KR20180116204A - Endosomal escape motif enabling antibody to escape from endosomes and uses thereof - Google Patents

Abstract

The present invention relates to endo-escape structural motifs imparting endosomal escape ability to an antibody and to uses thereof. More specifically, the present invention relates to the endo-escape structural motifs imparting endosomal escape ability to a cytosol-penetrating antibody (Cytotransmab), a light chain variable region and/or a heavy chain variable region comprising the endo-escape structural motifs, an antibody comprising the light chain variable region and/or the heavy chain variable region, a pharmaceutical composition for treatment, prevention or diagnosis of cancer comprising the antibody, and a method for producing the antibody having enhanced endo-escape ability.

Description

Endosomal escape motif enabling antibody to escape from endosomes and uses thereof {Endosomal escape motif enabling antibody to escape from endosomes and uses thereof}

The present invention relates to an endosomes escape structure motif and its utilization that gives the endosomes escape ability to the antibody. In more detail, the present invention provides an endosomes escape structure motif to give endosomes escape ability to a cytosol-penetrating antibody (Cytotransmab), a light chain variable region including the endosomes escape structure motif and/or heavy chain variable It relates to a region, an antibody comprising the light chain variable region and/or the heavy chain variable region, a pharmaceutical composition for treatment, prevention or diagnosis of cancer comprising the antibody, and a method of producing an antibody with improved endosomes escape ability.

Existing antibodies cannot penetrate directly into living cells due to their large size and hydrophilicity. Therefore, most of the existing antibodies specifically target proteins or cell membrane proteins secreted to the outside of cells. General antibodies and high molecular biopharmaceuticals have limitations in that they cannot bind to and inhibit various disease-related substances inside the cytoplasm because they cannot pass through hydrophobic cell membranes. In general, commercial antibodies that specifically bind to intracellular substances used in experiments for the study of mechanisms such as cell growth and specific inhibition cannot be directly processed on living cells, and in order to bind with intracellular substances, amphiphilic glycosides A pretreatment process is required to form a perforation in the cell membrane through a cell membrane permeabilization process using saponin.

Antibodies have high specificity, high affinity, and bind to a target protein, and many therapeutic antibodies have been developed as targets for proteins secreted to cell membranes or extracellularly. Antibodies targeting cell membrane proteins can bind to cell membrane proteins and then enter the inner cell through the endosomal pathway through receptor-mediated endocytosis, initially as early endosomes. Go and go through several routes. Specifically, 1) most of the antibodies go from the initial endosome to the late endosome, fused to the lysosome, and go to a path that is completely destroyed by acidic conditions and proteolytic enzymes, and 2) Some antibodies bind to the FcRn (neonatal Fc receptor) receptor in an initial endosome acidic condition, and may go through a pathway that goes out of the cell again through a recycling endosome pathway. Therefore, most of the antibodies are destroyed through the lysosome pathway by strong binding to the target membrane protein. As the endosomes mature in the endosome pathway, the inside is gradually acidified by a proton pump.The pH of the initial endosome is 5.5-6.5, the pH of the end endosome is 5.0-6.0, and the pH of the lysosome is 3.5. -4.5, and many proteolytic enzymes are activated inside the endosome, and proteins internalized from the outside are destroyed inside the endosome. Therefore, when the antibody moves along the endosome pathway after intracellular internalization by the receptor, before going to the lysosome, it must escape from the target membrane protein and escape by making a perforation in the endosome in order to escape from the early or late endosome and locate it in the cytoplasm. do.

It is known that viruses and toxins among the substances present in the natural system inside cells actively penetrate into living cells through endocytosis. In order for a substance that has penetrated into the cell by intracellular internalization to exhibit activity in the cytoplasm, the process of escaping from the endosome to the cytoplasm, “endosomal escape”, is essential. Although the endosomal escape mechanism is not yet clearly identified, there are three main hypotheses about the endosomal escape mechanism so far. The first hypothesis is the mechanism of forming perforations in the endosome membrane, and substances such as cationic amphiphilic peptides in the endosome membrane bind to the double lipid membrane of negatively charged cells, resulting in internal stress or contraction of the inner membrane. As a result, it forms a barrel-stave pore or a toroidal channel (Jenssen et al., 2006), and is called a pore formation mechanism. The second hypothesis is a mechanism in which endosomes burst by the proton sponge effect. Substances with amine groups by protonated amine groups increase the osmotic pressure of the endosomes through a high buffering effect. It can collapse (Lin and Engbersen, 2008). The third hypothesis maintains the shape of a hydrophilic coil at neutral, but in acids such as endosomes, a specific motif that transforms into a hydrophobic helical structure is fused with the endosomes membrane, and the hypothesis that viruses and toxins containing the motif escape the endosomes. It was named as a lipid membrane fusion mechanism. The three hypotheses have been proposed as endosomes escape mechanisms after intracellularization of viral proteins and plant/bacteria-derived toxin proteins, but such endosomes escape mechanisms have not been specifically elucidated in antibodies.

The phenomenon commonly observed in the above endosomes escape mechanism is that endosomes escape occurs under acidic pH conditions with low pH, which is an environment of endosomes and lysosomes. In the case of a protein whose function changes according to pH, its structure changes according to pH. The phenomenon that causes this change was called the Tanford transition (Qin et al., 1998). Negatively charged amino acids (aspartic acid (D), glutamic acid (E)) and hydrophobic amino acids (methionine (M), leucine (L), isoleucine (I)) under neutral pH conditions ) Has no interaction, but as the pH decreases, the carboxyl acids (COO-) of the negatively charged amino acids are hydrogenated (protonation) and become hydrophobic. Hydrophobic interaction becomes possible. As a result, the distance between the two amino acids becomes close, and the overall structure and function of the protein changes. For example, Nitrophorin 4, a nitrogen transport enzyme, has an open structure under neutral hydrogen ion concentration conditions, but performs a nitrogen transport function as it changes to a closed structure due to a hydrophobic interaction between aspartic acid and leucine as the pH decreases. (Di Russo et al., 2012). However, until now, the pH-dependent structural change has not been found in antibodies, and in particular, has not been reported in antibodies that undergo intracellular internalization.

Prior literature

1. Republic of Korea Patent Publication 10-2012-0026408

Therefore, the present inventors were studying the endosomes escape mechanism of the cytoplasmic penetration antibody (Cytotransmab) in the form of a complete immunoglobulin that penetrates into the cell and distributes in the cytoplasm. It was found that there is a specific region that plays a decisive role in imparting an endosomes, and based on this, an endosomes escape structure motif capable of remarkably improving the endosomes escape ability of an antibody was developed, and the present invention was completed. .

Therefore, the present invention is a light chain variable region and / or heavy chain variable region, the light chain variable region and / or heavy chain variable region including the endosomes escape structure motif, the endosomes escape structure motif for imparting endosomes escape ability to the antibody. An object comprising an antibody, a pharmaceutical composition for treating, preventing or diagnosing cancer comprising the antibody, and a method for producing an antibody having improved endosomes escape ability.

