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Fully-humanized anti-human interleukin 17A single-chain antibody

A technology of interleukin and antibody, which is applied in the field of medicine, can solve problems such as inability to effectively cause CDC

Active Publication Date: 2014-12-24
INST OF MATERIA MEDICA CHINESE ACAD OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In therapeutic applications, fully human antibodies can overcome many shortcomings of mouse monoclonal antibodies in clinical applications: such as inducing anti-mouse antibody (HAMA) reactions in the human body, inability to effectively induce CDC and ADCC, etc.

Method used

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  • Fully-humanized anti-human interleukin 17A single-chain antibody
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  • Fully-humanized anti-human interleukin 17A single-chain antibody

Examples

Experimental program
Comparison scheme
Effect test

preparation example 1

[0089] Preparation Example 1 Construction of Large Capacity Natural Phage Antibody Library

[0090] 1.1 Preparation of phagemid pHLS

[0091] 1) On the basis of the phagemid pUC119 vector (purchased from TAKARA company), design the gene sequence, such as figure 1 , using the whole gene synthesis technology to construct the phagemid pHLS.

[0092]2) Transfer the plasmid into XL1-blue (invitrogen) competent cells, pick a single colony, and insert 5mL2YT-A + In the culture solution (16g peptone, 10g yeast powder, 5g sodium chloride, add water to 1000mL, pH7.0, containing 100μg / mL ampicillin), transfer the next day to 200mL 2YT-A + culture solution overnight.

[0093] 3) Use the Qiagen plasmid extraction kit to extract the plasmid according to the kit instructions. Finally, the DNA concentration was detected in an ultraviolet spectrophotometer, and the final concentration was about 0.3 μg / mL.

[0094] 1.2 Extraction of total RNA from lymphocytes

[0095] 1) A total of 65 sam...

preparation example 2

[0178] Preparation Example 2 Screening of fully human anti-IL-17A single chain antibody

[0179] 2.1 Preparation of helper phage

[0180] 1) Pick a single XL1-BLUE strain and inoculate it into 40mL SB (containing 10ug / mL tetracycline), and culture overnight at 37°C with shaking.

[0181] 2) Dilute 1:500 into 10mL of the same SB medium the next day, and culture with shaking at 37°C for 1h.

[0182] 3) Pick a single M13K07 phage plaque, inoculate it into the above 10mL bacterial solution, and incubate with shaking at 37°C for 2h.

[0183] 4) Add it to the SB medium containing 10 μg / mL tetracycline and 70 μg / mL kanamycin to 500 mL, and culture overnight at 37°C with shaking.

[0184] 5) Cultivate until OD600 is about 1, centrifuge at 12000rpm at 4°C for 15min. The supernatant was taken, aseptically divided into test tubes, 50ml per tube, and stored at 4°C.

[0185] 2.2 Titration of phage virus species

[0186] 1) Prepare 6 LB culture plates without any resistance.

[0187] ...

preparation example 3

[0208] Preparation Example 3 Screening and Identification

[0209] 3.1 Selection of random clones

[0210] 1) Prepare 2YT medium, along with 100 μg / mL ampicillin and 10 μg / mL tetracycline, and add it to a 96-well deep-well culture plate, about 600uL per empty.

[0211] 2) On the culture plate output from the third round and the fourth round of screening, a toothpick randomly selects colonies and inserts them into a 96-well deep-well culture plate. Incubate overnight at 37°C with shaking.

[0212] 3) The next day, transfer to a new 96-well deep-well culture plate containing 600uL medium at 1:10, and culture with shaking at 37°C for 3 hours. Add helper phage, incubate at 37°C for 20 minutes, and culture at 30°C for 8 hours with shaking.

[0213] 4) Centrifuge at 3000 rpm for 10 minutes. The supernatant was used as the scFv phage solution to be tested.

[0214] 3.2 Polyclonal phage ELISA

[0215] 1) Dilute human IL-17A recombinant protein and bovine serum albumin (BSA) to 1...

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Abstract

The invention discloses a fully-humanized anti-human interleukin 17A single-chain antibody, relates to the technical field of genetically engineered antibodies and specifically relates to a fully-humanized antibody fragment which is genetically engineered, screened, expressed and provided with special affinity with interleukin 17A (IL-17A). The genetically engineered antibody fragment is connected with a heavy chain region and a light chain region end to end by virtue of a soft connecting fragment. By constructing a great-capacity natural phage antibody library, an antibody fragment with special adsorption capacity on IL-17A is obtained by biological elutriation. The antibody fragment is constructed into a full-length antibody, so that the reaction of IL-17A on interleukin 6(IL-6) released by a human fibrosarcoma cell HT1080 can be inhibited by virtue of eukaryotic secretory expression and affinity purification. The antibody fragment disclosed by the invention can be used for detecting and treating rheumatoid arthritis.

Description

technical field [0001] The invention relates to a method for constructing a natural phage antibody library and a single-chain antibody that can specifically bind to human interleukin 17A obtained from the antibody library. It belongs to the field of medical technology. Background technique [0002] Human interleukin-17 (human interleukin-17, hIL-17) was isolated from an activated T-cell hybridoma in 1993 and was originally called cytotoxic T-cell antigen 8 (CTLA8). At present, it is known that IL-17, namely IL-17A, is mainly secreted by Th-17 cells, and there are 6 members (IL-17A-F) in the IL-17 family, which are composed of 150-180 amino acids, usually in two exist in the form of aggregates. It can lead to the increase of chemotactic cytokines such as IL-8, monocyte chemoattractant-1 (MCP-1) and GRO-α, thereby promoting the recruitment of neutrophils and monocytes. In addition, IL-17 strengthens the local inflammatory response by stimulating the production of IL-6 and P...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24A61K39/395A61P19/02A61P29/00A61P37/02G01N33/68
Inventor 胡卓伟孙巍林珩米粟解静
Owner INST OF MATERIA MEDICA CHINESE ACAD OF MEDICAL SCI
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