Cellular Mechanisms of CNS Repair by Natural Autoreactive Monoclonal Antibodies

Abstract

Natural autoreactive monoclonal IgMs have demonstrated potential as therapeutic agents for CNS disease. These antibodies bind surface antigens on specific CNS cells activating intracellular repair-promoting signals. IgMs that bind to surface antigens on oligodendrocytes enhanced remyelination in animal models of multiple sclerosis. IgMs that bind to neurons stimulate neurite outgrowth and prevent neuron apoptosis. The neuron-binding IgMs may have utility in CNS axon- or neuron-damaging diseases such as amyotrophic lateral sclerosis, stroke, spinal cord injury or secondary progressive multiple sclerosis. Recombinant remyelination-promoting IgMs have been generated for formal toxicology studies and, after FDA approval, a Phase I clinical trial. Natural autoreactive monoclonal antibodies directed against CNS cells represent novel therapeutic molecules to induce repair of the nervous system.

Natural autoreactive (NA) monoclonal IgM antibodies can promote central nervous system (CNS) protection and repair. These repair-promoting IgMs have characteristics of classic NA antibodies. For example, they are generally of the IgM isotype; encoded by germline genes with few somatic mutations; and polyreactive with low affinity with a range of structurally unrelated, self and non-self antigens, specifically cytoskeleton, nuclear proteins and DNA ¹. In addition, NA monoclonal antibodies that promote CNS protection and repair bind specifically to surface plasma membrane antigens, which activate intracellular signals that promote neuron or glial cell survival ^{2, 3} and cross the blood-brain-barrier to accumulate within injured regions of the CNS⁴. Other independent investigators have also demonstrated that human NA monoclonal antibodies cross the blood-brain barrier and localize to normal and injured CNS tissues in vivo ⁵. The therapeutic human IgMs bind to membrane antigens, which are destroyed following treatments that disrupt cellular architecture including chemical/biochemical fixation, dehydration, solubilization, extraction or digestion and physical/mechanical crush forces or extreme temperatures ^{6, 7}. The human IgMs demonstrate specific affinity only when membranes are maintained under live physiological conditions ^6–8. Cell signals are activated through direct antibody-protein-glycolipid binding interactions. ^{2, 3, 9–11}.

Identification of Oligodendrocyte Binding Antibodies That Promote CNS Repair

We employed a novel strategy to identify human monoclonal antibodies that promote remyelination ¹². Monoclonal antibodies were isolated from the sera of patients with monoclonal gammopathy. Selection criteria included a serum monoclonal immunoglobulin concentration of greater than 3 g/dL and a lack of neurologic or antibody-associated pathologies. We screened antibodies for binding to myelin in live CNS tissue slices and to the surface of live oligodendrocytes in culture ¹². Six of 52 serum-derived human IgMs (sHIgM) and zero of 50 serum-derived human IgGs (sHIgG) bound in these assays. Two IgMs (sHIgM22 and sHIgM46) promoted significant remyelination in vivo ¹². A recombinant version of sHIgM22, rHIgM22, was engineered by cloning the antibody variable region DNA sequence into an expression vector ^{9, 13}. rHIgM22 promoted myelin repair in the Theiler's virus infection-induced model of MS equal to the serum-derived form ⁹. Gram quantities of GMP-grade rHIgM22 have been purified for formal toxicology studies prior to Phase I clinical trials. Our development of rHIgM22 established an infrastructure for rapid translation of additional human antibodies from basic science to clinical therapies.

We successfully used the same strategy to identify additional human IgMs for testing in other models of neurologic injury and disease. Two neuron-binding antibodies (sHIgM12 and sHIgM42) stimulated neurite extension ¹⁰. A dendritic cell-binding antibody (B7DC XAb) mediated melanoma tumor clearance from lungs ¹⁴. Several beta-amyloid (Aβ^1–40 and Aβ^1–42)-binding human antibodies will be tested in animal models of Alzheimer’s disease. This strategy for identifying human Abs that directly signal cells has the potential to generate therapeutic antibodies for a broad range of human diseases.

Specific Antibody-glycolipid-protein Interactions Mediate rHIgM22 Binding to Both Myelin and Oligodendrocyte Membrane Surface

Several mouse IgMs, including A2B5, O1, O4, HNK-1, SCH79.08 and SCH94.03, bind oligodendrocytes and promote remyelination in mouse models of multiple sclerosis (Table) ⁷. A2B5, O1, and O4 bind to surface glycolipid antigens on less-differentiated oligodendrocytes ^15–17. HNK-1, SCH79.08, SCH94.03 and rHIgM22 bind to antigens on the surface of relatively mature oligodendrocytes and myelin. We hypothesized that antibody-mediated remyelination required binding to oligodendrocyte membrane glycolipids.

