Strategic insights into αOX40 agonist and αTim3 blockade combination therapy

Introduction. Binding of OX40-ligand expressed on Antigen-Presenting Cells, to its receptor OX40 expressed on T cells, provides a co-stimulatory signal to T cells. It is reasonable to hypothesize that strengthening this signal using anti-OX40 (αOX40) agonist monotherapy would increase T cells anti-tumor function. Unfortunately, the results from clinical trials showed no benefit to cancer patients. Nevertheless, pre-clinical studies revealed synergistic effects between αOX40 agonist and anti-PD1 checkpoint blockade. Here, we aimed to deepen our understanding of the proximal effects of αOX40 agonist therapy on tumor-infiltrating leukocytes, in order to identify new potential therapeutic combinations.

Materials and Methods. Mice bearing established B16 tumors were administered twice with αOX40 agonist antibody or its isotype control. 24 hours after the second injection, tumors were excised and digested, leukocytes were isolated, stained with fluorescent antibodies and sorted. Single-cell libraries were processed by 10x, and sequenced for further informatics analysis.

Results and Discussion. As expected, no change in tumor growth was observed, at this early time point, after only two αOX40 agonist injections. The treatment mostly affected T cells (both regulatory and CD8⁺), which up-regulated the OX40 pathway. The abundance of regulatory T cells decreased, while their suppressive gene signature elevated. Activated CD8⁺ T cells upregulated the transcriptional programs of cytotoxicity, co-stimulation and co-inhibition, and compensatory upregulation of intracellular regulatory genes occurred. These changes were accompanied by CD8⁺ T cells up-regulation of co-inhibitory molecules Ctla4 and Tim3. This observation prompted us to propose a novel combination therapy between OX40 agonist and Tim3 antagonist. Testing this combination therapy in melanoma mouse model resulted in prolonged survival. An indirect effect of the treatment was observed in several subpopulations of dendritic cells and macrophages, which shifted towards a more pro-inflammatory state with increased chemotaxis.

Conclusion. Ultimately, by providing deeper understanding of proximal events of TILs upon αOX40 treatment, we proposed a rationale for a novel combination therapy in human clinical trials