Targeted Pubic Neoantigen Immunotherapy Using Encapsulated Nanoparticles, Directed by Radiation

Abstract

We tested a treatment combining radiotherapy with pubic neoantigen nanoparticles of CAND1 and ADGRF5-II (120 ± 56 nm) in nanocapsules detectable by computed tomography (CT) (545± 24 nm) with IFN γ or PD-L1 antibody (Ab), which released their contents upon radiation exposure. We performed two radiotherapy sessions: 1) induction of MHC-I and II in tumor cells and dendritic cells (DC) by the first round of radiation with concurrently released IFN γ from nanoparticles; 2) targeted neoantigen immunotherapy with PD-L1 blockade by the second round of radiation, elicited by CAND1-specific CD8+T-cell and ADGRF5-II-specific CD4+T-cell, related to induced MHC-I and MHC-II in session 1, respectively. For session one, nanocapsules were generated by mixing iopamiron and 15 mg IFN γ with a 1.0 mL solution containing 4.0% alginate, 3.0% hyaluronate, and 1 μg/mL P-selectin. This mixture was sprayed into 0.5 mmol/l FeCl2 supplemented with 1 µg/mL anti-VEGFR-1/2 Ab. For session two, Mixture of 110 fmol CAND1 and fmol ADGRF5-II, were encapsulated into poly lactic-co-Glycolic acid (PLGA) particle, using nanoprecitation method. Those particles were encapsulated by mixing into above alginate-hyaluronate cocktail with 400 mg PD-L1 antibody (Ab), then sprayed into 0.5 mmol/l FeCl2 supplemented with P-selectin Ab. In session one, 1 × 1010 nanocapsules were intravenously injected into BALB/c mice exhibiting primary 4TI mammary carcinoma in the left hind leg and lung metastases. Tumor accumulation was monitored by CT. Subsequently, 10 or 20 Gy 60Co γ-radiation was administered to primary tumors and lung metastasis. In session two, 1 × 1010 nanocapsules were injected i.v. and allowed to interact with P-selectin for 24 h; a further 10 or 20 Gy 60Co γ-radiation was administered to tumors and lung metastasis. In session one, CT imaged the accumulation of anti-VEGFR-1/2 nanocapsules around primary and metastatic tumors, which helped determine the timing of the first radiation. The nanocapsules released P-selectin and IFN γ in response to first radiation. P-selectin was deposited on tumor vessels. Radiation and released IFN γ elicited MHC-I and II on tumor cells and DCs. In session two, released CAND1 and ADGRF5-II from PLGA particle, which were emitted from nanocapsules upon second radiation, were loaded onto MHC-I and II. Subsequently, two kinds of tertiary complex were formed on tumor cells and DC: 1) MHC-I-CAND1-CD8+T-cell receptor (TCR); and 2) MHC-II- ADGRF5-II-CD4+TCR, which destroyed tumor cells, in collaboration with released PD-L1 Ab. Those T-cell mediated cell killing was magnified via enhanced DC-mediated T-cell priming by the two kinds of tertiary complex on DC. Those treatments resulted in EF 1.5 and 86% reduction of new metastasis formation. Our encapsulated pubic neoantigen particle will lead to quick and effective neoantigen immunotherapy.