The term “endosome escape ability” as used herein refers to the ability to escape endosomes to be located in the cytoplasm after actively infiltrating living cells through intracellular internalization, and the term “endosome escape structure motif )” refers to a region that imparts endosomes escape ability to an internalized complete immunoglobulin-type cytoplasmic infiltrating antibody by a structural change in response to an acidic hydrogen ion concentration.

The term "amino acid" as used herein, in a broad sense, includes naturally occurring L α-amino acids or their residues, as well as D-amino acids and chemically modified amino acids. For example, it includes the amino acid mimics and analogs described above, and in the present invention, the mimics and analogs may include functional equivalents. All amino acid sequences provided by the present invention are shown using Kabat numbers.

The term "complete immunoglobulin type antibody" as used herein has a structure having two full-length light chains and two full-length heavy chains, and each light chain has a heavy chain and a disulfide bond (SS- bond) The constant region of the antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, Subclasses include gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

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

The term "light chain" as used herein refers to a full-length light chain and fragments thereof comprising a variable region domain VL and a constant region domain CL comprising an amino acid sequence having a sufficient variable region sequence to impart specificity to an antigen. All inclusive.

One. Endosome Escape structure motif

According to an embodiment of the present invention,

It is located in the light chain variable region, the heavy chain variable region, or the CDR3 (Complementarity determining region-3) of the light and heavy chain variable regions of the antibody, and contains the following amino acid sequence for imparting endosomes escape ability after the antibody is internalized into the cell. Endosomal escape motif (Endosomal escape motif) is disclosed.

-X1-X2-X3-

(In the above formula,

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

In the endosomes escape structure motif according to the present invention, the endosomes escape structure motif may be located in a light chain variable region, a heavy chain variable region or a light chain variable region and a heavy chain variable region. In the endosomes escape structure motif according to the present invention, the X1-X2-X3 may be made of WWH, WYW, YWW, WYH or YWH amino acids. 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. The X1-X2-X3 may be located at amino acids 96, 97, and 98 from the N terminal of the heavy chain variable region. In addition, X1-X2-X3 may be located at amino acids 92, 93, and 94 from the N-terminus of the light chain variable region, and at amino acids 96, 97, and 98 from the N-terminus of the heavy chain variable region.

In the endosomes escape structure motif according to the present invention, the endosomes escape structure motif is located in the light chain variable region, the CDR3 is QQYWWHMYT (SEQ ID NO: 1), QQYWYWMYT (SEQ ID NO: 2), QQYYWWMYT (SEQ ID NO: 3), QQYWYHMYT (SEQ ID NO: 4)? It may include an amino acid sequence selected from the group consisting of QQYYWHMYT (SEQ ID NO: 5).

In the endosomes escape structure motif according to the present invention, the endosomes escape structure 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: 8) and YWYWMDL It may include an amino acid sequence selected from the group consisting of (SEQ ID NO: 9).

In the endosomes escape structure motif according to the present invention, the endosomes escape structure motif protrudes from the surface of the cytoplasmic penetration antibody at an acidic hydrogen ion concentration to perforate the endosomes cell membrane, more specifically to form a temporary and reversible perforation. 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,

A light chain variable region that imparts endosomes escape capability to an antibody is disclosed, including an endosomes escape structure motif in CDR3 (Complementarity determining region-3). The light chain variable region is:

i) CDR1 of SEQ ID NO: 10,

ii) CDR2 of SEQ ID NO: 11, and

iii) may include a CDR3 comprising an endosomes escape structure motif comprising the following amino acid sequence.

-X1-X2-X3-

(In the above formula,

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

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. The X1-X2-X3 may be located at amino acids 92, 93, and 94 from the N terminal 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 (SEQ ID NO: 5). It may include an amino acid sequence selected from.

In the light chain variable region according to the present invention, the light chain variable region is a structural transformation inducing part that induces a structural change of an endosome escape structural motif, and the first amino acid (a1) from the N-terminus and 95 interacts with the first amino acid. May further include the th amino acid (a95). A1 may be Aspartate (D) or Glutamate (E), and a95 may be Methionine (M), Leucine (L) or Isoleucine (I). The distance between a1 and a95 in the structure conversion induction part changes according to the hydrogen ion concentration inside the endosome, and accordingly, the structure of the endosome escape structure motif may be changed.

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,

A heavy chain variable region is disclosed that includes an endosomes escape structure motif in CDR3 (Complementarity determining region-3) and imparts endosomes escape ability to an antibody. The light chain variable region is:

i) CDR1 of SEQ ID NO: 12,

ii) CDR2 of SEQ ID NO: 13, and

iii) It may contain an endosomes escape structure motif comprising the following amino acid sequence.

-X1-X2-X3-

(In the above formula,

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

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. The X1-X2-X3 may be located at amino acids 96, 97, and 98 from the N terminal of the heavy chain variable region.

In the heavy chain variable region according to the present invention, the CDR3 contains 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). 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 an endosome escape structure motif, and the first amino acid (a1) from the N-terminus and 102 interacts with the first amino acid. May further include the second amino acid (a102). A1 may be Aspartate (D) or Glutamate (E), and a102 may be Methionine (M), Leucine (L) or Isoleucine (I). The distance between a1 and a102 in the structure conversion induction part changes according to the hydrogen ion concentration inside the endosome, and accordingly, the structure of the endosome escape structure motif may be changed.

In the heavy chain variable region according to the present invention, the heavy chain variable region may include 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, including an endosomes escape structure motif in CDR3 (Complementarity determining region-3), which imparts endosomes escape ability to an antibody.

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

The heavy chain variable region may include CDR1 of SEQ ID NO: 12, CDR2 of SEQ ID NO: 13, and CDR3 selected from the group consisting of SEQ ID NOs: 6 to 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 part that induces a structural change of an endosome escape structure motif, and the first amino acid (a1) and the first amino acid from the N-terminus It may further include an interacting 95th amino acid (a95). A1 may be Aspartate (D) or Glutamate (E), and a95 may be Methionine (M), Leucine (L) or Isoleucine (I). The distance between a1 and a95 in the structure conversion induction part changes according to the hydrogen ion concentration inside the endosome, and accordingly, the structure of the endosome escape structure motif may be changed. The light chain variable region may include 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 structure conversion inducing part that induces a structural change of an endosome escape structure motif, and the first amino acid (a1) and the first amino acid from the N-terminus It may further include an interacting 102 th amino acid (a102). A1 may be Aspartate (D) or Glutamate (E), and a102 may be Methionine (M), Leucine (L) or Isoleucine (I). The distance between a1 and a102 in the structure conversion induction part changes according to the hydrogen ion concentration inside the endosome, and accordingly, the structure of the endosome escape structure motif may be changed. The heavy chain variable region may include an amino acid sequence selected from the group consisting of SEQ ID NOs: 19 to 22.

