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.

Image‐guided intratumoral imigel ± cryoablation can overcome immunotherapy resistance to induce complete regression in distant tumor metastases. By optimizing this platform for interventional radiologists, who currently do most deep percutaneous intratumoral injections and cryoablation therapies, the technology reduces the hurdles for translating personalized cancer vaccines.

1. Introduction

In cancer treatment, the holy grail of locoregional therapies remains the promise of an abscopal effect. “Abscopal”, latin for away, refers to the phenomenon that a local therapy could induce therapeutic effect distantly in metastases, presumably secondary to immune activation. In interventional oncology, we have rarely seen the abscopal effect among patients referred for percutaneous cryoablation, a technique where a probe is placed under CT or ultrasound guidance and is used to freeze‐thaw tumors. The abscopal effect seen here has been theorized from research models to be secondary to the neo‐antigen burst release, and multiple pre‐clinical studies, including our own have shown increased immunostimulatory effects particularly for immunotherapy sensitive patients.^{[ 1} , ² , ^{3 ]} For example, at Massachusetts General Hospital, we identified 18 patients with metastatic disease treated with immunotherapy within 90 days before or up to 30 days after percutaneous cryoablation.^{[ 4 ]} Of those, one patient with metastatic melanoma demonstrated a particularly sustained abscopal effect following percutaneous cryoablation of a pulmonary metastasis (Figure 1A) manifested by regression of metastases in the contralateral lung and retroperitoneal lymph nodes. The combination with immunotherapy is actively being studied in clinical trials.^{[ 1} , ^{5 ]} For cryoablation, there is a significant need to find a method to make this rare but exciting abscopal effect more reproducible.^{[ 3} , ⁶ , ⁷ , ⁸ , ⁹ , ¹⁰ , ^{11 ]}

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).

One potential method is to inject intratumoral immunoadjuvants into a single site of disease peri‐cryoablation to activate the innate immune system to sample the neo‐antigen burst.^{[ 3} , ⁶ , ^{7 ]} The innate immune system in turn activates and educates the adaptive immune system, namely, the subset of cytotoxic CD8+ T cells that are primarily responsible for immune‐mediated tumor killing, ideally attacking tumor throughout the body.^{[ 8 ]} “Training” of the immune system to combat metastases following a local intervention has also been reported as an “in situ cancer vaccine”.^{[ 6} , ^{9 ]} Following tumor recognition, CD8+ antitumoral activity is in turn maintained by systemic checkpoint inhibitor therapy.^{[ 6 ]} While other groups have explored capturing antigens to augment the abscopal effect^{[ 10 ]} or modulating the freeze area in cryoablation with nanoparticles,^{[ 11 ]} we hypothesized that combining intratumoral immunoadjuvants with cryoablation may improve the reproducibility of the rarely clinically seen abscopal effect. Most immunoadjuvants are small molecules, with rapid diffusion away from the tumor region. Although several immunoadjuvants for intra‐tumoral injection are currently in clinical trials, including STING and TLR7/8/9 agonists, only a few have arrived in clinic.^{[ 12} , ¹³ , ¹⁴ , ¹⁵ , ¹⁶ , ^{17 ]} Among small molecules, only imiquimod is a clinically approved TLR7 agonist that increases dendritic cell activation and is clinically approved as a topical cream for treatment of basal cell carcinoma. We reasoned this drug could act as a proof‐of‐concept drug with high translational potential as many intratumoral drugs in development are hydrophobic small molecules.^{[ 8} , ¹⁸ , ^{19 ]} In preclinical studies, imiquimod in combination with immunotherapy has shown some efficacy albeit requiring daily 50 µL injections with 1 mg mL⁻¹ imiquimod across 6 days showed improved survival.^{[ 14} , ^{20 ]} We hypothesized a logarithmic release of imiquimod over a similar number of days, that is, a larger release times around the ablation, followed by a logarithmic release, may improve the abscopal rate of cryoablation. Following prior studies, the mechanism of action is expected to be due to dendritic cell recruitment from TLR 7 activation followed by T‐cell activation and cell mediated killing.^{[ 14} , ²¹ , ^{22 ]}

Further, a significant hurdle to clinical translation remains the need for multiple repeated injections and lack of imaging confirmation. The injection of visceral organs or deep lymph nodes by interventional radiologists requires CT‐ or ultrasound guidance and procedural sedation.^{[ 7} , ¹⁸ , ^{23 ]} Without some imaging agent embedded, it is challenging to verify the targeted delivery to delivery or assess for off‐target delivery during or after the procedure – which is critical for the evaluation of therapeutic efficacy and therapeutic window.^{[ 18} , ^{24 ]} For example, in a previously published instance of a radio‐opaque intratumoral therapy the authors demonstrated that therapy was often delivered off‐target despite correct needle placement.^{[ 18} , ^{24 ]} To address these challenges we developed an injectable, thermoresponsive, locally resident and imageable viscous gel to contain and deliver a high concentration of imiquimod, optimized for concurrent delivery with cryoablation. In two different syngeneic murine dual tumor models of checkpoint inhibitor resistant cancer, we tested whether intratumoral Imigel injection rate significantly improves the abscopal rate of cryoablation.

