Tumor-Specific Antibody, Cetuximab, Enhances the In Situ Vaccine Effect of Radiation in Immunologically Cold Head and Neck Squamous Cell Carcinoma.

In head and neck squamous cell carcinoma (HNSCC) tumors that over-expresses huEGFR, the anti-EGFR antibody, cetuximab, antagonizes tumor cell viability and sensitizes to radiation therapy. However, the immunologic interactions between cetuximab and radiation therapy are not well understood. We transduced two syngeneic murine HNSCC tumor cell lines to express human EGFR (MOC1- and MOC2-huEGFR) in order to facilitate evaluation of the immunologic interactions between radiation and cetuximab. Cetuximab was capable of inducing antibody-dependent cellular cytotoxicity (ADCC) in MOC1- and MOC2-huEGFR cells but showed no effect on the viability or radiosensitivity of these tumor cells, which also express muEGFR that is not targeted by cetuximab. Radiation enhanced the susceptibility of MOC1- and MOC2-huEGFR to ADCC, eliciting a type I interferon response and increasing expression of NKG2D ligands on these tumor cells. Co-culture of splenocytes with cetuximab and MOC2-huEGFR cells resulted in increased expression of IFNγ in not only NK cells but also in CD8+ T cells, and this was dependent upon splenocyte expression of FcγR. In MOC2-huEGFR tumors, combining radiation and cetuximab induced tumor growth delay that required NK cells, EGFR expression, and FcγR on host immune cells. Combination of radiation and cetuximab increased tumor infiltration with NK and CD8+ T cells but not regulatory T cells. Expression of PD-L1 was increased in MOC2-huEGFR tumors following treatment with radiation and cetuximab. Delivering anti-PD-L1 antibody with radiation and cetuximab improved survival and resulted in durable tumor regression in some mice. Notably, these cured mice showed evidence of an adaptive memory response that was not specifically directed against huEGFR. These findings suggest an opportunity to improve the treatment of HNSCC by combining radiation and cetuximab to engage an innate anti-tumor immune response that may prime an effective adaptive immune response when combined with immune checkpoint blockade. It is possible that this approach could be extended to any immunologically cold tumor that does not respond to immune checkpoint blockade alone and for which a tumor-specific antibody exists or could be developed.

In situ vaccination at a peripheral tumor site augments response against melanoma brain metastases.

BACKGROUND: Immune checkpoint inhibition (ICI) alone is not efficacious for a large number of patients with melanoma brain metastases. We previously established an in situ vaccination (ISV) regimen combining radiation and immunocytokine to enhance response to ICIs. Here, we tested whether ISV inhibits the development of brain metastases in a murine melanoma model. METHODS: B78 (GD2+) melanoma 'primary' tumors were engrafted on the right flank of C57BL/6 mice. After 3-4 weeks, primary tumors were treated with ISV (radiation (12 Gy, day 1), α-GD2 immunocytokine (hu14.18-IL2, days 6-10)) and ICI (α-CTLA-4, days 3, 6, 9). Complete response (CR) was defined as no residual tumor observed at treatment day 90. Mice with CR were tested for immune memory by re-engraftment with B78 in the left flank and then the brain. To test ISV efficacy against metastases, tumors were also engrafted in the left flank and brain of previously untreated mice. Tumors were analyzed by quantitative reverse transcription-PCR, immunohistochemistry, flow cytometry and multiplex cytokine assay. RESULTS: ISV+α-CTLA-4 resulted in immune memory and rejection of B78 engraftment in the brain in 11 of 12 mice. When B78 was engrafted in brain prior to treatment, ISV+α-CTLA-4 increased survival compared with ICI alone. ISV+α-CTLA-4 eradicated left flank tumors but did not elicit CR at brain sites when tumor cells were engrafted in brain prior to ISV. ISV+α-CTLA-4 increased CD8+ and CD4+ T cells in flank and brain tumors compared with untreated mice. Among ISV + α-CTLA-4 treated mice, left flank tumors showed increased CD8+ infiltration and CD8+:FOXP3+ ratio compared with brain tumors. Flank and brain tumors showed minimal differences in expression of immune checkpoint receptors/ligands or Mhc-1. Cytokine productions were similar in left flank and brain tumors in untreated mice. Following ISV+α-CTLA-4, production of immune-stimulatory cytokines was greater in left flank compared with brain tumor grafts. CONCLUSION: ISV augmented response to ICIs in murine melanoma at brain and extracranial tumor sites. Although baseline tumor-immune microenvironments were similar at brain and extracranial tumor sites, response to ISV+α-CTLA-4 was divergent with reduced infiltration and activation of immune cells in brain tumors. Additional therapies may be needed for effective antitumor immune response against melanoma brain metastases.

Tumor-Specific Inhibition of In Situ Vaccination by Distant Untreated Tumor Sites.

In situ vaccination is an emerging cancer treatment strategy that uses local therapies to stimulate a systemic antitumor immune response. We previously reported an in situ vaccination effect when combining radiation (RT) with intratumor (IT) injection of tumor-specific immunocytokine (IC), a fusion of tumor-specific antibody and IL2 cytokine. In mice bearing two tumors, we initially hypothesized that delivering RT plus IT-IC to the "primary" tumor would induce a systemic antitumor response causing regression of the "secondary" tumor. To test this, mice bearing one or two syngeneic murine tumors of B78 melanoma and/or Panc02 pancreatic cancer were treated with combined external beam RT and IT-IC to the designated "primary" tumor only. Primary and secondary tumor response as well as animal survival were monitored. Immunohistochemistry and quantitative real-time PCR were used to quantify tumor infiltration with regulatory T cells (Treg). Transgenic "DEREG" mice or IgG2a anti-CTLA-4 were used to transiently deplete tumor Tregs. Contrary to our initial hypothesis, we observed that the presence of an untreated secondary tumor antagonized the therapeutic effect of RT + IT-IC delivered to the primary tumor. We observed reciprocal tumor specificity for this effect, which was circumvented if all tumors received RT or by transient depletion of Tregs. Primary tumor treatment with RT + IT-IC together with systemic administration of Treg-depleting anti-CTLA-4 resulted in a renewed in situ vaccination effect. Our findings show that untreated tumors can exert a tumor-specific, Treg-dependent, suppressive effect on the efficacy of in situ vaccination and demonstrate clinically viable approaches to overcome this effect. Untreated tumor sites antagonize the systemic and local antitumor immune response to an in situ vaccination regimen. This effect is radiation sensitive and may be mediated by tumor-specific regulatory T cells harbored in the untreated tumor sites. Cancer Immunol Res; 6(7); 825-34. ©2018 AACR.