KR20180129514A - Cytosol-Penetrating Antibodies and Uses Thereof - Google Patents

Abstract

The present invention relates to a cytosol-penetrating antibody and a use thereof. Specifically, the present invention relates to a light chain variable region and/or a heavy chain variable region which is positioned in a cytosol with drastically improved endosomal escape efficiency by grasping a structural mechanism causing an endosomal escape which is an escape from an endosome to a cytosol after cellular internalization through a cell membrane protein of a living cell; a cytosol penetrating antibody including the same; a method for manufacturing the same; and a use thereof. The cytosol-penetrating antibody or an antigen-binding fragment thereof penetrates into a living cell to be in cytosol without a specific external protein delivery system.

Description

Cytosol-Penetrating Antibodies and Uses Thereof < RTI ID = 0.0 >

The present invention relates to a cytoplasmic penetration antibody and a use thereof, and more particularly to a cytoplasmic cytoplasmic penetration antibody and a use thereof, and more particularly to a cytoplasmic cytoplasmic penetration antibody and a use thereof, An endosomal escape-inducing motif capable of enhancing the endosomal escape efficiency of a cytoplasmic penetration antibody (Cytotransmab) distributed in the cytoplasm, a light chain variable region and / or a heavy chain variable region comprising the same, a cytoplasmic penetration antibody comprising the same, Lt; / RTI >

General antibodies and polymer biopharmaceuticals have limitations in that they can not pass through hydrophobic membranes and can not interfere with various disease-related substances inside the cytoplasm. In addition, existing antibodies can not penetrate directly into living cells due to their large size and hydrophilicity.

Thus, most of the existing antibodies specifically target proteins or cell membrane proteins secreted extracellularly.

In addition, commercially available antibodies that specifically bind to intracellular substances used in experiments for mechanism studies such as cell growth, specific inhibition, etc., can not be directly treated on living cells, and in order to bind with intracellular substances, A pretreatment process is required to form perforations in the cell membrane through permeabilization of the cell membrane using saponin, which is a glycoside.

A number of therapeutic antibodies have been developed which target proteins secreted into cell membranes or extracellular regions by their properties of binding to the target protein with high specificity and high affinity. Antibodies that target cell membrane proteins can bind to cell membrane proteins and then enter the cells through the endosomal pathway through receptor-mediated endocytosis of membrane receptor proteins.

This process first passes through various pathways after the early endosome process. 1) Most antibodies go through the endosomes in late endosomes, go to lysosomes and go to pathways that are completely destroyed by acidic atmospheric conditions and proteolytic enzymes, and 2) The antibody binds to FcRn (neonatal Fc receptor) receptors under acidic conditions in the initial endosomes and can go through the recycling endosome pathway and back out of the cell.

Therefore, most of the antibodies are strongly bound to the target membrane protein and are mostly destroyed through the lysosome pathway. As the endosomes mature in the endosomal pathway, acidification occurs gradually by a proton pump. The pH of the endosomes is 5.5-6.5, the endosomal pH is 4.5-5.5, the pH of the lysosomes is pH (Quadir MA et al., 2014; Li S et al., 2014), many protein hydrolytic enzymes are activated inside endosomes, and externally internalized proteins are destroyed inside the endosome.

As a result, when the antibody moves along the endosomal pathway after intracellular internalization by the receptor, it is separated from the target membrane protein in order to escape from the endo- or endo-endosome before lysozyme, Should be.

It is known that viruses and toxins among substances existing in nature inside the cell actively penetrate into living cells through cell internalization (endocytosis). The endosomal escape of endosomes to the cytoplasm is essential for a substance that penetrates into cells by cell internalization to be active in the cytoplasm. Although the endosomal escape mechanism has not yet been elucidated yet, there are three hypotheses for endosomal escape mechanism.

The first hypothesis is that endomembrane-like materials such as cationic amphiphilic peptides bind to the negative dendritic cell lipid membrane, resulting in internal stress or endocytosis, Which forms a barrel-stave pore or toroidal channel (Jenssen et al., 2006) and is called a pore formation mechanism.

The second hypothesis is that the protonated sponge effect causes the endosomal opening of the endosomal substance, which causes the substance with an amine group to be protonated by the protonated amine group, (Lin and Engbersen, 2008).

The third hypothesis is that a certain motif, which is retained in the form of a hydrophilic coil in neutrality but is transformed into a hydrophobic helical structure in an acid such as endosomal, fuses with endosomes, and the motif-containing virus and toxin escape the endosomes , And a membrane fusion mechanism (Karen J. Cross et al., 2001). Although these three hypotheses have been proposed as endosome escape mechanisms after cell internalization of viral proteins and plant / bacterial derived toxin proteins, such endosome escape mechanisms have not been elucidated in antibodies.

The common phenomenon observed in endosomal escape mechanism is that endosomal escape occurs at acidic pH conditions, endosomes and lysozyme environments. In the case of a protein whose function changes depending on pH, the structure changes in accordance with pH. Although the negatively charged amino acids (aspartic acid (D), glutamic acid (E)) and hydrophobic amino acids (methionine (M), leucine (L), isoleucine (I)) do not interact at neutral pH conditions, Carboxylic acids (COO-) on the side chains of negatively charged amino acids protonate and become hydrophobic (Korte et al., 1992) and then undergo hydrophobic interaction with surrounding hydrophobic amino acids . As a result, the distance between two amino acids becomes closer, and the structure and function of the whole protein changes. The phenomenon that causes this change is called the Tanford transition (Qin et al., 1998).

For example, nitrogen transport enzyme Nitrophorin 4 has an open structure at neutral hydrogen ion concentration, but as the pH is lowered from neutral (pH 7.4) to slightly acidic (pH 6.0), the hydrophobic nature of aspartic acid and leucine (Di Russo et al., 2012). As a result of the interaction,

However, up to now, these pH-dependent structural changes have not been revealed in antibodies, particularly in antibodies that do not undergo cell-internalization.

In one example, antibody engineering modifications to induce pH-dependent antigen binding in conventional antibody techniques include introducing histidine (H) into the antigen binding site, i.e., CDRs (Complementary Determining Regions) or random mutations including histidine Lt; / RTI > (Bonvin P et al., 2015). Both methods, however, do not cause structural changes and have a limitation in that library-based screening must be preceded in order to induce pH-dependent antigen binding.

In order to increase the effect of the substance showing activity in the cytoplasm, ultimately the amount of the substance located in the cytoplasm must be increased. Therefore, studies have been made to improve the endosomal escape ability. Such studies have been mainly conducted in cell-penetrating peptides (CPP). Although some cell penetrating peptides have been reported to be located in the cytoplasm through the endosomal escape pathway, there is no detailed study of the precise endosome escape mechanism and the endosome escape efficiency is very low and only about 0.1-4% of the cellular internalized peptides It is known to be distributed in cytoplasm.

Under these technical backgrounds, the inventors of the present application have identified endosomal escape structural motifs that can penetrate into cells and enhance the endosomal escape efficiency of cytoplasmic penetration antibodies (Cytotransmab) (Choi et al., 2014) distributed in the cytoplasm , It was confirmed that a light chain or heavy chain variable region having an endosomal cleavage structure motif with improved endosomal elimination ability and an antibody including the variable region and an antigen binding fragment thereof could be developed.

Further, the inventors of the present application confirmed that it is possible to produce a cytoplasmic penetration antibody having endosomal escape ability by grafting such endosomal cleavage structure motif to other kinds of light chain or heavy chain variable region, and completed the present invention .

The information described in the Background section is intended only to improve the understanding of the background of the present invention and thus does not include information forming a prior art already known to those skilled in the art .

An object of the present invention is to provide a cytoplasmic penetration antibody or antigen-binding fragment thereof having endosomal elimination ability.

It is another object of the present invention to provide a nucleic acid encoding the antibody or antigen-binding fragment thereof.

Another object of the present invention is to provide a vector comprising the nucleic acid, a cell transformed with the vector, and a method for producing the same.

It is another object of the present invention to provide an antibody-drug conjugate comprising said antibody or antigen-binding fragment thereof.

It is another object of the present invention to provide a composition for delivering an active substance in cytoplasm containing the cytoplasmic penetration antibody or an antigen-binding fragment thereof.

It is still another object of the present invention to provide a method for producing the cytoplasmic penetration antibody or antigen-binding fragment thereof.

In order to achieve the above object, the present invention provides a light chain variable region comprising a light chain variable region and / or a heavy chain variable region comprising a sequence represented by the following general formula in CDR3,

X1-X2-X3-Z1

Here, X1-X2-X3 is an endosomal elimination ability motif,

X1-X2-X3 is selected from the group consisting of tryptophan (W), tyrosine (Y), histidine (H) and phenylalanine (F)

Z1 is selected from the group consisting of methionine (M), isoleucine (I), leucine (L), histidine (H), aspartic acid (D) and glutamic acid (E)

Wherein the light chain variable region and / or heavy chain variable region comprising Z1 undergoes a change in the characteristics of the antibody at endosomal acidic pH conditions,

Wherein the antibody exhibits an ability to escape from the endosomes to the cytoplasm through a change in the characteristics of the antibody, or an antigen-binding fragment thereof.

In the cytoplasmic penetration antibody or antigen-binding fragment thereof according to the present invention, the first amino acid of the light chain variable region and / or the heavy chain variable region is asparaginic acid (D) or glutamic acid (E).

The present invention also provides a nucleic acid encoding a nucleic acid encoding said cytoplasmic penetration antibody or antigen-binding fragment thereof.

The present invention also provides a vector comprising said nucleic acid.

The present invention also provides a cell transformed with said vector.

The present invention also provides a composition for delivering an active substance in cytoplasm, comprising the cytoplasmic penetration antibody or an antigen-binding fragment thereof.

The present invention also relates to the use of the X1-X2-X3 endo-escape motif (X1-X2-X3 as tryptophan (W), tyrosine (Y), histidine (H) and phenylalanine (F) To CDR3 of the cytosol infiltrating antibody or antigen-binding fragment thereof, comprising the step of grafting the antibody to CDR3.

Since the cytoplasmic penetration antibody or antigen-binding fragment thereof comprising the light chain variable region and / or the heavy chain variable region comprising the endosomal escape motif according to the present invention is located in the cytoplasm through living cell infiltration, ultimately, without a special external protein delivery system The antibody or antigen-binding fragment thereof may penetrate into living cells and be distributed to the cytoplasm.

The cytoplasmic penetration antibodies or antigen-binding fragments thereof according to the present invention may be used in combination with a variety of human light chain variable region or heavy chain variable region such as a cytoplasmic penetration antibody comprising a light chain variable region or a heavy chain variable region located in the cytoplasm Or an antigen-binding fragment thereof, penetrates into cells and is distributed in the cytoplasm, and does not show nonspecific cytotoxicity to cells.

Based on the endosomal escape mechanism of the high-efficiency cytoplasmic penetration antibody or its antigen-binding fragment according to the present invention, an antibody library and a mutant design for enhancing endosomal escape ability can be performed.

The endosomal escape motif contained in the cytoplasmic penetration antibody or antigen-binding fragment thereof according to the present invention may be introduced into other antibodies to provide the endosomal escape ability.

In addition, the cytoplasmic penetration antibody or antigen-binding fragment thereof according to the present invention can be utilized as a carrier for transferring the active material into the cytoplasm of living cells, and can be utilized as a pharmaceutical composition for the treatment and prevention of diseases.

FIG. 1 shows the results of a pulse-chase and observing by confocal microscopy in order to observe the transport process and stability of cytotransmab TMab4 or TAT penetrated into cells. Results.
FIG. 2A shows the result of observing the cytoplasmic permeability according to presence or absence of the inhibitor of the cytoplasmic penetration antibody TMab4 or the cytotoxic peptide TAT by a confocal microscope.
2B is a histogram of FITC (green fluorescence) fluorescence of the confocal microscope photograph of FIG. 2A.
FIG. 2C shows the result of observing the migration of the cytoplasmic permeabilizing antibody TMab4 or cytotoxic peptide TAT to the cytoplasm according to the presence or absence of the inhibitor using a calcein using a confocal microscope.
FIG. 2 (d) is a histogram of the calcein fluorescence of the confocal microscope photograph of FIG. 2 (c).
FIG. 3A shows Western blotting of inhibition of heparanase expression by siRNA (short interfering RNA).
FIG. 3B shows the result of observing the superposition of the cytoplasmic penetration antibody and the lysosome by confocal microscopy to suppress the expression of heparanase.
FIG. 3C shows the result of observing the migration of the cytoplasmic penetration antibody to the cytoplasm using a confocal microscope as a result of inhibiting the expression of heparanase.
FIG. 4 is an overall schematic diagram showing the overall trafficking process until the cytoplasmic penetration antibody is intracellularized and located in the cytoplasm.
FIG. 5 is a result of confirming the antibody directly by fluorescence labeling in a Ramos cell line through a confocal microscope to determine whether a cytoplasmic penetration antibody can be passed through the cell membrane according to pH and induce cell membrane passage of another substance.
FIG. 6A is a result of confirming the ability of trypan blue, which has no transmembrane ability by cytoplasmic penetration antibody according to pH, to be obtained by perforation or by optical microscope in Ramos cell line.
6B is a graph showing a quantitative comparison of the number of cells acquiring a trypan blue.
FIG. 7A is a result of confirming the cell membrane perforation produced by the cytoplasmic penetration antibody under the condition of pH 5.5 by transient, reversible phenomenon or optical microscope.
FIG. 7B is a graph showing a quantitative comparison of the number of cells acquiring a trypan blue.
FIG. 8 shows the results of analysis of the cell membrane binding of the cytoplasmic penetration antibody and the control antibody, adalimumab, by a flow cytometer (FACS).
Fig. 9 shows the results of analysis of the cell membrane lipid flip-flap inducing ability of the cytoplasmic penetration antibody according to the pH and the control antibody anti-dalmatum by the flow cytometer (FACS).
10 is a schematic diagram showing a perforation formation model of a cytoplasmic penetration antibody expected through the above experiments.
Figure 11 is a diagram showing the amino acids exposed to structural changes and the structural changes due to structural change of cytoplasmic penetration antibody according to pH based on the WAM modeling structure of the light chain variable region (VL) of the cytoplasmic penetration antibody .
FIG. 12 is a graph showing the results of a comparison between the first amino acid aspartic acid and the 95th amino acid methionine in the light chain variable region (VL), which induces a change in the characteristics of the cytoplasmic penetration antibody at acidic pH, according to pH by mutations in which alanine, glutamic acid, The number of cells obtained was quantitatively compared.
Figure 13 shows the number of cells that obtained trypan blue according to pH by mutations in which amino acids that are likely to be involved in endosomal elimination among the amino acids of the light chain variable region (VL) CDR3 of the cytoplasmic penetration antibody were respectively replaced with alanine As shown in Fig.
FIG. 14A is a result of confirming the cytoplasmic permeability of a mutant obtained by substituting the human germline sequence with the CDR1 and CDR2 of the cytoplasmic penetration antibody light chain variable region (VL) which binds to the HSPG receptor and plays a role of cytoplasmic penetration.
FIG. 14B shows the number of cells that obtained trypan blue according to pH by a mutation in which a human germline sequence was substituted with CDR1 and CDR2, which is a cytoplasmic penetrating antibody light chain variable region (VL) that binds to HSPG receptor and plays a role of cytoplasmic penetration. FIG.
FIG. 15A shows the results of 12% SDS-PAGE analysis under reducing or nonreducing conditions after purification of expected mutants that improve cytoplasmic penetration antibody endosomal elimination ability.
FIG. 15B shows the result of observing through the confocal microscope that the cytoplasmic penetration ability of the anticipated mutation enhancing the cytoplasmic penetration antibody endosomal elimination ability is maintained.
FIG. 16 is a graph showing quantitative comparison of the number of cells acquiring the trypan blue according to the pH by the cytoplasmic penetration antibody wild type and expected endothelialization enhancing ability mutation in accordance with pH.
17A is a result of observing the migration of the cytoplasmic penetrating antibody wild type or the endosomal escape enhancing mutant TMab4-WYW into the cytoplasm using a calixain microscope.
FIG. 17B is a histogram of calcine fluorescence quantified in the confocal microscope photograph of FIG. 17A. FIG.
FIG. 18 is a schematic diagram showing a process in which GFP fluorescence is observed due to complementary binding of an improved segmented green fluorescent split protein when the cytosolic infiltrating antibody wild type or endosomal enhancing ability mutation is located in the cytoplasm.
FIG. 19 shows the analysis of GFP11-SBP2 fused cytoplasmic penetration antibody wild type and endosomal escape enhancing mutation through purification after 12% SDS-PAGE under reducing or nonreducing conditions.
20A is a result of observing GFP fluorescence by a confocal microscope for complementary binding of an improved segmented green fluorescent protein of GFP11-SBP2 fused cytoplasmic antibody wild type and endosomal escape enhancing mutation.
FIG. 20B is a graph quantifying GFP fluorescence in the confocal microscope photograph of FIG. 20A. FIG.
FIG. 21A is a graph showing the cell membrane binding of a mutant substituted with arginine, isoleucine, and glycine, which are amino acids having opposite properties to tryptophan, using a flow cytometer (FACS).
FIG. 21B is a graph showing quantitative comparison of the numbers of cells that have obtained trypan blue according to pH by mutation substituted with arginine, isoleucine, and glycine, which are amino acids having opposite properties to tryptophan.
FIG. 21C shows the results of observing the migration of a mutant substituted with arginine, isoleucine and glycine, which are amino acids having opposite properties to tryptophan, into the cytoplasm of the cytoplasm using a calixain, and quantifying the calcein fluorescence of the confocal microscope photograph It is a bar graph.
22A is a schematic diagram illustrating the construction of an anti-tubulin cytoplasmic penetration antibody in the form of a complete IgG to confirm the activity of an endosomal export enhancing cytoplasmic penetration antibody mutation.
Figure 22b shows the results of 12% SDS-PAGE analysis under reducing or nonreducing conditions after purification of the anti-tubulin cytoplasmic penetration antibody in complete IgG form.
FIG. 22C is a result of confocal microscopy to confirm whether or not the anti-tubulin cytoplasmic penetration antibody in a complete IgG form overlaps with tubulin, a cytoskeleton located in the cytoplasm.
23A is a schematic diagram illustrating the construction of a RAS target cytoplasmic penetration antibody in the form of a complete IgG to confirm the activity of an endosomal escape-enhancing mutation.
Figure 23B shows the results of 12% SDS-PAGE analysis under reducing or nonreducing conditions after purification of RAS target cytoplasmic permeabilizing antibodies in the form of complete IgG.
FIG. 23C shows the result of performing an enzyme linked immunosorbent assay (ELISA) for measuring the affinity of the K-RAS mutant K-RAS G12D in the form in which GppNHp is bound and in the form in which GDP is combined.
FIG. 24 shows the result of confocal microscopy to confirm whether overlapping of the intracellularly activated H-RAS G12V mutation with the RAS target cytoplasmic penetration antibodies of the complete IgG type.
25A is a graph showing the quantitative comparison of the number of cells that have obtained trypan blue according to the pH of a mutant in which a first amino acid aspartic acid is substituted with various amino acids in a light chain variable region (VL) It is a graph.
FIG. 25B shows the quantitative comparison of the number of cells obtained from trippine blue according to the pH of the mutant in which the 95th amino acid methionine was substituted with various amino acids, the light chain variable region (VL) It is a graph.
Figure 26a is a graph comparing quantitatively the number of cells harboring trypan blue according to the pH of mutants designed for the purpose of inducing a further acidic pH perception change.
FIG. 26B is a bar graph showing the movement of the mutant designed to induce additional acidic pH-dependent characteristic changes with a confocal microscope using calcein and quantifying the calcein fluorescence of the confocal microscope photograph.
FIG. 27 is a graph comparing quantitatively the number of cells acquiring trypan blue according to the pH of mutants having different numbers of amino acids constituting CDR3 of the cytosol penetrating antibody light chain variable region. FIG.
28A is a schematic diagram illustrating the construction of a RAS target cytoplasmic penetration antibody in the form of a complete IgG into which an improved endosomal escape motif is introduced into an existing therapeutic antibody light chain variable region.
FIG. 28B shows fluorescence microscopic observation of the reduction or elimination of HSPG binding capacity and cytoplasmic permeability of a RAS target cytoplasmic penetration antibody of a complete IgG form into which an improved endosomal escape motif was introduced into the existing therapeutic antibody light chain variable region.
FIG. 28C is a graph showing a quantitative comparison of the number of cells that have acquired trypan blue due to acidic pH by the RAS target cytoplasmic penetration antibody of the complete IgG type into which an improved endosomal escape motif is introduced into the existing therapeutic antibody light chain variable region to be.
29A is a graph showing the relationship between the form of GPSNHp binding of the K-RAS G12D K-RAS mutant of the RAS target cytoplasmic penetration antibody in the form of a complete IgG into which an improved endosomal motif was introduced into the existing therapeutic antibody light chain variable region, And the results of ELISA for measuring the affinity in the form.
29B is a schematic diagram illustrating the construction of a RAS target cytoplasmic penetration antibody in the form of a complete IgG incorporating an improved endosomal escape motif in an RGD10 peptide fused conventional therapeutic antibody light chain variable region.
FIG. 29c shows the overlapping of the RG-like G-12V mutant with a RG-like target cell-penetrating antibody in the form of a complete IgG into which an improved endosomal escape motif was introduced into an existing therapeutic antibody light chain variable region fused with an RGD10 peptide, The result is confirmed by a microscope.
30A is a schematic diagram illustrating the construction of a cytoplasmic penetration antibody having a cytoplasmic penetration antibody light chain variable region eliminating endosomal elimination ability and an improved endosomal escape motif introduced heavy chain variable region.
FIG. 30B is a graph comparing the quantitative comparison of the number of cells acquiring trypan blue according to the pH by a cytoplasmic penetration antibody having a cytoplasmic penetration antibody light chain variable region eliminating endosomal elimination ability and an improved endosomal escape motif-introduced heavy chain variable region Graph.
FIG. 30C shows the effect of complementary binding of an improved segmented green fluorescent protein of a cytoplasmic penetration antibody with a GFP11-SBP2 fused, endosomal exporting antibody light chain variable region and an improved endosomal exporting heavy chain introduced heavy chain variable region GFP fluorescence was observed through a confocal microscope.
30D is a result of observing the migration of a cytoplasmic penetration antibody having an endosomal infiltrating antibody light chain variable region and an improved endosomal exporting motif introduced heavy chain variable region to the cytoplasm using a calcein using a confocal microscope .
31A shows the quantitative comparison of the number of cells that have obtained trypan blue according to the pH of the mutant in which the first amino acid glutamic acid having a variable heavy chain variable region (VH) Graph.
FIG. 31B shows the quantitative comparison of the number of cells that obtained trypan blue according to the pH of a mutant in which a 102nd amino acid leucine was substituted with various amino acids, which is a heavy chain variable region (VH) that induces a characteristic change of a cytoplasmic penetration antibody at an acidic pH of 5.5 It is a graph.
Figure 32A shows the quantitative comparison of the number of cells harboring trypan blue according to the pH of a complete IgG type cytoplasmic penetration antibody containing an endosomal escape motif-introduced heavy chain variable region and / or a light chain variable region substituted with three tryptophan It is a graph.
FIG. 32B shows the results of observing the cytoplasmic transfer of a complete IgG-type cytoplasmic penetration antibody containing an endosomal escape motif-introduced heavy chain variable region and / or a light chain variable region substituted by three tryptophan with a calixain using a confocal microscope Confocal microscope This is a bar graph that quantifies the calcein fluorescence of a photograph.
33A is a schematic diagram illustrating the construction of a complete IgG-type cytoplasmic penetration antibody incorporating an improved endosomal escape motif in an existing therapeutic antibody light chain variable region fused with an improved endosomal exported heavy chain variable region and an EpCAM target peptide.
Figure 33B shows the migration of the intact cytoplasmic penetration antibody to the cytoplasm of the existing therapeutic antibody light chain variable region fused with the modified endosome-evoked motif-introduced heavy chain variable region and the EpCAM target peptide, into which the improved endosomal escape motif was introduced This is a bar graph obtained by observing with a confocal microscope using Calcine and quantifying the calcein fluorescence of the confocal microscope photograph.
Figure 33c shows a schematic representation of the effect of the endogenous exocytosis motif on the existing therapeutic antibody light chain variable region fused with the EpCAM target peptide. This graph is a graph comparing quantitatively the number of cells that have acquired plate blue.
FIG. 34A is a schematic diagram illustrating the construction of a complete IgG-type cytoplasmic penetration antibody into which an improved endosomal escape motif is introduced into an existing therapeutic antibody heavy chain variable region.
FIG. 34B is a graph comparing quantitatively the number of cells acquiring trypan blue according to pH by a complete IgG-type cytoplasmic penetration antibody into which an improved endosomal escape motif was introduced into an existing therapeutic antibody heavy chain variable region.
FIG. 35 is a graph comparing quantitatively the number of cells obtained with trypan blue according to pH by a complete IgG-type cytoplasmic penetration antibody containing an aspartic acid-introduced heavy chain variable region and / or a light chain variable region.
FIG. 36A shows the result of observing the crystal of CT-59 Fab formed under the Index G1 condition with RI1000 (Rock Imager 1000; automatic protein crystal image analyzer).
36B is a structure showing a tertiary structure of CT-59 in which refinement and validation are completed using a pymol program. The first asparaginic acid (D) and the 95th methionine (M) are shown in yellow, and the amino acids 92 to 94 are shown in orange.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention, in one aspect, comprises a light chain variable region and / or a heavy chain variable region which comprises a sequence represented by the following general formula in CDR3,

