  Citation: Hartimath, S.V.; Ramasamy, B.; Xuan, T.Y.; Rong, T.J.; Khanapur, S.;Cheng, P.; Hwang, Y.Y.; Robins, E.G.; Goggi, J.L. Granzyme B PET Imaging in Response to In Situ Vaccine Therapy Combined with αPD1 in a Murine Colon Cancer Model. Pharmaceutics 2022, 14, 150. https://doi.org/10.3390/ pharmaceutics14010150 Academic Editors: Steve J. Archibald and Louis Allott Received: 12 November 2021 Accepted: 4 January 2022 Published: 8 January 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). pharmaceutics Article Granzyme B PET Imaging in Response to In Situ Vaccine Therapy Combined with αPD1 in a Murine Colon Cancer Model Siddesh V. Hartimath 1, * ,† , Boominathan Ramasamy 1,2,† , Tan Yun Xuan 1 , Tang Jun Rong 1 , Shivashankar Khanapur 1 , Peter Cheng 1 , You Yi Hwang 3 , Edward G. Robins 1,4 and Julian L. Goggi 1, * 1 Laboratory of Radiochemistry & Molecular Imaging (LRMI), Institute of Bioengineering and Bioimaging (IBB), A*STAR Research Entities, Helios, Singapore 138667, Singapore; Boominathan_Ramasamy@ibb.a-star.edu.sg (B.R.); Tan_Yun_Xuan@ibb.a-star.edu.sg (T.Y.X.); Tang_Jun_Rong@ibb.a-star.edu.sg (T.J.R.); Shivashankar@ibb.a-star.edu.sg (S.K.); Peter_Cheng@ibb.a-star.edu.sg (P.C.); edward_robins@ibb.a-star.edu.sg (E.G.R.) 2 Department of Pharmacology, Faculty of Pharmaceutical Sciences, UCSI University, Kuala Lumpur 56000, Malaysia 3 FACS facility, Singapore Immunology Network (SIgN), A*STAR Research Entities, Immunos, Singapore 138665, Singapore; leon_hwang@immunol.a-star.edu.sg 4 Clinical Imaging Research Centre (CIRC), Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117599, Singapore * Correspondence: s_hartimath@ibb.a-star.edu.sg (S.V.H.); Julian_Goggi@ibb.a-star.edu.sg (J.L.G.) † These authors contributed equally to this work. Abstract: Immune checkpoint inhibitors (ICIs) block checkpoint receptors that tumours use for im- mune 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. [ 18 F]AlF-mNOTA-GZP was able to differentiate treatment responders from non-responders based on tumour uptake. Furthermore, [ 18 F]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. [ 18 F]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. Keywords: granzyme B; PET imaging; immunotherapy; CpG-ODN; [ 18 F]AlF-mNOTA-GZP 1. Introduction Immunotherapy has emerged as the fourth pillar of cancer treatment, along with chemotherapy, radiotherapy, and surgery. Immunotherapy exploits the host immune sys- tem, activating it to identify and destroy cancer cells [1]. Immune checkpoint inhibitors (ICIs) block the checkpoint receptors that tumours use to evade the immune system, damp- ening T cell activation. However, despite rapid advancements in immunotherapy, durable response rates to ICIs remain low, especially in colorectal cancers which are typically mi- crosatellite stable [2]. In order to address this, combination clinical trials are ongoing to assess which adjuvants can enhance responsiveness by increasing tumour immunogenicity. Pharmaceutics 2022, 14, 150. https://doi.org/10.3390/pharmaceutics14010150 https://www.mdpi.com/journal/pharmaceutics