Novel Anti-cd4o antibodies and use thereof
an anti-cd4o and anti-cd4o technology, applied in the field of new anti-cd4o antibodies, can solve the problems of failure of blood sugar control, non-normal expression of ligands, and inability to prevent complications, and achieve excellent anti-cancer effects
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[0127] Sequence of anti-CD40-antibody according to the present disclosure
[0128]The present inventors cloned nucleic acids encoding heavy and light chain fragments from hybridomas producing anti-CD40 antibodies to analyze the structure of antibodies produced according to the present disclosure.
[0129]Cloning and sequence determination were made as follows.
[0130]When using the RNA miniprep kit (Qiagen) according to the producer's method for cloning the gene, RNA was extracted from RM8G-1 hybridoma cells obtained in Example 1, and then PCR was performed to synthesize cDNA.
[0131]PCR conditions and cloning methods for the synthesis of heavy chain cDNA and light chain cDNA are as follows:
[0132]The CD40 antigen-specific antibody and RM8G-1 antibody gene were cloned using Mouse Ig-Primer Set (Millipore, Cat. #: 69831). PCR was performed on RNA isolated from RM8G-1 hybridoma using Mouse Ig-Primer Set. The PCR result was inserted into a pGem-T vector (Promega, Cat. #: A3600), and then the DNA ...
example 3
[0135] Determination of CD40 antigen epitope recognized by anti-CD40 antibody according to the present disclosure
[0136]The present inventors identified human CD40 antigen epitopes recognized by the anti-CD40 antibodies according to the present disclosure.
[0137]For epitope mapping, 277 peptides composed of 15 amino acids among the amino acid sequences of the human CD40 antigen were prepared and then the peptide microarray was performed using the prepared 277 peptides. One peptide is composed of 15 amino acids, 14 of which were duplicated. Since the peptide microarray plate was prepared in a duplicated manner, 78 negative control peptide spots which may not react with the total 554 peptide spots were prepared. One peptide microarray plate reacted with goat anti-IgG (H+L) Dylight680 antibody (1:5000) and we identified whether background reaction occurs. The other three microarray plates reacted with RM8G-1 at concentrations of 1 μg / ml, 10 μg / ml and 100 μg / ml, followed by washing, and t...
example 4
[0144] Antagonistic Effect of Anti-CD40 Antibodies in accordance with the Present Disclosure
[0145]The present inventors identified the antagonistic effect of the novel anti-CD40 antibody according to the present disclosure prepared in Example 1.
[0146]4-1. Inhibition of CD40-CD154 signaling by anti-CD40 antibodies according to the present disclosure
[0147]The present inventors identified the ability to inhibit CD40-CD154 signaling by the anti-CD40 antibody (RM8G-1) according to the present disclosure.
[0148]To identify the signaling inhibitory effects of RM8G-1, PBMC was isolated from the blood of rhesus monkey as a primate, and His-tag CD40L was reacted therewith. At this time, the reaction product was treated with RM8G-1 at a varying concentration, and they reacted at room temperature for 30 minutes. After washing, flow cytometry was performed by ice reaction thereof with anti-histidine-FITC antibody for 40 minutes. After flow cytometry, FITC expression is indicated as a mean-fluores...
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