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sp35 antibody and its use

A technology of antibodies and uses, applied in the field of neurology, can solve unmet problems

Active Publication Date: 2016-06-08
BIOGEN 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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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0492] Sp35 is involved in oligodendrocyte biology

[0493] Oligodendrocyte maturation goes through multiple developmental stages, from A2B5 progenitor cells (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).

[0494] 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 (BiogenIdee), Fyn (Santa Cruz Biotechnology) and phospho-Fyn (Biosource). Monoclonal ant...

Embodiment 2

[0509] Construction and purification of Sp35-Fc protein

[0510] 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 regions of human IgG1. The partial coding sequence of human Sp35 was obtained from clone 227.2 by PCR using forward primer 5'-CAGCAGGTCGACGCGGCCGCATGCTGGCGGGGGGCGT-3' (SEQ ID NO: 354) and reverse primer 5'-CAGCAGGTCGACCTCGCCCGGCTGGTTGGCCAACCAGCCGGGCGAGGTCGACCTCGAGG-3' (SEQ ID NO: 355).

[0511] This blunt-ended PCR product was subcloned into the SrfI site of the PCRSCRIPTAMP vector (Stratagene) to create PCRSCRIPTAMP-Sp35. The Sail fragment was isolated from PCRSCRIPTAMP-Sp35 and subcloned into PCRCAMP Ig vector (from Stratagene vector PCRSCRIPTAMP). In the PCRCAMPIg vector, the hinge and Fcgamma sequences were subcloned as SalI (5') into a NotI (3') fragment. This SalISp35 fragment was subcloned into the SalI site of the PCRCAMPIg vector, thereby fusing...

Embodiment 3

[0515] Production of Sp35-specific monoclonal antibodies

[0516] The anti-Sp35 antibody that specifically binds to the Sp35 polypeptide of the present invention can be prepared using the following methods and steps.

[0517] A. Antibody Screening Analysis

[0518] 1. ELISA analysis

[0519] 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) skim milk powder in 150 mM NACE) and 0.001% azide. Dilutions (eg, serial three-fold dilutions) of serum or hybridoma suspensions 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 secondary anti...

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Abstract

Endogenous Sp35 is a negative regulator of neuronal survival, axon regeneration, oligodendrocyte differentiation and myelination. Anti-Sp35 antibody molecules capable of blocking the function of endogenous Sp35 are useful in the treatment of neuronal and oligodendrocyte dysfunction. The present invention provides an antibody specific to Sp35, and a method of using the antibody as an antagonist of endogenous Sp35 function. The invention further provides monoclonal antibodies derived from specific hybridomas and phage libraries, nucleic acids encoding these antibodies, and vectors and host cells comprising these antibodies. The invention further provides a method of promoting oligodendrocyte survival and myelination in a vertebrate comprising administering to the vertebrate in need thereof 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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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28A61K39/00
CPCA61K31/70A61K38/16A61K39/395C07H21/00C07K14/00C07K16/00A61K2039/505C07K16/18C07K16/2803C07K2317/21C07K2317/24C07K2317/55C07K2317/56C07K2317/565C07K2317/92C07K2317/41C07K2317/52C07K2317/71C07K2317/76C07K2317/94A61P21/02A61P21/04A61P25/00A61P25/14A61P25/16A61P25/28A61P27/02A61P3/02A61P3/10A61P43/00A61P9/00A61P9/10
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 MA INC
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