X1-X2-X3-Z1

X1-X2-X3 is an endosomal elimination ability motif and each of X1-X2-X3 is selected from the group consisting of tryptophan (W), tyrosine (Y), histidine (H) and phenylalanine (F)

Z1 is selected from the group consisting of methionine (M), isoleucine (I), leucine (L), histidine (H), aspartic acid (D) and glutamic acid (E)

Wherein the light chain variable region and / or heavy chain variable region comprising Z1 undergoes a change in the characteristics of the antibody at endosomal acidic pH conditions,

Wherein the antibody exhibits an ability to escape from the endosome to the cytoplasm through a change in the characteristics of the antibody, or an antigen-binding fragment thereof.

In the present invention, " endosomal escape " may mean penetrating into living cells actively through cell internalization and then being located in the cytoplasm after leaving endosomes under acidic conditions.

In the present invention, the term " endosomal escape motif " includes a primary structure containing a specific amino acid sequence having a characteristic of inducing endosomal escape under acidic conditions, and a tertiary structure formed thereby, It can be used in combination with "motif holding endo escape". An antibody comprising a light chain variable region (VL) or heavy chain variable region (VH) comprising an "endomorphism motif" is capable of "cytoplasmic penetration" and a "cytoplasmic penetration antibody" Means that the antibody has escaped from the endosomes to the cytoplasm under acidic conditions, and can be mixed with " antibody having cytoplasmic penetration ability ".

In the present invention, Z1 constituting the endosomal escape motif X1-X2-X3-Z1 can be located at position 102 of the light chain variable region or in the amino acid position 102 of the heavy chain variable region according to Kabat numbering, (Methionine), isoleucine (I), leucine (L), aspartic acid (D) or glutamic acid (E) having a negatively charged amino acid and histidine (H) as a positively charged amino acid. (D) or glutamic acid (E) in which the first amino acid is negatively charged at the acidic pH condition of endosomes, and Z1 of the amino acid at position 102 in the light chain variable region or the amino acid at the 102nd position in the light chain variable region To induce a change in the properties of the endosomes to retain the ability to escape from the cytoplasm.

In the present invention, the first amino acid of the light chain variable region and / or the heavy chain variable region may include a cytoplasmic penetration antibody or an antigen-binding fragment thereof, which is characterized in that the first amino acid of the light chain variable region and / or the heavy chain variable region interacts with the Z1 at an endo-

Also at pH 7.4 the first amino acid interactions of the endosomal escape motif Z1 with the light chain variable region and / or the heavy chain variable region are altered as the endosomal acidic condition is changed to pH 5.5. That is, when Z1 is composed of methionine (M), isoleucine (I), leucine (L), or a negatively charged amino acid such as asparagine acid (D) or glutamic acid (E), which is a hydrophobic amino acid, the light chain variable region or the heavy chain variable region Hydrophobic interaction due to the protonation and hydrophobicity of the carboxylic acid of the side chain of amino acid which is negatively charged in the interaction with asparaginic acid (D) or glutamic acid (E), which is amino acid No. 1, .

In addition, methionine (M), isoleucine (I), leucine (L) and heavy chain variable (Z) amino acids Z1 are hydrophobic in inducing the pH dependent property change of the endosomal escape motif Z1 and the light chain variable region or the heavy chain variable region 1 amino acid, (D) or glutamic acid (E), which is a negatively charged amino acid, amino acid 1, which is the first amino acid, does not interact at neutral pH, but the hydrogenation of a negatively charged amino acid And a Tanford transition process, which is a phenomenon in which a distance between two amino acids is brought close to each other through hydrophobic interaction according to the protein structure and function.

In addition, when Z1 is composed of histidine (H), the pH changes from pH 7.4 to pH 5.5, so that the net charge of the amino acid side chain becomes positively charged. Asparaginic acid (D), the first amino acid in the light chain variable region or the heavy chain variable region, Or electrostatic interaction with glutamic acid (E).

In the examples of the present invention, in order to confirm the induction of the pH-dependent property change by the amino acid pair # 1 and # 95 of the light chain variable region, the endosomal elimination ability through the alanine substitution mutation was analyzed, and each alanine substitution mutation As a result of analyzing the endosomal elimination ability of each mutant in which the amino acid at position 95 was substituted with 20 amino acids, the endosomal elimination ability by pH was not observed. As a result, the light chain variable region 95 amino acid Showed pH-dependent endo-escape ability in mutants composed of methionine (M), leucine (L), isoleucine (I), aspartic acid (D), glutamic acid (E) and histidine (H).

Also, in the examples of the present invention, the mutation of 13 amino acids, which are mutually related to the heavy chain variable region 1 and amino acid pair 102, which are the pH-dependent characteristic change inducing amino acids discovered through the alanine substitution mutation experiment, As a result of analysis of the endosomal elimination ability, the heavy chain variable region 102 amino acid of the cytoplasmic penetration antibody according to the present invention contained methionine (M), leucine (L), isoleucine (I), aspartic acid (D), glutamic acid (E) (H), showed pH-dependent endosomal elimination.

Also, in one embodiment of the present invention, it is possible to further include an amino acid sequence represented by (a1 -...- an) (n is an integer of 1 to 10) between X3 and Z1. In one embodiment of the present invention, when an amino acid sequence further represented by (a1 -... an)) (n is an integer of 1 to 10), the length of CDR3 is increased while endosomal escape The change of the characteristic of the motif can be facilitated.

In the present invention, the endosomal escape motif comprises a light chain variable region; Heavy chain variable region; X2-X3 is a structure of X1-X2-X3-Z1 contained in the light chain variable region and the heavy chain variable region and each of X1-X2-X3 is a group consisting of tryptophan (W), tyrosine (Y), histidine (H) and phenylalanine .

In the present invention, the endosomal escape motif X1-X2-X3 is selected from the group consisting of Z1 and a light chain variable region or a heavy chain variable region amino acid 1 in response to a weakly acidic pH of the intracellular endosomes, for example, And the endotoxin excretion efficiency of the antibody whose characteristics have been changed by the interaction can be remarkably improved.

In the present invention, the endosomal escape motifs X1, X2 and X3 are hydrophilic heads of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) And amino acids that are easy to interact with at the tail.

Specifically, the average binding force between 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and the 20 amino acids was found to be higher than that of tryptophan (W), phenylalanine (F), tyrosine ), Isoleucine (I), cysteine (C), and methionine (M).

Specifically, the binding strength between the hydrophilic head of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and the 20 amino acids was determined using arginine (R), tryptophan (W), tyrosine (Y) (H), asparagine (N), glutamine (Q), lysine (K) and phenylalanine (F), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine ) Is higher in the order of tryptophan (W), phenylalanine (F), leucine (L), methionine (M), isoleucine (I), valine (V) and tyrosine (Y).

In the present invention, the amino acids constituting the endosomal escape motifs X1, X2, and X3 include tyrosine (Y) and histidine (H), which constitute the wild-type cytoplasmic penetration antibody, and 1-palmitoyl-2-oleoyl-sn (W) higher than tyrosine (Y), phenylalanine (F), and an average binding force with glycero-3-phosphatidylcholine (POPC).

In one embodiment of the present invention, amino acids which are easy to interact with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) were analyzed through literature review, and hydrophilic head and hydrophobic tail (W) endo-escape motifs X1, X2, and X3, which have the highest binding power, were analyzed. As a result, the improved cytoplasmic penetration antibody according to this nickname showed that tyrosine (X1, X2, Y), tyrosine (Y), and histidine (H).

In another embodiment, to determine whether the head or tail portion is important for interaction with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) for endosomal escape, (R), isoleucine (I), which is easy to bond with the tail portion, and glycine (G), which has a remarkably low interaction with lipid, are introduced into the motifs X1, X2 and X3, As a result of analyzing the excretion ability, endosomal escape ability of both mutants except the cytoplasmic penetration antibody introduced with tryptophan (W) according to the present invention was remarkably decreased. Thus, the interaction of the lipid hydrophilic head with the hydrophobic tail all contributes to endosome excretion.

In another embodiment, the endosomal export motif of the light chain variable region may comprise one or more, one or two tryptophan.

In order to increase the effect of the substance showing activity in the cytoplasm, ultimately the amount of the substance located in the cytoplasm must be increased. Therefore, studies have been conducted to improve the endosomal escape ability. These studies have been mainly carried out in cell-penetrating peptides (CPP), and in particular, the interaction with lipid membranes for the passage of cell lipid membranes is essential and a strategy for improving them has been introduced. For example, tryptophan has been added to the arginine-rich cell penetrating peptide between the N-terminus and the peptide.

However, this approach has not been made for antibodies. Tryptophan (W) is an amino acid that has excellent interaction between the hydrophilic head and the hydrophobic tail of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) , Interaction with the endomembranous membrane and endosomal escape may be increased.

Specifically, according to one embodiment of the present invention, the endosomal exporting motif X1-X2-X3 of the light chain variable region and / or the heavy chain variable region may comprise a sequence selected from the following group: WWH, WYW, YWW, WYH, YWH and WWW.

According to one embodiment of the present invention, the endosomal escape ability motif X1-X2-X3 of the light chain variable region and / or the heavy chain variable region is induced to interact at an endosomal acidic pH condition, .

As used herein, the term " endosolic acidic pH " refers to a range of pH 6.0 to 4.5, which meets the initial endosomal and terminal endosomal pH conditions and allows the modification of side-chain properties of aspartic acid (D) and glutamic acid It says.

CDR3 of the light chain variable region comprising the endosomal escape motif may comprise one or more sequences selected from the group consisting of SEQ ID NOS: 8-12, 51,

QQYWWHMYT (SEQ ID NO: 8);

QQYWYWMYT (SEQ ID NO: 9);

QQYYWWMYT (SEQ ID NO: 10);

QQYWYHMYT (SEQ ID NO: 11);

QQYYWHMYT (SEQ ID NO: 12)

QQYWWWMYT (SEQ ID NO: 51)

The light chain variable region comprising the endosomal escape motif may comprise, for example, a sequence of a light chain variable region selected from the group consisting of SEQ ID NOS: 1 to 5, 13 to 23, 25 to 37, 50, 60 to 64, Homologous, such as 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homologous.

X1-X2-X3 wherein each of X1-X2-X3 is selected from the group consisting of tryptophan (W), tyrosine CDR3, wherein CDR3 is selected from the group consisting of histidine (Y), histidine (H) and phenylalanine (F), wherein the Z1 of the heavy chain variable region interacts with the first amino acid at endo- Chain variable region that retains the ability to escape from the endosomes into the cytoplasm through changes.

The CDR3 of the heavy chain variable region including the motif-containing endo-escape motif may comprise one or more sequences selected from the group consisting of SEQ ID NOs: 46 to 49, 53 shown below:

GWYWMDL (SEQ ID NO: 46);

GWYWFDL (SEQ ID NO: 47);

GWYWGFDL (SEQ ID NO: 48);

YWYWMDL (SEQ ID NO: 49); And

GWWWMDL (SEQ ID NO: 53)

 The heavy chain variable region comprising the endosomal escape motif may have at least 80% homology with the sequence of the heavy chain variable region selected from the group consisting of SEQ ID NOs: 39 to 42, 52, 54 to 59, for example 85% 90%, 95%, 96%, 97%, 98%, 99% or 100%.

Further, in one embodiment of the present invention, it is possible to display the following general formula further including Z2 connected to X1.

Z2-X1-X2-X3-Z1

(Wherein Z2 is selected from the group consisting of glutamine (Q), leucine (L), and histidine (H)

When expressed as Z2-X1-X2-X3-Z1 as described above, the first amino acid of the light chain variable region and / or the heavy chain variable region interacts with the Z1 and / or Z2 to induce endosomal acidic pH- .

CDR3 of the light chain variable region comprising the endosomal escape motif may comprise the sequence of SEQ ID NO:

QHYWYWMYT (SEQ ID NO: 24)

As used herein, the term " antibody " encompasses not only the complete antibody form exhibiting target specific binding, but also antigen binding fragments of such antibodies.

A complete antibody is a structure having two full-length light chains and two full-length heavy chains, each light chain linked by a disulfide bond with a heavy chain. The heavy chain constant region has gamma (gamma), mu (mu), alpha (alpha), delta (delta) and epsilon (epsilon) types and subclasses gamma 1 (gamma 1), gamma 2 ), Gamma 4 (gamma 4), alpha 1 (alpha 1) and alpha 2 (alpha 2). The constant region of the light chain has kappa (kappa) and lambda (lambda) types.

An antigen binding fragment or an antibody fragment of an antibody refers to a fragment having an antigen binding function and includes Fab, F (ab ') 2, F (ab') 2 and Fv. Fabs in the antibody fragment have one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1) of a heavy chain. Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. The F (ab ') 2 antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Recombinant techniques for generating Fv fragments with minimal antibody fragments having only a heavy chain variable region and a light chain variable region are described in PCT International Publication Nos. WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 07086 and WO88 / 09344 Lt; / RTI > The double-chain Fv is a non-covalent linkage between the heavy chain variable region and the light chain variable region, and the single-chain Fv (scFv) is generally linked to the variable region of the heavy chain and the variable region of the light chain Is connected to the covalent bond or is directly connected at the C-terminal so that a dimer-like structure can be obtained like the double-chain Fv. Such an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, when the whole antibody is cleaved with papain, a Fab can be obtained, and when cut with pepsin, an F (ab ') 2 fragment can be obtained) It can also be produced through recombinant technology.

In one embodiment, an antibody according to the invention is in an Fv form (e.g., scFv) or it may be in the form of a complete antibody. The cytosol infiltrating antibody according to the present invention may be an IgG, IgM, IgA, IgD or IgE and may be, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA1, IgA5, Lt; RTI ID = 0.0 > IgG < / RTI > type monoclonal antibody.

In addition, the heavy chain constant region may be selected from any one of gamma (gamma), mu (mu), alpha (alpha), delta (delta) or epsilon (epsilon). The subclasses have gamma 1 (gamma 1), gamma 2 (gamma 2), gamma 3 (gamma 3), gamma 4 (gamma 4), alpha 1 (alpha 1) and alpha 2 (alpha 2). The light chain constant region may be kappa or lambda form.

As used herein, the term " heavy chain " refers to a variable region domain VH comprising an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen and a full length heavy chain comprising three constant region domains CH1, CH2 and CH3 And fragments thereof. The term " light chain " as used herein also refers to a full-length light chain comprising a variable region domain VL comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and a constant region domain CL and fragments thereof It means all.

The antibodies of the present invention can be used in combination with monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, short chain Fvs (scFV), single chain antibodies, Fab fragments, F (ab ') fragments, disulfide- And anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of such antibodies, and the like.

An " Fv " fragment is an antibody fragment that contains complete antibody recognition and binding sites. This region consists of one heavy chain variable domain and one light chain variable domain, for example, dimers substantially tightly covalently associated with scFv.

A "Fab" fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. F (ab ') 2 antibody fragments generally comprise a pair of Fab fragments covalently linked by their hinge cysteine near their carboxy ends.

A " single chain Fv " or " scFv " antibody fragment comprises the VH and VL domains of an antibody, which domains are within a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH domain and the VL domain such that the scFv can form the desired structure for antigen binding.

The monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, i. E. The same except for possible naturally occurring mutations in which individual antibodies that occupy the population may be present in minor amounts. Monoclonal antibodies are highly specific and are directed against a single antigenic site.

The non-human (e.g., murine) antibody of the "humanized" form is a chimeric antibody containing minimal sequence derived from non-human immunoglobulin. In most cases, the humanized antibody is a non-human species (donor antibody), such as a mouse, a rat, a rabbit or a non-human, having a desired specificity, affinity and ability to retain a residue from the hypervariable region of the recipient Is a human immunoglobulin (acceptor antibody) that has been replaced with a residue from the hypervariable region of the primate.

