Alan L. Kaganov Professor, Department of Biomedical Engineering Duke University, United States
Introduction:: Despite the clinical successes of checkpoint inhibitors over the past decade, their efficacy is largely correlated with the presence of tumor infiltrating lymphocytes resulting in poor outcomes for immunologically “cold” tumors1. IL-12 is a potent pro-inflammatory cytokine that can elicit strong Th1 responses and promote recruitment of immune cells to the tumor site2. However, IL-12 has not shown clinical efficacy because it induces severe-immune related adverse events (AE) in patients3. Our lab has developed a new strategy to deliver IL-12 that has the potential to reduce AEs by conjugating IL-12 to an elastin-like polypeptide (ELP) that exhibits tunable, thermally responsive phase behavior. Upon injection into the tumor, the IL-12-ELP fusion undergoes phase separation into an insoluble coacervate phase, which should release the fusion into the systemic circulation with near zero-order kinetics over a week to ten days based on our recent experience with similar ELP fusions4. To improve the specificity of IL-12 in the tumor environment, we have introduced a tumor-associated protease linker (TAPL) between the cytokine and ELP to exploit the overexpression of remodeling proteases in the tumor that can cleave the TAPL and release free IL-12 solely within the tumor5.
Materials and Methods:: IL-12 modified with a C-terminal LPETG sortase recognition site was produced by transfection of Expi293 cells (Thermo Fisher) and purified from the culture supernatant via affinity chromatography. Triglycine-TAPL-ELP was expressed in BL21 (DE3) E. coli and was purified from the soluble fraction of the cell lysate by inverse transition cycling, a purification technique that exploits the phase transition behavior of the ELP. IL-12 ELP was produced by sortase-mediated transpeptidation of IL-12 LPETG and triglycine-TAPL-ELP. Thermal phase behavior of the IL-12 ELP fusions was characterized by temperature dependent UV-Vis spectrophotometry. The coacervation—phase separation— of fluorescently labeled IL-12-ELP fusion mixed with excipient ELP was visualized by fluorescence microscopy. Cleavage of TAPL was confirmed by incubating tumor-associated proteases MMP2, MMP9, and UPa individually with the IL-12-ELP fusion and visualizing digested products on SDS-PAGE. Finally, the bioactivity of the IL-12-ELP fusion—after protease cleavage of IL-12 from its ELP tag— was measured in vitro by a HEK-Blue IL-12 reporter cell line.
Results, Conclusions, and Discussions:: The modification of IL-12 with a sortase recognition site does not impair IL-12 binding to IL-12R compared to native IL-12 ( Figure 1a
), though the fusion of IL-12 to TAPL-ELP results in an approximately 15-fold increase in IC 50
compared to IL-12. This decrease in affinity is expected because of the steric shielding effect by the ELP appended to the C terminus of IL-12. Upon cleavage by MMP2, the activity of the IL-12 matches IC 50
of the unconjugated IL-12 ( Figure 1b
). The thermal behavior of IL-12-ELP shows that the transition temperature of the ELP allows for physiologically relevant phase transitions, and brightfield microscopy shows that the IL-12-ELP fusion mixed with excipient ELP forms well defined coacervates ( Figure 2
). Future work includes testing the in vivo
activity of the IL-12-ELP fusion in immunologically “cold” tumor models such as CT-26 colorectal cancer and B16F10 melanoma to characterize the immune activation by IL-12, its biodistribution, and its effect on tumor regression. IL-12-ELP provides a promising new delivery method for IL-12 that sustains release of the drug within the tumor while limiting off target effects. By localizing immune cell recruitment, IL-12 ELP can also serve as an attractive local immunomodulator to further potentiate checkpoint inhibitors in poorly inflamed tumors.
Acknowledgements (Optional): :
References (Optional): : 1. Robert C. A decade of immune-checkpoint inhibitors in cancer therapy. Nat Commun. 2020 Jul 30;11(1):3801.
2. Nguyen KG, et al. Localized Interleukin-12 for Cancer Immunotherapy. Front Immunol. 2020 Oct 15;11:575597.
3. Leonard JP., et al. Effects of single-dose interleukin-12 exposure on interleukin-12-associated toxicity and interferon-gamma production. Blood. 1997 Oct 1;90(7):2541-8.
4. Varanko, A., Su, J., and Chilkoti, A. Elastin-Like Polypeptides for Biomedical Applications. Ann Rev Biomed Eng22, 343-369 (2020).
5. Mansurov A, et al. Masking the immunotoxicity of interleukin-12 by fusing it with a domain of its receptor via a tumour-protease-cleavable linker. Nat Biomed Eng. 2022 Jul;6(7):819-829.
