KR20170133933A - Method for inducing conformational changes in the complementarity determining regions of antibody - Google Patents

Abstract

The present invention relates to a method for inducing structural changes in complementary determining regions of a light chain variable area and/or a heavy chain variable area of an antibody, and a structural change inducing unit for the same. An antibody with improved endosome escape abilities according to the present invention penetrates into specific cells with high efficiency without a special external protein transfer system and is distributed in cytoplasm, thereby being usefully used for developing and manufacturing drugs and in fundamental medical research fields.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for inducing a structural change in an antibody complementarity determination region,

The present invention relates to a method for inducing a structural change of a complementarity determining region according to a change in hydrogen ion concentration in a light chain variable region and / or a heavy chain variable region of an antibody, and a structure conversion inducing unit therefor.

Existing antibodies do not penetrate directly into living cells due to their large size and hydrophilic character. Thus, most existing antibodies specifically target the secreted proteins or cell membrane proteins. General antibodies and polymer biopharmaceuticals have limitations in their ability to bind and inhibit various disease-related substances inside the cytoplasm because they are unable to pass through hydrophobic membranes. In general, 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, amphiphilic glycosides A preprocessing process is required to form perforations in the cell membrane through permeabilization of the cell membrane using saponin.

Antibodies have a high specificity and high affinity for binding to target proteins, and many therapeutic antibodies have been developed to target proteins secreted into cell membranes or cells. Antibodies that target cell membrane proteins can bind to cell membrane proteins and then enter the cell through the endosomal pathway through membrane-receptor-mediated endocytosis, first to the early endosome Go through several paths. Specifically, 1) most of the antibodies are fused to lysosomes through the endo-endosomes in the endo-endosomes and go to pathways that are completely destroyed by acidic conditions and proteolytic enzymes; 2) Some antibodies bind to FcRn (neonatal Fc receptor) receptors in early endosomal conditions and may 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 most of them are destroyed through the lysosome pathway. As the endosomes mature in the endosome pathway, acidification occurs gradually by a proton pump. The pH of the endosomes is 5.5-6.5, the endosomes are pH 5.0-6.0, the pH of the lysosomes is 3.5 -4.5, and many protein hydrolytic enzymes are activated inside the endosome, and externally internalized proteins are destroyed inside the endosome. Therefore, when the antibody moves along the endosomal pathway after intracellular internalization by the receptor, it must escape from the endo- or endo-endosome prior to lysosomes and be separated from the target membrane protein to create a perforation in the endosome do.

It is known that viruses and toxins among substances existing in nature inside the cell actively penetrate into living cells through cell internalization (endocytosis). In order for a substance that penetrates into the cell by cell internalization to become active in the cytoplasm, the process of escape from the endosomes to the cytoplasm, "endosomal escape", is essential. Although the endosomal escape mechanism has not yet been elucidated, 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 the pore formation mechanism. The second hypothesis is that the protonated sponge effect causes endosomes to be released by the protonated amine group, which causes the substance with an amine group to react with the endosomal membrane (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. Although the above three hypotheses have been proposed as an endosome escape mechanism after cell internalization of viral proteins and plant / bacterial derived toxin proteins, such endosomal escape mechanisms have not been specifically identified in antibodies.

The common phenomenon observed in endosomal escape mechanism is that endosomal escape occurs under acidic pH conditions where endosomal and lysosomal environment pH is low. In the case of a protein whose function changes depending on pH, the structure changes in accordance with pH. The phenomenon that causes this change is called the Tanford transition (Qin et al., 1998). (Aspartic acid, Glutamic acid, E) and hydrophobic amino acids (Methionine, M, Leucine, L, Isoleucine, I) ) Have no interaction, but the carboxyl acids (COO-) of the side chain of the negatively charged amino acid become hydrogenated (protonation) and become hydrophobic as the pH becomes lower, and the hydrophobic amino acids Hydrophobic interaction can be performed. As a result, the distance between two amino acids becomes closer, and the structure and function of the whole protein changes. For example, Nitrophorin 4, which is a nitrogen-transporting enzyme, has an open structure at the neutral hydrogen ion concentration, but as the pH is lowered, the nitrogen transport function is performed as the hydrophobic interaction of aspartic acid and leucine changes into a closed structure (Di Russo et al., 2012). However, to date, the pH-dependent structural changes are not known in antibodies.

Precedent literature

1. Korean Patent Publication No. 10-2012-0026408

Accordingly, the present inventors investigated the endosomal elimination mechanism of a complete immunoglobulin-type cytotransmucible antibody (Cytotransmab) distributed in the cytoplasm and penetration into the cell, and found that the endosomal escape mechanism Of the present invention, and based on this, the structural transformation site of the antibody that induces the structural change of the endosomal escape motif of the antibody is identified and the present invention is completed Respectively.

Therefore, the present invention is based on the finding that the interaction of the two amino acid residues in the light chain variable region and / or the heavy chain variable region of the antibody is induced according to the change of the hydrogen ion concentration in the endosome after the antibody is introduced into the cell, And a structural change inducing unit that induces a structural change of a complementarity determining region of a light chain variable region and / or a light chain variable region of the light chain variable region.

The term " endosomal escape ability ", as used herein, refers to the ability to penetrate living cells actively through cell internalization and to leave the endosomes to be located in the cytoplasm, and the term " endosomal escape motif ) Refers to a region which imparts endosomal elimination capability to a cytoplasmic penetration antibody of a complete immunoglobulin type which is internalized by structural change in response to an acidic hydrogen ion concentration.

As used herein, the term " structural transformation inducing moiety " refers to: 1) two residues in which the interaction of two amino acid residues in the light chain variable region and / or the heavy chain variable region of the antibody is induced, And a complementarity determining region where structural change occurs. The "structural transformation inducing moiety" may be present in the light chain variable region, the heavy chain variable region, or the light chain variable region and the heavy chain variable region of the antibody at the same time.

As used herein, the term "amino acid" encompasses L-amino acids or residues thereof that occur naturally in the broad concept, as well as D-amino acids and chemically modified amino acids. For example, the above-mentioned amino acid mimetics and the like are included, and the mimetics and the like in the present invention may include functional equivalents. All amino acid sequences provided in the present invention are shown using Kabat numbers.

As used herein, the term " complete immunoglobulin type antibody " has a structure with two full-length light chains and two full-length heavy chains, each light chain having a disulfide bond (SS- The constant region of the antibody is divided into a heavy chain constant region and a light chain constant region. The heavy chain constant region has gamma (gamma), mu (mu), alpha (alpha), delta (delta) and 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 constant region of the light chain has kappa (kappa) and lambda (lambda) types.

As used herein, the term "heavy chain" includes a variable region domain VH comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen and three constant region domains CH1, CH2 and CH3 It includes both full-length heavy chains and fragments thereof.

The term "light chain" as used herein refers to a light chain light chain comprising a variable region domain VL comprising the amino acid sequence having a sufficient variable region sequence to confer antigen specificity and a constant region domain CL, All included.

One. Endo  Escape structure motif

According to an embodiment of the present invention,

(CDR3) of the light chain variable region, the heavy chain variable region, or the light chain and heavy chain variable region of the antibody, and includes the following amino acid sequence for imparting endosomal elimination ability after the antibody is intracellularly introduced An endosomal escape motif is initiated.

-X1-X2-X3-

(Wherein,

X1, X2 and X3 are independently selected from the group consisting of Phenylalanine (F), Tyrosine (Y), Histidine (H) or Tryptophan (W), wherein at least one of X1, X2 and X3 is Tryptophan Included)

In the endosome escape sequence motif according to the present invention, the endosome escape sequence motif may be located in the light chain variable region, the heavy chain variable region or the light chain variable region and the heavy chain variable region. The X1-X2-X3 may be composed of WWH, WYW, YWW, WYH, or YWH amino acid, in the endosomal structure motif according to the present invention. More specifically, X1-X2-X3 may be located at amino acids 92, 93 and 94 from the N-terminus of the light chain variable region. X1-X2-X3 may be located at the 96th, 97th and 98th amino acids from the N-terminus of the heavy chain variable region. The X1-X2-X3 may also be located at positions 96, 97 and 98 from the N-terminus of the light chain variable region, and from the N-terminus of the heavy chain variable region.