The above-mentioned "human antibody" means a molecule derived from human immunoglobulin, wherein all the amino acid sequences constituting the antibody including the complementarity determining region and the structural region are composed of human immunoglobulin.

The other chain (s) may be derived from another species or may be derived from another antibody class or subgroup, such as a portion of the heavy chain and / or light chain derived from a particular species or identical or homologous to a corresponding sequence in an antibody belonging to a particular antibody class or subclass, Quot; chimeric " antibodies (immunoglobulins) identical or homologous to the corresponding sequences in antibodies belonging to the subclass as well as fragments of said antibodies exhibiting the desired biological activity.

As used herein, a "variable domain" refers to light and heavy chain portions of an antibody molecule comprising complementary determining regions (CDRs; ie, CDR1, CDR2, and CDR3), and the amino acid sequence of the framework region (FR). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

&Quot; Complementarity determining regions "(CDRs; i.e., CDR1, CDR2, and CDR3) refer to the amino acid residues of an antibody variable domain that are necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2 and CDR3.

A "framework region" (FR) is a variable domain residue other than a CDR residue. Each variable domain typically has four FRs identified as FR1, FR2, FR3, and FR4.

In another aspect, the present invention relates to a cytoplasmic active substance-containing composition comprising a cytoplasmic penetration antibody or an antigen-binding fragment thereof.

The active substance may be in the form of being fused or bound to an antibody and the active substance may be selected from the group consisting of, for example, peptides, proteins, toxins, antibodies, antibody fragments, RNA, siRNA, DNA, small molecule drugs, nanoparticles and liposomes But is not limited thereto.

The protein may be an antibody, antibody fragment, immunoglobulin, peptide, enzyme, growth factor, cytokine, transcription factor, toxin, antigenic peptide, hormone, a protein, a protein, a protein, a protein, a signaling protein, a storage protein, a membrane protein, a transmembrane protein, an internal protein, an external protein, Protein, and the like.

The RNA or ribonucleic acid is a type of nucleic acid that forms a nucleotide chain structure based on ribose, which is a type of pentose, and has a structure in which a single helix is twisted and a part of DNA is transcribed. In one embodiment, the RNA may be selected from rRNA, mRNA, tRNA, miRNA, snRNA, snoRNA, aRNA, but is not limited thereto.

The siRNA (Small Interfering RNA) is an RNA interference substance having a small size composed of dsRNA. It plays a role of degrading by binding with mRNA having a target sequence, and as a therapeutic agent for diseases or experimentally, And is used extensively herein by utilizing the activity of inhibiting the expression of a protein that is translated by the target mRNA.

The DNA or deoxyribonucleic acid is a kind of nucleic acid. The DNA or deoxyribonucleic acid is a kind of nucleic acid and has two kinds of backbone chain in which a phosphate group is bonded to a monosaccharide deoxyribose, purine, and pyrimidine It is composed of a nucleobase and is a substance that stores genetic information of a cell.

The small molecule drug is used extensively herein to represent an organic compound, an inorganic compound or an organometallic compound having a molecular weight of less than about 1000 daltons and having activity as a therapeutic agent for diseases. The small molecule drugs used in the present invention include oligopeptides and other biomolecules having a molecular weight of less than about 1000 daltons.

The nanoparticle refers to a particle composed of materials having a diameter of 1 to 1000 nm, and the nanoparticle is a metal / metal core composed of metal nanoparticles, a metal nanoparticle core, and a metal shell surrounding the core Metal core shell composite consisting of a shell / shell composite, a metal / non-metal core shell consisting of a metal nanoparticle core and a non-metallic shell surrounding the core, or a non-metallic 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 the present invention is not limited thereto and the base metal may be silica, polystyrene, latex, But it is not limited thereto.

The liposome is comprised of one or more lipid bilayer membranes that surround the aqueous inner compartment, which can self associate. Liposomes can be specified by the membrane type and its size. Small uniamella vesicles (SUV) may have a single membrane and have diameters of 20 nm to 50 nm. Large uniamella vesicles (LUV) may have a diameter of 50 nm or greater. Oligo lamella large vesicles and multi-lamellar large vesicles can have a diameter of greater than 100 nm with multiple, generally concentric, membrane layers. Liposomes with several non-concentric membranes, the various small vesicles contained within larger vesicles, are called multivesicular vesicles.

The term " fusion " or " binding " refers to the integration of two molecules having different functions or structures, including all physical, chemical or biological methods by which the tumor penetrating peptide can bind to the protein, small molecule drug, nanoparticle or liposome Lt; / RTI > fusion. The fusion may preferably be with a linker peptide, which can relay fusion with the active substance at various positions of the antibody light chain variable region, antibody, or fragment thereof of the invention.

In another aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the cytoplasmic penetration antibody or an antigen-binding fragment thereof and a cytoplasmic active substance transferred therefrom.

It is possible to impart the property of remaining in the cytoplasm by penetrating into the cell using the above active substance without affecting the high specificity and high affinity of the antigen of the antibody. Thus, it is classified as a target substance for the treatment of diseases using the small molecule drug The present invention can be expected to have high efficacy in the treatment and diagnosis of tumors and disease-related factors which are present in the cytoplasm and constitute a complex complex interaction between a protein and a protein on a wide and flat surface.

According to one embodiment of the present invention, it is possible to selectively inhibit KRAS mutation, which is a major drug resistance-related factor of existing various tumor therapeutic agents, and anticancer activity can be expected through concurrent treatment with existing therapeutic agents.

The cancer is selected from the group consisting of squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma of the lung, peritoneal cancer, skin cancer, skin or intraocular melanoma, rectal cancer, Pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colon cancer, colon cancer, colon cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, adenocarcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, Endometrial or uterine cancer, salivary gland cancer, renal cancer, liver cancer, prostate cancer, mucin cancer, thyroid cancer, liver cancer, and head and neck cancer.

When the composition is prepared with a pharmaceutical composition for the prevention or treatment of cancer, the composition may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the composition include those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, But are not limited to, crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. 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, muscle injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration and intrathecal administration. When administered orally, the protein or peptide is extinguished and the oral composition should be formulated to coat the active agent or protect it from degradation from above. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.

The appropriate dosage of the pharmaceutical composition for prevention or treatment of cancer may be determined by factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, Can be prescribed in various ways. The preferred dose of the composition is 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 caused by angiogenesis.

The composition may be prepared in unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily practiced by those skilled in the art, or may be manufactured by inserting it into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents. In addition, the composition may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with a conventional therapeutic agent. On the other hand, since the composition includes an antibody or an antigen-binding fragment, it can be formulated as an immunoliposome. Liposomes containing antibodies can be prepared according to methods well known in the art. The immunoliposome is a lipid composition comprising phosphatidylcholine, cholesterol and polyethylene glycol-derivatized phosphatidylethanolamine and can be prepared by reverse phase evaporation. For example, a Fab 'fragment of an antibody can be conjugated to a liposome via a disulfide-replacement reaction. A chemotherapeutic agent such as doxorubicin may be further included in the liposome.

In another aspect, the present invention provides a cancer diagnostic composition comprising the cytoplasmic penetration antibody or an antigen-binding fragment thereof and a cytoplasmic active substance transmitted thereto.

Said " diagnosis " means identifying the presence or characteristic of pathophysiology. The diagnosis in the present invention is to confirm the onset and progress of cancer.

The complete immunoglobulin type antibody and fragment thereof can be combined with a phosphor for molecular imaging to diagnose cancer through imaging.

The phosphor for molecular imaging refers to all substances that generate fluorescence and preferably emits red or near-infrared fluorescence, and more preferably a phosphor having a high quanta yield. However, the present invention is not limited thereto .

The fluorescent material for molecular imaging is preferably a fluorescent substance, fluorescent protein or other image-forming substance capable of binding with the tumor-permeable peptide specifically binding to the antibody of the complete immunoglobulin form and the fragment thereof, but is not limited thereto.

The fluorescent material is preferably fluorescein, BODYPY, Trtramethylrhodamine, Alexa, Cyanine, allopicocyanine, or a derivative thereof. It does not.

The fluorescent protein is preferably a Dronpa protein, a fluorescent coloring gene (EGFP), a red fluorescent protein (DsRFP), a Cy5.5 fluorescent protein or other fluorescent protein that exhibits near infrared fluorescence, but is not limited thereto .

Other imaging materials are preferably iron oxide, radioactive isotope, etc., but are not limited thereto, and can be applied to image equipment such as MR and PET.

In another aspect, the present invention relates to a nucleic acid encoding said cytoplasmic penetration antibody or antigen-binding fragment thereof.

The nucleic acid is a polynucleotide, and the " polynucleotide " is a polymer of a deoxyribonucleotide or a ribonucleotide in the form of a single strand or a double strand. RNA genomic sequences, DNA (gDNA and cDNA) and RNA sequences transcribed therefrom, and include analogs of natural polynucleotides unless otherwise specified.

The polynucleotide includes a nucleotide sequence encoding a light chain variable region (VL) and a heavy chain variable region (VH) having improved endolepta release capability as described above, as well as a sequence complementary to the sequence. The complementary sequence includes not only perfectly complementary sequences but also substantially complementary sequences. This can be achieved, for example, under stringent conditions known in the art, for example, as SEQ ID NOS: 1 to 5, 13 to 23, 25 to 37, 50, 60 to 64 and SEQ ID NOS: 39 to 42, 52, 54 to 59 The light chain variable region (VL) having any one of the sequences selected from the group consisting of the heavy chain variable region (VH), and the sequence capable of hybridizing with the nucleotide sequence encoding the heavy chain variable region (VH).

The nucleic acid may be modified. Such modifications include addition, deletion or non-conservative substitution or conservative substitution of nucleotides. The nucleic acid encoding the amino acid sequence is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence. The above substantial identity is determined by aligning the nucleotide sequence with any other sequence as closely as possible, and analyzing the aligned sequence using algorithms commonly used in the art to obtain a sequence having at least 80% homology, At least 90% homology or at least 95% homology.

The DNA encoding the antibody can be easily separated or synthesized using conventional procedures (for example, by using an oligonucleotide probe capable of specifically binding to the DNA encoding the heavy chain and the light chain of the antibody).

In another aspect, the present invention relates to a method for inhibiting the expression of X1-X2-X3 endosomal exporting motifs (each of X1-X2-X3 is selected from the group consisting of tryptophan (W), tyrosine (Y), histidine (H) and phenylalanine (F) To a CDR3 of a light chain and / or heavy chain variable region, or a method for producing an antigen-binding fragment thereof.

The heavy chain variable region (VH) was replaced with the heavy chain variable region (VH) whose endosomal elimination ability was improved by replacing the light chain variable region (VL) of the existing conventional antibody with the light chain variable region (VL) ≪ / RTI > or an antigen-binding fragment thereof.

In one embodiment, endosomal breakthrough activity penetrates into cells using an enhanced cytoplasmic penetration light chain variable region (VL) and endosomal exudate-imparted cytoplasmic penetration heavy chain variable region (VH), resulting in a complete immunoglobulin form Lt; RTI ID = 0.0 > of: < / RTI >

The light chain variable region (VL) and the light chain constant region (VL) are substituted with the light chain variable region (VL) and the light chain variable region (VL) Cloning a nucleic acid in which a region (VH) is substituted with a heavy chain variable region (VH) to which endosomal elimination capability is imparted, and transforming and expressing the vector in a host cell; And recovering the expressed antibody or antigen-binding fragment.

Through this, it is possible to produce a complete immunoglobulin type antibody having endocytotic capability and improved cytoplasmic permeability. In addition, a vector expressing a heavy chain including a heavy chain variable region capable of recognizing a specific protein in the cell is transformed to transform the vector into an intracellular protein and express an antibody capable of binding to a specific protein distributed in the cytoplasm .

In the present invention, the vector may be a vector system in which a light chain and a heavy chain are simultaneously expressed in one vector, or a system in which each of them is expressed in a separate vector. In the latter case, both vectors can be introduced into host cells via co-transfomation and targeted transformation.

As used herein, the term " vector " means 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, retroviral vectors, and adeno-associated viral vectors. The vector that can be used as the recombinant vector may be a plasmid (for example, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14 , pGEX series, pET series and pUC19), phage (e.g.,? gt4? B,? -charon,?? z1 and M13) or viruses (e.g. CMV, SV40 and the like).

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

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

The recombinant vector may be constructed with prokaryotic or eukaryotic cells as hosts. For example, when the vector used is an expression vector and a prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (for example, pL? Promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.) , A ribosome binding site for initiation of translation, and a transcription / translation termination sequence. When the eukaryotic cell is used as a host, the origin of replication that functions in the eukaryotic cells contained in the vector is f1 replication origin, SV40 replication origin, pMB1 replication origin, adeno replication origin, AAV replication origin, CMV replication origin, and BBV replication origin But is not limited thereto. Also, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, The cytomegalovirus (CMV) promoter and the tk promoter of HSV) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

Yet another aspect of the present invention is to provide a host cell transformed with said recombinant vector.

The host cell can be any host cell known in the art and includes, for example, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X 1776, E. coli W3110, Bacillus subtilis, Bacillus strain, and Enterobacteriaceae such as Salmonella typhimurium, Serratia marcesensus and various Pseudomonas spp. When transformed into a cell, Saccharomyce cerevisiae, insect cells, plant cells and animal cells such as SP2 / 0, CHO (Chinese hamster ovary) K1, CHO DG44, PER.C6, W138 , BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN and MDCK cell lines.

Yet another aspect of the present invention can provide a method for producing a complete immunoglobulin type antibody that penetrates into a cell and is located in the cytoplasm, including the step of culturing the host cell.

The insertion of the recombinant vector into a host cell can be carried out by using an insertion method well known in the art. For example, when the host cell is a prokaryotic cell, the CaCl ₂ method or the electroporation method can be used. When the host cell is a eukaryotic cell, the microinjection method, the calcium phosphate precipitation method, the electroporation method, Liposome-mediated transfection methods, and gene bombardment, but the present invention is not limited thereto. When E. coli and other microorganisms are used, the productivity is higher than that of animal cells, but it is not suitable for the production of intact Ig-type antibody due to glycosylation problem. However, since the antigen binding fragments such as Fab and Fv It can be used for production.

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

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example  1. cytosolic infiltration antibody ( Cytotransmab ) ≪ / RTI > expression and purification.

The cytoplasmic penetration antibody was purified for use in development to identify and enhance the endosomal escape mechanism of the cytoplasmic penetration antibody.

Specifically, in order to construct a heavy chain expression vector for production in the form of a monoclonal antibody in the form of a complete IgG, a heavy chain variable region (humanized hT0 VH: SEQ ID NO: 38) of an antibody fused with DNA encoding a secretion signal peptide at the 5 ' ) And heavy chain constant region (CH1-hinge-CH2-CH3) were cloned into NotI / HindIII vector in pcDNA3.4 (Invitrogen) vector, respectively.

In order to construct a vector expressing a light chain, a light chain comprising a cytoplasmic penetration light chain variable region (hT4 VL: SEQ ID NO: 65) and a light chain constant region (CL) fused with a DNA encoding a secretion signal peptide at the 5 ' The coding DNA was cloned into NotI / HindIII in pcDNA3.4 (Invitrogen) vector, respectively.

The light chain, heavy chain expression vector was transiently transfected to express and purify the protein. In a shake flask, HEK293-F cells (Invitrogen) growing in suspension in serum-free FreeStyle 293 expression medium (Invitrogen) were transfected with a mixture of plasmid and Polyethylenimine (PEI) (Polyscience). HEK293-F cells were seeded in 100 ml of the medium at a density of 2 X 10 ⁶ cells / ml and cultured at 150 rpm and 8% CO ₂ at 200 mL transfection in a shaking flask (Corning). The appropriate heavy and light chain plasmids were diluted to a total of 250 μg (2.5 μg / ml) with 125 μg heavy chain and 125 μg light chain in 10 ml FreeStyle 293 expression medium (Invitrogen) to produce each monoclonal antibody and 750 μg PEI (7.5 μg / ml) Mixed with 10 ml of diluted culture medium and reacted at room temperature for 10 minutes. Thereafter, the reaction mixture was added to the cells inoculated with 100 ml of the previously cultured medium and cultured at 150 rpm and 8% CO ₂ for 4 hours. The remaining 100 ml of FreeStyle 293 expression medium was further added for 6 days.

The protein was purified from the cell culture supernatant collected with reference to a standard protocol. Antibodies were applied to 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 the sample was immediately neutralized with 1 M Tris buffer. The eluted antibody fractions were concentrated by dialysis and the buffer was exchanged with PBS (pH 7.4). Purified proteins were quantified using absorbance and extinction coefficient at 280 nm wavelength.

Example  2. After cell internalization of cytoplasmic penetration antibody Trafficking  Confirm.

The trafficking was confirmed until the developed cytoplasmic penetration antibody was located in the cytoplasm after cell internalization. This can be an important clue to the mechanism by which endosomes escape to locate in the cytoplasm.

FIG. 1 shows the result of observing the transport process and stability of the cytoplasmic penetration antibody TMab4 or the cell penetrating peptide TAT introduced into the cells by a pulse-chase and confocal microscopy.

Specifically, a cover slip was inserted into a 24-well plate, and 2.5 x 10 ⁴ HeLa cells per well were added to 0.5 ml of medium containing 10% FBS and cultured for 12 hours at 5% CO ₂ and 37 ° C. Once the cells were stabilized, pcDNA3.4-flag-rab11 was transiently transfected. Opti-MEM media (Gibco) was used to maximize transient transfection efficiency. 500 ng of pcDNA3.4-flag-rab11 to be transiently transfected was reacted with 2 μl of Opti-MEM media, 50 μl of Lipofectamine 2000 (Invitrogen, USA) for 20 minutes at room temperature and added to each well. After addition of 450 μl of antibiotic-free DMEM medium, the cells were cultured at 37 ° C and 5% CO ₂ for 6 hours, replaced with 500 μl of DMEM medium containing 10% FBS, and cultured for 24 hours. Subsequently, 0.5 ml of the new medium was treated with 3 μM of TMab4 for 30 minutes at 37 ° C, washed three times with PBS, and cultured at 37 ° C for 0 hours, 2 hours, and 6 hours. Then, the medium was removed, washed with PBS, and proteins attached to the cell surface were removed with a weakly acidic solution (200 mM glycine, 150 mM NaCl pH 2.5). After washing with PBS, 4% paraformaldehyde was added and the cells were fixed at 25 ° C for 10 minutes.

Then, the cells were washed with PBS, and incubated in a buffer solution containing 0.1% saponin, 0.1% sodium azide, and 1% BSA in PBS at 25 ° C for 10 minutes to form holes in the cell membrane. After washing with PBS, the mixture was reacted at 25 ° C for 1 hour with a buffer solution containing 2% BSA in PBS to inhibit nonspecific binding. Then, the cells were stained with an antibody (Sigma) that specifically recognizes human Fc to which FITC (green fluorescence) or TRITC (red fluorescence) is bound. Rab5 is anti-rab5, which labels rab5, the marker of the early endosomal. Anti-flag antibodies that mark the flag-tag of rab11, a marker of the endogenous recycling end, were reacted for 1 hour at 25 ° C and the secondary antibody with TRITC (red fluorescence) or FITC (green fluorescence) was reacted at 25 ° C for 1 hour . Endogenous endosomes and lysosomes were treated with LysoTracker Red DND-99 1 mM directly into cultured cells 30 min before cell fixation. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. Unlike TAT, TMab4 was located in the initial endosomes up to 2 hours, but was not transported to lysosomal or recycling endosomes.

Example  3. Initial Endo  Acidification Endo  Identification of effects on escape.

In order to obtain more clear evidence that the cytoplasmic penetrating antibodies according to the present invention escape the endosomes from the initial endosomes, they were tested using inhibitors.

Specifically, the inhibitor used is wortmannin, which inhibits maturation from the endosomes to the endosomes, bafilomycin, which inhibits endosomal oxidation, and brefeldin A, which inhibits endosomal vesicles and vesicles.

FIG. 2A shows the result of observing cytoplasmic infiltration ability with or without an inhibitor of the cytoplasmic penetration antibody TMab4 or the cell penetrating peptide TAT according to the present invention by a confocal microscope.