3. Endosome Escape ability Improved antibody

According to another embodiment of the present invention,

An antibody comprising a light chain variable region, a heavy chain variable region, or a light chain variable region and a heavy chain variable region, which imparts endosome escape capability to the antibody, including an endosome escape structure motif in CDR3 (Complementarity determining region-3) is disclosed.

In the antibody with improved endosomes escape ability according to the present invention, the antibody may be a complete immunoglobulin type antibody or fragment thereof. The antibody may be a mouse, chimeric, human, or humanized antibody. In addition, the antibody may be IgG, IgM, IgA, IgD or IgE, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA1, IgA5, or IgD type, most preferably IgG It may be a type of monoclonal antibody.

In the antibody having improved endosomes escape ability according to the present invention, the antibody may be located in the cytoplasm by endosomes escape after infiltrating through cell internalization. The antibody can target cytoplasm, nucleus, mitochondria, endoplasmic reticulum or intracellular polymers. 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 phosphoric acid 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 with improved endosomes escape ability according to the present invention; And

A pharmaceutical composition for preventing or treating cancer comprising a bioactive molecule selected from the group consisting of peptides, proteins, small molecule drugs, nanoparticles, and liposomes fused to the antibody is disclosed.

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

In the pharmaceutical composition for preventing or treating cancer according to the present invention, the cancer is squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, skin cancer, skin or intraocular black Tumor, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovary It may be selected from the group consisting of cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colon cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, and head and neck cancer. .

5. Pharmaceutical composition for diagnosis of cancer

According to another embodiment of the present invention,

An antibody with improved endosomes escape ability according to the present invention; And

A pharmaceutical composition for diagnosis of cancer comprising a bioactive molecule selected from the group consisting of peptides, proteins, small molecule drugs, nanoparticles, and liposomes fused to the antibody is disclosed.

As used herein, the term “diagnosis” refers to ascertaining the presence or characteristics of a pathophysiology. The diagnosis in the present invention is to confirm the onset and course of cancer.

In the pharmaceutical composition for diagnosis of cancer according to the present invention, the cytoplasmic penetration antibody may be combined with a molecular imaging phosphor, a fluorescent protein, or other imaging material in order to diagnose cancer through an image. The phosphor may include fluorescein, BODYPY, tetramethylrhodamine, 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 gene (EGFP), a red fluorescent protein (DsRFP), or a cyanine fluorescent substance that exhibits near-infrared fluorescence, but is not limited thereto. The other imaging material may include iron oxide or a radioactive isotope, but is not limited thereto.

6. Endosome Escape ability Methods of making improved antibodies

According to another embodiment of the present invention,

Disclosed is a method of producing an antibody with improved endosomes escape ability, comprising the step of grafting a CDR3 comprising an endosomes escape structure motif comprising the following amino acid sequence to the CDR3 of the antibody.

-X1-X2-X3-

(In the above formula,

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

In the method for producing an antibody with improved endosomes escape ability according to the present invention, the X1-X2-X3 may be composed of WWH, WYW, YWW, WYH or YWH amino acids.

In the method for producing an antibody with improved endosomes escape ability 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 include 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 (SEQ ID NO: 5). .

In the method for producing an antibody with improved endosomes escape according to the present invention, the X1-X2-X3 may be located at amino acids 96, 97 and 98 from the N-terminus of the heavy chain variable region. The CDR3 may include 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 with improved endosomes escape ability according to the present invention, the antibody with improved endosomes escape ability may include a light chain variable region selected from the group consisting of SEQ ID NOs: 23 to 28.

The antibody with improved endosomes escape ability according to the present invention penetrates into a specific cell with high efficiency without using a special external protein delivery system and distributes it in the cytoplasm, making it useful not only for drug development and manufacturing, but also for basic medical science research fields. Can be used. More specifically, the antibody with improved endosomes escape ability according to the present invention exists in the cytoplasm, which is currently classified as a target material for disease treatment using small molecule drugs, and forms a structural complex interaction between the protein and the protein through a wide and flat surface. And it can be expected a high effect in the treatment and diagnosis of disease-related factors.

