Granzyme B PET Imaging of Combined Chemotherapy and Immune Checkpoint Inhibitor Therapy in Colon Cancer

Abstract

Purpose: Chemotherapeutic adjuvants, such as oxaliplatin (OXA) and 5-fluorouracil (5-FU), that enhance the immune system, are being assessed as strategies to improve durable response rates when used in combination with immune checkpoint inhibitor (ICI) monotherapy in cancer patients. In this study, we explored granzyme B (GZB), released by tumor-associated immune cells, as a PET imaging-based stratification marker for successful combination therapy using a fluorine-18 (¹⁸F)-labelled GZB peptide ([¹⁸F]AlF-mNOTA-GZP).

Methods: Using the immunocompetent CT26 syngeneic mouse model of colon cancer, we assessed the potential for [¹⁸F]AlF-mNOTA-GZP to stratify OXA/5-FU and ICI combination therapy response via GZB PET. In vivo tumor uptake of [¹⁸F]AlF-mNOTA-GZP in different treatment arms was quantified by PET, and linked to differences in tumor-associated immune cell populations defined by using multicolour flow cytometry.

Results: [¹⁸F]AlF-mNOTA-GZP tumor uptake was able to clearly differentiate treatment responders from non-responders when stratified based on changes in tumor volume. Furthermore, [¹⁸F]AlF-mNOTA-GZP showed positive associations with changes in tumor-associated lymphocytes expressing GZB, namely GZB+ CD8+ T cells and GZB+ NK+ cells.

Conclusions: [¹⁸F]AlF-mNOTA-GZP tumor uptake, driven by changes in immune cell populations expressing GZB, is able to stratify tumor response to chemotherapeutics combined with ICIs. Our results show that, while the immunomodulatory mode of action of the chemotherapies may be different, the ultimate mechanism of tumor lysis through release of Granzyme B is an accurate biomarker for treatment response.

Keywords: Checkpoint inhibitors; Chemotherapy; Granzyme B; Lymphocytes; Tumor.

Fig. 1.

Structure of [¹⁸F]AlF-mNOTA-GZP.

Fig. 2.

Comparison of therapeutic effect of chemotherapies, ICIs and combinations on change in tumor volume. A. Schematic representation of timeline shows dosing regimen. Mice (n=10–15) were i.p. treated with control IgG, αPD1, OXA, 5-FU or combinations of αPD1+OXA or αPD1+5-FU on days 6, 9, and 12 post tumor implantation. B. Individual tumor volume of CT26 tumor-bearing mice on days 6, 9, 12, 15, 19, and 21 post tumor implantation. C. Average tumor volume of CT26 tumor-bearing mice on days 6, 9, 12, 15, 19, and 21 post tumor implantation. Data are represented as mean ± S.D. D. Average tumor volume of CT26 tumor-bearing mice on days 6, 9, 12, 15, 19, and 21 post tumor implantation. Data are shown post separation of TNR group and represented as % change in tumor volume from the first day of assessment and are indicated as mean ± S.D. (TNR, treated non-responder).

Fig. 3.

A. Representative maximum intensity projection PET/CT images of [¹⁸F]AlF-mNOTA-GZP tumor uptake in CT26 tumors: Treated non-responders (TNR), ⍺PD1 monotherapy, OXA monotherapy, combined ⍺PD1 + OXA, 5-FU monotherapy and combined ⍺PD1 + 5-FU treated animals. Yellow dashed line indicates tumor boundary. Mice administered [¹⁸F]AlF-mNOTA-GZP (~10 MBq intravenously), and images acquired from 60-80 mins post tracer injection. B. In vivo assessment of [¹⁸F]AlF-mNOTA-GZP tumor uptake from PET-CT defined volumes of interest (VOI) from individual mice subjected to ICI/ chemo/ ombination therapy. Significant increases in [¹⁸F]AlF-mNOTA-GZP tumor uptake was observed in treatment arms with ⍺PD-1, OXA, 5-FU, ⍺PD1 + OXA and ⍺PD1 + 5-FU when compared to treated non-responders (TNR, n=10 mice/ group; *P<0.05; **P<0.01 comparing TR to TNR and ^#P<0.05 comparing chemo-ICI TR vs chemo alone; data shown as mean %ID/g ± S.E.M.). C. [¹⁸F]AlF-mNOTA-GZP tumor uptake in CT26 TRs and TNRs (****P<0.0001, data shown as individual %ID/g).

Fig. 4.

Multicolour flow cytometry analysis of immune cell profile of the tumor from CT26 tumor-bearing mice at day 14 post-induction of ICI monotherapy or combination therapies. Percentages of (i) CD8+ T cells relative to CD3+ cells, (ii) GZB+ CD8+ TILS relative to total CD8+ TILS, (iii) NK+ cells relative to total CD45+ cells (iv) GZB+ NK+ cells relative to total NK+ cells (v) GZB+ cells relative to CD45+ and (vi) F4/80+ relative to total CD45+ cells across all treatment arms. Data are shown as individual values with mean ± S.D. and are representative of n=5-10 mice/ group. * P<0.05; ** P<0.01 compared to TNR.