Unlocking the Potential of Cytokines for Cancer Immunotherapy

Cytokines are small soluble proteins that play a role in many biological processes, including growth, differentiation, apoptosis, and inflammation. In the context of cancer, the pro-inflammatory, -apoptotic, and –cytotoxic properties of cytokines such as Interleukin-2 (IL-2) and Interferon-alpha prompted their FDA approval as therapies for renal cell carcinoma and melanoma, among others. However, harnessing the therapeutic potential of cytokines in cancer treatment has been limited by the severe adverse effects resulting from their required high-dose systemic use (Berraondo et al. 2018). Therefore, strategies to improve targeting the effects of cytokines on the tumor microenvironment are needed to unlock their full immunotherapeutic benefits.

Jeffrey Hubbell’s team at the Pritzker School of Molecular Engineering, University of Chicago recently envisioned a strategy to enable preferential activation of a modified form of IL-12 at tumor sites. In that approach, the team engineered a masked form of IL-12. By adding an IL-12 receptor (Interleukin-12Rβ1Q20–A261) domain mask to the IL-12 molecule, connected via a linker sensitive to tumor-protease cleavage, the team ensured the localized activation of IL-12 within the tumor microenvironment. Preclinical studies demonstrated that this strategy could induce strong anti-tumor activity in various mouse tumor models, such as melanoma, colon adenocarcinoma, and triple-negative breast cancer while decreasing toxicity and adverse reactions (Mansurov et al. 2022). Additionally, in recently published work, David Baker’s team at the University of Washington focused on improving IL-2’s therapeutic benefits and has devised yet another innovative strategy to target its immunostimulatory activity to the tumor site (Quijano-Rubio et al. 2022).

Splitting an IL-2 mimetic to reduce toxicity

Among the various biological functions of IL-2, its role in the expansion of NK and T cells makes it an ideal candidate to leverage in combination with cellular cancer immunotherapy. Therefore, different strategies have been developed to reduce therapeutic IL-2 dosing and limit its toxicity. For instance, IL-2 PEGylation prolongs its circulation time, helping minimize therapeutic dosing. Alternatively, engineering IL-2 to favor interaction with lower affinity receptors, such as those in T cells, helps target its effects. Lastly, leveraging computational modeling strategies and mutant libraries, the Baker lab in 2019 designed a stable IL-2 mimetic with preferential binding to the IL-2 receptor βγc heterodimer (IL-2Rβγc). The de-novo IL-2 mimic, neoleukin-2/15 (Neo-2/15), was shown to bind with high affinity to the IL-2 receptor βγc, strongly activating T cells, and importantly, not interacting with IL-2Rα (CD25), which has been linked with the adverse effects of IL-2 cytokine therapy (Silva et al. 2019).

IL-2 and IL-15 receptors,signaling pathways,immunomodulatory functions

IL-2 and IL-15 receptors, signaling pathways, and immunomodulatory functions. Beyond the unique binding of IL-2 and IL-15 to their respective alpha receptors, these cytokines share binding to the IL-2/15Rβγc receptor complex, therefore, supporting the activation of similar downstream signaling pathways (i.e., JAKs and STATs). In contrast, de novo Neo-2/15 consists of helical structures (H1, H3, H2’, and H4) that interact with IL-2/15Rβ (H3), IL-2Rγ (H4), or with both receptors (H1). However, Neo-2/15 does not bind to either the IL-2 or IL-15 alpha receptors. Retrieved from Yang and Lundqvist, 2020. https://creativecommons.org/licenses/by/4.0/

In their most recent work, Baker and colleagues have devised a strategy that would further improve the safety of IL-2 and potentially any other cytokine used as immunotherapy. Rather than masking IL-2 as done previously with IL-12, Baker and colleagues resorted to splitting the neoleukin-2/15 protein. By separating one of the alpha-helical structures (i.e., H1) from the rest of the neoleukin-2/15 molecule (i.e., H3, H2’, and H4), the team successfully generated fragments that could only reconstitute in the presence of the IL-2Rβ, IL-2Rγ receptors.

Next, Baker’s team leveraged in vitro assays using tumor and NK cell line co-cultures to demonstrate the ability of the split neoleukin-2/15 molecule to induce a targeted immune response. To this end, cell co-cultures were incubated with neoleukin-2/15 fragments, each fused to domains targeting relevant tumor cell antigens, such as HER2 and EGFR. Co-expression of HER2 and EGFR on the surface of tumor cells enabled bringing together the neoleukin-2/15 fragments and supported transactivation of NK cells through binding to the IL-2Rβγ. Significantly, split neoleukin-2/15 fragments could induce even more robust transactivation under conditions conducive to forming tumor-specific immunological synapses.

Engineered split IL-2 mimetic

Engineered split IL-2 mimetic. Split neoleukin-2/15 fragments fused with tumor cell targeting molecules developed by the Baker team become reconstituted upon binding to the IL-2 receptors, enabling the activation of immune cells while in close proximity to tumor cells (Quijano-Rubio et al. 2022). Created with BioRender.

Lastly, preclinical studies in mice demonstrated that the already lower toxicity of neoleukin-2/15, compared to IL-2, was further reduced in animals dosed with split neoleukin-2/15. Moreover, in a melanoma tumor model, dosing with the split neoleukin-2/15 fragments, each fused to anti-PD-L1 nanobodies, showed greater anti-tumor efficacy than neoleukin-2/15.

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