SP35 antibodies and uses thereof

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

Inactive Publication Date: 2015-04-22
BIOGEN MA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 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

[0475] Sp35 involved in oligodendrocyte biology

[0476] 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 detect 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).

[0477] 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 beta III tubulin (Covance), Sp35 (Biogen Idee), Fyn (Santa Cruz Biotechnology) and phospho-Fyn (Biosource). Monoclonal ant...

Embodiment 2

[0492] Construction and purification of Sp35-Fc fusion protein

[0493] To study the biological function of Sp35, a construct was prepared that fused 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 can be obtained by using forward primer 5'-CAG CAG GTC GAC GCGGCC GCA TGC TGG CGG GGG GCG T-3' (SEQ ID NO: 354) and reverse primer 5'-CAG CAG GTC GAC CTC GCC PCR for CGG CTG GTT GGCCAA CCA GCC GGG CGA GGT CGA CCT CGA GG-3' (SEQ ID NO: 355) was obtained from clone 227.2.

[0494] 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 fragments from Sail (5') to Notl (3'). This SalI Sp35 fragment was s...

Embodiment 3

[0498] Production of Sp35-specific monoclonal antibodies

[0499] The anti-Sp35 antibody that specifically binds to the Sp35 polypeptide of the present invention can be prepared using the following methods and steps.

[0500] A. Antibody Screening Analysis

[0501] 1. ELISA analysis

[0502] Sp35-Fc (0.5 μg dissolved in 50 μl of 0.1 M sodium bicarbonate buffer pH 9.0) was added to each well of a 96-well MaxiSorp plate (Nunc). Plates were incubated at 37°C for 1 hour or at 4°C for 16 hours. Non-specific binding sites on the plate 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 . Dilutions (eg, serial three-fold dilutions) of serum or hybridoma supernatants are added to each row on the plate and incubated at 25°C for 1 hour. After three washes with PBS, 50 [mu]l of a 1:10,000 dilution of a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Jackson ImmunoResearch Inc....

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Abstract

Endogenous Sp35 is a negative regulator for neuronal survival, axon regeneration, oligodendrocyte differentiation and myelination (Negative Regulator). 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

technical field [0001] 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 [0002] 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 factors such as surface viscosity, growth factors, neurotransmitters, and electric fields. Nerve cones typically grow at a rate of one or two millimeters per day. Growth cones explore the frontal and lateral regions through extensions classified as lamellipodia and filopodia. When the extension touches an unsuitable surface, it will retract. When the extension touches a suitable growing surface, it will continue to ex...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/00C07K14/00C12N15/113
CPCC12N15/1138C07K2317/21A61K47/48684C07K2319/30C07K2317/55C07K2316/96C07K16/2803C07K16/18C07K2317/33C12N2310/14C07K16/28A61K47/48538A61K2039/505C07K2317/76C12N2310/111C07K2317/24C07K2317/75A61P21/02A61P25/00A61P25/04A61P25/14A61P25/16A61P25/28A61P27/02A61P3/02A61P43/00A61P9/00A61P9/10A61K39/395
Inventor 米莎R·布莱克·佩平斯凯邵朝晖克里斯蒂林恩·P·格莱夫
Owner BIOGEN MA INC
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