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Drug Delivery

Drug Delivery - Poster Session E

Poster X12 - Remodeling the tumor immune microenvironment with tunable supramolecular hydrogels

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Over the last few decades, cancer immunotherapy has shown remarkable progress, particularly in treating "hot" immune-infiltrated tumors such as melanoma and bladder cancer. This progress is due to revolutionary approaches like immune checkpoint inhibitors (ICI) and adoptive cell transfer therapy. However, "cold" immune-suppressive tumors remain a significant challenge due to the immune system’s inability to infiltrate their tumor microenvironment (TME). New clinical strategies aim to remodel these TMEs, transforming them from "cold" to "hot." A noteworthy catalyst to achieve this is the interleukin protein IL15, a pro-inflammatory cytokine that activates and expands immune cells like natural killer (NK) cells and T cells. Another promising immunostimulatory candidate is the tumor necrosis factor receptor CD40 agonist (CD40a), which stimulates the expansion of tumor antigen-specific cytotoxic CD8+ T cells by acting on myeloid cells such as antigen-presenting cells. The main challenge with these drugs however is their tolerability, often leading to toxic side effects due to off-target effects. Hence, there is a crucial need to balance immune activation efficacy with toxicity. To address these challenges, we are developing injectable hydrogels engineered for controlled and localized release of these immunomodulatory agents. Additionally, we seek to enhance the spatial presentation of IL15 within our hydrogels to favor trans-signaling, which has shown to improve IL15 treatment efficacy by further stimulating NK and CD8+ T cells and inducing memory tumor-specific T cells, crucial for an effective anti-tumor response.

Materials and

Methods:  Liposome Preparation: Liposomes were synthesized using thin film hydration method, followed by three rounds of extrusion (400nm, 200nm and 100nm filters) to achieve a uniform size distribution. The lipid composition was DNPC:DNPG:Cholestorol:NTA-Ni at a molar ratio of 9:1:2:0.379, respectively. The resulting liposomes exhibited an average particle size of 144nm (PDI=0.16). Lipids were purchased from Avanti. Liposomes were then complexed with the desired IL15 cytokines. In-vitro IL15 Detection: The detection of IL15 was conducted using a colorimetric enzyme assay on HEK-Blue CD122/CD132 reporter cells (InVivogen) according to the manufacturer’s protocol. Hydrogel Preparation: Dodecyl-Modified Hydroxypropyl Methylcellulose (HPMC-C12) was synthesized in lab following in-house published protocol. Liposome-based HPMC-C12 gels were prepared by syringe mixing at a 2:10 w/v % ratio, adhering to good manufacturing practices. In Vivo Tumor Model: Female C57BL/6 mice (Jackson Laboratory) were subcutaneously inoculated with 3x10^5 B16F10 cells on each flank. Two weeks post-inoculation, tumors were treated intratumorally with 30μL of the prepared hydrogels. Treatment groups included non-loaded hydrogel, IL15 loaded hydrogel and Il15 + CD40a loaded hydrogels. Mice were then sacrificed 3 days post-treatment, and tumors were harvested for histological analysis. Additionally, mice received a single intraperitoneal dose of 200μL of PDL1 at a concentration of 250ug/mL.

Results, Conclusions, and Discussions: Drug-carrying hydrogels have the potential to overcome the general toxicity observed with highly immunostimulatory agents. Our lab has experience in generating such hydrogels based on hydrophobic interactions between liposomes and biopolymers (Fig. 1A). Furthermore, these gels are injectable and able to regain their structural integrity following shear stress from a needle (Fig. 1B). This allows for controlled and localized administration of immunostimulatory agents. Additionally, as these gels are modular, we can tune their mechanical properties by modifying the size and composition of their components (Fig. 1C-D).
IL15, a proinflammatory and immunostimulatory agent of great interest in cancer therapy, has been shown to be more effective when induced via trans-signaling. Trans-signaling, unlike cis-signaling, requires the targeted cell to bind not only to the soluble IL15 protein but also its IL15 receptor complex, thereby increasing its binding affinity and activating downstream pathways. As our gels allow for various release modalities based on the cargo interaction with the liposomal component, we evaluated IL15 activation in HEK Blue cells using soluble and liposome-tethered forms of IL15 complexes (Fig. 2).
Using a colorimetric enzyme assay to assess IL15 detection, we observed an increased response rate and a lower EC50 in the tethered IL15 group compared to soluble IL15 group (Fig. 2A-B). Next, we complexed IL15 to IL15RA, forming a superagonist, and tethered this to our liposomes. Similar to the previous experiment, we observed an increased response rate and decreased EC50 for tethered superagonist in comparison to its soluble form (Fig. 2C-D). These results suggest enhanced signaling when IL15 complexes are spatially presented on our liposomes.
Bringing the discussion back to cis vs trans signaling, we then compared the responses between tethered IL15 and tethered superagonist (Fig. 2E-F). Here, we observed an increase response rate for the tethered superagonist group compared to the tethered IL15 group. While the current assay is unable to distinguish between cis- and trans-signaling, it is evident that the superagonist complex elicited a stronger response than its IL15 counterpart.
Having demonstrated that our liposome-tethered superagonist showed the strongest IL15 response, we incorporated these into hydrogels for ongoing in vivo TME remodeling studies.

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