In the endosomal escape structure motif according to the present invention, the endosomal escape structure motif is located in the light chain variable region, and the CDR3 is QQYWWHMYT (SEQ ID NO: 1), QQYWYWMYT (SEQ ID NO: 2), QQYYWWMYT (SEQ ID NO: 4)? QQYYWHMYT (SEQ ID NO: 5).

In the endosomal escape structure motif according to the present invention, the endosomal cleavage structural motif is located in the heavy chain variable region, and the CDR3 is GWYWMDL (SEQ ID NO: 6), GWYWFDL (SEQ ID NO: 7), GWYWGFDL (SEQ ID NO: 9).

In the endosomal escape structure motif according to the present invention, the endosomal escape structure motif may protrude from the acidic hydrogen ion concentration to the surface of the cytoplasmic penetration antibody to form perforations, more specifically, temporary and reversible perforations in the endosomal membrane have.

2. Light chain variable region  And / or Heavy chain variable region

(1) light chain variable region

According to another embodiment of the present invention,

CDR3 (Complementarity determining region-3) discloses a light chain variable region that confer endosomal escape ability to an antibody, including an endosomal cleavage motif. Wherein the light chain variable region comprises:

i) CDR1 of SEQ ID NO: 10,

ii) CDR2 of SEQ ID NO: 11, and

iii) CDR3 comprising an endosomal escape structural motif comprising the amino acid sequence shown below.

-X1-X2-X3-

(Wherein,

X1, X2 and X3 are independently selected from the group consisting of Phenylalanine (F), Tyrosine (Y), Histidine (H) or Tryptophan (W), wherein at least one of X1, X2 and X3 is Tryptophan Included)

In the light chain variable region according to the present invention, X1-X2-X3 may be composed of WWH, WYW, YWW, WYH or YWH amino acids. X1-X2-X3 may be located at amino acids 92, 93 and 94 from the N-terminus of the light chain variable region.

In the light chain variable region according to the present invention, the CDR3 is a group consisting of QQYWWHMYT (SEQ ID NO: 1), QQYWYWMYT (SEQ ID NO: 2), QQYYWWMYT (SEQ ID NO: 3), QQYWYHMYT (SEQ ID NO: 4) and QQYYWHMYT , ≪ / RTI >

In the light chain variable region according to the present invention, the light chain variable region is a structural transformation inducing portion inducing a structural change of an endosomal cleavage structure motif. The light chain variable region includes a first amino acid (a1) from the N terminus and a 95th amino acid Th amino acid (a95). The a1 may be Aspartate (D) or Glutamate (E), and the a95 may be Methionine (M), Leucine (L), or Isoleucine (I). The distance between the a1 and the a95 in the structure conversion inducing portion changes according to the endosomal inner hydrogen ion concentration, thereby changing the structure of the endosome escape structure motif.

In the light chain variable region according to the present invention, the light chain variable region may include an amino acid sequence selected from the group consisting of SEQ ID NOS: 14 to 18.

(2) heavy chain variable region

According to another embodiment of the present invention,

CDR3 (Complementarity determining region-3) discloses a heavy chain variable region that encompasses endosomal escape structural motifs and confer endosomal escape ability to the antibody. Wherein the light chain variable region comprises:

i) CDR1 of SEQ ID NO: 12,

ii) CDR2 of SEQ ID NO: 13, and

iii) an endosome escape structural motif comprising the amino acid sequence shown below.

-X1-X2-X3-

(Wherein,

X1, X2 and X3 are independently selected from the group consisting of Phenylalanine (F), Tyrosine (Y), Histidine (H) or Tryptophan (W), wherein at least one of X1, X2 and X3 is Tryptophan Included)

In the heavy chain variable region according to the present invention, X1-X2-X3 may be composed of WWH, WYW, YWW, WYH or YWH amino acids. X1-X2-X3 may be located at the 96th, 97th and 98th amino acids from the N-terminus of the heavy chain variable region.

In a heavy chain variable region according to the invention, said CDR3 comprises an amino acid sequence selected from the group consisting of GWYWMDL (SEQ ID NO: 6), GWYWFDL (SEQ ID NO: 7), GWYWGFDL (SEQ ID NO: 8) and YWYWMDL can do.

In the heavy chain variable region according to the present invention, the heavy chain variable region is a structural transformation inducing portion that induces a structural change of the endosome-excretory structural motif. The heavy chain variable region has a first amino acid (a1) from the N- Th amino acid (a102). The a1 may be Aspartate (D) or Glutamate (E), and the a102 may be Methionine (M), Leucine (L), or Isoleucine (I). The distance between a1 and a102 in the structure conversion inducing portion changes according to the endosomal inner hydrogen ion concentration, thereby changing the structure of the endosome escape structure motif.

In the heavy chain variable region according to the present invention, the heavy chain variable region may comprise an amino acid sequence selected from the group consisting of SEQ ID NOS: 19 to 22.

(3) light chain variable region and heavy chain variable region

A light chain variable region and a heavy chain variable region are disclosed that include an endosomal cleavage structural motif in CDR3 (Complementarity determining region-3) to confer endosomal elimination capability to the antibody.

Wherein the light chain variable region comprises CDR1 of SEQ ID NO: 10, CDR2 of SEQ ID NO: 11, and CDR3 selected from the group consisting of SEQ ID NOs: 1-5,

The heavy chain variable region may comprise CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 13, and CDR3 selected from the group consisting of SEQ ID NOs: 6-9.

 In the light chain variable region and the heavy chain variable region according to the present invention, the light chain variable region is a structural transformation inducing portion inducing a structural change of an endosomal cleavage structure motif, wherein the light chain variable region comprises a first amino acid (a1) And may further include an interacting 95th amino acid (a95). The a1 may be Aspartate (D) or Glutamate (E), and the a95 may be Methionine (M), Leucine (L), or Isoleucine (I). The distance between the a1 and the a95 in the structure conversion inducing portion changes according to the endosomal inner hydrogen ion concentration, thereby changing the structure of the endosome escape structure motif. The light chain variable region may comprise an amino acid sequence selected from the group consisting of SEQ ID NOS: 14 to 18.

In the light chain variable region and the heavy chain variable region according to the present invention, the heavy chain variable region is a structural transformation inducing portion that induces a structural change of an endosomal escape structural motif. The structural change inducing portion includes a first amino acid (a1) And may further comprise an interacting 102nd amino acid (a102). The a1 may be Aspartate (D) or Glutamate (E), and the a102 may be Methionine (M), Leucine (L), or Isoleucine (I). The distance between a1 and a102 in the structure conversion inducing portion changes according to the endosomal inner hydrogen ion concentration, thereby changing the structure of the endosome escape structure motif. The heavy chain variable region may comprise an amino acid sequence selected from the group consisting of SEQ ID NOS: 19 to 22.

3. Endo Ability to escape  Improved Antibodies

According to another embodiment of the present invention,

An antibody is disclosed that comprises a light chain variable region, a heavy chain variable region, or a light chain variable region and a heavy chain variable region, wherein the complementarity determining region (CDR3) comprises an endosomal cleavage structural motif to confer endosomal elimination capability to the antibody.

In the antibody with improved endosomal eliminability according to the present invention, the antibody may be a complete immunoglobulin type antibody or a fragment thereof. The antibody may be a mouse, chimeric, human, or humanized antibody. The antibody may also be of the IgG, IgM, IgA, IgD or IgE type and may be of the IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA1, IgA5 or IgD type, Lt; / RTI > type monoclonal antibody.