Specifically, when the cells were stabilized, HeLa cell line was prepared as in Example 2, and wortmannin 200 nM, bafilomycin 200 nM, and brefeldin A 7 μM were incubated for 30 minutes. Then, 2 μM of PBS, TMab4, and TAT was incubated at 37 ° C. for 6 hours. The cells were washed with PBS and a weak acid solution in the same manner as in Example 2, and then subjected to cell fixation, cell perforation, and blocking. TMab4 was stained with an antibody that specifically recognizes human Fc to which FITC (green fluorescence) is linked. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. In TMab4, bafilomycin - treated cells did not show green fluorescence in cytoplasm but all showed spot - like fluorescence.

FIG. 2B is a histogram of FITC (green fluorescence) fluorescence of the confocal microscope photograph of FIG. 2A.

Specifically, 20 cells were designated in each condition using Image J software (National Institutes of Health, USA), and the mean value of obtained fluorescence was plotted.

FIG. 2C shows the result of observing the migration of the cytoplasmic penetrant antibody TMab4 or cytotoxic peptide TAT according to the present invention to the cytoplasm according to the presence or absence of the inhibitor, using a calixain microscope.

Specifically, the HeLa cell line was prepared in the same manner as in Example 2, and wortmannin 200 nM, bafilomycin 200 nM, and brefeldin A 7 μM were cultured for 30 minutes in serum-free culture medium. Subsequently, 2 μM of PBS, TMab4, and 20 μM of TAT were incubated at 37 ° C. for 6 hours. After 4 hours, the wells containing PBS or antibody were treated with 150 [mu] M calcine and incubated at 37 [deg.] C for 2 hours. After washing with PBS and weakly acidic solution in the same manner as in Example 2, cells were fixed.

The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. The intracellular penetrating antibodies TMab4 and TAT according to the present invention exhibited green calcein fluorescence which was spread from the endosomes to the cytoplasm. However, in the case of TMab4, only the bafilomycin - treated cells could not observe green calcein fluorescence scattered in the cytoplasm, unlike cells treated with other inhibitors.

FIG. 2 (d) is a histogram showing the calcein fluorescence of a confocal microscope photograph. Specifically, 20 cells were designated in each condition using Image J software (National Institutes of Health, USA), and the mean value of obtained fluorescence was plotted.

Example 4. Identification of the effect of HSPG degradation on endosomal escape in early endosomes.

The cytoplasmic penetration antibody is intracellularized through binding to HSPG on the cell surface. At this time, the cells are internalized with pro-heparanase. Pro-heparanase is activated by endosomal oxidation (Gingis-Velitski et al., 2004). Activated heparanase degrades HSPG and the cytoplasmic penetration antibody can be freely present inside the endosome.

FIG. 3A shows Western blotting of inhibition of heparanase expression by siRNA (short interfering RNA).

Specifically, 1 x 10 ⁴ HeLa cells per well were added to 6-well plates in 1 ml of medium containing 10% FBS and cultured at 37 ° C in 5% CO ₂ for 12 hours. After incubation for 24 hours, siRNA is transiently transfected. 500 ng of each siRNA targeting inhibition of heparanase expression was added to each tube with 3.5 μl of Opti-MEM media (Gibco), Lipofectamine 2000 (Invitrogen, USA) for 20 minutes After reacting at room temperature, they were added to each well. After addition of 500 μl of antibiotic-free DMEM media, the cells were cultured at 37 ° C and 5% CO ₂ for 6 hours, then exchanged for 1 ml of DMEM medium containing 10% FBS, and cultured for 72 hours.

After incubation, add lysis buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% SDS, 1 mM EDTA, Inhibitor cocktail (Sigma)) to obtain cell lysates. Cell lysates were quantitated using the BCA protein assay kit (Pierce). The SDS-PAGE gel was transferred to a PVDF membrane and reacted with an antibody (SantaCruz) that recognizes heparanase and β-actin, respectively, for 2 hours at 25 ° C. Secondary antibody (SantaCruz) And then detected. The analysis was performed using ImageQuant LAS4000 mini (GE Healthcare).

FIG. 3B shows the result of observing the superposition of the cytoplasmic penetration antibody and the lysosome by confocal microscopy to suppress the expression of heparanase.

Specifically, in the same manner as in Example 2, HeLa cells inhibited the expression of heparanase and control HeLa cells were prepared, and 3 μM of TMab4 and 20 μM of FITC-TAT were treated at 37 ° C. for 30 minutes, And the cells were cultured at 37 ° C for 2 hours. The cells were washed with PBS and a weak acid solution in the same manner as in Example 2, and then subjected to cell fixation, cell perforation, and blocking.

TMab4 was stained with an antibody that specifically recognizes human Fc to which FITC (green fluorescence) is linked. LAMP-1-labeled anti-LAMP-1 (santa cruz), a lysosomal marker, was reacted at 25 ° C for 1 hour and secondary antibody to which TRITC (red fluorescence) was connected was reacted at 25 ° C for 1 hour. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. When TMab4 inhibited the expression of heparanase, the overlap with LAMP-1 was confirmed.

FIG. 3C shows the result of observing the migration of the cytoplasmic penetration antibody to the cytoplasm using a confocal microscope as a result of inhibiting the expression of heparanase.

Specifically, HeLa cells inhibiting heparanase expression and control HeLa cells were prepared in the same manner as in Example 2, and 2 μM of TMab4 and 20 μM of TAT were cultured at 37 ° C. for 6 hours. After 4 hours, the wells containing PBS or antibody were treated with 150 [mu] M calcine and incubated at 37 [deg.] C for 2 hours. After washing with PBS and weakly acidic solution in the same manner as in Example 2, cells were fixed. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. In the cells inhibited by heparanase expression, it was not possible to observe the calcein fluorescence spread to cytoplasm by TMab4.

FIG. 4 is a schematic diagram showing the overall trafficking process until the cytosol penetrating antibody according to the present invention is internalized and located in the cytoplasm.

Example  5. Cytoplasmic penetration by pH IgG  Form Monoclonal antibody  Confirmation of influx of antibody by cell membrane passage.

In order for the cytoplasmic penetration antibody of the present invention to be located in the cytoplasm after the cell is internalized, it is essential that the endosome is escaped. Until now, there has been no report on the endosomal escape of the antibody. In order to elucidate the mechanism of endosomal escape, we first experimented with the pH of endosomes.

The inner phospholipid layer and the outer phospholipid layer of the early endosomes are similar in composition (Bissig and Gruenberg, 2013) and the major constituents are 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine )to be. Thus, the outer membrane phospholipid layer of the Ramos cell line, which does not express HSPG, was identified with the inner phospholipid layer of the early endosomes.

FIG. 5 is a result of confirming the antibodies directly through fluorescence labeling in a Ramos cell line through a confocal microscope to determine whether a cytoplasmic penetration antibody can be passed through the cell membrane according to the pH and can induce cell membrane passage of another substance.

Specifically, 200 μl of a 0.01% poly-L-lysine solution was added to a 24-well plate in order to attach a cover slip and a floating cell, ramos, to the plate, followed by reaction at 25 ° C for 20 minutes. After washing with PBS, 5 x 10 ⁴ ramos cells per well were added to 0.5 ml of medium containing 10% FBS and cultured at 37 ° C for 30 minutes. Cell adhesion was confirmed and 200 μl of pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4), pH 5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5) was directly labeled with PBS and fluorescent reagent DyLight- 10 μM of TMab4, 10 μM of unlabeled TMab4, and 2 μM of the control antibody, adalimumab, directly labeled with DyLight-488, and incubated at 37 ° C for 2 hours. Adalimumab, used as a control, is a therapeutic antibody that targets an extracellular cytokine.

After washing with PBS in the same manner as in Example 2, cells were fixed. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. Fluorescence of TMab4 directly labeled with DyLight-488 was observed at pH 5.5, and green FITC fluorescence was observed in cells treated with adalimumab directly labeled with TMab4 and DyLight-488 at pH 5.5. It was confirmed that the cytoplasmic penetration antibody flows through the cell membrane at an acidic pH, and other substances can be introduced in addition to oneself.

In addition, the morphology of the cell membrane was maintained even though the material was introduced from the outside.

Example  6. pH-dependent cytoplasmic penetration of antibodies Trip plate blue  Confirmation of formation of perforation by acquisition.

As a result of the known endosomal escape mechanism, the endosomal material exits the endosome while maintaining the shape of the endosome, and the most potent endosome escape mechanism, which can be realized in a complete IgG form, is thought to be perforation formation.

The experiment of Example 5 was similarly conducted to confirm the morphology of the cell membrane when the cytoplasmic penetration antibody passed through the cell membrane.

FIG. 6A is a result of confirming the ability of obtaining trypan blue having no membrane permeability by perforation by a cytoplasmic penetrating antibody according to pH, or by observing the Ramos cell line through an optical microscope.

Specifically, 5 x 10 ⁴ ramos cells were attached to each well in the same manner as in Example 5 on a 24-well plate. Cell adhesion was checked and 200 μl of a pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) and an initial endosomal pH of pH 5.5 buffer (HBSS, 50 mM MES pH 5.5) were added to TMab4, adalimumab 1 μM and 10 μM, and incubated at 37 ° C for 2 hours. After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope.

6B is a graph showing a quantitative comparison of the number of cells acquiring a trypan blue. Specifically, the number of cells obtained from the trypan blue of all cells was counted as a percentage. A total of more than 400 cells were counted and the mean value was plotted.

In FIG. 6B, it was confirmed that trypan blue was obtained in proportion to the concentration in cells to which TMab4, a cytoprotective antibody according to the present invention, was added only under the condition of pH 5.5. In addition, it was confirmed that the cell membrane form when the cytoplasmic penetration antibody passed through the cell membrane was maintained.

Example 7. Confirmation of Transient and Reversible Perforation Formation by Cytoplasmic Penetration Antibody

In the case of peptides known to exhibit a perforation mechanism due to the existing endosomal cleavage mechanism, the alpha-helical structure of the peptide is known to form a perforation through the cell membrane.

However, the antibody did not have an alpha-helical structure, and it was almost impossible to form a perforation through the cell membrane as a whole. Therefore, it was hypothesized that the cell membrane would be recovered reversibly after the antibody had temporarily excreted the endosome by forming a perforation, and the experiment was conducted to prove this.

FIG. 7A is a result of confirming whether the cell membrane perforation produced by the cytoplasmic penetration antibody under the condition of pH 5.5 is a transient or reversible phenomenon through an optical microscope.

Specifically, 5 x 10 ⁴ ramos cells were attached to each well in the same manner as in Example 5 on a 24-well plate. 10 μM of TMab4 was added to 200 μl of pH 5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5) in order to confirm the cell adhesion and maintain the initial endo-pH of 5.5, and incubated at 37 ° C. for 2 hours. In addition, the buffer is removed and replaced with a new medium, followed by incubation for 2 hours to allow the cells to recover. After that, the plate was carefully washed with PBS, and then 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed per well and observed under a microscope.

FIG. 7B is a graph showing a quantitative comparison of the number of cells acquiring a trypan blue. A total of more than 400 cells were counted and the mean value was plotted. In FIG. 7B, in the case of TMab4-added cells having the endosomal escape function according to the present invention at pH 5.5, trypan blue accumulation was observed immediately after the addition of TMab4, but in the case of the cells having recovery time in the medium, . In other words, perforation formation by cytoplasmic penetration antibody was confirmed to be transient and reversible phenomenon.

Example  8. Complete penetration of cytoplasm by pH IgG  Form Monoclonal antibody  Membrane binding and lipid membrane flip-flop confirmation.

The perforation mechanism is a mechanism in which the perforation is formed while maintaining the overall shape of the membrane, and the endosomal material exits to the cytoplasm through the perforation. In order to form perforations, it is known that endo-phosphate interacts with the inner phospholipid layer first, and then membrane perforation is formed by the flip-flop mechanism (H. D. Herce et al., 2009)

Therefore, in order to induce endosomal escape by perforation in the early endosomes, we first tried to confirm membrane binding due to endosomal oxidation of the antibody.

FIG. 8 shows the result of analyzing the cell membrane binding of the cytoplasmic penetration antibody and the control antibody adalimumab according to the pH condition with a flow cytometer (FACS).

Specifically, 1x10 ⁵ Ramos cells are prepared for each sample. Cells were washed with PBS and incubated with pH 7.4 buffer (TBS, 2% BSA, 50 mM HEPES pH 7.4), pH 5.5 buffer (TBS, 2% BSA, 50 mM MES pH 5.5), 5 μM of TMab4 and adalimumab were added and incubated at 4 ° C for 1 hour. After washing with each pH buffer, TMab4 and adalimuma were reacted with antibodies specific for human Fc to which FITC (green fluorescence) was linked for 30 minutes at 4 ° C. After washing with PBS and analyzed by flow cytometer, it was confirmed that only TMab4 binds to the cell membrane at pH 5.5.

FIG. 9 shows the results of analysis of the cell membrane lipid flip-flap inducing ability of the cytoplasmic penetration antibody according to the pH condition and the control antibody anti-dalmatum with the flow cytometer (FACS).

Specifically, 1x10 ⁵ Ramos cells are prepared for each sample. Cells were washed with PBS and then resuspended in pH 7.4 buffer (TBS, 2% BSA, 50 mM HEPES pH 7.4) to maintain the pH at 7.4 in the cytoplasmic condition, pH 5.5 buffer TBS, 2% BSA, 50 mM MES pH 5.5), and incubated at 4 ° C for 1 hour.

After washing with each pH buffer, Annexin-V with FITC (green fluorescence) was reacted at 25 ℃ for 15 minutes. Annexin-V targets phosphatidylserine, a lipid present only in the cell membrane. Annexin-V can bind lipid to the outside only when cell membrane lipid flip-flops occur. After the reaction, the cells were washed with PBS and analyzed by flow cytometry. As a result, it was confirmed that Annexin-V binds only TMab4 at pH 5.5.

10 is a schematic diagram showing a perforation formation model of a cytoplasmic penetration antibody expected through the above experiments.

Example 9. pH-dependent property change prediction logic

It was predicted that the cytoplasmic penetration antibody according to the present invention exhibited a difference in cytoplasmic penetration characteristics depending on pH conditions, which was caused by a change in the interaction between antibody residues depending on the pH condition.

In order to prove this hypothesis, it was found that aspartic acid (D) and glutamic acid (E) in the amino acids decreased in pH from neutral pH 7.4 to pH 5.0, and thus proton was added, resulting in loss of negative charge and hydrophobicity (Korte et al , 1992).

Specifically, hydrophobic aspartic acid (D) and glutamic acid (E) formed hydrophobic interactions with methionine (M), leucine (L) and isoleucine (I), which are inherently hydrophobic amino acids . (Tan et al., 1998). In order to induce such changes in pH-dependent properties, we define the Tanford transition as a phenomenon in which surrounding amino acids are influenced by newly formed interactions to induce structural deformation (Di Russo et al., 2012).

(H), aspartic acid (D), and glutamic acid (E), which are the amino acids that can show a difference at pH 7,4 and pH 5.0 in the light chain variable region through the cytoplasmic penetration light chain variable region hT4 VL structure, The hydrophobic amino acids methionine (M), isoleucine (I) and leucine (L) were investigated.

Among these, candidate amino acids having a distance between the side chains of two amino acids were found to be less than 6-7 Å, and the first and 95th amino acid pairs from the N terminal were selected as candidate candidates for the carbon transition effect.

Among the 95th amino acid pair, the 95th amino acid is an amino acid present in VL-CDR3, which is a unique sequence of hT4 VL, a cytoplasmic penetration light chain variable region, VL-CDR3 loop structure in the rat brain.

The amino acid constituting the VL-CDR3 loop, which is a structural change induced by the amino acids # 1 and # 95 in hT4 VL, the cytoplasmic penetration light chain variable region, is a primary constituent of 1-palmitoyl- It was confirmed that tyrosine (Y), which is easy to interact with oleoyl-sn-glycero-3-phosphatidylcholine (POPC), has a very high specific gravity (Morita et al., 2011).

11 is a graph showing the amino acids involved in structural changes and the amino acids exposed by structural changes by predicting the structural change of the cytoplasmic penetration antibody according to the pH condition based on the WAM modeling structure of the light chain variable region (VL) of the cytoplasmic penetration antibody to be.

In order to confirm the induction of the pH dependent property change by the pair of amino acids # 1 and # 95 and the resulting endosomal escape, alanine (A) substitution of amino acid 1 and amino acid 95 was constructed.

Further, in order to confirm the pH-dependent change in the amino acid sequence of the first and 95 amino acid pairs and to thereby confirm the endosomal escape of the amino acid sequence, the first and 95th amino acids were substituted with glutamic acid (E), leucine (L ) Mutants were constructed.

Table 1 shows the names and sequences of mutations constructed using the overlap PCR technique.

Cloning was carried out in the same manner as in Example 1, and expression and purification were carried out in HEK293F cell line.

Example 10. Identification of pH-dependent property changes of cytoplasmic penetration antibody mutations.

FIG. 12 shows a mutation (TMab4-D1A) in which asparaginic acid (D), which is the first amino acid of light chain variable region (VL) involved in inducing structural change of cytoplasmic penetrating antibodies at acidic pH, is substituted with alanine (A) Mutants (TMab4-M95A) in which methionine (M) is substituted with alanine (A), mutants (TMab4-D1E) in which glutamic acid (E) is substituted for asparaginic acid (A) as the first amino acid, methionine (M) (TMab4-M95L) obtained by substituting leucine (L) for the presence of trypan blue in accordance with the change of pH condition.

Specifically, ramos cells were adhered to the plate in the same manner as in Example 5, and then pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) was used to maintain the cytoplasmic pH condition of 7.4, the initial endo- To maintain the pH at 5.5, 10 μM of TMab4, adalimuma, TMab4-D1A, TMab4-M95A, TMab4-D1E and TMab4-M95L was added to 200 μl of pH 5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5) And cultured for 2 hours.

After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. The number of cells that acquired trypan blue in all cells was counted as a percentage. A total of more than 400 cells were counted and the mean value was plotted.

Unlike TMab4, mutants TMab4-D1A and TMab4-M95A were found to have little trypan blue acquisition. TMab4-D1E, and TMab4-M95L showed similar trypan blue acquisitions as TMab4. That is, it was confirmed that the first amino acid and the 95th amino acid play an important role in endo escape.

Example  11. Antibodies to cytoplasm Endo Ability to escape  Exploring contributing amino acids and motifs

From the results of the experiments of the previous examples, it was confirmed that the pH dependent antibody characteristics were changed by the interaction of amino acids 1 and 95 of the cytoplasmic penetration antibody, and it was confirmed that endosomal elimination ability was induced by the change of the characteristics.

In order to confirm the endosomal escape by the change of the pH-dependent characteristics, alanine (Alanine, A) -substituted mutants were constructed for amino acids which are expected to directly interact with phospholipids among the amino acids constituting VL-CDR3.

Specifically, structural modeling revealed that mutations were made at alanine (Alanine, A) substitutions at amino acid positions 92, 93 and 94, which are expected to be exposed to the surface.

Table 2 shows the names and sequences of the mutations constructed using the overlap PCR technique.

Cloning was carried out in the same manner as in Example 1, and expression and purification were carried out in HEK293F cell line.

FIG. 13 shows the results of obtaining a trypan blue according to pH by mutants in which amino acids 92, 93 and 94 which are likely to be involved in endosomal elimination among the amino acids of CDR3 in the light chain variable region (VL) were replaced with alanine This is a graph that quantitatively compares the number of cells.

Specifically, ramos cells were adhered to the plate in the same manner as in Example 5, and then the plate was washed with a pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) to maintain the cytoplasmic pH of 7.4, To keep the pH at 5.5, 200 μl of pH 5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5) was added to buffer, TMab4, TMab4-Y91A, TMab4-Y92A, TMab4-Y93A, TMab4-H94A, TMab4- 10 [mu] M and incubated at 37 [deg.] C for 2 hours.

After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. The number of cells that acquired trypan blue in all cells was counted as a percentage. A total of more than 400 cells were counted and the mean value was plotted. TMab4-Y92A, TMab4-Y93A, and TMab4-H94A significantly decreased the trypan blue acquisition compared to TMab4. In particular, TMab4-AAA rarely had trypan blue acquisitions. On the other hand, TMab4-Y91A and TMab4-Y96A confirmed trypan blue acquisition similar to that of TMab4. In other words, it was confirmed that 92, 93 and 94 amino acids contributed greatly to endosomal escape.

Example  12. Cytoplasmic Penetration Antibody Light chain  Variable area ( VL ) CDR1 and CDR2 Endo Escape ability  Check contributions.

Previous experiments have demonstrated that light chain variable region (VL) CDR3 is involved in endosome excretion. Next, experiments were carried out to investigate the effect of (VL) CDR1 and CDR2, which are responsible for cell internalization, on endosomal escape.

