NATure BIoMedIcAl eNgINeerINgdeveloped an approach for in-cell site-specific protein phosphorylation to synthesize bioactive proteins fused with a phosphorylated alum-binding peptide (ABP) tag. We used this approach to produce a series of ABP-labelled cytokines, which rapidly adsorbed to alum after simple mixing, and upon i.t. injection were retained in tumours for more than a week. Applied to the cytokine IL-12, this approach dramatically increased i.t. retention of the cytokine and eliminated systemic toxicities seen upon i.t. injection of the free drug, while also increasing anti-tumour efficacy. Moreover, a single i.t. dose of alum-anchored IL-12 elicited strong IFN-γ-dependent collaboration between innate and adaptive immune cells, producing robust systemic anti-tumour responses in multiple poorly immunogenic preclinical models when combined with systemic checkpoint blockade therapy. ResultsTargeted phosphorylation via an in-cell approach is robust. A single kinase, Fam20C, is responsible for phosphorylation of

Adipose tissue engineered models are needed to enhance our understanding of disease mechanisms and for soft tissue regenerative strategies. Perfusion systems generate more physiologically relevant and sustainable adipose tissue models, however adipocytes have unique properties that make culturing them in a perfusion environment challenging. In this paper we describe the methods involved in the development of two perfusion culture systems (2D and 3D) to test their applicability for long term in vitro adipogenic cultures. It was hypothesized that a silk protein biomaterial scaffold would provide a 3D framework, in combination with perfusion flow, to generate a more physiologically relevant sustainable adipose tissue engineered model than 2D cell culture. Consistent with other studies evaluating 2D and 3D culture systems for adipogenesis we found that both systems successfully model adipogensis, however 3D culture systems were more robust, providing the mechanical structure required to contain the large, fragile adipocytes that were lost in 2D perfused culture systems. 3D perfusion also stimulated greater lipogenesis and lipolysis and resulted in decreased secretion of LDH compared to 2D perfusion. Regardless of culture configuration (2D or 3D) greater glycerol was secreted with the increased nutritional supply provided by perfusion of fresh media. These results are promising for adipose tissue engineering applications including long term cultures for studying disease mechanisms and regenerative approaches, where both acute (days to weeks) and chronic (weeks to months) cultivation are critical for useful insight.

Microneedles represent an exciting departure from the existing parenteral injection paradigm for drug delivery, particularly for the administration of vaccines. While the benefit of delivering vaccine antigens to immunocompetent tissue in the skin has been established, there have been varying degrees of success using microneedles to do so. Here, we investigate the use of silk fibroin protein to produce microneedles and evaluate their ability to deliver vaccines against influenza, Clostridium dif f icile, and Shigella. Fibroin protein from the silkworm Bombyx mori possesses suitable properties for use in a microneedle system, including all-aqueous processing, mechanical strength in dried formats, biocompatibility, and the ability to temperature stabilize biomacromolecules. As such, this biomaterial combines the processing and biocompatibility advantages of a dissolving microneedle system with the product stability and mechanical strength of coated insoluble microneedles. Through successful vaccination of mice against three separate antigens, we establish that silk fibroin is well-suited for use as a solid-coated microneedle delivery system and offers long-term potential similar to that of the leading microneedle biomaterials.

Introduction: Successful translation of novel cancer immunotherapies could be accelerated by using tumor models that recapitulate human disease and receive relevant standard-of-care treatments. Some cancers that naturally arise in pet dogs share critical features with human disease including tumor heterogeneity (immune infiltration, mutational burden, metastatic progression) and patient characteristics (outbred population, immunocompetence, age, microbiome). Investigating novel immunotherapies alongside radiation, chemotherapy, or surgical treatment of spontaneous canine cancers meaningfully informs their clinical translation. Methods: We have developed interleukin -2 and -12 cytokine fusion proteins that bind and anchor to collagen after intratumoral administration and have shown their superior activity and tolerability in murine tumor models (Momin 2019). Here in pet dogs, we test these cytokines for safety and efficacy in two cancer types. Soft tissue sarcomas (n=10) were treated with intratumoral IL-2 and -12 using different dose schedules prior to surgical resection with subsequent tumor tissues assessed by histology for immune infiltrates and by Nanostring for whole tumor transcriptomics. Oral malignant melanomas (n=10) were treated with 9 Gy radiation at day 0, and on day 1 treated with intratumoral injection of the cytokines Q2W for 6 total doses, with dose escalation performed in cohorts of 3. Plasma was collected for PK/PD analysis of drug and IFN-g post-injection, with serial CBC/chemistry profiles and CT scans for assessment of safety and radiologic response, respectively. Results: Intratumoral dosing of collagen binding cytokines was well tolerated by 19/20 dogs, with transient body temperature elevation observed 24-72 hours post-injection and 1 episode of grade 3 transaminase elevation. Magnitude of fever did not correlate with peak serum IFN-g or cytokine dose level. At the lowest dose level (17 ug/kg IL-2, 40 ug/m2 IL-12), 1/3 pet dogs with oral melanomas achieved CR by RECIST criteria, and 3/3 pet dogs achieved near CR at the 2x dose level. Transcriptomic analysis of treated sarcomas revealed enrichment for antigen processing and T cell function two days after treatment. Moreover, IDO1 was upregulated in treated tumors, which suggests its systemic inhibitors combined with the locally-delivered cytokines may potentiate stronger responses. Conclusions: This data supports the safety and efficacy of intratumorally delivered IL-2 and IL-12 collagen binding cytokines and the ability to combine with standard-of-care resection or radiation therapy. We are continuing to enroll dogs to evaluate safety and efficacy at higher dose levels, including response at metastatic sites. Canine cancers bridge the gap between murine and human disease, accelerating the build-test-learn design cycle for novel immunotherapies and treatment combinations. Citation Format: Jordan Stinson, Allison Sheen, Jordan Hampel, Noor Momin, Rebecca Bernstein, Rebecca Kamerer, Timothy M. Fan, K. Dane Wittrup. Treatment of canine soft tissue sarcomas and melanomas with intratumoral collagen-anchored IL-2 and IL-12 is safe and effective [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 4171.

RINV worsened QOL and was experienced even after treatment was completed; physicians should therefore be cognizant and monitor patients in the week following radiotherapy. Concomitant chemoradiotherapy should potentially be included in the moderate emetogenic risk category.