阅读说明：本技术 一种靶向神经生长因子的单克隆抗体及其应用 (Monoclonal antibody of targeted nerve growth factor and application thereof ) 是由 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 于 2019-09-30 设计创作，主要内容包括：本发明涉及一种靶向神经生长因子(NGF)的单克隆抗体及其应用。所述单克隆抗体包含：重链可变区,其包含SEQ ID NO：1或SEQ ID NO：7所示的CDR-H1、SEQ ID NO：2或SEQ ID NO：8所示的CDR-H2和SEQ ID NO：3或SEQ ID NO：9所示的CDR-H3；和轻链可变区,其包含SEQ ID NO：4或SEQ ID NO：10所示的CDR-L1、SEQ ID NO：5或SEQ ID NO：11所示的CDR-L2和SEQ ID NO：6或SEQ ID NO：12所示的CDR-L3。本发明的单克隆抗体可应用于NGF分子检测试剂及治疗性抗体的研发。(The invention relates to a monoclonal antibody of a targeted Nerve Growth Factor (NGF) and application thereof. The monoclonal antibody comprises: a heavy chain variable region comprising SEQ ID NO: 1 or SEQ ID NO: 7, CDR-H1, SEQ ID NO: 2 or SEQ ID NO: 8 and the CDR-H2 shown in SEQ ID NO: 3 or SEQ ID NO: 9 CDR-H3; and a light chain variable region comprising SEQ ID NO: 4 or SEQ ID NO: 10, CDR-L1, SEQ ID NO: 5 or SEQ ID NO: 11 and CDR-L2 shown in SEQ ID NO: 6 or SEQ ID NO: CDR-L3 shown in FIG. 12. The monoclonal antibody can be applied to the research and development of NGF molecular detection reagents and therapeutic antibodies.)

1. A monoclonal antibody targeting Nerve Growth Factor (NGF) comprising:

a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 sequences; and

a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 sequences,

wherein the content of the first and second substances,

CDR-H1 is selected from SEQ ID NO: 1 or SEQ ID NO: 7;

CDR-H2 is selected from SEQ ID NO: 2 or SEQ ID NO: 8;

CDR-H3 is selected from SEQ ID NO: 3 or SEQ ID NO: 9, and

CDR-L1 is selected from SEQ ID NO: 4 or SEQ ID NO: 10;

CDR-L2 is selected from SEQ ID NO: 5 or the sequence shown in SEQ ID NO: 11;

CDR-L3 is selected from SEQ ID NO: 6 or SEQ ID NO: 12, or a sequence shown in figure 12.

2. The monoclonal antibody of claim 1, comprising:

a heavy chain variable region comprising SEQ ID NO: 1, CDR-H1, SEQ ID NO: 2 and CDR-H2 shown in SEQ id no: 3 and CDR-H3, and

a light chain variable region comprising SEQ ID NO: 4, CDR-L1, SEQ ID NO: 5 and CDR-L2 shown in SEQ id no: CDR-L3 shown in FIG. 6.

3. The monoclonal antibody of claim 1, comprising:

a heavy chain variable region comprising SEQ ID NO: 7, CDR-H1, SEQ ID NO: 8 and the CDR-H2 shown in SEQ id no: 9 CDR-H3, and

a light chain variable region comprising SEQ ID NO: 10, CDR-L1, SEQ ID NO: 11 and CDR-L2 and seq id NO: CDR-L3 shown in FIG. 12.

4. The monoclonal antibody of claim 1, wherein said monoclonal antibody comprises the amino acid sequence of SEQ ID NO: 13 and SEQ ID NO: 14, or a light chain variable region as shown in fig. 14.

5. The monoclonal antibody of claim 1, wherein said monoclonal antibody comprises the amino acid sequence of SEQ ID NO: 15 and SEQ ID NO: 16, or a light chain variable region as shown in figure 16.

6. Use of a monoclonal antibody according to any one of claims 1 to 5 for the preparation of an analgesic or an NGF molecular detection reagent.

7. An analgesic composition comprising at least the monoclonal antibody of any one of claims 1 to 5.

8. An NGF molecule detecting reagent comprising at least the monoclonal antibody according to any one of claims 1 to 5.

Technical Field

The invention belongs to the field of biochemistry, and particularly relates to a monoclonal antibody of a targeted Nerve Growth Factor (NGF) and application thereof.

Background

Nerve Growth Factor (NGF) is a small secreted protein belonging to the neurotrophin family, 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 comprises 2 α subunits, 1 β subunits, 2 γ subunits, together forming a structure of α 2 β γ 2. The beta subunit is a 118 amino acid homodimeric polypeptide, the amino acid composition and sequence of which is highly conserved among different ethnic groups (similarity between human and murine NGF is about 90%). NGF can exert biological activity only when the β subunit is present alone. The gamma subunit has protein hydrolyzing activity and belongs to the trypsin-like kallikrein family. Although the alpha subunit is highly similar to the gamma subunit, the alpha subunit has no enzymatic activity. The receptors for NGF are the p75 neurotrophin receptor (NTR) and the tyrosine kinase receptor a (trka). The p75NTR belongs to the Tumor Necrosis Factor (TNF) receptor family and consists of a cysteine-rich extracellular domain, a transmembrane domain, and an intracellular death domain. In which the intracellular death domain can be phosphorylated to bind to a variety of death-related signaling proteins. p75NTR has no tyrosine kinase activity and is not associated with a G protein-coupled signaling pathway. The affinity of p75NTR for NGF was lower. Compared with p75NTR, TrkA has higher affinity to NGF, has tyrosine kinase activity and is mainly selectively expressed on pain neurons. Similar to most tyrosine kinase receptors, activation of TrkA relies on ligands to induce formation of the relevant receptor dimerization.

