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Antibody to be cross-linked to human and mouse sema3A, and use thereof

A semaphore, antibody technology, applied in the direction of antibodies, anti-animal/human immunoglobulin, etc., can solve the problems of high growth rate of cancer cells, poor prognosis of patients, and no anti-cancer drugs

Active Publication Date: 2018-07-17
SAMSUNG LIFE PUBLIC WELFARE FOUND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Also, it has been reported that in specific cancers with high semaphorin 3A, the growth rate of cancer cells is high, and cancer cell metastasis is promoted due to increased migration of cancer cells, and the prognosis of patients is poor
Currently, there is no anti-cancer agent that inhibits semaphorin 3A as an anti-cancer target, so an anti-semaphorin 3A antibody that inhibits related signal transmission by neutralizing semaphorin 3A will become a new anti-cancer treatment strategy

Method used

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  • Antibody to be cross-linked to human and mouse sema3A, and use thereof
  • Antibody to be cross-linked to human and mouse sema3A, and use thereof
  • Antibody to be cross-linked to human and mouse sema3A, and use thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0106] Example 1: Screening using human semaphorin 3A recombinant protein

[0107] The synthetic scFv antibody fragment phage library (Yang et al., Mol. Cells. 27:225-235, 2009) made in the past was used to identify scFv antibody fragments that bind to human semaphorin 3A through phage display screening. The phage display screening process is shown in Figure 1.

[0108] In detail, in order to recover the phagemid vector introduced into the Escherichia coli host ER2537 in the form of phage, the lower library samples were cultured in 400 ml of medium (SB / ampicillin / 2% glucose) 2 hours. If the O.D600 absorbance reaches about 0.5 to 0.7, centrifuge at 5000g for 20 minutes to remove the supernatant, suspend it in 400ml of the second medium (SB / ampicillin), and then add 10 12 pfu (plaque forming unit) helper phage (VCSM13), incubated for 1 hour. After that, 70 μg / ml of kanamycin antibiotic (an antibiotic gene introduced into the helper phage) was added, and cultured overnight at ...

Embodiment 2

[0111] Example 2: ELISA and sequence analysis for subsequent screening of anti-semaphorin 3A scFv

[0112] The phage particles recovered in the fourth screening were confirmed as colonies in the medium by infecting the host cell ER2537. The colonies were collected and inoculated on a 96-well plate containing 200 μl of SB / ampicillin medium, and cultured at 37° C. for 2 to 3 hours. Then, in order to induce the expression of scFv-pⅢ protein, each well was treated with isopropyl-β-D-thiogalactopyranoside (IPTG, Isopropylβ-D-1-thiogalactopyranoside) at a final concentration of 1 mM at a temperature of 30°C. Incubate overnight. After centrifuging the cultured well plate at 3000 rpm to remove the supernatant, 40 μl of TES solution (20% w / v sucrose, 50 mM Tris, 1 mM EDTA, pH 8.0) and left at 4°C for 30 minutes to lyse the cells. Afterwards, 60 μl of 0.2X TES solution was treated and placed at 4° C. for 30 minutes. After osmotic pressure was used to decompose the cells, the plate wa...

Embodiment 3

[0114] Example 3: Production of anti-semaphorin 3A scFv protein and confirmation of semaphorin 3A binding ability

[0115] The basic structure of the phagemid can be found at Figure 6It was confirmed in the above procedure that in the case of the host cell ER2537 described in the procedure above, scFv could not be expressed alone due to suppression of the amber codon (amber codon (UAG)) located in front of phage pIII. Therefore, an expression strain (TOP10F') as a non-suppressor strain was used to transform the phagemid into the expression strain. Afterwards, it was confirmed by DNA sequence analysis that each phagemid did not produce a mutation and was introduced into the expression strain. After the expression strain was taken as a colony, it was inoculated in 3 ml of LB / ampicillin medium and incubated at a temperature of 37°C. Incubate overnight. After that, transfer 3 ml of the overnight culture solution to 400 ml of medium (SB / ampicillin) and culture at OD600 until it ...

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Abstract

The present invention provides an antibody having the ability to be cross-linked to human Sema3A and mouse Sema3A. The antibody of the present invention can be used as a therapeutic antibody for inhibiting Sema3A in various carcinomas in which Sema3A expression is high, such as glioblastoma, pancreatic cancer and liver cancer. Since Sema3A is considered to be a therapeutic target of diabetic retinopathy, autoimmune arthritis, neuropathic pain and osteoporosis, the antibody of the present invention or an antigen binding fragment thereof can be used as an agent, in addition to being used as an anticancer drug, for treating associated diseases. The antibody of the present invention inhibits the growth of cancer cells derived from various carcinomas, by using high anti-Sema3A binding and Sema3A function inhibition caused thereby, and inhibits the migration of cancer cells by inhibiting the phosphorylation of ERK among the downstream signaling materials of Sema3A, thereby being very effective in cancer prevention and treatment.

Description

technical field [0001] This patent application claims Korean Patent Application No. 10-2015-0149272 filed with the Korean Patent Office on October 27, 2015 and Korean Patent Application No. 10-2016-0123233 filed with the Korean Patent Office on September 26, 2016 Priority, the entire content of the above-mentioned patent application is hereby incorporated into this specification as a reference. [0002] The present invention relates to antibodies cross-linked with human and mouse semaphorin 3A and uses thereof. Background technique [0003] Semaphorin 3A is a secreted protein composed of immunoglobulin-like (Ig-like, immunoglobulin-like) C2-type domain, PSI domain, and Sema domain. It is known to bind to NRP1 and PLXNA1 and induce related signal transmission. In addition, it has been reported that in specific cancers with high semaphorin 3A, the growth rate of cancer cells is high, and cancer cell metastasis is promoted due to increased migration of cancer cells, and the pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/18A61K39/395
CPCA61K39/395C07K16/18
Inventor 南都铉申镛在李在铉
Owner SAMSUNG LIFE PUBLIC WELFARE FOUND
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