IDENTIFICATION OF THE ANTIGEN RECOGNIZED BY RHIGM22, A REMYELINATION-PROMOTING HUMAN MONOCLONAL ANTIBODY

Oligodendrocytes (OLs) are the myelin-forming cells of the central nervous system (CNS). They synthesize large amounts of plasma membrane and extends multiple processes that individually wrap around axons generating a multilayered stack of membranes tightly attached at their cytosolic and external surfaces, i.e. myelin. The myelin membrane provides electric insulation of axons and dictates the clustering of the sodium channels at the nodes of Ranvier and the organization of the node itself, allowing saltatory nerve conduction. A number of neurological diseases of the CNS are characterized by destruction of oligodendrocytes with consequent damage or loss of the myelin sheath. In most experimental models, the normal response to this is remyelination, a process mediated by oligodendrocyte precursor cells (OPC) that ultimately leads to functional recovery. However in human diseases, and in specific in multiple sclerosis (MS), this process is inefficient and fails to successfully counteract the accumulation of lasting axonal damage and increasing brain atrophy, thus resulting in motor and neurological deficits. The development of strategies aimed to increase the efficiency of the remyelination process is therefore an important therapeutic goal. One of these strategies involves the use of CNS reactive antibodies to promote remyelination. One of these antibodies, rHIgM22, is able to bind to oligodendrocytes and myelin in vitro. Moreover, rHIgM22 is able to enter the CNS, accumulate at lesion site and promote remyelination in mouse models of chronical demyelination. As a matter of fact, this antibody has recently passed a phase I clinical trial for treatment of MS. rHIgM22 binds to CNS tissues with a pattern very similar to that of the anti-sulfatide antibody O4, and binding of rHIgM22 is abolished in CNS tissue slices from CST (-/-) mice, suggesting that rHIgM22 binding to myelin requires the presence of a product of cerebroside sulfotransferase, possibly sulfatide. However the exact identity of the antigen recognized by this antibody remains to be elucidated. The binding of rHIgM22 to purified lipids and to lipid extracts from various sources, including wild type, ASM (-/-), CST (+/-) and CST (-/-) mice brains, mouse mixed glial cells (MGC), mouse astrocytes, rat rHIgM22+ oligodendrocytes (OL), rat microglia, and mouse myelin, has been tested using TLC immunostaining assays and SPR experiments with lipid monolayers with different composition. The results obtained show that rHIgM22 binds to sulfatide, and, to a lesser extent, to lysosulfatide in vitro, while it does not bind to other myelin sphingolipids, including galactosylceramide and sphingomyelin, suggesting that sulfatide at the oligodendrocyte surface might be important for the binding of rHIgM22 to the surface of these cells and to myelin. The binding affinity for both sulfatide and its deacylated derivate is low, even if the binding is specific. On the other hand, our data shows that the binding affinity of rHIgM22 for sulfatide can be modulated by the presence of other lipids suggesting a possible role of the membrane microenvironment in the recognition of the antigen by rHIgM22. In addition, rHIgM22 also reacts with phosphatidic acid, and with an unknown molecule present in lipid extracts from various sources, including CST knock-out mice brains, MGC, and isolated astrocytes and microglia. The exact identity of this antigen has yet to be confirmed but preliminary data suggests it might be a form of phosphatidylethanolamine with a free amino group and multiple hydroxylation in the fatty acid residues. Remarkably, this antigen is also present in the extracts from mixed glial cultures, which do not contain mature O4-positive oligodendrocytes, and also in isolated astrocytes and microglia suggesting that other glial cells in addition to oligodendrocytes might be important in the response to rHIgM22. All this suggests that not only sulfatide, but also other membrane lipids might play a role in the binding of rHIgM22 to oligodendrocytes and other cell types. Moreover, binding of rHIgM22 to intact cells might require a complex molecular arrangement, and, in particular, sulfatide might be part of the functional rHIgM22 antigen localized at the cell surface.