WO2016013870A1 - Method for positioning, in cytoplasm, antibody having complete immunoglobulin form by penetrating antibody through cell membrane, and use for same - Google Patents

Abstract

The present invention relates to a method for positioning, in a cytoplasm, an antibody having a complete immunoglobulin form, by means of active cell penetration. In addition, the present invention relates to a light chain variable region (VL) that induces the active penetration of a cell membrane of a living cell by an antibody having a complete immunoglobulin form and the positioning of the antibody in the cytoplasm, and an antibody comprising same. Also, the present invention relates to a bioactive molecule fused to the antibody. Also, the present invention 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 the light chain variable region and the antibody. The present invention also relates to a method for preparing an antibody that is made to penetrate through to the inside of a cell and positioned in the cytoplasm.

Description

Method and use of the antibody in the form of a complete immunoglobulin penetrates the cell membrane

The present invention relates to a method for locating an antibody in the form of a complete immunoglobulin in the cytoplasm by actively penetrating the cell membrane of living cells.

The present invention also relates to light chain variable regions (VLs) and antibodies comprising the same, which induce a complete immunoglobulin form of the antibody to penetrate the cell membrane and be located in the cytoplasm.

The present invention also relates to a bioactive molecule selected from the group consisting of peptides, proteins, small molecule drugs, nanoparticles and liposomes fused to the antibody.

The present invention also relates to a composition for preventing, treating or diagnosing cancer, comprising a bioactive molecule selected from the group consisting of the antibody, or peptides, proteins, small molecule drugs, nanoparticles, and liposomes fused thereto.

The present invention also relates to a polynucleotide encoding the light chain variable region and the antibody.

The present invention also provides a method for preparing an antibody located in the cytoplasm by penetrating into a cell, the light chain variable region of the antibody comprising the step of infiltrating into the living cell into a light chain variable region having a characteristic of being located in the cytoplasm. It is about.

Fully immunoglobulin-type antibodies form a Y-shaped highly stable structure (molecular weight, 150 kDa) consisting of two heavy chain (50 kDa) proteins and two light chain (25 kDa) proteins. have. The light and heavy chains of antibodies are divided into variable regions with different amino acid sequences and constant regions with the same amino acid sequence, and the CH1, hinge, CH2, and CH3 domains exist in the heavy chain constant region. In the light chain constant region, a Ckappa or Cλ domain exists. In the heavy and light chain variable regions of an antibody, the amino acid sequence is particularly different for each antibody, which is also called complementarity determining regions (CDRs) because it constitutes a site for binding to the antigen. Looking at the three-dimensional structure of an antibody, these CDRs have a loop shape on the surface of the antibody, and there is a framework region (FR) that structurally supports it under the ring. There are three ring structures in the heavy and light chains, respectively, and these six ring region structures merge with each other and are in direct contact with the antigen. The heavy chain constant region (Fc) of the antibody ensures a long half-life in the blood through binding to the neonatal Fc receptor (FcRn), which, unlike small molecule drugs, can last for a long time in the body. In addition, specific cells for cells overexpressing markers through antibody-dependent cellular cytotoxicity and complement-dependent cellular cytotoxicity through binding to Fc gamma receptors, etc. Induction of death is possible. In the case of antibodies developed in several species for the purpose of treating various diseases in humans recently, various humanization methods such as CDR-grafting with human antibody FR (framework) to overcome immunogenicity have improved treatment effects. You can expect

Conventional antibodies do not penetrate directly into living cells due to their large size and hydrophilic character. Thus, most existing antibodies specifically target extracellularly secreted proteins or cell membrane proteins (Kim SJ et al., 2005). In general, antibodies and polymer biopharmaceuticals cannot pass through hydrophobic cell membranes, and thus cannot bind to and inhibit various disease-related substances in the cytoplasm. In general, commercial antibodies that specifically bind to intracellular substances used in experiments such as cell growth, specific inhibition, etc. cannot be directly processed on living cells, and amphiphilic glyco to bind to intracellular substances. Pretreatment is necessary to form perforations in the cell membrane through permeabilization using saponin as a side. Small molecules, nucleic acids, or nanoparticles can be transported into living cells using a variety of reagents, electroporation, or thermal shock, but for proteins and antibodies, most of the reagents and experimental conditions described above are unique. It can adversely affect tertiary structure, resulting in loss of activity. Intracellular antibodies (intrabody) that specifically bind intracellular proteins and inhibit their activity have been developed, but they also have no activity to penetrate the cell membranes of living cells, making them applicable only for gene therapy applications. Future applications are very limited (Manikandan J et al., 2007).

In contrast to polymer materials such as various types of antibody fragments including recombinant immunoglobulin antibodies and recombinant proteins as described above, small molecule materials effectively penetrate into living cells using small size and hydrophobic characteristics. Is easy. However, small molecule drugs require a hydrophobic pocket on the surface of the target material for specific binding to various disease-associated substances in the cell, and the target material having such a hydrophobic pocket is a drug of the entire disease-related substance in the cell. Because they are around 10%, they do not specifically target most intracellular pathogenic proteins (Imai K et al., 2006).

In many diseases, including cancer, mutations and abnormal overexpression of enzymes or transcription, signaling-related proteins that play an important role in intracellular protein-protein interactions (PPI) occur. In particular, it is difficult to specifically inhibit by small molecule drugs, as described above, for disease-related substances having a structural complex interaction through a wide and flat surface among these proteins (Blundell et al., 2006). For example, RAS, one of the important cytoplasmic tumor-related factors that currently lacks effective therapeutic agents, acts as a molecular switch that delivers extracellular signals to intracellular signaling through cell membrane receptors. About 30%, mainly colon cancer and pancreatic cancer, are always activated by intracellular carcinogenic mutants, and these carcinogenic mutations are known to be the major tumor-related factors by conferring strong resistance to conventional chemotherapy (Scheffzek K et al. , 1997).

In order to overcome the current technical limitations, various studies have been conducted to impart the ability to penetrate into living cells high molecular materials such as antibody fragments or recombinant proteins that can effectively inhibit protein-protein interactions. The basic amino acid sequence and hydrophobic Protein transduction domains (PTDs) with amphipathic properties have been found to have cell penetrating ability against living cells (Leena N et al., 2007) and to recognize intracellular specific proteins using the same. To this end, many attempts have been made to fuse permeation domains with various types of antibody fragments genetically. However, most of the fusion proteins are not secreted from animal cells or only a very small amount is discharged into the supernatant (NaKajima O et al., 2004), and the fusion protein with the arginine-rich protein permeation domain is the host's Purin protease. Has a productive problem of being vulnerable (Chauhan A et al., 2007). In addition, there is a problem in that development of therapeutic antibodies is difficult due to poor penetration efficiency into cells of the fusion protein (Falnes P et al., 2001). In order to overcome the expression problem, after purifying the protein, the cell permeation domain is fused through chemical covalent bond or biotin-streptavidin, etc. have.

In addition, studies using some autoantibodies have reported that antibody and single-chain variable region (scFv) antibody fragments can be penetrated into cells through cell internalization. Autoantibodies are anti-DNA antibodies commonly found in humans and mice with autoimmune diseases, some of which have the ability to penetrate intracellularly into living cells (Michael R et al. , 1995; Michael P et al., 1996; Jeske Zack D et al., 1996). Most of the cell-infiltrating autoantibodies reported to date are located in the nucleus after being introduced into the cell, and studies are being actively conducted to see the effect through the fusion with specific proteins active in the nucleus (Weisbart et al., 2012). . However, cell penetration into living cells using autoantibodies has a limit that cannot finally inhibit the specific binding and activity of various disease-related substances in the cytoplasm inside the nucleus.

Representative substances having the property of penetrating into the cells among the natural polymer materials are viruses (HIV, HSV), toxins (choleratoxin, diphtheria toxin). They are known to penetrate into cells by endocytosis, an active intracellular transport mechanism. These intracellularizations are largely classified into three pathways, including clathrin-induced endocytosis, which is involved in ligand-induced cellular internalization, or by caveolae, which is found in some toxins such as choleratoxin, dextran, and ebolavirus. There is macropinocytosis found in the back. The endocytosis involving clathrin and caveolae begins primarily when membrane receptors bind to specific ligands. Clathrin is located on the inner surface of the cell membrane. When the substance binds to the receptor, the clathrin protein forms a fibrous shell, forming a vesicle and moving the vesicle into the cytoplasm. Caveolae interacts with the caveolin-1 protein to form oligomers, creating a stable vesicle called caveosome that migrates into the cytoplasm. Macropinocytosis protrudes through a portion of the cell membrane, envelops the material, forms macropinosomes, and migrates into the cytoplasm (Gerber et al., 2013). Substances that have been infiltrated cytoplasm through these internalization pathways are mostly degraded through the lysosomal pathway in the absence of additional endosomal escape mechanisms.

In order to avoid degradation through the lysosomal pathway as described above, viruses, toxins, and the like have a mechanism of escape from the endosome to the cytoplasm. Although the endosomal escape mechanisms are not clear yet, there are three major hypotheses about the endosomal escape mechanisms. The first hypothesis is a mechanism for forming holes in the endosomal membrane, in which substances such as cationic amphiphilic peptides in the endosomal membrane bind to the negatively charged cellular double lipid membrane, resulting in internal stress or inner membrane contraction. To form a barrel-stave pore or toroidal channel (Jenssen et al., 2006), and the second hypothesis is that the endosome is bursted by the proton sponge effect. The protonated amine group may cause the endosomal membrane to collapse by increasing the osmotic pressure of the endosome through the high buffering effect of the amine group (Lin and Engbersen, 2008). Third, it is hypothesized that certain motifs, which remain neutral in the form of hydrophilic coils, but which are transformed into hydrophobic helical structures in acids such as endosomes, escape the endosomes through fusion with the endosomal membrane (Horth et al., 1991). . However, the above hypothesis lacks research results to prove the endosomes escape mechanism for various substances in the natural world.

Therefore, the present inventors have developed a humanized light chain variable region (VL) single domain that penetrates into the cell and is distributed in the cytoplasm. In addition, in order to construct a stable immunoglobulin-type antibody, light chain variable region monodomain (VL) antibody fragments having cytoplasmic penetration ability can be easily interacted with various human heavy chain variable regions (VH) and intracellularly. Improved to maintain invasive and cytoplasmic activity, a fully immunoglobulin-type antibody (Cytotransmab) has been developed that penetrates into the cell and distributes into the cytoplasm.

In addition, the present inventors selected a heavy chain variable region (VH) library to select a heavy chain variable region (VH) having a specific binding ability to the activated RAS through the selection, the complete immunoglobulin infiltrating into the cell and distributed in the cytoplasm By immobilizing the heavy chain variable region (VH) of the (immunoglobulin) type of antibody, it is a complete immunoglobulin that can specifically penetrate the living cells and bind to activated RAS in the cytoplasm to inhibit cell growth signaling. ) Anti-RAS cytoplasmic penetration antibody (iMab: internalizing & interfering monoclonal antibody).

Moreover, the present inventors confirmed that the anti-RAS cytoplasmic monoclonal antibody penetrated into various RAS mutant-dependent cancer cell lines, and showed inhibition of cell growth by RAS mutation-specific neutralization in the cytoplasm, and the antibody showed tumor tissue. Even in the form of a fusion of a peptide for imparting specificity, it was confirmed that the present invention exhibits an activity of specifically inhibiting activated RAS in RAS mutant-dependent tumors without adversely affecting cytoplasmic penetration and activated RAS neutralizing ability. .

Therefore, the present invention is to infiltrate the cell membrane of living animal cells through the process of endocytosis and actively immobilize the complete immunoglobulin-type antibody in the cytoplasm using the endosomal escape mechanism To provide a way.

In addition, the present invention is to be placed in the cytoplasm using the endosomal escape mechanism after the complete immunoglobulin form of the antibody actively penetrates the cell membrane of live animal cells through the process of endocytosis It is to provide an inducing light chain variable region (VL) and an antibody comprising the same.

The present invention also provides a bioactive molecule selected from the group consisting of peptides, proteins, small molecule drugs, nanoparticles and liposomes fused to the antibody.

The present invention also provides a composition for preventing, treating or diagnosing cancer, comprising a bioactive molecule selected from the group consisting of the antibody, or a peptide, a protein, a small molecule drug, a nanoparticle, and a liposome fused thereto.

The present invention also provides a polynucleotide encoding the light chain variable region and the antibody.

In addition, the present invention includes the step of infiltrating into the cell, comprising the step of actively infiltrating the light chain variable region of the antibody into a light chain variable region having the characteristic of being located in the cytoplasm by actively inducing and endosomes escape mechanism into the living cells It is to provide a method for producing an antibody located in the cytoplasm.

In order to achieve the above object, the present invention provides a method for actively infiltrating a cell membrane of a living cell through endocytosis and placing it in the cytoplasm of an antibody of a complete immunoglobulin form, wherein the antibody has a cytoplasmic infiltration ability. The branch provides a light chain variable region (VL).

Hereinafter, the present invention will be described in detail.

The method of the present invention is to induce the complete immunoglobulin (antimunoglobulin) type of antibody to be located in the cytoplasm by endocytosis to actively penetrate the cell membrane of living cells and induce endosomal escape mechanism By means of the variable light chain variable region (VL), antibodies in the form of complete immunoglobulin (immunoglobulin) can penetrate the cell membranes of living cells and be located in the cytoplasm.

That is, the antibody of the present invention is an antibody of the complete immunoglobulin type that exhibits both the ability to penetrate into the cells of living cells and to be located in the cytoplasm, and the antibody light chain variable region corresponding to some fragments thereof. As itself, it exerts its ability to penetrate inside the cell and locate in the cytoplasm.

