Background The clinical outcome of combination immunotherapy could be amplified hinging on the elimination of adverse effects associated with systemic off-target immune activation. Combination immunotherapy-induced toxicities, some of which are lifelong or life-threatening, typically necessitate treatment discontinuation. To this end, intratumoral immunotherapy delivery can augment in situ bioavailability and prevent systemic dissemination, thus enhancing therapeutic index. However, variable intratumoral injection techniques and leakage into the systemic circulation due to bolus administration are clinical concerns. To address these challenges, we present a long-acting intratumoral delivery seed for sustained in situ release of immunotherapeutics. In situ delivery enables the use of a multi-drug immunotherapeutic cocktail, which is likely clinically unfeasible due to systemic administration associated toxicities.

Methods The long-acting intratumoral delivery seed comprises of biodegradable polymers, poly(ε-caprolactone) (PCL) and poly (D, L-lactic-co-glycolic acid) (PLGA), which are used in United States Food and Drug Administration approved therapeutics. The intratumoral delivery seed is cylindrical, comparable to a grain of rice, with a hollow central core acting as the drug reservoir. Intratumorally insertion adopts the one-time minimally-invasive clinical trocar procedure used for brachytherapy seed. Controlled drug release occurs autonomously through the nanoporous material directly into the tumor. We used up to 5-drug immunotherapeutics combination, consisting of CD40 agonist and CTLA-4 antagonist antibodies, interleukin-12, STING agonist, and resiquimod. We evaluated treatment efficacy in murine models of triple negative breast cancer (4T1) and pancreatic cancer (KPC). Tumor volume, body weight and temperature were monitored.

Results Sustained intratumoral delivery of the immunotherapeutic cocktail led to complete regression of 4T1 tumors in 80% of mice. Tumor rechallenge study showed 100% long-term rejection, substantiating that durable antitumor immunity was attained. In a KPC bilateral tumor model, where only one tumor received treatment with the intratumoral delivery seed, an abscopal response was achieved in the untreated lesion. This highlights the potency of the long-acting intratumoral delivery seed for in situ treatment and concomitant activation of systemic antitumor immunity. Immune analyses demonstrated immunomodulation within the tumor locally as well as systemically. Moreover, the long-acting intratumoral delivery seed obviated toxicities commonly associated with systemic treatment, suggestive of safe and effective treatment.

Conclusions The long-acting intratumoral delivery seed offers the advantage of simultaneous multi-drug combination delivery in a sustained manner without inducing toxicities. Further, it could present an effective and safe approach for neoadjuvant immunotherapy or long-term treatment of inoperable tumors. Moreover, drug- and tumor-agnosticity allow for broad application across different cancers and drug combination permutations.

Acknowledgements This work is supported by Nancy Owens Breast Cancer Foundation and Houston Methodist Research Institute (A.G., C.Y.X.C.).

Ethics Approval Animal experiments were conducted following the Animal Welfare Act and the National Institutes of Health Guide for Care and Use of Laboratory Animals, with protocols approved by the Institutional Animal Care and Use Committee at the Houston Methodist Research Institute.