Identification of Human Monoclonal Antibodies Specific for Human SOD1 Recognizing Distinct Epitopes and Forms of SOD1

Abstract

Mutations in the gene encoding human SOD1 (hSOD1) can cause amyotrophic lateral sclerosis (ALS) yet the mechanism by which mutant SOD1 can induce ALS is not fully understood. There is currently no cure for ALS or treatment that significantly reduces symptoms or progression. To develop tools to understand the protein conformations present in mutant SOD1-induced ALS and as possible immunotherapy, we isolated and characterized eleven unique human monoclonal antibodies specific for hSOD1. Among these, five recognized distinct linear epitopes on hSOD1 that were not available in the properly-folded protein but were available on forms of protein with some degree of misfolding. The other six antibodies recognized conformation-dependent epitopes that were present in the properly-folded protein with two different recognition profiles: three could bind hSOD1 dimer or monomer and the other three were specific for hSOD1 dimer only. Antibodies with the capacity to bind hSOD1 monomer were able to prevent increased hydrophobicity when mutant hSOD1 was exposed to increased temperature and EDTA, suggesting that the antibodies stabilized the native structure of hSOD1. Two antibodies were tested in a G93A mutant hSOD1 transgenic mouse model of ALS but did not yield a statistically significant increase in overall survival. It may be that the two antibodies selected for testing in the mouse model were not effective for therapy or that the model and/or route of administration were not optimal to produce a therapeutic effect. Therefore, additional testing will be required to determine therapeutic potential for SOD1 mutant ALS and potentially some subset of sporadic ALS.

Protease-resistant SOD1 aggregates in amyotrophic lateral sclerosis demonstrated by Paraffin Embedded Tissue (PET) blot

“…Bars represent 50 μm (a-f, h-l) and 100 μm (g). [9,[30][31][32]. It has to be demonstrated if it is possible to differentiate between different misfolded SOD1 forms with the PET-blot approach, e.g.…”

Identification of a Misfolded Region in Superoxide Dismutase 1 That Is Exposed in Amyotrophic Lateral Sclerosis

“…The extent to which SOD1 bound to C4F6 was determined using curves generated by Bio-layer Interferometry, which is based on light interference on the biosensor tip in the presence of SOD1 compared with an internal reference. To generate binding curves, the C4F6-coated tips were immersed into wells containing 12, 15, or 25 M of the respective SOD1 variant in PBS (pH 7.4) until the binding reached equilibrium (ϳ120 s) followed by immersion into PBScontaining wells for dissociation as described (28). Dissociation constants (K d ) were determined by fitting the generated curves using the ForteBio software with a 1:1 binding model.…”

“…Wild-type SOD1-The C4F6-SOD1 binding interaction was quantified in nondenaturing conditions using the Octet system, which utilizes Bio-Layer Interferometry technology to accurately determine binding affinities (28). No binding interaction was detected between C4F6 and WT SOD1; thus, the apparent dissociation constant (K d ) is below the limit of detection (Table 1).…”

Applying the tools of chemistry (mass spectrometry and covalent modification by small molecule reagents) to the detection of prions and the study of their structure

“…119 Some of the lysine residues of SOD1 were shown to be components of the epitopes of anti-SOD1 monoclonal antibodies. 120 As has been shown with prions, these or other antibodies (with lysinecontaining epitopes) may be useful in developing a Western blotbased detection of conformation dependent differences in small molecule reactivity between the normal and pathogenic forms of amyloid-β, tau, α-synuclein, or SOD1.…”