ObjectiveWe assessed whether famotidine improved inflammation and symptomatic recovery in outpatients with mild to moderate COVID-19.DesignRandomised, double-blind, placebo-controlled, fully remote, phase 2 clinical trial (NCT04724720) enrolling symptomatic unvaccinated adult outpatients with confirmed COVID-19 between January 2021 and April 2021 from two US centres. Patients self-administered 80 mg famotidine (n=28) or placebo (n=27) orally three times a day for 14 consecutive days. Endpoints were time to (primary) or rate of (secondary) symptom resolution, and resolution of inflammation (exploratory).ResultsOf 55 patients in the intention-to-treat group (median age 35 years (IQR: 20); 35 women (64%); 18 African American (33%); 14 Hispanic (26%)), 52 (95%) completed the trial, submitting 1358 electronic symptom surveys. Time to symptom resolution was not statistically improved (p=0.4). Rate of symptom resolution was improved for patients taking famotidine (p<0.0001). Estimated 50% reduction of overall baseline symptom scores were achieved at 8.2 days (95% CI: 7 to 9.8 days) for famotidine and 11.4 days (95% CI: 10.3 to 12.6 days) for placebo treated patients. Differences were independent of patient sex, race or ethnicity. Five self-limiting adverse events occurred (famotidine, n=2 (40%); placebo, n=3 (60%)). On day 7, fewer patients on famotidine had detectable interferon alpha plasma levels (p=0.04). Plasma immunoglobulin type G levels to SARS-CoV-2 nucleocapsid core protein were similar between both arms.ConclusionsFamotidine was safe and well tolerated in outpatients with mild to moderate COVID-19. Famotidine led to earlier resolution of symptoms and inflammation without reducing anti-SARS-CoV-2 immunity. Additional randomised trials are required.

The substrate promiscuity of microbial transglutaminase (mTG) has been exploited in various applications in biotechnology,inparticular for the attachment of alkylamines to glutamine-containing peptides and proteins.H ere,w e expand the substrate repertoire to include hydrazines,h ydrazides,a nd alkoxyamines,resulting in the formation of isopeptide bonds with varied susceptibilities to hydrolysis or exchange by mTG.F urthermore,w ed emonstrate that simple unsubstituted hydrazine and dihydrazides can be used to install reactive hydrazide handles onto the side chain of internal glutamine residues.The distinct hydrazide handles can be further coupled with carbonyls,i ncluding ortho-carbonylphenylboronic acids, to form site-specific and functional bioconjugates with tunable hydrolytic stability.T he extension of the substrate scope of mTG beyond canonical amines thus substantially broadens the versatility of the enzyme,providing anew approach to facilitate novel applications. Scheme 1. Transamidation reaction catalyzed by microbial transglutaminase (mTG) using an amine or an a-effect nucleophile a) hydrazine, b) hydrazide, or c) alkoxyaminea sthe acyl acceptor substrate.

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The secreted protein Cystatin C (CyC) is a cysteine protease inhibitor of incompletely characterized biomedical function, used clinically for estimation of glomerular filtration rate. Plasma CyC is elevated in many patients, especially when they receive glucocorticoid (GC) treatment. Here we empirically connect GCs with systemic regulation of CyC. First, we leveraged genome-wide association and structural equation modeling to determine the genetics of the latent trait CyC production in UK Biobank. Using multi-modal genomic, transcriptional, and experimental approaches, we demonstrated that CyC is a direct target of GC receptor, with GC-responsive CyC secretion exhibited by macrophages and cancer cells in vitro. Elevated serum CyC levels were positively correlated with GC levels in a murine model of cancer progression. Consistent with the coupling of CyC levels to GC signaling in a disease relevant manner, CyC predicted elevated all-cause and cancer-specific mortality in humans. These associations were orthogonally confirmed by a polygenic score (PGS) capturing germline predisposition to CyC production. This PGS predicted checkpoint immunotherapy failure in a combined clinical trial cohort of 685 metastatic cancer patients, with available germline exome sequencing. Taken together, our results demonstrate that CyC captures biomedically-relevant variations in endogenous GC activity, raising the possibility that CyC may be a direct effector of GC-induced immunosuppression and therefore a target for combination cancer immunotherapy.

