Background Activation of immune networks within the tumor microenvironment (TME) through the delivery of pro-inflammatory cytokines has shown potential for promoting tumor cell recognition and killing. However, systemic targeting of these cytokines has been limited by indiscriminate toxicity. Alternative approaches, such as masked cytokines cleaved by tumor-associated MMPs, have yet to demonstrated clinical efficacy. Attenuated cytokines functioning in cis have shown promise in pre-clinical studies, but translating their therapeutic windows to humans remains a major challenge. IL-18, which can to mobilize both innate and adaptive immunity, has emerged as a strong candidate for a platform technology due to its synergistic effects with various cytokines. However, its tight regulation by IL-18BP has hindered clinical success.
Methods First, we generated IL-18 mutants resistant to IL-18BP and utilized its natural pro-peptide to engineer a tunable pro-cytokine with cis-activity. Cleavage sites were introduced between the modified pro-peptide and the mature cytokine, allowing proteases like Granzymes released in lytic synapses between cytotoxic lymphocytes and tumor cells to trigger cytokine activation. The IL-18 cassette, with or without an attenuated cis-acting IL-15 motif, was genetically fused to antibody-like proteins that either facilitated direct bridging between cytotoxic-lymphocytes and tumor-cells or promoted their natural docking. These proteins included a γ9δ2-engager, an NKG2D-engager, cetuximab, trastuzumab, and ADCC-competent atezolizumab, and nivolumab. By targeting lymphocytes like NK cells and γ9δ2 T cells capable of distinguishing normal from transformed cells, we enabled immune surveillance which avoids on-target off-tumor toxicity to determine the optimal sites for IL-18 activation.
Results Fusion of IL-18 to antibody-like proteins, facilitating bridging between cytotoxic lymphocytes and tumor cells, enhanced tumor cell killing, Granzyme release, and target-dependent IFN-γ release. This enhanced activity occurred in a stepwise manner, driven by both cis and trans activities (figure 1). Trans activity depended on proteolysis of the Granzyme cleavage site(s), resulting in IL-18 release. This triggered a positive-biofeedback loop in which IL-18 promoted further Granzyme production, more potent cytotoxicity and additional IL-18 release. Fusion of IL-15 to these antibody-like proteins enabled targeted cis-based immune cell proliferation independent of tumor cells.
Conclusions We have developed a modular and highly expressible platform for systemic delivery of a cis-acting and masked IL-18 variant to tumor-cells, leveraging natural immune surveillance mechanisms. Co-fusion of cis-acting IL-15 and IL-18 combines their beneficial properties, including expansion, persistence, and memory. This innovative approach to targeted and combined cytokine delivery holds great promise as a new and disruptive treatment paradigm for cancer.
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