UC Berkeley

Over the past decade, there have been many advances made in the field of cancer immunotherapy, one of the major therapies discovered is that of checkpoint blockades. While these therapies have vastly improved patient outcomes, many cancers do not respond to checkpoint blockade highlighting the need for further development of new therapeutics. Most current cancer immunotherapies are based on mobilizing CD8 T cell responses. However, many types of tumors evade CD8 T cell recognition by displaying few or no antigens, or losing expression of MHC I. These considerations underlie the need for complementary therapies that mobilize other antitumor effector cells. NK cells are cytotoxic innate-like lymphocytes that are hardwired to recognize stressed cells, such as tumor cells and certain infected cells, and mediate spontaneous killing of MHC I-deficient tumor cells. CD4 T cells recognize tumor cells through epitopes displayed on MHC II and have been shown to be capable of eliciting antitumor responses against tumor cells. Identifying new immunotherapies, aimed at targeting NK and CD4 cells for antitumor responses, could potentially activate responses against CD8 T cell-resistant tumors.

Cyclic dinucleotides (CDNs) activate the cGAS-STING pathway of the innate immune system and are candidates as immunotherapy agents. Intratumoral CDN injections induce type I IFNs and inflammatory cytokines that amplify the CD8 T cell response and induce tumor regression. Recently, CDN therapy was shown to induce long-term tumor regressions in some MHC I-deficient tumor models, mediated primarily by NK cells and in some cases, CD4 T cells. However, in harder-to-treat tumor models, CDN therapy shows few to no long-term remissions and early phase clinical trials of STING agonists in patients have not yielded sustained clinical remissions indicating a need for improvements in this immunotherapy approach.

My thesis work aims to examine the potential to extend the efficacy of CDN therapy, by combining CDN with the IL-2 superkine, H9-MSA, to target and activate NK cells and CD4 T cells in the tumor microenvironment against MHC I-deficient tumors and CD8 T cells against MHC I WT tumors. Chapter 3 addresses the synergy observed with CDN and H9-MSA therapy in mobilizing powerful NK cell antitumor responses against MHC I-deficient tumors. CDN/H9-MSA therapy markedly enhanced tumor rejection of two hard-to-treat MHC I-deficient tumor models. These responses were mediated by NK cells and in some cases CD4 T cells and were accompanied by increased recruitment to and sustained activation of NK cells in the tumor. This combination therapy regimen activated NK cells systemically, as shown by antitumor effects distant from the site of CDN injection and enhanced cytolytic activity of splenic NK cells against tumor cell targets ex vivo. H9-MSA also showed strong synergy with another innate activator, CpG. The CDN/H9-MSA therapy showed promising effects in treating tumors in mice with a complex microbiota, a known factor to negatively influence immunotherapies.

CD4 T cell antitumor responses were also induced by CDN or the CDN/H9-MSA combination therapy against two different MHC I-deficient tumor models. Chapter 4 explores the role of tumor specific CD4 T cells induced by CDN therapy alone or CDN/H9-MSA combination therapy, independently of NK cells. CDN treatment increased systemic levels of tumor specific CD4 T cells, with a Th1-like phenotype. Mice treated with CDN/H9-MSA induced potent CD4 T cell and CD4/CD8 T cell responses against an MHC I-deficient tumor model in the absence of NK cells. The antitumor CD4 T cell response did not rely on MHC II expression on the tumor cells to elicit tumor rejection. Tumor-specific priming of these CD4 T cells showed enhanced cytokine production in combination therapy treated mice. Finally, this chapter explores the CDN/H9-MSA combination therapy regimen synergistically mobilizing powerful CD8 T cell responses in the case of MHC I+ tumors, both syngeneic and carcinogen (methylcholanthrene, MCA)-induced models, suggesting the generality of the approach.

The final chapter of my thesis focuses on attempts to improve CDN/H9-MSA therapy in both syngeneic and MCA-induced sarcoma models. With many factors in the tumor microenvironment influencing the immune responses, we endeavored to amplify the impact of tumor infiltrating NK cells and T cells through various forms of activation or through the inhibition of suppressive factors found in the TME. Blockade of immunosuppressive metabolites and checkpoint receptors did not enhance CDN/H9-MSA therapy against MHC I-deficient tumors. However, addition of checkpoint blockade to the CDN/H9-MSA therapy regimen greatly increased the survival of mice in the spontaneous MCA-induced sarcoma model.

Overall, the work in this thesis demonstrates the impact of a novel combination therapy in mobilizing powerful NK and T cell-mediated antitumor activity, providing important justification for evaluating approach of combining a STING agonist with the IL-2 superkine, for treating human cancers that are refractory to available treatment options.