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

Abstract

BackgroundImmune 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.MethodsB78 (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.ResultsISV+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.ConclusionISV 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.

Low-Dose Radiation Potentiates the Propagation of Anti-Tumor Immunity against Melanoma Tumor in the Brain after In Situ Vaccination at a Tumor outside the Brain

“…We are now evaluating this treatment approach in patients with metastatic melanoma in a phase I/ II trial (NCT03958383). Although this ISV regimen consistently results in a potent systemic anti-tumor immune response in mice leading to complete response at distant tumor sites that are not directly treated with radiation or intratumoral injection (12), we recently reported that this treatment is much less effective in controlling tumor that is present in the brain at time of treatment (13). This is consistent with prior studies demonstrating the restrictive effects of the brain tumor microenvironment on anti-tumor immunity (14)(15)(16)(17)(18)(19).…”

“…C57BL/6-Tg (Foxp3 DTR/EGFP) 23.2 Spar/Mmjax ''DEREG'' mice were purchased from the Jackson Laboratory (MMRRC; Bar Harbor, ME). Treg depletion with diphtheria toxin was achieved following a prior methodology, as reported elsewhere (13,27,30) by daily intraperitoneal (i.p.) injection of 1 lg diphtheria toxin (MilliporeSigma, St. Louis, MO) diluted in PBS for 2 days at day 14 and 15 postirradiation, to ensure presence of melanoma brain tumor prior to Treg depletion.…”

“…We previously reported that the ISV effect of radiation therapy can be augmented in preclinical studies by combination with local injection of tumor-specific antibody and the immune-stimulatory cytokine IL2 (12,13). This combined-modality ISV regimen rendered immunologically ''cold'' tumors responsive to immune checkpoint blockade; and, when combined with anti-CTLA-4 therapy, this regimen resulted in much more effective tumor clearance and activation of tumor-specific immune memory than radiation alone (12).…”

“…While moderate-to high-dose (8-12 Gy/fraction) hypofractionated radiation may be optimal for priming an ISV effect (26), we have been investigating the potential capability of low-to-moderate-dose radiation (2)(3)(4) to enhance the propagation of such an effect. Although ISV may prime a more effective T-cell response, when these effector T cells circulate to nonirradiated tumors, particularly those in immune-restrictive locations like the brain, they encounter a suppressive tumor microenvironment and poorly susceptible tumor cells (13,27). In mice with multiple extracranial tumors, we recently observed that delivery of radiation to the secondary melanoma tumor sites enhances the efficacy of ISV or ISV þ anti-CTLA-4 directed at a single tumor site (27).…”

Utilizing Immunocytokines for Cancer Therapy

“…This reaction was dependent on ADCC and T cell infiltration, and it conferred protection against rechallenge. Even greater anti-tumor responses were seen when radiation, immunocytokine, and anti-CTLA-4 blockade were combined in immunocytokine antigen-positive tumors [ 156 , 157 ]. However, the presence of a secondary untreated tumor negatively impacted the immunocytokine/radiation efficacy in primary treated pancreatic and melanoma tumors.…”