1 is a result of observation with a confocal microscopy by performing a pulse-chase experiment to observe the transport process and stability of cytransmab TMab4 or cell-penetrating peptide TAT introduced into cells.
2 is a result of confirming through a confocal microscope in a Ramos cell line by directly fluorescently labeling an antibody whether Cytotransmab can be introduced through the cell membrane according to the pH, and whether it can induce the passage of other substances through the cell membrane.
3A is a result of confirming through an optical microscope in a Ramos cell line whether trypan blue without membrane permeability can be obtained by perforation by Cytotransmab according to pH.
3B is a graph for quantitative comparison of the number of cells obtaining trypan blue.
Figure 4a is a result of confirming through an optical microscope whether the perforation of the cell membrane produced by Cytotransmab under the condition of pH 5.5 is a temporary and reversible phenomenon.
4B is a graph for quantitative comparison of the number of cells obtaining trypan blue.
5 is a result of analyzing the cell membrane binding of Cytotransmab and the control antibody adalimumab according to pH by flow cytometry (FACS).
6 is a result of analyzing the cell membrane lipid flip-flop induction ability of Cytotransmab and the control antibody adalimumab according to pH by flow cytometry (FACS).
7 is a schematic diagram showing a perforation model of Cytotransmab predicted through the above experiments.
8 is a diagram showing amino acids involved in structural changes and amino acids exposed by structural changes by predicting structural changes of Cytotransmab according to pH based on the WAM modeling structure of the light chain variable region (VL) of Cytotransmab.
Figure 9a is a light chain variable region (VL) involved in inducing the structural change of Cytotransmab at acidic pH, 1st amino acid aspartic acid, 95th amino acid methionine substituted with alanine, glutamic acid, leucine, respectively, through a confocal microscope. This is the result of confirmation.
Figure 9b is a light chain variable region (VL) involved in inducing the structural change of Cytotransmab at acidic pH, 1st amino acid aspartic acid, 95th amino acid methionine, respectively, alanine, glutamic acid, trypan blue according to the pH of the mutations substituted with leucine It is a graph that quantitatively compares the number of obtained cells.
FIG. 10 is a quantitative comparison of the number of cells obtaining trypan blue according to pH by mutations in which amino acids that may be involved in endosome escape among amino acids of the CDR3 of the light chain variable region (VL) are replaced with alanine, respectively. It is a graph.
Figure 11a is a Cytotransmab endosomes after the purification of the expected to improve the escape performance of the mutation was analyzed through 12% SDS-PAGE in reducing or non-reducing conditions.
Figure 11b is a result of observing through a confocal microscope whether the cytoplasmic penetration ability of the expected mutant to improve Cytotransmab endosomes escape ability is maintained.
12 is a graph quantitatively comparing the number of cells that obtained trypan blue according to pH by the expected mutation of Cytotransmab wild type and endosomes escape ability improvement according to pH according to pH.
13A is a result of observing the migration of Cytotransmab wild-type or endosomes escape ability enhancing mutant TMab4-WYW to the cytoplasm with a confocal microscope using calcein.
13B is a bar graph quantifying calcein fluorescence of the confocal micrograph of FIG. 12A.
Figure 14a is a graph analyzed by flow cytometry (FACS) to the cell membrane binding of the mutations respectively substituted with arginine, isoleucine, and glycine, which are amino acids having properties opposite to tryptophan.
14B is a graph quantitatively comparing the number of cells obtaining trypan blue according to pH by mutations respectively substituted with arginine, isoleucine, and glycine, which are amino acids having properties opposite to tryptophan.
14C is a bar graph showing the migration of the mutants substituted with arginine, isoleucine, and glycine, which are amino acids having properties opposite to tryptophan, to the cytoplasm using calcein using a confocal microscope, and quantifying calcein fluorescence in the confocal micrograph. to be.
Figure 15a is a schematic diagram illustrating the construction of Cytotransmab having a light chain variable region from which the endosomes escape ability has been removed and a heavy chain variable region introduced with an improved endosomes escape ability motif.
Figure 15b is a graph quantitatively comparing the number of cells obtaining trypan blue according to pH by Cytotransmab having a light chain variable region from which endosomes escape ability has been removed and a heavy chain variable region introduced with an improved endosomes escape ability motif.
16 is a schematic diagram illustrating a process in which GFP fluorescence is observed due to complementary binding of the improved split green fluorescent split protein when cytotransmab is located in the cytoplasm.
FIG. 16 is an analysis of GFP11-SBP2 fused cytotransmab through 12% SDS-PAGE in reducing or non-reducing conditions after purification.
17A is a result of observing GFP fluorescence through a confocal microscope by complementary binding of the improved split green fluorescent protein of the GFP11-SBP2 fused cytotransmab wild-type and the endosomes escape ability-enhancing mutation.
17B is a graph quantifying GFP fluorescence of the confocal micrograph of FIG. 20A.
Figure 18a is a graph analyzed by flow cytometry (FACS) to the cell membrane binding of the mutations respectively substituted with arginine, isoleucine and glycine, which are amino acids having properties opposite to tryptophan.
Figure 19a is a schematic diagram illustrating the construction of a complete IgG form of anti-tubulin Cytotransmab in order to confirm the activity of the enhanced mutation endosomes escape ability.
19B is an analysis of the complete IgG form of anti-tubulin Cytotransmab through 12% SDS-PAGE under reducing or non-reducing conditions after purification.
FIG. 19C is a result of confirming whether or not overlapping of the complete IgG form of anti-tubulin Cytotransmab and tubulin, a cytoskeleton located in the cytoplasm, with a confocal microscope.
Figure 20a is a schematic diagram illustrating the construction of a complete IgG form of anti-RAS iMab in order to confirm the activity of the enhanced mutation endosomes escape ability.
FIG. 20B is an analysis of complete IgG form of anti-RAS iMab through 12% SDS-PAGE under reducing or non-reducing conditions after purification.
Figure 20c is a result of confirming the overlap of the complete IgG form of anti-RAS iMab and the intracellular activated H-RAS G12V mutation with a confocal microscope.
Figure 21a is a schematic diagram illustrating the construction of an anti-RAS iMab in the form of a complete IgG containing a monoclonal antibody skeleton with improved endosomes escape capability.
Figure 21b is a result of observing through a fluorescence microscope whether the HSPG binding ability and cytoplasmic penetration ability of the anti-RAS iMab in the form of a complete IgG containing a monoclonal antibody skeleton with improved endosomes escape ability is reduced or eliminated.
Figure 21c is a graph quantitatively comparing the number of cells obtained trypan blue according to the pH by a complete IgG form of anti-RAS iMab containing a monoclonal antibody skeleton that has been given improved endosomes escape ability.
22A is a result of performing ELISA for measuring the affinity of the K-RAS mutant K-RAS G12D in the GTP-bound form and the GDP-bound form.
Figure 22b is a schematic diagram illustrating the construction of an anti-RAS iMab in the form of a complete IgG comprising a monoclonal antibody backbone with improved endosomes escape ability fused with RGD10 peptide.
Figure 22c is a result of confirming with a confocal microscope whether the anti-RAS iMab in the form of a complete IgG containing a monoclonal antibody skeleton with improved endosomes escape ability fused with RGD10 peptide and the intracellular activated H-RAS G12V mutation to be.
Figure 23a is a schematic diagram illustrating the construction of a complete IgG-type cytoplasmic penetration antibody including a heavy chain variable region introduced with an improved endosomes escape motif and a light chain variable region having a monoclonal antibody skeleton that has an improved endosomes escape ability.
Figure 23b is a complete IgG-type cytoplasmic penetrating antibody including a heavy chain variable region with an improved endosomes escape motif and a light chain variable region having a monoclonal antibody skeleton that has improved endosomes escape ability to transfer to the cytoplasm. Using a confocal microscope, it is a histogram that quantifies the calcein fluorescence of the confocal micrograph.
Figure 23c is a trypan blue according to pH by a fully IgG-type cytoplasmic penetrating antibody comprising an improved endosomes escape motif-introduced heavy chain variable region and a light chain variable region having a monoclonal antibody skeleton that gives improved endosomes escape ability. It is a graph that quantitatively compares the number of acquired cells.

Hereinafter, the present invention will be described in more detail through 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 Expression and purification

In order to construct a heavy chain expression vector for production in the form of a complete IgG type monoclonal antibody, the heavy chain variable region (humanized hT0 VH) and the heavy chain constant region (CH1-) of the antibody are fused with DNA encoding the secretion signal peptide at the 5'end. The DNA encoding the heavy chain containing hinge-CH2-CH3) was cloned into pcDNA3.4 (Invitrogen) vector as NotI/HindIII, respectively. In addition, in order to construct a vector expressing the light chain, DNA encoding a light chain including a cytoplasmic penetrating light chain variable region (hT4 VL) and a light chain constant region (CL) in which DNA encoding a secretion signal peptide is fused at the 5'end is respectively included. The pcDNA3.4 (Invitrogen) vector was cloned as NotI/HindIII.

