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

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.

Effective antitumor immunity in mice requires activation of the type I interferon (IFN) response pathway. IFNα and IFNβ therapies have proven promising in humans, but suffer from limited efficacy and high toxicity. Intratumoral IFN retention ameliorates systemic toxicity, but given the complexity of IFN signaling, it was unclear whether long-term intratumoral retention of type I IFNs would promote or inhibit antitumor responses. To this end, we compared the efficacy of IFNα and IFNβ that exhibit either brief or sustained retention after intratumoral injection in syngeneic mouse tumor models. Significant enhancement in tumor retention, mediated by anchoring these IFNs to coinjected aluminum-hydroxide (alum) particles, greatly improved both their tolerability and efficacy. The improved efficacy of alum-anchored IFNs could be attributed to sustained pleiotropic effects on tumor cells, immune cells, and nonhematopoietic cells. Alum-anchored IFNs achieved high cure rates of B16F10 tumors upon combination with either anti-PD-1 antibody or interleukin-2. Interestingly however, these alternative combination immunotherapies yielded disparate T cell phenotypes and differential resistance to tumor rechallenge, highlighting important distinctions in adaptive memory formation for combinations of type I IFNs with other immunotherapies.

Confining cytokine exposure to the tumors would greatly enhance cancer immunotherapy safety and efficacy. Immunocytokines, cytokines fused to tumor-targeting antibodies, have been developed with this intention, but without significant clinical success to date. A critical limitation is uptake by receptor-expressing cells in the blood, that decreases the dose at the tumor and engenders toxicity. Small-format immunocytokines, constructed with antibody fragments, are hypothesized to improve tumor specificity due to rapid systemic clearance. However, effective design criteria for small-format immunocytokines need further examination. Here we engineer small interleukin-2 (IL-2) immunocytokines fused to nanobodies with nanomolar to picomolar affinities for the tumor-specific EIIIB domain of fibronectin (also known as EDB). Upon intravenous delivery into immunocompetent mice, such immunocytokines led to similar tumor growth delay as size-matched untargeted IL-2. Intratumoral delivery imparted improved survival dependent on affinity to EIIIB. Intratumoral administration offers a promising avenue to deliver small-format immunocytokines, given effective affinity for the tumor microenvironment.

Purpose: Cytokine therapies such as IL2 and IL12 suffer from impractically small therapeutic windows driven by their on-target, off-tumor activity, limiting their clinical potential despite potent antitumor effects. We previously engineered cytokines that bind and anchor to tumor collagen following intratumoral injection, and sought to test their safety and biomarker activity in spontaneous canine soft-tissue sarcomas (STS). Experimental Design: Collagen-binding cytokines were canine-ized to minimize immunogenicity and were used in a rapid dose-escalation study in healthy beagles to identify a maximum tolerated dose. Ten client-owned pet dogs with STS were then enrolled into trial, receiving cytokines at different intervals prior to surgical tumor excision. Tumor tissue was analyzed through IHC and NanoString RNA profiling for dynamic changes within treated tumors. Archived, untreated STS samples were analyzed in parallel as controls. Results: Intratumorally administered collagen-binding IL2 and IL12 were well tolerated by STS-bearing dogs, with only Grade 1/2 adverse events observed (mild fever, thrombocytopenia, neutropenia). IHC revealed enhanced T-cell infiltrates, corroborated by an enhancement in gene expression associated with cytotoxic immune function. We found concordant increases in expression of counter-regulatory genes that we hypothesize would contribute to a transient antitumor effect, and confirmed in mouse models that combination therapy to inhibit this counter-regulation can improve responses to cytokine therapy. Conclusions: These results support the safety and activity of intratumorally delivered, collagen-anchoring cytokines for inflammatory polarization of the canine STS tumor microenvironment. We are further evaluating the efficacy of this approach in additional canine cancers, including oral malignant melanoma.