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 Granzyme targeting Restricted Interaction Peptide specific to family member B (GRIP B) to measure secreted granzyme B (GZMB) biochemistry with positron emission tomography. A proteolytic cleavage of 64Cu-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 64Cu-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 64Cu-GRIP B and tumor imaging studies in germline GZMB knockout mice were applied to show that 64Cu-GRIP B is specific for GZMB proteolysis. Furthermore, we explored a potential noncytotoxic function for GZMB by applying 64Cu-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.
Highlights
IntroductionThe human granzymes consist of five serine proteases (A, B, H, K, M) that are expressed primarily within the secretory vesicles (i.e., granules) of lymphocytes involved in the host defense, namely, natural killer (NK) and cytotoxic T cells (CTLs).[1] In these cell types, granzymes are best understood to be proapoptotic effectors against problematic cells, for example, cancer cells or cells infected with pathogens
The human granzymes consist of five serine proteases (A, B, H, K, M) that are expressed primarily within the secretory vesicles of lymphocytes involved in the host defense, namely, natural killer (NK) and cytotoxic T cells (CTLs).[1]
We show that granzyme biochemistry can be detected in vivo with nuclear imaging
Summary
The human granzymes consist of five serine proteases (A, B, H, K, M) that are expressed primarily within the secretory vesicles (i.e., granules) of lymphocytes involved in the host defense, namely, natural killer (NK) and cytotoxic T cells (CTLs).[1] In these cell types, granzymes are best understood to be proapoptotic effectors against problematic cells, for example, cancer cells or cells infected with pathogens. The secretion of granzymes (including A and B) in abundance during conditions unrelated to host defense has suggested noncytotoxic signaling functions.[11] These data align with a developing narrative suggesting that dysregulated granzyme secretion and proteolysis may contribute to the pathobiology of chronic human disorders, for example, systemic autoimmune diseases.[12] the canon that granzymes are primarily cytotoxic effectors is being challenged by a more complex biological model in which secreted granzymes can persist in extracellular space to perform noncytotoxic signaling functions
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call