Abstract Body:

Purpose/Background: Melanoma patients with brain metastases have very high mortality rates, with a median survival of 4-16 months [1,2]. In recent years, immune checkpoint inhibitors, such as those that target programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), have revolutionized cancer treatment. However, only a subset of patients responds to combined checkpoint blockade immunotherapies, and methods to effectively monitor the in vivo immune dynamics associated with the success or failure of this therapeutic approach are limited. Antibody-based PET (immunoPET) is a promising strategy that can address this unmet need. Here, we investigate inducible T-cell costimulatory receptor (ICOS), known to be highly upregulated on both activated CD4⁺ and CD8⁺ T cell subsets, as a potential marker of T cell-mediated immunotherapy response [3]. Specifically, we assess the ability of an ICOS-targeted immunoPET tracer to monitor T cell activation after anti-PD-1/anti-CTLA-4 monoclonal antibody (mAb) therapies in a melanoma murine model of brain metastasis.

Methods: Female C57BL/6 mice (n=30) were injected subcutaneously on the right flank with 0.25×10⁶ B16F1 melanoma cells to generate extracranial tumors. Two days after extracranial tumor inoculation, 5×10³ B16F1 melanoma cells were stereotactically injected into the striatum. Four days after intracranial tumor inoculation, mice were randomized into treatment (n=16) and vehicle (n=14) groups based on intracranial tumor bioluminescence signal. On days 5, 7 and 9 post-intracranial tumor inoculation, mice in the treatment group were given anti-PD-1 and anti-CTLA-4 therapies (200 µg via intraperitoneal injection). Expression of icos was assessed in treatment (n=7), vehicle (n=10), and sham (n=4) mice using quantitative polymerase chain reaction (qPCR) on day 9 after intracranial tumor implantation. Murine ICOS mAb was DFO-conjugated, radiolabeled with ⁸⁹Zr-oxalate, and administered intravenously on day 11 post-intracranial tumor inoculation. PET/CT/MR imaging was performed 72 hours post-⁸⁹Zr-DFO-ICOS mAb injection (50 µCi [1 µCi/µL, mass dose injected; 7 µg]). Mice were then perfused, and organs of interest were analyzed via gamma counting and high-resolution autoradiography. Another cohort of metastatic brain tumor mice (n=10) were used for flow cytometry.

Results: ICOS immunoPET revealed significantly elevated signal in both intracranial and extracranial tumors following anti-PD-1/anti-CTLA-4 treatment, suggesting presence of ICOS⁺-activated T cells after combination therapy with these two immune checkpoint inhibitors (Fig. 1). Whole body PET/CT images in both treatment and vehicle groups also showed signal in the blood pool and liver, but not in bones, consistent with mAb tracer circulation and clearance. PET imaging results were also corroborated by gamma counting and autoradiography, and H&E staining showed localized radioactive signal to the tumor site (Suppl. Fig. 1). A dramatic relative increase in ICOS mRNA expression was observed in brains of therapy-treated mice compared to those of vehicle-treated mice (Suppl. Fig. 2A). Flow cytometry revealed significant upregulation of ICOS on activated CD8⁺ T cells in the brain of therapy-treated mice versus vehicle-treated mice, corresponding with increased binding of ⁸⁹Zr-DFO-ICOS mAb observed in PET images in the intracranial tumor of mice that received the combination immunotherapy (Suppl. Fig. 2B). Combination therapy with anti-PD-1 and anti-CTLA-4 mAbs was effective in halting tumor progression, resulting in significantly smaller tumor volumes in treated mice compared with untreated mice starting on day 12 post-intracranial tumor inoculation (Suppl. Fig. 2C-D). There is also a strong inverse correlation between tumor response (measured as a decrease in tumor volume) and tracer uptake in the intracranial tumor on day 14 versus day 9 after implantation (Suppl. Fig. 2E).

Conclusion: ImmunoPET imaging of ICOS is a promising approach for monitoring T cell activation in response to anti-PD-1 and anti-CTLA-4 immunotherapies in the context of melanoma brain metastasis. ICOS immunoPET imaging has high potential to have significant clinical impact by aiding the stratification of patients for cancer immunotherapies in addition to monitoring and optimizing treatment efficacy in patients.

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