The light chain variable region (VL) CDR1 and CDR2 were replaced with a CDR sequence having an amino acid sequence that does not contain the cationic patch sequence of CDR1 that has the same number of amino acids as the human germline sequence or is responsible for cellular internalization. At this time, amino acids known to be important for the stability of existing light chain variable regions were conserved.

Table 3 shows the names and sequences of the mutations constructed using gene synthesis.

Cloning was carried out in the same manner as in Example 1, and expression and purification were carried out in HEK293F cell line.

FIG. 14A shows the result of confirming the cytoplasmic infiltration ability of the mutant in which the light chain variable region (VL) CDR1 and CDR2, which bind to the HSPG receptor and play the role of cytoplasmic penetration, was substituted with the human germline sequence.

Specifically, the HeLa cell line was prepared in the same manner as in Example 2, and when cells were stabilized, 2 μM of PBS, TMab4, TMab4-01, TMab4-02, and TMab4-03 were cultured at 37 ° C for 6 hours. The cells were washed with PBS and a weak acid solution in the same manner as in Example 2, and then subjected to cell fixation, cell perforation, and blocking.

TMab4 was stained with antibodies specific for human Fc to which Alexa-488 (green fluorescence) was linked. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. In all three mutants, the intracellular fluorescence was decreased compared to the wild type TMab4, and in particular, the intracellular fluorescence was not observed in the case of TMab4-03.

FIG. 14B is a graph comparing the quantitative comparison of the number of cells obtained with trypan blue according to pH by mutation in which the light chain variable region (VL) CDR1 and CDR2 binding to the HSPG receptor and carrying the cytoplasmic permeability were replaced with the human germline sequence Graph.

Specifically, ramos cells were adhered to the plate in the same manner as in Example 5, and then the plate was washed with a pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) to maintain the cytoplasmic pH of 7.4, Buffer, TMab4, TMab4-01, TMab4-02, and TMab4-03 (10 μM) were added to 200 μl of pH 5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5) After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. The number of cells that acquired trypan blue in all cells was counted as a percentage. A total of more than 400 cells were counted and the mean value was plotted. The mutations TMab4-01 and TMab4-03 confirmed trypan blue acquisitions similar to those of TMab4. In other words, it proved that the regions responsible for cell internalization (VL-CDR1 and VL-CDR2) and the region responsible for endosome excretion (VL-CDR3) are separated in the light chain variable region.

Example  13. Antibodies to cytoplasm Endo Escape ability  Enhancement logic

The changes in VL-CDR3 characteristics due to the interaction of amino acid pair # 1 and # 95 of hT4 VL, the cytoplasmic penetration light chain variable region, lead to improved solvent accessibility for binding to phospholipids of early endosome endometrium, an early mechanism of endosomal escape The expected amino acids are amino acids 92, 93 and 94, tyrosine (Y), tyrosine (Y), and histidine (H), respectively.

These amino acids are easy to interact with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), a major component of the endosomal internal phospholipid layer.

It was confirmed that the three amino acids expected to be exposed according to the change of the pH condition were involved in the interaction with the endosomal inner phospholipid layer and the endosomal escape, and in order to improve the ratio of endosomal cytoplasmic penetration antibody, 92 , 93, and 94 amino acids were prepared.

The mutagenesis logic is based on the identification of amino acids that are easy to interact with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) (Morita et al., 2011) 92, 93, and 94 amino acids, respectively.

Specifically, the average binding force between 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and the 20 amino acids was found to be higher than that of tryptophan (W), phenylalanine (F), tyrosine ), Isoleucine (I), cysteine (C), and methionine (M).

Specifically, the binding strength between the hydrophilic head of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) and the 20 amino acids was determined using arginine (R), tryptophan (W), tyrosine (Y) (H), asparagine (N), glutamine (Q), lysine (K) and phenylalanine (F), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine ) Is higher in the order of tryptophan (W), phenylalanine (F), leucine (L), methionine (M), isoleucine (I), valine (V) and tyrosine (Y).

Based on these results, tryptophan (W) is the most easily accessible amino acid to POPC (Morita et al., 2011). Therefore, in the present invention, a strategy of replacing one or two amino acids with tryptophan (T) is adopted.

Tables 4, 5, and 6 show the light chain variable region sequences of expected mutants that enhance the endosomal elimination ability of human antibodies having cytoplasmic penetration ability. Table 4 shows the whole sequence of the human antibody light chain variable region according to the Kabat numbering. Tables 5 and 6 show the CDR1, CDR2 sequence or CDR3 sequence alone in the antibody sequence of Table 4 above.

Example 14 . Cytosolic infiltration antibody endosome Improvement of escape ability Expected expression of expected mutants Purification and confirmation of maintenance of cytoplasmic infiltration ability

To construct a vector expressing the light chain as in Example 1 at all times for the expression of the mutant animal cell which is expected to improve the cytosolic infiltrating antibody endosomal elimination ability, the cytoplasmic penetration light chain in which the DNA encoding the secretory signal peptide is fused at the 5 ' A light chain comprising a light chain variable region (hT4-WWH VL, hT4-WYW VL, hT4-YWW VL, hT4-WYH VL, hT4-YWH VL) and a light chain constant region (CL) Were cloned into pcDNA3.4 (Invitrogen) vector, NotI / HindIII, respectively.

Then, an animal expression vector coding for the humanized hT0 VH and a light chain-encoded animal vector containing the constructed endosomal enhancer enhanced expected light chain variable region was simultaneously transiently transfected into HEK293F protein expressing cells . Thereafter, improvement of cytoplasmic penetration antibody endosomal elimination ability was performed. The expected mutation was purified in the same manner as in Example 1.

FIG. 15A shows the results of 12% SDS-PAGE analysis under reducing or nonreducing conditions after purification of expected mutants that improve cytoplasmic penetration antibody endosomal elimination ability.

Specifically, the molecular weight of about 150 kDa was confirmed under non-reducing conditions and the molecular weight of the heavy chain of 50 kDa and the light chain of 25 kDa were shown under reducing conditions. This shows that the mutants expected to improve the expression of purified cytoplasmic penetration antibody endosomal elimination are monolithic in solution state and do not form dendritic cells or oligomers through non-natural disulfide bonds.

FIG. 15B shows the result of observing through the confocal microscope that the cytoplasmic penetration ability of the anticipated mutation enhancing the cytoplasmic penetration antibody endosomal elimination ability is maintained.

Specifically, when the cells were stabilized, a HeLa cell line was prepared in the same manner as in Example 2, and 2 μM of PBS, TMab4, TMab4-WWH, TMab4-WYW, TMab4-YWW, TMab4-WYH and TMab4- Lt; / RTI >

The cells were washed with PBS and a weak acid solution in the same manner as in Example 2, and then subjected to cell fixation, cell perforation, and blocking. Each antibody was stained with an antibody that specifically recognizes human Fc to which FITC (green fluorescence) is linked. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. All 5 mutants were confirmed to retain cytoplasmic permeability.

Example 15. Cellular infiltration antibody endosomes Identification of pH dependence of expectant mutants with improved escape ability .

FIG. 16 is a graph comparing quantitatively the number of cells acquiring a trypan blue according to a pH due to a cytoplasmic penetration antibody wild type and an endosomal enhancer improvement expected mutation according to pH.

Specifically, ramos cells were adhered to the plate in the same manner as in Example 5, and then the plate was washed with a pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) at a cytoplasmic pH, a pH 5.5 buffer (HBSS ), TMAB4-WYW, TMab4-YWW, TMab4-WYH, and TMab4-YWH were added to 200 쨉 l of 50 mM MES pH 5.5 for 2 hours at 37 째 C. After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. The number of cells that acquired trypan blue in all cells was counted as a percentage. A total of more than 400 cells were counted and the mean value was plotted. Among the five mutants, the rate of acquisition of the trypan blue was increased in TMab4-WYW, TMab4-YWW, TMab4-WYH, and TMab4-YWH, and TMab4-WYW was found to be pH dependent.

As the wild-type cytoplasmic permeability was maintained, TMab4-WYW with increased pH-dependent trypan blue acquisition was selected as the final clone.

Example 16. Cellular infiltration antibody endosomes Enhancement of escape ability Confirmation of cytoplasmic location of mutation .

17A is a result of observing the migration of the cytoplasmic penetrating antibody wild type or the endosomal escape enhancing mutant TMab4-WYW into the cytoplasm using a calixain microscope.

Specifically, the HeLa cell line was prepared as in Example 2, and 0.1 μM, 0.5 μM, and 1 μM of PBS, TMab4, and TMab4-WYW were cultured at 37 ° C. for 6 hours in serum-free culture medium. After 4 hours, the wells containing PBS or antibody were treated with 150 [mu] M calcine and incubated at 37 [deg.] C for 2 hours. After washing with PBS and weakly acidic solution in the same manner as in Example, cells were fixed. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. TMab4-WYW showed strong intense green color fluorescence even at lower concentrations than TMab4.

FIG. 17B is a histogram of calcein fluorescence quantified in the confocal microscope photograph of FIG. 17A. FIG.

Specifically, 20 cells were designated in each condition using Image J software (National Institutes of Health, USA), and the mean value of obtained fluorescence was plotted.

Example  17. Identification of the cytoplasmic location of cytoplasmic penetration monoclonal antibodies through complementary binding of an improved segmented green fluorescent protein

FIG. 18 is a schematic diagram illustrating a process in which GFP fluorescence is observed by complementary binding of an improved segmented green fluorescent protein when the cytoplasmic penetration antibody is located in the cytoplasm.

Specifically, we used a complementary system of an improved segmented green fluorescent protein to directly confirm that the cytoplasmic penetration antibody is located in the cytoplasm. If the green fluorescent protein GFP is divided into fragments of 1-10 and 11 fragments, the fluorescent character is removed and if the two fragments are close together, fluorescence can be recovered (Cabantous et al ., 2005).

Using these properties, GFP fragments 1-10 were expressed in the cytoplasm and GFP fragment 11 was fused to the heavy chain C-terminus of the cytoplasmic penetration antibody. In addition, to complement complementary binding between GFP fragments, streptavidin (Streptavidin) and streptavidin-binding peptide 2 (SBP2) with high affinity are fused to GFP fragments, respectively. Thus, the observation of GFP fluorescence disproves that the cytoplasmic penetration antibody is located in the cytoplasm.

Example  18. GFP11 - SBP2  Expression and purification of fused cytoplasmic penetration antibodies.

GFP11-SBP2 GFP11-SBP2 was fused genetically to the C-terminus of the heavy chain using three GGGGS linkers for the expression of animal cells in fused cytoplasmic infiltration antibodies. Then, transient transfection of HEK293F protein-expressing cells with an animal expression vector in which an expression vector encoding GFP11-SBP2 and an endogenous light chain encoding GFP11-SBP2 are expressed is simultaneously transfected into the HEK293F protein expressing cells, Respectively. Subsequently, purification of the GFP11-SBP2 fused cytoplasmic penetration antibody was carried out in the same manner as in Example 1.

FIG. 19 shows the analysis of GFP11-SBP2-fused cytoplasmic penetration antibody after purification and 12% SDS-PAGE under reducing or nonreducing conditions.

Specifically, the molecular weight of about 150 kDa was confirmed under non-reducing conditions and the molecular weight of the heavy chain of 50 kDa and the light chain of 25 kDa were shown under reducing conditions. This indicates that the expressing purified GFP11-SBP2 fused cytoplasmic penetration antibody exists as a single entity in solution and does not form a dendritic or oligomer through an unnatural disulfide bond.

Example  19. GFP11 - SBP2  Depending on the cytoplasmic location of the fused cytoplasmic infiltration antibody GFP  Check fluorescence.

20A is a result of observing GFP fluorescence by a confocal microscope for complementary binding of an improved segmented green fluorescent protein of GFP11-SBP2 fused cytoplasmic antibody wild type and endosomal escape enhancing mutation.

Specifically, a transformed HeLa cell line stably expressing SA-GFP1-10 was prepared in the same manner as in Example 2, and cells were stabilized. PBS, TMab4-GFP11-SBP2, TMab4-WYW-GFP11- 0.4, 0.6, 0.8, 1.6 and 3.2 μM were incubated at 37 ° C for 6 hours.

After washing with PBS and weakly acidic solution in the same manner as in Example 2, cells were fixed. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. TMab4-WYW showed strong GFP fluorescence even at a lower concentration than TMab4.

FIG. 20B is a graph quantifying GFP fluorescence in the confocal microscope photograph of FIG. 20A. FIG.

Specifically, 20 cells were designated in each condition using Image J software (National Institutes of Health, USA), and the mean value of obtained fluorescence was plotted.

GFP11-SBP2 fused complete IgG-like cytoplasmic penetration antibody and endosome-releasing ability located inside the cytoplasm The experiment was conducted to quantitatively show the intracellular concentration and the endosomal escape efficiency of the enhanced cytoplasmic penetration antibody.

Table 7 below is a table showing intracellular concentration and endosomal escape efficiency of GFP11-SBP2 fused complete IgG-type cytoplasmic penetration antibody and endosomal escape-enhancing cytoplasmic penetration antibody located in cytoplasm.

Example  20. Endo Escape ability  In-depth analysis of enhanced cytoplasmic infiltration antibody-lipid interaction

The light chain variable region CDR3 92 of the wild type cytoplasmic penetration antibody was substituted with tryptophan for amino acid 94, which confirmed that endosomal elimination ability was improved.

Experiments were conducted to confirm that the enhancement of endosomal outgrowth was due to improved interaction with the lipid. Tryptophan (W) is an amino acid that facilitates the interaction of the lipid hydrophilic head with the hydrophobic tail. (R) which is easy to interact with hydrophilic hair, isoleucine (I) which is easy to interact with hydrophobic tail, glycine (G) which has very weak interaction with lipid, It is possible to see whether the interaction with

To elucidate the interaction between lipid peroxidation and lipid peroxidation, a mutation was constructed in which tryptophan was substituted with arginine (R), isoleucine (I), and glycine (G), respectively.

Table 8 shows the names and sequences of the mutations constructed using the overlap PCR technique.

Cloning was carried out in the same manner as in Example 1, and expression and purification were carried out in HEK293F cell line.

Example  21. In-depth analysis of interactions between cytoplasmic penetration antibody and lipid

FIG. 21A is a graph showing the cell membrane binding of a mutant substituted with arginine, isoleucine, and glycine, which are amino acids having opposite properties to tryptophan, using a flow cytometer (FACS).

Specifically, 1x10 ⁵ Ramos cells are prepared for each sample. Cells were washed with PBS and then incubated with pH 7.4 buffer (TBS, 2% BSA, 50 mM HEPES pH 7.4) and pH 5.5 buffer (TBS, 2% BSA, 50 mM MES pH 5.5) , TMab4-WYW, TMab4-RYR, TMab4-IYI and TMab4-GYG were added and incubated at 4 ° C for 1 hour. After washing with each pH buffer, TMab4, TMab4-WYW, TMab4-RYR, TMab4-IYI and TMab4-GYG were reacted with antibodies specific for human Fc with FITC (green fluorescence) . After washing with PBS and analyzed by flow cytometer, it was confirmed that only TMab4 binds to the cell membrane at pH 5.5.

FIG. 21B is a graph comparing quantitatively the number of cells acquiring a trypan blue according to a pH condition by a mutation substituted with arginine, isoleucine and glycine, which are amino acids having opposite properties to tryptophan.

Specifically, ramos cells were adhered to the plate in the same manner as in Example 5, and then the plate was washed with a pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) at a cytoplasmic pH, a pH 5.5 buffer (HBSS , TMab4-RYR, TMab4-IYI, and TMab4-GYG were added to 200 [mu] l of 50 mM MES pH 5.5 for 2 hours at 37 [deg.] C. After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. The number of cells that acquired trypan blue in all cells was counted as a percentage. A total of more than 400 cells were counted and the mean value was plotted. TMab4-RYR, TMab4-IYI, and TMab4-GYG compared to TMab4-WYW decreased the trypan blue acquisition.

FIG. 21C shows the results of observing the migration of a mutant substituted with arginine, isoleucine and glycine, which are amino acids having opposite properties to tryptophan, into the cytoplasm of the cytoplasm using a calixain, and quantifying the calcein fluorescence of the confocal microscope photograph It is a bar graph.

Specifically, the HeLa cell line was prepared in the same manner as in Example 2, and 0.5 μM of TMab4, TMab4-WYW, TMab4-RYR, TMab4-IYI and TMab4-GYG were cultured at 37 ° C for 6 hours. After 4 hours, the wells containing PBS or antibody were treated with 150 [mu] M calcine and incubated at 37 [deg.] C for 2 hours. After washing with PBS and weakly acidic solution in the same manner as in Example 2, cells were fixed. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. In the cells treated with TMab4-RYR, TMab4-IYI, and TMab4-GYG, the green calcein fluorescence scattered in the cytoplasm was weaker than the cells treated with TMab4-WYW.

Therefore, it was confirmed that the interaction between lipid hydrophilic head and hydrophobic tail is involved in endosome excretion, and that endosomal escape ability was improved when it was substituted with tryptophan for this reason.

Example  22. Complete IgG  Lt; RTI ID = 0.0 & Tubulin  Expression of cytoplasmic infiltration antibody, purification

Endosome excretion Enhanced mutations will eventually increase the amount of antibody located in the cytoplasm, so that proteins located inside the cytoplasm can be more effectively targeted.

22A is a schematic diagram illustrating the construction of an anti-tubulin cytoplasmic penetration antibody in the form of a complete IgG to confirm the activity of an endosomal escape-enhancing mutation.

For the expression of animal cells in an anti-tubulin cytoplasmic penetration monoclonal antibody in the form of a complete IgG, as in Example 1, a DNA encoding a secretion signal peptide was fused at the 5 'end and a heavy chain variable that specifically binds to the cytoskeleton tubulin Region and the heavy chain constant region (CH1-hinge-CH2-CH3) was cloned into pcDNA3.4 (Invitrogen) vector with NotI / HindIII (Laurence et al., 2011).

Then, an animal expression vector in which an expression vector encoding the cytoplasmic penetration light chain and endosomal export enhancing cytoplasmic infiltration light chain and a heavy chain variable region that specifically binds to the constructed tubulin is encoded is expressed in HEK293F protein expressing cells At the same time, transient transfection was performed. Subsequently, the purification of the anti-tubulin cytoplasmic penetration antibody in the form of complete IgG proceeded as in Example 1.

Figure 22b shows the results of 12% SDS-PAGE analysis under reducing or nonreducing conditions after purification of anti-tubulin cytoplasmic permeabilizing antibodies in the form of complete IgG.

Specifically, the molecular weight of about 150 kDa was confirmed under non-reducing conditions and the molecular weight of the heavy chain of 50 kDa and the light chain of 25 kDa were shown under reducing conditions. This shows that the purified anti-tubulin cytoplasmic penetration antibody in the form of a complete IgG exists as a single entity in solution and does not form a duplex or an oligomer through an unnatural disulfide bond.

Example  23. Complete IgG  Lt; RTI ID = 0.0 & Tubulin  Of cytoplasmic penetration antibody Cytoskeleton Tubulin and  Identification of specific binding.

FIG. 22C shows the confluence of an anti-tubulin cytoplasmic penetrating antibody in the form of a complete IgG with tubulin, a cytoskeleton located in the cytoplasm, by confocal microscopy.

Specifically, an HeLa cell line was prepared in the same manner as in Example 2, and 3 μM of PBS, TuT4, and TuT4-WYW was added to 500 μl of a medium containing 10% FBS and cultured at 37 ° C. for 6 hours. The cells were washed with PBS and a weak acid solution in the same manner as in Example 2, and then subjected to cell fixation, cell perforation, and blocking.

Tubulin, a cytoskeleton, was reacted with anti-tubulin antibody (santa cruz) at 25 ° C for 1 hour, and secondary antibody with TRITC (red fluorescence) was reacted at 25 ° C for 1 hour. Each antibody was stained with an antibody that specifically recognizes human Fc to which FITC (green fluorescence) is linked. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope.

As shown in FIG. 22C, TuT4-WYW of green fluorescence was superimposed on the cytoplasmic portion where the tubulin of red fluorescence was located, while TuT4 was not superimposed.

Example  24. Complete IgG  Type RAS target cytoplasmic penetration antibody expression, purification and affinity analysis of K-RAS mutants.

In addition to tubulin, the cytoskeleton, experiments were conducted to determine whether proteins within other cytoplasm could be efficiently targeted.

23A is a schematic diagram illustrating the construction of a Ras-targeted cytoplasmic penetration antibody in the form of a complete IgG to confirm the activity of an endosomal escape-enhancing mutation.