2. Results

2.1. Development of a Gel Depot for Hydrophobic Immunoadjuvant Imiquimod (Imigel)

We investigated multiple polymeric systems for the short‐term controlled release of imiquimod. Given the constraints of injecting during an interventional oncology procedure, we looked for a thermoresponsive polymer with biocompatibility, injectability, and the ability to carry a high dose of drug in a relatively small volume. To increase the translatability of the formulation, we focused on immunoadjuvants with proven clinical relevance, particularly imiquimod, and approved polymers with benign safety profiles. PLGA‐PEG‐PLGA is an amphiphilic triblock‐copolymer composed of relatively hydrophobic (PLGA) and hydrophilic elements (PEG), which has reached clinical trials for the delivery of paclitaxel‐based chemotherapy as Oncogel.^{[ 25 ]} The triblock structure enables two critical features of our controlled release formulation: first, it self‐assembles into micellar nanoparticles around hydrophobic drugs, thereby dramatically increasing their aqueous solubility, and second, around body temperature this micellar structure creates a viscous gel (Figure 2A, Figure S1, Supporting Information). Both the gel formation and the drug encapsulation are largely guided by the PLGA copolymer structure, where the ratio of the lactic and glycolic acid monomers guides the hydrophobicity of the PLGA blocks. Lactic acid (LA) has a methyl side group, rendering it more hydrophobic than glycolic acid (GA). Capitalizing on this molecular handle to tune the polymer's amphiphilic properties, we explored PLGA‐PEG‐PLGA polymers with varying LA:GA ratios to tune hydrophobicity, and thus gel behavior, drug encapsulation, and drug release, ​​with the goal of a multiday release to sustain a therapeutic effect as observed in previous studies.^{[ 14 ]}

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.

Imiquimod has a relatively low solubility in saline (around 0.002 mg mL⁻¹) and reported 1 mg mL⁻¹ solutions only exist as suspensions. These concentrations are well below the necessary 6 mg mL⁻¹ based on the sum aggregate dose of daily imiquimod over a 6‐day course that previously demonstrated improved survival.^{[ 14} , ^{20 ]} The use of PLGA1.5k‐PEG1.5k‐PLGA1.5k micelles was able to uniformly solubilize imiquimod at around 6 mg mL⁻¹ and thus in much larger concentrations than water, phosphate buffered saline (PBS), 10% DMSO/water or 10% Ethanol/water (Figure 2B). Molecular weight and LA:GA ratios were confirmed by gel permeation chromatography (GPC) and by nuclear magnetic resonance (NMR) spectroscopy (Figure S2, Supporting Information). Increasing the hydrophobicity of the PLGA block by tuning the LA:GA ratio influences gelling behavior, where LA:GA = 1:1 does not exhibit significant gelling due to insufficient hydrophobicity, whereas 3:1 and 5:1 show gelling points conveniently around physiological temperatures, as evidenced visually (Figure 2D) and by rheometry (Figure 2E). With increasing hydrophobicity (higher LA:GA ratio), particle size at room temperature decreases (Figure 2F). Lower Critical Solution Temperature (LCST) marks the phase transition of thermosensitive polymers, which is ≈33.1 °C for LA:GA = 3:1, while 1:1 did not exhibit an LCST and remained liquid up to 40 °C.

Drug release from PLGA‐PEG‐PLGA occurs first via diffusion until the polymer undergoes hydrolysis and thereby releases drug (43). Paralleling literature, we find that the rate of release depends on polymer hydrophobicity, tuned by the ratio of LA:GA within the gel's PLGA block.^{[ 27 ]} Similarly, we find changing the LA:GA ratio also modulates the drug release kinetics of imiquimod, where LA:GA = 1:1 leads to an almost instantaneous (burst) release of all drug while the more hydrophobic variants retain imiquimod for longer. The ratio can be used to obtain a logarithmic 4–5‐day release (Figure 2F). The differences in plateau release amount may be a reflection of difference in imiquimod solubilization efficiency: all imigel formulations were prepared with 25% w/v PLGA‐PEG‐PLGA (1 gram polymer in 4 mL saline) and 6 mg mL⁻¹ imiquimod (24 mg added to the batch), however, the more hydrophobic formulations seem to uptake slightly less imiquimod, which would be congruent with the smaller micelle size observed. Smaller micellar sizes and accordingly lower drug loads have been reported for increasingly hydrophobic copolymer fractions of PEG‐polyester copolymers before.^{[ 28 ]} An increased hydrophobic core packing density can result in smaller micelles and thus lower drug load. Comparing the different formulations used herein, LA:GA = 3:1 was chosen as the optimal tradeoff between gelation (1:1 and 2:1 did not gel) and drug load (5:1 showed less encapsulation). Relative release rate of imiquimod from LA:GA 3:1 imigel shows minimal changes with acidic pH (5 or 6.8) (Figure S3, Supporting Information). Although polymers can be designed for a linear release, we opted to design for a logarithmic release profile, hypothesizing that having a relative majority delivered early would aid sampling of neo‐antigens released during ablation.

