Monoclonal antibody targeting nerve growth factor and application thereof

A nerve growth factor and monoclonal antibody technology, applied in the field of biochemistry, can solve problems such as hallucinations, drug abuse, abuse and addiction, and achieve good application prospects

Active Publication Date: 2020-01-14
DARTSBIO PHARM LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Nowadays, opioids such as morphine are used clinically for analgesia. However, these drugs have serious side effects such as respiratory depression, depression, and hallucinations, and are easy to cause abuse and addiction, and cause social problems such as drug abuse.

Method used

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  • Monoclonal antibody targeting nerve growth factor and application thereof
  • Monoclonal antibody targeting nerve growth factor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Production and screening of murine antibodies

[0033] BALB / c mice were immunized multiple times with His-tagged NGF, and mice with high serum titers were obtained. The splenocytes of the mouse with the highest titer are fused with SP20 myeloma cells to form hybridoma cells, and then screened for binding. The screening method is "direct ELISA", that is, the human antigen (extracellular region) is coated on the ELISA plate , incubated with the culture supernatant, and then chromogenically screened using biotin-labeled goat anti-mouse Fc antibody, Strepavidin-HRP, and TMB substrate. Through combination screening, a large number of binding clones were obtained. Finally, the two antibodies with the strongest affinity were selected and named: 1D4 and 5F2.

Embodiment 2

[0034] Example 2: Sequence analysis of murine antibodies

[0035] The light and heavy chain cDNAs of 1D4 and 5F2 were cloned and sequenced. The sequences were analyzed using tools on AbYsis combined with manual experience, and the framework regions, complementarity determining regions (CDRs) and invariable regions were annotated. A Genebank protein sequence homology search was performed on the variable regions of 2D3 (by comparing the framework regions and CDR regions), and the results showed that the framework regions were highly homologous to those of various mouse antibodies. No protein sequences homologous to the CDR regions of 1D4 and 5F2 were found. Therefore, 1D4 and 5F2 are completely new murine antibodies whose sequences have not been reported yet.

[0036] The sequencing results of the light and heavy chain variable regions of 1D4 and 5F2 are as follows:

[0037] CDR-H1 (SEQ ID NO: 1 or SEQ ID NO: 7);

[0038] CDR-H2 (SEQ ID NO: 2 or SEQ ID NO: 8);

[0039] CDR-...

Embodiment 3

[0048] Example 3: Antigen affinity analysis

[0049] Coat 1ug / ml antibody protein on the ELISA plate overnight at 4°C, block with blocking solution at room temperature the next day to avoid non-specific binding, then add biotinylated antigen to be detected and incubate at room temperature, add horseradish peroxide Enzyme-labeled streptavidin (Streptavidin-HRP) was used as a chromogenic substrate, and TMB was used to develop color, and the absorbance was measured at 450nm. The corresponding binding curves were made on Prism. The results showed that the affinities of 1D4 and 5F2 to the antigen were 5.2 and 2.9 nM (see figure 1 ), which has a similar affinity to the NGF antibody molecule Tanezumab of Pfizer and Eli Lilly and Company of the United States.

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Abstract

The invention relates to a monoclonal antibody targeting a nerve growth factor (NGF) and application thereof. The monoclonal antibody contains a heavy chain variable region including CDR-H1 shown in SEQ ID NO:1 or SEQ ID NO:7, CDR-H2 shown in SEQ ID NO:2 or SEQ ID NO:8 and CDR-H3 shown in SEQ ID NO:3 or SEQ ID NO:9, and a light chain variable region including CDR-L1 shown in SEQ ID NO:4 or SEQ IDNO:10, CDR-L2 shown in SEQ ID NO:5 or SEQ ID NO:11 and CDR-L3 shown in SEQ ID NO:6 or SEQ ID NO:12. The monoclonal antibody disclosed by the invention can be applied to research and development of NGFmolecular detection reagents and therapeutic antibodies.

Description

technical field [0001] The invention belongs to the field of biochemistry, in particular, the invention relates to a monoclonal antibody targeting nerve growth factor (NGF) and application thereof. Background technique [0002] Nerve growth factor (NGF) is a small secreted protein belonging to the neurotrophin family that is present not only in the nervous system but also in other tissues throughout the body. NGF exists in the form of precursor (Pro-NGF) and is activated by endonucleases. NGF contains 2 α subunits, 1 β subunit, and 2 γ subunits, which together form the structure of α2βγ2. The β subunit is a homodimeric polypeptide composed of 118 amino acids, and its amino acid composition and sequence are highly conserved among different races (the similarity between human and mouse NGF is about 90%). Only when the β subunit exists alone, NGF can exert biological activity. The γ subunit has the activity of hydrolyzing proteins and belongs to the trypsin-like kallikrein f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28A61K39/395A61P25/04G01N33/68
CPCA61P25/04C07K16/2863C07K2317/56C07K2317/565C07K2317/76C07K2317/92G01N33/68G01N2333/48
Inventor 王春河李欢欢刘国键陈淦均李三泰温家明陈艺丽王琪林以均王米娜
Owner DARTSBIO PHARM LTD
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