Figure 1 shows the activity in the cells of the antibody or antibody light chain variable region of the present invention.

The antibody may be a chimeric, human, or humanized antibody.

In addition, the antibody may be IgG, IgM, IgA, IgD or IgE, for example IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA1, IgA5, or IgD type, most preferably IgG Type of monoclonal antibody.

A full immunoglobulin type antibody has a structure having two full length light chains and two full length heavy chains, each light chain having a heavy chain constant region of a heavy chain and disulfide bond (SS-bond) antibody. It is divided into light chain constant region and heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) type, and subclasses gamma 1 (γ1), gamma 2 (γ 2), gamma 3 (γ 3), gamma 4 (γ 4), alpha 1 (α 1), and alpha 2 (α 2). The constant regions of the light chains have kappa (κ) and lambda (λ) types.

"Heavy chain" refers to a full-length heavy chain and fragment thereof comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and three constant region domains CH1, CH2 and CH3 Are interpreted to include all. In addition, the term “light chain” includes both the full-length light chain and fragments thereof comprising the variable region domain VL and the constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to the antigen. It is interpreted as meaning.

The cytoplasmic penetrating ability may be located in the cytoplasm by endosome escape after actively infiltrating living cells through endocytosis.

In one aspect of the present invention, the light chain variable region having cytoplasmic penetration ability is CDR1 consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 7 and 10 or a sequence having at least 90% homology thereto; And

CDR3 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 9, and 12 or a sequence having at least 90% homology thereto.

Sequence information of the CDR1, CDR2, and CDR3 is as follows.

[Correction under Rule 91 11.08.2015]

More preferably, the light chain variable region may further include a CDR2 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 8, and 11 or a sequence having at least 90% homology thereto.

In one embodiment of the present invention, the light chain variable region may include CDR1 of SEQ ID NO: 4, CDR2 of SEQ ID NO: 5, and CDR3 of SEQ ID NO: 6.

In another embodiment of the present invention, the light chain variable region may include CDR1 of SEQ ID NO. 7, CDR2 of SEQ ID NO. 8, and CDR3 of SEQ ID NO.

In another aspect of the present invention, the light chain variable region may include CDR1 of SEQ ID NO: 10, CDR2 of SEQ ID NO: 11, and CDR3 of SEQ ID NO: 12.

In one aspect of the present invention, the light chain variable region may be one in which the second and fourth amino acids from the N terminus of the light chain variable region are substituted with leucine (L) and methionine (M), respectively.

This is to replace the important second and fourth residues in order to obtain the CDR structure of the cytoplasmic permeability among the residues included in the CDR (Vernier zone) located in the FR (framework).

In addition, in one aspect of the present invention, the light chain variable region includes amino acids 9, 10, 13, 17, 19, 21, 22, 42, 45, 58, 60, 79, and 85 from the N terminus of the light chain variable region. each

Serine (S), Serine (S), Alanine (A), Valine (V), Aspartic acid (D), Valine (V), Isoleucine (Isoleucine, I ), Trionine (T), lysine (Lysine, K), lysine (Lysine, K), valine (V), serine (S), glutamine (Q) and trionine (Threonine, It may be substituted with T).

It is very stable among humanized therapeutic antibodies on the market after FDA approval, and FR (Framework) is analyzed through sequence analysis of the light chain variable region of Trastuzumab (Herceptin) consisting of the heavy chain variable region of VH3 subgroup and the light chain variable region of Vκ1 subgroup. A total of 14 residues located at) were found to have been replaced.

In another aspect of the present invention, the light chain variable region may be one in which the 89th and 91th amino acids are substituted with glutamine (Q) and tyrosine (Yyrosine, Y) from the N terminus of the light chain variable region, respectively.

This was confirmed by substituting the two residues located in the mouse-derived CDR3 of the existing cytoplasmic penetration light chain variable region through analysis of the interface between the human antibody variable region (VH-VL interface).

In a preferred aspect of the present invention, the light chain variable region may be composed of amino acids selected from the group consisting of SEQ ID NO: 1, 2, and 3.

Sequence information of the sequence number is as follows.

Cytoplasmic Penetration Humanized Light Chain Variable Region (VL) Monodomain Name and Sequence

However, all residue numbers specified in SEQ ID NOs provided herein were used Kabat number (Kabat EA et al., 1991).

In one aspect of the invention, the antibody penetrates the membrane, so that the cell located in the cytoplasm may be a live animal cell. That is, the antibody may be one in which the antibody actively penetrates live animal cells.

In addition, in one aspect of the present invention, the antibody may target not only the cytoplasm but also various organelles and molecules present in the cell, but not limited to, for example, cytoplasm, nucleus, mitochondria , Endoplasmic reticulum, and intracellular macromolecules.

In addition, in one aspect of the present invention, the intracellular polymer may be a protein, lipid, DNA or RNA. More specifically, the protein may be related to control of cell growth, cell proliferation, cell cycle, DNA repair, DNA preservation, transcription, replication, translation or intracellular migration, wherein the protein is a phosphate group, a carboxylic acid group, a methyl group And may be modified, activated, or mutated by sulfate, lipid, hydroxyl, or amide groups.

In the most preferred embodiment of the invention, the antibody may be to specifically bind to target activated RAS in the cytoplasm. The activated RAS is a tumor-associated factor to which GTP is bound, and the RAS may be in a mutant form. The RAS mutations are various forms related to the disease, but there is no limitation in the type thereof. For example, the RAS mutations may be substituted mutations of Glycine No. 12, Glycine No. 13, glutamine No. 61 of KRas, HRas, and NRas.

In one aspect of the present invention, the binding force to the activated RAS in the cytoplasm may be due to the heavy chain variable region (VH) of the antibody.

In one aspect of the invention, the heavy chain variable region is

The CDR1 of SEQ ID NO: 14 or an amino acid sequence having at least 90% homology with it;

CDR2 of SEQ ID NO: 15 or an amino acid sequence having a homology of 90% or more; And

CDR3 of SEQ ID NO: 16 or an amino acid sequence having 90% or more homology thereto;

Sequence information of the sequence number is as follows.

In a more preferred embodiment of the invention, the heavy chain variable region may consist of the amino acid of SEQ ID NO: 13.

Sequence information of the sequence number is as follows.

The heavy chain variable region that specifically binds to and inhibits the RAS was screened by the following method.

In one embodiment of the present invention, a library in which artificial mutations are induced for a total of 18 residues in CDR1, CDR2, and CDR3 regions while the constructed human heavy chain variable region (VH) and heavy chain constant region (CH1) are fused. By selection.

In addition, in one embodiment of the present invention using a library in which the human heavy chain variable region (VH) and the heavy chain constant region (CH1) is fused to the cytoplasmic penetration humanized light chain variable region (VL) is activated even in the state coupled to (GTP is The heavy chain variable region capable of binding specifically to RAS) was selected.

In one embodiment of the present invention, KRas G12D, which is an activated (GTP coupled) RAS mutant, was used as a target molecule. In one embodiment of the present invention, carcinogenesis-related RAS mutations occur mainly at residues 12, 13 and 61, and residues 12 and 13 are located in the P-loop of the RAS protein and are bound to the RAS protein. Hydrolysis of GTP affects the binding of GAP (GTPase-activating protein), which induces protein structural changes in an inactive form. In addition, residue 61 binds to the hydrolytic activity of GAP and prevents GTP hydrolysis. Therefore, various carcinogenic RAS mutations have the same region as the RAS G12D mutation and signal transduction (Switch I, Switch II). It is not limited to.

In addition, in one embodiment, NRas and HRas have 85% or more similarity between residues of the catalytic domain 1 to 165 called KRas and G domain, and the site Switch I (which binds to the lower signaling material) 32-38), Switch II (59-67) domains are 100% identical. However, the C-terminal hypervariable regions 165 to 189 have a similarity of 15% but are not limited to activated KRas G12D used as target molecules because they do not structurally affect downstream signaling.

In one embodiment of the present invention using the yeast cell surface expression system cytoplasmic infiltration after the initial screening for RAS activated (GTP coupled) in the state in which the heavy chain variable region (VH) and heavy chain constant region (CH1) is expressed A light chain comprising a light chain variable region (VL) and a light chain constant region (CL) was selected in the form of the bound Fab through yeast and yeast conjugation expressing and secreting a light chain.

In addition, one aspect of the present invention provides a light chain variable region (VL) that induces a complete immunoglobulin form of antibody to penetrate the cell membrane and located in the cytoplasm.

In one embodiment of the present invention, the light chain variable region is CDR1 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 7, and 10 or a sequence having a homology of 90% or more; And

CDR3 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 9, and 12, or may include a sequence having at least 90% homology thereto.

In addition, in one embodiment, the light chain variable region (VL) may be a second and fourth amino acid is substituted with leucine (L) and methionine (Methionine, M) from the N terminal of the light chain variable region, respectively. .

In another embodiment of the present invention, the light chain variable region includes amino acids 9, 10, 13, 17, 19, 21, 22, 42, 45, 58, 60, 79, and 85 from the N terminus of the light chain variable region. each

Serine (S), Serine (S), Alanine (A), Valine (V), Aspartic acid (D), Valine (V), Isoleucine (Isoleucine, I ), Trionine (T), lysine (Lysine, K), lysine (Lysine, K), valine (V), serine (S), glutamine (Q) and trionine (Threonine, It may be substituted with T).

In another embodiment of the present invention, the light chain variable region may be one in which the 89th and 91th amino acids are substituted with glutamine (Glutamine, Q) and tyrosine (Y) from the N terminus of the light chain variable region, respectively.

In addition, in a preferred embodiment of the present invention, the light chain variable region may be composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, and 3.

The cell penetration capacity of the light chain variable region according to the present invention may be due to endosomal escape after infiltrating through endocytosis.

In addition, an aspect of the present invention provides an antibody comprising the light chain variable region.

In an embodiment of the present invention, the antibody may be located in the cytoplasm through the cell membrane, and the antibody may be a chimeric, human, or humanized antibody. The antibody may be selected from the group consisting of IgG, IgM, IgA, IgD and IgE. In addition, the antibody may be to target the cytoplasm, nucleus, mitochondria, endoplasmic reticulum, intracellular macromolecule (organelle macromolecule), the intracellular polymer may be protein, lipid, DNA or RNA. The protein may be related to control of cell growth, cell proliferation, cell cycle, DNA repair, DNA preservation, transcription, replication, translation or intracellular migration. In a preferred embodiment of the present invention, the antibody may be specifically binding to activated RAS in the cytoplasm, it may be by a heavy chain variable region (VH) that specifically binds to the activated RAS in the cytoplasm. The activated RAS may be in mutated form.

In addition, the heavy chain variable region according to an embodiment of the present invention

The CDR1 of SEQ ID NO: 14 or an amino acid sequence having at least 90% homology with it;

CDR2 of SEQ ID NO: 15 or an amino acid sequence having a homology of 90% or more; And

CDR3 of SEQ ID NO: 16 or an amino acid sequence having 90% or more homology thereto;

In a more preferred embodiment of the invention, the heavy chain variable region may consist of the amino acid of SEQ ID NO: 13.

In addition, an aspect of the present invention provides a bioactive molecule selected from the group consisting of peptides, proteins, small molecule drugs, nanoparticles and liposomes, fused to the antibody.

The protein may be an antibody, a fragment of an antibody, an immunoglobulin, a peptide, an enzyme, a growth factor, a cytokine, a transcription factor, a toxin, an antigenic peptide, a hormone, a carrier protein, a motor function protein, a receptor , Signaling proteins, storage proteins, membrane proteins, transmembrane proteins, internal proteins, external proteins, secreted proteins, viral proteins, glycoproteins, truncated proteins, protein complexes, or chemical Modified proteins, and the like.

In a specific embodiment of the present invention provides a form in which the RGD4C peptide is fused to the N-terminus of the light chain variable region of a complete immunoglobulin-type antibody that specifically binds to and inhibits RAS activated (which binds to GTP) through cellular infiltration. do. In one embodiment of the present invention, the light chain variable region N-terminus and the RGD4C peptide are preferably fused to (G ₄ S) ₁ linker, but are not limited thereto.

Small molecule drugs in the present invention are used broadly herein to denote organic compounds, inorganic compounds or organometallic compounds having a molecular weight of less than about 1000 Daltons and having activity as therapeutic agents for the disease. Small molecule drugs herein include oligopeptides and other biomolecules having a molecular weight of less than about 1000 Daltons.

In the present invention, the nanoparticle (nanoparticle) means a particle made of a material having a diameter of 1 to 1000 nm, the nanoparticle is composed of a metal nanoparticle, a metal nanoparticle core and a metal shell surrounding the core It may be a metal / nonmetal coreshell composed of a metal / metal coreshell composite, a metal nanoparticle core and a nonmetal shell surrounding the core, or a nonmetal / metal coreshell composite composed of a nonmetal nanoparticle core and a metal shell surrounding the core. . According to one embodiment, the metal may be selected from gold, silver, copper, aluminum, nickel, palladium, platinum, magnetic iron and oxides thereof, but is not limited thereto, and the nonmetal may be silica, polystyrene, latex, and acrylic. It may be selected from the rate-based material, but is not limited thereto.

