Percutaneous Intratumoral Immunoadjuvant Gel Increases the Abscopal Effect of Cryoablation for Checkpoint Inhibitor Resistant Cancer

Abstract

Percutaneous cryoablation is a common clinical therapy for metastatic and primary cancer. There are rare clinical reports of cryoablation inducing regression of distant metastases, known as the "abscopal" effect. Intratumoral immunoadjuvants may be able to augment the abscopal rate of cryoablation, but existing intratumoral therapies suffer from the need for frequent injections and inability to confirm target delivery, leading to poor clinical trial outcomes. To address these shortcomings, an injectable thermoresponsive gel-based controlled release formulation is developed for the FDA-approved Toll-like-receptor 7 (TLR7) agonist imiquimod ("Imigel") that forms a tumor-resident depot upon injection and contains a contrast agent for visualization under computed tomography (CT). The poly-lactic-co-glycolic acid-polyethylene glycol-poly-lactic-co-glycolic acid (PLGA-PEG-PLGA)-based amphiphilic copolymer gel's underlying micellar nature enables high drug concentration and a logarithmic release profile that is additive with the neo-antigen release from cryoablation, requiring only a single injection. Rheological testing demonstrated the thermoresponsive increase in viscosity at body temperature and radio-opacity via microCT. Its ability to significantly augment the abscopal rate of cryoablation is demonstrated in otherwise immunotherapy resistant metastatic tumors in two aggressive colorectal and breast cancer dual tumor models with an all or nothing response, responders generally demonstrating complete regression of bilateral tumors in 90-day survival studies.

Keywords: cancer immunotherapy; drug delivery; image guided therapy; imiquimod; intratumoral immunoadjuvants.

Figure 1

Concept of image‐guided intratumoral treatment inducing an abscopal effect. A) Example of the abscopal effect following percutaneous lung cryoablation (a physical immunostimulatory technique) leading to sustained reduction in tumors of hilum and portal lymphadenopathy. B) Locations and types of cancers amenable to local image guided drug delivery. C) Treatment paradigm of CT‐guided intratumoral immunogel, with or without cryoablation, in presence of systemic checkpoint inhibition immunotherapy leading to metastases regression. The local immunostimulation induces local immune cells to release cytokines and recruit dendritic cells to sample these tumor antigens. T‐cells are activated by the antigen presenting cells which then induce cancer cell death at distant sites (the “abscopal” effect).

Figure 2

Development of an imiquimod‐bearing, depot‐forming, injectable micellar hydrogel. A) Chemical structure of hydrophilic and hydrophobic portions of the PLGA‐PEG‐PLGA copolymer, where LA:GA ratios can be tuned to alter gelling behavior and release. B) Solubility of imiquimod is significantly higher encapsulated in PLGA‐PEG‐PLGA (LA:GA = 1:1, 3:1 and 5:1 shown here) than in 10% EtOH/DI, 10%DMSO/DI, PBS, or DI water. C) Schematic of micellar formation to enable imiquimod solubility and release. D) Phase transition of the gel at different temperatures centered around body temperature for LA:GA ratio of 3:1. E) Quantification of viscosity change over temperature as a function of LA:GA ratios, 1:1, 3:1, and 5:1, N = 3. F) Dynamic light scattering measurements of micellar nanoparticle size with difference in LA:GA ratio and temperature of Imigel loaded with 6 mg/mL of imiquimod, Z‐avg N = 3. G) Imiquimod release (HPLC‐UV data) into PBS over time at different LA:GA ratios, 1:1, 2:1, 3:1, and 5:1, N = 4.

Figure 3

Evaluation of Imigel injectability and imageability. A) Evaluation of injectability, force of extrusion of certain volume of Imigel through a 22‐gauge 15 cm needle at 20 °C (room temperature) and 37 °C as compared to normal saline and a literature‐based maximum hand injection force of 50 N, n = 3. B) Visualization of the injected Imigel containing 100 mg mL⁻¹ Iopamidol contrast agent into a dorsal flank tumor (right) versus naïve tumor (left). C) H&E staining demonstrating cleft in the tumor created by gel injection (arrow). Representative image of n = 3. Scale represents 200 µm.

Figure 4

Survival studies demonstrating the abscopal effect of Imigel in immunotherapy‐resistant dual tumor models. A) Murine experimental model in dual tumor balb/c mice receiving concomitant intraperitoneal (systemic) injection of checkpoint inhibition (CPI), on days 3, 6, and 9. On Day 8, tumors measuring at least 6 mm were randomized into treatment categories. B) In a CT‐26 cancer model, a single Imigel depot achieved similar survival as 6 daily injections, with or without the use of cryoablation, n = 8–18 mice per arm. C) Similarly, the CPI‐resistant EMT‐6 breast cancer model showed synergistic enhancement in survival when Imigel was used. Cryoablation only did not lead to survival, n = 8–19 mice per arm. All animals that survived to 90 days had full regression of the ipsilateral treated and contralateral untreated tumors demonstrating the abscopal effect. All animals that did not survive had at least one tumor that grew to 2 cm and were euthanized.

Figure 5

Evaluation of size and immunity markers in local and distal tumor of the CT‐26 model. A) Tumor growth curves for ipsilateral (treated) and contralateral (untreated, metastasis model) tumors, comparing systemic checkpoint inhibition (CPI) immunotherapy with CPI + Imigel, split into responders and non‐responders. B) Flow cytometry results, showing unchanged CD8 T cells in ipsilateral and contralateral tumors, while demonstrating a significant change in number of CD44+/PD1+ CD8 T cells with Imigel treatment compared to CPI alone (p = 0.01), and also with CPI + 6x daily imiquimod (p = 0.04). Note systemic CPI alone are the same data points in both contralateral and ipsilateral, given there is no specifically treated tumor. C) Granzyme B immunofluorescence (IF, orange dots) and DAPI (blue) staining of tumors, and H&E images of the studied regions, for the different cohorts as in (B). D) Cytokine analysis of ipsilateral and contralateral CT‐26 tumors, compared to serial imiquimod in absolute fold change, where white is equivalent, red higher, and blue lower.