For expression of animal cells of Ras-targeted cytoplasmic penetration antibody in the form of complete IgG, DNA encoding secretion signal peptide is fused at the 5 'end as in Example 5, and specific binding to K-RAS in which GTP is coupled in a heavy chain variable region- DNA encoding the heavy chain variable region (RT11 VH) and heavy chain constant region (CH1-hinge-CH2-CH3) was cloned into pcDNA3.4 (Invitrogen) vector, NotI / HindIII.

Then, a heavy chain-encoding animal expression vector comprising an expression vector encoding the cytoplasmic penetration light chain and the endosomal export enhancing cytoplasmic penetration light chain and a heavy chain variable region specifically binding to the constructed GTP-bound K-RAS Transient transfection was simultaneously performed on HEK293F protein expressing cells. Then, purification of the Ras target cytoplasmic penetration antibody in the form of complete IgG proceeded in the same manner as in Example 1.

FIG. 23B shows the result of 12% SDS-PAGE analysis under reducing or non-reducing conditions after purification of Ras target cytoplasmic penetration antibody in the form of complete IgG.

Specifically, the molecular weight of about 150 kDa was confirmed under non-reducing conditions and the molecular weight of the heavy chain of 50 kDa and the light chain of 25 kDa were shown under reducing conditions. This indicates that the purified purified full IgG form of Ras target cytoplasmic penetration antibody is present as a monolayer in the solution state and does not form a dendritic or oligomer through an unnatural disulfide bond.

FIG. 23C shows the result of performing ELISA for measuring the affinity of the K-RAS mutant K-RAS G12D in a form in which GTP is bound and GDP is combined.

Specifically, since GTP is highly hydrolyzed, it is difficult to maintain the form of K-RAS G12D in which GTP is bound. Thus, GTP-like K-RAS G12D antigens were constructed using GTPNHp, which is a non-hydrolyzable GTP analogue, so as to have a stable K-RAS structure such as GTP. Binding of the GppNHp-bound form of the target molecule K-RAS G12D to the GDP-bound form was carried out in 96 well EIA / RIA plates (COSTAR Corning) for 1 hour at 37 ° C and then washed with 0.1% Tween 20 137 mM NaCl, 12 mM phosphate, 2.7 mM KCl) (SIGMA) for 10 minutes three times. 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), rinse with 0.1% PBST for 10 minutes three times. Subsequently, the IgG-type cytoplasmic penetrating antibodies TMab4, RT11, and RT11-WYW are bound and washed with 0.1% PBST for 10 minutes three times. Conjugated anti-human mAb (SIGMA) conjugated with chlorine-derived AP as a labeling antibody. pNPP (p-nitrophenyl palmitate) (Sigma) to determine the absorbance at 405 nm.

Affinity analysis for K-RAS mutation confirmed that there was almost no difference in affinity between wild type RT11 and mutant RT11-WYW. In the case of TMab4 used as a negative control, it was not bound, and in the K-RASs bound with GDP, not all clones were bound.

Example  25. Perfect IgG  Form of anti-RAS cytotransmab  of Intracellular GTP Combined  Identification of specific binding to K-RAS.

FIG. 24 shows the confluence of the Ras-targeted cytoskeleton-penetrating antibody in the form of a complete IgG and the intracellularly-activated H-RAS G12V mutation by a confocal microscope.

Specifically, fibronectin (sigma) was coated on a 24-well plate, and NIH3T3 cell line expressing mCherry (red fluorescence) H-RAS G12V was diluted to 0.5 ml per 2 x 10 ⁴ wells, After culturing at 37 ° C and 5% CO 2, TMAB4, RT11, and RT11-WYW were treated with 2 μM and cultured at 37 ° C for 12 hours. Then, the cells were stained under the same conditions as in Example 2 and observed with a confocal microscope.

As shown in Fig. 24, green fluorescence RT11 and RT11-WYW were superimposed on the intracellular portion where the activated RAS of red fluorescence was located, whereas TMab4 was not superimposed.

As a result of the above experiment, it was confirmed that the activated RAS in the cell and the complete IgG-type Ras-targeted cytoplasmic penetration antibody were specifically bound, and the degree of overlap was in the order of RT11-WYW and RT11.

Example  26. The D1- M95  Characteristic Analysis.

In order to analyze the characteristics of the first amino acid aspartic acid (D) and the 95th amino acid methionine (M) in the light chain variable region (VL) leading to the change in the characteristics of the cytoplasmic penetration antibody according to the pH, Was replaced with glutamic acid (E), alanine (A), and arpaargin (N) present in the germline-derived sequence, and the 95th amino acid located in CDR3 was replaced with 20 amino acids to construct a mutation.

When constructing this mutation, the 87th amino acid tyrosine was substituted with phenylalanine to improve the protein expression yield, which was reduced by the improved endosomal motility. Phenylalanine is an amino acid capable of enhancing the interface between a light chain variable region and a heavy chain variable region by facilitating interaction with an amino acid having an aromatic ring located in the skeleton of the heavy chain variable region and a hydrophobic amino acid. The light chain variable region having the 87th amino acid replaced with phenylalanine and the 92-94th amino acid as the improved endosomal exporting motif WYW was designated as 'hT4-3'. Therefore, the antibody having the cytoplasmic penetration complete IgG form including the light chain variable region was named 'TMab4-3'.

Each mutation was subjected to cloning and expression and purification in an HEK293F cell line in the same manner as in Example 1.

25A is a graph showing the results of quantitative determination of the number of cells acquiring trypan blue according to the pH of a mutant in which a first amino acid aspartic acid was substituted with various amino acids in a light chain variable region (VL) involved in inducing a structural change of a cytoplasmic penetrating antibody at an acidic pH FIG.

Specifically, the adherent cell pgsD-677 cell line (1 x 10 ⁴ ) which does not express the HSPG receptor is cultured in a 24-well plate. The next day, pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) was added to maintain the pH at cytoplasmic pH conditions as in Example 5, pH 5.5 buffer (HBSS , TMab4-3 D1A, TMab4-3 D1E, and TMab4-3 D1N were added to 200 쨉 l of the culture medium (Welgene, 50 mM MES pH 5.5) and incubated at 37 째 C for 2 hours. After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. Then, carefully wash with PBS, and add 50 μl of 1% SDS (sodium dodecyl sulfate) to dissolve the cells. It was transferred to a 96-well plate and absorbance was measured at a wavelength of 590 nm.

TMab4-3 D1E showed almost similar degree of trypan blue acquisition, while TMab4-3 D1A and TMab4-3 D1N mutations decreased trypan blue acquisition.

25B is a graph showing the number of cells that have obtained trypan blue according to the pH of a mutant in which methionine, which is the 95th amino acid of the light chain variable region (VL), is involved in inducing a structural change of the cytoplasmic penetrating antibody at acidic pH, .

Specifically, pgsD-677 cells were prepared in the same manner as in Example 26, and a pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) at a cytoplasmic pH was used as in Example 5, And 1 μM of 19 mutants of TMab4-3 and TMab4-3 M95X were added to 200 μl of 5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5) and incubated at 37 ° C for 2 hours. After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. Then, carefully wash with PBS, and add 50 μl of 1% SDS (sodium dodecyl sulfate) to dissolve the cells.

It was transferred to a 96-well plate and absorbance was measured at a wavelength of 590 nm. Compared with TMab4-3, TMab4-3 M95L, M95I, and M95H showed a similar degree of trypan blue acquisition, while mutations of TMab4-3 M95A, M95S, M95V, M95G, and M95P reduced trypan blue acquisition. In addition, TMab4-3 M59D and M59E increased pH-dependent trypan blue acquisitions, while TMab4-3 M95K and M95R mutants increased trypan blue acquisition at neutral pH.

It was confirmed that the interaction between the hydrophobic amino acids having a long side chain length and the negatively charged amino acid and histidine (H) is most effective for recognizing the acidic pH and causing the structural change.

When the amino acid at position 95 of the light chain variable region is composed of methionine (M), isoleucine (I), leucine (L) or an aspartic acid (D) or glutamic acid (E) (D) or glutamic acid (E), which is the first amino acid in the variable region of the heavy chain, is a hydrophobic interaction due to the protonation and hydrophobic character of the carboxylic acid of the amino acid side chain having a certain negative charge (Di Russo et al., 2012).

In addition, when the amino acid at position 95 of the light chain variable region is composed of histidine (H), the net charge of the amino acid side chain is positively charged as the pH is changed from pH 7.4 to 5.5, and the light chain variable region or the heavy chain variable region 1 It is expected to induce endosomal escape through electrostatic interaction with the amino acid aspartic acid (D) or glutamic acid (E).

Example  27. 'Change of pH cognitive properties Induced amino acid  Introduction mutation design

In addition to the existing D1-M95, the present inventors intend to introduce amino acids that can induce endosomal elimination through interaction with pH conditions.

Through structural modeling analysis, the 90th and 91st amino acids, which can interact with the first amino acid, aspartic acid (D), were selected as possible candidates. The 90th amino acid was replaced with histidine (TMab4-3 Q90H) and the 91st amino acid was replaced with histidine (TMab4-3 Y91H) so as to be able to interact at acidic pH conditions.

In addition, the 91th amino acid, which is capable of further interaction with the second hydrophobic amino acid, was replaced with aspartic acid (TMab4-3Y91D).

In order to interact with the negatively charged first amino acid, the second amino acid is replaced with the negatively charged glutamic acid (E), and the 90th amino acid is replaced with leucine (L) to interact with the hydrophobic 95th amino acid. (TMab4-3 L2E Q90L), and the second amino acid can also be replaced with the negatively charged glutamic acid (E) to interact with the negatively charged first amino acid and interact with the hydrophobic 95th amino acid The 97th amino acid was replaced with isoleucine (I) (TMab4-3 L2E T97I).

Table 9 below shows the names and sequences of the mutations constructed using the overlap PCR technique.

Each mutation was subjected to cloning and expression and purification in an HEK293F cell line in the same manner as in Example 1.

Example  28. 'Change of pH cognitive properties Induced amino acid  Introduction mutation Endo Escape ability  Improvement confirmation.

FIG. 26A is a graph comparing quantitatively the number of cells acquiring a trypan blue according to the pH of a target mutation for inducing a change in pH-perception characteristic. FIG.

Specifically, cells were prepared in the same manner as in Example 26, and then the cells were treated with a pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) at a cytoplasmic pH, a pH 5.5 buffer (Welgene, 50 mM MES pH 5.5) were added 0.5 μl or 1 μM of seven kinds of mutants such as TMab4-3 and TMab4-3 D1E-M95L, followed by incubation at 37 ° C for 2 hours. After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. Then, carefully wash with PBS, and add 50 μl of 1% SDS (sodium dodecyl sulfate) to dissolve the cells. It was transferred to a 96-well plate and absorbance was measured at a wavelength of 590 nm.

Compared with TMab4-3, the TMab4-3 Q90H mutation showed a high trypan blue acquisition at 0.5 μM. In addition, the experiment was performed using the TMab4-3 Q90H mutation, which showed a significant difference from wild type.

FIG. 26B is a histogram showing the movement of the design mutation to the cytoplasm of the pH-dependent cognitive change-inducing mutation observed with a confocal microscope using calcein and quantifying the calcein fluorescence of the confocal microscope photograph.

Specifically, in the same manner as in Example 2, an HeLa cell line was prepared and TMab4-3, TMab4-3 Q90H 0.25, 0.5, and 1 μM were cultured at 37 ° C for 6 hours. After 4 hours, the wells containing PBS or antibody were treated with 150 [mu] M calcine and incubated at 37 [deg.] C for 2 hours. After washing with PBS and weakly acidic solution in the same manner as in Example 2, cells were fixed. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope.

Compared with cells treated with TMab4-3, TM9 cells treated with TMab4-3 Q90H exhibited enhanced green calcein fluorescence in cytoplasm. Thus, in addition to the 95th amino acid in the light chain variable region of the cytoplasmic penetration antibody, it was confirmed that the 90th amino acid interacted with the amino acid No. 1 to induce the endosomal elimination by the change of the pH-dependent interaction.

Table 10 below shows the light chain variable region CDR3 sequence of the mutant enhanced endosomal export ability by inducing additional pH perception change.

Example 29. Detection of endosomal cleavage according to CDR3 length of light chain variable region

CDR3 in the light chain variable region is composed of 9 amino acids of 85% or more. Depending on the number and composition of amino acids, the structure of the CDR3 ring differs. In the present invention, a mutation consisting of 8, 10, and 11 amino acids constituting CDR3 was constructed in order to analyze how the endo-escape ability changes according to the number of amino acids and the constitution.

Table 11 shows the names and sequences of the mutations constructed using the overlap PCR technique.

Each mutation was subjected to cloning and expression and purification in an HEK293F cell line in the same manner as in Example 1.

FIG. 27 is a graph comparing quantitatively the number of cells acquiring the trypan blue according to the pH of the mutants in which the number of amino acids constituting CDR3 in the light chain variable region were different. FIG.

Specifically, cells were prepared in the same manner as in Example 26, and then the cells were treated with a pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) at a cytoplasmic pH, a pH 5.5 buffer (Welgene), 50 mM MES pH 5.5), and 1 μM of mutants such as TMab4-3 and TMab4-3 L8-1 were added and cultured at 37 ° C for 2 hours. After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. Then, carefully wash with PBS, and add 50 μl of 1% SDS (sodium dodecyl sulfate) to dissolve the cells. It was transferred to a 96-well plate and absorbance was measured at a wavelength of 590 nm.

Compared with TMab4-3, the increase in the number of amino acids led to an increase in trypan blue acquisition at neutral pH. This is because the number of amino acids is increased and the entire CDR3 ring structure is reduced. It is considered that WYW, which is an endosomal escape motif that binds to the endosomal cell membrane, is externally exposed for endosomal escape, resulting in acquisition of a trypan blue at neutral pH .

In addition, in the above experimental results, the distance between the residues 92, 93 and 94, which affects the endosomal escape through binding with the amino acid 95 and the phospholipid, which induces the endosomal escape through the change of the pH- It was confirmed that the characteristics of endorphin escape motif were maintained.

Example  30. Conventional Therapeutic Antibodies In the light chain variable region  improved Endo Escape ability  Motif assignable logic.

Among the therapeutic antibodies currently on the market, there are many types of monoclonal antibodies that target cell surface receptors, particularly cell surface receptors in which intracellular production occurs. However, in the past, such antibodies have not been able to migrate to the cytoplasm and are then released out of the cell since they do not lose their binding with the antigen and have no endosomal elimination ability. Therefore, if endosomal elimination ability can be given to a receptor-target antibody in which intracellular cleavage occurs, it can be applied to a wider range than the present invention.

In addition, it is expected that the overall expression yield can be increased by using the stable framework of the commercially available therapeutic antibody, and it is possible to eliminate the affinity with HSPG, which is not a tumor tissue specific receptor, It has advantages.

The sequence of the light chain variable region of the receptor target antibody in which intracellular cleavage occurs and the sequence of the light chain variable region of the cytoplasmic penetration antibody are compared in order to give an improved endosomal motif. The first amino acid is negatively charged, and the CDR3 ring structure The major amino acid residues in the skeleton that can affect the amino acid sequences of the candidate light chain variable regions are summarized.

A mutation was constructed in which the CDR3 sequence of the candidate light chain variable region was replaced with the CDR3 sequence of the cytoplasmic penetration antibody.

Table 12 shows the names and sequences of the mutations constructed using the gene synthesis technique.

28A is a schematic diagram illustrating the construction of a RAS target cytoplasmic penetration antibody in the form of a complete IgG into which an improved endosomal escape motif is introduced into an existing therapeutic antibody light chain variable region.

In the same manner as in Example 1, heavy-chain-encoded animal expression vectors containing a heavy chain variable region specifically binding to GTP-bound K-RAS undergoing light chain variable region cloning were simultaneously transiently transfected into HEK293F protein expressing cells transient transfection). Then, the purification of the anti-RAS cytotransmab in the form of complete IgG proceeded as in Example 1.

Example 31. Enhanced endosomes in existing therapeutic antibody light chain variable regions Confirmation that the escape motif can be given .

FIG. 28B shows fluorescence microscopic observation of the reduction or elimination of HSPG binding capacity and cytoplasmic permeability of a RAS target cytoplasmic penetration antibody of a complete IgG form into which an improved endosomal escape motif was introduced into the existing therapeutic antibody light chain variable region.

Specifically, HeLa cell lines were prepared in the same manner as in Example 2, and when the cells were stabilized, PBS, RT11-3, RT11-Neci-WYW, RT11-Nimo-WYW, RT11-Pani-WYW, RT11- RT11-Lumr-WYW, RT11-Emib-WYW 1 [mu] M were incubated at 37 [deg.] C for 6 hours.

The cells were washed with PBS and a weak acid solution in the same manner as in Example 2, and then subjected to cell fixation, cell perforation, and blocking. Each antibody was stained with an antibody that specifically recognizes human Fc to which FITC (green fluorescence) is linked. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. No fluorescence was observed in the whole cell in the RAS target cytoplasmic penetration antibody of the complete IgG type including the monoclonal antibody backbone with six improved endosomal elimination functions.

FIG. 28C is a graph showing a quantitative comparison of the number of cells that have acquired trypan blue due to acidic pH by the RAS target cytoplasmic penetration antibody of the complete IgG type into which an improved endosomal escape motif is introduced into the existing therapeutic antibody light chain variable region to be.

Specifically, ramos cells were adhered to the plate in the same manner as in Example 5, and then the plate was washed with a pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) at a cytoplasmic pH, a pH 5.5 buffer (HBSS RTI-Nym-WYW, RT11-Pan-WYW, RT11-Pert-WYW, RT11-Lumr-WYW, RT11-Emib-WYW, 1 [mu] M and incubated at 37 [deg.] C for 2 hours. After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope.

The number of cells that acquired trypan blue in all cells was counted as a percentage. A total of more than 400 cells were counted and the mean value was plotted. Similar to RT11-3, trypan blue accumulation was observed in the case of RAS-targeted cytoplasmic penetration antibody in the form of a complete IgG containing the therapeutic antibody backbone with 5 improved endotoxin excreta except RT11-Pert.

Example  32. Improved Endo Escape ability  Of the complete IgG form of the RAS target cytoplasmic penetration antibody containing the therapeutic antibody backbone GTP Combined  Confirmation of specific binding with K-RAS.

29A is a graph showing the relationship between the form of GPSNHp binding of the K-RAS G12D K-RAS mutant of the RAS target cytoplasmic penetration antibody in the form of a complete IgG into which an improved endosomal motif was introduced into the existing therapeutic antibody light chain variable region, And the results of ELISA for measuring the affinity in the form.

Concretely, the GDP-bound form of the target molecule K-RAS G12D and GDP-bound form was bound to 96-well EIA / RIA plate (COSTAR Corning) for 1 hour at 37 ° C, and then 0.1% PBST , pH 7.4, 137 mM NaCl, 12 mM phosphate, 2.7 mM KCl) (SIGMA) for 3 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), the cells were washed 3 times with 0.1% PBST for 10 minutes. After that, RTG-type RAS target cytoplasmic penetration antibodies RT11-3, RT11-Neci-WYW, RT11-Nimo-WYW, RT11-Pani-WYW, RT11-Pert-WYW, RT11-Lumr-WYW, RT11-Emib-WYW And then washed three times with 0.1% PBST for 10 minutes. Conjugated anti-human mAb (SIGMA) conjugated with chlorine-derived AP as a labeling antibody. pNPP (p-nitrophenyl palmitate) (Sigma) to determine the absorbance at 405 nm.

In the affinity analysis for K-RAS mutation, there was a difference in affinity between RT11-3 and RT11-3 in the case of RAS-labeled cytoplasmic infiltrating antibody of complete IgG type including the therapeutic antibody framework which gave 5 improved endosomal elimination functions except RT11-Nimo Not all clones were bound to K-RASs that were unseen and combined with GDP used as a negative control.

29B is a schematic diagram illustrating the construction of a RAS target cytoplasmic penetration antibody in the form of a complete IgG incorporating an improved endosomal escape motif in an RGD10 peptide fused conventional therapeutic antibody light chain variable region.

RGD10 peptide, which is specific for integrin (Integrin αvβ3), which is overexpressed in neovascular cells and various tumors, can be introduced to enable cell infiltration because the RAS target cytoplasmic penetration antibody with improved endosomal motif introduced RAS - Genetic engineering fusion was performed using two linkers of GGGGS at the light chain N-terminus. In the case of the RGD10 peptide, it has an affinity similar to that of the RGD4C peptide, but only one disulfide bond is formed by two cysteines and is characterized by the possibility of genetic engineering fusion.