In the present invention, liposomes are composed of one or more lipid bilayer membranes surrounding an aqueous internal compartment that can associate themselves. Liposomes can be specified by membrane type and size. Small unilamellar vesicles (SUVs) have a single membrane and may have a diameter of 20 nm to 50 nm. Large uni-lamellar vesicles (LUV) may have a diameter of 50 nm or more. Oligolamella large vesicles and multilamellar large vesicles have multiple, generally concentric, membrane layers and may be 100 nm or more in diameter. Liposomes with multiple asymmetrical membranes, ie several small vesicles contained within larger vesicles, are called multivesicular vesicles.

In the present invention, "fusion" refers to the integration of two molecules having different or the same function or structure, and any physical, chemical or biological method in which the tumor-penetrating peptide can bind to the protein, small molecule drug, nanoparticle or liposome. By fusion. The fusion may preferably be by a linker peptide, which may relay fusion with the bioactive molecule at various positions in the antibody light chain variable region, antibody, or fragment thereof.

The present invention also provides a pharmaceutical composition for preventing or treating cancer, including a bioactive molecule selected from the group consisting of the antibody, or a peptide, a protein, a small molecule drug, a nanoparticle, and a liposome fused thereto.

The high specificity and high affinity of the antigen of the human antibody heavy chain variable region (VH) is influenced by using a bioactive molecule selected from the group consisting of an antibody according to the present invention or peptides, proteins, small molecule drugs, nanoparticles, and liposomes fused thereto. It is able to penetrate into the cell and remain in the cytoplasm without being known, and it is present in the cytoplasm that is currently classified as a target for the treatment of diseases using small molecule drugs. A high effect can be expected in the treatment and diagnosis of tumor and disease related factors that make up complex interactions. In addition, while it is possible to selectively inhibit the KRas mutation, which is a major drug resistance-related factor of various existing tumor therapeutic agents, effective anticancer activity can be expected through concurrent treatment with existing therapeutic agents.

The cancer includes squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, anal muscle cancer, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, Parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocyte lymphoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colon cancer, Endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulva cancer, thyroid cancer, liver cancer and head and neck cancer.

When the composition is prepared as a pharmaceutical composition for preventing or treating cancer, the composition may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the composition are conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, fine Crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components.

The pharmaceutical composition for preventing or treating cancer may be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration. In oral administration, because proteins or peptides are digested, oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the composition may be administered by any device in which the active substance may migrate to the target cell.

Suitable dosages of the pharmaceutical compositions for the prophylaxis or treatment of cancer are dependent on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to response of the patient. It can be prescribed in various ways. Preferred dosages of the compositions are in the range of 0.001-100 mg / kg on an adult basis. The term "pharmaceutically effective amount" means an amount sufficient to prevent or treat cancer or to prevent or treat a disease due to angiogenesis.

The composition may be prepared in unit dose form or formulated into a multi-dose container by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods readily available to those skilled in the art. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers. In addition, the composition may be administered as a separate therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. On the other hand, since the composition comprises an antibody or antigen-binding fragment, it can be formulated as an immune liposome. Liposomes comprising the antibody can be prepared according to methods well known in the art. The immune liposomes can be prepared by reverse phase evaporation as a lipid composition comprising phosphatidylcholine, cholesterol and polyethyleneglycol-derivatized phosphatidylethanolamine. For example, Fab 'fragments of antibodies can be conjugated to liposomes via disulfide-replacement reactions. Chemotherapeutic agents such as doxorubicin may further be included in the liposomes.

The present invention also provides a composition for diagnosing cancer comprising a bioactive molecule selected from the group consisting of the antibody, or a peptide, a protein, a small molecule drug, a nanoparticle, and a liposome fused thereto.

As used herein, the term “diagnosis” refers to identifying the presence or characteristic of pathophysiology. Diagnosis in the present invention is to determine the onset and progress of cancer.

The complete immunoglobulin form of the antibody and fragments thereof may be combined with a phosphor for molecular imaging to diagnose cancer through imaging.

The fluorescent material for molecular imaging refers to any material generating fluorescence, and preferably emits red or near-infrared fluorescence, and more preferably, a phosphor having a high quantum yield is more preferred, but is not limited thereto. .

The molecular imaging phosphor is preferably, but not limited to, a phosphor, a fluorescent protein or other imaging material capable of binding to a tumor-penetrating peptide specifically binding to the antibody and fragments thereof of the complete immunoglobulin form.

The phosphor is preferably fluorescein, BODYPY, tetramethylrhodamine, Alexa, cyanine, allopicocyanine or derivatives thereof, but not limited thereto. Do not.

The fluorescent protein is preferably, but not limited to, Dronpa protein, fluorescent color gene (EGFP), red fluorescent protein (DsRFP), Cy5.5 or other fluorescent protein which is a near infrared fluorescence.

Other imaging materials are preferably iron oxide, radioisotopes, etc., but are not limited thereto, and may be applied to imaging equipment such as MR and PET.

In another aspect, the present invention provides a polynucleotide encoding the light chain variable region or an antibody or fragment thereof.

The "polynucleotide" is a polymer of deoxyribonucleotides or ribonucleotides present in single- or double-stranded form. It encompasses RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom and includes analogs of natural polynucleotides unless specifically stated otherwise.

The polynucleotide includes not only the nucleotide sequence encoding the light chain variable region described above, but also a complementary sequence to the sequence. Such complementary sequences include sequences that are substantially complementary, as well as sequences that are substantially complementary. This means a sequence that can hybridize under stringent conditions known in the art, for example, with a nucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOS: 1-3.

The polynucleotide may also be modified. Such modifications include addition, deletion or non-conservative substitutions or conservative substitutions of nucleotides. The polynucleotide encoding the amino acid sequence is to be interpreted to also include a nucleotide sequence showing substantial identity to the nucleotide sequence. The substantial identity is at least 80% homology when the nucleotide sequence is aligned with any other sequence as closely as possible and the aligned sequence is analyzed using algorithms commonly used in the art. A sequence exhibiting at least 90% homology or at least 95% homology.

In addition, an aspect of the present invention includes the step of replacing the light chain variable region of the antibody with a light chain variable region having a characteristic of infiltrating into the living cell and located in the cytoplasm,

It is possible to provide a method for producing an antibody that penetrates into living cells and is located in the cytoplasm.

In addition, one embodiment of the present invention is to replace the light chain variable region (VL) of the existing full immunoglobulin form of the antibody with the cytoplasmic penetration light chain variable region (VL), the monoclonal antibody of the substituted full immunoglobulin form It is possible to provide a method of having the same characteristics as the cellular penetration of a monoclonal antibody in the form of a complete immunoglobulin having the cytoplasmic penetration ability.

In an embodiment of the present invention, an example of a method for producing a fully immunoglobulin-type antibody which penetrates into the cell using the cytoplasmic infiltration light chain variable region (VL) and distributes the cytoplasm is as follows.

(1) a nucleic acid substituted with a humanized light chain variable region (VL) that penetrates the light chain variable region (VL) into the cytoplasm in a light chain including the human light chain variable region (VL) and the human light chain constant region (CL) Preparing a cytoplasmic infiltrating light chain expression vector which has been cloned;

(2) a nucleic acid encoding a heavy chain including a heavy chain variable region (VH) and a heavy chain constant region (CH1-hinge-CH2-CH3) for interacting with the prepared light chain to express the antibody in the form of a complete immunoglobulin preparing a heavy chain expression vector cloned from the acides);

(3) co-transforming the prepared light and heavy chain expression vectors into animal cells for protein expression to express the complete immunoglobulin type antibody including intracellular infiltration and cytoplasmic residual humanized light chain variable region (VL); And

(4) purifying and recovering the antibody in the form of a complete immunoglobulin having the expressed cytoplasmic permeability.

The method can express a vector expressing a light chain and a vector expressing a heavy chain to prepare antibodies in the form of complete immunoglobulins having cytoplasmic penetrating ability. In addition, through transformation with a vector expressing a heavy chain comprising a heavy chain variable region capable of recognizing a specific protein within a cell, the antibody may penetrate into the cell and be distributed in the cytoplasm to express an antibody capable of binding to a specific protein. Can be. The vector can be either a vector system in which the light and heavy chains are simultaneously expressed in one vector or a system in which each is expressed in a separate vector. In the latter case, both vectors can be introduced into the host cell via co-transfomation and targeted transformation.

As used herein, the term "vector" means a means for expressing a gene of interest in a host cell. For example, viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors are included. Vectors that can be used as the recombinant vector are plasmids often used in the art (eg, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14). , pGEX series, pET series, pUC19 and the like), phage (e.g., λgt4λB, λ-Charon, λΔz1 and M13, etc.) or viruses (e.g., CMV, SV40, etc.).

In the recombinant vector, the light chain variable region, the light chain constant region (CL), the heavy chain variable region (VH), and the heavy chain constant region (CH1-hinge-CH2-CH3) provided in the present invention may be operably linked to a promoter. The term "operatively linked" means a functional bond between a nucleotide expression control sequence (eg, a promoter sequence) and another nucleotide sequence. Thus, the regulatory sequence can thereby regulate transcription and / or translation of the other nucleotide sequence.

The recombinant vector can typically be constructed as a vector for cloning or a vector for expression. The expression vector may be a conventional one used in the art to express foreign proteins in plants, animals or microorganisms. The recombinant vector may be constructed through various methods known in the art.

The recombinant vector may be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vector used is an expression vector and the prokaryotic cell is a host, a strong promoter (for example, a pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.) capable of promoting transcription It is common to include ribosome binding sites and transcription / detox termination sequences for initiation of translation. In the case of using a eukaryotic cell as a host, replication origins that operate in eukaryotic cells included in the vector include f1 origin, SV40 origin, pMB1 origin, adeno origin, AAV origin, CMV origin, and BBV origin. Including but not limited to. In addition, promoters derived from the genome of mammalian cells (eg, metallothionine promoters) or promoters derived from mammalian viruses (eg, adenovirus late promoters, vaccinia virus 7.5K promoters, SV40 promoters, Cytomegalovirus (CMV) promoter and tk promoter of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

Another aspect of the invention can provide a host cell transformed with the recombinant vector.

The host cell may use any host cell known in the art, and prokaryotic cells include, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, Strains of the genus Bacillus, such as E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus thuringiensis, and enterococci and strains such as Salmonella typhimurium, Serratia marsons and various Pseudomonas species. In the case of transformation into cells, as a host cell, yeast (Saccharomyce cerevisiae), insect cells, plant cells and animal cells, for example, SP2 / 0, Chinese hamster ovary K1, CHO DG44, PER.C6, W138 , BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN and MDCK cell lines and the like can be used.

Another aspect of the present invention can provide a method for producing a complete immunoglobulin-type antibody in the cytoplasm to penetrate into the cell, comprising culturing the host cell.

Insertion of the recombinant vector into the host cell, can be used insertion methods well known in the art. For example, when the host cell is a prokaryotic cell, a CaCl ₂ method or an electroporation method may be used. When the host cell is a eukaryotic cell, a micro-injection method, calcium phosphate precipitation method, electroporation method, Liposome-mediated transfection and gene bombardment may be used, but is not limited thereto. When using microorganisms such as E. coli, productivity is higher than that of animal cells, but is not suitable for the production of intact Ig-type antibodies due to glycosylation problems. However, antigen-binding fragments such as Fab and Fv Can be used for production.

The method of selecting the transformed host cell can be easily carried out according to methods well known in the art using a phenotype expressed by a selection label. For example, when the selection marker is a specific antibiotic resistance gene, the transformant can be easily selected by culturing the transformant in a medium containing the antibiotic.

In addition, an aspect of the present invention is to the activated (GTP coupled) tumor-associated factor RAS located in the cytoplasm through cytoplasmic infiltration using the antibody of the complete immunoglobulin form that penetrates into the living cell and distributed in the cytoplasm It is possible to provide a method for preparing a complete immunoglobulin form of antibody that inhibits specific binding and activity.

In one embodiment of the above aspect, the activated (GTP conjugated) GRA which is penetrated into the animal cell using the heavy chain variable region (VH) having RAS specific binding ability and distributed in the cytoplasm and located in the cytoplasm Antibodies in the form of fully immunoglobulin that specifically bind to bound RAS can be prepared by the following method.

(1) Heavy chain expression by cloning of nucleic acid containing human heavy chain variable region (VH) and heavy chain constant region (CH1-hinge-CH2-CH3) that specifically bind to activated (GTP-bound) RAS Preparing a vector;

(2) The co-transformation of the prepared heavy chain expression vector and the cell infiltration light chain expression vector into animal cells for protein expression infiltrate into living cells and cytoplasmic distribution to completely recognize specific activated RAS (GTP-coupled). Expressing an antibody in an immunoglobulin form; And

(2) Purifying and recovering an antibody of a complete immunoglobulin form having cytoplasmic penetrating ability to specifically recognize the expressed activated (GTP bound) RAS.

According to the method of locating an antibody of the complete immunoglobulin form provided by the present invention in the cytoplasm through living cell infiltration, the antibody can be infiltrated into living cells and distributed in the cytoplasm without a special external protein delivery system. have.

In particular, in the present invention, in order to implement a fully immunoglobulin-type antibody having stable cytoplasmic penetration ability, an antibody having a property of being able to remain in the cytoplasm through cytoplasmic penetration while being easily interacted with various human heavy chain variable regions (VH) It provides a light chain variable region, the antibody of the complete immunoglobulin form comprising the light chain variable region is distributed in the cell and cytoplasm, and does not show nonspecific cytotoxicity to the cell. Activated (GTP-coupled) located in the cytoplasm of the living cell through substitution of the heavy chain variable region (VH) of the antibody to the activated (GTP-coupled) RAS specific heavy chain variable region (VH). RAS can inhibit the target and its activity.