The light chain and heavy chain expression vectors were expressed and purified using transient transfection. In a shake flask, HEK293-F cells (Invitrogen) suspended in serum-free FreeStyle 293 expression medium (Invitrogen) were transfected with a mixture of plasmid and polyethyleneimine (Polyethylenimine, PEI) (Polyscience). Upon 200 mL transfection into a shake flask (Corning), HEK293-F cells were seeded in 100 ml of medium at a density of ^{2 × 10 6} cells/ml, and cultured at _{150 rpm and 8% CO 2.} For the production of each monoclonal antibody, the heavy and light chain plasmids were diluted in 10ml FreeStyle 293 expression medium (Invitrogen) with 125μg of heavy chain and 125μg of light chain with a total of 250μg (2.5 μg/ml), and 750 μg (7.5 μg/ml) of PEI. It was mixed with 10 ml of diluted medium and reacted at room temperature for 10 minutes. Thereafter, the reacted mixed medium was put into the cells seeded at _{100 ml and incubated at 150 rpm and 8% CO 2} for 4 hours, and then the remaining 100 ml of FreeStyle 293 expression medium was added and cultured for 6 days. Protein was purified from the cell culture supernatant collected by referring to the standard protocol. The antibody was applied to a Protein A Sepharose column (GE healthcare) and washed with PBS (pH 7.4). After eluting the antibody at pH 3.0 using 0.1 M glycine buffer, the sample was immediately neutralized using 1 M Tris buffer. The eluted antibody fraction was concentrated by exchanging the buffer solution with PBS (pH 7.4) through a dialysis method. The purified protein was quantified using absorbance and extinction coefficient at a wavelength of 280 nm.

Example 2. Cytotransmab TMab4 After intracellularization Trafficking Confirm

Coverslip was placed in a 24-well plate, and 2.5 x 10 ⁴ HeLa cells per well were added to 0.5 ml of a medium containing 10% FBS, and cultured under the conditions of _{5% CO 2 and 37 degrees for 12 hours.} When the cells were stabilized, pcDNA3.4-flag-rab11 was transiently transfected. Opti-MEM media (Gibco) was used to maximize the efficiency of transient transfection. 500 ng of pcDNA3.4-flag-rab11 to be transiently transfected was reacted with 50 µl of Opti-MEM media and 2 µl of Lipofectamine 2000 (Invitrogen, USA) on a tube at room temperature for 20 minutes, and then added to each well. In addition, 450 µl of antibiotic-free DMEM media was added and incubated at 37°C and 5% CO2 for 6 hours, exchanged for 500 µl of DMEM medium containing 10% FBS, and cultured for 24 hours. Thereafter, each well was treated with 3 μM of TMab4 in 0.5 ml of a new medium at 37° C. for 30 minutes, then rapidly washed three times with PBS, and cultured at 37° C. for 0 hours, 2 hours, and 6 hours by adding the medium. Then, the medium was removed, washed with PBS, and then proteins attached to the cell surface were removed with a weakly acidic solution (200mM glycine, 150mM NaCl pH 2.5). After washing with PBS, 4% paraformaldehyde was added, and the cells were fixed for 10 minutes at 25°C. After washing with PBS, 0.1% saponin, 0.1% sodium azide, and 1% BSA were added to PBS and incubated for 25 degrees for 10 minutes in a buffer solution to form a hole in the cell membrane. After washing with PBS again, the reaction was performed for 1 hour at 25 degrees in a buffer solution to which 2% BSA was added to PBS to inhibit non-specific binding. Then, FITC (green fluorescence) or TRITC (red fluorescence) was stained with an antibody (Sigma) that specifically recognizes the bound human Fc. Rab5 is an anti-rab5 that labels rab5, a marker of early endosomes. Anti-flag antibodies that label the flag-tag of rab11, a marker of the recycling endosome, were reacted for 1 hour at 25 degrees, and a secondary antibody to which TRITC (red fluorescence) or FITC (green fluorescence) was linked was reacted at 25 degrees for 1 hour. . Endosomes and lysosomes were directly treated with LysoTracker® Red DND-99 1mM 30 minutes before cell fixation. Then, the nuclei were stained (blue fluorescence) using Hoechst33342 and observed with a confocal microscope. As a result, it was confirmed that, unlike TAT, TMab4 was located in the initial endosomes up to 2 hours and then was not transported to lysosomes or recycling endosomes (FIG. 1).

Example 3. According to pH Cytotransmab TMab4 Endosome Confirmation of antibody inflow by escape

Using the outer phospholipid layer of the cell membrane of the Ramos cell line, which is known to be similar to the inner phospholipid layer of the initial endosome, the inflow of Cytotransmab TMab4 into the cytoplasm by the escape of the endosome was confirmed. In a 24-well plate, a coverslip was placed, and 200 μl of a 0.01% poly-L-lysine solution was added to attach the floating cells, Ramos, to the plate, and reacted for 20 minutes at 25°C. After washing with PBS, 5 x 10 ⁴ Ramos cells per well were added to 0.5 ml of a medium containing 10% FBS and incubated for 30 minutes at 37°C. Confirm cell adhesion and label directly with PBS and fluorescent reagent DyLight-488 in 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) After adding 10 μM of one TMab4 and 10 μM of unlabeled TMab4 and 2 μM of the control antibody adalimumab directly labeled with DyLight-488, and incubating for 2 hours at 37°C, using a confocal microscope using the same method as in Example 2. Observed. As a result, it was confirmed that fluorescence of TMab4 directly labeled with DyLight-488 was observed under pH 5.5 condition, and green FITC fluorescence was observed in cells treated with adalimumab directly labeled with TMab4 and DyLight-488 under pH 5.5 condition. In addition, it was confirmed that TMab4 flows through the cell membrane at an acidic pH, and other substances, not itself, can also be introduced (FIG. 2).

Example 4. According to pH Cytotransmab TMab4 Confirmation of cell membrane morphology by passage

Using the same method as in Example 3, an experiment to confirm trypan blue without membrane permeability was conducted by Cytotransmab according to pH, observed with an optical microscope, and the number of cells obtaining trypan blue was determined. It was quantified (a total of 400 or more cells were counted and the average value was shown). As a result, it was confirmed that trypan blue was obtained in proportion to the concentration in the cells to which TMab4 was added only under the pH 5.5 condition, so that when TMab4 passed through the cell membrane, a perforation was formed (FIG. 3).

In order to check whether the perforation formed by TMab4 was a temporary and reversible phenomenon, the cells were allowed to have a recovery time, and then the cells were observed with an optical microscope, and the number of cells obtaining trypan blue was quantified (a total of 400 Count the number of cells or more and represent the average value). As a result, trypan blue acquisition was observed in the cells to which TMab4 was added under the condition of pH 5.5, but in the case of cells having a recovery time in the medium, trypan blue acquisition did not occur. Accordingly, it was confirmed that the puncture formation by TMab4 is a temporary and reversible phenomenon (FIG. 4).

In addition, in order to confirm the membrane binding and lipid membrane flip-flop mechanism of the TMab4, TMab4 and the control antibody adalimumab, an antibody specifically recognizing human Fc to which FITC (green fluorescence) is linked, and Annexin-V to which FITC (green fluorescence) is linked After reacting with, it was analyzed by flow cytometry (FACS). As a result, it was confirmed that only TMab4 was bound to the cell membrane under pH 5.5 condition (FIG. 5), and that only TMab4 was bound to Annexin-V under pH 5.5 condition, resulting in cell membrane lipid flip-flop (FIG. 6).

