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Inhibition of immune checkpoints, including cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed death-1 (PD-1), and its ligand PD-L1, has demonstrated exciting and durable remissions across a spectrum of malignancies. Combinatorial regimens blocking complementary immune checkpoints further enhance the therapeutic benefit. The activity of these agents for patients with glioblastoma, a generally lethal primary brain tumor associated with significant systemic and microenvironmental immunosuppression, is not known. We therefore systematically evaluated the antitumor efficacy of murine antibodies targeting a broad panel of immune checkpoint molecules, including CTLA-4, PD-1, PD-L1, and PD-L2 when administered as single-agent therapy and in combinatorial regimens against an orthotopic, immunocompetent murine glioblastoma model. In these experiments, we observed long-term tumor-free survival following single-agent anti–PD-1, anti–PD-L1, or anti–CTLA-4 therapy in 50%, 20%, and 15% of treated animals, respectively. Combination therapy of anti–CTLA-4 plus anti–PD-1 cured 75% of the animals, even against advanced, later-stage tumors. In long-term survivors, tumor growth was not seen upon intracranial tumor rechallenge, suggesting that tumor-specific immune memory responses were generated. Inhibitory immune checkpoint blockade quantitatively increased activated CD8+ and natural killer cells and decreased suppressive immune cells in the tumor microenvironment and draining cervical lymph nodes. Our results support prioritizing the clinical evaluation of PD-1, PD-L1, and CTLA-4 single-agent targeted therapy as well as combination therapy of CTLA-4 plus PD-1 blockade for patients with glioblastoma. Cancer Immunol Res; 4(2); 124–35. ©2015 AACR.

Supplementary Methods

Supplemental Figure 1. Bioluminescence plots of individual animals following treatment with immune checkpoint blocking mAbs. Supplemental Figure 2. Immune cell infiltrates within intracranial GL261-luc2 tumor following treatment with isotype control IgG or combination therapy with CTLA-4 plus PD-1 mAb therapy. Supplemental Figure 3: Treatment antibody and detection antibody for PD-1 do not share same the epitope. Supplemental Figure 4. Representative immunohistochemistry results of intratumoral CD3+, CD4+ and CD8+ T cell infiltrates following immune checkpoint therapy with mAbs against CTLA-4, PD-1 and both CTLA-4 and PD-1. Supplemental Figure 5. Immune cell infiltration within the draining cervical lymph node (cLN) of GL261-luc2 tumor-bearing mice following treatment with isotype control IgG or combination therapy with mAbs against CTLA-4 plus PD-1. Supplemental Figure 6. Comparisons of chemokine expression levels in sera of mice treated with checkpoint blockade. Supplemental Figure 7: Expression of checkpoint ligands and receptors on GL261-Luc cells in vitro. Supplementary Table 1. Arrangement of chemokines on the Mouse Chemokine Antibody Array.

Supplementary Figure Legends

Di-2-pyridylketone-4,4,-dimethyl-3-thiosemicarbazone (Dp44mT) is being developed as an iron chelator with selective anticancer activity. We investigated the mechanism whereby Dp44mT kills breast cancer cells, both as a single agent and in combination with doxorubicin. Dp44mT alone induced selective cell killing in the breast cancer cell line MDA-MB-231 when compared with healthy mammary epithelial cells (MCF-12A). It induces G1 cell cycle arrest and reduces cancer cell clonogenic growth at nanomolar concentrations. Dp44mT, but not the iron chelator desferal, induces DNA double-strand breaks quantified as S139 phosphorylated histone foci (γ-H2AX) and Comet tails induced in MDA-MB-231 cells. Doxorubicin-induced cytotoxicity and DNA damage were both enhanced significantly in the presence of low concentrations of Dp44mT. The chelator caused selective poisoning of DNA topoisomerase IIα (top2α) as measured by an in vitro DNA cleavage assay and cellular topoisomerase-DNA complex formation. Heterozygous Nalm-6 top2α knockout cells (top2α+/−) were partially resistant to Dp44mT-induced cytotoxicity compared with isogenic top2α+/+ or top2β−/− cells. Specificity for top2α was confirmed using top2α and top2β small interfering RNA knockdown in HeLa cells. The results show that Dp44mT is cytotoxic to breast cancer cells, at least in part, due to selective inhibition of top2α. Thus, Dp44mT may serve as a mechanistically unique treatment for cancer due to its dual ability to chelate iron and inhibit top2α activity. [Cancer Res 2009;69(3):948–57]

Supplementary Figure 1 from The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase IIα in Breast Cancer Cells

Supplementary Figure Legend and Methods from The Iron Chelator Dp44mT Causes DNA Damage and Selective Inhibition of Topoisomerase IIα in Breast Cancer Cells