Adjuvant therapy with agonistic antibodies to CD134 (OX40) increases local control after surgical or radiation therapy of cancer in mice

Abstract

The tumor recurrence from residual local or micrometastatic disease remains a problem in cancer therapy. In patients with soft tissue sarcoma and the patients with inoperable nonsmall cell lung cancer, local recurrence is common and significant mortality is caused by the subsequent emergence of metastatic disease. Thus, although the aim of the primary therapy is curative, the outcome may be improved by additional targeting of residual microscopic disease. We display in a murine model that surgical removal of a large primary sarcoma results in local recurrence in approximately 50% of animals. Depletion of CD8 T cells results in local recurrence in 100% of animals, indicating that these cells are involved in the control of residual disease. We further show that systemic adjuvant administration of αOX40 at surgery eliminates local recurrences. In this model, αOX40 acts to directly enhance tumor antigen-specific CD8 T-cell proliferation in the lymph node draining the surgical site, and results in increased tumor antigen-specific cytotoxicity in vivo. These results are also corroborated in a murine model of hypofractionated radiation therapy of lung cancer. Administration of αOX40 in combination with radiation significantly extended the survival compared with either agent alone, and resulted in a significant proportion of long-term tumor-free survivors. We conclude that αOX40 increases tumor antigen-specific CD8 T-cell cytotoxic activity resulting in improved endogenous immune control of residual microscopic disease, and we propose that adjuvant αOX40 administration may be a valuable addition to surgical and radiation therapy for cancer.

Figure 1. Role of CD8 T cells in local recurrence following sarcoma surgery and influence of αOX40 therapy on local recurrence

a) MCA205 tumors were established s.c. in the flank of C57BL/6 mice and were surgically removed when they reached 7–10 mm in diameter. One day prior to the operation, mice began receiving weekly injections of 200μg of control (○) or CD8-depleting (●) antibody and followed for local tumor recurrence. b) MCA205 tumors were established s.c. in the flank of C57BL/6 mice and were surgically removed when they reached 7–10 mm in diameter. At the time of the operation mice received a single injection of 250μg of control (○) or αOX40 (●) antibody and followed for local tumor recurrence. c) C57BL/6 mice bearing MCA205 tumors were treated with 250μg aOX40 or control Ig on day 3 and day 7 following tumor challenge. Draining lymph node cells were harvested on day 12 and activated in vitro. These cells were used in a ^[51]Cr release cytotoxicity assay to determine specific cytotoxicity against MCA205 (circles) or non-specific cytotoxicity against 3LL (squares). The graph shows the mean cytotoxicity at a range of effector: target ratios calculated from 3 individual mice per group treated with control Ig (empty shapes) or αOX40 (filled shapes).

Figure 2. Loss of tumor antigen-specific priming following sarcoma surgery

MCA205ova tumors were established s.c. in the flank of C57BL/6 mice and were surgically removed when they reached 7–10mm in diameter. a) At the time of the operation or b) three days following the operation, mice received adoptive transfer of 1×10⁶ naïve CFSE labeled OT1 T cells i.v.. Four days following adoptive transfer, the tumor-draining lymph node (DLN) and a non-draining lymph node (NDLN) were harvested and FACS analyzed for the proportion of OT1 T cells. b) Representative FACS plots identifying CD8⁺Thy1.1⁺ OT1 T cells or CFSE dilution and CD25 expression in gated CD8⁺Thy1.1⁺ OT1 T cells. c) Graphs showing percentage of CD8 T cells that are Thy1.1⁺ OT1 cells in DLN or NDLN 4 days following adoptive transfer. d) Graphs showing Mean Fluorescence Intensity (MFI) of CFSE Thy1.1⁺ OT1 cells in DLN or NDLN 4 days following adoptive transfer. Each symbol represents one animal. Key: NS - not significant; * - p<0.05; ** - p<0.01; *** - p<0.001.