In the antibody having enhanced endo-escape ability according to the present invention, the antibody may be located in the cytoplasm by intrusion of the endosome after intracellularization. The antibody can target the cytoplasm, nucleus, mitochondria, endoplasmic reticulum, or intracellular polymer. The intracellular polymer may be a protein, a substrate, DNA or RNA. The protein may be related to cell growth, cell proliferation, cell cycle, DNA repair, DAN conservation, transcription, replication, translation or control of intracellular migration. The protein may be modified or activated with a phosphate group, a carboxylic acid group, a methyl group, a sulfate group, a lipid, a hydroxyl group or an amide group.

4. Pharmaceutical composition for preventing or treating cancer

According to another embodiment of the present invention,

An antibody having enhanced endo-escape ability according to the present invention; And

Disclosed are pharmaceutical compositions for the prevention or treatment of cancer comprising a bioactive molecule selected from the group consisting of peptides, proteins, small molecule drugs, nanoparticles and liposomes fused to the antibody.

The term " fusion ", as used herein, refers to the integration of two molecules of different function or structure, including but not limited to any physical, chemical, or biological Fusion method. The fusion may preferably be with a linker peptide, which can relay the fusion with the bioactive molecule at various positions of the antibody light chain variable region, antibody, or fragment thereof of the invention.

The pharmaceutical composition for preventing or treating cancer according to the present invention is a pharmaceutical composition for preventing or treating cancer, wherein the cancer is squamous cell carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, skin cancer, The present invention relates to the use of a compound of formula I or a pharmaceutically acceptable salt or solvate thereof for the treatment or prophylaxis of a cancer selected from the group consisting of a malignant tumor, a rectum, a rectum, an anal canal, an esophagus cancer, a small intestine cancer, an endocrine cancer adenocarcinoma, And may be selected from the group consisting of cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, colon cancer, endometrial or uterine cancer, salivary cancer, kidney cancer, liver cancer, prostate cancer, mucin cancer, thyroid cancer, liver cancer and head and neck cancer .

5. Diagnostic pharmaceutical composition of cancer

According to another embodiment of the present invention,

An antibody having enhanced endo-escape ability according to the present invention; And

Disclosed are diagnostic pharmaceutical compositions comprising a bioactive molecule selected from the group consisting of peptides, proteins, small molecule drugs, nanoparticles and liposomes fused to the antibody.

As used herein, the term "diagnosis" means identifying the presence or characteristic of pathophysiology. The diagnosis in the present invention is to confirm the onset and progress of cancer.

In the pharmaceutical composition for diagnosing cancer according to the present invention, the cytoplasmic penetration antibody can be combined with a fluorescent material for a molecular imaging, a fluorescent protein or other imaging substance to diagnose cancer through imaging. The fluorescent material may include fluorescein, BODYPY, Trtramethylrhodamine, Alexa, Cyanine, allopicocyanine, or derivatives thereof, , But is not limited thereto. The fluorescent protein may include, but is not limited to, a Dronpa protein, a fluorescent coloring gene (EGFP), a red fluorescent protein (DsRFP), or a cyanine fluorescent material Cy5.5 that exhibits near infrared fluorescence. The other imaging material may include, but is not limited to, iron oxide or radioactive isotopes.

6. Endo Ability to escape  Method for producing improved antibodies

According to another embodiment of the present invention,

A method for producing an antibody having enhanced endosomal escape ability, comprising the step of grafting CDR3 comprising an endosomal escape structural motif comprising the amino acid sequence shown below in the CDR3 of the antibody.

-X1-X2-X3-

(Wherein,

X1, X2 and X3 are independently selected from the group consisting of Phenylalanine (F), Tyrosine (Y), Histidine (H) or Tryptophan (W), wherein at least one of X1, X2 and X3 is Tryptophan Included)

In the method of the present invention, X1-X2-X3 may be composed of WWH, WYW, YWW, WYH or YWH amino acids.

In the method for producing an antibody having enhanced endosomal eliminability according to the present invention, X1-X2-X3 may be located at amino acids 92, 93 and 94 from the N-terminus of the light chain variable region. The CDR3 may comprise an amino acid sequence selected from the group consisting of QQYWWHMYT (SEQ ID NO: 1), QQYWYWMYT (SEQ ID NO: 2), QQYYWWMYT (SEQ ID NO: 3), QQYWYHMYT (SEQ ID NO: 4) and QQYYWHMYT .

In the method of the present invention, X1-X2-X3 may be located at the 96th, 97th and 98th amino acids from the N-terminal of the heavy chain variable region. The CDR3 may comprise an amino acid sequence selected from the group consisting of GWYWMDL (SEQ ID NO: 6), GWYWFDL (SEQ ID NO: 7), GWYWGFDL (SEQ ID NO: 8) and YWYWMDL (SEQ ID NO: 9).

In the method for producing an antibody having improved endosomal eliminability according to the present invention, the antibody having improved endosomal eliminability may include a light chain variable region selected from the group consisting of SEQ ID NOS: 23 to 28.

INDUSTRIAL APPLICABILITY The antibody having enhanced endo-escape ability according to the present invention is useful for medical drug development and manufacturing as well as basic medical research fields by infiltrating a bioactive molecule into a specific cell with high efficiency without using a special external protein delivery system Can be used. More specifically, the antibody having improved endo-escape ability according to the present invention is a tumor which is present in the cytoplasm classified as a target substance for the treatment of diseases using a small molecule drug, and has a structure complex interaction through a wide flat surface between protein and protein And high efficacy in the treatment and diagnosis of disease-related factors can be expected.

