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Antibodies cross-linked to human and mouse semaphorin 3a and uses thereof

A semaphore and antibody technology, which is applied in the direction of antibodies, anti-animal/human immunoglobulins, etc., can solve the problems of no anticancer agents, high growth rate of cancer cells, and poor prognosis of patients, so as to achieve effective prevention and treatment, The effect of inhibiting migration

Active Publication Date: 2021-08-27
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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  • Antibodies cross-linked to human and mouse semaphorin 3a and uses thereof
  • Antibodies cross-linked to human and mouse semaphorin 3a and uses thereof
  • Antibodies cross-linked to human and mouse semaphorin 3a and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1: Screening using human semaphorin 3A recombinant protein

[0104] 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.

[0105] 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. Thereafter, 70 μg / ml of kanamycin antibiotic was added, and cultured overnight at a temperature of 30° C., so that the phage library cou...

Embodiment 2

[0108] Example 2: ELISA and sequence analysis for subsequent screening of anti-semaphorin 3A scFv

[0109] 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

[0111] Example 3: Production of anti-semaphorin 3A scFv protein and confirmation of semaphorin 3A binding ability

[0112] The basic structure of the phagemid can be found at Figure 6 It 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 invention provides an antibody capable of cross-linking human semaphorin 3A and mouse semaphorin 3A. The antibody of the present invention can be used as an antibody therapeutic agent for inhibiting semaphorin 3A in various cancers such as glioblastoma, pancreatic cancer, and liver cancer in which semaphorin 3A is highly expressed. Semaphorin 3A is considered to be a therapeutic target for diabetic retinopathy, autoimmune arthritis, neuropathic pain, and osteoporosis, so the antibody or antigen-binding fragment of the present invention can be used not only as an anticancer therapeutic agent, but also as a multifunctional therapeutic agent for related diseases. The antibody of the present invention has high anti-semaphorin 3A binding and semaphorin 3A function inhibitory properties based on it, so it inhibits the growth of cancer cells derived from various cancers, by controlling the phosphorylation of ERK in the downstream signaling substance of semaphorin 3A It is very effective for the prevention and treatment of cancer by inhibiting the migration of cancer cells by inhibiting chemical transformation.

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 Patents(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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