Based on the results of the analysis of the expression yield, endosomal elimination ability, and affinity for Ras, the RGD10 peptide was fused to the N-terminus of the light chain variable region of RT11-Pani-WYW and RT11-Neci-WYW, Respectively.

FIG. 29c shows the overlapping of the RG-like G-12V mutant with a RG-like target cell-penetrating antibody in the form of a complete IgG into which an improved endosomal escape motif was introduced into an existing therapeutic antibody light chain variable region fused with an RGD10 peptide, The result is confirmed by a microscope.

Specifically, the SW480 cell line, a human colorectal cancer cell line having a K-RAS G12V mutation, was diluted in 0.5 ml of 2x10 ⁴ cells per well of a 24-well plate and cultured at 37 ° C and 5% CO ₂ for 12 hours RT-11-i3, RT11-i-Neci-WYW and RT11-i-Pani-WYW were treated and cultured at 37 ° C for 12 hours. Then, the antibody and the nucleus were labeled with the same conditions as in Example 2, and the Ras labeled antibody was stained with a secondary antibody after the reaction at 37 ° C for 1 hour and observed with a confocal microscope.

As shown in FIG. 29C, RT11-i3, RT11-i-Neci-WYW and RT11-i-Pani-WYW of green fluorescence were superimposed on the intracellular portion where the activated RAS of red fluorescence was located.

As a result of the above experiment, it was confirmed that a complete IgG type RAS target cytoplasmic penetration antibody into which the activated RAS in the cell and the improved endosomal escape motif were introduced specifically binds.

Example  33. Heavy chain  Variable area CDR  improved Endo Escape ability  Motif assignable logic.

The heavy chain variable region and the light chain variable region have a structure common in that they are composed of three CDRs having a ring structure and a beta-window structure. Therefore, it was concluded that the induction motif of endotoxin releasing ability according to the pH-dependent interaction change of the light chain variable region could be given to the heavy chain variable region.

This phenomenon can be reproduced in the heavy chain variable region. The heavy chain variable region sequence and the tertiary structure analysis showed that the CDR3 improved ending at a distance capable of interacting with glutamic acid (E), the first amino acid of the heavy chain variable region I was able to move a little escape motif.

The number of amino acids in CDR3 of the wild type heavy chain variable region was 11, and it was judged that the pH-dependent phenomenon like the light chain variable region was hard to occur because the structure was already exposed to the center portion of the ring already on the surface. Accordingly, the number of amino acids in CDR3 was reduced to 7 or 8 while retaining the sequence in part.

Also, it was determined that the 102nd amino acid of the heavy chain variable region was located at a suitable distance as the amino acid capable of interacting with the 1st amino acid of the heavy chain variable region at the initial endophore pH 5.5, and this amino acid was substituted with leucine (L) .

A mutation was constructed by introducing the improved endosomal motility into the CDR3 of the heavy chain variable region.

Tables 13, 14 and 15 below show the heavy chain variable region mutant sequence in which the designed endosuppression motif was transferred to the heavy chain variable region. Table 13 shows the whole sequence of the human antibody light chain variable region according to Kabat numbering. Tables 14 and 15 show the CDR1, CDR2 sequence or CDR3 sequence alone in the antibody sequence of Table 13 above.

30A is a schematic diagram illustrating the construction of a cytoplasmic penetration antibody having a light chain variable region from which endosomal elimination ability is removed and an improved endosomal exporting motif introduced heavy chain variable region.

In order to evaluate the endosomal elimination ability of the heavy chain variable region introduced with the improved endosomal motility motif, WYW, which is the 92-94th amino acid in the existing light chain variable region, was substituted with AAA Three consecutive alanines) to eliminate its function. In the same manner as in Example 1, the heavy chain variable region was cloned and the expression and purification were carried out in the HEK293F cell line together with the light chain including the light chain variable region in which endosomal elimination ability was removed.

The TMab4 is abbreviated as CT in order to more clearly name the cytoplasmic penetration antibody containing the heavy chain variable region into which the improved endosomal motility was introduced. That is, TMab4-AAA is CT-AAA.

Example 34. Enhanced endosomes in the CDRs of the heavy chain variable region Confirmation that the escape motif can be given .

30B is a graph comparing quantitatively the number of cells acquiring trypan blue according to pH by a cytoplasmic penetration antibody having a light chain variable region from which endosomal elimination ability is removed and an endosomal elimination ability motif-introduced heavy chain variable region.

Specifically, ramos cells were adhered to the plate in the same manner as in Example 5, and then the plate was washed with a pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) at a cytoplasmic pH, a pH 5.5 buffer (HBSS ), 50 mM MES pH 5.5), and incubated at 37 ° C for 2 hours with 1 μM of TMab4-WYW, TMab4-AAA, CT01-AAA, CT02-AAA, CT03-AAA and CT04-AAA. After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. The number of cells that acquired trypan blue in all cells was counted as a percentage. A total of more than 400 cells were counted and the mean value was plotted. In the CT01-AAA, CT02-AAA, CT03-AAA, and CT04-AAA mutants, approximately half of the trypan blue acquisition was observed compared to TMab4-3. However, in the CT04-AAA mutation, trypan blue accumulation was also observed at neutral pH.

Figure 30c shows GFP fluorescence < RTI ID = 0.0 > (GFP) < / RTI > fluorescence by complementary binding of an improved segmented green fluorescent protein of a cytoplasmic penetration antibody with a light chain variable region that has been fused with GFP11-SBP2 and has an improved endosomal export motif introduced heavy chain variable region Were observed through a confocal microscope.

Specifically, a transformed HeLa cell line stably expressing SA-GFP1-10 was prepared in the same manner as in Example 2. After the cells were stabilized, PBS, CT01-AAA-GFP11-SBP2, CT02-AAA-GFP11-SBP2 , CT03-AAA-GFP11-SBP2, and CT04-AAA-GFP11-SBP2 were incubated at 37 占 폚 for 6 hours. After washing with PBS and weakly acidic solution in the same manner as in Example 2, cells were fixed. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. It was confirmed that green GFP fluorescence was observed in the cells treated with CT01-AAA-GFP11-SBP2, CT02-AAA-GFP11-SBP2, CT03-AAA-GFP11-SBP2 and CT04-AAA-GFP11-SBP2.

FIG. 30D shows the result of observing the movement of the cytoplasmic penetration antibody having a light chain variable region from which endosomal elimination ability has been removed and an improved endosomal exporting motif-introduced heavy chain variable region into the cytoplasm using a calcein using a confocal microscope.

Specifically, an HeLa cell line was prepared in the same manner as in Example 2, and CT01-AAA, CT02-AAA, CT03-AAA 0.2, 0.5, and 1 μM were cultured at 37 ° C for 6 hours. After 4 hours, the wells containing PBS or antibody were treated with 150 [mu] M calcine and incubated at 37 [deg.] C for 2 hours. After washing with PBS and weakly acidic solution in the same manner as in Example 2, cells were fixed. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. In the cells treated with CT01-AAA and CT02-AAA, the green calcein fluorescence scattered in the cytoplasm was similar to the cells treated with TMab4. On the other hand, in the cells treated with CT03-AAA, the green calcein fluorescence scattered in the cytoplasm was weaker than the CT fluorescence.

Therefore, it was confirmed that the endosomal escape is possible even when the improved endosomal motility is given to the heavy chain variable region, and finally it is located inside the cytoplasm.

Example  35. In the heavy chain variable region  Characterization of E1-L102 to induce structural changes according to pH.

In order to further analyze the characteristics of the first amino acid glutamic acid, the 102nd amino acid leucine, the first amino acid located in the antibody backbone is arpa Alanine, glutamine, and the 102nd amino acid located in CDR3 was constructed by substituting 12 amino acids that showed trypan blue acquisition at neutral or acid pH conditions in the light chain variable region. Each mutation was subjected to cloning and expression and purification in an HEK293F cell line in the same manner as in Example 1.

31A is a graph showing the number of cells obtained by obtaining trypan blue according to the pH of a mutant in which a first amino acid glutamic acid (first amino acid glutamic acid) was substituted with a heavy chain variable region (VH) involved in inducing a structural change of a cytoplasmic penetration antibody at acidic pH, FIG.

Specifically, as in Example 26, the pgsD-677 cell line (1 × 10 ⁴ ) was cultured in a 24-well plate. On the next day, 200 μl of a pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) and an initial endosomal pH pH 5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5) , CT01-AAA E1A, CT01-AAA E1D, and CT01-AAA E1Q 1 μM, and incubated at 37 ° C for 2 hours. After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. Then, carefully wash with PBS, and add 50 μl of 1% SDS (sodium dodecyl sulfate) to dissolve the cells. It was transferred to a 96-well plate and absorbance was measured at a wavelength of 590 nm. CT01-AAA E1D showed a similar degree of trypan blue acquisition, while CT01-AAA E1A and CT01-AAA E1Q mutations decreased trypan blue acquisition.

FIG. 31B shows the number of cells obtained by obtaining trypan blue according to the pH of the mutant in which the 102nd amino acid leucine of the heavy chain variable region (VH) involved in the induction of the structural change of the cytoplasmic penetrating antibody at acidic pH was replaced with various amino acids was quantitatively FIG.

Specifically, pgsD-677 cells were prepared in the same manner as in Example 26, and a pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) at a cytoplasmic pH was used as in Example 5, The CT01-AAA and CT01-AAA L102X mutants were incubated with 1 μM of 19 mutants in 200 μl of 5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5) for 2 hours at 37 ° C. After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. Then, carefully wash with PBS, and add 50 μl of 1% SDS (sodium dodecyl sulfate) to dissolve the cells. It was transferred to a 96-well plate and absorbance was measured at a wavelength of 590 nm. Compared with CT01-AAA, CT01-AAA L102I, L102M, and L102H showed a similar degree of trypan blue acquisition, while CT01-AAA L102K and L102R mutants increased trypan blue acquisition at neutral pH.

In this case, amino acid 102 in the variable region of the light chain variable region, like the amino acid 95 of the light chain variable region, recognizes the initial endosomal pH of 5.5, and in order to cause the endosomal escape through the interaction change, a hydrophobic amino acid having a long side chain, It was confirmed that the interaction between the amino acid and the histidine (H) was most effective.

Example  36. Three With tryptophan Endo  Build an escape motif mutation.

In order to improve endosomal escape ability of the endotoxin having two tryptophan, endosomal escape motif having three tryptophan was constructed by replacing all 92-94 amino acids with tryptophan.

Tables 16 and 17 below show a light chain variable region mutant sequence incorporating endosomal escape motifs each having 3 tryptophan. Table 16 shows the entire sequence of the human antibody light chain variable region according to Kabat numbering. Table 17 shows the CDR3 sequence alone in the antibody sequence of Table 16 above.

Tables 18 and 19 below show the heavy chain variable region mutant sequences incorporating endosomal escape motifs each having 3 tryptophan. Table 18 shows the entire sequence of human antibody heavy chain variable region according to Kabat numbering. Table 19 shows the CDR3 sequence alone in the antibody sequence of Table 18 above.

At this time, in order to evaluate the endosomal elimination ability of the heavy chain variable region or the light chain variable region including the endorphin releasing ability motif composed of three tryptophan, a heavy chain variable region or a light chain variable region containing no endosomal elimination motif The HEK293F cell line was tested for expression and purification in the same manner as in Example 1 above.

Example  37. Consisting of two or three tryptophan Endo Escape ability  Motif introduction Heavy chain variable region  And Light chain variable region  Including complete IgG  Endosomes of cytoplasmic infiltration antibodies in the form Escape ability  Improvement confirmation.

As a strategy to improve the endosomal escape ability, both the heavy chain variable region and the light chain variable region were given an endosomal export motif. Thus, a complete IgG-type cytoplasmic penetration antibody will contain a total of four endosomal export motifs.

The heavy chain variable region and the light chain variable region including the endosomal exporting motif composed of two or three tryptophan were tested and expressed in the same manner as in Example 1 in the HEK293F cell line.

Figure 32A shows the quantitative comparison of the number of cells harboring trypan blue according to the pH of a complete IgG type cytoplasmic penetration antibody containing an endosomal escape motif-introduced heavy chain variable region and / or a light chain variable region substituted with three tryptophan It is a graph.

Specifically, pgsD-677 cells were prepared in the same manner as in Example 26, and a pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) at the cytoplasmic pH was prepared in the same manner as in Example 5, 3, CT-3_WWW, CT01-AAA, CT01_WWW-AAA, CT01-3, CT01_WWW-3_WWW 0.5 or 1 μM was added to 200 μl of pH 5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5) And cultured for 2 hours. After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. Then, carefully wash with PBS, and add 50 μl of 1% SDS (sodium dodecyl sulfate) to dissolve the cells. It was transferred to a 96-well plate and absorbance was measured at a wavelength of 590 nm.

CT-3_WWW, CT01_WWW-AAA, and CT01_WWW-3_WWW show significantly improved tripping blue acquisition compared to CT-3, CT01-AAA and CT01-3 which include the existing endomorphism motif. In addition, CT01-3 and CT01_WWW-3_WWW, which contain endosome-exiting motifs in both the heavy and light chain variable regions, show higher tripping blue gain compared to CT-3 and CT01-AAA.

FIG. 32B shows the results of observing the cytoplasmic transfer of a complete IgG-type cytoplasmic penetration antibody containing an endosomal escape motif-introduced heavy chain variable region and / or a light chain variable region substituted by three tryptophan with a calixain using a confocal microscope Confocal microscope This is a bar graph that quantifies the calcein fluorescence of a photograph.

Specifically, HeLa cell lines were prepared in the same manner as in Example 2, and CT-3, CT-3_WWW, CT01-AAA, CT01_WWW-AAA, CT01-3, CT01_WWW-3_WWW 0.25, 0.5, Lt; / RTI > After 4 hours, the wells containing PBS or antibody were treated with 150 [mu] M calcine and incubated at 37 [deg.] C for 2 hours. After washing with PBS and weakly acidic solution in the same manner as in Example 2, cells were fixed. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope.

Compared with cells treated with CT-3, CT01-AAA and CT01-3 containing the existing endomorphism motif, a green knife spreading in the cytoplasm in cells treated with CT-3_WWW, CT01_WWW-AAA and CT01_WWW-3_WWW Cine fluorescence was strongly observed. Compared with CT-3 and CT01-AAA, CT01-3 and CT01_WWW-3_WWW containing endosomal escape motifs in both the heavy chain and light chain variable regions were strongly observed in the cytoplasmic green calcein fluorescence .

Endotoxin motifs with three tryptophan motifs improved endotoxin excretion ability than existing endotoxin motifs and enhanced endotoxin escape ability even when endotoxin motifs were given to the heavy chain variable region and the light chain variable region Respectively.

Example  38. Improved Endo  Introduction of escape motif Heavy chain  Variable areas and improved Endo  Having a therapeutic antibody backbone that gives escape ability Light chain  Complete with variable regions IgG  Confirmation of enhancement of endosomal elimination ability of cytoplasmic infiltration antibody of form.

In order to confirm the enhancement of endosomal escape ability when the endosomal escape motif was given to both the heavy chain variable region and the light chain variable region, an improved endosomal escape motif introduction therapeutic antibody framework Were also expressed and purified in HEK293F cell line.

Specifically, a complete IgG-type cytoplasmic penetration antibody comprising a modified endosome-derived motif-introduced heavy chain variable region and a light chain variable region having a therapeutic antibody framework imparted with improved endosomal elimination ability has a cell infiltration capability, EpCAM target circular peptides that are specific for EpCAM overexpressed on the cell membrane surface in various tumors, including colon cancer, were genetically engineered to fuse two light chain GGGGS linkers to the N-terminus of the light chain (US 2015/0246945 A1).

33A is a schematic diagram illustrating the construction of a complete IgG-type cytoplasmic penetration antibody incorporating an improved endosomal escape motif in an existing therapeutic antibody light chain variable region fused with an improved endosomal exported heavy chain variable region and an EpCAM target peptide.

Specifically, in the same manner as in Example 1, a heavy chain comprising an improved endosomal export heavy chain variable region and an animal expression vector having a light chain encoding a monoclonal antibody light chain variable region imparting improved endosomal elimination ability Were simultaneously transiently transfected into HEK293F protein expressing cells. Thereafter, purification of the cytoplasmic penetration antibody in the form of complete IgG proceeded as in Example 1.

Figure 33B shows the migration of the intact cytoplasmic penetration antibody to the cytoplasm of the existing therapeutic antibody light chain variable region fused with the modified endosome-evoked motif-introduced heavy chain variable region and the EpCAM target peptide, into which the improved endosomal escape motif was introduced This is a bar graph obtained by observing with a confocal microscope using Calcine and quantifying the calcein fluorescence of the confocal microscope photograph.

Specifically, in the same manner as in Example 2, a human colon cancer cell line HCT116 cell line having a K-RAS G13D mutation was prepared and CT-ep41, CT01-ep41 0.1, 0.25, and 0.5 μM were cultured at 37 ° C for 6 hours. After 4 hours, the wells containing PBS or antibody were treated with 150 [mu] M calcine and incubated at 37 [deg.] C for 2 hours. After washing with PBS and weakly acidic solution in the same manner as in Example 2, cells were fixed. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. In the cells treated with CT01-ep41, green calcein fluorescence scattered in the cytoplasm was stronger than that of CT-ep41 treated cells.

Figure 33c shows a schematic representation of the effect of the endogenous exocytosis motif on the existing therapeutic antibody light chain variable region fused with the EpCAM target peptide. This graph is a graph comparing quantitatively the number of cells that have acquired plate blue.

Specifically, ramos cells were adhered to the plate in the same manner as in Example 5, and then pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) was added to maintain the pH at 7.4, the cytoplasmic pH, To keep the pH at 5.5, CT-ep41, CT01-ep41 0.5, and 1 μM were added to 200 μl of a pH 5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5) and incubated at 37 ° C for 2 hours. After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. The number of cells that acquired trypan blue in all cells was counted as a percentage. A total of more than 400 cells were counted and the mean value was plotted. In the case of CT01-ep41, it was observed that the acquisition of trypan blue increased in a concentration-dependent manner compared to CT-ep41.

Therefore, when the endosomal escape motif was introduced into the heavy chain variable region and the light chain variable region, the endosomal escape ability was improved compared to the case where the endosomal escape motif was present only in the light chain variable region.

Example  39. Existing Therapeutic Antibodies In the heavy chain variable region  improved Endo Escape ability  Motif assignable logic.

It is expected that the overall expression yield can be increased by using the stable framework of the commercially available therapeutic antibody in the same manner as the logic for imparting the endosomal eliminative motif to the existing therapeutic antibody light chain variable region, Endothelial motility was also imparted to the existing therapeutic antibody heavy chain variable region to confirm that it could function as an endotoxin.

A mutation was constructed in which the CDR3 of the candidate heavy chain variable region was replaced with the CDR3 of the cytoplasmic penetrating antibody.

Table 20 shows the names and sequences of the mutations constructed using the gene synthesis technique.

FIG. 34A is a schematic diagram illustrating the construction of a complete IgG-type cytoplasmic penetration antibody into which an improved endosomal escape motif is introduced into an existing therapeutic antibody heavy chain variable region.

In the same manner as in Example 1, heavy chain variable region cloning was carried out to express and purify the HEK293F cell line together with the light chain containing the monoclonal antibody light chain variable region introduced with the improved endosseous motility motif.

Example 40. Monoclonal antibody Improved Endosomes in the heavy chain variable region Confirmation that the escape motif can be given .

FIG. 34B is a graph comparing quantitatively the number of cells acquiring a trypan blue according to pH by a complete IgG-type cytoplasmic penetration antibody into which an improved endosomal escape motif was introduced into an existing therapeutic antibody heavy chain variable region.

Specifically, pgsD-677 cells were prepared in the same manner as in Example 26, and a pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) at a cytoplasmic pH was used as in Example 5, The cells were incubated at 37 ° C for 2 hours in the presence of CT01-ep41, Hu01-ep41, Her01-ep41, 0.5 and 1 μM of Ava01-ep41 in 200 μl of 5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5). After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. Then, carefully wash with PBS, and add 50 μl of 1% SDS (sodium dodecyl sulfate) to dissolve the cells. It was transferred to a 96-well plate and absorbance was measured at a wavelength of 590 nm. All mutations showed a trippin blue acquisition similar to CT01-ep41.

Example  41. Construction of heavy chain variable region and light chain variable region with asparaginic acid introduced for the purpose of improving physical properties of cytoplasmic penetration antibody.