FIG. 1 shows the result of observing the transport process and stability of cytransmab TMab4 or TAT infiltrated into cells by a pulse-chase and confocal microscopy.
FIG. 2 shows the result of confirming the antibody directly through fluorescence labeling in a Ramos cell line through a confocal microscope to determine whether a cytotransmab can be introduced through the cell membrane according to pH and induce cell membrane passage of another substance.
FIG. 3A shows results obtained by perforating trypan blue without membrane permeability by Cytotransmab according to pH or by optical microscope in Ramos cell line.
FIG. 3B is a graph showing a quantitative comparison of the number of cells acquiring trypan blue.
FIG. 4A is a result obtained by observing the cell membrane perforation produced by Cytotransmab under the condition of pH 5.5, which is transient and reversible, through an optical microscope.
FIG. 4B is a graph showing a quantitative comparison of the number of cells acquiring the trypan blue.
FIG. 5 shows the results of analysis of cell membrane binding of cytotransmab and control antibody adalimumab by pH with a flow cytometer (FACS).
FIG. 6 shows the results of analysis of cell membrane lipid flip-flop induction ability of Cytotransmab and control antibody adalimumab by pH using a flow cytometer (FACS).
FIG. 7 is a schematic diagram showing a perforation formation model of Cytotransmab predicted through the above experiments.
FIG. 8 is a diagram showing amino acids exposed to structural changes and structural changes due to structural change of Cytotransmab according to pH, based on WAM modeling structure of light chain variable region (VL) of Cytotransmab.
9A is a graph showing the cytoplasmic infiltration ability of mutants in which light chain variable region (VL) first amino acid aspartic acid, 95th amino acid methionine substituted with alanine, glutamic acid and leucine, respectively, involved in induction of structural change of Cytotransmab at acidic pH, The result is confirmed.
FIG. 9b shows the results of a comparison between the first amino acid aspartic acid, the 95th amino acid methionine of light chain variable region (VL) involved in inducing a structural change of Cytotransmab at acidic pH, The number of cells obtained was quantitatively compared.
FIG. 10 is a graph showing the quantitative comparison of the number of cells acquiring 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 CDR3 in the light chain variable region (VL) It is a graph.
Figure 11a shows the results of 12% SDS-PAGE analysis under reducing or nonreducing conditions after purification of expected mutations that improved the cytotransmab endosomal export.
Fig. 11B shows the result of observing through the confocal microscope whether cytoplasmic infiltration ability of expected mutation enhancing ability of Cytotransmab endosomal exudation is maintained.
FIG. 12 is a graph showing quantitative comparison of the number of cells acquiring the trypan blue according to the pH by the Cytotransmab wild type and the endosomal enhancing ability expected mutation according to the pH.
FIG. 13A shows the results of observing the migration of cytotransmab wild type or endosomal escape enhancing mutant TMab4-WYW into the cytoplasm using a calcein using a confocal microscope.
FIG. 13B is a bar graph quantifying calcein fluorescence of the confocal microscope photograph of FIG. 12A. FIG.
FIG. 14A 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. 14B is a graph comparing quantitative comparison of the number of cells acquiring trypan blue according to pH by mutation substituted with arginine, isoleucine and glycine, which are amino acids having opposite properties to tryptophan.
FIG. 14C is a graph showing the movement of a mutant substituted with arginine, isoleucine, and glycine, which are amino acids having opposite properties to tryptophan, to the cytoplasm of a cytoplasm using a calcein and a bar graph plotting the calcein fluorescence of a confocal microscope photograph to be.
15A is a schematic diagram illustrating the construction of a Cytotransmab construct having a light chain variable region eliminating endosomal elimination ability and an improved endosomal exporting motif introduced heavy chain variable region.
15B is a graph comparing quantitatively the number of cells acquiring trypan blue depending on the pH by the Cytotransmab having the light chain variable region from which endosomal elimination ability was removed and the heavy chain variable region containing the improved endosomal elimination ability motif.
FIG. 16 is a schematic diagram illustrating a process in which GFP fluorescence is observed by complementary binding of an improved segmented green fluorescent split protein when the cytotransmab is located in the cytoplasm.
Figure 16 shows the results of 12% SDS-PAGE analysis under reducing or nonreducing conditions after purification of GFP11-SBP2 fused cytotransmab.
FIG. 17A shows the result of observing GFP fluorescence through a confocal microscope due to the complementary binding of the improved segmented green fluorescent protein of the GFP11-SBP2-fused cytotransmab wild type and endosomal escape enhancing mutation.
FIG. 17B is a graph quantifying GFP fluorescence in the confocal microscope photograph of FIG. 20A. FIG.
FIG. 18A 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. 19A is a schematic diagram illustrating the construction of an anti-tubulin Cytotransmab in the form of a complete IgG to confirm the activity of endosomal enhancing mutations.
Figure 19b shows the result of 12% SDS-PAGE analysis under reducing or nonreducing conditions after purification of anti-tubulin Cytotransmab in the form of complete IgG.
FIG. 19C shows the result of confocal microscopy to determine whether the anti-tubulin Cytotransmab in the form of a complete IgG overlaps with tubulin, a cytoskeleton located in the cytoplasm.
20A is a schematic diagram illustrating the construction of an anti-RAS iMab in the form of a complete IgG to confirm the activity of an endosomal enhancing mutation.
Figure 20b shows the results of 12% SDS-PAGE analysis under reducing or nonreducing conditions after purification of anti-RAS iMab in the form of complete IgG.
FIG. 20C shows the confluence of a complete IgG-type anti-RAS iMab and an intracellularly activated H-RAS G12V mutation by a confocal microscope.
21A is a schematic diagram illustrating the construction of an anti-RAS iMab in the form of a full IgG containing a monoclonal antibody backbone that provides improved endosomal eliminability.
FIG. 21B shows fluorescence microscopic observation of the HSPG binding capacity and cytoplasmic permeability of the anti-RAS iMab of the complete IgG form including the monoclonal antibody backbone with improved endosomal elimination ability reduced or eliminated.
FIG. 21C is a graph comparing quantitatively the number of cells acquiring the trypan blue according to the pH by the anti-RAS iMab of the complete IgG form including the monoclonal antibody backbone with improved endosomal elimination ability.
22A is a result of performing an 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.
22B is a schematic diagram illustrating the construction of an anti-RAS iMab in the form of a full IgG comprising a monoclonal antibody backbone with RGD10 peptide fused enhanced endosomal elimination capability.
FIG. 22C shows the confluency of an intracellularly-activated H-RAS G12V mutation with an anti-RAS iMab in the form of a complete IgG including a monoclonal antibody backbone having an improved endosomal elimination ability fused with an RGD10 peptide to be.
23A is a schematic diagram illustrating construction of a cytoplasmic penetration antibody in the form of a complete IgG comprising a light chain variable region having an improved endosomal introduction heavy chain variable region and a monoclonal antibody framework imparting improved endosomal elimination ability.
FIG. 23B shows the migration of the intact cytoplasmic penetration antibody into the cytoplasm of a full IgG form containing a light chain variable region having an improved endosome-introduced motif-introduced heavy chain variable region and a monoclonal antibody framework imparting improved endosomal elimination ability, , And the calcein fluorescence of the confocal microscope photograph is quantified.
FIG. 23C shows the effect of pH of the complete IgG-type cytoplasmic penetration antibody containing the modified endosome-derived motif-introduced heavy chain variable region and the light chain variable region having the monoclonal antibody framework imparted with improved endosomal elimination ability, This is a graph comparing quantitatively the number of cells obtained.

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

< Example >

Example  One. Cytotransmab TMab4 &Lt; / RTI &gt;

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) and a heavy chain constant region (CH1-VH) of the antibody fused with a DNA encoding a secretion signal peptide at the 5 ' hinge-CH2-CH3) was cloned into pcDNA3.4 (Invitrogen) vector, NotI / HindIII, respectively. Further, in order to construct a vector expressing a light chain, a DNA encoding a light chain comprising a cytoplasmic penetration light chain variable region (hT4 VL) and a light chain constant region (CL) fused with a DNA encoding a secretion signal peptide at the 5 ' Cloned into pcDNA3.4 (Invitrogen) vector with NotI / HindIII.

The light and heavy chain expression vectors were 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). A 200mL transfection when, HEK293-F cells in shake flasks (Corning) 2 × 10 ⁶ to sowing in 100ml culture medium at a density of cells / ml, were incubated at 150 rpm, 8% CO _2. 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 protein was quantified using absorbance and extinction coefficient at 280 nm wavelength.

Example  2. Cytotransmab TMab4 After cell internalization Trafficking  Confirm

The cover slip was placed in 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 transiently transfected pcDNA3.4-flag-rab11 were incubated with 2 μl of Opti-MEM media (50 μl) and Lipofectamine 2000 (Invitrogen, USA) for 20 min at room temperature and added to each well. In addition, 450 μl of DMEM media without antibiotics was added and cultured at 37 ° C and 5% CO 2 for 6 hours. Then, the cells were replaced with 500 μl of DMEM medium containing 10% FBS and cultured for 24 hours. Then, 0.5 ml of fresh medium was treated with 3 μM of TMab4 for 30 minutes at 37 ° C., followed by rapid washing with PBS three times. The medium was added to the wells and cultured at 0, 2, and 6 hours at 37 ° C. 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, cells were fixed for 10 minutes at 25 ° C after addition of 4% paraformaldehyde. Then, the cells were washed with PBS and incubated with PBS containing 0.1% saponin, 0.1% sodium azide, and 1% BSA at 25 ° C for 10 minutes to form holes in the cell membrane. After washing with PBS, the reaction was carried out 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. As a result, TMab4, unlike TAT, was located in the initial endosomes up to 2 hours and was not transported to the lysosomal or recycling endosomes (Fig. 1).

