X-Ray Cristallography Characterization of Human CD44 Epitopes Releasing Differentiation Blockage in Acute Myeloid Leukemia Blasts.

CD44 is a highly glycosylated type-1 transmembrane protein with multiple isoforms, including the standard one, CD44s (80kDa), which is the most abundant on hematopoietic cells, and alternatively spliced variant isoforms (CD44v). CD44 is strongly expressed on Acute Myeloid Leukemia (AML) blasts, which are immature myeloid cells which terminal differentiation is blocked at various stages, defining the distinct AML subtypes. In our laboratory, we have previously shown that several specific monoclonal antibodies (mAbs) directed to the CD44s isoform, can release the differentiation blockage of primary AML blasts (Charrad et al., Nature Medicine 1999), in all AML subtypes. These results indicated the possibility of using such mAbs to develop a CD44-targeted differentiation therapy that would be efficient in all AML subtypes. Our present aim is to characterize the molecular structure of the CD44 epitopes involved in the release of AML differentiation blockage, by using X-ray crystallography. In order to obtain high levels of soluble CD44 protein and to preserve its native conformational properties, we have constructed distinct fusion proteins comprising the glycosylated CD44s extracellular domain plus the human IgG1 Fc fragment (CD44-Fc). We have also added a polyhistidine tag, which allows to easily purify them by using Ni2+- and protein G affinity, and a TEV specific recognition sequence to cleave off the fusion partner. Eight distinct CD44-Fc constructions have been prepared, transiently expressed in COS-7 cells and the one displaying both the highest affinity for the anti-CD44 mAbs and the highest expression level, has been transfected into CHO-K1 cells. Using a stable cell clone, we have succeeded to produce large amounts of CD44-Fc (100 to 200 mg/L), in serum-free culture medium, using CELLine bioreactor flasks. This optimization of the CD44 production combined with an efficient three-step purification process (successive specific binding to G protein, to Ni2+, concluded by highly resolutive gel filtration), has allowed us to produce, for the first time, a sufficient quantity of highly pure and glycosylated CD44s extracellular domain for structural studies. SPR (Surface Plasmon Resonance) analysis have shown that the 2 differentiation-inducing anti-CD44 mAbs analyzed so far, differ in their dynamics of interaction with the CD44 protein. The molecular structure of the epitopes specifically mapped by differentiation-inducing anti-CD44 mAbs, as revealed by X-ray crystallography, will be presented and discussed, with the perspective of developing an efficient CD44-targeted differentiation therapy in AML.