A desired feature for application of this technology in the clinic is the ability to be injectable through at least a 20‐gauge needle, considering 18‐ to 22‐gauge needles are common in clinical use. Figure 3A demonstrates the injectability through a 22‐gauge 15 cm Chiba needle of the room temperature (20 °C) version of the gel, whereas injectability requires higher force at 37 °C. Force measurements demonstrate that either scenario requires forces well below the maximum 50 N suggested for an average healthcare worker's hand injection.^{[ 29 ]}

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.

To facilitate delivery confirmation and for the clinician to be able to assess technical success of the procedure, we explored the feasibility of incorporation of a radio‐opaque contrast agent into the gel. Imigel incorporated 100 mg mL⁻¹ of hydrophilic Iopamidol, an iodinated contrast agent, to enable the visualization of the injection in mice under micro‐CT immediately after injection (Figure 3B). The concentration of iodine required depends on CT scanner clinically but has been reported as low as 2 mg mL⁻¹, which can likely be optimized to the use case and organ.^{[ 30 ]} Immediate post‐injection histologic evaluation of resected tumor post‐injection demonstrates how the injected gel forms a depot within the tumor, creating a cleft in the tumor tissue (Figure 3C).

2.2. Imigel Significantly Improves the Abscopal Efficacy of Cryoablation in Both Colorectal and Breast Cancer Immunotherapy Resistant Mouse Models

We used syngeneic dual flank implanted tumor models for assessment of Imigel's performance in increasing the response rate to traditional checkpoint inhibitor (CPI) therapy in CPI‐resistant tumor models, where CPI alone leads to zero response (i.e., no survival), and compare the results also to a combination with cryoablation and the 6x daily injection scenario. CT‐26 tumors are a micro‐satellite stable murine‐derived colon cancer line that is known to be moderately responsive to CPI therapy.^{[ 31} , ^{32 ]} EMT‐6 is a murine breast cancer that is poorly responsive to CPI.^{[ 33 ]} In these dual tumor models, intra‐tumoral therapy is applied to only one of the two implanted tumors (i.e., the ipsilateral tumor), while response is monitored in both the intervened and non‐intervened (contralateral) tumor (Figure 4A–C). In all cases the two tumors implanted in each mouse were of the same cell line, to create a consistent and readily assessed model of distant disease. In general, all treatments were done in the background of systemic checkpoint inhibitor therapy.

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.

In both the CT‐26 and EMT‐6 model, we see a significant increase in 90‐day survival and abscopal rate of mice when cryoablation is combined with imigel co‐injection compared to cryoablation and CPI. For the CT‐26 model, cryoablation + imigel + CPI had a 90‐day survival percentage of 57% (10/19) compared to CPI alone, 0% (0/10) (p < 0.0001 Log rank test), or CPI + cryoablation of 21% (4/19) (p = 0.0003 Log rank test).

For the EMT‐6 model, at 90 days cryoablation + imigel + CPI demonstrated a higher 90‐day survival (5.6%, 1/18) compared to CPI alone (0%, 0/7) or CPI + cryoablation (0%, 0/8), (p = .0003 Log rank test). (Figure 4c).

Intriguingly, in each model we also see significant effect from the imigel itself plus checkpoint inhibition without cryoablation. At 90 days, in the CT‐26 model, the addition of Imigel to checkpoint inhibition immunotherapy significantly increases 90‐day survival to 46% (6/13) compared to the control experiments CPI alone 0% (0/10) (p = .0045 Log rank test). Similarly, addition of the gel (without imiquimod) + CPI has a significant effect with a 90 day 35% (11/31) compared to CPI alone, (p = .02, Log rank test). In the CT‐26 model, the imigel + CPI versus gel + CPI are not significantly different (46% vs 35% 90‐day survival p = .54, Log rank test). Adding cryoablation to imigel however does have a significant increase in survival compared to CPI+gel alone (57% vs 35% p = 0.04 Log rank test) suggesting some degree of an additive effect of the triple combination. As expected, Imigel acts similarly to serial imiquimod injection. For example, the efficacy with imigel is similar to the efficacies with 6 daily injections of imiquimod alone. (CPI with cryoablation and serial imiquimod, 46.7% (7/15) 90‐day survival) and (serial imiquimod with CPI, 57% (8/14) 90‐day survival) (Figure 4B). Again, all mice that survived to 90 days had full tumor regression, while CPI only and treatment non‐responders had significant tumor growth until euthanasia (Figure 5A).

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.