Example  3. pH dependent Cytotransmab TMab4 of Endo  Confirmation of antibody entry by excretion

The intracytoplasmic layer of the Ramos cell line known to resemble the inner phospholipid layer of early endosomes was used to confirm cytoplasmic entry into the cytoplasm by endosomal escape of Cytotransmab TMab4. In a 24-well plate, 200 μl of a 0.01% poly-L-lysine solution is added to the plate to attach Ramos, a floating cell, to the plate and allowed to react for 20 minutes at 25 ° C. After washing with PBS, 5 × 10 ⁴ Ramos cells per well were added to 0.5 ml of medium containing 10% FBS and incubated 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 control antibody adalimumab directly labeled with DyLight-488 were added and cultured at 37 ° C. for 2 hours. Then, using a confocal microscope using the same method as in Example 2 Respectively. As a result, 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. Also, it was confirmed that TMab4 flows through the cell membrane at an acidic pH, and that not only itself but also other substances can be introduced (FIG. 2).

Example  4. pH dependent Cytotransmab TMab4 Identification of cell membrane morphology by passage of

Using the same method as in Example 3, trypan blue confirmation experiment without membrane permeability was carried out by Cytotransmab according to pH, and the result was observed with an optical microscope, and the number of cells obtained with trypan blue (Representing a mean value by counting more than 400 cells in total). As a result, it was confirmed that the trypan blue was obtained in proportion to the concentration in the TMab4-added cells only in the pH 5.5 condition, so that perforation was formed when the TMab4 passed through the cell membrane (FIG. 3).

In order to confirm whether the puncture formed by TMab4 was transient and reversible, the cells were allowed to have recovery time and observed with an optical microscope to quantify the number of cells obtained with trypan blue (total of 400 Number of cells or more). As a result, trypan blue accumulation was observed in TMab4-added cells at pH 5.5, but no recovery of trypan blue was observed in cells with recovery time in the medium. As a result, it was confirmed that the perforation by TMab4 was transient and reversible (Fig. 4).

In order to confirm the membrane binding and lipid membrane flip-flop mechanism of TMab4, TMab4 and control antibody adalimumab were incubated with an antibody that specifically recognizes human Fc to which FITC (green fluorescence) is linked and an Annexin-V And analyzed by flow cytometry (FACS). As a result, it was confirmed that only TMab4 was bound to the cell membrane at pH 5.5 (FIG. 5), and membrane lipid flip-flop occurred due to binding of Annexin-V only to TMab4 at pH 5.5 (FIG. 6).

The perforation model of Cytotransmab TMab4 expected through this example is shown in Fig.

Example  5. pH dependent Cytotransmab TMab4 Confirmation of structural change of

(1) Prediction model of structural change of Cytotransmab TMab4 by pH

Whether the passage of the cytotransmab through the membrane according to the pH according to Example 4 was caused by the change in the pH dependent Cytotransmab structure due to the Tanford transition was examined. Amino acids such as asparaginic acid (D) and glutamic acid (E) are added protonically as the pH decreases, resulting in loss of negative charge and hydrophobicity. Hydrophobic aspartic acid (D) and glutamic acid (E) This phenomenon is referred to as a tan-transition (Qin et al., 1998).

The hT4 VL structure, which is a cytoplasmic penetration light chain variable region, was used to investigate amino acids capable of exhibiting a carbon transition, and the first and 95th amino acid pairs from the N-terminus having a side-chain distance of less than 7 ~ As the candidates to represent. The 95th amino acid is an amino acid existing in CDR-L3 which is a unique sequence of hT4 VL which is a cytoplasmic penetration light chain variable region, and it is confirmed that it is possible to induce the change of CDR-L3 ring structure by interaction with the first amino acid . FIG. 8 shows the amino acids that are involved in the structural changes and the amino acids exposed by the structural changes by predicting the structural change of the Cytotransmab according to the pH based on the WAM modeling structure of the light chain variable region (VL) of the Cytotransmab.

(2) Confirmation of endosomal escape by amino acid pair showing the effect of the tan-pod transition

(A) and glutamic acid (E) or leucine (L) having properties similar to those of the amino acid are substituted for the first and the 95th amino acid pair to induce the pH-dependent structural change and thereby the endosomal escape. One mutation was constructed as shown in Table 1 below.

[Table 1]

Then, in the same manner as in Example 1, expression and purification were carried out in HEK293F cell line. Then, the presence or absence of the mutated antibody due to passage of the cell membrane through the pH was observed through a trypan blue confirmation experiment, The number of cells obtained was quantitated (averaging over 400 cells total). As a result, the TMab4-D1A and TMab4-M95A mutations showed little trypan blue acquisition unlike TMab4, while the TMab4-D1E and TMab4-M95L mutations showed similar trypan blue acquisitions to TMab4. As a result, it was confirmed that the first amino acid and the 95th amino acid play an important role in endo escape (FIG. 9).

(3) Of Cytotransmab Endo Ability to escape  Exploring contributing amino acids and motifs

In order to confirm the endosomal escape according to the change of the CDR3 loop structure, amino acids showing possibility of being exposed to the surface among the amino acids constituting CDR3 were selected and constructed as shown in Table 2 below in which alanine (A) Using the same method, the influx of the mutant antibody was observed through the trypan blue confirmation experiment and the number of cells that obtained the trypan blue was quantified (a total of 400 or more cells was counted to represent the average value).

[Table 2]

As a result, mutations of TMab4-Y92A, TMab4-Y93A, and TMab4-H94A significantly reduced trypan blue acquisition compared to TMab4. In particular, mutations of TMab4-AAA rarely produced trypan blue accumulation. On the other hand, TMab4-Y91A and TMab4-Y96A showed similar trypan blue acquisitions as TMab4. As a result, it was confirmed that amino acids 92, 93, and 94 greatly contribute to endosomal escape (FIG. 10).

Example  6. Endo Ability to escape  Improved Light chain variable region  Have Cytotransmab Manufacturing

TMAB4-WYW, TMab4-YWW, TMab4-WYH, TMab4-YWH (TM), and TMab4-YWH for the 92nd tyrosine (Y), 93rd tyrosine (Y) and 94th histidine (H) amino acid confirmed through Example 4, , TMab4-RYR, TMab4-IYI and TMab4-GYG mutants were prepared, and the same method as in Example 4 was used to confirm the endosomal escape of the mutant-containing antibody.

(1) TMab4-WWH, TMab4-WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH mutants

[Table 3]

After the purification of Cytotransmab including TMab4-WWH, TMab4-WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH mutations, analysis by 12% SDS-PAGE under reducing or nonreducing conditions revealed that Had a molecular weight of 150 kDa and had a molecular weight of the heavy chain of 50 kDa and light chain of 25 kDa under reducing conditions (FIG. 11A). Thus, the Cytotransmab including TMab4-WWH, TMab4-WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH mutations are present in a single state in solution and do not form duplexes or oligomers through non- .

The cytoplasmic permeability of Cytotransmab including TMab4-WWH, TMab4-WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH mutations was observed through a confocal microscope using the same method as in Example 2, It was confirmed that cytoplasmic permeability was maintained in all the mutants (Fig. 11B).

In order to confirm the pH-dependent endosomal elimination ability of Cytotransmab including TMab4-WWH, TMab4-WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH mutations, Experiments were performed and the number of cells harvested from Trypan blue was quantified (averaging over 400 cells total). As a result, TMab4-WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH increased the trypan blue acquisition ratio among the five mutants, and it was confirmed that TMab4-WYW had a pH-dependent trypan blue acquisition (Fig. 12). Thus, TMab4-WYW with increased pH-dependent trypan blue capture was selected as the final clone, while retaining the cytoplasmic permeability of the wild-type.

