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Abstract
Background IL-2 and IL-12 synergistically trigger the stimulation and proliferation of T and NK cells to mediate anti-tumor immunity. Although aldesleukin, a high-dose IL-2 intravenous (IV) infusion regimen, has been approved for the treatment of melanoma and renal cell carcinoma, adoption has been hindered by frequent grade 3 and 4 severe adverse events. No IL-12 therapy has been approved yet due to toxicity. Cullinan Amber is developing a fusion protein that uniquely combines in one polypeptide both IL-2 and IL-12 with a collagen-binding domain to reduce toxicity and increase efficacy following intra-tumoral (IT) administration via retention in the tumor microenvironment.
Methods Proteins were expressed in HEK293 cells. Collagen binding was measured by ELISA. IL-2 and IL-12 bioactivity was evaluated by CTLL-2 proliferation and HEK-Blue IL-12 reporter cells. In vivo studies were conducted in B16F10, MC38, and CT26 syngeneic tumor models. Systemic Amber construct concentrations were determined by ELISA.
Results ”Amber” constructs, comprised of IL-2, IL-12, and a collagen-binding domain, were produced and confirmed to retain bioactivity. B16F10 tumor-bearing mice injected with Amber IT had systemic Amber levels <5% as compared to mice administered the same dose IV. When IL-2/IL-12 fusion proteins lacking a collagen-binding domain were injected IT in B16F10-bearing mice, 60% of mice needed to be euthanized due to severe body weight loss, while Amber-treated mice did not lose body weight. In the checkpoint-refractory B16F10 and MC38 models, Amber demonstrated 95% tumor growth inhibition (figure 1a) and 100% CRs (figure 1b), respectively. 90% of the mice cured of their primary MC38 tumors were protected from re-challenge (figure 1b). Notably, 70% CRs were observed in the MC38 model even after a single-dose treatment of Amber. Similar data was obtained in the CT26 model. Amber treatment of mice bearing large 500 mm3 MC38 tumors resulted in dramatic tumor shrinkage (figure 1c). In mice bearing two MC38 tumors, only one of which was treated IT, 100% of treated tumors and 90% of distal untreated tumors were eliminated when Amber was combined with an anti-PD1 antibody (figure 1d), demonstrating a robust abscopal response.
Efficacy of amber constructs in syngeneic tumor models
Conclusions The use of collagen-binding domains for tumor retention enables the safe and effective delivery of IL-2 and IL-12 in a single multifunctional molecule. Taken together, the preclinical data suggests that Amber constructs may show robust single-agent activity in clinical trials against checkpoint-refractory tumors with minimal toxicity, as well as the potential to significantly deepen anti-tumor responses in combination with checkpoint inhibitor therapy.