Figure 3. Influence of αOX40 therapy on tumor antigen-specific T cell proliferation and tumor antigen-specific cytotoxicity following sarcoma surgery

a) MCA205ova tumors were established s.c. in the flank of C57BL/6 mice and were surgically removed when they reached 7–10mm in diameter. At the time of the operation mice received adoptive transfer of 1×10⁶ naïve CFSE-labeled Thy1.1⁺ OT1 T cells i.v. and 250μg control Ig or αOX40 i.p., and the tumor draining lymph node (DLN) and a non-draining lymph node (NDLN) were harvested at various times following surgery. b) Graphs show the time course of the percentage of CD8⁺Thy1.1⁺ OT1 cells in the draining and non-draining lymph nodes following surgery. c) Representative dot plots 5 days following surgery, gating on CD8⁺Thy1.1⁺ cells and showing CFSE dilution versus CD62L expression in the lymph node draining the surgical site of parental MCA205, MCA205ova treated with control Ig, or MCA205ova treated with αOX40. d)i) MCA205ova tumors were established s.c. in the flank of C57BL/6 mice and were surgically removed when they reached 7–10mm in diameter. At the time of the operation, mice received adoptive transfer of 1×10⁶ naïve OT1 T cells i.v. and 250μg control Ig or αOX40 i.p.. Four or six days later mice received adoptive transfer of 5×10⁶ CFSE^hi SIINFEKL-pulsed splenocytes and CFSE^lo control splenocytes in a 1:1 ratio. Naïve control mice also received adoptive transfer of target cells to determine background levels of cytotoxicity. Four hours later, draining lymph nodes were harvested and FACS analyzed for the proportion of CFSE^hi to CFSE^lo cells. ii) Representative histograms showing the proportions of CFSE^lo and CFSE^hi targets in draining lymph nodes day 4 following surgery, and day 6 following surgery. iii) Graph showing in vivo antigen-specific cytotoxicity calculated as described in the materials and methods. d) MCA205ova tumors were established s.c. in the flank of wild-type or OX40^−/− C57BL/6 mice and were surgically removed when they reached 7–10mm in diameter. At the time of the operation mice received adoptive transfer of 1×10⁶ naïve wild-type or OX40^−/− Thy1.1⁺ OT1 T cells i.v.. Four days following adoptive transfer, the DLN was harvested and FACS analyzed for the proportion of OT1 T cells. Graphs showing percentage of CD8 T cells that are Thy1.1⁺ OT1 cells in DLN 4 days following adoptive transfer. Key: * - p<0.05

Figure 4. Tumor recurrence involves loss of tumor-specific T cell function in the tumor

MCA205H12ova tumors were established s.c. in the flank of C57BL/6 mice and were surgically removed at 12 days. At surgery, mice received adoptive transfer of 1×10⁶ naïve CFSE-labeled Thy1.1+ OT1 T cells i.v. and 250μg control Ig or αOX40 i.p and followed for local tumor recurrence. In 1 (of 5) control mice the tumor recurred locally. The tumor, blood, draining lymph node (DLN) and non-draining lymph node (NDLN) were harvested and analyzed for the percentage of CD8⁺Thy1.1⁺ OT1 cells in each site. b) Tumor and DLN cells were stimulated with 1μg/ml SIINFEKL for 6 hours in the presence of secretion inhibitors, and analyzed for cytokine production by intracellular cytokine staining. c) Primary cell cultures of the recurrent tumor were established and tested for expression of H2Kb by flow cytometry. d) Naïve OT1 T cells were co-cultured for 48 hours with MCA205, MCA205 pulsed with SIINFEKL peptide, parental MCA205ova or the primary culture of recurrent MCA205ova. Antigen-specific recognition of presented ovalbumin by OT1 was determined by expression of the activation markers CD69 and CD25.

Figure 5. Role of CD8 T cells in local recurrence following radiation and influence of αOX40 therapy on local recurrence

a) 3LL tumors were established s.c. in the leg of C57BL/6 mice and treated at 5–7 days with 20Gy focal radiation. Tumors and the tumor draining lymph node were harvested i) 2 days or ii) 7 days following treatment. Graphs show the proportion of lymph node or tumor-infiltrating cells that are CD8⁺, or the proportion of CD8⁺ cells that express CD25. b) 3LL tumors were established for 5–7 days and treated with 3×20Gy focal radiation over 10 days. One day prior to the first dose of radiation mice began weekly treatment with CD8-depleting or control antibody. Graph shows ii) mean leg thickness or iii) survival of tumor-bearing mice. c) 3LL tumors were established for 5–7 days and treated with 3x20Gy focal radiation over 10 days. One day after the first dose of radiation mice received a single dose of αOX40 or control antibody. Graph shows ii) mean leg thickness or iii) survival of tumor-bearing mice. Key: NS - not significant; * - p<0.05; ** - p<0.01; *** - p<0.001.