In Vivo Measurement of Granzyme Proteolysis from Activated Immune Cells with PET

Abstract

The biology of human granzymes remains enigmatic in part due to our inability to probe their functions outside of in vitro assays or animal models with divergent granzyme species. We hypothesize that the biology of human granzymes could be better elaborated with a translational imaging technology to reveal the contexts in which granzymes are secreted and biochemically active in vivo. Here, we advance toward this goal by engineering a G ranzyme targeting R estricted I nteraction P eptide specific to family member B (GRIP B) to measure secreted granzyme B (GZMB) biochemistry with positron emission tomography. A proteolytic cleavage of 64 Cu-labeled GRIP B liberates a radiolabeled form of Temporin L, which sequesters the radioisotope by binding to adjacent phospholipid bilayers. Thus, at extended time points postinjection (i.e., hours, not seconds), tissue biodistribution of the radioisotope in vivo reflects relative units of the GZMB activity. As a proof of concept, we show in three syngeneic mouse cancer models that 64 Cu-GRIP B detects GZMB from T cells activated with immune checkpoint inhibitors (CPI). Remarkably, the radiotracer detects the proteolysis within tumors but also in lymphoid tissue, where immune cells are activated by a systemic CPI. Control experiments with an uncleavable analogue of 64 Cu-GRIP B and tumor imaging studies in germline GZMB knockout mice were applied to show that 64 Cu-GRIP B is specific for GZMB proteolysis. Furthermore, we explored a potential noncytotoxic function for GZMB by applying 64 Cu-GRIP B to a model of pulmonary inflammation. In summary, we demonstrate that granzyme biochemistry can be assessed in vivo using an imaging modality that can be scaled vertically into human subjects.

Radionuclide Imaging of Cytotoxic Immune Cell Responses to Anti-Cancer Immunotherapy

“…Rapid renal excretion was demonstrated through biodistribution studies in nude BALB/c mice. A significantly higher tracer uptake was seen in the tumors and spleens of CT26 mice treated with anti-PD-1/anti-CTLA-4 than in a control group, and post-treatment tracer accumulation correlated with a decrease in tumor volume in wild-type mice treated with ICI [102]. One of the advantages that 64 Cu-GRIP B has over the 18 F and 68 Ga probes is that the unique targeting method allows the imaging at a later time point (>4h), improving the contrast with background [101,102].…”

“…Recently, a restricted interaction peptide (RIP) that consists of a labelled antimicrobial peptide fragment that integrates in the cell membrane, a cleavage sequence (recognized by granzyme B), and a peptide masking domain, was designed by Zhao et al [102]. The group chose the octapeptide IEPDVSQV as cleavage site, due to the high specificity of IEPD for granzyme B over other human granzymes and the increased catalytic activity observed after addition of VSQV.…”

Large library docking for novel SARS-CoV-2 main protease non-covalent and covalent inhibitors

“…A fluorescence-quenched substrate with the sequence H 2 N(d-Arg)(d-Arg)-K(MCA)-ATLQAIAS-K(DNP)-COOH was synthesized via the Fmoc solid-phase peptide synthesis as previously described 49 . Kinetic measurements were carried out in Corning black 384-well flat-bottom plates and read on a BioTek H4 multimode plate reader.…”

Role of Serine Proteases at the Tumor-Stroma Interface

“…In particular, in vivo imaging approaches with activatable probes utilizing GzmBcleavable IEPD or IEFD linker allow to directly report functional readouts of immune cell (e.g. CTLs and NK cells) infiltration, activation, and cytotoxicity in tumors after immunotherapy treatments (138)(139)(140)(143)(144)(145)(146). Advanced protease-responsive technologies would enable more comprehensive exploration of proteolytic networks in cancer and provide the next generation of clinical modalities for cancer chemotherapy and immunotherapy.…”