The movement of TMab4-WYW selected as the final clone into the cytoplasm was observed with a confocal microscope using a calcein using the same method as in Example 2, and the image was analyzed using Image J software (National Institutes of Health, USA) (Representing the average value of fluorescence obtained after 20 cells were designated in each condition). As a result, it was confirmed that the above-mentioned TMab4-WYW showed strong intensity of green calcein fluorescence even at a lower concentration than that of TMab4 (FIG. 13).

(2) TMab4-RYR, TMab4-IYI and TMab4-GYG mutations

[Table 4]

To confirm whether cell membrane binding of Cytotransmab including the TMab4-RYR, TMab4-IYI and TMab4-GYG mutations occurred according to pH, the same method as in Example 3 was used and then analyzed by flow cytometry (FACS) Respectively. As a result, it was confirmed that only TMab4 binds to the cell membrane at pH 5.5 (Fig. 14A).

In order to confirm the pH-dependent endosomal elimination ability of Cytotransmab including TMab4-RYR, TMab4-IYI and TMab4-GYG mutations, a trypan blue confirmation experiment was performed using the same method as in Example 5, The number of cells obtained was quantified and compared to TMab4-WYW (representing a mean of more than 400 cells total). As a result, compared to TMab4-WYW, TMab4-RYR, TMab4-IYI and TMab4-GYG were found to decrease the trypan blue acquisition (Fig. 14B).

In addition, the migration of the cytotransmab including the TMab4-RYR, TMab4-IYI, and TMab4-GYG mutations to the cytoplasm was observed using a confocal microscope using calcein using the same method as in Example 2, Respectively. As a result, in the cells treated with TMab4-RYR, TMab4-IYI and TMab4-GYG, the intensity of the green calcein fluorescence scattered in the cytoplasm was weaker than that of the cells treated with TMab4-WYW (FIG. Thus, it has been proved that endosomal escape ability can be improved by substituting tryptophan (W) at amino acids 92, 93 and 94. The amino acid sequences of Cytotransmab including TMab4-WWH, TMab4-WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH mutations are shown in SEQ ID NOS: 14-18.

Example  7. Endo Ability to escape  Improved The heavy chain variable region  Have Cytotransmab Manufacturing

(1) Confirmation of the motif area

As in the case of Example 5, the amino acid capable of exhibiting the carbon transition was examined through the sequence and structure of the heavy chain variable region, and the first amino acid from the N-terminus and the 102nd amino acid pair located at CDR3 were selected as candidates capable of exhibiting the carbon- And the 96th, 97th and 98th amino acids were confirmed to be the endothermic escape structure motif region using the same method as in Example 6 above. On the other hand, the number of amino acids in CDR3 of the wild type heavy chain variable region is 11, and the center portion of the ring is exposed to the surface of the structure, so that the number of amino acids of CDR3 is 7 or 8 to facilitate the pH- Respectively.

(2) Confirming endotoxin excretion of Cytotransmab

In order to confirm the endosomal elimination ability of the TMAB4-WYW selected in Example 6 above, TMab4-AAA in which the endosomal cleavage motif was removed from the light chain variable region was prepared, and the 96th , 97th and 98th amino acids were substituted with WYW to prepare Cytotransmab TMab4-HW1-AAA, TMab4-HW2-AAA, TMab4-HW3-AAA and TMab4-HW4-AAA (FIG.

[Table 5]

HW1-AAA (HT0-HW1 VH), TMab4-HW2-AAA (HT0-HW2 VH), TMab4-HW1-AAA (HT0-HW3 VH), and TMab4- Cell permeability is determined by the number of cells that have undergone the trippin blue confirmation experiment and acquired trypan blue (representing averages by counting more than 400 cells total). As a result, trypan blue acquisition was observed at pH 5.5 in both TMab4-HW1-AAA, TMab4-HW2-AAA, TMab4-HW3-AAA and TMab4-HW4-AAA mutants (FIG. Thus, it has been proved that endosomal escape is possible even when the improved endosomal structural motif is given to the heavy chain variable region, and finally it can be located inside the cytoplasm.

< Experimental Example >

Experimental Example  One. GFP11 - SBP2  Fused Cytotransmab Of the cytoplasm GFP  Fluorescence confirmation

FIG. 16 is a schematic diagram showing a process in which GFP fluorescence due to complementary binding of an improved segmented green fluorescent protein is observed when Cytotransmab is located in the cytoplasm. Specifically, we used a complementary system of improved segmented green fluorescent proteins to directly confirm that the cytotransmab 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 cytotransmab. Streptavidin and streptavidin-binding peptide 2 (SBP2) having high affinity were fused to GFP fragments, respectively, in order to complement complementary binding between GFP fragments. Thus, the observation of GFP fluorescence disproves that the cytotransmab is located in the cytoplasm.

(1) Expression and purification of GFP11-SBP2 fused cytotransmab

GFP11-SBP2 GFP11-SBP2 was fused genetically to the heavy chain C-terminus using GGGGS 3 linkers for the expression of animal cells in fused cytotransmabs. Then, an animal expression vector in which an expression vector encoding GFP11-SBP2 and a heavy chain encoding an endogenous light chain and endosomal enhancer-enhancing cytoplasmic penetration light chain were simultaneously transfected into HEK293F protein expressing cells (transient transfection ). The purification of the GFP11-SBP2 fused cytotransmab was carried out in the same manner as in Example 1, and the results of 12% SDS-PAGE under reducing or non-reducing conditions are shown in FIG.

As a result, a molecular weight of about 150 kDa was confirmed under non-reducing conditions, and a molecular weight of a heavy chain of 50 kDa and a light chain of 25 kDa were shown under reducing conditions. This indicates that the purified purified GFP11-SBP2 fused cytotransmab is present as a single entity in solution and does not form a duplex or an oligomer through an unnatural disulfide bond.

(2) Confirmation of GFP fluorescence by cytoplasmic location of GFP11-SBP2 fused cytotransmab

When the cells were stabilized, transformed HeLa cell lines stably expressing SA-GFP1-10 were prepared in the same manner as in Example 2. Cells were stained with PBS, TMab4-GFP11-SBP2, TMab4-WYW-GFP11-SBP2 0.2, 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, cells were immobilized in the same manner as in Example 2. The nuclei were stained (blue fluorescence) using a Hoechst 33342, observed with a confocal microscope, and quantitated (representing the average value of fluorescence obtained after specifying 20 cells in each condition). As a result, it was confirmed that TMab4-WYW showed strong intensity of green GFP fluorescence even at a lower concentration than TMab4 (FIG. 18).

In addition, the intracellular concentration of endotoxin-enhanced cytotransmab and endotoxin excretion efficiency of cytotransmab of GFP11-SBP2 fused complete IgG form located in cytoplasm are compared with each other in Table 6 below.

[Table 6]

Experimental Example  2. Complete IgG  Lt; RTI ID = 0.0 & Tubulin Cytotransmab Of target proteins in cells

(1) Construction of anti-tubulin Cytotransmab in complete IgG form

An anti-tubulin Cytotransmab in the form of a complete IgG containing a recombinant endosomal structural motif was prepared as shown in the schematic diagram shown in Fig. 19A. In order to express an animal cell of an anti-tubulin cytoplasmic penetration monoclonal antibody in a complete IgG form, a DNA encoding a secretion signal peptide is fused at the 5 'end as in Example 1, and a heavy chain that specifically binds to tubulin, The DNA encoding the heavy chain including the variable region and the heavy chain constant region (CH1-hinge-CH2-CH3) was cloned into pcDNA3.4 (Invitrogen) vector as NotI / HindIII. Then, an animal expression vector in which the heavy chain encoding a heavy chain variable region that specifically binds to the constructed animal expression vector coding for TMab4-WYW and the constructed heavy chain variable region was transiently transfected into HEK293F protein expressing cells transfection). Then, purification of the anti-tubulin Cytotransmab in the form of complete IgG was carried out in the same manner as in Example 1, and 12% SDS-PAGE was analyzed on reducing and non-reducing conditions after purification. As a result, it was shown to have a molecular weight of about 150 kDa under non-reducing conditions, and it was confirmed to have a molecular weight of a heavy chain of 50 kDa and a light chain of 25 kDa in a reducing condition (Fig. 19B). As a result, it was confirmed that an anti-tubulin Cytotransmab in the form of a purified purified full IgG was present as a single substance in a solution state and did not form a dendritic cell or an oligomer through an unnatural disulfide bond.

