Biomaterial-Facilitated Immunotherapy for Established Oral Cancers

Abstract

We evaluated a peptide-based immunotherapy termed SynerGel: an injectable, biomaterial-based platform for intratumoral drug delivery. A drug-mimicking peptide hydrogel named L-NIL-MDP was loaded with an antitumor cyclic dinucleotide (CDN) immunotherapy agonist. The biomaterial combines inducible nitric oxide synthase (iNOS) inhibition with controlled delivery of CDNs, demonstrating between 4- and 20-fold slower drug release than commercially available hydrogels. SynerGel allowed for immune-mediated elimination of established treatment-resistant oral tumors in a murine model, with a median survival of 67.5 days compared with 44 days in no-treatment control. This report details findings for a promising therapy showing improved efficacy over previous hydrogel systems.

Keywords: bioactive peptide biomaterial; controlled drug release; immunotherapy; intratumoral hydrogel.

Figure 1.

(A, C) Drug release profiles of cyclic dinucleotide (CDN) ML RR-S2 CDA from various hydrogels systems over 24 hours, comparing commercially available alginate, hyaluronic acid (HyAcid), collagen, and Matrigel systems to synthesized multidomain peptides (MDPs) K2-MDP, E2-MDP, O5-MDP, and L-NIL-MDP, with quantification of release profiles shown in (B). All release data are shown as means ± S.D. with n = 3. Data for K2-MDP and collagen hydrogels are reproduced from our previous study for comparison. (D) Rheological storage modulus (G’) of studied MDP hydrogels, showing bulk material strength was not predictive of drug release properties.

Figure 2.

(A-C) Viability of mouse oral cancer (MOC1) cells cultured on L-NIL-MDP hydrogels. All cell-gel experiments were cultured under 200 μL of media and processed for live-dead viability assays at time-points of days 1, 3, and 7 (green live cell-Calcein AM; red dead cells-Ethidium-homodimer I; blue nuclei-Hoechst 33342). (D) Masson’s trichrome stained subcutaneous histology of K2-MDP hydrogel implant three days post injection in vivo at 4X magnification. (E-F) 40X magnification images of the black boxes in panel D, with image E on implant edge, and image F in implant core. (G) L-NIL-MDP hydrogel implant three days post injection at 4X magnification. (H-I) 40X magnification images of the black boxes in panel G, with image H on implant edge, and image I in implant core. Example areas of undegraded or non-infiltrated hydrogel have been marked by asterisks for clarity.

Figure 3.

MOC1 tumor growth curves. (A) Mean tumor growth until time of first mouse euthanization, (B-G) Individual tumor growth curves for controls and L-NIL-MDP+CDN treated mice bearing established MOC1 oral tumors over course of study. Tumor rechallenge was at day 100 post-inoculation (indicated by dotted line). The number of tumor-bearing mice that were euthanized is listed above each plot. L-NIL-MDP+CDN (SynerGel) treated mice had decreased tumor growth. a, p<0.01 vs K2-MDP; b, p<0.0001 vs. HBSS (Hank’s balanced salt solution), K2-MDP, L-NIL-MDP; #, p<0.01 vs. K2-MDP+CDN; c, p<0.0001 vs. HBSS, K2-MDP, K2-MDP+CDN and L-NIL-MDP.

Figure 4.

Survival curves for mice bearing established MOC1 oral tumors. Kaplan-Meier curves of the different experimental groups is based on euthanasia timepoints due to tumor burden. Intratumoral injections (IJ) were given when tumors were at 4-5 mm, represented on the x-axis as 5mm(IJ). Survivor rechallenge (RC) was done at post day 100 time point. L-NIL-MDP+CDN (SynerGel) treated mice had improved survival. SynerGel increased survivorship from 20% to 33% compared to CDN alone. Log-rank test; **, p< 0.01 vs. HBSS; ^^, p< 0.01 vs. K2-MDP; ##, p< 0.01 vs. L-NIL-MDP.