IFNγ PET Imaging as a Predictive Tool for Monitoring Response to Tumor Immunotherapy

Abstract

IFNγ is an attractive target for imaging active antitumor immunity due to its function in the T-cell signaling axis. Here, we test an IFNγ immuno-PET (immunoPET) probe for its capacity to identify adaptive immunotherapy response after HER2/neu vaccination in both spontaneous salivary and orthotopic neu⁺ mouse mammary tumors. IFNγ immunoPET detected elevated cytokine levels in situ after vaccination, which inversely correlated with tumor growth rate, an indicator of response to therapy. In a model of induced T-cell anergy where CD8 T cells infiltrate the tumor, but upregulate PD-1, IFNγ tracer uptake was equivalent to isotype control, illustrating a lack of antitumor T-cell activity. The IFNγ immunoPET tracer detected IFNγ protein sequestered on the surface of tumor cells, likely in complex with the IFNγ receptor, which may explain imaging localization of this soluble factor in vivo Collectively, we find that the activation status of cytotoxic T cells is annotated by IFNγ immunoPET, with reduced off-target binding to secondary lymphoid tissues compared with imaging total CD3⁺ tumor-infiltrating lymphocytes. Targeting of soluble cytokines such as IFNγ by PET imaging may provide valuable noninvasive insight into the function of immune cells in situ Significance: This study presents a novel approach to monitor therapeutic outcomes via IFNγ-targeted positron emission tomography. Cancer Res; 78(19); 5706-17. ©2018 AACR.

Figure 1.. Validation of specificity of ⁸⁹Zr-anti-IFN-γ.

A) BALB/c mice treated with CpG-ODN and imaged with the tracer 72 h p.i. displayed higher uptake in the spleen compared to control (Ctrl) untreated cohorts (n=3 each). B) Tissue distribution of ⁸⁹Zr-anti-IFN-γ at 72 h p.i. demonstrated lower probe accumulation in the spleen upon competitive saturation with 10× cold AN-18 mAb (n=4 each). C) Binding of ⁸⁹Zr-anti-IFN-γ receptor-localized IFN-γ was tested in vitro. TUBO cells were incubated with ⁸⁹Zr-anti-IFN-γ alone (n=5), or with recombinant IFN-γ (rIFN-γ) and washed before addition of ⁸⁹Zr-anti-IFN-γ (n=5). Activity was measured by a gamma counter and adjusted for cell count.

Figure 2.. PET evaluation of immunotherapy response in orthotopic TUBO mammary tumors.

A) Tumor volume was monitored in both untreated control (Ctrl, n=11, left) and vaccinated (Vx, n=12, right) tumors. TUBO cells were inoculated 10 days prior to the start of vaccinations, given on days 0 and 14. PET imaging was conducted on day 15 (Ctrl) and 21 (Vx). B) Representative whole body maximum intensity projections (MIP, top row) and planar (bottom row) images of control (left panels, n=6) and vaccinated (right panels, n=6) mice with ⁸⁹Zr-anti-IFN-γ tracer (left). White circle = tumor, L = liver, H = heart, S = spleen, Th = Thymus. Tumor VOIs were measured for each mouse with a ⁸⁹Zr labeled rat IgG isotype control included for each treatment group (n=3, untreated control; n=6, vaccinated control). C) MIP image (top panels) and planar sections (bottom panels) of ⁸⁹Zr-anti-CD3 images in control (left, n=5) and vaccinated mice (middle, n=6). A non-specific ⁸⁹Zr labeled Armenian hamster IgG isotype control was used to measure tumor VOI in a separate group of untreated mice (right, n = 3).

Figure 3.. Ex Vivo validation of immunotherapy response in TUBO-bearing mice.

Tumors were removed after imaging and validated. A) Total RNA obtained from Ctrl (n=11) and Vx (n=13) tumor tissue was analyzed by qPCR with primers specific to CD3 (left), CD8 (middle), and IFN-γ (right). Cultured TUBO cells serve as control (n=2). B) IFN-γ ELISA was conducted with protein lysates of TUBO tumor segments of control Ctrl (n=10) and Vx (n=11) mice. C) HER2 and neu-specific IgG were measured in serum by flow cytometry (Ctrl: n=13, Vx: n=14). D) HER2 and neu-responsive T cells were measured by IFN-γ ELISPOT (Ctrl: n=6, Vx: n=7).

Figure 4.. PET detection of anti-tumor immunity in spontaneous tumor-bearing NeuT mice.

A) Control, untreated mice (Ctrl: n=6) were imaged by PET after palpable tumors were permitted to grow 31 days. For vaccinated mice (Vx: n=7), upon detection of palpable spontaneous salivary tumors, regulatory T cells (Treg) were depleted 10 d prior to the first vaccination. Mice received two HER2/neu DNA vaccinations 14 d apart. PET imaging was conducted 7 days after the final vaccination. B) Representative whole body maximum intensity projections (MIP, top row) and planar (bottom row) images of control (left panels, n=3) and HER2/neu DNA-vaccinated (right panels, n=4) mice with ⁸⁹Zr-anti-IFN-γ tracer (left). White circle = tumor, L = liver, S = spleen. Tumor VOIs were calculated for each mouse. C) Representative CD3 PET images of MIP (top) and planar sections (bottom) are shown for Ctrl (left) vs. Vx groups (right).

Figure 5.. Ex vivo validation of anti-tumor immunity in spontaneous tumor-bearing NeuT mice.

A) Total RNA was isolated from tumor tissue and qPCR analysis for CD3, CD8, and IFN-γ was conducted (Ctrl: n=3, Vx n=5) B) Serum HER2 (Ctrl, n=5; Vx, n=8) and neu-specific IgG (Ctrl, n=5; Vx n=8) was measured by flow cytometry. C) HER2 and neu-responsive T cells were measured by IFN-γ ELISPOT (n=5 each).

Figure 6.. IFN-γ PET depicts response to ITx.

A) Tumor volume was monitored in TUBO-bearing vaccinated BALB/c mice (n=11). TUBO cells were inoculated 13 days prior to the start of vaccinations, to allow for variability in tumor volumes at treatment onset. Vaccines were given on days 0 and 14. PET imaging was conducted on day 28. B) Weekly tumor growth rate, calculated by regression analysis of log tumor growth, versus ⁸⁹Zr-anti-IFN-γ tracer uptake is plotted for each mouse and evaluated by Pearson’s correlation. C) Tumor growth was monitored during passive immunotherapy with anti-neu mAb 7.16.4, given as 5 doses at 1.5 mg i.p. every 3–4 days as indicated beginning 15 days after tumor inoculation. ⁸⁹Zr-anti-IFN-γ (n=5) or ⁸⁹Zr-rat-IgG control (n=6) PET imaging was conducted 30 days after treatment onset. D) Tumor VOIs were calculated for ⁸⁹Zr-anti-IFN-γ or ⁸⁹Zr-rat-IgG tracers in 7.16.4 treated TUBO-bearing mice. E) Intratumoral localization of CD8 was analyzed by IHC on FFPE tissue (400×). H&E sections are included. F) Control and 7.16.4-treated tumors (n=4 each) were dissociated and analyzed for T cell infiltration by flow cytometry by staining for CD45 and CD8. G) PD-1 expression was analyzed by flow cytometry on CD8+ tumor infiltrates from (F).