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Human anti-SOD1 antibodies

One-SOD1, SOD1 technology, applied in the field of human antibodies and their fragments, derivatives and variants, can solve the problem of unknown exact mechanism of SOD1 aggregation

Active Publication Date: 2013-10-30
NEURIMMUNE HLDG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The precise mechanism leading to SOD1 aggregation is unknown

Method used

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  • Human anti-SOD1 antibodies
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  • Human anti-SOD1 antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0293] Example 1: Target Validation and Binding Specificity of Human SOD1-Antibodies

[0294] To validate SOD1 as a putative target for the isolated antibodies, a direct ELISA assay was performed as described above. For exemplary recombinant human NI-204.10D12, NI-204.12G7, NI-204.9F6 and NI-204.10A8 antibodies, 96-well microplates (Costar, Corning, USA) were prepared by mixing with recombinant human SOD1 (biomol, Hamburg, Germany) Or coated with BSA (Sigma-Aldrich, Buchs, Switzerland) diluted to a concentration of 3.3 μg / ml, in carbonate ELISA coating buffer (15 mM Na 2 CO 3 , 35mM NaHCO 3 , pH9.42), and tested the affinity of the antibody. Exemplary NI-204.10D12, NI-204.12G7, NI-204.9F6 and NI-204.10A8 antibodies specifically bind to human SOD1 by ELISA. No binding to BSA was observed; see figure 2 .

[0295] In order to determine the half maximal effective concentration (EC 50 ), additional direct ELISA experiments with different antibody concentrations were perform...

Embodiment 2

[0297] Example 2: EC 50 Analysis of coating concentrations for increasing human superoxide dismutase 1 (SOD1), thus preferentially forming conformational epitopes

[0298] To determine the binding capacity of NI-204.10D12, NI-204.10A8, NI-204.12G7 and NI-204.9F6 for conformational epitopes, direct ELISA experiments were performed using four different coatings in the coating buffer Human recombinant SOD1 (Biomol, Hamburg, Germany) at concentrations (0.1; 1; 10 or 30 μg / ml). Primary antibodies human NI-204.10D12, human NI-204.10A8, human NI-204.9F6 and mouse monoclonal antibody SOD-172B1 (Santa Cruz Biotechnology, Santa Cruz, USA) were diluted to the indicated concentrations ( Figure 4 ) and incubated for 1 hour at room temperature. Binding was determined using a goat anti-mouse IgG antibody (Jackson Immunological Research, Newmarket, UK) or a donkey anti-human IgGγ antibody conjugated to HRP (Jackson Immunological Research, Newmarket, UK), followed by a standard colorimetric...

Embodiment 3

[0302] Example 3: Binding analysis of physiological SOD1 dimers and misfolded / aggregated SOD1 in vitro

[0303] Metal-catalyzed oxidation reactions were used to induce superoxide dismutase 1 aggregates in vitro (according to Rakhit et al., J Biol Chem. 279 (2004), 15499-504). Wild-type SOD1 from human erythrocytes (dimers) and all other reagents were purchased from Sigma-Aldrich (Buches, Switzerland). For aggregation, 10 μM human SOD1 was incubated in 10 mM Tris-acetate buffer, pH 7.0, containing 4 mM ascorbic acid and 0.2 mM CuCl 2 , at 37 °C for 48 h. As a control, 10 μM human SOD1 was incubated in 10 mM tris-acetate buffer, pH 7.0, at 37°C for 48 hours.

[0304] In coating buffer (15mM Na 2 CO 3 , 35mM NaHCO 3 , pH 9.42), 96-well microplates (Corning) were coated with physiological human SOD1 dimer or in vitro pooled human SOD1 dimer. Non-specific binding sites were blocked for 1 hour at room temperature with PBS / 0.1% -20 with 2% BSA (Sigma-Aldrich, Buchs, Switzerla...

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Abstract

Provided are novel human copper-zinc superoxide dismutase, also known as superoxide dismutase 1 or SOD1, specific antibodies as well as fragments, derivatives and variants thereof as well as methods related thereto. Assays, kits, and solid supports related to antibodies specific for SOD1 are also disclosed. The antibody, immunoglobulin chain(s), as well as binding fragments, derivatives and variants thereof can be used in pharmaceutical and diagnostic compositions for SOD1 targeted immunotherapy and diagnosis, respectively.

Description

technical field [0001] The present invention broadly relates to novel molecules that specifically bind to superoxide dismutase [Cu-Zn], also known as superoxide dismutase 1 or SOD1, in particular to proteins that recognize SOD1 and misfolded / Aggregated forms of human antibodies and fragments, derivatives and variants thereof. In addition, the present invention relates to pharmaceutical and diagnostic compositions comprising such binding molecules, antibodies and mimetic compounds thereof, both of which are useful for recognizing SOD1 and misfolded / aggregated SOD1 in plasma and CSF (cerebrospinal fluid). diagnostic tool, as well as passive vaccination strategies also in the treatment of conditions associated with misfolded / aggregated SOD1 such as amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, or Charcot's disease. All are valuable. Background technique [0002] Protein accumulation, modification and aggregation are caused by many neurodegenerative...

Claims

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Application Information

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IPC IPC(8): C07K16/40A61K39/00
CPCC07K2317/24C07K16/40G01N2800/28C07K2317/34C07K2317/21A61K51/10A61K2039/505A61K39/3955A61K39/0005A61K45/06C12N9/0089G01N33/573A61K47/48646C07K14/435C07K2317/30A61K49/085C07K2317/92G01N33/6893G01N2800/285G01N33/577G01N2333/90283C12Y115/01001A61P21/00A61P21/02A61P43/00A61K47/6871C07K2317/33C07K2317/56C07K2317/565
Inventor 法比奥·孟亚轲玛丽亚格雷西亚·布雷肯孟亚轲简·格林罗格·尼奇克里斯托夫·霍克杜巴斯·韦尔特乔丹·麦克富斯马塞尔·麦尔斯
Owner NEURIMMUNE HLDG
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