Humanized interleukin-22-resistant genetically engineered antibody and application thereof

A humanized and antibody technology, applied in the field of biomedicine, can solve problems such as rapid clearance and ineffective activation of effector systems

Active Publication Date: 2016-05-25
武汉原谷生物科技有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, there are many problems in the application of mouse-derived antibodies in human therapy: (1) they cannot effectively activate the effector systems related to complement and Fc receptors in the human body; (2) they are recognized by the human immune system to produce human anti-mouse antibodies (HAMA ); (3) is quickly cleared in the human circulatory system

Method used

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  • Humanized interleukin-22-resistant genetically engineered antibody and application thereof
  • Humanized interleukin-22-resistant genetically engineered antibody and application thereof
  • Humanized interleukin-22-resistant genetically engineered antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Preparation of human IL-22

[0038] The human IL-22 gene expression vector is constructed by using genetic engineering technology (the coding nucleotide sequence of human IL-22 includes SEQ ID NO.7, and the amino acid coding sequence includes SEQ ID NO.8). We constructed human IL-22 cDNA into pTXB1 expression vector, transformed competent E.coliBL21(DE3), obtained a single clone on LB(Amp+) agar plate, and cultured overnight at 37°C. On the next day, they were transferred to 1LLB training set at a ratio of 1:100. Shake the bacteria at 37°C to A600=0.5-0.7, and add IPTG (1mM) to induce expression. Shake the bacteria at 37°C for 6-8 hours, centrifuge, and remove the supernatant. Resuspend the bacteria in 50ml Column buffer (Column buffer formula: 20mMTris-HCl, 1mM EDTA, 1mMDTT, pH 7.425°C), ultrasonically disrupt, and take the supernatant of ultrasonically disrupted bacteria to pass through the column. Wash the column with 100ml Column buffer, and then wash i...

Embodiment 2

[0039] Example 2 Screening of humanized anti-IL-22 antibodies

[0040] 1. Positive antibodies from phage antibody library screening

[0041] Phage surface display technology is a new technology that uses phage to express foreign genes established and developed in recent years. It uses the modified phage as a carrier, and inserts the gene fragment to be selected into the gene region of the phage coat protein, so that foreign polypeptides or proteins are expressed and displayed on the surface of the phage, and then the expression of specific peptides or proteins is screened by the affinity enrichment method. Phage. It realizes the conversion of genotype and phenotype, and provides a high-efficiency screening system, so it will have a profound impact in many basic and applied research fields. Because this technology has the potential to produce humanized antibodies, it has attracted many scholars to invest in this research, which has led to the rapid development of phage antibo...

Embodiment 3

[0047] The preparation of embodiment 3 recombinant antibodies

[0048] 1. Construction of expression recombinant antibody plasmid

[0049] The light chain of the obtained Fab antibody was cloned into the pAC-K-Fc vector (catNo. PROGENPR3003), and then the Fd segment of the heavy chain was cloned to construct a recombinant antibody expression vector.

[0050] 2. Transfection and recombinant virus titer determination:

[0051] Sf9 cells (PharmagenBaculoGoldco-transfection kit) were transfected, and the specific transfection method was detailed in the transfection instructions. After culturing at 27°C for 4-5 days, the supernatant was collected and the virus titer was determined.

[0052] 3. Secretion expression and purification of recombinant antibody IgG:

[0053] Sf9 cells were infected with the virus, incubated at 27°C for 2 hours, replaced with SF-900ⅡSFM serum-free medium, continued to culture at 27°C for 5 days, and collected the supernatant. The supernatant was purifi...

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Abstract

The invention relates to the technical field of biology, and particularly discloses a humanized interleukin-22-resistant genetically engineered antibody and a preparation method and application thereof. By means of the genetic engineering technology and the phage display technology, the antibody capable of being combined with human IL-22 can be directly screened out of a human antibody gene pool, and the antibody gene is obtained and expressed. The antibody can be used for treating autoimmune diseases related to Th17/IL-17 and detecting IL-22.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and specifically relates to a preparation method of a humanized anti-interleukin-22 (interleukin-22, IL-22) genetically engineered antibody and its use in Th17 / IL-17 related autoimmune diseases. Background technique [0002] Th17 cells are a newly discovered T cell subset in recent years, mainly secreting IL-17A, IL-17F, IL-21 and IL-22. The main role of Th17 cells is to participate in host defense against fungi and bacteria, but many studies have found that Th17 / IL-17 plays a role in many autoimmune diseases such as psoriasis, lupus erythematosus, multiple sclerosis, inflammatory bowel disease and other diseases and tumors. play an important role in the development of 1 . [0003] The main function of Th17 / IL-17 is to fight against bacterial and fungal infections, but in the process of activating host defense, it is often accompanied by a local inflammatory response dominated by inflammato...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/24A61K39/395A61P19/02A61P29/00A61P37/02A61P1/00A61P3/10G01N33/68
CPCC07K16/244C07K2317/565C07K2317/622
Inventor 王晨辉
Owner 武汉原谷生物科技有限责任公司
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