(2) Specific binding of the anti-tubulin Cytotransmab in complete IgG form to the cytoskeletal tubulin

Confocal microscopy was used to confirm the overlap between anti-tubulin Cytotransmab in the form of a complete IgG and tubulin in the cytoplasm. As a result, TuT4-WYW of green fluorescence was superimposed in a fibrous shape on the cytoplasmic part where the tubulin of red fluorescence was located, whereas TuT4 was not overlapped (Fig. 19C). This demonstrated that the intracellular cytoskeletal tubulin and the complete IgG form of the anti-tubulin Cytotransmab specifically bind.

Experimental Example  3. Perfect IgG  Form of anti-RAS iMab Of target proteins in cells

(1) Production of anti-RAS iMab in complete IgG form

As shown in the schematic diagram shown in Fig. 20 (a), a complete IgG-type anti-RAS iMab containing a recombinant endosomal structural motif was prepared. RAS iMab of the full IgG form was fused with DNA encoding secretion signal peptide at the 5 'end as in Example 5, and specific binding to K-RAS to which GTP was coupled in a heavy chain variable region-dependent manner 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-encoded animal expression vector containing a heavy chain variable region that specifically binds to K-RAS in which the TMab4-WYW-encoding animal expression vector and the constructed GTP-bound K-RAS are specifically bound is transiently transfected into HEK293F protein- Transient transfection. Then, purification of the anti-RAS iMab in the form of complete IgG was carried out in the same manner as in Example 1, and 12% SDS-PAGE was analyzed under reducing and non-reducing conditions after purification. As a result, it was found to have a molecular weight of about 150 kDa under non-reducing conditions, but it was confirmed that under reducing conditions, the molecular weight of the heavy chain was 50 kDa and the light chain was 25 kDa. As a result, it was confirmed that the anti-RAS iMab in the form of fully purified IgG in the form of a purified IgG exists as a single body in a solution state, and does not form a dendritic cell or an oligomer through an unnatural disulfide bond (Fig. 20B).

(2) Specific binding of K-RAS to intracellular GTP-bound anti-RAS iMab in complete IgG form

Confirmation of overlap between anti-RAS iMab in the form of complete IgG and intracellularly activated H-RAS G12V mutation was confirmed using confocal microscopy. As a result, green fluorescence RT11 and RT11-WYW were superimposed on the intracellular part where red fluorescence activated RAS was located, while TMab4 was not overlapped (Fig. 20C). Thus, it was demonstrated that the intracellularly activated RAS and the complete IgG form of anti-RAS iMab specifically bind to each other.

Experimental Example  4. Improved Endo  Of the monoclonal antibody to which the excretory structural motif is grafted Endo Escape ability  Confirm

Among the currently available monoclonal antibodies, there are many types of monoclonal antibodies that target cell surface receptors, particularly cell surface receptors that undergo intracellular processing. Since these antibodies are not intracellularly bound, they do not lose their binding to the antigen, they do not migrate to the cytoplasm because they do not have endosomal elimination ability, and then they are released from the cells again. Therefore, when endosomal elimination ability can be given to the receptor-target antibody in which intracellular cleavage occurs, it is possible to apply it to a wider range than the present invention.

In order to give improved endosomal motility, we compared the sequence of the light chain variable region of the receptor-target antibody with the sequence of the light chain variable region of the cytotransmab in which the intracellular cleavage occurs and found that the first amino acid is negatively charged and affects the CDR3 loop structure The major amino acid residues in the skeleton are summarized in the same candidate light chain variable region sequence. The mutations that grafted CDR3 of the candidate light chain variable region to the CDR3 of the cytotransmab were constructed as shown in Table 7 below. When constructing this mutation, the 87th amino acid tyrosine was substituted with phenylalanine to increase the protein expression yield decreased 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.

[Table 7]

(1) Production of anti-RAS iMab in complete IgG form

A schematic diagram illustrating the construction of an anti-RAS iMab in the form of a complete IgG containing a monoclonal antibody backbone with improved endosomal eliminability is shown in Figure 21A. 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 transiently transfected into HEK293F protein expressing cells (transient transfection). Then, purification of the anti-RAS iMab in the form of complete IgG proceeded as in Example 1 above.

(2) confirmation of the ability to impart improved endosomal motility motifs to the monoclonal antibody backbone

HeLa cell lines were prepared as described in Example 2, and when cells were stabilized, 1 μM of PBS, RT11-WYW, RT11-Neci, RT11-Nimo, RT11-Pani, RT11- Lt; / RTI &gt; for 6 hours. The cells were washed with PBS and a weakly acidic solution and then subjected to cell fixation, cell perforation, and blocking in the same manner as in Example 2. 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. As a result, no fluorescence was observed in the cells in all of the anti-RAS iMabs of the complete IgG form including the monoclonal antibody backbone with six improved endosomal elimination functions (FIG. 21B).

In addition, the number of cells acquiring trypan blue according to the pH by the anti-RAS iMab of the complete IgG form including the monoclonal antibody backbone with the improved endosomal elimination ability was quantitatively compared (total of 400 or more cells Numbered and expressed as an average value). As a result, a trypan blue acquisition similar to that of RT11-WYW was observed in the case of the complete IgG type anti-RAS iMab containing the monoclonal antibody backbone with five improved endosomal elimination functions except RT11-Pert (Fig. 21c) .

Experimental Example  5. Improved Endo Escape ability  Complete with monoclonal antibody backbone IgG  Form of anti-RAS iMab's GTP end Combined  Confirmation of specific binding with K-RAS

(1) Determination of the affinity of the K-RAS mutant K-RAS G12D in the form in which GTP is combined with GDP

The GTP-bound form of the target molecule, K-RAS G12D, and the form bound to GDP were bound to 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. Then, the IgG type monoclonal antibodies RT11-WYW, RT11-Neci, RT11-Nimo, RT11-Pani, RT11-Pert, RT11-Lumr and RT11-Emib are combined 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. The affinity analysis for K-RAS mutation showed a difference in affinity with RT11-WYW in the case of anti-RAS iMab in the form of a complete IgG including a monoclonal antibody backbone with five improved endosomal elimination functions except for RT11-Nimo , And all clones did not bind to K-RAS conjugated with GDP used as a negative control (Fig. 22A)

(2) Production of full IgG type anti-RAS iMab containing monoclonal antibody backbone with RGD10 peptide fused improved endosomal elimination ability

The anti-RAS iMab in the form of a complete IgG containing a monoclonal antibody backbone with improved endothelial capacity has been shown to have specificity for integrin overexpressed in neovascular cells and various tumors Were genetically fused with two GGGGS linkers at the N-terminus of the light chain. 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. On the basis of the expression yield, endosomal elimination ability and affinity analysis test for Ras, the RGD10 peptide was fused to the N-terminus of the light chain variable region of RT11-Pani and RT11-Neci, which are excellent candidate antibodies (Fig. 22B ).

