Authors: Castano Ana , Tang Li , Kudchodkar Sagar , Darrell Irvine , Maus Marcela 
Published: March 2017

Abstract
Adoptive cell transfer (ACT) with autologous tumor-reactive T-cells has recently shown great promise in the treatment of leukemia in clinical trials. Effective control of solid tumors, remain challenging. Glioblatoma multiforme (GM) is a lethal disease in need of clinically effective therapies. Ninety-seven percent of tumors in the ‘classical’ subtype carry extra copies of the epidermal growth factor receptor (EGFR) gene, and most have higher than normal expression of EGFR. In the present study we utilize a novel carrier-free delivery strategy to specifically support a EGFR directed chimeric antigen receptor (CAR) T-cell with recombinant Interleukin-15 super agonist (IL15-Sa), in a heterotopic mouse model of GM.
CAR-T cell design: The CAR-T cell was designed based on the heavy and light chains of cetuximab to form a single-chain variable fragment, which was fused to a portion of the extracellular and transmembrane domains of human CD8 alpha, followed by the intracellular domains of 4-1BB and CD3 zeta. The bicistronic vector also encoded truncated human CD19 as a selectable marker. The plasmid coding hu EGFRscFv-BBz-CAR was synthesized and lentivirus packaging was made. Isolated T cells were derived from leukapheresis products and stimulated with Dynabeads Human T Activator CD3/CD28. T cell were cultured and were transduced and or left untransduced one day following bead stimulation, and then T cells were expanded for 10 days and cryopreserved. Surface expression of the CAR was confirmed and quantified with biotinylated human EGFR protein and by staining for CD19. CAR-T cells phenotype was identified.
Coupling of NGs to T cells: anti CD45 antibody (aCD45) against highly expressed cell surface phosphatase CD45 was used as an anchor to stably link human IL-15Sa as a drug cargo/nanogel (NG) to the T cell surface. The aCD45/IL-15-Sa-NG was added to CAR T cells.
In vivo therapy study using human Chimeric Antigen Receptor (CAR)-T cells: Luciferase-expressing U-87 MG human glioblastoma cells (1.0 × 106) were injected s.c. into NSG mice on Day 0. Animals received i.v. adoptive transfer of activated CAR-T cells (2.6×106 total T-cells -38% transduction equating to 106 CAR-T-cells)) alone or with surface coupled NGs on Day 7. In other groups, free IL-15Sa was injected i.v. after ACT at equivalent dose. Tumor area and body weight were measured every two days. Mice were also imaged for bioluminescence to monitor the tumor growth and were euthanized when body weight loss was beyond 20%, or tumor area reached 150 mm2 or the animal had become moribund.
Findings: CAR T-cells backpacked with IL-15Sa-NGs were compared to CAR T-cells alone or T-cells supplemented with an equivalent systemic dose of free IL-15Sa. NG-backpacked CAR T-cells eradicated tumors in 80% of animals. By contrast, transfer of CAR-T cells alone had a small impact on tumor growth and survival, which did not reach statistical significance; responses were marginally improved by the addition of free IL-15Sa. We have demonstrated NG delivery of cytokines has the potential to enhance CAR T-cell therapy in a mice model of GM. This strategy should be broadly applicable in ACT strategies employing natural, TCR-engineered, or CAR T-cell therapies, and illustrates a general approach to couple the action of a synthetic drug delivery carrier to the function of therapeutic cells.
Citation Format: Ana Castano, Li Tang, Sagar Kudchodkar, Irvine Darrell, Marcela Maus. Enhancing T-cell therapy through TCR signaling-responsive nanogel drug delivery. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B59.