Methods and compositions for detecting amyotrophic lateral sclerosis
Inventors
Chakrabartty, Avijit • Rakhit, Rishi • Cashman, Neil R.
Assignees
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Publication Number
US-8513387-B2
Publication Date
2013-08-20
Expiration Date
Abstract
The invention provides binding proteins that bind to misfolded or monomeric SOD1, and not to native homodimeric SOD1. The invention also includes methods of diagnosing, detecting or monitoring amyotrophic lateral sclerosis in a subject. In addition, the invention provides methods of identifying substances for the treatment or prevention of amyotrophic lateral sclerosis and kits using the binding proteins of the invention.
Core Innovation
The invention provides binding proteins, including the SEDI antibody, that selectively bind exposed epitopes on misfolded or monomeric SOD1 and do not bind native homodimeric SOD1. The described epitope sequences include ACGVIGI (SEQ ID NO:1), RLACGVIGI (SEQ ID NO:2), and GGRLACGVIGIGGKG (SEQ ID NO:3), corresponding to a dimer-interface epitope exposure state.
The invention further provides diagnostic and monitoring approaches for amyotrophic lateral sclerosis (ALS) by contacting subject samples with the binding proteins and detecting binding as an indicator of misfolded or monomeric SOD1. The document also outlines a drug-identification approach by comparing binding levels between treated samples and controls, and includes a kit concept for ALS detection/monitoring.
Specificity and in vivo relevance are described using discrimination of monomer versus dimer on Western blot and SDS-PAGE, peptide competition specificity, and immunoprecipitation of misfolded SOD1 from ALS mouse models (G85R, G93A, G37R) with minimal non-specific binding. Immunohistochemical localization is described as predominantly to motor neurons and perivacuolar/vacuolated mitochondria, with temporal appearance prior to symptom onset, and the presence of the marker in a human SOD1-ALS case (A4V mutation).
Claims Coverage
The independent claims cover three core inventive areas: (i) isolated antibodies or binding fragments with selective binding to misfolded or monomeric SOD1 and to a dimer-interface epitope, (ii) epitope-defined antibodies that bind ACGVIGI and/or RLACGVIGI epitopes, and (iii) a method of making such antibodies using defined SOD1-fragment immunogens. Across the claim families, additional dependent features include labeled formats, defined sample-contact detection/monitoring methods, and kit embodiments for ALS detection/monitoring.
Selective binding to misfolded or monomeric SOD1 with no binding to native homodimeric SOD1
An isolated antibody or binding fragment thereof that binds to misfolded or monomeric SOD1, wherein the antibody or binding fragment thereof does not bind to native homodimeric SOD1.
Dimer-interface epitope binding on SOD1
The antibody or binding fragment thereof binds to an epitope on the dimer interface of SOD1.
Epitope-defined antibody binding to ACGVIGI and/or RLACGVIGI
An isolated antibody or binding fragment thereof that binds to an epitope selected from ACGVIGI (SEQ ID NO:1), and/or RLACGVIGI (SEQ ID NO:2).
Antibody manufacturing using defined SOD1-fragment immunogens
A method of making an antibody that binds to misfolded or monomeric SOD1 comprising immunizing a host with immunogens comprising isolated SOD1 fragments comprising the sequence ACGVIGI (SEQ ID NO:1); and the sequence RLACGVIGI (SEQ ID NO:2); and/or the sequence GGRLACGVIGIGGKG (SEQ ID NO:3); and isolating from said host an antibody that binds misfolded or monomeric SOD1.
Overall, the claim set is directed to epitope-specific isolated antibodies or binding fragments that distinguish misfolded or monomeric SOD1 from native homodimeric SOD1 by binding a dimer-interface epitope, including epitope-defined binding to ACGVIGI and/or RLACGVIGI, and to generating these antibodies using immunogens that comprise defined SOD1-fragment sequences.
Stated Advantages
Selectively bind exposed epitopes on misfolded or monomeric SOD1 while not binding native homodimeric SOD1.
Provide an indicator for ALS detection and monitoring based on detecting binding to misfolded or monomeric SOD1.
Enable comparison of binding levels between treated samples and controls for drug identification.
Demonstrate specificity and minimal non-specific binding in described experimental validation.
Show immunohistochemical localization to motor neurons and perivacuolar/vacuolated mitochondria, including temporal appearance prior to symptom onset and presence in a human SOD1-ALS case (A4V mutation).
Documented Applications
ALS detection and monitoring by contacting subject samples with the binding proteins and detecting binding as an indicator of misfolded or monomeric SOD1.
Drug identification by comparing binding levels between treated samples and controls.
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