In order to enhance endosomal escape of the cytoplasmic penetrating antibodies, endosomal escape motifs were introduced into the CDR3 of the heavy chain variable region and the light chain variable region. At this time, the hydrophobic amino acids tryptophan (W) and tyrosine (Y), which constitute the endo-excretory motif, are highly hydrophobic. To counteract this hydrophobicity, we have constructed a mutation that replaces the endosomal escape motif and the adjacent amino acid with negatively charged aspartic acid. Mutations were constructed by introducing aspartic acid into the framework and CDR regions of the antibody variable region to increase overall stability of the antibody and reduce protein aggregation due to high hydrophobicity of the antibody (Perchiacca et al., 2011; Dudgeon et al., 2012).

Since the 32nd, 33rd, and 58th amino acids of the heavy chain variable region are adjacent to the endosome escape motif of the heavy chain variable region or light chain variable region, this amino acid was substituted with aspartic acid. This was named CT11 VH (F32D, S33D), CT12 VH (F32D, S33D, Y58D).

In addition, since the 27th, 50th, and 51st amino acids of the light chain variable region are adjacent to the endosomal escape motif of the heavy chain variable region or the light chain variable region, the amino acid was substituted with aspartic acid. This was named hT4-60 VL (L27bD), hT4-61 VL (W50D) hT4-62 VL (W50D, A51D) hT4-63 VL (L27Bd, W50D, A51D).

At this time, the heavy chain variable region and the light chain variable region, which became the template of the mutation, were designated as CT01 VH and hT4-59 VL, respectively, in which the endosomal escape motif was introduced with high yield in the animal cell expression system.

Tables 21 and 22 below show heavy chain variable region or light chain variable region mutant sequences in which asparaginic acid is introduced into the framework and CDR regions of the antibody variable region.

In the same manner as in Example 1, each heavy chain variable region was cloned, and an improved endosseous motility-introduced monoclonal antibody was expressed and purified in a HEK293F cell line together with a light chain variable region containing the light chain variable region.

Example  42. Introduction of asparaginic acid Heavy chain variable region  And / or Light chain variable region  Confirming the enhancement of endosomal excretion of intact IgG-containing cytoplasmic penetrating antibodies.

FIG. 35 is a graph comparing quantitatively the number of cells obtained with trypan blue according to pH by a complete IgG-type cytoplasmic penetration antibody containing an aspartic acid-introduced heavy chain variable region and / or a light chain variable region.

Specifically, pgsD-677 cells were prepared in the same manner as in Example 26, and a pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4) at a cytoplasmic pH was used as in Example 5, 5, 6, 7, 8, 9, 12, 12, and 12 were added to 200 μl of a buffer (HBSS (Welgene), 50 mM MES pH 5.5) And incubated at 37 ° C for 2 hours. After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl of each was dispensed into each well and observed under a microscope. Then, carefully wash with PBS, and add 50 μl of 1% SDS (sodium dodecyl sulfate) to dissolve the cells. It was transferred to a 96-well plate and absorbance was measured at a wavelength of 590 nm. All mutations showed a trippan blue acquisition similar to that of CT01-ep41 tested in the previous example. Therefore, it was confirmed that even when aspartic acid was introduced, endosomal elimination ability did not decrease. Antibody stability tests on the antibodies will be conducted at a later date.

Example 43. Analysis of cytoplasmic penetration antibody structure

In order to clarify the structure of IgG-type cytoplasmic penetration antibody, we used CT-59 antibody with high production yield of cytoplasmic penetration antibody with endosomal elimination ability at endosomal acidic pH condition. This is an IgG-type cytoplasmic penetration antibody composed of a heavy chain variable region and a light chain variable region consisting of hT0 VH and hT4-59 VL, respectively.

To identify the tertiary structure, purity of IgE-type CT-59 produced using the existing HEK293 cell line was treated with papain, followed by separation of high purity Fab using Protein A column and size exclusion chromatograpy Crystals for structural characterization were formed on screening buffer Index G1 conditions (0.2 M NaCl, 0.1 M Tris, pH 8.5, 25% (w / v) PEG 3350) on a Mosquito-LCP instrument. The final pH of the cytoplasmic infiltration antibody and the screening buffer is 8.1.

FIG. 36A shows the result of observing the crystal of CT-59 Fab formed under the Index G1 condition with RI1000 (Rock Imager 1000; automatic protein crystal image analyzer).

X-ray diffraction data were acquired using a 5C beamline (Pohang Accelerator Laboratory, PAL) and indexing and scaling through the HKL2000 package (HKL Research Inc., USA) followed by molecular replacement (MR) The initial electron density map was obtained. In order to use the MR method, three-dimensional structure data of a protein having a structure similar to that of CT-59 is required. By using the FFAS site (http://ffas.sanfordburnham.org/ffas-cgi/cgi/ffas.pl) Structure was used as a model. The initial phase information of the CT-59 Fab was acquired using CCP4, and based on the obtained initial phase information, using the COOL (Crystallographic Object-Oriented Toolkit, http://www.biop.ox.ac.uk/coot/) model construction work has been carried out and Refmac5 (http://www.ccp4.ac.uk/html/refmac5.html) and PHENIX (Python-based Hierarchical Environment for Integrated Xtallography, http://www.phenix-online.org/) ) software to complete the refinement and validation work (see FIG. 36B).

As a result, the high-resolution tertiary structure of 1.8 Å was identified at the final pH of 8.1, and the distance between the side chains of the first asparaginic acid (D) and the 95th methionine (M) in the light chain variable region of CT- 6.87 Å.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the invention is not limited thereby. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Orum Therapeutics <120> Cytosol-Penetrating Antibodies and Uses Thereof <130> DP-2017-0135-KR <150> KR 10-2016-0065365 <151> 2016-05-27 <150> KR 10-2016-0065379 <151> 2016-05-27 <160> 78 <170> KoPatentin 3.0 <210> 1 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-WWH VL <400> 1 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Trp Trp His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 2 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-WYW VL <400> 2 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 3 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-YWW VL <400> 3 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Trp Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 4 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-WYH VL <400> 4 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Trp Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 5 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-YWH VL <400> 5 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Trp His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 6 Lys Ser Ser Gln Ser Leu Phe Asn Ser Arg Thr Arg Lys Asn Tyr Leu   1 5 10 15 Ala     <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 7 Trp Ala Ser Thr Arg Glu Ser   1 5 <210> 8 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> hT4-WWH VL <400> 8 Gln Gln Tyr Trp Trp His Met Tyr Thr   1 5 <210> 9 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> hT4-WYW VL <400> 9 Gln Gln Tyr Trp Tyr Trp Met Tyr Thr   1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> hT4-YWW VL <400> 10 Gln Gln Tyr Tyr Trp Trp Met Tyr Thr   1 5 <210> 11 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> hT4-WYH VL <400> 11 Gln Gln Tyr Trp Tyr His Met Tyr Thr   1 5 <210> 12 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> hT4-YWH VL <400> 12 Gln Gln Tyr Tyr Trp His Met Tyr Thr   1 5 <210> 13 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-RYR VL <400> 13 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Arg Tyr Arg Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 14 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-IYI VL <400> 14 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Ile Tyr Ile Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 15 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-GYG VL <400> 15 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Gly Tyr Gly Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 16 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 VL <400> 16 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln                  85 90 95 Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 17 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 D1E-M95L VL <400> 17 Glu Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Trp Tyr Trp Leu Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 18 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 Y91H VL <400> 18 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 His Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 19 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 Y91D VL <400> 19 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Asp Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 20 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 L2E Q90L VL <400> 20 Asp Glu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Leu                  85 90 95 Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 21 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 L2E T97I VL <400> 21 Asp Glu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Trp Tyr Trp Met Tyr Ile Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 22 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 Q90H M95A VL <400> 22 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His                  85 90 95 Tyr Trp Tyr Trp Ala Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 23 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 Q90H VL <400> 23 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His                  85 90 95 Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 24 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> hT4 3 Q90H VL <400> 24 Gln His Tyr Trp Tyr Trp Met Tyr Thr   1 5 <210> 25 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 L8-1 VL <400> 25 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln                  85 90 95 Tyr Trp Tyr Trp Met Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg     <210> 26 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 L8-2 VL <400> 26 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln                  85 90 95 Trp Tyr Trp Met Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys             100 105 110 Arg     <210> 27 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 L10-1 VL <400> 27 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln                  85 90 95 Tyr Trp Tyr Trp Pro Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu             100 105 110 Ile Lys Arg         115 <210> 28 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 L10-2 VL <400> 28 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Trp Tyr Trp Leu Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu             100 105 110 Ile Lys Arg         115 <210> 29 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 L10-3 VL <400> 29 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Trp Tyr Trp Tyr Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu             100 105 110 Ile Lys Arg         115 <210> 30 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 L11-1 VL <400> 30 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Trp Tyr Trp Leu Tyr Met Tyr Thr Phe Gly Gln Gly Thr Lys Val             100 105 110 Glu Ile Lys Arg         115 <210> 31 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 L11-2 VL <400> 31 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Pro Trp Tyr Trp Pro Met Tyr Thr Phe Gly Gln Gly Thr Lys Val             100 105 110 Glu Ile Lys Arg         115 <210> 32 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Necitumumab-WYW VL <400> 32 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly   1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr              20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile          35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro  65 70 75 80 Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr Trp Tyr Trp Met Tyr                  85 90 95 Thr Phe Gly Gln Gly Thr Lys Ala Glu Ile Lys Arg             100 105 <210> 33 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Panitumumab-WYW VL <400> 33 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr              20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Trp Tyr Trp Met Tyr                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg             100 105 <210> 34 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> Lumretuzumab-WYW VL <400> 34 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly   1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Asn Ser              20 25 30 Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Phe Cys Gln Gln                  85 90 95 Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile             100 105 110 Lys Arg         <210> 35 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> Nimotuzumab-WYW VL <400> 35 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Asn Ile Val His Ser              20 25 30 Asn Gly Asn Thr Tyr Leu Asp Trp Tyr Gln Gln Thr Pro Gly Lys Ala          35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro      50 55 60 Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile  65 70 75 80 Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr                  85 90 95 Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Leu Gln Ile Thr             100 105 110 Arg     <210> 36 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Emibetuzumab-WYW VL <400> 36 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Val Ser Ser Val Ser Ser Ile              20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu          35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Ser Ser Arg Phe Ser      50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln  65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Trp Tyr Trp Met                  85 90 95 Tyr Thr Ply Gly Gly Gly Thr Lys Val Glu Ile Lys Arg             100 105 <210> 37 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Pertuzumab-WYW VL <400> 37 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly              20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Ser Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Tyr Trp Tyr Trp Met Tyr                  85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105 <210> 38 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> HT0 VH <400> 38 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ala Ile Asn Pro Tyr Asn Asp Gly Asn Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Lys Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Ala Tyr Lys Arg Gly Tyr Ala Met Asp Tyr Trp Gly Gln             100 105 110 Gly Thr Thr Val Thr Val Ser Ser         115 120 <210> 39 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> HT0-01 VH <400> 39 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ala Ile Asn Pro Tyr Asn Asp Gly Asn Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Lys Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Thr Val             100 105 110 Thr Val Ser Ser         115 <210> 40 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> HT0-02 VH <400> 40 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ala Ile Asn Pro Tyr Asn Asp Gly Asn Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Lys Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Trp Tyr Trp Phe Asp Leu Trp Gly Gln Gly Thr Thr Val             100 105 110 Thr Val Ser Ser         115 <210> 41 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> HT0-03 VH <400> 41 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ala Ile Asn Pro Tyr Asn Asp Gly Asn Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Lys Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Trp Tyr Trp Gly Phe Asp Leu Trp Gly Gln Gly Thr Thr             100 105 110 Val Thr Val Ser Ser         115 <210> 42 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> HT0-04 VH <400> 42 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ala Ile Asn Pro Tyr Asn Asp Gly Asn Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Lys Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Tyr Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Thr Val             100 105 110 Thr Val Ser Ser         115 <210> 43 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> CDR1 <400> 43 Ser Tyr Val Met His   1 5 <210> 44 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 <400> 44 Ala Ile Asn Pro Tyr Asn Asp Gly Asn Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 45 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HT0 VH <400> 45 Gly Ala Arg Lys Arg Gly Tyr Ala Met Asp Tyr   1 5 10 <210> 46 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HT0-01 VH <400> 46 Gly Trp Tyr Trp Met Asp Leu   1 5 <210> 47 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HT0-02 VH <400> 47 Gly Trp Tyr Trp Phe Asp Leu   1 5 <210> 48 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HT0-03 VH <400> 48 Gly Trp Tyr Trp Gly Phe Asp Leu   1 5 <210> 49 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HT0-04 VH <400> 49 Tyr Trp Tyr Trp Met Asp Leu   1 5 <210> 50 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-3 WWW VL <400> 50 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln                  85 90 95 Tyr Trp Trp Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 51 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> hT4 3 WWW VL <400> 51 Gln Gln Tyr Trp Trp Trp Met Tyr Thr   1 5 <210> 52 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> HT0-01 WWW VH <400> 52 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr              20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ala Ile Asn Pro Tyr Asn Asp Gly Asn Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Lys Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Trp Trp Trp Met Asp Leu Trp Gly Gln Gly Thr Thr Val             100 105 110 Thr Val Ser Ser         115 <210> 53 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HT0-01 WWW VH <400> 53 Gly Trp Trp Trp Met Asp Leu   1 5 <210> 54 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Humira-01 VH <400> 54 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr              20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val      50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Lys Gly Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 55 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Herceptin-01 VH <400> 55 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr              20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ser Arg Gly Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 56 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Avastin-01 VH <400> 56 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr              20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe      50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Lys Gly Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 57 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> CT10 VH <400> 57 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Phe              20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Tyr Ile Ser Arg Thr Ser His Thr Thyr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 58 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> CT11 VH <400> 58 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Asp Asp Asp Phe              20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Tyr Ile Ser Arg Thr Ser His Thr Thyr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 59 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> CT12 VH <400> 59 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Asp Asp Asp Phe              20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Tyr Ile Ser Arg Thr Ser His Thr Thr Asp Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Arg Gly Trp Tyr Trp Met Asp Leu Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser         115 <210> 60 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-59 VL <400> 60 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser              20 25 30 Arg Asp Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln                  85 90 95 Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 61 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-60 VL <400> 61 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Asp Leu Asn Ser              20 25 30 Arg Asp Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln                  85 90 95 Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 62 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-61 VL <400> 62 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser              20 25 30 Arg Asp Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln                  85 90 95 Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 63 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-62 VL <400> 63 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser              20 25 30 Arg Asp Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Asp Asp Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln                  85 90 95 Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 64 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-63 VL <400> 64 Asp Ile Gln Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Asp Leu Asn Ser              20 25 30 Arg Asp Gly Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Asp Asp Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln                  85 90 95 Tyr Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 65 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4 VL <400> 65 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 66 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-D1A VL <400> 66 Ala Leu Val Met Thr Gln Ser Ser Ser Leu Ser Ala Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 67 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-M95A VL <400> 67 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Tyr His Ala Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 68 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-D1E VL <400> 68 Glu Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 69 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-M95L VL <400> 69 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Tyr His Leu Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 70 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-Y91A VL <400> 70 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Ala Tyr Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 71 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-Y92A VL <400> 71 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Ala Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 72 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-Y93A VL <400> 72 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Ala His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 73 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-H94A VL <400> 73 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Tyr Ala Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 74 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-AAA VL <400> 74 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Ala Ala Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 75 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-Y96A VL <400> 75 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Phe Asn Ser              20 25 30 Arg Thr Arg Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Tyr His Met Ala Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 76 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> hT4-01 VL <400> 76 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Gly Leu Ser Ser Tyr              20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile          35 40 45 Tyr Trp Ala Ser Thr Leu Glu Ser Gly Val Ser Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro  65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Tyr His Met Tyr                  85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg             100 105 <210> 77 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-02 VL <400> 77 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser              20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 78 <211> 114 <212> PRT <213> Artificial Sequence <220> <223> hT4-03 VL <400> 78 Asp Leu Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Val Gly   1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser              20 25 30 Asp Asp Gly Asn Thr Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys          35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Leu Ser Tyr Arg Ala Ser Gly Val      50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr  65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln                  85 90 95 Tyr Tyr Tyr His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg        

Claims (20)

A light chain variable region and / or a heavy chain variable region comprising a sequence represented by the following general formula in CDR3,
X1-X2-X3-Z1
Here, X1-X2-X3 is an endosomal elimination ability motif,
X1-X2-X3 is selected from the group consisting of tryptophan (W), tyrosine (Y), histidine (H) and phenylalanine (F)
Z1 is selected from the group consisting of methionine (M), isoleucine (I), leucine (L), histidine (H), aspartic acid (D) and glutamic acid (E)
Wherein the light chain variable region and / or heavy chain variable region comprising Z1 undergoes a change in the characteristics of the antibody at endosomal acidic pH conditions,
Wherein the antibody exhibits an ability to escape from the endosome to the cytoplasm through a change in the characteristics of the antibody, or an antigen-binding fragment thereof.
The cytoplasmic penetration antibody or the antigen-binding fragment thereof according to claim 1, wherein the first amino acid of the light chain variable region and / or the heavy chain variable region is aspartic acid (D) or glutamic acid (E).
The cytoplasmic penetrating antibody or antigen-binding fragment thereof according to claim 1, wherein the first amino acid of the light chain variable region and / or the heavy chain variable region interacts with the Z1 at an endothermic acidic pH condition to induce a change in the property.
The cytoplasmic penetration antibody or the antigen-binding fragment thereof according to claim 1, wherein the light chain variable region and / or the heavy chain variable region endogenous escape ability motif X1-X2-X3 comprises at least one tryptophan.
3. The cytoplasmic penetration antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region and / or the heavy chain variable region endosomal exporting motif X1-X2-X3 comprises a sequence selected from the group consisting of:
WWW, WWH, WYW, YWW, WYH, YWH
(Tryptophan (W), tyrosine (Y) and histidine (H) above)
The method of claim 1, wherein
Wherein the antibody further comprises an amino acid sequence represented by (a1- ... an) (wherein n is an integer of 1 or more and 10 or less) between X3 and Z1.
The method according to claim 1,
And Z2 linked to X1 is further represented by the general formula below.
Z2-X1-X2-X3-Z1
(Wherein Z2 is selected from the group consisting of glutamine (Q), leucine (L), and histidine (H)
The method according to claim 7, wherein the first amino acid of the light chain variable region and / or the heavy chain variable region interacts with the Z1 and / or Z2 at an acidic pH condition of the endosome to induce a change in the property. Or an antigen-binding fragment thereof.
8. The method of claim 7,
And further comprising an amino acid sequence represented by (b1 -...-bn) (wherein n is an integer of 1 or more and 10 or less) between the X1 and the Z2, or an antigen-binding fragment thereof.
The cytopenic antibody or the antigen-binding fragment thereof according to claim 1, wherein the light chain variable region CDR3 is at least one selected from the group consisting of SEQ ID NOS: 8 to 12, 24 or 51.
4. The cytoplasmic penetration antibody or the antigen-binding fragment thereof according to claim 1, wherein the heavy chain variable region CDR3 is at least one selected from the group consisting of SEQ ID NOS: 46 to 49 and 53.
2. The antibody of claim 1, wherein the antibody comprises a cytoplasmic penetration antibody comprising a sequence having 80% or more homology with the sequence of the light chain variable region selected from the group consisting of SEQ ID NOS: 1 to 5, 13 to 23, 25 to 37, 50, Or an antigen-binding fragment thereof.
The antibody according to claim 1, which comprises a heavy chain variable region sequence having 80% or more homology with a sequence selected from the group consisting of SEQ ID NOs: 39 to 42, 52, 54 to 59, or an antigen-binding fragment thereof .
The cytoplasmic penetration antibody or antigen-binding fragment thereof according to claim 1, which is a complete immunoglobulin G form.
17. A nucleic acid encoding the cytoplasmic penetration antibody or antigen-binding fragment thereof of any one of claims 1 to 14.
17. A vector comprising the nucleic acid of claim 15.
A cell transformed with the vector of claim 15.
15. A composition for delivering an active substance in a cytoplasm containing the cytoplasmic penetration antibody or the antigen-binding fragment thereof according to any one of claims 1 to 14.
16. The composition of claim 15, wherein the active material is at least one selected from the group consisting of peptides, proteins, toxins, antibodies, antibody fragments, RNAs, siRNAs, DNAs, small molecule drugs, nanoparticles and liposomes.
(X1-X2-X3 is selected from the group consisting of tryptophan (W), tyrosine (Y), histidine (H) and phenylalanine (F) Or a grafting step of transferring the antibody to the CDR3 of the heavy chain variable region, or a method for producing the antigen binding fragment of the cytoplasmic penetration antibody according to any one of claims 1 to 14.

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