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SP35 antibodies and uses thereof

An antibody and monoclonal antibody technology, applied in the field of neurology, can solve unmet problems

Active Publication Date: 2013-10-23
BIOGEN IDEC MA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] There remains an unmet need for molecules and methods capable of inhibiting NgR1-mediated growth cone atrophy and neurite outgrowth

Method used

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  • SP35 antibodies and uses thereof
  • SP35 antibodies and uses thereof
  • SP35 antibodies and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0569] Sp35 is involved in oligodendrocyte biology

[0570] Oligodendrocyte maturation goes through multiple developmental stages, from A2B5 progenitors (expressing A2B5), to premyelinating oligodendrocytes (expressing O1 and O4) and finally to mature myelinating oligodendrocytes (expressing O1 , O4 and MBP). Thus, by monitoring the presence and absence of A2B5, O1, O4 and MBP markers, it is possible to determine the developmental stage of a given cell and assess the role of Sp35-Fc in oligodendrocyte biology. A general review of oligodendrocyte biology can be found, for example, in Baumann and Pham-Dinh, Physiol. Rev. 81:871-927 (2001).

[0571] Monoclonal antibodies against O4, MBP and CNPase were purchased from Sternberger Monoclonals; antibodies against APC (clone CC-1; ref.29) were purchased from Calbiochem. Other antibodies were directed against βIII-tubulin (Covance), Sp35 (Biogen Idee), Fyn (Santa Cruz Biotechnology) and phospho-Fyn (Biosource). Monoclonal antibod...

Embodiment 2

[0586] Construction and purification of Sp35-Fc protein

[0587] To study the biological function of Sp35, a construct can be prepared that fuses the extracellular portion of human Sp35 (residues 1-532) with the hinge and Fc region of human IgG1. The partial coding sequence of human Sp35 can be obtained by using forward primer 5'-CAG CAG GTC GAC GCG GCC GCA TGC TGG CGG GGG GCG T-3' (SEQ ID NO:354) and reverse primer 5'-CAG CAG GTC GAC CTC PCR for GCC CGG CTG GTT GGC CAA CCA GCC GGG CGA GGT CGA CCT CGA GG-3' (SEQ ID NO: 355) was obtained from clone 227.2.

[0588] This blunt ended PCR product was subcloned into the SrfI site of the PCR SCRIPT AMP vector (Stratagene) to create PCR SCRIPT AMP-Sp35. The SalI fragment was isolated from PCR SCRIPT AMP-Sp35 and subcloned into PCRCAMP Ig vector (from Stratagene vector PCR SCRIPT AMP). In the PCRCAMP Ig vector, the hinge and Fc gamma sequences were subcloned as SalI (5') into a NotI (3') fragment. This SalI Sp35 fragment was subclon...

Embodiment 3

[0592] Production of Sp35-specific monoclonal antibodies

[0593] The anti-Sp35 antibody that specifically binds to the Sp35 polypeptide of the present invention can be prepared using the following methods and steps.

[0594] A. Antibody Screening Analysis

[0595] 1. ELISA analysis

[0596] 96-well MaxiSorp TM Tablet (Nunc TM ) was added to each microwell of Sp35-Fc (0.5 μg dissolved in 50 μl of 0.1 M sodium bicarbonate buffer at pH 9.0). Plates were incubated at 37°C for 1 hour or at 4°C for 16 hours. Non-specific binding sites on plates were blocked with 25 mM HEPES pH 7.4 containing 0.1% BSA, 0.1% ovalbumin, 0.1% (5% (w / v) nonfat dry milk in 150 mM NACE) and 0.001% azide point. Serum or dilutions of the hybridoma suspension (eg, serial three-fold dilutions) are added to each row of that on the plate, followed by incubation at 25°C for 1 hour. After washing three times with PBS, 50 μl of a 1:10,000 dilution of a horseradish peroxidase-conjugated goat anti-mouse secon...

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Abstract

Endogenous Sp35 is a negative regulator for neuronal survival, axon regeneration, oligodendrocyte differentiation and myelination. Molecules that block endogenous Sp35 function, such anti-Sp35 antibodies can be used as therapeutics for the treatment of neuron and oligodendrocyte dysfunction. The present invention provides antibodies specific for Sp35, and methods of using such antibodies as antagonists of endogenous Sp35 function. The invention further provides specific hybridoma and phage library-derived monoclonal antibodies, nucleic acids encoding these antibodies, and vectors and host cells comprising these antibodies. The invention further provides methods of promoting oligodendrocyte survival and myelination in a vertebrate, comprising administering to a vertebrate in need of such treatment an effective amount of an anti-Sp35 antibody.

Description

[0001] This application is an invention patent with the international application number PCT / US2008 / 000316 and the international application date of January 9, 2008. After the PCT international application entered the Chinese national phase, the application number is 200880007685.5, and the invention name is "SP35 antibody and its use". Divisional application of the application. technical field [0002] The present invention relates to neurology, neurobiology and molecular biology. The invention further relates to molecules and methods for the treatment of neurobiological diseases, disorders and injuries such as spinal cord injuries. Background technique [0003] Axons and dendrites extend outward from neurons. The tip of the extending axon or neurite contains a specialized region called a growth cone. Growth cones sense the local environment and guide axon growth towards neuronal target cells. Growth cones respond to environmental cues such as surface adhesion, growth fa...

Claims

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Application Information

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IPC IPC(8): C07K16/28A61K39/00
CPCC07K2317/24C07K2317/55C07K2317/52C07K2317/71C07K2317/21C07K2317/94C07K16/2803A61K2039/505C07K2316/96C07K2317/56C07K2317/565C07K2317/41C07K16/18C07K2317/76C07K2317/92A61K31/70A61K38/16A61K39/395C07H21/00C07K14/00C07K16/00A61P21/02A61P21/04A61P25/00A61P25/14A61P25/16A61P25/28A61P27/02A61P3/02A61P3/10A61P43/00A61P9/00A61P9/10
Inventor 米莎R·布莱克·佩平斯凯邵朝晖埃伦·A·加伯斯蒂芬·D·米克拉
Owner BIOGEN IDEC MA INC
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