Abstract
BackgroundImmune checkpoint inhibitors (ICIs) for solid tumors, including those targeting programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), have shown impressive clinical efficacy, however, most patients do not achieve durable responses. One major therapeutic obstacle is the immunosuppressive tumor immune microenvironment (TIME). Thus, we hypothesized that a strategy combining tumor-directed radiation with TIME immunomodulation could improve ICI response rates in established solid tumors.MethodsUsing a syngeneic mouse model of human papillomavirus (HPV)-associated head and neck cancer, mEER, we developed a maximally effective regimen combining PD-1 and CTLA-4 inhibition, tumor-directed radiation, and two existing immunomodulatory drugs: cyclophosphamide (CTX) and a small-molecule inducible nitric oxide synthase (iNOS) inhibitor, L-n6-(1-iminoethyl)-lysine (L-NIL). We compared the effects of the various combinations of this regimen on tumor growth, overall survival, establishment of immunologic memory, and immunologic changes with flow cytometry and quantitative multiplex immunofluorescence.ResultsWe found PD-1 and CTLA-4 blockade, and radiotherapy alone or in combination, incapable of clearing established tumors or reversing the unfavorable balance of effector to suppressor cells in the TIME. However, modulation of the TIME with cyclophosphamide (CTX) and L-NIL in combination with dual checkpoint inhibition and radiation led to rejection of over 70% of established mEER tumors and doubled median survival in the B16 melanoma model. Anti-tumor activity was CD8+ T cell-dependent and led to development of immunologic memory against tumor-associated HPV antigens. Immune profiling revealed that CTX/L-NIL induced remodeling of myeloid cell populations in the TIME and tumor-draining lymph node and drove subsequent activation and intratumoral infiltration of CD8+ effector T cells.ConclusionsOverall, this study demonstrates that modulation of the immunosuppressive TIME is required to unlock the benefits of ICIs and radiotherapy to induce immunologic rejection of treatment-refractory established solid tumors.
Highlights
Immune checkpoint inhibitors (ICIs) for solid tumors, including those targeting programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), have shown impressive clinical efficacy, most patients do not achieve durable responses
ΑPD-1 and/or αCTLA-4 showed only minor tumor growth and survival improvements and even in combination remained incapable of promoting tumor rejection (Fig. 1b and Additional file 1: Figure S1A)
Additional assessment of the tumor-draining lymph node (tdLN) showed similar lymphocyte percentages for all ICI treated groups as well, with only minor increases in CD8+ T cell percentages for αCTLA-4 monotherapy treated mice (Additional file 1: Figure S1C). These data suggest that αPD-1 and αCTLA-4, alone or in combination, promote only minor treatment benefit in established mEER tumors, likely due to their inability to overcome the highly immunosuppressive tumor immune microenvironment (TIME)
Summary
Immune checkpoint inhibitors (ICIs) for solid tumors, including those targeting programmed cell death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), have shown impressive clinical efficacy, most patients do not achieve durable responses. In 2011 the first ICI, a monoclonal antibody targeting cytotoxic T lymphocyteassociated protein 4 (anti-CTLA-4 or αCTLA-4), was approved for use in advanced melanoma and followed in 2014 by another ICI targeting programmed cell death protein 1 (anti-PD-1 or αPD-1) [2,3,4,5] Both αCTLA-4 and αPD-1 are currently clinically approved or under investigation for use in numerous solid tumor malignancies [6]. Radiation promotes substantial lymphodepletion [19, 20] and immunosuppressive effects, including impaired T cell reactivity; diminished antigen presentation; and elevation of circulating immunosuppressive cells [21, 22] This suggests that additional therapeutic combinations may be required to unmask the maximum benefit of ICIs
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