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Method for suppressing ras activated in cell by using antibody having cytoplasm penetration capacity and complete immunoglobulin form, and use for same

An immunoglobulin and antibody technology, applied in the composition of preventing, treating or diagnosing cancer, treating cancer or tumor, and the antibody field comprising the variable region of the heavy chain, which can solve the problems of insufficient research, weak host resistance, fusion Problems such as low cell penetration efficiency of proteins

Inactive Publication Date: 2017-08-01
ORUM THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, most fusion proteins are not secreted from animal cells and are only released into the supernatant in very small amounts (NaKajima O et al., 2004) and fusion proteins with arginine-rich protein transduction domains have the problem that It is weakly resistant to host furin during production (Chauhan A et al., 2007)
Additionally, there is a problem with low cell penetration efficiency of fusion proteins, making it difficult to develop these fusion proteins into therapeutic antibodies (Falnes P et al., 2001)
However, studies proving endosomal escape mechanisms of various naturally occurring substances based on the above assumptions are still insufficient

Method used

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  • Method for suppressing ras activated in cell by using antibody having cytoplasm penetration capacity and complete immunoglobulin form, and use for same
  • Method for suppressing ras activated in cell by using antibody having cytoplasm penetration capacity and complete immunoglobulin form, and use for same
  • Method for suppressing ras activated in cell by using antibody having cytoplasm penetration capacity and complete immunoglobulin form, and use for same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0200] Example 1: Heavy chain variable binding specifically to GTP-bound KRas by a highly diverse human VH library Region (VH) to select

[0201] figure 1 A schematic diagram showing the strategy for inducing cytotoxicity specific for Ras mutant cells by using the following monoclonal antibody (Anti-Ras GTP iMab: Internalization and Interfering Monoclonal Antibody) by incorporating the heavy chain of IgG type cytotransmab The variable region (VH) (only capable of penetrating the cytosol) was constructed by replacing the variable region (VH) of the heavy chain that specifically binds to GTP-bound KRas and the antibody penetrated cells and remained in the cytosol Binds specifically to GTP-bound Ras.

[0202] figure 2 A schematic diagram showing the construction by replacing the heavy chain variable region (VH) of an intact IgG-type cytotransmab capable of penetrating only the cytosol with a heavy chain variable region (VH) that specifically binds GTP-bound KRas Methods o...

Embodiment 2

[0205] Example 2: Preparation of GTP-bound KRas G12D protein

[0206] Expression and purification in E. coli for the preparation of GTP-bound KRas G12D antigen for library screening and affinity analysis is described in detail in a previously reported paper (Tanaka T et al., 2007).

[0207] Specifically, residues 1-188 of the CAAX motif comprising wild-type KRas and mutants KRas G12D, KRas G12V, and KRas G13D (listed with higher to lower mutation frequencies) were identified by using the restriction enzymes BamHI / EcoRI. The coding DNA was cloned into the Escherichia coli expression vector pGEX-3X. In this application, the expression vector was designed with T7 promoter-GST-KRas. All KRas mutations were induced using overlapping PCR techniques and expression vectors were constructed using the methods described above. The pGEX-3X-KRas vector was transformed into E. coli by electroporation and selected in selection medium. The selected Escherichia coli were cultured in LB me...

Embodiment 3

[0209] Example 3: Selection of the heavy chain variable region (VH) specific for GTP-bound KRas G12D

[0210] Figure 15 A schematic diagram showing the library screening strategy to obtain humanized antibody heavy chain variable single domains with high affinity only for the GTP-bound KRas G12D protein.

[0211] Specifically, the GTP-bound KRas G12D purified in Example 14 was biotinylated (EZ-LINK TM Sulfo-NHS-LC-Biotinylation Kit (Pierce Inc., USA)) and then reacted with the heavy chain variable region library displayed on the yeast cell surface for 1 hour at room temperature. A library of heavy chain variable regions reacted with biotinylated GTP-conjugated KRas G12D on the surface of yeast cells was reacted with streptavidin (Microbead TM (Miltenyi Biotec) was reacted at 4°C for 20 minutes and then MACS (Magnetic Activated Cell Sorting) was used to enrich yeasts exhibiting heavy chain variable regions with high affinity for GTP-KRAS G12D. The selected library display ye...

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Abstract

The present invention relates to a method for suppressing RAS activated in a cell by using an antibody having cytoplasm penetration capacity and complete immunoglobulin form. In addition, the present invention relates to a heavy chain variable region (VH) that induces bonding, with RAS activated in a cytoplasm, by means of the active penetration, by an antibody having complete immunoglobulin form, of the cytoplasm of a living cell by endocytosis and endosomal escape, and an antibody comprising same. Also, the present invention relates to a method for treating cancer or tumors or for inhibiting the growth of cancer or tumor cells by using the antibody. The present invention also relates to a method for screening a heavy chain variable region that specifically bonds to RAS in a cytoplasm. The present invention also relates to a bioactive molecule fused to the antibody. The present invention also relates to a composition for preventing, treating or diagnosing cancer, comprising the antibody or the bioactive molecule fused to same. The present invention also relates to a polynucleotide that codes a light chain variable region and the antibody.

Description

technical field [0001] The present invention relates to methods of inhibiting intracellularly activated (GTP-bound) RAS using antibodies of the intact immunoglobulin type capable of penetrating the cytosol and to uses thereof. [0002] In addition, the present invention relates to a heavy chain variable region (VH) that induces an antibody of the intact immunoglobulin type to penetrate the cytosol and bind to activated RAS in the cytosol and to an antibody comprising said heavy chain variable region (VH) . [0003] In addition, the present invention relates to methods of using said antibodies to inhibit the growth of cancer or tumor cells and to methods of treating cancer or tumors. [0004] In addition, the present invention relates to methods of screening heavy chain variable regions that specifically bind to RAS in the cytosol. [0005] In addition, the present invention relates to biologically active molecules fused to said antibodies and selected from the group consisti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395C07K16/32C07K16/46A61K47/68C12N15/13G01N33/53A61P35/00
CPCC07K16/32C07K16/44G01N33/566G01N33/5748A61K2039/505C07K2317/30C07K2317/569G01N2500/04C07K2317/21C07K2317/24C07K2317/56C07K2317/73C07K2317/76C07K2317/77C07K2317/92C07K2319/00C12Y306/05002A61P35/00A61P43/00A61K39/395A61K47/50C07K16/46G01N33/53C07K7/06C07K16/40C07K2317/565G01N33/6854G01N2333/914
Inventor 金容星崔东起辛承敏金圣勋
Owner ORUM THERAPEUTICS INC
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