Fig. 7 shows a model for forming a perforation of Cytotransmab TMab4 expected through this example.

Example 5. pH dependent Cytotransmab TMab4 Checking for structural changes

(1) Prediction model for structural change of Cytotransmab TMab4 according to pH

It was investigated whether the passage of the cell membrane of Cytotransmab according to the pH according to Example 4 was caused by a change in the structure of the pH-dependent Cytotransmab due to the Tanford transition. Amino acids such as aspartic acid (D) and glutamic acid (E) are protonated as the pH decreases, thereby losing a negative charge and becoming hydrophobic, and aspartic acid (D) and glutamic acid (E), which have become hydrophobic, are naturally hydrophobic amino acids and There is an increase in the interaction of the two, which is called the Tanford transition (Qin et al., 1998).

After examining amino acids capable of representing Tanford transition through the hT4 VL structure, which is a cytoplasmic penetrating light chain variable region, among them, the first and 95th amino acid pairs from the N-terminus where the distance between the side chains of the two amino acids is less than 7-8 Å have the effect of the Tanford transition. Was selected as a candidate capable of representing. The 95th amino acid is an amino acid present in CDR-L3, a sequence specific to hT4 VL, which is a cytoplasmic light chain variable region, and the possibility of inducing a change in the CDR-L3 ring structure by interaction with the first amino acid was confirmed. . Based on the WAM modeling structure of the light chain variable region (VL) of Cytotransmab, the structural change of Cytotransmab is predicted according to pH, and amino acids involved in the structural change and amino acids exposed by the structural change are shown in FIG. 8.

(2) Confirmation of escape of endosomes by amino acid pairs showing Tanford transition effect

Substitution with alanine (A) and glutamic acid (E) or leucine (L) having properties similar to those of the amino acid in order to confirm the induction of pH-dependent structural change by the 1st and 95th amino acid pairs and thereby escape of the endosomes. One mutation was constructed as shown in Table 1 below.

[Table 1]

Then, after expression and purification in the HEK293F cell line in the same manner as in Example 1, the introduction of the antibody having the mutation by passage through the cell membrane according to pH was observed through a trypan blue confirmation experiment, and trypan blue was observed. The number of acquired cells was quantified (a total of 400 or more cells were counted and the average value was shown). As a result, TMab4-D1A and TMab4-M95A mutations rarely obtained trypan blue unlike TMab4, and TMab4-D1E and TMab4-M95L mutations obtained trypan blue similar to TMab4. As a result, it was confirmed that the first amino acid and the 95th amino acid play an important role in the escape of the endosomes (FIG. 9).

(3) Cytotransmab of Endosome To escape ability Search for contributing amino acids and motifs

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

[Table 2]

As a result, TMab4-Y92A, TMab4-Y93A, and TMab4-H94A mutations significantly decreased trypan blue acquisition compared to TMab4, and in particular, TMab4-AAA mutation hardly caused trypan blue acquisition. On the other hand, TMab4-Y91A and TMab4-Y96A obtained trypan blue similar to TMab4 was observed. As a result, it was confirmed that the 92, 93, and 94 th amino acids greatly contributed to the escape of the endosomes (FIG. 10).

Example 6. Endosome Escape ability Improved Light chain variable region Having Cytotransmab Produce

TMab4-WWH, TMab4-WYW, TMab4-YWW, TMab4-WYH, TMab4-YWH for the 92 th tyrosine (Y), 93 th tyrosine (Y) and 94 th histidine (H) amino acids identified in Example 4 , TMab4-RYR, TMab4-IYI and TMab4-GYG mutations were prepared, and it was confirmed whether or not the antibody containing the mutation escaped endosomes using the same method as in Example 4.

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

[Table 3]

After purification of Cytotransmab containing the TMab4-WWH, TMab4-WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH mutations, analysis by 12% SDS-PAGE under reducing or non-reducing conditions, about It had a molecular weight of 150 kDa, and was found to have a molecular weight of 50 kDa of a heavy chain and a molecular weight of 25 kDa of a light chain under reducing conditions (FIG. 11A). Thus, the TMab4-WWH, TMab4-WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH mutations containing Cytotransmab exists in a single body in solution, and does not form a duplex or oligomer through a non-natural disulfide bond. Confirmed.

The cytoplasmic penetration ability of Cytotransmab containing the 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 the cytoplasmic penetration ability was maintained in all of the branch mutations (FIG. 11B).

To confirm the pH-dependent endosomes escape ability of Cytotransmab containing the TMab4-WWH, TMab4-WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH mutations, trypan blue was confirmed using the same method as in Example 5 The experiment was performed and the number of cells obtaining trypan blue was quantified (a total of 400 or more cells were counted and the average value was indicated). As a result, among the five mutations, TMab4-WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH increased the trypan blue acquisition rate, of which TMab4-WYW was confirmed that pH-dependent trypan blue acquisition occurred. (Fig. 12). Therefore, TMab4-WYW, which had increased pH-dependent trypan blue acquisition while maintaining the wild-type cytoplasmic penetration ability, was selected as the final clone.

In addition, the migration of TMab4-WYW selected as the final clone to the cytoplasm was observed with a confocal microscope using calcein using the same method as in Example 2, and Image J sofrtware (National Institutes of Health, USA) was used. And quantified (represents the average value of fluorescence obtained after designating 20 cells in each condition). As a result, it was confirmed that the TMab4-WYW showed strong intensity of green calcein fluorescence even at a lower concentration than TMab4 (FIG. 13).

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

[Table 4]

In order to determine whether the cell membrane binding of Cytotransmab containing the TMab4-RYR, TMab4-IYI and TMab4-GYG mutations occurs according to pH, the same method as in Example 3 was used, and then flow cytometry (FACS) was used. Observed. As a result, it was confirmed that only TMab4 binds to the cell membrane under the condition of pH 5.5 (FIG. 14A).

To confirm the pH-dependent endosome escape ability of Cytotransmab containing the TMab4-RYR, TMab4-IYI and TMab4-GYG mutations, a trypan blue confirmation experiment was performed using the same method as in Example 5, and trypan blue was used. The number of cells obtained was quantified and compared with TMab4-WYW (a total of 400 or more cells were counted and the average value was shown). As a result, it was confirmed that TMab4-RYR, TMab4-IYI, and TMab4-GYG decreased trypan blue acquisition compared to TMab4-WYW (FIG. 14B).

