Use of direct fluorescence labeling and confocal microscopy to determine the biodistribution of two protein therapeutics, Cerezyme® and Ceredase®

Abstract

Efficient targeting of therapeutic reagents to tissues and cell types of interest is critical to achieving therapeutic efficacy and avoiding unwanted side effects due to offtarget uptake. To increase assay efficiency and reduce the number of animals used per experiment during preclinical development, we used a combination of direct fluorescence labeling and confocal microscopy to simultaneously examine the biodistribution of two therapeutic proteins, Cerezyme and Ceredase, in the same animals. We show that the fluorescent tags do not interfere with protein uptake and localization. We are able to detect Cerezyme and Ceredase in intact cells and organs and demonstrate colocalization within target cells using confocal microscopy. In addition, the relative amount of protein internalized by different cell types can be quantified using cell type-specific markers and morphometric analysis. This approach provides an easy and straightforward means of assessing the tissue and cell type-specific biodistribution of multiple protein therapeutics in target organs using a minimal number of animals.

Biotherapeutic target or sink: analysis of the macrophage mannose receptor tissue distribution in murine models of lysosomal storage diseases

“…These data are consistent with the observed staining patterns described above and are not significantly different from analogous measurements made using images acquired from wild-type controls (see Table 2). Our results generally correlate with previous data documenting the cell type and tissue distribution of both placental and recombinant glucocerebrosidase when given for ERT (Friedman et al 1999;Piepenhagen et al 2010). In addition to glucocerebrosidase, we examined the tissue and cell type distribution of IV-administered recombinant human acid α-glucosidase.…”

“…In addition, the cell type and tissue expression pattern of MMR generally correlates with the biodistribution of recombinant human β-glucocerebrosidase (Piepenhagen et al 2010), acid α-glucosidase (Fig. 4), and α-galactosidase A (Schiffmann et al 2000) when any of these enzymes is intravenously administered.…”

“…The presence of appropriate receptors on target cells is beneficial, whereas their presence on nontarget cells can generate nonproductive "sinks," which require higher therapeutic doses and may lead to off-target side effects. Detailed biodistribution studies of exogenous therapeutic enzymes in a variety of LSD mouse models (Gaucher, Fabry, and Pompe) have revealed that the majority of administered enzyme is taken up by endothelial cells and macrophages in liver and spleen (Friedman et al 1999;Piepenhagen et al 2010). Targeting to these tissues is effective in Gaucher and Niemann-Pick, where these tissues are the major sites of disease.…”

X-ray and Biochemical Analysis of N370S Mutant Human Acid β-Glucosidase

“…It is interesting that the 2-3-fold increase in GCase levels we observed after 24 h in the presence of NN-DNJ is comparable with the increase originally reported using Gaucher patient fibroblasts (20) as well as subsequent studies in patient fibroblasts using several different competitive inhibitors (17,21), suggesting that the mechanism(s) of action is similar in all cases. In our experiments, both proteins were already folded and targeted to lysosome via the mannose receptor, so an effect on protein folding or transport from the ER to lysosome could be ruled out in this case (36). The competitive inhibitor isofagomine has been reported to increase the activity and lysosomal level of GCase in patient fibroblasts by several mechanisms (17).…”

“…The N370S GCase purified from insect cells naturally contains pauci-mannose glycan structures, and no further processing is required. Because imiglucerase is delivered directly to the endosomal/lysosomal compartment via the mannose receptor on the cell surface (36), it is possible to study the effect of NN-DNJ on lysosomal GCase while removing any effect on folding or ER degradation. Endogenous rat GCase is not recognized by the polyclonal antibody used in this experiment (Fig.…”