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Single-chain antibody of fully-human anti-human interleukin-21 receptor and application thereof

A single-chain antibody and human interleukin technology, applied in the field of bioengineering, can solve the problems of incomplete elimination of immunogenicity and reduction of therapeutic value

Inactive Publication Date: 2012-07-04
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although humanization can reduce the immunogenicity of murine antibodies to a large extent, it cannot completely remove its immunogenicity, so it will inevitably neutralize the antibody and reduce its therapeutic value in clinical treatment

Method used

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  • Single-chain antibody of fully-human anti-human interleukin-21 receptor and application thereof
  • Single-chain antibody of fully-human anti-human interleukin-21 receptor and application thereof
  • Single-chain antibody of fully-human anti-human interleukin-21 receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Screening of fully human anti-IL-21R single-chain antibody

[0036] Dilute the IL-21R extracellular region protein (purchased from Beijing Sino Biological Technology Co., Ltd.) to 100 μg / ml with coating buffer (100 mM Tris, NaCl 150 mM, pH 9.0), take 4 ml and add it to the immunotube, and wrap it at room temperature. overnight; the next day, discard the supernatant, and quickly wash the tube 3 times with PBS; fill the immunotube with 2% MPBS (PBS containing 2% skimmed milk), and block at 37°C for 2 hours; discard the blocking solution, and quickly wash with PBS tube 3 times; the phage antibody library (10 12 ~10 13 p.f.u) (Dana library, donated by Harvard Medical School) was suspended in 4ml 2% MPBS and added to the immunotube. After repeated inversion at room temperature for 30 minutes, it was allowed to stand at room temperature for more than 90 minutes; the supernatant was discarded and replaced with PBS containing 0.1% Tween-20 Wash the tube 10 times, th...

Embodiment 2

[0037] Example 2 Soluble expression and separation and purification of anti-IL-21R single-chain antibody

[0038] Select the correctly sequenced TG1 strain, collect phagemids, infect and express Escherichia coli HB2151 according to routine operations (antibody drug engineering, P51), culture to OD600nm≈0.6, add IPTG at a final concentration of 0.1mM, induce overnight at 20°C, 12% SDS -PAGE detection of induced expression results.

[0039] Centrifuge at 6000rpm at 4°C for 5min to collect the cells; resuspend the cells in PBS, add 1mmol / L phenylmethylsulfonyl fluoride, and ultrasonically break (ultrasonic 2s, interval 2s, 10min in total); centrifuge at 12000rpm at 4°C for 20min, collect clear; supernatant nickel affinity chromatography column (purchased from GE), with different concentrations of imidazole elution; 12% SDS-PAGE detection of purified results (see Figure 4 ), the target protein was stored at -20°C, named C2, its amino acid sequence was SEQ ID NO: 1 and SEQ ID NO:...

Embodiment 3

[0040] Western Blot identification of embodiment 3 anti-IL-21R single chain antibody

[0041] Purified C2 was subjected to denaturing SDS-PAGE electrophoresis with a separation gel concentration of 12%; 4°C, 100mA constant current transfer for 2 hours, and the protein was transferred to a PVDF membrane (purchased from Millipore); after the transfer, the membrane was transferred at 5% Block overnight at 4°C in MTBS (TBS containing 5% skimmed milk); dilute anti-His mouse antibody (purchased from Millipore) with 5% MTBS at 1:2000, incubate at 37°C for 1.5h, wash with TBS 3 times, each time for 5min; Dilute HRP-anti-Mouse secondary antibody (purchased from Lianke Biotech) with 5% MTBS at 1:5000, incubate at 37°C for 1.5h, wash with TBS 3 times, 5min each time; use DAB to develop color. (See Figure 5 )

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Abstract

The invention belongs to the technical field of genetic engineering antibody, and particularly discloses a single-chain antibody C2 of a fully-human anti-human interleukin-21 receptor, and a preparation method and application thereof. The invention also discloses amino acid sequences of immunoglobulin molecules in a heavy chain variable region and a light chain variable region of the C2, including the sequences corresponding to complementarity determining regions CDR1, CDR2 and CDR3. The invention also provides a method for expressing the C2, which comprises the following steps of: screening the single chain antibody C2 of the fully-human anti-human interleukin-21 receptor from a natural human phage antibody library; and obtaining the single chain antibody through secretion and expression of a prokaryotic system, and nickel column affinity chromatography and purification. The single-chain antibody can be specifically bonded to the human interleukin-21 receptor, can inhibit the activation of the interleukin-21 receptor, is applicable to the treatment of interleukin-21 receptor-related diseases including rheumatoid arthritis, autoimmune diseases such as transplantation rejection and other immune system diseases, and also can be coupled with detectable substances and therapeutic agents.

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a high-affinity fully human single-chain antibody that can specifically bind to human interleukin-21 receptors. Background technique [0002] Human interleukin 21 (IL-21) is a recently discovered type I cytokine with a "four-helix bundle" structure, which is similar in structure to IL-2, IL-4, and IL-15. IL-21 is mainly produced by activated CD4 + T cell production plays an important role in lymphocyte maturation and proliferation. [0003] Human interleukin 21 receptor (IL-21R) is a type I cytokine receptor expressed by lymphoid tissues, especially NK, B and T cells, and is a specific receptor for cytokine IL-21. The human interleukin-21 receptor gene is located at 16p11, and the ORF of its cDNA encodes 538 amino acids. IL-21R protein is composed of leader sequence, WSXWS motif, transmembrane domain, extracellular domain and intracellular domain, and its structure is ...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N15/63C12N1/21G01N33/577A61K39/395A61P37/02A61P35/00A61P35/02
Inventor 王旻吴沁航张娟王彤罗辰
Owner CHINA PHARM UNIV
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