Table 1.

Properties of CNS-Reactive Signaling Antibodies

Antibody1	CNS Surface Membrane Target	CNS Cell Signals 2	Repair
A2B5 MIgM	c-series gangliosides (GT3, GQ1c and GP1c)	Ca2+	Remyelination
HNK-1 MIgM	Sulfated Glucuronosyl Glycosphingolipids	Ca2+	Remyelination
O1 MIgM	Galactosyl Cerebroside (GalC)	Ca2+	Remyelination
O4 MIgM	Sulfatide (GalC-Sulfate)	Ca2+, P-MAPK, caspase-3I, P-Srcfk	Remyelination
SCH94.03 MIgM	Myelin	Ca2+	Remyelination
79.08 MIgM	Myelin basic protein (MBP)	Ca2+	Remyelination
rHIgM22	Myelin	Ca2+, P-MAPK, caspase-3I, P-Srcfk	Remyelination
sHIgM46	Myelin	Ca2+	Remyelination
rHIgM12	Neuronal Membrane	Ca2+, caspase-3I	Neuroprotection, Neurite outgrowth
sHIgM42	Neuronal Membrane	Ca2+, caspase-3I	Neuroprotection, Neurite outgrowth
sHIgM39 isotype control	None	No	None

¹hybridoma (h), serum (s) recombinant (r), mouse (M), human (H), isotype control (control)

²calcium influx (Ca²⁺), Inhibition of caspase-3 activation (I-caspase-3), MAPK phosphorylation (P-MAPK), Src family kinase phosphorylation (P-Src-fk)

Binding IgMs to CNS tissue from glycolipid knock out mice demonstrates that the molecules bound by rHIgM22 in CNS myelin depend upon components of the glycolipid synthesis pathway. Binding of rHIgM22 to the plasma membrane requires the presence of a substrate of cerebroside sulfotransferase (CST). Normally, the cerebroside galactosyltransferase (CGT) enzyme converts ceramide to galactosylcerebroside (GalC) in oligodendenrocytes. Then the enzyme CST converts GalC to sulfatide. Immunofluorescence of live CNS tissue slices demonstrates the strong affinity of rHIgM22 for densely myelinated axons in wild type mice (Figure 1). rHIgM22 and antibodies against myelin oligodendrocyte glycoprotein (MOG), expressed on mature myelin, bound to densely myelinated fiber tracts and to individual myelinated axons in the cerebellum. In contrast, rHIgM22 affinity for white matter tracts was abolished in CNS tissue from mice lacking sulfatide (cst ^(−/−))¹⁸. Similarly O4 antibody which labels sulfatide was absent in cst ^(−/−) mice. In addition, rHIgM22 binding was not detected in other sulfatide-expressing tissues including peripheral nervous system myelin and Schwann cells. These data support the hypothesis that rHIgM22 binding depends upon one or more CST-sulfated antigens present exclusively on the surface myelin of oligodendrocytes. rHIgM22 may target sulfatide or a number of sulfated antigens within the CNS including glucosylcerebroside sulfate, lactosylceramind-3-sulfate, seminolipid, bis-sulphoganglio tetraosylceramide, bis-sulphoganglio triaosylceramide. The data support the hypothesis that rHIgM22 recognizes a complex on oligodendrocytes dependent upon one or more sulfated antigens at the myelin plasma membrane.

Figure 1. rHIgM22-binding interactions are dependent upon a CST substrate.

Images (100x) of O4 (sulfatide; A,G), rHIgM22 (B,H) or MOG (C, I) reactivity shown using live cerebellar sections (200µm) subsequently fixed, FITC immunostained and DAPI counterstained (D–F, J–L). Wild-type showed reactivity to all in contrast cst ^(−/−) showed MOG but lacked O4 and rHIgM22 reactivity.

Oligodendrocytes express a repertoire of integrins during specific stages of development. Oligodendrocyte myelination correlates with the expression of laminin receptor α6β1 and vitronectin/fibronectin receptors αvβ1, αvβ3, αvβ5 and αvβ8 ¹⁹. Recent studies in collaboration with Drs Jens Watzlawik and Richard Pagano at Mayo Clinic demonstrate a co-localization of rHIgM22 with β integrins on the plasma membrane of mature oligodendrocytes.