The interesting effect of the gel alone seen in the CT‐26 model does not replicate in EMT‐6, with CPI + gel having a 0% survival effect (0/12), at 90 days. Similarly, in the EMT‐6 model there was limited 90‐day survival when using CPI alone (0%,0/7) or CPI + cryoablation (0%, 0/8). Instead, the breast cancer model appears to require imiquimod for efficacy of inducing an abscopal effect. Imigel significantly improves response to 20% (3/15), and triple combination therapy (immunotherapy + cryoablation + Imigel) had a 5.6% (1/18) 90‐day survival compared to gel (p < 0.0001 Log Rank Test), CPI alone(p < .0001 Log rank test), or CPI + cryoablation (p = .0003 Log rank test) (Figure 4c). Imigel + CPI versus triple combination therapy was not significantly different. For toxicity, no mice demonstrated significant weight loss or signs of systemic sepsis like findings.

2.3. CT‐26 Model Intratumoral Flow Cytometry and Cytokine Release Analysis

When evaluating tumor growth curves, interestingly, in both models, this increase in survival is due to an all‐or‐nothing abscopal response, with either complete regression in the bilateral tumors (treated and untreated) or almost no effect for imigel + CPI (Figure 5a). Almost all of the imiquimod exposed treatment groups demonstrate this complete regression effect for the majority of treated mice if they responded. A few mice in the CT‐26 or EMT‐6 model for cryoablation + imigel or cryoablation + serial imiquimod demonstrate later onset recurrence in the contralateral tumors (Figure S4, Supporting Information)

Given these results, we explored the immune response profile of immune stimulation from CT‐26 colorectal cancer models from imigel application and systemic checkpoint inhibitor therapy. Evaluation of the ipsilateral injected and contralateral non‐injected tumors by flow cytometry demonstrates no significant increase in the absolute number of CD8+ T cells relative to CPI‐only treated mice. However, we do see a significant increase in the percentage of activated CD8+ T cells, among both imiquimod and Imigel treated mice as assessed by the percentage of dual CD44+PD1+ T‐cells among CD8+T‐cells. A significant increase in T cell activation is seen in both injected and non‐injected tumors with Imigel (p = 0.01 ipsilateral and p = 0.03 contralateral) compared to CPI only (Figure 5B). Granzyme B can be used as a marker of CD8 T activation. Immunofluorescence of this marker qualitatively, but non‐quantitatively, demonstrates increase in cytotoxic cell activation within bilateral tumors with imigel treatment (Figure 5C). Quantifying this visual result is limited by the potential sampling bias from slice artifact.

In evaluating cytokines released by the treatment, of note, imiquimod itself is known to induce a significant increase in IL‐1, IL‐1RA, IL‐6, IL‐8, IL‐10, IL‐12p40, TNF, IFN‐alpha, G‐CSF, among others.^{[ 34 ]} As such, we evaluated the effect of the gel and cryoablation compared to serial imiquimod ipsilateral fold change release compared to ipsilateral serial imiquimod. Compared to imiquimod alone, imigel notably increases TNF‐alpha (1.9×) (pro‐inflammatory), IL‐4(3.9×), and IL‐5 (2.5×) (an eosinophilic response, respectively), while adding cryoablation demonstrates an increase in pro‐inflammatory cytokines TNF‐alpha (1.9×), IL‐6 (11.8×), and CXCL‐2 (3×), also eosinophilic IL‐5 (2.6×), IL‐4 (2.5×), and pro‐macrophage inflammatory markers CCL‐4 (3×) and IL‐25 (2.4×) (Figure 5D). Comparatively in the contralateral cytokine evaluation, we see a general mild decrease in cytokines in imigel and with cryoablation compared to serial imiquimod. This may be due to the differing release profile of Imigel versus daily imiquimod. The cytokine release suggests that the gel and cryoablation are complementary with the improved efficacy from the robust IL‐6 response seen in cryoablation.

3. Discussion

Cryoablation as a locoregional therapy has offered the tantalizing promise of a local therapy inducing distant effects on metastatic disease. This effect, though, has been rare clinically, particularly among checkpoint inhibitor resistant cancers, both pre‐clinically and clinically. Co‐delivered imigel provides a potential method to overcome local barriers to the anti‐tumoral immune response, facilitate systemic anti‐tumoral immunity and increase the abscopal effect of percutaneous cryoablation + CPI for patients with metastatic disease.

While many intratumoral immunotherapy drugs and drug types are in development, their effective delivery remains underappreciated. Limitations to intratumoral immunotherapy have become clear, which include the leaking of drug away from target, the need for frequent injections, and the inability to confirm on‐target delivery of therapy.^{[ 18} , ^{24 ]} We have addressed these limitations by the development of an injectable controlled‐release immunoadjuvant depot with the capacity to enable image‐guided delivery and confirmation, which thereby can unlock the full potential of existing immunoadjuvant drugs and those in development. The use of our imageable depot‐forming hydrogel can be expanded to many intratumoral drugs in development, especially hydrophobic small molecules.^{[ 8} , ¹⁸ , ^{19 ]} In this study, we optimized our drug delivery around co‐delivery with an ablative therapy, with the goal for a logarithmic release to coincide with antigen release.

