Anti-IL-17A single-domain antibody and application thereof

A single-domain antibody, application-specific technology, applied in the direction of antibodies, resistance to vector-borne diseases, and anti-animal/human immunoglobulins, etc., can solve the problem of lack of anti-IL-17A single-domain antibody products, and achieve a wide range of affinity. , The effect of reducing production cost and simple transformation

Active Publication Date: 2022-04-22
REGENECORE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] At present, there is still a lack of anti-IL-17A single domain antibody products with strong affinity and medicinal value in the prior art

Method used

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  • Anti-IL-17A single-domain antibody and application thereof
  • Anti-IL-17A single-domain antibody and application thereof
  • Anti-IL-17A single-domain antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0099] Example 1: Preparation of human IL-17A recombinant extracellular domain protein:

[0100] The human recombinant ectodomain protein used in this patent is obtained by the company's own expression and purification. The design scheme of the human IL-17A recombinant ectodomain protein expression vector is as follows:

[0101] (1) The coding sequence of IL-17A was retrieved from NCBI, its accession number is NM_002190.2, the amino acid sequence code generated by this sequence is NP_002181.1, and the Uniprot ID is Q16552.

[0102] (2) The amino acid sequence corresponding to NP_002181.1 was analyzed by TMHMM and SMART website respectively.

[0103] (3) The analysis results showed that IL-17A is a secreted protein, and positions 1-23 are the signal peptides of the protein.

[0104](4) The nucleotide sequence encoding amino acids 24-155 of IL-17A protein was cloned into the vector pcDNA3.4 by means of gene synthesis.

[0105] (5) Perform Sanger sequencing on the constructed v...

Embodiment 2

[0106] Example 2: Construction of single domain antibody library against IL-17A protein:

[0107] 1 mg of the recombinant human IL-17A protein purified in Example 1 was mixed with an equal volume of Freund's complete adjuvant to immunize an Inner Mongolia Alxa Bactrian camel once a week for a total of 7 consecutive immunizations, except for the first time. In addition to immunization, the other six times were immunized animals with equal volume of 1 mg IL-17A protein mixed with incomplete Freund's adjuvant.

[0108] After the animals were immunized, 150 mL of camel peripheral blood lymphocytes were extracted, and the RNA of the cells was extracted. cDNA was synthesized from the extracted total RNA, and VHH (variable region of antibody heavy chain) was amplified by nested PCR reaction using cDNA as template.

[0109] Then, the pMECS vector and the VHH fragment were digested with restriction endonucleases, respectively, and then the digested fragment and the vector were linked....

Embodiment 3

[0111] Example 3: Single domain antibody screening against IL-17A protein:

[0112] Take 200 μL of the recombinant TG1 cells in Example 2 and culture them in 2×TY medium. During this period, 40 μL of the helper phage VCSM13 was added to infect the TG1 cells, and the cells were cultured overnight to amplify the phages. The next day, the phages were precipitated with PEG / NaCl, and the expanded cells were collected by centrifugation. Bacteriophage.

[0113] Dilute in 100 mM NaHCO pH 8.3 3 500 μg of IL-17A protein was coupled to the ELISA plate, placed at 4 °C overnight, and a negative control well (medium control) was set up; the next day, 200 μL of 3% skim milk was added, and it was blocked at room temperature for 2 hours; , add 100 μl of amplified phage library (approximately 2 × 10 11 phage particles) at room temperature for 1 h; after 1 h, wash 15 times with PBS+0.05% Tween-20 to wash off unbound phage.

[0114] The phage that specifically binds to the IL-17A protein was d...

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Abstract

The invention belongs to the field of immunology, and relates to an anti-IL-17A single-domain antibody and application thereof. The single-domain antibody is composed of heavy chains, and the heavy chains comprise a heavy chain CDR1 as shown in any one of SEQ ID NO: 19 to SEQ ID NO: 25, a heavy chain CDR2 as shown in any one of SEQ ID NO: 26 to SEQ ID NO: 32, and a heavy chain CDR3 as shown in any one of SEQ ID NO: 33 to SEQ ID NO: 41. Compared with the prior art, the invention has the beneficial effects that the specific single-domain antibody aiming at IL-17A is screened by using a biological genetic engineering technology, the affinity of the antibody is better, specific cells can be prevented from releasing cytokines, and the antibody has good binding activity and certain druggability through prokaryotic expression and eukaryotic expression.

Description

technical field [0001] The present invention relates to a single-domain antibody capable of specifically binding to IL-17A (hereinafter, abbreviated as "IL-17A single-domain antibody"), a pharmaceutical composition containing the single-domain antibody as an active ingredient, and its pharmaceutical therapeutic use . Background technique [0002] Psoriasis is a relatively common chronic inflammatory skin disease, commonly known as psoriasis. The disease is prone to recurrence or aggravation in winter, and is mostly relieved in spring and autumn. The global prevalence rate is about 2% to 3%. Among them, 1 / 3 of psoriatic patients have psoriatic arthritis (Psoriaticarthritis, PsA), often accompanied by There are joint swelling, stiffness and movement disorders, some of which can involve the spine, and in severe cases can cause disability, which has a serious impact on the physical and mental health of patients. Psoriasis is classified according to its clinical characteristics...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24C12N15/13A61K39/395A61P17/06A61P37/02G01N33/68
CPCC07K16/244A61P17/06G01N33/6869C07K2317/569C07K2317/565C07K2317/567C07K2317/76C07K2317/24C12N15/70C12N15/85C12N5/0686C12N5/0682A61K39/3955A61K47/68C07K2317/31C12N2510/00C12N2800/107G01N2333/54A61K2039/505Y02A50/30
Inventor 苏志鹏王乐飞张云孟巾果谢维
Owner REGENECORE BIOTECH CO LTD
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