Penetration into the living cells and remaining in the cytoplasm without affecting the antigen specificity and high affinity of the human antibody heavy chain variable region (VH) using the full immunoglobulin type antibody having cytoplasmic penetration ability according to the present invention Treatment of tumors and disease-related factors that exist within the cytoplasm that is currently classified as a target for the treatment of diseases using small molecule drugs, and that form structurally complex interactions between proteins and proteins through broad and flat surfaces. And high effect can be expected in the diagnosis. In addition, while it is possible to selectively inhibit the KRas mutation, which is a major drug resistance-related factor of various existing tumor therapeutic agents, effective anticancer activity can be expected through concurrent treatment with existing therapeutic agents.

1 is a schematic diagram showing the concept of a fully immunoglobulin-type antibody located in the cytoplasm through cell infiltration named cytotransmab.

FIG. 2A is a sequence analysis including clones used in the improvement process from m3D8 VL, a mouse-derived light chain variable region monodomain, to cytoplasmic penetration humanized light chain variable region monodomain hT3 VL, which has been improved to achieve stable binding with humanized antibody heavy chain variable region. Data.

Figure 2b is a diagram comparing the model structure using the WAM modeling of m3D8 VL and humanized light chain variable region monodomain hT0 VL, mutant hT2 VL, hT3 VL using a superimposing method.

Figure 3a is a result of observing the cytoplasmic penetration capacity of the light chain variable region single domain by confocal microscopy (confocal microscopy).

Figure 3b is a result of observation by confocal microscopy to verify the cytoplasmic penetration mechanism of the light chain variable region monodomain.

Figure 4a is a light chain variable region (VL) and amino acid sequence analysis of the existing human antibody Adalimumab (Humira) and humanized antibody Bevacizumab (Avastin) to confirm the applicability to various human antibody heavy chain variable region of hT3 VL to be.

Figure 4b is the result of further analysis of the interface residues between the variable region for stable cytotransmab construction through the optimized binding of the human antibody heavy chain variable region.

5 is a general schematic diagram of a method for substituting a humanized light chain variable region having cytoplasmic penetration capacity for a light chain variable region having no cell permeability for cytotransmab construction.

Figure 6a is analyzed by reducing or non-reducing SDS-PAGE after cytotransmab purification.

FIG. 6B shows a size exclusion chromatography column (Superdex) to confirm that the cytotransmab is monolithic in its natural environment via high performance liquid chromatography (HPLC) (The Agilent 1200 Series LC systems and Modules) (Agilent). ^TM 200 10 / 300GC) (GE Healthcare).

FIG. 6C shows an ELISA (enzyme linked immunosorbent assay) for measuring affinity for a target molecule by a heavy chain variable region of cytotransmab (TMab4, HuT4, AvaT4) and IgG form of antibody Bevacizumab (Avastin) Adalimumab (Humira) The result is.

Figure 6d is a result of agarose gel nucleic acid hydrolysis verification experiment to confirm the nucleic acid hydrolytic ability in cytotransmab through human light chain variable region (hT4) substitution having a cell penetrating ability grafting the CDRs of autoimmune mouse-derived antibody.

Figure 7a is a light chain variable region hT4 VL having a cytoplasmic penetration ability in order to verify the cytoplasmic penetration ability of the light chain variable region substituted by the cytotransmabs, focusing on one or two cells in various cell lines and observed by confocal microscopy.

Figure 7b is a result of confirming the cytoplasmic penetration ability to a plurality of cells by lowering the lens magnification in order to confirm the cell penetration efficiency in the experiment to verify cellular penetration through confocal microscopy of Figure 7a.

Figure 8a is a result of observing the degree of cell penetration according to the concentration of TMab4 under a confocal microscope.

Figure 8b is the result of observing the degree of cell penetration according to the time zone treated with TMab4 by confocal microscopy.

Figure 9a is a graphical representation of the evaluation of cell growth inhibition in vitro by treatment with cytotransmab in HeLa and PANC-1 cell lines.

Figure 9b shows a photograph confirming the extent of cell growth inhibition in vitro by treating cytotransmab in HeLa and PANC-1 cell line.

FIG. 10 is a result of observing with a confocal microscope through a pulse-chase experiment to observe the transport process and stability of TMab4 introduced into the cell.

Figure 11a is a result of observation with a confocal microscope using calcein to indirectly confirm the transfer of cytotransmab TMab4 or HuT4 into the cytoplasm.

FIG. 11B is a bar graph quantifying calcein fluorescence of the confocal micrograph of FIG. 11A.

12A is a schematic diagram illustrating the process of observing GFP fluorescence due to complementary binding of split-GFP when cytotransmab is located in the cytoplasm.

12B shows Western blot expression of streptavidin-GFP1-10 in the constructed stable cell line.

12C shows the results of observing GFP fluorescence by split GFP complementary binding of GFP11-SBP2 fused cytotransmab through confocal microscopy.

Figure 13 illustrates the construction of anti-RasGTP iMab by replacing the heavy chain variable region (VH) of the complete IgG format Cytotransmab with only cytoplasmic permeability with a heavy chain variable region (VH) that specifically binds to GTP-coupled KRas. It is a schematic diagram.

Figure 14 shows the application of IgG format cytotransmab with cytoplasmic penetrating ability. Cell penetration and intracellular distribution of GTP in which the heavy chain variable region (VH) is replaced with the heavy chain variable region (VH) that specifically binds to GTP-coupled KRas Schematic diagram of a strategy for inducing specific cytotoxicity against Ras mutant cells using monoclonal antibodies (anti-Ras · GTP iMab: internalizing & interfering monoclonal antibodies) that specifically bind to bound Ras.

15 is a schematic diagram illustrating a library selection strategy for obtaining a humanized antibody heavy chain variable region monodomain having high affinity specifically for KRTP G12D protein to which GTP is bound.

FIG. 16 is a flow cytometric analysis of binding ability of GTP-coupled KRas G12D alone and competitive conditions with GDP-coupled KRas G12D in the selection step to obtain specific high affinity to the above-described GTP-bound KRas G12D. Data.

FIG. 17 is an analysis of 12% SDS-PAGE in reducing or non-reducing conditions after purification of anti-Ras.GTP iMab RT4.

FIG. 18 shows the results of ELISA for measuring affinity between GTP-bound and GDP-bound forms of wild KRas and KRas mutants KRas G12D, KRas G12V, KRas G13D.

19 shows the results of affinity analysis of anti-Ras.GTP iMab RT4 for GTP binding to KRAS G12D using SPR (BIACORE 2000) (GE healthcare).

20 is a result of observing with confocal microscopy to confirm the cytoplasmic penetration capacity of anti-Ras.GTP iMab RT4.

FIG. 21 shows the results of in vitro evaluation of cell growth inhibition by treatment with anti-Ras.GTP iMab RT4 in NIH3T3, NIH3T3 KRas G12V, and NIH3T3 HRas G12V cell lines.

22 shows the results of evaluation of non-adherent cell growth inhibition in NIH3T3 HRas G12V cell line.

FIG. 23 shows confocal microscopy results of overlapping of anti-Ras.GTP iMab RT4 with intracellular activated HRas G12V mutant.

FIG. 24 shows the results of overlapping with anti-Ras.GTP iMab and KRas G12V mutants bound to intracellular GTP.

FIG. 25 shows the results of in vitro evaluation of cell growth inhibition by treatment of RGD-TMab4 and RGD-RT4 in HCT116 and PANC-1 cell lines.

Figure 26a is an experimental result confirming the tumor growth inhibitory effect of RGD-fused anti-Ras.GTP iMab RT4 in mice transplanted with HCT116 cell line.

Figure 26b is a graph measuring the weight of rats to identify non-specific side effects of RGD fused anti-Ras.GTP iMab RT4.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1 Cellular Infiltration Humanized Light Chain Variable Region (VL) Single Domain Development Logic

Figure 1 is a schematic diagram showing the concept of a monoclonal antibody of the complete IgG form located in the cytoplasm through the cell infiltration named cytotransmab, in order to understand the cytoplasmic penetration of the humanized antibody light chain variable region in order to implement this, the light chain variable derived from the existing mouse Reference was made to the correlation of cytoplasmic permeability of the region single domain m3D8 VL with CDRs belonging to the light chain variable region fragment (Lee et al., 2013).

FIG. 2A is a sequence analysis including clones used in the improvement process from m3D8 VL, a mouse-derived light chain variable region monodomain, to cytoplasmic penetration humanized light chain variable region monodomain hT3 VL, which has been improved to achieve stable binding with humanized antibody heavy chain variable region. Data.

Specifically, the cytoplasmic permeability of m3D8 VL, a mouse-derived light chain variable region monodomain, and hT0 VL humanized using CDR-grafting technology, is compared to the cytoplasmic cytoplasm of the light chain variable region (VL). The characteristics that lost the penetration ability was confirmed.

Accordingly, in order to restore the cytoplasmic penetrating ability of the humanized antibody light chain variable region monodomain, the CDR Vernier zone was compared in the light chain variable region FR (framework) to improve the structure of CDR1 similar to that of m3D8 VL. It was. As a result, it was confirmed that residues 2 and 4 were different from m3D8 VL having mouse-derived cytoplasmic penetration ability. In particular, since residues 2 and 4 play a role in the upper core, which is a Vernizer zone and have a great influence on the CDR1 structure, hT2 VL was induced by inducing a CDR1 structure similar to m3D8 VL through a return mutation to hT0 VL (see FIG. 2A).

Afterwards, in order to develop a light chain variable region complementary to various human antibody heavy chain variable regions for stable cytotransmab construction, and to preserve cytoplasmic penetrating ability, to simulate pairs between VH3 and Vκ1 subgroups, which occupy a high proportion of stable antibodies. , The comparative analysis of the light chain variable region FR (framework) of the monoclonal antibody Trastuzumab (Herceptin) with the subgroups of VH3 and Vκ1, and the FR (framework) of the hT2 VL was performed to compare the 14 residues with Trastuzumab (Herceptin). HT3 VL was developed by mutating with the light chain variable region FR (framework) sequence (see FIG. 2A).

Figure 2b is a diagram comparing the model structure using the WAM modeling of m3D8 VL and humanized light chain variable region monodomain hT0 VL, mutant hT2 VL, hT3 VL using a superimposing method. As described above, it was confirmed that the residue shift mutation of No. 4 and 4 of hT0 VL reduced the CDR1 region structural difference with m3D8 VL.

Example 2. Humanized light chain variable region (VL) single domain expression and purification with cytoplasmic penetration

Humanized light chain variable region (VL) single domain was purified in order to compare the actual cytoplasmic penetration ability of hT2 VL, hT3 VL designed in the above example.

Specifically, a pIg20 vector containing a Pho A signal peptide at the N-terminus and a protein A tag at the C-terminus of the light chain variable region single domain having cytoplasmic permeability was cloned using NheI / BamHI restriction enzymes. BL21 (DE3) plysE was transformed using electroporation. After incubation at 600 rpm in absorbance 600 nm at 180 rpm, 37 ° C in LBA medium containing 100 ug / ml ampicillin, the final concentration was 20 at 23 ° C after treatment with 0.5 mM IPTG (Isoprophy β-D-1-thiogalactopyronoside). Time expression. After expression, the supernatant was collected by centrifugation at 8,000 rpm for 30 minutes using a high-speed centrifuge, and then reacted with IgG Sepharose resin (GE Healthcare). The resin was washed with 50 ml of TBS (Tris-HCl, 137 mM NaCl, 2.7 mM KCl, pH 7.4) followed by further washing with 5 ml of 5 mM NH ₄ Ac, pH 5.0 buffer. Then, the protein was eluted from the resin with 0.1 M HAc, pH 3.0 buffer, and the buffer was replaced with TBS, pH 7.4 using the dialysis method, and the protein concentration was measured and analyzed through the BCA (bicinchoninic acid (Pierce)) method. Purity of the protein was confirmed through.

Example 3 Cellular Infiltration Cellular infiltration capacity and cell infiltration mechanism validation of humanized light chain variable region (VL) single domain.

Figure 3a is a result of observing the cytoplasmic penetration capacity of the light chain variable region single domain by confocal microscopy (confocal microscopy).

Specifically, in order to verify the cytoplasmic penetration of m3D8 VL, hT0 VL, hT2 VL, hT3 VL, coverslips were placed in a 24-well plate, and 5 × 10 ⁴ HeLa cell lines in each well were added with 10% FBS (Fetal bovine Serum). 0.5 ml of medium was incubated at 37 ° C for 5% CO ₂ for 12 hours. When the cells were stabilized, 10 ml of m3D8 VL, hT0 VL, hT2 VL or hT3 VL in 0.5 ml of fresh medium was incubated for 6 hours at 37 ° C and 5% CO ₂ . Then, after removing the medium and washed with PBS, proteins attached to the cell surface with a weak acid solution (200 mM glycine, 150 mM NaCl pH 2.5), and after washing the PBS, at 25 degrees after 4% paraformaldehyde addition The cells were fixed for 10 minutes. The cells were washed with PBS, incubated for 25 minutes in a buffer containing 0.1% saponin, 0.1% sodium azide, and 1% BSA in PBS for 10 minutes to form pores in the cell membrane. After washing with PBS again, it was reacted for 1 hour at 25 degrees with a buffer containing 2% BSA added to PBS to inhibit nonspecific binding. Treating Rabbit-IgG (Sigma) Recognizing Protein A Tag of Light Chain Variable Domain Single Domain 25 After staining for 2 hours in the Figure, washed three times with PBS and treated with anti-red rabbit antibody (sigma) connected with red fluorescence (TRITC) 25 The reaction was carried out for 1 hour. Finally, the nuclei were stained (blue fluorescence) using Hoechst33342 and observed by confocal microscopy. Except for hT0 VL, m3D8 VL, hT2 VL, and hT3 VL all confirmed cellular infiltration.

