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A kind of anti-il-17a single domain antibody and use thereof

A single-domain antibody, humanized antibody technology, applied in the direction of antibodies, resistance to vector-borne diseases, anti-animal/human immunoglobulins, etc., to achieve a wide range of affinities, lower production costs, and simple transformation.

Active Publication Date: 2022-06-14
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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  • A kind of anti-il-17a single domain antibody and use thereof
  • A kind of anti-il-17a single domain antibody and use thereof
  • A kind of anti-il-17a single domain antibody and use 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 extracellular domain protein used in this patent is obtained by the company's own expression and purification. The design scheme of the human IL-17A recombinant extracellular domain protein expression vector is as follows:

[0101] (1) The coding sequence of IL-17A was retrieved in NCBI, its accession number is NM_002190.2, the amino acid sequence accession number generated by this sequence encoding 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 websites, respectively, for the transmembrane region and the extracellular end of the protein.

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

[0104](4) Cloning the nucleotide sequence encoding amino acids 24-155 of IL-17A protein int...

Embodiment 2

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

[0107] Mix 1 mg of the human recombinant IL-17A protein purified in Example 1 with an equal volume of complete Freund's adjuvant, and immunize a Bactrian camel from Alxa, Inner Mongolia, once a week, for a total of 7 consecutive immunizations, except for the first Except for immunization, the remaining six times were immunized with 1 mg IL-17A protein mixed with Freund's incomplete adjuvant in equal volumes. The immunization process was to stimulate the camels to produce antibodies against IL-17A protein.

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

[0109] Then, the pMECS vector and the VHH fragment were respectively digested ...

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, add 40 μL of helper phage VCSM13 to infect TG1 cells, and culture overnight to amplify the phages. The next day, use PEG / NaCl to precipitate the phages, and collect the amplified cells by centrifugation. Amplified phage.

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

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

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Abstract

The invention belongs to the field of immunology, and relates to an anti-IL-17A single-domain antibody and its application. The single domain antibody is composed of a heavy chain, and the heavy chain includes the heavy chain CDR1 shown in any one of SEQ ID NO: 19-SEQ ID NO: 25, the heavy chain shown in any one of SEQ ID NO: 26-SEQ ID NO: 32 Heavy chain CDR2 and heavy chain CDR3 shown in any one of SEQ ID NO:33-SEQ ID NO:41. Compared with the prior art, the beneficial effect of the present invention is: the present invention screens out the single-domain antibody specific for IL-17A using biogenetic engineering technology, the antibody has better affinity, and can block specific cells from releasing cytokines, and pass prokaryotic Both expression and eukaryotic expression have good binding activity and certain druggability.

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 a therapeutic use thereof . Background technique [0002] Psoriasis is a relatively common chronic inflammatory skin disease, commonly known as psoriasis. The disease tends to recur or aggravate in winter, and it is usually relieved in spring and autumn. The global prevalence is about 2% to 3%. Among them, 1 / 3 of psoriasis patients have psoriatic arthritis (PsA), often accompanied by There are joint swelling and pain, stiffness and movement disorders, some of which can involve the spine, and 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 features, which mainly include the following type...

Claims

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Application Information

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Patent Type & Authority Patents(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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