Enhancing Anti-Tumor Immunity and Responses to Immune Checkpoint Blockade By Suppressing Interleukin-10 in Chronic Lymphocytic Leukemia
Authors: Rivas Jacqueline R. , Alhakeem Sara S. , Eckenrode Joseph M. , Zhang Yinan , Collard James P. , Hildebrandt Gerhard C. , Fleischman Roger A. , Chen Li , Thorson Jon S. , Leggas Markos , Bondada Subbarao 
Published: November 2019

B-cell Chronic Lymphocytic Leukemia (CLL) is the most common leukemia in the Western world, accounting for nearly one third of all leukemia cases. In CLL abnormal B-cells accumulate in the blood and lymphoid organs leading to serious immune dysfunction. This immune suppression is in part due to CLL-produced mediators that downregulate T-cell responses, such as the regulatory cytokine Interleukin-10 (IL-10). We previously found that eliminating T-cell IL-10 signaling enhanced their ability to control CLL growth in vivo. Therefore, we investigated the potential for IL-10 blockade to enhance the anti-tumor activity of CD8+ T-cells.
In our studies we use human CLL cells as well as the Eμ-Tcl1 mouse model of CLL, in which the oncogene Tcl1 is expressed under the immunoglobulin VH promoter and µ-enhancer. IL-10 production by CLL cells depends on the transcription factor Sp1, and we found that the Sp1 inhibitor mithramycin (MTM) suppresses CLL IL-10 production. However, MTM is not well tolerated in vivo, so we synthesized novel, less toxic analogues of MTM to test for IL-10 suppression. One of these MTM analogues similarly suppresses mouse and human CLL IL-10 with little to no effect on effector T-cell cytokines and viability. Therefore, we treated mice with this analogue in the adoptive transfer model of Eμ-Tcl1, and later combined this with anti-PD-L1 checkpoint blockade to determine its effects on anti-tumor immunity.
Here we show that this MTM analogue enhances the efficacy of anti-CLL T-cells in vivo by suppressing CLL IL-10 production, allowing for increased CD8+ T-cell proliferation, effector memory cell prevalence, and CD8+ interferon-γ (IFN-γ) production. Treatment slowed the growth of Eμ-TCL1 CLL cells in the spleen and blood and reduced the spread of CLL to the bone marrow. Furthermore, suppressing IL-10 in this manner improved responses to anti-PD-L1 treatment, decreasing the burden of CLL cells and the functionality of CD8+ T-cells in comparison to anti-PD-L1 alone. The overall number and frequency of CD8+ T-cells was higher in double treated mice, with more IFN-γ+ CD8+ cells, more effector memory cells, and fewer exhausted T-cells.
This paradigm shifting approach is novel as current therapies for CLL do not target IL-10 and it may increase the efficacy of T-cell-based immunotherapies in human CLL. T-cell-based immunotherapies have experienced limited success in trials with CLL, and since there is no cure for this disease, our approach may provide a new avenue for combination therapies. Moreover, IL-10 blockade could be applicable to other B-cell malignancies and even solid tumors where T-cell suppression plays a significant role.

Disclosures
Hildebrandt: Axim Biotechnologies: Equity Ownership; Kite Pharma: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other; Sangamo: Equity Ownership; Novartis: Equity Ownership; Axim Biotechnologies: Equity Ownership; Juno Therapeutics: Equity Ownership; Kite Pharma: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Novartis: Equity Ownership; Insys Therapeutics: Equity Ownership; Abbvie: Equity Ownership; GW Pharmaceuticals: Equity Ownership; Cardinal Health: Equity Ownership; Immunomedics: Equity Ownership; Endocyte: Equity Ownership; Clovis Oncology: Equity Ownership; Cellectis: Equity Ownership; Aetna: Equity Ownership; CVS Health: Equity Ownership; Celgene: Equity Ownership; Bluebird Bio: Equity Ownership; Bristol-Myers-Squibb: Equity Ownership; crispr therapeutics: Equity Ownership; IDEXX laboratories: Equity Ownership; Johnson & Johnson: Equity Ownership; Pfizer: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Procter & Gamble: Equity Ownership; Vertex: Equity Ownership; Bayer: Equity Ownership; Scotts-Miracle: Equity Ownership; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Travel; Jazz Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel, Research Funding; Takeda: Research Funding; Pharmacyclics: Research Funding; Astellas: Other: Travel.