Anti-nerve growth factor antibodies and methods of preparing and using the same

一种抗体、供体的技术，应用在抗生长因子免疫球蛋白、化学仪器和方法、神经系统疾病等方向，能够解决抗原结合亲和性与亲合力降低等问题

Inactive Publication Date: 2014-04-30
NEXVET AUSTRALIA PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Likewise, the generation of equinized forms of anti-NGF antibodies using CDR grafting or related techniques may result in the production of neutralizing antibodies and may further show a reduction in antigen-binding affinity and avidity

Method used

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  • Anti-nerve growth factor antibodies and methods of preparing and using the same
  • Anti-nerve growth factor antibodies and methods of preparing and using the same
  • Anti-nerve growth factor antibodies and methods of preparing and using the same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0234] Example 1 - Production of Antibodies

[0235] Complete antibody sequences were generated by combining the equinized light chain variable domain sequences and heavy chain variable domain sequences of SEQ ID NO: 1 and SEQ ID NO: 2 with a C-terminal equine constant heavy chain or constant light chain, respectively . The equinized αD11 VH domain was combined with two different equine heavy chain constant domains, HC2 (IgG2) and HC6 (IgG6); and the equinized αD11 VL domain was combined with the equine kappa light chain constant domain. The sequences of the full-length mature antibody chains are shown in SEQ ID No: 4 (light chain with kappa constant domain) and SEQ ID No: 6 (heavy chain with HC2 constant domain).

Embodiment 2

[0237] Example 2 - Determination of Antibody Binding to Murine NGF and Horse NGF

[0238] The equinized heavy chain cDNA and light chain cDNA were transfected into CHO cells, and the supernatant was collected and reacted with equine NGF or mouse NGF in ELISA format. After incubation and washing steps, bound horse antibodies were detected by the reactivity of a goat-anti-horse IgG-specific polyclonal antibody conjugated to horseradish peroxidase (HRP) and visualized using TMB. The optical density of the resulting product was measured at 450 nm and compared with the supernatant from mock blank vector transfection ( figure 1 Denoted as "Mock (Mock)" in the optical density) for comparison.

[0239] figure 1 The graph in the figure shows the result. Binding of HC2 (HC2 constant domain) equinized antibody (designated eqN-HC2+eqN-kLC-1 ) to mouse NGF is shown. In the second part of the graph, the binding of the eqN-HC2+eqN-kLC-1 antibody comprising the eqN-KlC-1 light chain and...

Embodiment 3

[0240] Example 3 - Purification of equinized antibodies

[0241] The supernatant obtained from Example 2 was purified using a protein A column, separated by SDS-PAGE, and tested for reactivity with anti-equine IgG polyclonal antibody HRP. SDS-PAGE gels were also stained with Coomassie blue to detect heavy and light chains. Anti-horse IgG polyclonal antibody preparations primarily recognize horse heavy chains. The results are shown in figure 2 A and B.

[0242] Results show purification of horse anti-NGF with type 2 heavy chain by protein A as shown by western blot formed with anti-horse polyclonal antibody HRP. Peak fractions were analyzed by Coomassie-stained SDS-PAGE. Degradation of the heavy and light chains was evident using SDS-PAGE. Coomassie blue stained gel ( figure 2 B) Shows the presence of heavy and light chains as well as intact antibody (MW 70).

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Abstract

A method of preparing an antibody suitable for use in an equine is provided. Also provided are equinised antibodies which specifically bind to equine neuronal growth factor (NGF) and neutralise the ability of equine NGF to bind to the p75 or TrkA equine NGF receptor. The invention extends to nucleic acids encoding same and to methods of treating pain and arthritis in an equine using said antibodies and / or nucleic acids.

Description

technical field [0001] The present invention relates to antibodies and fragments thereof that act as antagonists of equine nerve growth factor. The invention also extends to methods of making these antibodies and fragments and the therapeutic use of these antibodies and fragments in the treatment of nerve growth factor-related conditions in horses, such as pain, pain-related conditions and conditions leading to the onset of pain. Background technique [0002] Nerve growth factor (NGF) is a naturally occurring secreted protein composed of α-polypeptide chains, β-polypeptide chains, and γ-polypeptide chains. NGF is a member of the neurotrophic factor family involved in many different actions. NGF promotes the survival and differentiation of sensory and sympathetic neurons and signals through two classes of membrane-bound receptors, the low-affinity NGF receptor p75 and the high-affinity NGF receptor transmembrane tyrosine kinase TrkA. Binding of NGF to TrkA or p75 leads to u...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22C07K16/46
CPCC07K16/467C07K16/22C07K2317/24C07K2317/20C07K2317/74C07K2317/76A61P19/00A61P19/02A61P25/04A61P29/00A61P35/00A61P43/00
Inventor 大卫·吉尔芮
Owner NEXVET AUSTRALIA PTY LTD
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