In addition, the migration of Cytotransmab containing the TMab4-RYR, TMab4-IYI and TMab4-GYG mutations to the cytoplasm was observed with a confocal microscope using calcein using the same method as in Example 2. Compared. As a result, it was confirmed that in the cells treated with TMab4-RYR, TMab4-IYI and TMab4-GYG, the intensity of green calcein fluorescence spread across the cytoplasm was weaker than that of the cells treated with TMab4-WYW (FIG. 14C). Accordingly, it was proved that the endosomes escape ability can be improved when the 92, 93 and 94 th amino acids are substituted with tryptophan (W). The amino acid sequences of Cytotransmab containing the TMab4-WWH, TMab4-WYW, TMab4-YWW, TMab4-WYH and TMab4-YWH mutations are shown in SEQ ID NOs: 14 to 18.

Example 7. Endosome Escape ability Improved Heavy chain variable region Having Cytotransmab Produce

(1) Confirmation of endosomes escape structure motif region

As in Example 5, an amino acid capable of representing the Tanford transition was investigated 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 in CDR3 were candidates that could exhibit the Tanford transition effect. After selecting as, it was confirmed that the 96th, 97th, and 98th amino acids were endosomes escape structure motif regions using the same method as in Example 6. On the other hand, the number of amino acids in the CDR3 of the wild-type heavy chain variable region is 11, and since the center of the ring is exposed to the surface of the structure, the number of amino acids in the CDR3 is 7 or 8 to facilitate the occurrence of a pH-dependent phenomenon. I did.

(2) Confirmation of endosomes escape ability of Cytotransmab

In order to confirm the endosomes escape ability in the heavy chain variable region of TMab4-WYW finally selected in Example 6, TMab4-AAA from which the endosomes escape structure motif was removed from the light chain variable region was prepared, and the 96 th of the heavy chain variable region , Cytotransmab TMab4-HW1-AAA, TMab4-HW2-AAA, TMab4-HW3-AAA and TMab4-HW4-AAA were prepared by substituting WYW for 97 th and 98 th amino acids (FIG. 15A).

[Table 5]

Of the TMab4-HW1-AAA (HT0-HW1 VH), TMab4-HW2-AAA (HT0-HW2 VH), TMab4-HW3-AAA (HT0-HW3 VH), and TMab4-HW4-AAA (HT0-HW4 VH) Trypan blue confirmation experiment was performed to determine the cytoplasmic penetration ability, and the number of cells that obtained trypan blue was counted (a total of 400 or more cells were counted and the average value was indicated). As a result, TMab4-HW1-AAA, TMab4-HW2-AAA, TMab4-HW3-AAA, and TMab4-HW4-AAA mutants all observed trypan blue acquisition at pH 5.5 (FIG. 15B). Therefore, it was proved that endosomes escape is possible even if the improved endosomes escape structure motif is given to the heavy chain variable region, and can be finally located inside the cytoplasm.

< Experimental example >

Experimental example One. GFP11 - SBP2 Fused Cytotransmab According to the cytoplasmic location GFP Fluorescence confirmation

Fig. 16 shows a schematic diagram illustrating a process in which GFP fluorescence is observed due to the complementary binding of the improved split green fluorescent protein when Cytotransmab is located in the cytoplasm. Specifically, a complementary system of improved split green fluorescent protein was used to directly confirm that cytotransmab is located in the cytoplasm. When the green fluorescent protein GFP is divided into 1-10 and 11 fragments, the fluorescent property is removed, and if the distance between the two fragments is close and combined, the fluorescent property can be restored (Cabantous et al. ., 2005). Using this characteristic, GFP fragments 1-10 were expressed in the cytoplasm, and GFP fragments 11 were fused to the C-terminus of the heavy chain of cytotransmab. In addition, in order to complement the complementary binding between GFP fragments, streptavidin and streptavidin-binding peptide 2 (SBP2) having high affinity were fused to GFP fragments, respectively. Thus, the observation of GFP fluorescence disprove that cytotransmab is located in the cytoplasm.

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

For the expression of GFP11-SBP2 fused cytotransmab in animal cells, GFP11-SBP2 was genetically fused to the C-terminus of the heavy chain using three GGGGS linkers. Then, the animal expression vector encoding the cytoplasmic penetrating light chain and the endosomes escape ability enhancement cytoplasmic penetrating light chain and the animal expression vector encoding the heavy chain fused with GFP11-SBP2 were simultaneously transient transfection into HEK293F protein-expressing cells. ). Purification of the GFP11-SBP2 fused cytotransmab was performed in the same manner as in Example 1, and the 12% SDS-PAGE result in reducing or non-reducing conditions is shown in FIG. 17.

As a result, a molecular weight of about 150 kDa was confirmed under non-reducing conditions, and a molecular weight of 50 kDa of a heavy chain and a molecular weight of 25 kDa of a light chain were shown under reducing conditions. This shows that the expression-purified GFP11-SBP2 fused cytotransmab exists as a monolith in a solution state, and does not form duplexes or oligomers through unnatural disulfide bonds.

(2) GFP fluorescence confirmation according to the cytoplasmic location of GFP11-SBP2 fused cytotransmab

When the cells are stabilized by preparing a transformed HeLa cell line stably expressing SA-GFP1-10 as in Example 2, PBS, TMab4-GFP11-SBP2, TMab4-WYW-GFP11-SBP2 0.2, 0.4, 0.6 , 0.8, 1.6, 3.2 μM were incubated at 37 degrees for 6 hours. After washing with PBS and a weakly acidic solution through the same method as in Example 2, the cells were fixed. Then, the nuclei were stained (blue fluorescence) using Hoechst33342 and observed with a confocal microscope, and this was quantified (represents the average value of fluorescence obtained after designating 20 cells in each condition). As a result, it was confirmed that the strong intensity of green GFP fluorescence was observed in TMab4-WYW even at a lower concentration than TMab4 (FIG. 18).

In addition, GFP11-SBP2 located inside the cytoplasm of the complete IgG form of the fused cytotransmab and endosomes escape function improved cytotransmab in the cytoplasm concentration and endosomes escape efficiency are compared and shown in Table 6 below.

[Table 6]

Experimental example 2. Complete IgG Form of anti- Tubulin Cytotransmab Confirmation of target ability of intracellular protein

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

As shown in the schematic diagram shown in Fig. 19a, a complete IgG type anti-tubulin Cytotransmab containing a recombinant endosome escape structure motif was constructed. A heavy chain that specifically binds to tubulin, a cytoskeleton, is fused with DNA encoding a secretion signal peptide at the 5'end as in Example 1 for the expression of a complete IgG-type anti-tubulin cytoplasmic infiltrating monoclonal antibody in animal cells. DNA encoding a heavy chain containing a variable region and a heavy chain constant region (CH1-hinge-CH2-CH3) was cloned into pcDNA3.4 (Invitrogen) vector as NotI/HindIII. Then, the animal expression vector encoding the TMab4-WYW and the animal expression vector encoding the heavy chain including the heavy chain variable region that specifically binds to the constructed tubulin were simultaneously transiently transfected into HEK293F protein-expressing cells. transfection). Then, purification of the complete IgG form of anti-tubulin Cytotransmab was performed in the same manner as in Example 1, and after purification, 12% SDS-PAGE was analyzed under reducing and non-reducing conditions. As a result, it was found to have a molecular weight of about 150 kDa under non-reducing conditions, and it was confirmed that it had a molecular weight of 50 kDa of heavy chain and 25 kDa of light chain under reducing conditions (FIG. 19B). Accordingly, it was confirmed that the expression-purified complete IgG form of anti-tubulin Cytotransmab exists as a monolith in a solution state, and does not form a duplex or oligomer through an unnatural disulfide bond.

