Abstract P006: Partial tumor volume irradiation by 177Lu-PNT6555 induces diverse immune responses in a syngeneic murine tumor model

Abstract Purpose/Objective: Radiopharmaceutical therapy (RPT) enables the systemic delivery of an inherently heterogeneous dose of radiation. The RPT dose heterogeneity is created by a tumor’s non-uniform perfusion and the variation in RPT target expression. Radiation therapy is known to immunomodulate the tumor microenvironment (TME) in a dose-dependent manner. The hypothesis is that RPT with a heterogeneous distribution, delivering a low to high dose gradient in a single TME, will activate multiple dose dependent immune effects that are more effective in promoting priming of adaptive immunity compared to a more homogenous distribution of RPT. Methods: RPT was administered using PNT6555, a fibroblast activation protein alpha targeting agent, and uptake was confirmed with B16 murine melanoma. The B16 cell line is engineered to overexpress mouse fibroblast activation protein alpha (mFAP). The engineered B16 was implanted either alone to form a tumor with 100% mFAP expression (B16-mFAP+) or in a mixture with the parental cell line to create a heterogeneous expression of mFAP (mixed B16-mFAP+/B16-mFAP-). Utilizing the SPECT/CT data and ex vivo biodistribution, image-based dosimetry for 177Lu-PNT6555 was performed using the Monte-Carlo-based RAPID platform. C57BL/6 mice bearing B16-mFAP+ or mixed B16-mFAP+/mFAP- tumors were treated with non-radiolabeled PNT6555, 5, 10, 20 or 40 Gy on day 0. Tumors were harvested on day 7 post-treatment. RT-qPCR was performed to evaluate the type I interferon response and tumor immune susceptibility markers. Mice bearing B16-mFAP+ or mixed B16-mFAP+/mFAP- received a mean tumor dose of 5 Gy 177Lu-PNT6555. Mice were monitored for tumor growth and overall survival. In a separate cohort, tumors and tumor draining lymph nodes (TDLN) were harvested at days 4, 7, and 12 post-injection. Flow cytometry was performed on the tumor to evaluate immune infiltration and TDLN to evaluate dendritic cell and B cell activation. Statistical significance was determined by a one-way ANOVA (flow) or Kaplan-Meier with log-rank testing (therapy). Results: Optimal upregulation of immune-related genes by 177Lu-PNT6555 occurred at 2.5 Gy for Icam, 10 Gy for Ifnβ1, and 20 Gy for H2-K1 (murine gene coding MHCI). A heterogeneous delivery of 5 Gy to mice bearing mixed B16-mFAP+/mFAP- resulted in upregulation of all three genes in a single TME. For the therapy study, a heterogeneous distribution of 5 Gy 177Lu-PNT6555 significantly increased overall survival compared to a homogenous distribution. Flow cytometry revealed a significant increase in dendritic cell activation by CD80 and MHCII expression in the 5 Gy 177Lu-PNT6555 with heterogeneous distribution. Additionally, there was a significant increase in infiltration of CD4+ cells, CD8+ cells, NK cells and B cells as well as preservation of CD8+ cell in the 5 Gy 177Lu-PNT6555 with heterogeneous distribution. Conclusions: These preclinical studies demonstrate the capacity of a heterogeneous dose of RPT to enhance the immunomodulatory effect of RPT and improve overall survival in a monotherapy setting. Citation Format: Maya E. Takashima, Ohyun Kwon, Shin Hye Ahn, Anthony Belanger, Won Jong Jin, Charlotte Sawicki, Paul A. Clark, Tessa Chen, Ria Kumari, Bryan Bednarz, Zachary S. Morris. Partial tumor volume irradiation by 177Lu-PNT6555 induces diverse immune responses in a syngeneic murine tumor model.[abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Translating Targeted Therapies in Combination with Radiotherapy; 2025 Jan 26-29; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(2_Suppl):Abstract nr P006