Target polypeptide of Nav1.9, antibody and antibody fragment bound with polypeptide, and related medicine composition

一种抗体片段、抗体的技术，应用在治疗疼痛,瘙痒和咳嗽领域，能够解决中枢神经副作用、心脏毒性、电压门控钠离子通道亚型缺乏等问题，达到治疗和缓解疼痛、好靶向性、克服副作用的效果

Active Publication Date: 2018-09-14
POPULAS BIOPHARMACEUTICAL WUHAN LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, chemical small molecules (such as carbamazepine, lidocaine, mexiletine, etc.) are widely used clinically as voltage-gated sodium ion channel inhibitors to treat pain, but they lack sufficient effect on voltage-gated sodium ion channel subtypes. Selective and thus vulnerable to cardiotoxicity and CNS side effects

Method used

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  • Target polypeptide of Nav1.9, antibody and antibody fragment bound with polypeptide, and related medicine composition
  • Target polypeptide of Nav1.9, antibody and antibody fragment bound with polypeptide, and related medicine composition
  • Target polypeptide of Nav1.9, antibody and antibody fragment bound with polypeptide, and related medicine composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 [antigen synthesis]

[0034] According to the amino acid sequence of Nav1.9 (GenBank No.NP_001274152) and the crystal structure model, the hydrophilicity and antigenicity were analyzed, and the DVEFSGEDNAQRIT sequence was screened out. Its hydrophilicity and antigenicity met the requirements of the antigen, and an automatic synthesizer was used. Artificially synthesized DVEFSGEDNAQRIT (SEQ ID NO.9) polypeptide.

[0035] Specific steps are as follows:

[0036] (1) Connect -COOH of the first AA to Cl-Resin with DIEA, and then block the unreacted functional groups on the resin with MeOH;

[0037] (2) washing with DMF;

[0038] (3) Use Pip to remove -NH in the first AA 2 The protecting group Fmoc makes -NH 2 exposed;

[0039] (4) washing with DMF;

[0040] (5) Activate the -COOH of the second AA with DIC+HOBT, and then condense to -NH in the first AA 2 on, forming an amide bond;

[0041] (6) washing with DMF;

[0042] (7) Use Pip to remove -NH in the seco...

Embodiment 2

[0049] Example 2【Preparation of Monoclonal Cell Line】

[0050] 2.1 Animal immunity

[0051] Prepare Freund's complete adjuvant Sigma, F5881 and Freund's incomplete adjuvant (Sigma, F5506). Using the end-SH of polypeptide C2363BB030-1, the polypeptide was coupled to the carrier protein KLH as an immunogen.

[0052] Five 8-week-old female BALB / c (animal numbers: #1939, #1940, #1941, #1942, #1943) were selected for three times of intraperitoneal immunization to stimulate the body to produce an immune response to produce antibodies. Primary immunization: 50 μg / monkey, followed by secondary immunization three weeks later, with a dose of 50 μg / bird; 2 weeks after the second immunization, the third immunization was performed, with a dose of 50 μg / bird, within 1 day of the third immunization. A week later, blood was drawn for antibody testing.

[0053] 2.2 Animal serum ELISA detection

[0054] 2.2.1 Instruments and equipment:

[0055] Plate washer: Beijing Nanhua ZDMX

[0056] M...

Embodiment 3

[0097] Example 3【Antibody Sequencing】

[0098] To determine the monoclonal antibody sequence, select one of the monoclonal 51H10D12 Perform sequencing. Total RNA was isolated from hybridoma cells according to the technical manual of TRIzol reagent. Total RNA was then reverse transcribed into cDNA using isotype-specific antisense primers or universal primers, following the PrimeScript™ First Strand cDNA Synthesis Kit technical manual. Antibody fragments of VH and VL were amplified according to GenScript's Rapid Amplification of cDNA Ends (RACE) standard operating procedure (SOP) method. The amplified antibody fragments were cloned individually into standard cloning vectors. Colony PCR was performed to screen for clones with inserts of the correct size. Sequence at least 5 colonies with inserts of the correct size. The sequences of the different clones were compared to determine the consensus sequence of these clones.

[0099] Thus, the DNA sequence of VH was determined, ...

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Abstract

The invention provides target polypeptide of Nav1.9, an antibody and an antibody fragment bound with the polypeptide, and the related medicine composition. The target polypeptide is S3-4 ring of a voltage sensor paddle of a III-rd structural domain of a voltage gate sodium ion channel alpha subunit. The antibody of the antibody fragment can deactivate a voltage sensor valve and sodium ions cannotenter nerve cells normally, so that the effects of performing treatment and relieving pain are achieved.

Description

technical field [0001] The present invention relates to a target (polypeptide) of Nav1.9, an antibody and / or antibody fragment that specifically recognizes the above target (polypeptide), and a pharmaceutical composition comprising the above antibody and / or antibody fragment for treating pain and itching and cough. Background technique [0002] Pain originates from the nociceptors of the peripheral nervous system, and as a kind of free nerve endings, it is widely distributed in the skin, muscles, joints and visceral tissues of the whole body, and it can convert thermal, mechanical or chemical stimuli into actions The potential is transmitted to the cell body part of the dorsal root ganglia (DRG) through nerve fibers, and finally transmitted to the higher nerve center, thereby causing pain. The generation and conduction of action potentials in neurons depend on voltage-gated sodium channels (voltage-gated sodium channels, VGSCs) located on the cell membrane. When the cell m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K14/705A61K39/395A61P29/00
CPCC07K14/705C07K16/28A61K2039/505C07K2317/24C07K2317/565A61P25/02A61P29/00C07K2317/76A61P29/02C07K2317/52
Inventor 杨代常
Owner POPULAS BIOPHARMACEUTICAL WUHAN LTD
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