Then, the SW480 cell line was diluted to 0.5 ml with 2x104 cells per well and cultured in a 24-well plate for 12 hours at 37 ° C in 5% CO2. RT11-i-WYW, RT11-i-Neci, RT11- i-Pani was treated with 1 μM and cultured at 37 ° C for 12 hours. Then, the antibody and the nucleus were labeled under the same conditions as in Example 2, and the Ras labeled antibody was stained with a secondary antibody after the reaction at 1 hour and 37 degrees, and observed with a confocal microscope. As a result, RT11-i-WYW, RT11-i-Neci and RT11-i-Pani of green fluorescence were superimposed on the intracellular part where the activated RAS of red fluorescence was located. As a result of the above experiment, it was confirmed that the anti-RAS iMab of the full IgG form including the monoclonal antibody backbone with the activated RAS in the cell and the improved endosomal elimination ability binds specifically (FIG. 22C).

EXPERIMENTAL EXAMPLE 6 Improved endo- escape motif introduction The heavy chain variable region and the improved endo Given the ability to escape make monoclonal antibodies endosomes escape performance improvement of the full IgG form of the cellular penetration antibody comprising a light chain variable region having the skeleton.

(1) Fabrication of complete IgG type cytoplasmic penetration antibody

FIG. 23A is a schematic diagram illustrating the construction of a complete IgG-type cytoplasmic penetration antibody including a light chain variable region having an improved endo-excretory motif-introduced variable region and a monoclonal antibody framework imparting improved endosomal elimination ability. Using the same method as in Example 1, a light chain containing the heavy chain comprising the improved endosomal exporting heavy chain variable region and the light chain variable region having the monoclonal antibody framework imparting improved endosomal elimination ability Expression vectors 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.

(2) Confirmation of endo escape ability

Introduction of the improved endogenous exocytotic motif The migration of the cytoplasmic infiltrating antibody of the full IgG form containing the light chain variable region and the light chain variable region having the monoclonal antibody backbone imparted with improved endosomal elimination ability to the cytoplasm was investigated by using calcein Focus microscopy was used to quantify the calcein fluorescence of the confocal microscope photographs. As a result, in the cells treated with each concentration of TMab4-HW1-ep41, green calcein fluorescence scattered in the cytoplasm was stronger than that of TMab4-ep41 treated cells (Fig. 23B).

Improved endotoxin-induced motif introduction Complete-IgG-type cytoplasmic infiltration antibody containing a light chain variable region and a light chain variable region having a monoclonal antibody backbone giving improved endosomal elimination ability Were quantitatively compared. As a result, in the case of TMab4-HW1-ep41, it was observed that the trypan blue acquisition was increased in a concentration-dependent manner compared to TMab4-ep41 (FIG. 23C).

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.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> METHOD FOR INDUCING CONFORMATIONAL CHANGES IN THE COMPLEMENTARITY          DETERMINING REGIONS OF ANTIBODY <130> 2016-PP-30932 <160> 28 <170> Kopatentin 2.0 <210> 1 <211> 9 <212> PRT <213> VL CDR3 <400> 1 Gln Gln Tyr Trp Trp His Met Tyr Thr   1 5 <210> 2 <211> 9 <212> PRT <213> VL CDR3 <400> 2 Gln Gln Tyr Trp Tyr Trp Met Tyr Thr   1 5 <210> 3 <211> 9 <212> PRT <213> VL CDR3 <400> 3 Gln Gln Tyr Tyr Trp Trp Met Tyr Thr   1 5 <210> 4 <211> 9 <212> PRT <213> VL CDR3 <400> 4 Gln Gln Tyr Trp Tyr His Met Tyr Thr   1 5 <210> 5 <211> 9 <212> PRT <213> VL CDR3 <400> 5 Gln Gln Tyr Tyr Trp His Met Tyr Thr   1 5 <210> 6 <211> 7 <212> PRT <213> VH CDR3 <400> 6 Gly Trp Tyr Trp Met Asp Leu   1 5 <210> 7 <211> 7 <212> PRT <213> VH CDR3 <400> 7 Gly Trp Tyr Trp Phe Asp Leu   1 5 <210> 8 <211> 8 <212> PRT <213> VH CDR3 <400> 8 Gly Trp Tyr Trp Gly Phe Asp Leu   1 5 <210> 9 <211> 7 <212> PRT <213> VH CDR3 <400> 9 Tyr Trp Tyr Trp Met Asp Leu   1 5 <210> 10 <211> 17 <212> PRT <213> VL CDR1 <400> 10 Lys Ser Ser Gln Ser Leu Phe Asn Ser Arg Thr Arg Lys Asn Tyr Leu   1 5 10 15 Ala     <210> 11 <211> 7 <212> PRT <213> VL CDR2 <400> 11 Trp Ala Ser Thr Arg Glu Ser   1 5 <210> 12 <211> 5 <212> PRT <213> VH CDR1 <400> 12 Ser Tyr Val Met His   1 5 <210> 13 <211> 17 <212> PRT <213> VH CDR2 <400> 13 Ala Ile Asn Pro Tyr Asn Asp Gly Asn Tyr Tyr Ala Asp Ser Val Lys   1 5 10 15 Gly     <210> 14 <211> 114 <212> PRT <213> hT4-WWH 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 Trp Trp His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 15 <211> 114 <212> PRT <213> hT4-WYW 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 Trp Tyr Trp Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 16 <211> 114 <212> PRT <213> hT4-YWW 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 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> 17 <211> 114 <212> PRT <213> hT4-WYH VL <400> 17 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> 18 <211> 114 <212> PRT <213> hT4-YWH 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 Tyr Tyr Trp His Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile             100 105 110 Lys Arg         <210> 19 <211> 116 <212> PRT <213> HT0-HW1 VH <400> 19 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> 20 <211> 116 <212> PRT <213> HT0-HW2 VH <400> 20 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> 21 <211> 117 <212> PRT <213> HT0-HW3 VH <400> 21 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> 22 <211> 116 <212> PRT <213> HT0-HW4 VH <400> 22 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> 23 <211> 108 <212> PRT <213> Neci-WYW VL <400> 23 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> 24 <211> 108 <212> PRT <213> Pani-WYW VL <400> 24 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> 25 <211> 114 <212> PRT <213> Lumr-WYW VL <400> 25 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> 26 <211> 113 <212> PRT <213> Nimo-WYW VL <400> 26 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> 27 <211> 109 <212> PRT <213> Emib-WYW VL <400> 27 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> 28 <211> 108 <212> PRT <213> Pert-WYW VL <400> 28 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

Claims (8)

A first amino acid (a1) and an nth amino acid (an, n being 80 or more and less than 140) from the N-terminus of the light chain variable region, the heavy chain variable region or the light chain variable region and the heavy chain variable region,
A structural transformation induction unit inducing a conformational change of complementarity determining regions (CDRs) by interaction of a1 and an.
(Provided that the amino acid position is according to the Kabat number)

The method according to claim 1,
Wherein the structural change of the CDRs is induced by a change in the distance between a1 and an.
3. The method of claim 2,
The change in the distance between a1 and an is the result of induction of interaction between two periods depending on the hydrogen ion concentration. The distance between a1 and an in an acid ion concentration (d1) and the distance between a1 and an in neutral ion concentration and the value of d1 / d2, which is the ratio of (d2), is greater than 0 and less than 1.
The method of claim 3,
And a conformational change induction unit that induces a conformational change of an adjacent CDR3 when the value of d1 / d2 is less than 1.
5. The method of claim 4,
Wherein when the value of d1 / d2 is less than 1, structural change of CDR3 adjacent to an is reversibly perforated on an endosomal membrane after the antibody is intracellularly introduced.
5. The method of claim 4,
Wherein when the value of d1 / d2 is less than 1, structural change of CDR3 adjacent to an makes a difference in the binding ability of the antibody to the antigen according to the hydrogen ion concentration,
The method of claim 3,
Wherein the a1 is Aspartate (D) or Glutamate (E), and the an is Methionine (M), Leucine (L) or Isoleucine (I).
8. The method of claim 7,
Wherein the an is the light chain variable region is a95,
And the heavy chain variable region is a102.

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