Interestingly, the ability of imigel itself to induce an abscopal effect itself was a welcome surprise. By results of the survival study, the contribution of imiquimod versus cryoablation appears to be different based on the tumor type. In the CT‐26 colorectal cancer model, the combination of cryoablation and imigel appears additive with at least some effect from the gel itself. In the EMT‐6 model, the effect of imigel is nearly identical to that of cryoablation + imigel itself. These results are consistent with cryoablation and imigel being potentially additive and complimentary with imigel and the gel providing a significant boost.

Imigel is expected to hit less regulatory costs for translation given the decades of clinical experience with imiquimod. The mechanism of TLR 7 agonists such as imiquimod are well known. Briefly, these innate markers activate dendritic cells and other antigen presenting cells within tumors.^{[ 35} , ^{36 ]} When these cells are activated within a tumor they travel to tumor‐draining lymph nodes to in turn activate cytotoxic CD8 T cells. Cytotoxic T cells then are directed to tumor to mediate tumor‐cell killing. Our evaluation of the tumor infiltrate similarly matches these expectations of increases in activated CD‐8 T‐cells. Interestingly, our all‐or‐nothing abscopal response model provides a unique pre‐clinical model to study what other stochastic factors are occurring to explain why some tumors completely regressed and others did not and is currently an active area of study.

Our cytokine analysis suggests both a pro‐inflammatory and interesting eosinophilic response from cryoablation and the gel itself over the usual effects of imiquimod, consistent with prior reports of the need for eosinophilic response for cryoablation immunotherapy efficacy,^{[ 37 ]} and it is interesting that the gel itself can induce that response, possibly explaining its ability to also reproduce an abscopal effect even without imiquimod in the CT‐26 model. The eosinophilic effect could potentially be explained with the immunogenicity of certain polymers including PEGs.^{[ 38 ]} In comparison, other groups have focused on augmenting cryoablation via trapping antigens nearby with nanoparticles to augment the abscopal effect, or shaping the ice‐ball with nanomaterial adjuncts, a complementary approach.^{[ 10} , ^{11 ]} Given the gel appears to be mimicking the eosinophilic response seen from cryoablation, this study provides the basis for future work in optimizing percutaneous gel depots to potentially get the same effect from cryoablation itself without needing the second ablation therapy.

A notable challenge with working with imiquimod is its poor aqueous solubility, which limits large concentrations in saline to be injected as suspensions with impractical heterogeneity.^{[ 14} , ^{36 ]} Prior attempts at intratumoral imiquimod delivery using PLGA microparticles have been limited to doses of around 0.1–0.4 mg mL⁻¹ and required combination with photoablation for efficacy, thereby limiting use for the much more commonly seen deeper lesions.^{[ 39 ]} Multiple groups have attempted to use higher activity but non‐clinical TLR7/8 agonists such as resiquimod (R848) or MEDI9197 to get around these solubility limitations, however, even using polymers such as Pluronics (PEO‐PPO‐PEO) or PLGA nanoparticles, multiple injections were required, for example ten injections with drug loadings ranging from 20–75 ug of resiquimod or novel variants thereof per injection.^{[ 21} , ⁴⁰ , ⁴¹ , ⁴² , ⁴³ , ^{44 ]} We were able to deliver a large depot of imiquimod through the encapsulating, micellar structure of PLGA‐PEG‐PLGA (increasing its aqueous solubility by ≈2000‐fold). This polymer has been used previously for chemotherapy delivery and has been well tolerated clinically, though the formulation has not been shown to have significant efficacy.^{[ 25 ]} Previous evaluations of PLGA‐PEG‐PLGA thermo‐gels for chemotherapy formulations encountered challenges with achieving full‐tumor coverage.^{[ 26} , ^{45 ]} However, in the context of concurrent treatment with checkpoint inhibitors, complete tumor coverage is not necessary, as it is the subsequent activation of cytotoxic CD8 T cells that mediates tumor killing, locally, and distally. Combining a logarithmic release of imiquimod with an ablative therapy, a single dose therapy may be able to be found.

The radio‐opaque intratumoral imigel enables clinical translation for deeper lesions. We were able to show the imigel is able to concurrently incorporate an iodinated contrast agent, Iopamidol, and demonstrate optimal injection into the tumor ideally avoiding issues of mis‐targeting as reported clinically.^{[ 18} , ^{24 ]} This visualization opens the door for intratumoral immunotherapy to deep visceral tumors where mis‐targeting, as reported, can be significantly more problematic.^{[ 18} , ^{24 ]}

Limitations for the study include utilization of a murine tumor model with tumor implantation via xenograft. By selecting and treating tumors that had measured to 6 mm while immunotherapy was already active, tumors chosen were immunotherapy resistant to begin with, suggesting that our results likely under‐estimate the efficacy of combination therapy in immunotherapy naïve tumors. In addition, as compared to more novel immunoadjuvants, we utilize an FDA‐approved drug imiquimod, that has a broader activation profile than an even more targeted agent might have. This limitation reduced the biochemical control in situ but provides for likely greater translatability to patients.

