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Biomaterials induced host immune responses, and fibrotic overgrowth remains a major barrier to the long-term function of medical devices and biomaterial consisting of tissue grafts. Screening new biomaterials to identify anti-fibrotic formulation requires in vivo testing, which is challenging to multiplex and remains a significant obstacle to progress in this field. Herein, we synthesized a combinatorial chemically modified hydrogel library and developed a cellular barcoding method that enables high-throughput multiplexed in vivo screening of 20 formulations in a single mouse and 100 formulations in a single non-human primate. Our screening method consists of implanting a mixture of biomaterials and each barcoded with human umbilical vein epithelial cells (HUVEC) from different individual donors. Single nucleotide polymorphism (SNP) genotypes of the cells were utilized as readouts using next-generation sequencing (NGS) to pair the material identity with material performance. Screening of the library using a xenogeneic transplantation model identified three novel lead hydrogel formulations (Z4-A10, Z1-A3, and Z2-A19) with improved anti-fibrotic properties that enable long-term cell viability. Z4-A10 was used to encapsulate human islets and validated for long-term glycemic control in an STZ-induced C57BL/6J diabetic mouse model. Leads, Z1-A3 and B2-A17, were further validated as immunomodulating coatings for medical-grade catheters to prevent fibrosis and occlusion, highlighting the translation of our screening approach and findings to other medical devices. Our results suggest that the developed cellular barcoding method and in vivo multiplexed screening technique can be leveraged to identify biomaterials for a wide range of clinical applications. Significantly, our newly discovered leads can improve the long-term performance of medical devices and encapsulated cell-based therapeutics.

Pancreatic cancer is often diagnosed at advanced stages and responds poorly to chemotherapy. Because high tumor T cell infiltration corresponds with better clinical outcomes in pancreatic cancer patients, immunotherapy has gained significant interest over the last decade for the treatment of recurrent pancreatic cancer. IL-12 is a proinflammatory cytokine with pleiotropic effects including activation of CD8+ T cells and NK cells. Unfortunately, systemic high dose IL-12 administration led to severe toxicities in clinical trials which has limited further development of this cytokine as a cancer therapeutic. To address this limitation, we developed an implantable cytokine delivery platform to allow for local administration of IL-12. These cytokine factories, composed of genetically engineered epithelial cells encapsulated in biocompatible polymers, allow for safe and controlled dosing in vivo. Tumor adjacent administration of IL-12-based cytokine factories caused reduction of intraperitoneal pan02 tumor burden by 80% after only 1 week of treatment in mice with pancreatic cancer. Single cell RNAseq of the tumor adjacent immune cells at this time point showed 2x more T and NK cells present in the IL-12 treated mice than untreated mice suggesting modulation of the tumor microenvironment via immune cell infiltration. Importantly, the necessary IL-12 dose was well tolerated in all mice without signs of toxicity for 180 days. In efforts to evaluate the translatability of this platform, we further tested IL-12-based cytokine factories in a non-human primate. The cytokine factories produced a high local IL-12 concentration without substantial leakage into the systemic circulation and were well tolerated by the primates as shown by the lack of fever or weight loss, as well as the lack of renal or liver toxicity. Our findings highlight the therapeutic potential of IL-12 treatments when administered locally via cytokine factories in preclinical animal models. Further, these findings provide rationale for future development and clinical testing of cytokine factories for treatment of a wide range of metastatic peritoneal cancers in humans. Citation Format: Amanda Nash, Samira Aghlara-Fotovat, Bertha Castillo, Annie Nguyen, Andrew Cui, Andrea Hernandez, Peter Rios, Sofia Ghani, Ira Joshi, Chunyu Xu, Rahul Sheth, Weiyi Peng, Jose Oberholzer, Amir Jazaeri, Omid Veiseh. IL-12-based cytokine factories modulate tumor microenvironment to eradicate pancreatic tumors in mice and are well tolerated in non-human primates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3547.

Pro-inflammatory cytokines have been approved as a cancer immunotherapy for treatment of metastatic melanoma and renal carcinoma for over 30 years. However, widespread use of cytokine therapy in the clinic is limited by its short half-life in circulation and the associated toxicities that emerge as a result of high systemic exposure. To overcome these limitations, we developed a clinically translatable cytokine delivery platform, called cytokine factories, composed of genetically engineered epithelial cells encapsulated in biocompatible polymers. These cells are able to produce a wide range of natural cytokines (IL2, IL7, IL10, or IL12) and allow for controlled and predictable dosing in vivo. In vivo administration of cytokine factories created a high local cytokine concentration (IP space) without substantial leakage into the systemic circulation. Local, or tumor adjacent, administration of pro-inflammatory (IL-2-based) cytokine factories caused reduction of tumor burden by 70% after only 1 week of treatment when delivered as a monotherapy to mice with malignant pleural mesothelioma (MPM). Importantly, when administered in combination with local anti-PD1 injections, these cytokine factories lead to eradication of these highly aggressive tumors in 7/7 treated mice. To validate the translatability of this platform, we evaluated the safety profile in non-human primates. Significantly, this platform produced local and systemic T cell biomarker profiles that predict efficacy without renal, liver, or general toxicity in non-human primates. Our findings demonstrate the safety and efficacy of cytokine factories in preclinical animal models and provide rationale for future clinical testing for the treatment of metastatic peritoneal cancers in humans. Citation Format: Amanda Nash, Samira Aghlara-Fotovat, Andrea Hernandez, Bertha Castillo, Alexander Lu, Aarthi Pugazenthi, Peter Rios, Sofia Ghani, Ira Joshi, Douglas Isa, Chunyu Xu, Rahul Sheth, Weiyi Peng, Jose Oberholzer, Amir Jazaeri, Hee-Jin Jang, Bryan Burt, Hyun-Sung Lee, Ravi Ghanta, Omid Veiseh. Evaluation of implantable cytokine factories in combination with checkpoint inhibitors for eradication of malignant pleural mesothelioma (MPM) tumors in mice with safe and predictable dosing in non-human primates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4189.

Recombinant IL-2 has been approved for the treatment of melanoma and renal cancer. However, IL-2 therapy is limited by its short half-life in circulation and the associated toxicities that emerge as a result of high systemic exposure. To overcome these limitations, we developed a localized cytokine LOCOcyteTM platform composed of allogeneic and engineered cells which produce potent immune effector molecules for local delivery with temporal regulation of activity. Administration of AVB-001 in mice and non-human primates demonstrated that IL2 local concentration (IP space) was 100x higher than the systemic concentration. Additional studies in mice demonstrated dose-dependent levels of IL2 in the intraperitoneal cavity in mice. Treatment of peritoneal tumors in mice using a single administration of AVB-001 provided sustained eradication of peritoneal tumors in an ovarian cancer mouse model. Our data in mice confirmed local increases in activation (CD25+CD8+) (8.6 fold) and proliferation (Ki67+CD8+)(3.4 fold) of cytotoxic T cells within the IP space in comparison to sham mice. Finally, a single administration of AVB-001 led to therapeutic levels of IL2 in the IP space in NHP and produced local and systemic T cell biomarker profiles that predict efficacy, AVB-001 was well tolerated by all animals with no abnormal clinical observations during the study. Our findings demonstrate that the LOCOcyteTM platform has a dose-dependent PK/PD effect, is safe and efficacious in animal models and enables itself to deliver a diverse set of cytokines alone or in combination which is presently explored. Combined, our pre-clinical results provide a rationale for testing AVB-001 in ovarian cancer patients.