We propose that sulfated molecules and β integrin facilitate specific rHIgM22 binding to myelin and oligodendrocytes. In addition, the pentameric structure of the IgM molecule is necessary for remyelination and may be critical to cross-linking these antigens on the oligodendrocyte surface and inducing intracellular repair signals.

Identification of Neuron-Binding Antibodies for CNS Protection and Repair

Two neuron-binding human IgMs, sHIgM12 and sHIgM42, were identified using the strategy described earlier.¹⁰ These antibodies supported in vitro CNS neurite extension equal to the potent neurite stimulatory molecule laminin. Both IgMs bound to the surface of many types of cultured neurons but not to the surface of mature oligodendrocytes. Both IgMs stimulated neurite extension in the presence of CNS myelin, which normally inhibits outgrowth. These two IgMs are novel agents to promote neurite extension and are being tested in models of CNS disease where destructive of axons and neurons.

Antibody-mediated Membrane Rearrangement Initiates Signaling

We propose that NA antibodies activate endogenous cellular mechanisms to protect CNS neurons and oligodendrocytes. Remyelination-promoting IgM antibodies bind to the surface of oligodendrocytes. However, not all IgMs that bind to oligodendrocytes promote remyelination. Oligodendrocyte binding did not perfectly predict the remyelinating potential of an IgM in vivo. Therefore, IgM-mediated repair requires other mechanisms besides strict binding. Specific signaling events are also required. Antibody-mediated signaling in oligodendrocytes is mediated through membrane rearrangement and microdomain signaling ². Recent studies have demonstrated that mouse IgM antibody O4 and rHIgM22 bound to the surface of unfixed primary oligodendrocytes diffusely at 4°C. However, allowing membrane rearrangement at 15°C resulted in small punctate structures indicative of signaling microdomain clustering. sHIgM42 under the same conditions exhibited a similar punctate membrane pattern on neurons (Figure 2). Coincident with IgM-mediated membrane clustering on oligodendrocytes was the rapid activation of intracellular MAP kinases. The phosphorylation of several signaling proteins increased within 1 minute and persisted for 15 minutes in primary oligodendrocytes treated with rHIgM22.

Figure 2. Neuron-Binding Antibodies Demonstrate Temperature-Dependent, Surface Membrane Rearrangement.

sHIgM42 reactivity shown using live cerebellar granule neurons subsequently fixed, FITC immunostained and DAPI counterstained. sHIgM42 demonstrated binding interactions at 0°C (A) and membrane rearrangements at 15°C (B). Note formation of small punctuate domains along neurites at 15°C but not at 0°C.

Reparative IgMs Induce Rapid Calcium Influx and Inhibit Apoptosis

The in vivo remyelination-promoting ability of an antibody correlated with its ability to induce transient Ca²⁺ influx in oligodendrocytes in culture ^{3, 9}. rHIgM22 binding to the oligodendrocyte plasma membrane induced signaling cascades that down regulated caspase-3 activation to prevent apoptosis. In addition, rHIgM22 altered gene expression upon calcium influx through CNQX-sensitive AMPA channels in oligodendrocytes. The neuron-binding antibodies sHIgM12 and sHIgM42 demonstrated robust rescue of cultured neurons from H₂O₂-induced apoptosis.

Antibody-mediated Activation of CNS Protection and Repair

Strong parallels in character exist between remyelination promoting IgMs, rHIgM22 and O4, and the neurite outgrowth-promoting IgMs, sHIgM42 and sHIgM12. This suggests a common membrane-rearrangement mechanism that recruits signaling molecules into clustered microdomains and ultimately leads to specific cell responses in vitro and in vivo (Figure 3).

Figure 3. Mechanism of NA Monoclonal Antibody-mediated CNS Repair.

Pentameric interactions of antibody binding to plasma membrane glycolipids (A) and proteins (B) on live cells induce membrane rearrangements. Signaling molecule (C) activation of pathways including calcium flux, MAP kinase activation and blocking caspase-3 activation that converge on transcriptional regulation of genes promoting CNS protection and repair.

We propose this class of autoreactive antibodies activates the cellular process of CNS protection and repair through direct signaling cascades. Although each antibody reacts to unique cell-specific antigens, binding to the appropriate cells activates the target cell in a conserved manner. Defining the common signaling components regulating changes in specific cells may lead to an understanding of the underlying mechanism of antibody-induced repair and result in the design of better strategies to promote remyelination and protect axons.