Overall, a single intratumoral injection of our engineered logarithmic‐release Imigel in combination significantly increases the abscopal rate of cryoablation in two different immunotherapy resistant tumor models. As suggested by our immune‐assays, Imigel achieves these improved outcomes through increased activation of cytotoxic CD8 T cells, and in the ipsilateral tumor, an increased TNF‐alpha, IL‐4, and IL‐5 release. This strategy brings personalized therapy to cancer patients with metastatic disease, effectively immunizing them to their own cancer via a local therapy that could be done concurrently with standard of care percutaneous cryoablation.

4. Experimental Section

Statistical Analysis

In murine survival studies, statistical analysis was done using Kaplan‐Meier and Log‐rank tests. Any comparison of means was done using a 2‐tailed t‐Test. Statistical studies were done in Prism (Graphpad, Boston, MA)

Ethics

All studies were completed under an approved animal care and use protocol, 2017N000163, as well as an IRB protocol, 2020P003099, for the retrospective chart review.

Materials

PLGA (1500 g mol⁻¹)‐PEG (1500 g mol⁻¹)‐PLGA (1500 g mol⁻¹) copolymers with lactide‐to‐glycolide ratios (LA/GA) = 1:1, 2:1, 3:1, 5:1 were purchased from NanoSoft Polymers (Winston‐Salem, NC) and stored at 4 °C. NMR and GPC quality control data provided by NanoSoft Polymers. The copolymer, imiquimod (98%, Sigma‐Aldrich, St. Louis, MO), Iopamidol (99.8%, MedChemExpress (Monmouth Junction, NJ), and Dulbecco′s Phosphate Buffered Saline (DPBS, FisherScientific, Hampton, NH) were used as received.

Imigel Preparation

Typically, 1 g of PLGA‐PEG‐PLGA was dissolved in 4 ml 0.9% saline solution (Baxter, Deerfield, IL) to obtain a 25% weight/volume (w/v) concentration at 4 °C over 2 days and vortexed/stirred daily. Importantly, the normal saline cannot contain the preservative phenol, commonly found in preservative containing commercial saline solutions. After a homogeneous hydrogel solution was observed by stirring with a spatula, imiquimod was added at 24 mg to the batch, the dispersion was vortexed and allowed to dissolve at room temperature over 2 days. Some of the gels received Iopamidol as contrast agent at 100 mg mL⁻¹, which was left to disperse or dissolve over 1 day. The resulting Imigel or Imigel + Iopamidol was stored at room temperature, shielded from evaporation, and used within 1 week from full dissolution to prevent hydrolysis.  (Figure S1, Supporting Information)

In Vitro Release Studies

Drug release kinetics of imiquimod from various imigel formulations were studied by placing aliquots of drug‐loaded hydrogel into 50 mL conical tubes at 100 rpm at 37 °C, releasing drug into DPBS. 50 µL of Imigel were added to the bottom of 50 mL conical Falcon tubes or in a dialysis tubing (D‐tube Dialyzer Maxi, MWCO 3.5 kDa) at room temperature and brought to gel state by incubating at 37 °C for 30 min while DPBS as receiving fluid was preheated at the same temperature. After Imigel solidified, 50 mL DPBS (pH 7.4), Lactated Ringers (pH 6.8, Vetivex), or Acetate Buffer (pH 5, 0.03 m Acetic acid, and 0.07 m Sodium Acetate in DI water) were added slowly to the conical tubes. These containers were then incubated at 37 °C under shaking at 100 rpm. 400 µL samples were taken from the receiving fluid with a 1 mL syringe through 0.2 µm filters (PALL super membrane low protein binding non‐pyrogenic) to avoid accidental removal of gel. Concentration of imiquimod in the samples was assessed eluting the samples on an Agilent 1100 high‐performance liquid chromatography (HPLC) with UV detector at 242 nm over an Eclipse XD8‐C18 column  (150 × 4.6 mm, 3.5 µm pore size) with an acetonitrile/water gradient from 5/95 to 95/5 over 6 min at 1 mL mi⁻¹ n. Formic acid 0.1% (v/v) was added as stabilizer to both the organic and aqueous phases, using millipore water as the aqueous phase and ACN as the organic phase. The injection volume was kept constant at 5 µL. Concentrations were calculated from UV signals using a calibration curve of known concentration imiquimod standards in DMSO/Acetonitrile.

Rheometry

Gel rheology was characterized using a Discovery Hybrid Rheometer (TA instruments, New Castle, Delaware) using 20 mm parallel plates modified with 600 grit adhesive‐backed sandpaper to eliminate slip effects. Frequency sweeps at 10 and 40 °C were performed to measure storage and loss modulus at a strain of 1%. Flow sweeps were performed at varying shear rates from 0.1–100 1/s at 20° and 37° to compare viscosity and shear stress. These same flow sweeps were done after injection through an 18‐gauge needle for the extreme ratios of 1:1 and 5:1.

Injection Pressure Measurements

300 µL of Imigel at room temperature (≈20 °C) and after 30 min at 37 °C incubation, as well as normal saline at room temperature were compressed in a 3 mL syringe at 200 µL s⁻¹ through a 22‐gauge 15 cm Chiba needle using an Instron machine. A 22‐gauge 15 cm Chiba needle was considered clinically to be the likely most difficult and clinically relevant injection scenario.

