Patent EP-3173099-A1 (EP)
Inventor: KIM YONG SUNG
IPC: G01N33/53
Filed: 2015-07-22
Abstract: The present invention relates to a method for inhibiting intracellular activated RAS using an intact immunoglobulin-type antibody having the ability to penetrate the cytosol. The present invention also relates to a heavy-chain variable region (VH) which induces an intact immunoglobulin-type antibody to actively penetrate the cytosol of living cells through endocytosis and endosomal escape and to bind to activated RAS in the cytosol, and to an antibody comprising the same. The present invention also relates to a method of inhibiting the growth of cancer or tumor cells and a method of treating cancer or tumor, by use of the antibody. The present invention also relates to a method for screening a heavy-chain variable region that binds specifically to RAS in the cytosol. The present invention also relates to a biologically active molecule fused to the antibody and selected from the group consisting of peptides, proteins, small-molecule drugs, nanoparticles and liposomes. The present invention also relates to a composition for prevention, treatment or diagnosis of cancer, comprising: the antibody; or a biological active molecule fused to the antibody and selected from the group consisting of peptides, proteins, small-molecule drugs, nanoparticles and liposomes. The present invention also relates to a polynucleotide that encodes the light-chain variable region and the antibody. 
     According to the present invention, the method for inhibiting intracellular activated RAS using an intact immunoglobulin-type antibody having the ability to penetrate the cytosol is achieved by allowing the antibody to penetrate living cells and to specifically recognize activated (GTP-bound) RAS in the cytosol. Thus, the antibody can target activated (GTP-bound) RAS in the cytosol of living cells and inhibit the activity of the RAS. 
     Furthermore, the antibody light-chain variable region according to the present invention and an antibody comprising the same is able to penetrate living cells and localize in the cytosol, without having to use a special external protein delivery system. Particularly, the antibody light-chain variable region according to the present invention can easily interact with various human heavy-chain variable regions (VHs) and has the ability to penetrate the cytosol and remain in the cytosol, and an intact IgG-type monoclonal antibody comprising the light-chain variable region can penetrate cells and localize in the cytosol, and shows no cytotoxicity non-specific for target cells. 
     The antibody heavy-chain variable region according to the present invention and an antibody comprising the same can selectively inhibit mutations of the major drug resistance-related factor RAS of conventional various tumor therapeutic agents, and can exhibit synergistic anticancer activity with conventional therapeutic agents. In addition, the cytosol-penetrating, intact immunoglobulin-type antibody according to the present invention can penetrate cells and remain in the cytosol, without affecting the high specificity and affinity of a human antibody heavy-chain variable region (VH) for antigens, and thus can localize in the cytosol which is currently classified as a target in disease treatment based on small-molecule drugs, and at the same time, can exhibit high effects on the treatment and diagnosis of tumor and disease-related factors that show structurally complex interactions through a wide and flat surface between protein and protein.