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Antibody or antibody fragment specifically binding to voltage-gated sodium channel alpha subunit Nav1.7

A sodium ion channel and antibody fragment technology, which is applied in the field of antibodies and/or antibody fragments that specifically recognize the above-mentioned targets, can solve problems such as research difficulties, side effects that are difficult to overcome, and cardiotoxicity, and achieve overcoming side effects and good targeting , therapeutic and pain-relieving effects

Active Publication Date: 2021-10-22
POPULAS BIOPHARMACEUTICAL WUHAN LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Small chemical molecules (such as carbamazepine, lidocaine, mexiletine, etc.) are commonly used clinically as voltage-gated sodium channel inhibitors to treat pain, but they lack sufficient selectivity for voltage-gated sodium channel subtypes , thus having the disadvantages of cardiotoxicity and central nervous side effects
Recently, some small-molecule blockers (blockers) targeting Nav1.7 have entered clinical research. Due to the high homology of sodium ion channel subtypes, the selectivity of small-molecule blockers is poor, and their side effects are difficult to overcome
However, macromolecular blockers have high specificity, good stability, and less side effects, but because the voltage-gated sodium ion channel antigens that produce antibodies are not easy to prepare, research is very difficult

Method used

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  • Antibody or antibody fragment specifically binding to voltage-gated sodium channel alpha subunit Nav1.7
  • Antibody or antibody fragment specifically binding to voltage-gated sodium channel alpha subunit Nav1.7
  • Antibody or antibody fragment specifically binding to voltage-gated sodium channel alpha subunit Nav1.7

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Antigen Synthesis

[0048] According to the amino acid sequence of Nav 1.7 (GenBank No.NP_002968) and the functional region of the crystal structure ( figure 1 ) was analyzed for hydrophilicity and antigenicity, and the DSVNVDKQPKYEYS sequence was screened out, and its hydrophilicity and antigenicity met the requirements of the antigen, and the CDSVNVDKQPKYEYS (SEQ ID NO.9) polypeptide was artificially synthesized by using an automatic synthesizer.

[0049] Specific steps are as follows:

[0050] (1) Connect -COOH of the first AA to Cl-Resin with DIEA, and then block the unreacted functional groups on the resin with MeOH;

[0051] (2) washing with DMF;

[0052] (3) Use Pip to remove -NH in the first AA 2 The protecting group Fmoc makes -NH 2 exposed;

[0053] (4) washing with DMF;

[0054] (5) Activate -COOH of the second AA with DIC+HOBT, and then condense to -NH in the first AA 2 on, forming an amide bond;

[0055] (6) washing with DMF;

[0056] (7...

Embodiment 2

[0063] Embodiment 2 monoclonal cell line preparation

[0064] 2.1 Animal immunity

[0065] Prepare Freund's complete adjuvant Sigma, F5881 and Freund's incomplete adjuvant (Sigma, F5506). Using the end-SH of polypeptide C9797BL020-7, the polypeptide was coupled to the carrier protein KLH as an immunogen.

[0066] Five 8-week-old female BALB / c (animal numbers: #4061, #4062, #4063, #4064, #4065) 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.

[0067] 2.2 Animal serum ELISA detection

[0068] 2.2.1 Instruments and equipment:

[0069] Plate washer: Beijing Nanhua ZDMX

[0070] Mi...

Embodiment 3

[0103] Example 3 Detection of antibody immunogenicity

[0104] 3.1 Antigen preparation

[0105] 3.1.1: Vector construction and crude protein preparation

[0106] Synthesize 80 amino acid peptides containing the ion conduction pore module target of the DIVS3 domain of Nav1.7, construct the prokaryotic expressed His fusion protein expression vector, inoculate in 2000mL LB liquid medium (Kana resistance), at 37 degrees Shake culture overnight, when OD600 is about 0.6, lower the culture temperature to 30°C; add IPTG inducer to a final concentration of 0.1mM, continue shaking culture at 30°C for 8h; collect bacteria by centrifugation for 3min, resuspend in 50mL pre-cooled NTA-0 buffer solution, after 30 minutes in ice bath, sonicate the bacterial cells, centrifuge at 4°C for 50 minutes, and collect the precipitate (inclusion body); , centrifuge at 4°C for 10 min, remove the supernatant, and obtain the crude protein preparation;

[0107] 3.1.2 Protein denaturation and renaturatio...

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Abstract

The invention provides an antibody or an antibody fragment of a targeted cell membrane voltage-gated sodium channel alpha subunit Nav1.7, and the specific binding target of the antibody or the antibody fragment is an ion-conducting pore module (PM) of an S3 structural domain of an IV structural domain (Domain IV) of the voltage-gated sodium channel alpha subunit. The antibody or the antibody fragment thereof can inactivate the ion-conducting pore module so that sodium ions cannot normally enter nerve cells, and the effect of treating and relieving pain is achieved.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to an antibody and / or antibody fragment that specifically recognizes the above-mentioned target (polypeptide) with the ion conduction pore module of Nav1.7 as the target. Background technique [0002] Pain originates from the nociceptors of the peripheral nervous system, and the peripheral nerve tissue, as a kind of free nerve endings, is widely distributed in the skin, muscles, joints and visceral tissues of the whole body, and it can sense thermal, mechanical or chemical stimuli After that, it is transformed into an action potential, which 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 (VGSCs) located on the cell membrane. When the cell membrane is depolarized, the sodiu...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13C12N15/63A61K39/395A61P29/00
CPCC07K16/18C12N15/63A61P29/00C07K2317/565A61K2039/505
Inventor 杨代常
Owner POPULAS BIOPHARMACEUTICAL WUHAN LTD
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