Abstract PS3-12-28: Hydrogel-mediated local anti-CTLA-4 delivery potentiates immune activation after breast tumor cryoablation

Abstract Background: Triple-negative breast cancer (TNBC) is highly aggressive with few treatment options and a significant risk of recurrence and metastasis owing to immune suppressive signals. Cryoablation offers dual advantage of local tumor necrosis while simultaneously stimulating anti-tumor immune responses via releasing tumor-associated antigens offering systemic control. Combining cryoablation with immune checkpoint inhibitors (ICIs) fosters stronger anti-tumor immunity, including abscopal effects. Nevertheless, systemic administration of ICIs like anti-CTLA-4 lead to widespread immune activation and immune-related adverse events (irAEs). To enhance the efficacy of immunotherapy while reducing systemic toxicity, we developed an injectable crosslinked chitosan-alginate hydrogel for localized and sustained delivery of anti-CTLA-4 - vinylated Poly(vinyl alcohol) conjugate in the tumor bed. Methods: Pharmacokinetic studies were conducted in naïve BALB/c mice to compare local (mammary fat pad, MFP; injection) and systemic (intraperitoneal; IP) administration of an anti-CTLA-4 hydrogel formulation (200 µg for direct comparison). Mice were bled on days 1, 4, and 8, and were sacrificed on day 14 for necropsy, during which plasma and mammary fat pad tissues were analyzed for anti-CTLA-4 concentrations using ELISA. The peripheral blood was further examined for T cell (CD4+, CD8+) activation by ICOS expression. Therapeutic efficacy was evaluated in our bilateral 4T1 tumor-bearing cryoablation mouse model; the left tumor was cryoablated and mice were treated with 200 µg of anti-CTLA-4 either systemically (IP) or locally via the hydrogel. Tumor size, hydrogel retention, T cell activation, and systemic anti-CTLA-4 levels were assessed post-treatment on days 7 and 14. Results: Non-tumor bearing mice treated with gel-formulated anti-CTLA-4 through local injection into the MFP exhibited significantly lower circulating antibody levels compared to those receiving systemic (IP) administration, as measured by ELISA (p 0.05). At day 14, locally treated mice showed an approximately 10-fold increase in anti-CTLA-4 concentration within the mammary fat pad tissue and greater tissue mass relative to systemically treated controls (p 0.05). Flow cytometry analysis revealed elevated ICOS expression on CD4+ and CD8+ T cells in the blood at day 8 in both groups, indicating intact immunostimulatory activity of the antibody; by day 14, no significant differences in ICOS expression were observed. In a bilateral 4T1 breast cancer cryoablation mouse model, local administration of anti-CTLA-4 into the cryoablated tumor site resulted in significantly higher antibody retention at the tumor and lower systemic exposure compared to IP delivery. At day 7, locally treated tumors were larger and contained higher anti-CTLA-4 concentrations reflecting gel retention, while by day 14, tumors in both groups were nearly fully reabsorbed. Peripheral blood analysis confirmed lower plasma levels of anti-CTLA-4 in the locally treated group. Flow cytometry showed comparable ICOS expression on T cells from the PB across both groups at days 7 and 14, indicating equivalent T cell activation. Conclusions: Local delivery of anti-CTLA-4-vinylated PVA conjugates using a chitosan-alginate hydrogel after cryoablation enhanced retention of anti-CTLA-4 in the tumor and provided equivalent activation of T cells in the blood, reducing systemic antibody exposure. This strategy presents a promising approach to minimize irAEs without compromising therapeutic efficacy. Moreover, this hydrogel-based method requires additional validation with lower doses of anti-CTLA-4 to further de-escalate treatment-associated toxicity. It supports its future application in clinical settings to enhance combinatorial cryoablation and ICI therapy. Citation Format: F. Sardela de Miranda, Y. Dauletov, R. L. Babcock, M. F. Mahecha, P. J. Gukhool, S. P. Singh, H. S. Gill, R. Layeequr Rahman, M. W. Melkus. Hydrogel-mediated local anti-CTLA-4 delivery potentiates immune activation after breast tumor cryoablation abstract. In: Proceedings of the San Antonio Breast Cancer Symposium 2025; 2025 Dec 9-12; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2026;32(4 Suppl):Abstract nr PS3-12-28.