Dynamic Light Scattering

Particle size distributions were analyzed on a Malvern Zetasizer. 500–1000  µL aliquots of imigel formulations were evaluated in 70 µL plastic cuvettes (BRAND, 70–850 µL) at room temperature and 37 °C. Material parameters were used for PEG as the material and water as the solvent. Particle size averages and distributions were then taken and compared.

Injection Rheometry and DLS Measurements

Rheometry and DLS data were collected pre and post injection through an 18‐gauge needle. For the pre‐injection measurement, ≈800 µL of imigel (1:1, 5:1) were pipetted into a cuvette and measured in the DLS at 25 °C with the aforementioned settings. 500 µL of imigel were pulled up from the pre‐injection cuvette using an 18‐gauge needle and injected into a separate cuvette for DLS measurement with the aforementioned settings at 25 °C. These samples were then measured in the rheometer using the aforementioned settings.

MicroCT Scans

Mice (N = 3) were injected subcutaneously dorsally with CT‐26 cells (1–2 million cells) in Matrigel. After 14 days, the tumors were injected with 50 µL 6 mg mL⁻¹ imigel containing Iopamidol (100 mg mL⁻¹) and imaged with a microCT scanner to confirm the radio‐opacity of the gel and visibility in vivo.

Cell Culture

CT‐26 and EMT‐6 tumor cells lines (ATCC, Manassas, VA) were cultured for in vivo mouse models. These cells were cultured in flasks of T125 mL RPMI 1640 (Sigma Aldrich, St. Louis, MO) medium supplemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, St. Louis, MO) at 37 °C in a 5% CO₂ atmosphere.

Dual Tumor Model In Vivo Experiments

Per experimental arm 10–15 Balb c⁻¹ mice were injected with ≈10⁶ cells of CT‐26 or EMT‐6 cells, day 0 (Figure 3) in bilateral flanks. Immunotherapy (anti‐PD‐1 (3.9 mg mL⁻¹), anti‐CTLA‐4 (2.2 mg mL⁻¹) co‐immunotherapy was given intraperitoneally (Sigma‐ Aldrich) every 3 days from day 3–12. Mice with primary and contralateral tumors of at least 6 mm were chosen to be randomized to various treatment arms evaluating CPI with and without cryoablation, imiquimod drug‐only injection, PLGA‐PEG‐PLGA only injection, or imigel (3:1) injection. The imigel, gel alone, or serial imiquimod suspensions were injected as 50 µL injections using a 26‐gauge needle. For cryoablation, parameters included a 30 s freeze, followed by 2 min thaw, followed by a 30 s freeze with goal of ≈50% tumoral freezing. When using cryoablation, the drug formulations were injected 5 min post final freeze, with a mild pause during injection to ensure no push back via the tract into the remaining viable tumor. Imiquimod alone was prepared as a suspension by 30 min of sonication at 1 mg mL⁻¹ in normal saline. Injections for imiquimod alone were done as a total 50 µL 1 mg mL⁻¹ injections once per day for six total doses. Specifically, Imigel delivered a total of 300 µg in one injection in two 25 µL injection depots within the tumor at the same injection session for only one session. Imiquimod suspensions were delivered as two 25 µL depots at 1 mg mL⁻¹ (50 µg injection⁻¹ day⁻¹) over the course of 6 days leading to a total 300 µg dose. Mice were then followed for tumor size, with tumor measurements twice weekly for up to 90 days when the mice were sacrificed. Mice were sacrificed if the tumor grew to be at least 2 cm in size, ulcerated, or the mice showed other signs of stress, such as >20% weight loss.

Flow Cytometry and Cytokine Analysis

Additional groups of 4 mice were injected with CT‐26 and treated as above. 6 days post local intervention, mice were euthanized, and ipsilateral and contralateral tumors excised. In all cases tumor samples were minced and then incubated in a collagenase solution (1 mg mL⁻¹ in RPMI—1640) for ≈45 min at 37 °C. Tumors were processed into a single cell suspension using a 70 µm cell strainer.

Supernatant from the above process was analyzed for cytokine and chemokine levels. Cytokine and chemokine levels were measured using a preconfigured ProcartaPlex Multiplex Immunoassay (ThermoFisher Scientific), according to the manufacturer's instructions. Briefly, magnetic beads were added to a 96‐well plate and then washed with 1X Wash Buffer. Universal Assay Buffer was added to the wells, followed by the samples, antigen standards, and blanks (additional Universal Assay Buffer). The plate was incubated for 2 hours at room temperature on a plate shaker at 500 rpm. The wells were then washed with 1× Wash Buffer and detection antibody was added, followed by incubation for 30 min at room temperature on a plate shaker at 500 rpm. SAPE (streptavidin, R‐phycoerithrin conjugate) was then added, followed by incubation for 30 min at room temperature on a plate shaker at 500 rpm. The plate was then washed, and Reading Buffer was added to each well. The beads were resuspended by shaking the plate at 500 rpm for 5 min at room temperature. The plate was then run on a Luminex 200 instrument (Luminex Corporation).

