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Human-derived neutralizing antibody of follistatin-like protein and application of human-derived neutralizing antibody

A follistatin and antibody technology, which is applied in the field of biomedicine, can solve the problems of inability to accurately target key cytokines, lack of understanding of different types of skin fibrosis initiation factors and pathological mechanisms, and achieve the effect of high application advantages.

Active Publication Date: 2021-07-16
TIANJIN JIKUN MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reason why the research on skin fibrosis drugs has failed to make breakthroughs is that the initiating factors and pathological mechanisms of different types of skin fibrosis are not well understood, and the key cytokines involved in the etiology of skin fibrosis cannot be accurately identified

Method used

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  • Human-derived neutralizing antibody of follistatin-like protein and application of human-derived neutralizing antibody
  • Human-derived neutralizing antibody of follistatin-like protein and application of human-derived neutralizing antibody
  • Human-derived neutralizing antibody of follistatin-like protein and application of human-derived neutralizing antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Activity screening of human FSTL1 antibody

[0067] First, we screened 47 antibodies that bind to human Fstl1 protein from the phage display antibody library, cloned the antibody active sequence (sc-Fv) into the expression vector, transfected 293T cells, and collected the conditioned medium after 2-3 days ( Conditioned medium), which contains a high concentration of scFv, can be directly added to the CAGA-luciferase reporter cell line for luciferase reporter gene detection antibody to Fstl1 neutralizing activity. In the first round of antibody activity screening, we used the conditioned medium obtained by the above method to detect the activity of the anti-Fstl1 human antibody through the TGF-β1 luciferase reporter cell line, and the first round of screening was repeated three times for the luciferase reporter gene detection, screening results ( figure 1 ) shows the increase multiple of luciferase fluorescence intensity in the control group and the experime...

Embodiment 2

[0073] Example 2. Affinity detection of human FSTL1 neutralizing antibodies JK07 and JK04 and affinity comparison with mouse FSTL1 neutralizing antibodies.

[0074] In order to further select more excellent FSTL1 neutralizing antibodies, the two human antibodies JK07 and JK04 with neutralizing activity obtained through activity screening were studied for affinity with the target protein-human FSTL1 protein, using surface plasmon resonance SPR technology The results of the research affinity showed that the affinity of the JK07 antibody to the antigen was 6.17nM, and the affinity of the JK04 antibody to the antigen was 31.17nM; at the same time, the affinity of the human antibody was compared with the mouse FSTL1 neutralizing antibodies 2K6 and 4D22 obtained in previous studies, The affinity of the mouse-derived FSTL1 neutralizing antibody 2K6 to the antigen is 106.8nM, and the affinity of the mouse-derived FSTL1 neutralizing antibody 4D22 to the antigen is 113.1nM. According to ...

Embodiment 3

[0077] Example 3. FSTL1 neutralizing antibody (JK07) can alleviate bleomycin-induced lung fibrosis in mice.

[0078] Male C57BL / 6J (age 8-10 weeks) mice were injected with 2U / kg bleomycin through the trachea, and intraperitoneally injected respectively on the 2nd, 4th, 6th, 8th, 10th, and 12th day of bleomycin treatment 100ug, 200ug, 400ug of FSTL1 neutralizing antibody (JK07) or the corresponding subtype (IgG1), and 200mg / kg of pirfenidone was used as a positive control by intragastric administration 7 days after modeling, and the samples were sacrificed after 14 days of modeling. Detection of pulmonary fibrosis and collagen content.

[0079] Measuring method of dermal thickness: use O8 pathological slide scanner to collect H&E staining image data, use Viewpoint software to measure dermal thickness, the measurement unit is mm, each piece of skin is measured 5 times at different positions, take the average value for statistics, and then calculate the Bole The ratio of the ski...

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Abstract

An embodiment of the invention relates to a human-derived neutralizing antibody of follistatin-like protein and application of the human-derived neutralizing antibody. The human-derived neutralizing antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the following CDR: VH CDR1 with an amino acid sequence shown as SEQ ID NO:1, VH CDR2 with an amino acid sequence shown as SEQ ID NO:2 and VH CDR3 with an amino acid sequence shown as SEQ ID NO:3; and the light chain variable region comprises the following CDR: VL CDR1 with an amino acid sequence shown as SEQ ID NO:4, VL CDR2 with an amino acid sequence shown as SEQ ID NO:5 and VL CDR3 with an amino acid sequence shown as SEQ ID NO:6. The human-derived neutralizing antibody provided by the invention is an antibody capable of blocking the biological activity of FSTL1, thereby regulating various fibrotic diseases (such as idiopathic pulmonary fibrosis (IPF) and skin fibrosis).

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a human neutralizing antibody of a follistatin-like protein and an application thereof. Background technique [0002] The information disclosed in this Background section is only for enhancing the understanding of the general background of the present invention and should not be taken as an acknowledgment or any form of suggestion that the information constitutes the prior art that is already known to those skilled in the art. [0003] The essence of visceral fibrosis is the repair response after tissue damage to protect the relative integrity of tissues and organs. The main pathological changes of visceral fibrosis are the increase of fibrous connective tissue and the reduction of parenchymal cells in organ tissues. Continuous progress can lead to organ structural damage, functional decline, and even failure, which seriously threatens human health and life. play an important role in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18C12N15/13A61K39/395A61P17/00A61P11/00
CPCC07K16/18A61P17/00A61P11/00C07K2317/24C07K2317/56C07K2317/565C07K2317/567C07K2317/92A61K2039/505
Inventor 杨诚周红刚宁文李霄鹤张亮李建伦东超
Owner TIANJIN JIKUN MEDICAL TECH CO LTD