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Inducible T-cell costimulator (ICOS) is a homodimeric protein expressed on the surface of activated T-cells that is being investigated as a potential therapeutic target to treat various cancers. Recent studies have reported aberrant increases in the soluble form of ICOS (sICOS) in human serum in disease-state patients, primarily using commercial ELISA kits. However, results from our in-house immunoassay did not show these aberrant increases, leading us to speculate that commercial sICOS ELISAs may be prone to interference. We directly tested that hypothesis and found that one widely used commercial kit yields false-positives and is prone to human anti-mouse antibody (HAMA) interference. We then analyzed a panel of healthy, cancer, chronic hepatitis C virus, systemic lupus erythematosus, and diffuse cutaneous systemic sclerosis human serum using our in-house immunoassay and reported the measured sICOS concentrations in these populations. Since even well characterized immunoassay methods are prone to non-specific interference, we also developed a novel sICOS LC-MS/MS method to confirm the results. Using these orthogonal approaches, we show that sICOS is a low abundance soluble protein that cannot be measured above approximately 20 pg/mL in human serum.

Conjugation or fusion to AlbudAbs™ (albumin-binding domain antibodies) is a novel approach to extend the half-life and alter the tissue distribution of biological and small molecule therapeutics. To understand extravasation kinetics and extravascular organ concentrations of AlbudAbs in humans, we studied tissue distribution and elimination of a non-conjugated 89Zr-labeled AlbudAb in healthy volunteers using positron emission tomography/computed tomography (PET/CT). Methods A non-conjugated AlbudAb (GSK3128349) was radiolabeled with 89Zr and a single 1 mg (~ 15 MBq) dose intravenously administered to eight healthy males. 89Zr-AlbudAb tissue distribution was followed for up to 7 days with four whole-body PET/CT scans. 89Zr-AlbudAb tissue concentrations were quantified in organs of therapeutic significance, measuring standardized uptake value and tissue/plasma ratios. Plasma pharmacokinetics were assessed by gamma counting and LC-MS/MS of blood samples. Results 89Zr-AlbudAb administration and PET/CT procedures were well tolerated, with no drug-related immunogenicity or adverse events. 89Zr-AlbudAb rapidly distributed throughout the vasculature, with tissue/plasma ratios in the liver, lungs, and heart relatively stable over 7 days post-dose, ranging between 0.1 and 0.5. The brain tissue/plasma ratio of 0.025 suggested minimal AlbudAb blood-brain barrier penetration. Slowly increasing ratios in muscle, testis, pancreas, and spleen reflected either slow AlbudAb penetration and/or 89Zr residualization in these organs. Across all tissues evaluated, the kidney tissue/plasma ratio was highest (0.5–1.5 range) with highest concentration in the renal cortex. The terminal half-life of the 89Zr-AlbudAb was 18 days. Conclusion Evaluating the biodistribution of 89Zr-AlbudAb in healthy volunteers using a low radioactivity dose was successful (total subject exposure ~ 10 mSv). Results indicated rapid formation of reversible, but stable, complexes between AlbudAb and albumin upon dosing. 89Zr-AlbudAb demonstrated albumin-like pharmacokinetics, including limited renal elimination. This novel organ-specific distribution data for AlbudAbs in humans will facilitate a better selection of drug targets to prosecute using the AlbudAb platform and significantly contribute to modeling work optimizing dosing of therapeutic AlbudAbs in the clinic. Electronic supplementary material The online version of this article (10.1186/s13550-019-0514-9) contains supplementary material, which is available to authorized users.

Traditionally, trypsin and Lys-C proteases are used in combination to digest proteolytically resistant proteins. Here we describe use of Trypsin/Lys-C Mix for general digestion needs. Working simultaneously under conventional non-denaturing trypsin digestion conditions, Trypsin and Lys-C enhance proteolysis and provide multiple positive effects on protein mass spectrometry analysis including increased number of identified peptides and proteins, higher analytical reproducibility and more accurate protein quantitation. Effectively, by supplementing trypsin with Lys-C, we create improved trypsin.