2804 - Efficacy of Fas-4-1BB Immunomodulatory Fusion Proteins Engineered T-Cells in Applying to Targeted Pubic Neoantigen Immunotherapy Using Encapsulated Nanoparticles Directed by Radiation

S. Harada¹, and T. Sato²; ¹Iwate Medical University School of Medicine, Shiwa-gun, Yahaba, Japan, ²Japan Atomic Energy Association, Takasaki, Japan

Purpose/Objective(s): Fas-4-1BB immunomodulatory fusion proteins (IFP) engineered T-cells were primed with CAND1 and ADGRF5-II pubic neoantigen, through antigen presenting nanoparticles (AP-NPs) and dendritic cells (DCs). T-cells were applied to the CAND1 and ADGRF5-II neoantigen radioimuunotherapy under PD-L1 blockade, which was targeted by two radiation sessions, using computed tomography (CT) detectable nanocapsules (?545 ± 4 nm) that released their contents upon radiation exposure in 4TIFas^L+ tumor in BALB/c mice. Materials/

Methods: Fas-4-1BB was transduced to PI4 CD8⁺ and CD4⁺ T-cells, using retroviral vector (RV), and 1 X 10⁷ stimulated engineered T-cells were intravenously administered to mice and used for experiments 27 days after treatment. For session 1, poly-lactic-co-glycolic acid (PLGA) AP-NPs were coated with 110 fmol of CAND1 and ADGRF5-II neoantigen through nanoprecipitation method. Nanocapsules (hyaluronate-alginate nanocapsules; HA-NCs) were generated by combining 1 X 10¹⁰ AP-NPs and 15 µg IFN? with a 1.0 mL solution of 4.0% alginate, 3.0% hyaluronate, and 1 µg/mL P-selectin. This mixture was sprayed onto 0.5 mmol/L FeCl₂ supplemented with 1 µg/mL anti-VEGFR-1/2 antibody (Ab). For session 2, 110 fmol of CAND1 and ADGRFII with 400 µg PD-L1 Ab were encapsulated into P-selectin Ab labelled HA-NCs through the same electrospray and Fe-polymerization as session 1. In session 1, 1 × 10¹⁰ HA-NCs were intravenously administered into mice. Tumor accumulation was monitored by CT. Subsequently, 10 or 20 Gy ⁶⁰Co ?-radiation was administered to primary tumors and lung metastasis. In session 2, 1 × 10¹⁰ HA-NCs were intravenously administered and allowed to interact with P-selectin for 24 h; subsequently, radiation was likewise conducted.
Results: In session 1, CT image of anti-VEGFR-1/2 Ab accumulation labelled HA-NCs around primary and metastatic tumors, releasing P-selectin, IFN?, CAND1, and ADGRF5-II coated AC-NPs in response to session 1. P-selectin was deposited in tumor vessels. Radiation and released IFN? elicited MHC-I and II on tumor cells. CAND1/ADGRF5-II coated AC-NPs and DCs primed CD8⁺/CD4⁺ T-cells/Fas -4-1 BB. In session 2, CAND1 and ADGRF5-II were released from HA-NCs and captured to MHC-I and II, respectively. Subsequently, two tertiary complexes were formed on tumor cells; MHC-I-CAND1-primed CD8⁺T-cell receptor (TCR)/Fas-4-1BB and MHC-II-ADGRF5-II-CD4⁺TCR/Fas-4-1BB. Fas-4-1BB converted inhibitory/death signals into activating/survival signals in T-cells, enhancing CD8⁺/CD4⁺ T-cell function and persistence, in collaboration with released PD-L1 Ab. These treatments resulted in EF 1.8 and 77% reduction of new metastasis formation.
Conclusion: Our targeted pubic neoantigen therapy with Fas-4-BB IFP engineered T-cell results to effective radioimmunotherapy.