The substrate promiscuity of microbial transglutaminase (mTG) has been exploited in various applications in biotechnology,inparticular for the attachment of alkylamines to glutamine-containing peptides and proteins.H ere,w e expand the substrate repertoire to include hydrazines,h ydrazides,a nd alkoxyamines,resulting in the formation of isopeptide bonds with varied susceptibilities to hydrolysis or exchange by mTG.F urthermore,w ed emonstrate that simple unsubstituted hydrazine and dihydrazides can be used to install reactive hydrazide handles onto the side chain of internal glutamine residues.The distinct hydrazide handles can be further coupled with carbonyls,i ncluding ortho-carbonylphenylboronic acids, to form site-specific and functional bioconjugates with tunable hydrolytic stability.T he extension of the substrate scope of mTG beyond canonical amines thus substantially broadens the versatility of the enzyme,providing anew approach to facilitate novel applications. Scheme 1. Transamidation reaction catalyzed by microbial transglutaminase (mTG) using an amine or an a-effect nucleophile a) hydrazine, b) hydrazide, or c) alkoxyaminea sthe acyl acceptor substrate.

Immune checkpoint inhibitors (ICI) are highly effective for a minority of patients with cancer. Pre-clinical research into mechanisms of ICI resistance is reliant upon anti-PD-1 or anti-PD-L1 antibody treatment in mouse models of cancer. Common approaches utilize commercially-available rat isotype anti-PD-L1 antibodies given by intraperitoneal (IP) injection at relatively high doses (5mg/kg every 2-3 days), often for extended periods. However, there has been limited investigation into the pharmacokinetics of these antibodies with single and repeat dosing, which could inform rational dose scheduling. Non-species-matched antibodies are known to induce accelerated anti-drug immune responses, compared to species-matched antibodies. As a result, we hypothesized that matched-isotype anti-PD-L1 (based on the grafting of atezolizumab variable regions onto a mouse IgG2a scaffold) and unmatched-isotype rat IgG2b anti-PD-L1 would be associated with distinct pharmacokinetics on single and repeated dosing. We first confirmed that both matched and unmatched antibodies have nanomolar-range affinity for mouse PD-L1 by surface plasmon resonance (0.85nM and 0.37nM, respectively). Using a single IP injection in 8-week-old male C57BL/6J mice followed by repeated tail-vein plasma sampling and an indirect anti-PD-L1 ELISA, we detected a trend towards prolonged median half-life for the matched (n=17 mice) versus unmatched (n=19) antibody (91.5 versus 39.0 hours). We detected a particularly wide range of half-life measurements for matched antibody experiments (19.8 to 337.7 hours), suggesting that antibody clearance can be highly heterogeneous. Using repeated IP injection (2 doses, 14 days apart), we identified markedly accelerated antibody clearance for both antibodies resulting in comparable antibody half-lives after dose 2 (11.9 versus 12.5 hours, n=13 and n=8 mice, respectively), suggestive of a time-dependent anti-drug adaptive immune response. These findings have significant implications for in vivo antibody treatment experiments, demonstrating that repeat dosing is associated with rapid antibody clearance, irrespective of the antibody species. These data should be taken into consideration when planning longer-term antagonism of PD-1/PD-L1 signaling with antibodies in mice and strengthen the basis for short term window studies that examine the intratumoral immune biology. Furthermore, the high variability in antibody half-life suggests that antibody pharmacokinetics could confound ICI treatment response studies in vivo. Citation Format: Breanna Demestichas, Sam Kleeman, Matthew Chvasta, Tobias Janowitz. Comparative mouse pharmacokinetics of matched- and unmatched-isotype anti-PD-L1 antibodies. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5672.