For flow cytometry, using the cells harvested as above, cell viability was assessed by trypan blue staining, and 1 × 10⁶ viable cells were stained for flow cytometry analysis. Cells were washed with PBS and stained with a 1:1000 solution of ZOMBIE Aqua (from ZOMBIE Violet Fixable Viability Kit, Biolegend) and incubated on ice for 15 min. After washing twice with cell staining buffer (Biolegend), the following extracellular antibodies were added: CD45.2‐FITC (clone 30‐F11, Biolegend), CD3‐Pacific Blue (close 17A2, Biolegend), CD8a‐Brilliant Violet 421 (clone 53–6.7, Biolegend), CD44‐APC/Cy7 (clone IO7, Biolegend), PD‐1‐APC (clone 29F.1A12, Biolegend) at 100 µL total volume diluted with cell staining buffer and incubated for 20 min on ice in the dark. Cells were resuspended in cell staining buffer, and the samples were analyzed on an Aurora (Cytek Biotechnology). Data were gated using FlowJo software (FlowJo LLC). Doublets and cell clumps were excluded by gating along the 1:1 line for forward scatter height (FSC‐H) versus forward scatter area (FSC‐A). Cellular debris and dead cells were excluded by side scatter area (SSC‐A) versus FSC‐A and viability stain, and immune cells were then selected based on CD45 expression. Gating for individual markers was determined by fluorescent minus one (FMO) control panels and unstained controls. Gates were then confirmed using backgating.

Immunofluorescence

Immunohistochemical staining of formalin‐fixed paraffin‐embedded excised tissues was performed. Antigen retrieval was conducted in citrate buffer (EMD Millipore, Burlington, MA) and blocking was conducted with Dako Protein Block Serum‐Free buffer (Agilent, Santa Clara, CA). Cell nuclei were visualized with DAPI (1 µL/5 mL PBS) (BioLegend, San Diego, CA). Granzyme B expression was visualized with mouse monoclonal anti‐granzyme B antibody (200 µg mL⁻¹, diluted 1:100 in PBS) (sc 8022) conjugated to Alexa Fluor 546 (Santa Cruz Biotechnology, Dallas, TX). Fluorescent images were captured on Biotek Cytation 5 Imaging Reader (Agilent, Santa Clara, CA) with constant LED exposure, integration, and camera gain for all tissues.

Conflict of Interest

A.S., E.W., J.‐G.R., G.T., U.M., R.L. are co‐inventors on a patent application describing the Imigel system. A.S. is a founder of CareSignal, a digital health company, now Lightbeam Health, and receives consultancy fees from Boston Scientific. E.W. receives consultancy fees from Boston Scientific. Complete details of all relationships for profit, not for profit for G.T. can be found at the following link: https://www.dropbox.com/sh/szi7vnr4a2ajb56/AABs5N5i0q9AfT1IqIJAE‐T5a?dl=0&preview=summary‐COI‐traverso‐2023‐03‐07.pdf. To the best of his knowledge, R.L. has no competing interests in relation to this paper. For a complete list of R.L.’s general competing interest disclosures, please visit: https://www.dropbox.com/s/yc3xqb5s8s94v7x/Rev%20Langer%20COI.pdf?dl=0. Funding for this study was provided in part by a Boston Scientific Grant (E.W.), the Philips RSNA Research award (A.S.), Deshpande Grant (A.S.), Schlaeger Award (A.S.). U.M., E.W. are cofounders, shareholders, consultants (Scientific Advisory Board) of CytoSite BioPharma. All other authors declare they have no competing interests.

Author Contributions

A.S., J.‐G.R., and E.W.‐K. contributed equally to this work. Conceptualization: A.S., E.W., J.‐G.R., G.T., U.M., A.B. Methodology: A.S., E.W., J.R. Investigation: A.S., E.W., J.‐G.R., A.C., G.N., A.V., E.C., J.S. Visualization: A.S., J.‐G.R., A.C., F.J.F. Supervision: G.T., U.M., R.L. Writing—original draft: A.S., E.W., J.‐G.R., A.C. Writing—review and editing: A.S., E.W., J.‐G.R., A.C., G.N., A.B., A.V., G.T., U.M., R.L., F.J.F.

Supporting information

Supporting Information

Som A., Rosenboom J.‐G., Wehrenberg‐Klee E., Chandler A., Ndakwah G., Chen E., Suggs J., Morimoto J., Kim J., Mustafa A. R., Marcos‐Vidal A., Fintelmann F. J., Basu A., Langer R., Traverso G., Mahmood U., Percutaneous Intratumoral Immunoadjuvant Gel Increases the Abscopal Effect of Cryoablation for Checkpoint Inhibitor Resistant Cancer. Adv. Healthcare Mater. 2024, 13, 2301848. 10.1002/adhm.202301848

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.