Figure 3b is a result of observation by confocal microscopy to verify the cytoplasmic penetration mechanism of the light chain variable region monodomain.

Specifically, when the cells were stabilized by preparing a HeLa cell line as shown in FIG. 3A, Alexa Fluor 488-transferrin (TF, green fluorescence showing green fluorescence with 10 μM of m3D8 VL, hT2 VL, or hT3 VL in 0.5 ml of fresh medium was added to each well. ), FITC-cholera toxin B (Ctx-B, green fluorescence) or Oregon green-dextran (Dextran, green fluorescence) was diluted to 10 ug / ml and incubated at 37 degrees, 5% CO ₂ conditions for 2 hours. Since the light chain variable region single domain staining method is shown in Figure 3a. As shown in FIG. 3B, all of the light chain variable region monodomains overlapped with cholera toxin-B to confirm cellular penetration by Caveolae.

Example 4 Development of Humanized Light Chain Variable Region (VL) Single Domain Cellular Penetration Facilitates Interaction with Human Antibody Heavy Chain Variable Regions.

Figure 4a is a light chain variable region (VL) and amino acid sequence analysis of the existing human antibody Adalimumab (Humira) and humanized antibody Bevacizumab (Avastin) to confirm the applicability to various human antibody heavy chain variable region of hT3 VL to be.

Specifically, as a result of analyzing the VH-VL interface residues involved in the binding between the heavy chain and the light chain variable regions, the 89th lysine (K) and the 91st serine (SELIN) located at CDR3 of the VL domain. S) was identified as 89th glutamine (Q) and 91st tyrosine (Y) in human antibodies.

In order to construct an improvement strategy for the residues, the influence of CDRs of the VH-VL interface residue heavy chain variable region and the light chain variable region was analyzed in more detail.

Figure 4b is the result of further analysis of the interface residues between the variable region for stable cytotransmab construction through the optimized binding of the human antibody heavy chain variable region.

Specifically, the existing literature has approved hT3 VL and FDA based on data on the location of interface residues between human antibody variable regions, the frequency and binding of specific interface residues located on opposite variable regions, and the frequency of use of interface residues in human antibodies. The interface between the heavy and light chain variable regions of the therapeutic antibodies Bevacizumab (Avastin) and Adalimumab (Humira) was analyzed (Vargas-Madrazo and Paz-Garcia, 2003). As a result of analysis, residues 89 and 91 included in CDR3 involved in binding between variable regions in mouse-derived CDRs of hT3 VL are regions of high human antibody use and may affect the structure of CDR3 of heavy chain variable region (VH). It was confirmed that there is. The two residues were mutated to amino acids that are frequently used in human antibodies to develop hT4 VL that can be optimized for binding to human antibody heavy chain variable regions.

Tables 1 and 2 below show human antibody light chain variable region sequences having designed cytoplasmic penetrating ability. Table 1 is a table showing the entire sequence of the human antibody light chain variable region according to Kabat numbering, Table 2 is the content of the CDR sequence in the antibody sequence of Table 1 separately.

[Correction under Rule 91 11.08.2015]
Table 1

Cytoplasmic Penetration Human Antibody Light Chain Variable Region Complete Sequence.

[Correction under Rule 91 11.08.2015]
TABLE 2

CDR sequences of the cytoplasmic infiltrating human antibody light chain variable region.

Example 5 Cytoplasmic Penetration Cytotransmab Development and Expression Purification via Humanized Light Chain Variable Region (VL) Substitution.

5 is a general schematic diagram of a method for substituting a humanized light chain variable region having cytoplasmic penetration capacity for a light chain variable region having no cell permeability for cytotransmab construction.

Specifically, the heavy chain variable regions (Bevacizumab VH, Adalimumab VH, fused with DNA encoding the secretion signal peptide at the 5 'end to construct a heavy chain expression vector for production in the form of monoclonal antibodies in the form of complete IgG) DNA encoding the heavy chain comprising the humanized hT0 VH) and the heavy chain constant region (CH1-hinge-CH2-CH3) was cloned into NotI / HindIII in the pcDNA3.4 (Invitrogen) vector, respectively. In addition, the cytoplasmic infiltration light chain variable region (hT4 VL) or the light chain variable region (Bevacizumab VL, Adalimumab VL) and the light chain constant of the antibody fused with DNA encoding the secretion signal peptide at the 5 'end to construct the vector expressing the light chain. The DNA encoding the light chain comprising the region (CL) was cloned into NotI / HindIII in the pcDNA3.4 (Invitrogen) vector, respectively.

The light and heavy chain expression vectors were transiently transfected to express and purify proteins to compare yields. In shake flasks, HEK293-F cells (Invitrogen) suspended growing in serum-free FreeStyle 293 expression medium (Invitrogen) were transfected with a mixture of plasmid and Polyethylenimine (PEI) (Polyscience). Upon 200 mL transfection in a shake flask (Corning), HEK293-F cells were seeded in 100 ml of medium at a density of 2.0 × 10 ⁶ cells / ml and incubated at 150 rpm, 8% CO ₂ . To produce each monoclonal antibody, the appropriate heavy and light chain plasmids were diluted in 125 ml of heavy chain and 125 µg of light chain (250 µg / ml) in 10 ml FreeStyle 293 expression medium (Invitrogen), and 750 µg (7.5 µg / ml) of PEI was added. The mixture was mixed with diluted 10 ml of medium and reacted at room temperature for 10 minutes. Thereafter, the reacted mixed medium was added to the cells seeded with 100 ml, and then cultured at 150 rpm and 8% CO ₂ for 4 hours, and then the remaining 100 ml of FreeStyle 293 expression medium was added and cultured for 6 days. Proteins were purified from cell culture supernatants harvested with reference to standard protocols. The antibody was applied to a Protein A Sepharose column (GE healthcare) and washed with PBS (pH 7.4). The antibody was eluted at pH 3.0 with 0.1 M glycine buffer and then immediately neutralized with 1 M Tris buffer. The eluted antibody fraction was concentrated by exchanging buffer with PBS (pH7.4) through dialysis. Purified protein was quantified using absorbance and extinction coefficient at 280 nm wavelength.

Table 3 below shows the yield of protein produced per liter of culture volume of purified cytotransmab and monoclonal antibody. The results obtained three times were statistically processed, and ± represents the standard deviation value. The yield of the obtained protein was not significantly different from that of the wild type monoclonal antibody in the case of cytotransmab including hT4 VL modified to facilitate interaction binding with human heavy chain variable region (VH).

TABLE 3

Comparison of Purification Yields of Cytotransmab and Wild-type IgG Monoclonal Antibodies Adalimumab and Bevacizumab.

Through this, it was confirmed that the humanized light chain variable region (hT4 VL) further improving the interface residue was stably expressed and purified through optimized binding with the humanized antibody heavy chain variable region.

Figure 6a is analyzed by reducing or non-reducing SDS-PAGE after cytotransmab purification.

Specifically, the molecular weight of about 150 kDa was confirmed under non-reducing conditions, and the molecular weight of the heavy chain 50 kDa and the light chain 25 kDa was shown under the reducing conditions. This shows that expression-purified cytotransmabs and monoclonal antibodies are monolithic in solution and do not form duplexes or oligomers through unnatural disulfide bonds.

FIG. 6B shows a size exclusion chromatography column (Superdex) to confirm that the cytotransmab is monolithic in its natural environment via high performance liquid chromatography (HPLC) (The Agilent 1200 Series LC systems and Modules) (Agilent). ^TM 200 10 / 300GC) (GE Healthcare).

Specifically, an elution buffer (12 mM phosphate, pH 7.4, 500 mM NaCl, 2.7 mM KCl) (SIGMA) having a high salt was used to suppress nonspecific binding with the resin due to electrical attraction due to basic residues. The flow rate is 0.5 ml / min. Proteins used as protein size markers were dehydrogenase (150 kDa), albumin (66 kDa), carbonic anhydrase (29 kDa). In all monoclonal antibodies and cytotransmabs, one pole was measured and confirmed to exist as a monolith.

Example 6. Analysis of antibody affinity by heavy chain variable region of Cytotransmab and DNA hydrolysis by light chain variable region (VL)

Figure 6c shows the results of performing an enzyme linked immunosorbent assay (ELISA) to determine the affinity for the target molecules by the heavy chain variable region of cytotransmab (TMab4, HuT4, AvaT4) and monoclonal antibody Bevacizumab (Avastin) Adalimumab (Humira) to be.

Specifically, the target molecules VEGF-A, TNFα were bound to 96 well EIA / RIA plate (COSTAR Corning) for 1 hour at 37 degrees, and then 0.1% PBST (0.1% Tween20, pH 7.4, 137 mM NaCl, 12 mM phosphate, Wash 3 times with 2.7 mM KCl) (SIGMA) for 10 minutes. After binding for 1 hour with 5% PBSS (5% Skim milk, pH 7.4, 137 mM NaCl, 12 mM phosphate, 2.7 mM KCl) (SIGMA), washed three times for 10 minutes with 0.1% PBST. After combining the cytotransmab and monoclonal antibodies TMab4, Bevacizumab, Adalimumab, AvaT4, HuT4 and washed three times for 10 minutes with 0.1% PBST. The labeling antibody binds to an alkaline phosphatase-conjugated anti-human mAb (SIGMA) conjugated with goat derived AP. 405 nm absorbance was quantified by reaction with p-nitrophenyl palmitate (pNPP) (SIGMA).

As shown in Figure 6c, AvaT4, HuT4 was confirmed to lose the affinity for the target molecule. In the case of Adalimumab and TNFα, it was confirmed that all of the CDRs located in the heavy and light chains of the antigen recognition site are involved (Shi et al., 2013). In the case of Bevacizumab, CDR3 of the heavy chain variable region (VH) plays an important role in binding to the antigen, but when analyzed in Figure 8b, it was confirmed that Bevacizumab has a VH7 subgroup, and in the heavy chain variable region (VH) CDR3 Residue No. 96 in the light chain variable region of Bevacizumab was significantly different from hT4 VL (Charlotte et al., 2007).

Figure 6d is a result of agarose gel nucleic acid hydrolysis verification experiment to confirm the nucleic acid hydrolytic ability in cytotransmab through human light chain variable region (hT4) substitution having a cell penetrating ability grafting the CDRs of autoimmune mouse-derived antibody.

Specifically, purified purified pUC19 substrate (2.2 nM) and m3D8 scFv protein (0.5 μM and 0.1 μM) or cytotransmab and monoclonal antibodies TMab4, AvaT4, HuT4 (0.1 μM) in 10 μl total mixture volume In addition, the reaction was added to TBS reaction buffer (50 mM Tris-HCl, 50 mM NaCl, pH 7.4) (SIGMA). At this time, 2 mM MgCl ₂ was added to the TBS buffer and 50 mM EDTA (SIGMA) was added to the other buffer to be used as a control. The sample thus prepared was reacted at 37 degrees and observed after 1 hour.

As shown in FIG. 6D, it was confirmed that TMab4, AvaT4, and HuT4 had no nucleic acid hydrolytic ability at 0.1 μM. It can be seen that it will not cause nonspecific cytotoxicity if the cytoplasm remains through the cytoplasmic penetrating ability.

Example 7 Verification of Cytotransmabs Cytoplasmic Infiltration Capacity

Figure 7a is a light chain variable region hT4 VL having a cytoplasmic penetration ability in order to verify the cytoplasmic penetration ability of the light chain variable region substituted by the cytotransmabs, focusing on one or two cells in various cell lines and observed by confocal microscopy.

Specifically, 5 × 10 ⁴ HeLa, PANC-1, HT29, and MCF-7 cells per well were added to 0.5 ml of medium containing 10% FBS in a 24-well plate at 5% CO ₂ , 37 ° C for 12 hours. Incubated. When the cells were stabilized, each well was diluted with 1 μM of TMab4, Adalimumab (Humira), Bevacizumab (Avastin), HuT4, and AvaT4 in 0.5 ml of fresh medium and incubated at 37 ° C and 5% CO ₂ for 6 hours. Then, after removing the medium and washed with PBS, proteins attached to the cell surface was removed with a weak acid solution (200 mM glycine, 150 mM NaCl pH 2.5). After PBS wash, cells were fixed for 10 min at 25 degrees after 4% paraformaldehyde addition. Thereafter, the cells were washed with PBS, incubated for 25 minutes in a buffer solution containing 0.1% saponin, 0.1% sodium azide, and 1% BSA in PBS to form pores in the cell membrane. After washing with PBS again, it was reacted for 1 hour at 25 degrees with a buffer containing 2% BSA added to PBS to inhibit nonspecific binding. Next, the antibody (Sigma) that specifically recognizes human Fc to which FITC (green fluorescence) is bound is stained at 25 degrees for 1.5 hours, and the nucleus is stained using Hoechst33342 (blue fluorescence) and observed by confocal microscopy. did. Unlike the IgG-type monoclonal antibodies Adalimumab and Bevacizumab, which target the extracellular secreted protein, TMab4, HuT4 and AvaT4 were observed to have fluorescence inside the cells.

Figure 7b is a result of confirming the cytoplasmic penetration ability to a plurality of cells by lowering the lens magnification in order to confirm the cell penetration efficiency in the experiment to verify cellular penetration through confocal microscopy of Figure 7a.