NGF mainly functions to maintain the survival, proliferation and differentiation of nerve cells. Recent studies have shown that NGF also plays a role in inflammation and pain. NGF-mediated pain modulation is mainly mediated by binding to TrkA. When NGF is combined with TrkA on pain sensation end, another molecule of TrkA is combined with the NGF, and the formed NGF/TrkA complex promotes autophosphorylation of TrkA intracellular domain, so that a downstream path is activated to play a role. Through this signaling pathway, NGF activates the transient baroreceptor vanilloid receptor subtype 1(TRPV1) cation channel, which is stimulated by intracellular and extracellular machinery, temperature, chemicals, etc. After the channel is opened, a large amount of calcium ions can flow in, action potential is generated, pain signals are converted into electric signals, and the electric signals are transmitted to pain neurons to generate pain. NGF also promotes the expression of TRPV1 and transports it to the cell membrane. Therefore, NGF can effectively activate TRPV1 cation channel by the above 2 mechanisms, thereby causing pain. Chronic persistent pain was found to be associated with NGF-mediated production of TRPV1 agonist oxidized lipid activated TRPV1 cation channel. In addition, NGF can up-regulate the expression of pain neuron-associated genes, such as substance P, Nav1.8, BNDF, to promote sensitization of pain neurons. NGF also indirectly activates mast cells, releases pain mediators such as prostaglandins, bradykinin and histamine, stimulates pain terminals, and increases pain. Briefly, NGF produces pain primarily by mechanisms that activate or upregulate ion channels, upregulate expression of pain-associated genes, and promote the release of pain mediators by mast cells.

At present, opioid drugs such as morphine and the like are mostly used for analgesia clinically, however, the drugs have serious toxic and side effects such as respiratory depression, hallucination and the like, are easy to cause abuse and addiction, and cause social problems such as drug absorption and the like. The therapeutic efficacy of the NGF blocking antibody molecule is comparable to that of opiates, but the NGF blocking antibody molecule has no tolerance and addiction risk and small toxic and side effects, so that the NGF blocking antibody molecule has obvious advantages compared with the opiates and has high clinical application value. NGF antibodies can also be used in the detection of NGF molecules. Based on the above advantages of NGF antibodies, there is a need to develop NGF antibody molecules that can effectively block NGF binding to TrkA.

Disclosure of Invention

In view of the above problems, it is a technical object of the present invention to provide a monoclonal antibody targeting Nerve Growth Factor (NGF) and use thereof.

Accordingly, in one aspect, the present invention provides a monoclonal antibody targeting Nerve Growth Factor (NGF), comprising:

a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 sequences; and

a light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3 sequences,

wherein the content of the first and second substances,

CDR-H1 can be selected from SEQ ID NO: 1 or SEQ ID NO: 7;

CDR-H2 can be selected from SEQ ID NO: 2 or SEQ ID NO: 8;

CDR-H3 can be selected from SEQ ID NO: 3 or SEQ ID NO: 9; and is

CDR-L1 may be selected from SEQ ID NO: 4 or SEQ ID NO: 10;

CDR-L2 may be selected from SEQ ID NO: 5 or the sequence shown in SEQ ID NO: 11;

CDR-L3 may be selected from SEQ ID NO: 6 or SEQ ID NO: 12, or a sequence shown in figure 12.

In one embodiment, the monoclonal antibody comprises:

a heavy chain variable region comprising SEQ ID NO: 1, CDR-H1, SEQ ID NO: 2 and CDR-H2 shown in SEQ ID NO: 3 and CDR-H3, and

a light chain variable region comprising SEQ ID NO: 4, CDR-L1, SEQ ID NO: 5 and SEQ ID NO: CDR-L3 shown in FIG. 6.

In one embodiment, the monoclonal antibody comprises:

a heavy chain variable region comprising SEQ ID NO: 7, CDR-H1, SEQ ID NO: 8 and the CDR-H2 shown in SEQ ID NO: 9 CDR-H3, and

a light chain variable region comprising SEQ ID NO: 10, CDR-L1, SEQ ID NO: 11 and CDR-L2 shown in SEQ ID NO: CDR-L3 shown in FIG. 12.

In one embodiment, the monoclonal antibody comprises SEQ ID NO: 13 and SEQ ID NO: 14, or a light chain variable region as shown in fig. 14.

In one embodiment, the monoclonal antibody comprises SEQ ID NO: 15 and SEQ ID NO: 16, or a light chain variable region as shown in figure 16.

In another aspect, the invention provides the use of the monoclonal antibody for preparing an analgesic or an NGF molecular detection reagent.

In another aspect, an analgesic composition comprises at least the monoclonal antibody described above.

In still another aspect, an NGF molecule detecting reagent comprises at least the monoclonal antibody described above.

Advantageous effects

The affinity of the two NGF antibody molecules 1D4 and 5F2 screened by the invention is similar to that of the NGF antibody molecule Tanezumab in the prior art, and both can block the combination of NGF and TrkA, and the EC50 of 1D4 and 5F2 is 0.92 nM and 0.95nM respectively, which are superior to that of the NGF antibody molecule Tanezumab in the prior art, and the NGF antibody molecule Tanezumab has good application prospect in preparation of analgesics or NGF molecule detection reagents.

Drawings

FIG. 1 shows the results of affinity test of the antibodies 1D4 and 5F2 prepared in preparation example 1 of the present invention and Tanezumab, a positive control.

FIG. 2 shows the results of a test that the antibodies 1D4 and 5F2 prepared in preparation example 1 of the present invention and Tanezumab, a positive control, block the binding of NGF to TrkA.

Detailed Description

The following description of the present invention is provided by way of specific examples to enable those skilled in the art to better understand the present invention, however, these embodiments are not intended to limit the scope of the present invention.