Granzyme B PET Imaging Stratifies Immune Checkpoint Inhibitor Response in Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a notoriously difficult cancer to treat. The recent development of immune checkpoint inhibitors has revolutionised HCC therapy; however, successful response is only observed in a small percentage of patients. Biomarkers typically used to predict treatment response in other tumour types are ineffective in HCC, which arises in an immune-suppressive environment. However, imaging markers that measure changes in tumour infiltrating immune cells may supply information that can be used to determine which patients are responding to therapy posttreatment. We have evaluated [¹⁸F]AlF-mNOTA-GZP, a radiolabeled peptide targeting granzyme B, to stratify response to ICIs in a HEPA 1-tumours, a syngeneic model of HCC. Posttherapy, in vivo tumour retention of [¹⁸F]AlF-mNOTA-GZP was correlated to changes in tumour volume and tumour-infiltrating immune cells. [¹⁸F]AlF-mNOTA-GZP successfully stratified response to immune checkpoint inhibition in the syngeneic HEPA 1-6 model. FACS indicated significant changes in the immune environment including a decrease in immune suppressive CD4+ T regulatory cells and increases in tumour-associated GZB+ NK+ cells, which correlated well with tumour radiopharmaceutical uptake. While the immune response to ICI therapies differs in HCC compared to many other cancers, [¹⁸F]AlF-mNOTA-GZP retention is able to stratify response to ICI therapy associated with tumour infiltrating GZB+ NK+ cells in this complex tumour microenvironment.

Figure 1

Graph displaying tumour volumes in response to administration of ICI therapy. Mice (n = 10 − 15) were i.p. treated with control IgG, αPD1 monotherapy, αCTLA4 monotherapy, or combined αPD1 + αCTLA4 on days 6, 9, and 12 posttumour implantation. (a) Individual HEPA 1-6 tumour volumes posttumour implantation. (b) Average tumour volume and (c) % change in tumour volume of HEPA 1-6 tumour-bearing mice on days 3, 6, 9, 12, 16, and 19 posttumour implantation. Data are shown postseparation of TNR group and represented as mean ± S.D. (TNR: treated nonresponder).

Figure 2

(a) Maximum intensity projection images depicting [¹⁸F]AlF-NOTA-GZP tumour retention. Yellow dotted areas indicate the HEPA 1-6 tumour border. Mice were injected with [¹⁸F]AlF-NOTA-GZP (~10 MBq intravenously) and static images acquired from 60-80 mins postinjection. (b) Graph showing differences in [¹⁸F]AlF-NOTA-GZP tumour retention from each treatment arm. [¹⁸F]AlF-NOTA-GZP tumour retention was significantly increased in αPD-1, αCTLA4 and combined αPD1 + αCTLA4 treatment arms when compared to treated nonresponders (TNR, n = 6 − 8 mice/group; ^∗P < 0.05; ^∗∗P < 0.01 comparing TR to TNR; data shown as mean%ID/g ± S.E.M.). (c) Individual values for [¹⁸F]AlF-NOTA-GZP tumour retention in TRs and TNRs (^∗∗∗P < 0.001, data shown as individual %ID/g).

Figure 3

Multicolour flow cytometry analysis of HEPA 1-6 tumour-associated immune cells after treatment. Percentages of (a) CD3+ T cells relative to CD45+ cells, (b) CD4+ TILS relative to total CD3+ T cells, (c) CD4+ Treg cells relative to total CD4+ cells, and (d) GZB+ NK+ cells relative to total NK+ cells. Data are shown as individual values with mean ± S.D. and are representative of n = 4 − 5 mice/group. ^∗P < 0.05; ^∗∗P < 0.01 compared to TNR.