In the case of the cytotransmab in which the humanized light chain variable region having cytoplasmic penetration ability was introduced, it was confirmed that all cells were distributed in the cytoplasm through cytoplasmic infiltration.

Figure 8a is a result of observing the degree of cell penetration according to the concentration of TMab4 under a confocal microscope. HeLa cell lines were treated with 10 nM, 50 nM, 100 nM, 500 nM, 1 mM, 2 mM in TMab4 and incubated at 37 degrees for 6 hours. It prepared similarly to the method mentioned above, and observed with the confocal microscope. When TMab4 was incubated for 6 hours, green fluorescence was observed in cells from 100 nM. Since the concentration increased, the green fluorescence inside the cells increased.

Figure 8b is the result of observing the degree of cell penetration according to the time zone treated with TMab4 by confocal microscopy. HeLa cell line treated with 1 mM TMab4, incubated at 10 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours 24 hours, 48 hours and 37 degrees, respectively, and stained in the same manner as in the above example, and observed by confocal microscopy. did.

From 30 minutes, the intracellular TMab4 green fluorescence was visible and gradually increased, showing the strongest fluorescence at 6 hours. After that time, the gradual decreases, and at 48 hours the fluorescence is very weak.

Example 8. Cytotoxicity Evaluation of Cytotransmabs

In order to confirm that cytotransmab having cytoplasmic infiltration ability in Example 10 had cytotoxicity in vitro, HeLa, PANC-1 cell lines were treated with TMab4, HuT4, Adalimumab, AvaT4, and Bevacizumab for MTT assay ( sigma).

Specifically, 1 × 10 ⁴ cells (HeLa, PANC-1) per well in a 96 well plate were diluted in 0.1 ml of medium containing 10% FBS, respectively, and cultured at 12 hours, 37 degrees, and 5% CO ₂ conditions. Thereafter, 1 μM of TMab4, HuT4, Adalimumab, AvaT4, and Bevacizumab were treated for 20 hours or 44 hours, and then 20 μl of MTT solution (1 mg / ml PBS) was added and further incubated for 4 hours. Formed fomazan was dissolved in 200 μl of DMSO (Dimethyl Sulfoxide), and cell viability was determined by measuring absorbance at 595 nm with an absorbance meter.

Figure 9a is a graphical representation of the evaluation of cell growth inhibition in vitro by treatment with cytotransmab in HeLa and PANC-1 cell lines. Figure 9b shows a photograph confirming the extent of cell growth inhibition in vitro by treating cytotransmab in HeLa and PANC-1 cell line. As shown in FIGS. 9A and 9B, it was confirmed that all the antibodies did not show cytotoxicity. As confirmed in Example 6, it was confirmed that there is no cytotoxicity because the cytotransmabs have no nucleic acid hydrolytic activity unlike m3D8 scFv.

Example 9 Verification of Intracellular Transport and Degradation Mechanism of Cytotransmab

FIG. 10 is a result of observing with a confocal microscope through a pulse-chase experiment to observe the transport process and stability of TMab4 introduced into the cell.

Specifically, HeLa cell lines were prepared in the same manner as described above, TMab4 3 mM was treated at 37 degrees for 30 minutes, and then washed three times with PBS rapidly and medium was added at 2 hours, 6 hours, 18 hours and 37 degrees. Incubated. After washing with PBS and weakly acidic solution through the same method as in the above example, the cells were fixed, perforated, and blocked. TMab4 was stained with an antibody that specifically recognizes human Fc to which green fluorescence (FITC) or red fluorescence (TRITC) is linked. And anti-caveolin-1, an anti-caveolin-1 marker, and a vesicle marker, calnexin, which labels anti-EEA1, which is an early endosome marker (Eearly Endosome Antigen1), and a caveolin-1, which is a marker of a caveosome. Anti-58K Golgi (santa cruz) antibodies that label anti-calnexin or 58K Golgi protein, which is a Golgi marker, were reacted for 4 hours and 12 hours, and a secondary antibody connected with red fluorescent (TRITC) was reacted for 1 hour at 25 degrees. Lysosomes and transferrin were treated directly with cells incubated with LysoTracker® Red DND-99 1 mM or Alexa Fluor 488-transferrin (10 mg / ml) 30 minutes prior to cell immobilization. After completion of the staining process, analysis was carried out by confocal microscopy. TMab4 is more stably present in the cells than transferrin, and is cytoplasmic infiltrated by clathrin and placed in the early endosomes for up to 2 hours, after which it is not transported to lysosomes and does not overlap any organelles.

Figure 11a is the result of observing the transfer of cytotransmab TMab4 or HuT4 into the cytoplasm under confocal microscopy using calcein.

Specifically, HeLa cell lines were prepared in the same manner as described above, and 5 μM of PBS, TMab4, Adalimumab, and HuT4 were cultured in serum-free medium at 37 ° C. for 4 hours. After 4 hours, wells containing PBS or antibody were treated with 50 μM calcein and incubated for 2 hours at 37 ° C. After washing with PBS, the cells were fixed in the same manner as in the above example and observed under a confocal microscope. TMab4 and HuT4 released calcein from the endosomes and showed green calcein fluorescence in the cytoplasm. Adalimumab, on the other hand, did not show green fluorescence in the cytoplasm.

FIG. 11B is a bar graph quantifying calcein fluorescence of the confocal micrograph of FIG. 11A.

Specifically, 15 cells were designated under each condition using Image J software (National Institutes of Health, USA), and then the mean value of fluorescence obtained was plotted.

Example 10. Confirmation of cytoplasmic residual capacity of cytotransmab through GFP fragment recombination ability

12A is a schematic diagram illustrating the process of observing GFP fluorescence due to complementary binding of split-GFP when cytotransmab is located in the cytoplasm.

Specifically, the split-GFP complementary system was used to directly confirm that cytotransmab is located in the cytoplasm. Dividing the green fluorescent protein GFP into fragments 1-10 and 11 eliminates the fluorescent properties, and if the two fragments are closer together, they can restore the fluorescent properties (Cabantous et al. , 2005). Using this property, GFP fragments 1-10 are expressed in the cytoplasm, and GFP fragment 11 is fused to the heavy chain C-terminus of Cytotransmab. Thus, the observation of GFP fluorescence proves that Cytotransmab is located in the cytoplasm.

In addition, streptavidin-SBP2 (streptavidin binding peptide 2) with higher affinity was used to aid complementary binding of split GFP. (Barrette-Ng et al., 2013) Smaller SBP2 was genetically fused using GGGGS three linkers to the C-terminus of GFP 11 fragment. In addition, streptavidin was genetically fused to the N-terminus of GFP 1-10 fragments using GGGGS three linkers, and a stable transformed cell line expressing streptavidin-GFP1-10 was developed to implement the system.

Specifically, the DNA encoding Streptavidin-GFP1-10 was cloned into SalI / EcoRI into pLJM1 (Addgene) vector, which is a Lenti virus vector. In a cell culture dish, 3 x 10 ⁶ HEK293T cells were placed in 1 ml of medium containing 10% FBS and incubated at 5% CO ₂ , 37 ° C for 12 hours. Add 40 μl of Lipofectamine 2000 (Invitrogen, USA) to 600 μl of Opti-MEM media (Gibco) and carefully add the lentiviral vector and viral packaging vectors pMDL, pRSV, and pVSV-G (Addgene) for 20 minutes. After the reaction at room temperature it was added to the dish. In addition, 9 ml of antibiotic-free DMEM media was added and cultured at 37 ° C. for 5 hours at 5% CO 2, followed by exchanging 10 ml of DMEM medium containing 10% FBS, followed by incubation for 72 hours. After 60 hours, 1 × 10 ⁵ HeLa cells were added to 1 ml of medium containing 10% FBS and incubated at 37 ° C. and 5% CO ₂ for 12 hours. The medium in which the lentiviral vector is transiently transfected is filtered and added to the cell culture dish containing HeLa cells prepared in advance. Antibiotic resistance was used as a selection marker of the puromycin resistance gene.

12B shows Western blot expression of streptavidin-GFP1-10 in the constructed stable cell line.

Specifically, 1 x 10 ⁵ HeLa cells per well were plated into 6-well plates in 1 ml of medium containing 10% FBS and incubated at 37 degrees and 5% CO ₂ for 12 hours. After incubation, add a lysis buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% SDS, 1 mM EDTA, Inhibitor cocktail (sigma)) to obtain cell lysate. Cell lysates were quantified using the BCA protein assay kit (Pierce). The gels subjected to SDS-PAGE were transferred to PVDF membranes and reacted with antibodies (SantaCruz) that recognizes streptavidin and β-actin, respectively, at 25 ° C for 2 hours, and HRP-coupled secondary antibody (SantaCruz) at 25 ° C for 1 hour. After the reaction was detected. Analysis was performed using ImageQuant LAS4000 mini (GE Healthcare).

Example 11 Expression and Purification of GFP11-SBP2 Fused Cytotransmab

For expression of GFP11-SBP2 fused cytotransmab animal cells, GFP11-SBP2 was genetically fused to the heavy chain C-terminus using three GGGGS linkers. Afterwards, transient transfection of HEK293F protein-expressing cells simultaneously with the animal expression vector encoding GFP11-SBP2 and the animal expression vector encoding the cytoplasmic infiltration light chain and endosomal escape ability were performed. Was done. After the purification of GFP11-SBP2 fused cytoplasmic penetration monoclonal antibody was carried out in the same manner as in Example 5.

Example 12. Confirmation of GFP Fluorescence According to Cytoplasmic Location of GFP11-SBP2 Fusion Cytotransmab.

12C shows the results of observing GFP fluorescence by split GFP complementary binding of GFP11-SBP2 fused cytotransmab through confocal microscopy.

Specifically, in the same manner as in Example 7, HeLa cell lines were prepared, and when the cells were stabilized, PBS, TMab4-GFP11-SBP2 0.2, 0.4, 0.6, 0.8, and 1 μM were incubated at 37 degrees for 6 hours. After washing with PBS and weakly acidic solution in the same manner as in Example 7, cells were fixed. Nuclei were stained (blue fluorescence) using Hoechst33342 and observed with a confocal microscope. TMab4 was confirmed that 0.8, 1 μM GFP fluorescence was observed.

The results clearly confirm that cytotransmab TMab4 is located in the cytoplasm through cell infiltration.

Example 13. Selection of KRAS Specific Binding Heavy Chain Variable Region (VH) Bound GTP Through Highly Diversity Human VH Libraries

Figure 13 illustrates the construction of anti-RasGTP iMab by replacing the heavy chain variable region (VH) of the complete IgG format Cytotransmab with only cytoplasmic permeability with a heavy chain variable region (VH) that specifically binds to GTP-coupled KRas. It is a schematic diagram.

Figure 14 shows the application of IgG format cytotransmab with cytoplasmic penetrating ability. Cell penetration and intracellular distribution of GTP in which the heavy chain variable region (VH) is replaced with the heavy chain variable region (VH) that specifically binds to GTP-coupled KRas Schematic diagram of a strategy for inducing specific cytotoxicity against Ras mutant cells using monoclonal antibodies (anti-Ras · GTP iMab: internalizing & interfering monoclonal antibodies) that specifically bind to bound Ras.

In order to select a stable humanized heavy chain variable domain monodomain (VH) antibody fragment specifically bound to GTP-coupled KRas for introduction into the anti-Ras GTP iMab, a yeast expression VH library constructed from previous studies was used. (Baek and Kim, 2014).

Specifically, FR (framework) of the used library uses IGHV3-23 * 04, J _H 4, which are the most commonly used V genes in conventional antibodies, and a library having 9 residues in CDR3 length was used. The construction of the library and yeast surface expression methods are described in detail in a previously published paper (Baek and Kim, 2014).

Example 14 Preparation of KRas G12D Protein with GTP

The process of expression purification in Escherichia coli to prepare GTP-linked KRas G12D antigen for library selection and affinity analysis is described in detail in a previously published paper (Tanaka T et al., 2007).

Specifically, the DNA coding 1 to 188 containing the three mutant KRas G12D, KRas G12V, and KRas G13D CAAX motifs in the order of wild KRas and the high frequency of mutation are restricted to the pGEX-3X vector, an E. coli expression vector. Cloned using the enzyme BamHI / EcoRI. The expression vector was designed to have a T7 promoter-GST-KRas. All KRas mutations were induced by mutations using the overlap PCR technique, expression vectors were constructed using the above technique, and pGEX-3X-KRas vectors were transformed into E. coli by electroporation and selected in selective medium. . Selected Escherichia coli was cultured in LB medium in the presence of 100 μg / ml ampicillin antibiotic to absorbance of 0.6 at 37 to 600 nm, and then 0.1 mM IPTG was added for protein expression, followed by further incubation at 30 degrees for 5 hours. . Thereafter, E. coli was collected using a centrifuge, and then E. coli was pulverized using ultrasonic waves (SONICS). Only the supernatant from which E. coli pulverization was removed using a centrifuge was purified using Glutathione resin (Clontech), which specifically purified GST-tagged proteins. Elution after washing 50 ml with washing buffer (140 mM NaCl, 2.7 mM KCl, 10 mM NaH ₂ PO ₄ , 1.8 mM KH ₂ PO ₄ , 1 mM EDTA, 2 mM MgCl ₂ pH 7.4) (SIGMA) to wash Glutathione resin The protein was eluted using buffer (50 mM Tris-HCl pH8.0, 10 mM reduced glutathione, 1 mM DTT, 2 mM MgCl ₂ ) (SIGMA). The eluted protein was changed to a buffer (50 mM Tris-HCl pH8.0, 1 mM DTT, 2 mM MgCl ₂ ) (SIGMA) using a dialysis method. Purified protein was quantified using absorbance and extinction coefficient at 280 nm wavelength. SDS-PAGE analysis confirmed the purity of more than about 98%.

