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).

Often, patients fail to respond to immune checkpoint inhibitor (ICI) treatment despite favourable biomarker status. Numerous chemotherapeutic agents have been shown to promote tumour immunogenicity when used in conjunction with ICIs; however, little is known about whether such combination therapies lead to a lasting immune response. Given the potential toxicity of ICI-chemotherapy combinations, identification of biomarkers that accurately predict how individuals respond to specific treatment combinations and whether these responses will be long lasting is of paramount importance. In this study, we explored [18F]AlF-NOTA-KCNA3P, a peptide radiopharmaceutical that targets the Kv1.3 potassium channel overexpressed on T-effector memory (TEM) cells as a PET imaging biomarker for lasting immunological memory response. The first-line colon cancer chemotherapies oxaliplatin and 5-fluorouracil were assessed in a syngeneic colon cancer model, either as monotherapies or in combination with PD1, comparing radiopharmaceutical uptake to memory-associated immune cells in the tumour. [18F]AlF-NOTA-KCNA3P reliably separated tumours with immunological memory responses from non-responding tumours and could be used to measure Kv1.3-expressing TEM cells responsible for durable immunological memory response to combination therapy in vivo.

Immune checkpoint inhibitors (ICIs) block checkpoint receptors that tumours use for immune evasion, allowing immune cells to target and destroy cancer cells. Despite rapid advancements in immunotherapy, durable response rates to ICIs remains low. To address this, combination clinical trials are underway assessing whether adjuvants can enhance responsiveness by increasing tumour immunogenicity. CpG-oligodeoxynucleotides (CpG-ODN) are synthetic DNA fragments containing an unmethylated cysteine-guanosine motif that stimulate the innate and adaptive immune systems by engaging Toll-like receptor 9 (TLR9) present on the plasmacytoid dendritic cells (pDCs) and B cells. Here, we have assessed the ability of AlF-mNOTA-GZP, a peptide tracer targeting granzyme B, to serve as a PET imaging biomarker in response to CpG-ODN 1585 in situ vaccine therapy delivered intratumourally (IT) or intraperitoneally (IP) either as monotherapy or in combination with αPD1. [18F]AlF-mNOTA-GZP was able to differentiate treatment responders from non-responders based on tumour uptake. Furthermore, [18F]AlF-mNOTA-GZP showed positive associations with changes in tumour-associated lymphocytes expressing GZB, namely GZB+ CD8+ T cells, and decreases in suppressive F4/80+ cells. [18F]AlF-mNOTA-GZP tumour uptake was mediated by GZB expressing CD8+ cells and successfully stratifies therapy responders from non-responders, potentially acting as a non-invasive biomarker for ICIs and combination therapy evaluation in a clinical setting.

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.