The Uptake of Recombinant Glucocerebrosidases by Blood Monocytes From Type 1 Gaucher Disease Patients Is Heterogeneous

Gaucher disease (GD) is due to an inborn deficiency of glucocerebrosidase (GC), that leads to the accumulation of glucosylceramide in monocytes/macrophages, known as “Gaucher cells”, which are thought to be responsible for a wide range of symptoms. Imiglucerase (CEREZYME^®, Genzyme Corporation) (IMI) is the first line treatment of type 1 GD patients. Two new biosimilar agents, velaglucerase-alfa (VPRIV^®, Shire Human Genetic Therapies, Dublin, Ireland) (VEL) and taliglucerase-alfa (PROTALIX^®, Biotherapeutics, Pfizer) (TAL), have been described as being similar to IMI but differing slightly in glycan structure which could have an impact on macrophage uptake and thus on therapeutic efficacy. However, the ability of native GD cells to capture recombinant enzymes (REs) remains unknown. We used blood monocytes (Mo) from GD patients and healthy donors (HD) as a model to compare the uptake of the three REs.

After informed consent was obtained, cells were obtained from healthy donor blood (n=34). The left over part of biological samples collected for routine analysis from Gaucher patients (n=6) could be used for research because patients had been informed and did not verbally express any disagreement. Mononuclear cells were incubated with three concentrations of each RE (0.1, 0.5 and 1 U/ml) at 37°C for 30 minutes and one hour and washed twice with PBS to eliminate exogenous RE before evaluation of intra-cellular GC activity by standardized flow cytometry as previously described (Berger J. et al., Br J Haematol 2010)

Firstly, we confirmed that GD Mo (n=6) showed a marked enzyme deficiency (about 7% of the normal endogenous activity) as compared to normal glucocerebrosidase activity (GCA) (n=34) Then we observed a dose-dependent in vitro uptake of IMI, TAL and VEL in Mo from 4 untreated GD patients. However, the intra-monocyte (IMo) GCA of TAL was systematically lower than that of IMI and VEL.

Case analysis showed an inter-patient heterogeneity, with the highest increase of intra-monocyte enzyme activity for all REs in patient #2 and the lowest in patient #3; this observation was confirmed in vivo by analysis of Mo 15 min. after the end of the first IMI infusion (× 38 and × 9 endogeneous IMoGCA respectively) whereas patient #2 had received a lower dose of enzyme (45 U/kg/2 weeks vs 60 U/kg/2 weeks). Interestingly, patient #2 showed mild GD suffering only thrombocytopenia and asthenia while patient #3 had an aggressive form of GD, with bone disorders (aseptic osteonecrosis, bone infarction and pseudarthrosis after traumatic fracture). Patient #2 responded rapidly, with improved thrombocytopenia (68%) and increased hemoglobin level (+1.9g/dL) from M3 but hematological response for patient #3 could not be evaluated because initial parameters had been normal in this patient who had been splenectomized 20 years ago. Because of the chitotriosidase deficiency of patient #2, we used plasma CCL18 as a biomarker; its kinetic of decrease was clearly more rapid than for patient #3. Similarly, correction of glycosylated-ferritin was better than in patient #2. The two other patients with IMoGCA values close to that of patient #2 had non-progressive disease not requiring treatment (patient #1), or had thrombocytopenia that improved over the expected period of time (patient #4).

In conclusion, this study shows inter-patient variability in the ability of blood Mo to store recombinant enzymes which to our knowledge has not been previously reported. This was confirmed in vivo 15 min. after the start of the infusion. This variability could partially explain the heterogeneity of GD response to enzyme replacement therapy. In this small series, the least aggressive disease corresponded to the highest intra-monocyte GCA and the most aggressive disease to the least intra-monocyte GCA. Six-month follow-up showed differences in change in biological biomarkers suggesting a relationship between intra-monocyte GCA and disease response. Furthermore in this in vitro GD Mo model, all the compounds available are not similar, even if their chemical structures are only slightly different with no relationship with the expression of the CD206 (data not shown). Another RE influx mechanism could exist and influence enzyme replacement therapy. These findings could help in customizing replacement therapy.

Belmatoug:Genzyme: Consultancy; Shire: Research Funding. Berger:Shire: Consultancy; Genzyme: Consultancy, Research Funding.