Then, to bind the GTPλS (Millipore) or GDP (Millipore) substrate to the KRas protein, the reaction buffer (50 mM Tris-HCl pH8.0, 1 mM DTT, 5 mM MgCl _{2 with a} ratio of 1: 20 of KRas and the substrate) was used. , 30 mM in 15 mM EDTA) (SIGMA) at 30 degrees and then stored at -80 degrees after the addition of 60 mM MgCl ₂ to stop the reaction.

Example 15. Selection of heavy chain variable region (VH) specific for KRas G12D bound to GTP

15 is a schematic diagram illustrating a library selection strategy for obtaining a humanized antibody heavy chain variable region monodomain having high affinity specifically for KRTP G12D protein to which GTP is bound.

Specifically, heavy chain variable expressed on the surface of yeast cells after biotinylation of the GTP-linked KRas G12D purified in Example 14 (EZ-LINK ^TM Sulfo-NHS-LC-Biotinylation kit (Pierce Inc., USA)). The reaction was conducted with the zone library at room temperature for 1 hour. The heavy chain variable region library was reacted with the streptavidin Microbead ^TM (Miltenyi Biotec) for 20 minutes at 4 ° C on the surface of yeast cells reacted with KRas G12D conjugated with biotinylated GTP and then subjected to magnetic activated cell sorting (MACS). The yeast expressing the heavy chain variable region having high affinity to GRAS-bound KRAS G12D was enriched (enrichment). Yeast expressing the selected library was cultured in selective medium and SG-CAA + URA (20 g / L Galactose, 6.7 g / L Yeast nitrogen base without amino acids, 5.4 g / L Na2HPO ₄ , 8.6 g / L NaH ₂ PO ₄ , After inducing protein expression in 5 g / L casamino acids, 0.2 mg / L Uracil) (SIGMA) medium, biotinylation was not 10% higher than that of GTP-linked KRas G12D alone or GTP-linked KRas G12D alone. The conjugated KRas G12D antigen was reacted with a library-expressed yeast at room temperature for 1 hour, followed by reaction with PE conjugated streptavidin (Streptavidin-R-phycoerythrin conjugate (SA-PE) (Invitrogen) to produce FACS (Fluorescence). Suspension was confirmed by activated cell sorting (FACS Caliber) (BD biosciences). After selection of the next screening condition by FACS analysis, antigens were bound to yeast expressing the suspended library under the same conditions as above, and then suspended by FACS ariaII instrument. Humanized heavy chain variable chain libraries suspended by primary MACS and primary FACS screening were expressed on the yeast surface in the form of Fab after yeast conjugation with yeast secreting cytoplasmic infiltration light chain single chain (hT4 VL), followed by secondary FACS, 3 The second FACS screening process was conducted.

Specifically, in order to construct a yeast secreting cytoplasmic infiltrating light chain variable region (VL) to be conjugated with the heavy chain variable region (VH) library, DNA encoding hT4 VL having cytoplasmic permeability in pYDS-K, a light chain variable region yeast secreting vector, was constructed. PYDS-K-hT4 VL cloned using restriction enzymes NheI and BsiWI was transformed into YVH10 strain, a yeast conjugate strain of the mating α type by electroporation, to select medium SD-CAA + Trp (20 g / L Glucose, 6.7 g / L Yeast nitrogen base without amino acids, 5.4 g / L Na2HPO ₄ , 8.6 g / L NaH ₂ PO ₄ , 5 g / L casamino acids, 0.4 mg / L tryptophan) (SIGMA) One yeast and a yeast junction.

Specifically, in the case of yeast conjugation, when the absorbance at 600 nm is 1, there is 1 X 10 ⁷ yeast. In yeast cultured yeast expressing the heavy chain variable region library selected in KRas G12D bound GTP and yeast containing hT4 VL each 1.5 X 10 ⁷ and YPD (20 g / L Dextrose, 20 g / L) Wash three times with peptone, 10 g / L yeast extract, 14.7 g / L sodium citrate, 4.29 g / L citric acid, pH 4.5) (SIGMA), and then resuspend with 100 μl of YPD to prevent spreading on the YPD plate. After dropping, dry and incubate at 30 ° C for 6 hours. Then, the yeast smeared area dried with YPD medium was washed three times, and then cultured in selective medium SD-CAA medium at 24 ° C. for 24 hours so as to have a final yeast concentration of 1 × 10 ⁶ or less. SG-CAA medium was used to induce the expression of humanized antibody Fab fragments in the selected yeast, and the cells were suspended in second and third FACS by competing with 100 times GDP-linked KRas G12D at 100 nM concentration of KRas G12D.

FIG. 16 is a flow cytometric analysis of binding ability of GTP-coupled KRas G12D alone and competitive conditions with GDP-coupled KRas G12D in the selection step to obtain specific high affinity to the above-described GTP-bound KRas G12D. Data. Through this, the heavy chain variable region (VH) -dependent library selection capable of specific binding to GTP-coupled KRas G12D was confirmed.

RT4 clones were finally selected through individual clone analysis from a library having high affinity and specificity for GTP-coupled KRas G12D protein through high-speed screening.

Tables 4 and 5 show sequence information and sequence numbers of the heavy chain variable region RT4 binding to the activated RAS. Table 4 is a table showing the entire RT4 sequence according to Kabat numbering, Table 5 is the content of the CDR sequence in the antibody sequence of Table 4 separately.

Table 4

Full sequence of heavy chain variable region RT4 binding to activated RAS.

Table 5

CDR sequence of heavy chain variable region RT4 that binds to activated RAS.

Example 16 Anti-Ras.GTP iMab Expression, Purification and Affinity Analysis with KRas Mutations

By replacing the heavy chain variable region (VH) of the cytotransmab having the characteristics of cellular infiltration and cytoplasm with RT4 VH selected in Example 13, it is possible to specifically target Ras conjugated to intracellular GTP through cellular infiltration. In order to express the anti-Ras GTP iMab in animal cells, the DNA encoding the secretory signal peptide at the 5 'end is fused as in Example 5, and the RT4 heavy chain variable region and the heavy chain constant region specifically bind to GTP-coupled KRas. DNAs encoding heavy chains containing (CH1-hinge-CH2-CH3) were cloned into NotI / HindIII in pcDNA3.4 (Invitrogen) vectors, respectively. Thereafter, transgenic transfection of HEK293F protein-expressing cells simultaneously with the animal expression vector encoding the cytoplasmic penetration light chain and the heavy-chain encoded animal expression vector comprising a heavy chain variable region that specifically binds to the constructed GTP-coupled KRas. transient transfection). After the purification of anti-Ras.GTP iMab proceeded in the same manner as in Example 5.

FIG. 17 is an analysis of 12% SDS-PAGE in reducing or non-reducing conditions after purification of anti-Ras.GTP iMab RT4.

Specifically, the molecular weight of about 150 kDa was confirmed under non-reducing conditions, and the molecular weight of the heavy chain 50 kDa and the light chain 25 kDa was shown under the reducing conditions. This shows that the expression-purified anti-Ras.GTP iMab exists as a monolith in a non-covalently removed solution, and does not form duplexes or oligomers through unnatural disulfide bonds.

FIG. 18 shows the results of ELISA for measuring affinity between GTP-bound and GDP-bound forms of wild KRas and KRas mutants KRas G12D, KRas G12V, KRas G13D.

Specifically, the target molecule GTP-coupled KRas mutant and GDP-coupled KRas mutant were bound to 96 well EIA / RIA plate (COSTAR Corning) at 37 ° C. for 1 hour, and then 0.1% TBST (0.1% Tween20, pH 7.4, Wash three times for 10 min with 137 mM NaCl, 12 mM Tris, 2.7 mM KCl, 5 mM MgCl ₂ ) (SIGMA). Combine for 1 hour with 4% TBSB (4% BSA, pH7.4, 137 mM NaCl, 12 mM Tris, 2.7 mM KCl, 10 mM MgCl ₂ ) (SIGMA) and wash three times for 10 minutes with 0.1% TBST. Then, anti-Ras.GTP iMab RT4 was diluted with 4% TBSB, bound by concentration, and washed three times for 10 minutes with 0.1% PBST. The labeling antibody binds to an alkaline phosphatase-conjugated anti-human mAb (SIGMA) conjugated with goat derived AP. 405 nm absorbance was quantified by reaction with p-nitrophenyl palmitate (pNPP) (SIGMA).

Surface plasmon resonance (SPR) was performed to more quantitatively analyze the binding capacity of anti-Ras.GTP iMab RT4 to GTP-bound KRas G12D. A Biacore2000 instrument (GE healthcare) was used.

Specifically, anti-Ras.GTP iMab RT4 was diluted in 10 mM Na-acetate buffer (pH 4.0) and fixed to about 1100 response units (RU) in a CM5 sensor chip (GE healthcare). Tris buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 5 mM MgCl ₂ , 0.005% Tween 20) was analyzed at a flow rate of 30 μl / min, and GTP-coupled KRas G12D at 1000 nM to 62.5 nM Analyzed. After binding and dissociation analysis, regeneration of CM5 chip was performed by flowing a buffer solution (10 mM NaOH, 1M NaCl, pH10.0) at a flow rate of 30 μl / min for 1.5 minutes. Each sensorgram obtained by 3 minutes of association and 3 minutes of dissociation was normalized and subtracted to compare a blank cell to calculate affinity.

19 shows the results of affinity analysis of anti-Ras.GTP iMab RT4 for GTP binding to KRAS G12D using SPR (BIACORE 2000) (GE healthcare).

Example 17. Confirmation of cytoplasmic permeability of anti-Ras.GTP iMab RT4.

20 is a result of observing with confocal microscopy to confirm the cytoplasmic penetration capacity of anti-Ras.GTP iMab RT4. The cell penetrating ability of anti-Ras.GTP iMab RT4 was observed in cell lines with KRas mutations (PANC-1, HCT116) and cell lines with KRas wild type (HT29, HeLa).

Specifically, each cell line was placed in 0.5 ml of medium containing 10% FBS at 5 × 10 ⁴ per well in a 24 well plate and incubated at 5% CO ₂ , 37 ° C for 12 hours. When the cells were stabilized, each well was diluted with 1 μM of TMab4, RT4 in 0.5 ml of fresh medium and incubated at 37 ° C., 5% CO ₂ for 6 hours. After the process was the same as the dyeing process of Example 7. Intracellular fluorescence was observed in the anti-Ras.GTP iMabs, and the cytotransmab did not lose cytoplasmic permeability even after substitution with a heavy chain variable region that specifically binds to GTP-coupled KRas.

Example 18. Cytotoxicity Assessment of Anti-Ras.GTP iMab RT4

(1) Evaluation of Adhesion Cell Growth Inhibition of Anti-RasGTP iMab

FIG. 21 shows the results of in vitro evaluation of cell growth inhibition by treatment with anti-Ras.GTP iMab RT4 in NIH3T3, NIH3T3 KRas G12V, and NIH3T3 HRas G12V cell lines.

Specifically, NIH3T3 KRas G12V artificially overexpressing the wild-type KRas mouse fibroblast cell line NIH3T3 and Ras mutations to determine whether anti-Ras.GTP iMab has specific toxicity to KRas mutation-dependent cell lines in vitro. Inhibition of adherent cell growth was evaluated by treatment of NIH3T3 HRas G12V mutant cell line and KRas G13D mutant human pancreatic cancer cell line PANC-1 with TMab4 and RT4 1 μM, respectively.

Specifically, 2 × 10 ³ cells NIH3T3, PANC-1 per well in a 24-well plate were diluted in 0.5 ml of medium containing 10% FBS, respectively, and incubated at conditions of 12 hours, 37 degrees, and 5% CO ₂ . Thereafter, 1 μM TMab4 and RT4 were treated twice for 72 hours and observed for a total of 144 hours, and then the number of living cells was counted to compare the growth of the cells.

As shown in FIG. 21, while not toxic in TMab4, RT4 inhibited cell growth only in KRas mutant cell lines (NIH3T3 KRas G12V, NIH3T3 HRas G12V), and did not show toxicity in NIH3T3 cell lines. In addition, as a result of confirming the inhibition of cell growth in the KRas G13D mutant PANC-1 cell line, TMab4 was not toxic, while RT4 showed cell growth inhibition.

(2) Evaluation of Non-adherent Cell Growth Inhibition of Anti-RasGTP iMab RT4

22 shows the results of evaluation of non-adherent cell growth inhibition in NIH3T3 HRas G12V cell line.

Specifically, in order to confirm that anti-Ras.GTP iMab causes non-adherent cell growth inhibition in KRas mutant cell line, colony formation assay was measured in NIH3T3 HRas G12V mutant cell line. Specifically, first, 0.5 ml of 2x DMEM medium and 0.5 ml of 1% agarose solution were mixed and plated on a 12 well plate and hardened with 0.5% agarose gel. Thereafter, 0.05 ml of 2x DMEM medium 0.4 ml, 0.7 ml agarose 0.5 ml, and ³ ml of NIH3T3 HRas G12V cell lines 1x10 were mixed with 0.05 ml PBS, TMab4, RT4, and Lonafarnib (20 μM), and plated on a 0.5% agarose gel. Then, on a 0.35% agarose gel with PBS, TMab4, RT4, Lonafarnib 1 μM in 0.5 ml of 1x DMEM medium was treated on a total of 21 days at 3 day intervals. After 21 days, the cells were stained with nitro-blue tetrazolium (NBT) solution, and the colony count was counted.

