A novel [89Zr]-anti-PD-1-PET-CT to assess response to PD-1/PD-L1 blockade in lung cancer

Abstract

Background: Harnessing the anti-tumor immune system response by targeting the program cell death protein (PD-1) and program cell death ligand protein (PD-L1) axis has been a major breakthrough in non-small cell lung cancer (NSCLC) therapy. Nonetheless, conventional imaging tools cannot accurately assess response in immunotherapy-treated patients. Using a lung cancer syngeneic mouse model responder to immunotherapy, we aimed to demonstrate that [⁸⁹Zr]-anti-PD-1 immuno-PET is a safe and feasible imaging modality to assess the response to PD-1/PD-L1 blockade in NSCLC.

Materials and methods: A syngeneic mouse model responder to anti-PD-1 therapy was used. Tumor growth and response to PD-1 blockade were monitored by conventional 2-deoxy-2-[¹⁸F]fluoro-D-glucose ([¹⁸F]-FDG) PET scans. Additionally, tumor lymphocyte infiltration was analyzed by the use of an [⁸⁹Zr]-labeled anti-PD-1 antibody and measured as ⁸⁹Zr tumor uptake.

Results: Conventional [¹⁸F]-FDG-PET scans failed to detect the antitumor activity exerted by anti-PD-1 therapy. However, [⁸⁹Zr]-anti-PD-1 uptake was substantially higher in mice that responded to PD-1 blockade. The analysis of tumor-infiltrating immune cell populations and interleukins demonstrated an increased anti-tumor effect elicited by activation of effector immune cells in PD-1-responder mice. Interestingly, a positive correlation between [⁸⁹Zr]-anti-PD-1 uptake and the proportion of tumor-infiltrating lymphocytes (TILs) was found (Cor = 0.8; p = 0.001).

Conclusion: Our data may support the clinical implementation of immuno-PET as a promising novel imaging tool to predict and assess the response of PD-1/PD-L1 inhibitors in patients with NSCLC.

Keywords: PD-1 inhibition; immuno-PET; inhibitor of differentiation 1; lung adenocarcinoma; pseudoprogression.

Figure 1

[¹⁸F]-FDG does not detect the antitumor effect of PD-1 blockade in LLC tumors. (A) Representative PET images at days 6 and 13 of ¹[¹⁸F]-FDG uptake in LLC cells (LLC Sc) injected in Id1^+/+ (C57BL/6J) or Id1^-/- (IDKO) (n = 4) mice groups. (B) Quantification of [¹⁸F]-FDG SUVmax representing the fold change between the uptake at days 6 and 13 of the four mice groups described in (A). (C) Representative PET images of [¹⁸F]-FDG uptake at days 6 and 13 of Id1 silenced LLC cells (LLC sh-ID1) injected in Id1^+/+ (C57BL/6J) or Id1^-/- (IDKO) (n = 4) mice groups. (D) Quantification of [¹⁸F]-FDG SUVmax representing the fold change between the uptake at days 6 and 13 of the four mice groups described in (C). Error bars denote SD.

Figure 2

[⁸⁹Zr]-anti-PD-1 allows an accurate evaluation of tumor response to immunotherapy in a lung cancer mouse model. (A) Outline of the mouse model. LLC [sh_Control (Sc) and Id1 silenced cells (sh-ID1)] were subcutaneously injected in Id1 ^+/+ C57BL/6J (C57) and Id1 ^-/- C57BL/6J (IDKO) mice, and animals were treated with PBS or an anti-PD-1 mAb (days 7, 10; RPM-14 100mg, intraperitoneally) and with [⁸⁹Zr]-anti-PD-1 (day 14; 100mg, intraperitoneally) (n=4 mice per group). Tumor volume was measured using a caliper (mm³), using [¹⁸F]-FDG-PET scan analyses (days 6 and 13) or using [⁸⁹Zr]-anti-PD-1 PET scans analysis (days 15, 17 and 20 after LLC inoculation). (B) Representative PET-CT images of ⁸⁹Zr signal at days 15, 17 and 20 after LLC inoculation. (C) ⁸⁹Zr signal LLC Sc and sh-ID1 cells injected in C57 and IDKO treated with anti-PD-1 at days 15, 17, and 20 after LLC inoculation. Asterisks denote significance (*p < 0.05, **p < 0.005, ***p < 0.001), and error bars denote SD.

Figure 3

[⁸⁹Zr]-anti-PD-1 uptake correlates with immune T cell infiltration. (A) Representative IHC images illustrating: Left: CD3⁺ T cells; Middle: CD8⁺ T cells; Right: CD4⁺ T cells; Scale bar: 200μm. (B) Quantification of the relative stained area of: Left: CD3⁺ T cells; Middle: CD8⁺ T cells; Right: CD4⁺ T cells. (C) Correlation between ⁸⁹Zr uptake at day 20 after LLC inoculation and: Left: CD3⁺ T cells proportion area (Cor = 0.8); Right: CD8⁺ T cells proportion area (Cor = 0.8). Asterisks denote significance (*p < 0.05, ***p < 0.001), and error bars denote SD.

Figure 4

Id1 inhibition at the tumor-microenvironment promotes proinflammatory interleukin expression and T cell infiltration. (A) Representative IHC images illustrating CD3⁺ T cells, CD8⁺ T cells and Id1⁺ cells of LLC cells inoculated in C57 and IDKO mice. Scale bar: 200μm. (B) Left: Quantification of proportion of relative stained area of CD3⁺ T cells. Right: Quantification of the proportion of relative stained area of CD8⁺ T cells of tumor samples illustrated in (A). (C) Representative images of multiplex immunofluorescence staining panel with nuclei (white), Id1 (red), CD3 (green), CD8 (yellow) of LLC cells inoculated in C57 and IDKO mice. Scale bar: 200μm. (D) Left: Quantification of multiplex immunofluorescence staining of CD3⁺ T cells. Right: Quantification of multiplex immunofluorescence staining of CD8⁺ T cells of tumor samples illustrated in (C). (E) Relative mRNA expression levels of Il-1b, Ifn-γ and Tnf-α in LLC tumors in C57 and IDKO mice. Asterisks denote significance (*p < 0.05, ***p < 0.001), and error bars denote SD.