Cystatin C (CyC) is a secreted cysteine protease inhibitor that is commonly used as a marker of renal function, and whose biological functions remain insufficiently characterized. Plasma CyC is elevated relative to renal function in many patients, including those receiving glucocorticoid (GC) treatment and patients with cancer. Endogenous GCs are essential for life and are appropriately upregulated in response to systemic stress. Here we empirically connect GCs with the systemic regulation of CyC. Using measurements of CyC and creatinine, another established marker of renal function, we used genome-wide association and structural equation modeling to determine the genetics of the latent trait CyC production in UK Biobank, thereby uncoupling CyC from renal function. CyC production and a polygenic score (PGS) capturing germline predisposition to CyC production predicted elevated all-cause and cancer-specific mortality. Using phenome-wide association, we further identified an association between germline predisposition to CyC production and accelerated onset of metabolic syndrome. As a potential explanation for these associations, we provide multiple lines of evidence that CyC is a direct transcriptional target of GC receptor, with GC-responsive CyC secretion exhibited by macrophages (cf. monocytes) and co-opted by multiple cancer cell lines, potentially explaining selective CyC elevations in disease states. Using isogenic CyC-knockout tumors (CRISPR-Cas9), we discovered a markedly attenuated tumor growth in vivo that was associated with a significantly reduced fraction of non-epithelial cells. To investigate whether depletion of specific non-epithelial cells could explain this, we performed single-cell RNA sequencing, which revealed abrogated recruitment of Trem2+ macrophages in knockout tumors, subsequently validated by Trem2 immunohistochemistry in a non-overlapping cohort. Trem2+ macrophages have previously been linked to immune suppression and failure of cancer immunotherapy. Consistent with this, we show that the CyC production PGS predicted checkpoint immunotherapy failure in a combined clinical trial cohort of 685 metastatic cancer patients. Taken together, our results demonstrate that CyC may be a direct effector of GC-induced immunosuppression, acting through the recruitment of Trem2+ macrophages, and therefore could be a target for combination cancer immunotherapy. Citation Format: Sam O. Kleeman, Breanna Demestichas, Nicholas Mourikis, Miriam Ferrer, Qing Gao, Dominik Loiero, Yosef J. Riazat-Kesh, Sean Bankier, Dimitrios Chantzichristos, Gudmundur Johannsson, Claire Regan, Jonathan Preall, Viktor H. Koelzer, Brian R. Walker, Hannah Meyer, Tobias Janowitz. Cystatin C is glucocorticoid-responsive, directs recruitment of Trem2+ macrophages and predicts failure of cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2891.

Non-invasive imaging of tumors expressing reporter transgenes is a popular preclinical method for studying tumor development and response to therapy in vivo due to its ability to distinguish signal from tumors over background noise. However, the utilized transgenes, such as firefly luciferase, are immunogenic and, therefore, impact results when expressed in immune-competent hosts. This represents an important limitation, given that cancer immunology and immunotherapy are currently among the most impactful areas of research and therapeutic development. Here we present a non-immunogenic preclinical tumor imaging approach. Based on the expression of murine sodium iodide symporter (mNIS), it facilitates sensitive, non-invasive detection of syngeneic tumor cells in immune-competent tumor models without additional immunogenicity arising from exogenous transgenic protein or selection marker expression. NIS-expressing tumor cells internalize the gamma-emitting [99mTc]pertechnetate ion and so can be detected by SPECT (single photon emission computed to-mography). Using a mouse model of pancreatic ductal adenocarcinoma he-patic metastases in immune-competent C57BL/6 mice, we demonstrate that the technique enables the detection of very early metastatic lesions and longitudinal assessment of immunotherapy responses using precise and quantifiable whole-body SPECT/CT imaging.