Similar to the results of the adherent cell growth inhibition experiment, RT4 inhibited colony formation, whereas TMab4 did not show colony formation inhibition.

As a result, it was confirmed that anti-Ras.GTP iMab RT4 specifically binds to Ras mutations in the cytoplasm and inhibits adherent and non-adherent cell growth.

Example 19 Confirmation of Specific Binding of KR- with Intra-GTP of Anti-Ras.GTP iMab RT4

FIG. 23 shows confocal microscopy results of overlapping of anti-Ras.GTP iMab RT4 with intracellular activated HRas G12V mutant. FIG. 24 shows the results of overlapping with anti-Ras.GTP iMab and KRas G12V mutants bound to intracellular GTP.

Specifically, after fibronectin (sigma) was coated on a 24-well plate, 2 × 10 ⁴ cells per well were diluted in 0.5 ml of NIH3T3 cell lines expressing mCherry (red fluorescence) HRas G12V and mCherry (red fluorescence) KRas G12V, respectively. After incubation at 37 ° C. for 5% CO ₂ , the cells were treated with 2 μM of TMab4 and RT4 for 37 ° C. for 12 hours. After staining under the same conditions as in Example 7 was observed with a confocal microscope.

As shown in Figs. 23 and 24, the green fluorescent fluorescence RT4 was superimposed on the endothelial portion where the activated fluorescence of red fluorescence was located, whereas the TMab was not superimposed.

As a result of the experiment, it was confirmed that Ras combined with intracellular GTP and anti-Ras.GTP iMab RT4 specifically bind.

Example 20. Cytotoxicity Assessment of RGD-fused Anti-Ras.GTP iMab RT4

For in vivo experimentation, it is necessary to give tumor tissue specificity. Existing cytotransmabs bind to HSPG on the cell surface and do not have any other tumor tissue specificity, and thus are unlikely to inhibit tumor specific growth in vivo. In order to improve this, RGD4C peptide (CDCRGDCFC, SEQ ID NO: 17) having specificity for integrin (Integrin αvβ3) overexpressed in neovascular cells and various tumors was genetically fused to the light chain N-terminus using one GGGGS linker. The RGD4C peptide has a higher affinity than the existing RGD peptide, enables genetic engineering fusion, and maintains a specific structure of the RGD peptide even when fused to the N-terminus (Koivunen E et al., 1995). ).

FIG. 25 shows the results of in vitro evaluation of cell growth inhibition by treatment of RGD-TMab4 and RGD-RT4 in HCT116 and PANC-1 cell lines.

In vitro, RGD to human pancreatic cancer cell lines HCT116 and KRas G12D mutations carrying the KRas G13D mutation and PANC-1, the human pancreatic cancer cell lines carrying the KRas G13D mutation, to determine whether RGD-TMab4 and RGD-RT4 itself are cytotoxic. Treatment with -TMab4 and RGD-RT4, respectively, evaluated the extent of cell growth inhibition.

Specifically, 5 × 10 ³ cells HCT116 and PANC-1 per well in a 24 well plate were diluted in 0.5 ml of a medium containing 10% FBS, respectively, and cultured at 12 hours, 37 degrees, and 5% CO ₂ . Thereafter, 1 μM of RGD-TMab4 and RGD-RT4 were treated twice for 72 hours and observed for a total of 144 hours, and then the number of living cells was counted to compare the growth of the cells.

As shown in FIG. 25, RGD-TMab4 inhibited cell growth by about 40% and 50% in HCT116 and PANC-1 cell lines, respectively, about 20% and 15% RGD-RT4, respectively. In the previous study, RGD4C peptide was about 3 times lower in affinity than integrin αvβ3 for integrin αvβ5, but integrin αvβ3 was mainly overexpressed in neovascular cells, and integrin αvβ5 was expressed in various tumor cells, resulting in HCT116. , PANC-1 cell line shows αvβ5 activity and inhibits cell adhesion (Cao L et al., 2008).

Through this, it is difficult to confirm that the TMab4 fused with the RGD4C peptide shows cytotoxicity. In addition, when RGD-TMab4 and RGD-RT4 were compared, it was indirectly confirmed that even if the RGD peptide was bound, Ras-specific cell growth could be inhibited.

Example 21. Confirmation of Tumor Growth Inhibition of RGD-fused Anti-Ras.GTP iMab

Figure 26a is an experimental result confirming the tumor growth inhibitory effect of RGD-fused anti-Ras.GTP iMab RT4 in mice transplanted with HCT116 cell line. Figure 26b is a graph measuring the weight of rats to identify non-specific side effects of RGD fused anti-Ras.GTP iMab RT4.

Specifically, on the basis of the in vitro results of Example 20, in order to confirm tumor growth inhibition of RGD-RT4 in vivo, Balx / c nude mice with HCT116, a KRas G13D mutant human colon cancer cell line, 5x10 per mouse ^Six cells were injected subcutaneously, and after about 6 days, when the tumor volume reached about 50 mm ³ , PBS, RGD-TMab4 and RGD-RT4 were intravenously injected at 20 mg / kg, respectively. A total of nine intravenous injections were made every two days, and tumor volume was measured for 18 days using a caliper.

As shown in Figure 26a, RGD-TMab4 and RGD-RT4 inhibited the growth of cancer cells compared to the control group PBS, it was confirmed that RGD-RT4 inhibits tumor growth more effectively than RGD-TMab4. In addition, as shown in Figure 26b, it was confirmed that there is no change in body weight of the RGD-RT4 experimental group mice, and accordingly confirmed that there is no other toxicity.

Claims (44)

1. A method of placing an antibody in the form of a complete immunoglobulin in the cytoplasm through a cell membrane, wherein the antibody comprises a light chain variable region (VL) having cytoplasmic penetrating ability.
2. The method of claim 1, wherein the antibody is a chimeric, human, or humanized antibody.
3. The method of claim 1, wherein the antibody is selected from the group consisting of IgG, IgM, IgA, IgD and IgE.
4. The method of claim 1, wherein the cytoplasmic penetrating ability is by endosomal escape after penetrating through cell internalization.
5. The method according to claim 1, wherein the light chain variable region is

   CDR1 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 7 and 10 or a sequence at least 90% homologous thereto; And

   And a CDR3 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 9, and 12, or a sequence at least 90% homologous thereto.
6. The method according to claim 1, wherein the light chain variable region (VL), the second and fourth amino acids from the N terminus of the light chain variable region, wherein the leucine (L) and methionine (Methionine, M), respectively.

   (However, the amino acid position is according to Kabat number.)
7. The light chain variable region of claim 1, wherein the 9, 10, 13, 17, 19, 21, 22, 42, 45, 58, 60, 79, and 85th amino acids from the N terminus of the light chain variable region, respectively.

   Serine (S), Serine (S), Alanine (A), Valine (V), Aspartic acid (D), Valine (V), Isoleucine (Isoleucine, I ), Trionine (T), lysine (Lysine, K), lysine (Lysine, K), valine (V), serine (S), glutamine (Q) and trionine (Threonine, Substituted with T).

   (However, the amino acid position is according to Kabat number.)
8. The method of claim 1, wherein the light chain variable region is where the 89th and 91th amino acids from the N terminus of the light chain variable region are substituted with Glutamine (Q) and Tyrosine (Y), respectively.

   (However, the amino acid position is according to Kabat number.)
9. The method of claim 1, wherein the light chain variable region consists of amino acids selected from the group consisting of SEQ ID NOs: 1, 2, and 3. 6.
10. The method of claim 1, wherein the antibody actively penetrates living cells.
11. The method of claim 1, wherein the antibody targets the cytoplasm, nucleus, mitochondria, endoplasmic reticulum, organelle macromolecule.
12. The method of claim 11, wherein the intracellular polymer is a protein, lipid, DNA or RNA.
13. The method of claim 12, wherein the protein is associated with control of cell growth, cell proliferation, cell cycle, DNA repair, DNA integrity, transcription, replication, translation, or intracellular migration.
14. The method of claim 12, wherein the protein is modified or activated or mutated with a phosphoric acid group, a carboxylic acid group, a methyl group, a sulfate group, a lipid, a hydroxyl group, or an amide group.
15. The method of claim 1, wherein the antibody specifically binds to activated RAS in the cytoplasm.
16. The method of claim 15, wherein the binding is by heavy chain variable region (VH) that specifically binds to activated RAS in the cytoplasm.
17. The method of claim 15, wherein the activated RAS is in mutated form.
18. The method according to claim 16, wherein the heavy chain variable region is

    The CDR1 of SEQ ID NO: 14 or an amino acid sequence having at least 90% homology with it;

    CDR2 of SEQ ID NO: 15 or an amino acid sequence having a homology of 90% or more; And

    CDR3 of SEQ ID NO: 16 or an amino acid sequence having at least 90% homology thereto.
19. The method according to claim 16, wherein the heavy chain variable region consists of the amino acid of SEQ ID NO: 13.
20. Light chain variable region (VL) that induces the full immunoglobulin form of antibodies to penetrate the cell membrane and be located in the cytoplasm.
21. The method according to claim 20, wherein the light chain variable region is

    CDR1 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 7 and 10 or a sequence at least 90% homologous thereto; And

    And a CDR3 consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 9, and 12 or a sequence at least 90% homologous thereto.
22. The light chain variable region of claim 20, wherein the light chain variable region (VL) is a second and fourth amino acid substituted with leucine (L) and methionine (M), respectively, from the N terminus of the light chain variable region. (VL).

    (However, the amino acid position is according to Kabat number.)
23. The method according to claim 20, wherein the light chain variable region is 9, 10, 13, 17, 19, 21, 22, 42, 45, 58, 60, 79, and 85th amino acid from the N terminal of the light chain variable region, respectively

    Serine (S), Serine (S), Alanine (A), Valine (V), Aspartic acid (D), Valine (V), Isoleucine (Isoleucine, I ), Trionine (T), lysine (Lysine, K), lysine (Lysine, K), valine (V), serine (S), glutamine (Q) and trionine (Threonine, Light chain variable region (VL).

    (However, the amino acid position is according to Kabat number.)
24. The light chain variable region (VL) according to claim 20, wherein the light chain variable region is substituted with glutamine (Q) and tyrosine (Y) in the 89th and 91th amino acids from the N terminus of the light chain variable region, respectively. .

    (However, the amino acid position is according to Kabat number.)
25. The light chain variable region (VL) according to claim 20, wherein the light chain variable region consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, and 3.
26. The light chain variable region (VL) according to claim 20, wherein the cytoplasmic penetration ability is due to endosomal escape after infiltrating through cell internalization.
27. An antibody comprising the light chain variable region of any one of claims 20 to 26.
28. The antibody of claim 27, wherein the antibody is located in the cytoplasm through the cell membrane.
29. The antibody of claim 27, wherein the antibody is a chimeric, human, or humanized antibody.
30. The antibody of claim 27, wherein the antibody is selected from the group consisting of IgG, IgM, IgA, IgD and IgE.
31. The antibody of claim 27, wherein the antibody targets the cytoplasm, nucleus, mitochondria, endoplasmic reticulum, organelle macromolecule.
32. The antibody of claim 31, wherein the intracellular polymer is a protein, lipid, DNA or RNA.
33. The antibody of claim 32, wherein the protein is associated with control of cell growth, cell proliferation, cell cycle, DNA repair, DNA preservation, transcription, replication, translation, or intracellular migration.
34. The antibody of claim 32, wherein the protein is modified or activated or mutated with a phosphoric acid group, a carboxylic acid group, a methyl group, a sulfate group, a lipid, a hydroxyl group, or an amide group.
35. The antibody of claim 27, wherein the antibody specifically binds to activated RAS in the cytoplasm.
36. The antibody of claim 35, wherein the binding is by heavy chain variable region (VH) that specifically binds to activated RAS in the cytoplasm.
37. The antibody of claim 35, wherein the activated RAS is in mutated form.
38. The method according to claim 36, wherein the heavy chain variable region is

    The CDR1 of SEQ ID NO: 14 or an amino acid sequence having at least 90% homology with it;

    CDR2 of SEQ ID NO: 15 or an amino acid sequence having a homology of 90% or more; And

    The CDR3 of SEQ ID NO: 16 or an amino acid sequence having 90% or more homology thereto;
39. The antibody of claim 36, wherein the heavy chain variable region consists of the amino acid of SEQ ID NO: 13.
40. A bioactive molecule selected from the group consisting of peptides, proteins, small molecule drugs, nanoparticles, and liposomes, fused to an antibody of any one of claims 27 to 39.
41. 40. A pharmaceutical composition for preventing or treating cancer, comprising a bioactive molecule selected from the group consisting of the antibody of any one of claims 27 to 39, or a peptide, a protein, a small molecule drug, a nanoparticle, and a liposome fused thereto.
42. 40. A composition for diagnosing cancer, comprising a bioactive molecule selected from the group consisting of an antibody of any one of claims 27 to 39, or a peptide, protein, small molecule drug, nanoparticle, and liposome fused thereto.
43. 27. A polynucleotide encoding the light chain variable region of any one of claims 20 to 26 or the antibody of any one of claims 27 to 39.
44. Replacing the light chain variable region of the antibody with a light chain variable region having the property of infiltrating into the living cell and located in the cytoplasm,

    A method of making an antibody that penetrates into a cell and is located in the cytoplasm.

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