Fibroblast activation protein targeted radiotherapy induces an immunogenic tumor microenvironment and enhances the efficacy of PD-1 immune checkpoint inhibition

Abstract

Purpose: FAP is a membrane-bound protease under investigation as a pan-cancer target, given its high levels in tumors but limited expression in normal tissues. FAP-2286 is a radiopharmaceutical in clinical development for solid tumors that consists of two functional elements: a FAP-targeting peptide and a chelator used to attach radioisotopes. Preclinically, we evaluated the immune modulation and anti-tumor efficacy of FAP-2287, a murine surrogate for FAP-2286, conjugated to the radionuclide lutetium-177 (¹⁷⁷Lu) as a monotherapy and in combination with a PD-1 targeting antibody.

Methods: C57BL/6 mice bearing MCA205 mouse FAP-expressing tumors (MCA205-mFAP) were treated with ¹⁷⁷Lu-FAP-2287, anti-PD-1, or both. Tumor uptake of ¹⁷⁷Lu- FAP-2287 was assessed by SPECT/CT scanning, while therapeutic efficacy was measured by tumor volume and survival. Immune profiling of tumor infiltrates was evaluated through flow cytometry, RNA expression, and immunohistochemistry analyses.

Results: ¹⁷⁷Lu-FAP-2287 rapidly accumulated in MCA205-mFAP tumors leading to significant tumor growth inhibition (TGI) and longer survival time. Significant TGI was also observed from anti-PD-1 and the combination. In flow cytometry analysis of tumors, ¹⁷⁷Lu-FAP-2287 increased CD8⁺ T cell infiltration which was maintained in the combination with anti-PD-1. The increase in CD8⁺ T cells was accompanied by an induction of STING-mediated type I interferon response and higher levels of co-stimulatory molecules such as CD86.

Conclusion: In a preclinical model, FAP-targeted radiotherapy enhanced anti-PD-1-mediated TGI by modulating the TME and increasing the recruitment of tumor-infiltrating CD8⁺ T cells. These findings provide a rationale for clinical studies of combined ¹⁷⁷Lu-FAP-2286 radiotherapy and immune checkpoint inhibition in FAP-positive tumors.

Keywords: CD8; FAP; PD-1; STING; TRT; Theranostic.

Fig. 1

In vivo uptake of ¹⁷⁷Lu-FAP-2287 into FAP-positive MCA205-mFAP syngeneic tumors. Representative images of MCA205-mFAP tumors analyzed by FAP IHC (A, 200 μm) and ¹¹¹In-FAP-2287 ARG (B, 2 mm) are shown. SPECT images of one representative mouse at 4 different timepoints are shown (C). Quantification of ¹⁷⁷Lu-FAP-2287 biodistribution as mean ± SEM %ID/g (n = 6) in tumor and kidney at 3, 24, 48 and 72 h pi (D) and summary table of uptake in several organs and tumor to kidney ratios (E) are shown

Fig. 2

Tumor efficacy of ¹⁷⁷Lu-FAP-2287 and in combination with anti-PD-1 in MCA205-mFAP syngeneic tumor model. MCA205-mFAP tumor-bearing mice were treated with vehicle, ¹⁷⁷Lu-FAP-2287, anti-PD-1 antibody, or combination ¹⁷⁷Lu-FAP-2287 plus anti-PD-1 (n = 12 mice/group). Mean tumor volumes ± SEM during study period (A), Kaplan Meier survival curve (B) and a summary table (C) are shown

Fig. 3

Immune profiling of CD86⁺ myeloid subsets after treatment with ¹⁷⁷Lu-FAP-2287 and in combination with anti-PD-1 in MCA205-mFAP syngeneic tumor model. Individual percentage of CD45⁺ cells with bars as mean ± SEM on day 8 and 13 pi (A), individual percentage of Mo-MDSC and TAM and their CD86 MFI with bars as mean ± SEM on day 8 and 13 pi (B), and a summary table (C) are shown. Significant changes compared to vehicle in the summary table and between groups in the graph are denoted as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001

Fig. 4

Immune profiling of CD8⁺ T cells after treatment with ¹⁷⁷Lu-FAP-2287 and in combination with anti-PD-1 in MCA205-mFAP syngeneic tumor model. Individual percentage of T cells and CD8⁺ T cells with bars as mean ± SEM on day 8 and 13 pi (A), individual percentage of CD127⁺CD8⁺ cells and their CD127 MFI with bars as mean ± SEM on day 8 and 13 pi (B), and a summary table (C) are shown. Significant changes compared to vehicle in the summary table and between groups in the graph are denoted as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001

Fig. 5

IHC analysis of CD8⁺ T cells after treatment with ¹⁷⁷Lu-FAP-2287 and in combination with anti-PD-1 in MCA205-mFAP syngeneic tumor model. Representative images show CD8 staining in MCA205-mFAP tumors (A, 100 μm) and individual percentage of CD8⁺ cells by IHC quantification with bars as mean ± SEM are plotted for days 8 and 13 pi (B). Significant changes between groups are denoted as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001

Fig. 6

Differential expression analysis of MCA205-mFAP tumors after treatment with ¹⁷⁷Lu-FAP-2287 and in combination with anti-PD-1. Fold changes in gene expression versus vehicle are shown as volcano plots with statistically significant genes above the horizontal line, and highly differentially expressed genes on either side

Fig. 7

Molecular profiling of MCA205-mFAP tumors after treatment with ¹⁷⁷Lu-FAP-2287 and in combination with anti-PD-1. Individual normalized linear count with bars as mean ± SEM are plotted for FAP and several immune genes on days 8 and 13 pi (A). Significant changes between groups are denoted as * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. The heatmap was obtained by hierarchical clustering centered around genes with correlation distance and complete linkage, using ClustVis tools (https://bio.tools/clustvis). The color scale displays the row z-score: red color indicates high abundance while blue color low abundance (B). Groups are marked as VEH, vehicle; TRT, ¹⁷⁷Lu-FAP-2287; PD1, anti-PD-1, COM, combination