A central tenet of cancer immunotherapy is that the immune system actively recognizes and eliminates malignant cells (1). However, tumours develop mechanisms to evade the immune response, which hinder the efficacy of cancer immunotherapies. One such evasion mechanism is the generation of a highly acidic tumour microenvironment (TME), which contributes to blunt the effectiveness of antitumor immunity (2). Overproduction of lactic acid by the tumour cells, due to their high glycolytic activity, results in an acidic TME with pH values around 6.5, contrasting with pH:7.4 found in normal tissues (3). The acidic TME contributes to establishing an immune-suppressive environment by inhibiting: 1) the proliferation and cytotoxic activities of CD8 T cells; 2) the cytotoxic response against tumour cells of Natural Killer (NK) cells and 3) the differentiation of monocytes into dendritic cells (DCs), thereby affecting anti-tumour T cell responses. Moreover, Regulatory T cells (Treg), which contribute to establish tolerance and prevent anti-tumour responses, maintain a strong suppression of effector T cells within the TME, due to their ability to use lactic acid to fuel their activities.
Cytokines are soluble small proteins that control the immune response, with defects in cytokine activities resulting in worse prognosis and increased cancer progression (4, 5). Despite their potential to fight cancer, few cytokines have reached the clinic due to their severe systemic toxicities, emphasising the urgent need for more specific and less toxic cytokine-based therapies. Our laboratory is at the vanguard of current research using protein engineering to manipulate immuno-stimulatory properties of cytokines for cancer treatment (6-8). Preliminary observations from our laboratory show that the acidic TME inhibits cytokine immunoregulatory activities by preventing their binding to cognate receptors. In this project, we propose to use protein engineering to generate new cytokine variants with pH-switched sensitivities that exert their activities only at the TME. We believe that this revolutionary novel approach will result in a new generation of cytokine anti-cancer therapies with enhanced efficacy and reduced toxicity. Initially, we will focus on Interleukin (IL)-15, a cytokine that plays a central role in the anti-tumour response. IL-15 induces the proliferation and survival of T and NK cells and promotes the generation of cytotoxic lymphocytes (9). IL-15 therapies have shown encouraging results in the clinic, but their efficacy has been hindered by dose-limiting toxicity (10).
We will use yeast surface display to generate large IL-15 mutant libraries from which we will isolate IL-15 mutants binding the IL-15 receptor with higher affinity at acidic pH than at neutral pH. Our laboratory has extensive expertise in generating and screening cytokine yeast display libraries. Iterative rounds of negative selections at pH 7 followed by positive selections at pH 6 will be carried out at decreasing concentrations of the IL-15 receptor. Yeast binding the receptor only at pH 6 will be purified using magnetic beads. Single yeast clones will be isolated, and the IL-15-encoding sequences will be cloned and recombinantly expressed to further validate their activities. We will characterize the receptor binding kinetics of IL-15 variants via surface plasmon resonance studies, study their signalling potential using flow cytometry and proteomic approaches, and their ability to trigger robust gene expression programs via RNA-seq, ChIP-seq and ATAC-Seq studies. We will pursue the most interesting variants in relevant in vivo mouse models of cancer. Overall, I believe that these studies will provide a path to improving the efficacy and reducing systemic toxicity of high-dose IL-15 therapy, an established immunotherapy with presently limited efficacy.
1. G. L. Beatty et al., Clin Cancer Res 21, 687-692 (2015).
2. F. Erra Diaz et al., Mediators Inflamm 2018, 1218297 (2018).
3. E. Boedtkjer et al., Annu Rev Physiol 82, 103-126 (2020).
4. J. Lokau et al., Adv Protein Chem Struct Biol 116, 283-309 (2019).
5. A. V. Villarino et al., J Immunol 194, 21-27 (2015).
6. C. Gorby et al., Front Immunol 9, 2143 (2018).
7. C. Gorby et al., Sci Signal 13, (2020).
8. J. Martinez-Fabregas et al., Elife 8, (2019).
9. M. C. Sneller et al., Blood 118, 6845-6848.
10. T. O. Robinson et al., Immunol Lett 190, 159-168 (2017).