(2) Confirmation of specific binding to cytoskeleton tubulin of complete IgG form of anti-tubulin Cytotransmab

It was confirmed by using a confocal microscope whether the anti-tubulin Cytotransmab in the complete IgG form and the tubulin, a cytoskeleton located in the cytoplasm, overlap. As a result, it was observed that the green fluorescent TuT4-WYW was overlapped in a fibrous shape on the cytoplasm where the red fluorescent tubulin was located, whereas the TuT4 did not overlap (FIG. 19C). As a result, it was demonstrated that intracellular cytoskeleton tubulin and the complete IgG form of anti-tubulin Cytotransmab specifically bind.

Experimental example 3. Complete IgG Form of anti-RAS iMab Confirmation of target ability of intracellular protein

(1) Preparation of complete IgG form of anti-RAS iMab

As shown in Fig. 20 (a), a complete IgG form of anti-RAS iMab containing a recombinant endosome escape structure motif was prepared. For the expression of complete IgG-type anti-RAS iMab in animal cells, as in Example 5, DNA encoding a secretion signal peptide is fused at the 5'end, and specific binding to K-RAS to which GTP is bound in a heavy chain variable region DNA encoding the heavy chain including the heavy chain variable region (RT11 VH) and the heavy chain constant region (CH1-hinge-CH2-CH3) were cloned into pcDNA3.4 (Invitrogen) vector as NotI/HindIII, respectively. Thereafter, the animal expression vector encoding the TMab4-WYW and the heavy chain-encoded animal expression vector containing a heavy chain variable region that specifically binds to K-RAS to which the constructed GTP is bound were simultaneously transiently transfected into HEK293F protein-expressing cells. Transient transfection was performed. Next, purification of the complete IgG form of anti-RAS iMab was performed in the same manner as in Example 1, and after purification, 12% SDS-PAGE was analyzed under reducing and non-reducing conditions. As a result, it was found to have a molecular weight of about 150 kDa under non-reducing conditions, but it was confirmed to have a molecular weight of 50 kDa of heavy chain and a molecular weight of 25 kDa of light chain under reducing conditions. Accordingly, it was confirmed that the expression-purified complete IgG form of anti-RAS iMab exists as a monolith in solution, and does not form a duplex or oligomer through an unnatural disulfide bond (FIG. 20B).

(2) Confirmation of specific binding to GTP-bound K-RAS of complete IgG form of anti-RAS iMab

Whether or not overlapping of the complete IgG form of anti-RAS iMab with the intracellular activated H-RAS G12V mutation was confirmed using a confocal microscope. As a result, while the green fluorescent RT11 and RT11-WYW overlapped on the inner cell membrane where the red fluorescent activated RAS is located, TMab4 did not overlap (FIG. 20C). As a result, it was proved that the activated RAS in the cell and the anti-RAS iMab in the complete IgG form were specifically binding.

Experimental example 4. Improved Endosome The escape structure motif is grafted Of monoclonal antibodies Endosome Escape ability Confirm

Among the monoclonal antibodies currently on the market, there are many types of monoclonal antibodies that target cell surface receptors, particularly cell surface receptors where cell internalization occurs. After these antibodies are internalized into the cell, their binding to the antigen is not broken, and because they do not have the ability to escape endosomes, they cannot move to the cytoplasm and are spit out again. Therefore, if the endosomes escape ability can be given to the receptor target antibody in which cell internalization occurs, there is an advantage that it can be applied in a wider range than now.

In order to give an improved endosomes escape ability motif, the sequence of the light chain variable region of the receptor target antibody in which cell internalization takes place and the sequence of the light chain variable region of cytotransmab are compared, and the first amino acid is negatively charged and affects the CDR3 ring structure. Candidate light chain variable region sequences having the same main amino acids located in the backbone that can be given are summarized. A mutation for grafting the CDR3 of the candidate light chain variable region into the CDR3 of cytotransmab was constructed as shown in Table 7 below. When constructing this mutation, the 87th amino acid tyrosine was substituted with phenylalanine in order to increase the protein expression yield reduced by the improved endosomes escape ability motif. Phenylalanine is an amino acid that can enhance the interface between the light chain variable region and the heavy chain variable region by facilitating interaction with an amino acid having an aromatic ring and a hydrophobic amino acid located in the backbone of the heavy chain variable region.

[Table 7]

(1) Preparation of complete IgG form of anti-RAS iMab

A schematic diagram illustrating the construction of a complete IgG form of anti-RAS iMab containing a monoclonal antibody skeleton with improved endosomes escape capability is shown in FIG. 21A. In the same manner as in Example 1, the light chain variable region was cloned, and a heavy chain-encoded animal expression vector containing a heavy chain variable region specifically binding to GTP-bound K-RAS was simultaneously transiently transfected into HEK293F protein-expressing cells. (transient transfection) was performed. Then, purification of the complete IgG form of anti-RAS iMab was carried out in the same manner as in Example 1.

(2) Confirmation that improved endosomes escape ability motif can be given to the skeleton of monoclonal antibody

When the cells are stabilized by preparing a HeLa cell line in the same manner as in Example 2, PBS, RT11-WYW, RT11-Neci, RT11-Nimo, RT11-Pani, RT11-Pert, RT11-Lumr, RT11-Emib 1 μM were added to 37 Incubated for 6 hours in degrees. After washing with PBS and a weakly acidic solution through the same method as in Example 2, cell fixation, cell perforation, and blocking were performed. TMab4 was stained with an antibody that specifically recognizes human Fc to which FITC (green fluorescence) is linked. Then, the nuclei were stained (blue fluorescence) using Hoechst33342 and observed with a confocal microscope. As a result, in all of the anti-RAS iMab in the form of a complete IgG containing a monoclonal antibody skeleton with improved endosomes escape ability was not observed in all of the cells (Fig. 21b).

In addition, the number of cells that obtained trypan blue according to pH by a complete IgG form of anti-RAS iMab containing a monoclonal antibody skeleton that gave the improved endosomes escape ability was quantitatively compared (total of 400 or more cells Counted and expressed as an average value). As a result, in the case of a complete IgG form of anti-RAS iMab containing a monoclonal antibody skeleton with five improved endosomes, except for RT11-Pert, a trypan blue acquisition similar to that of RT11-WYW was observed (FIG. 21c ). .

Experimental example 5. Improved Endosome Escape ability Granted Monoclonal antibody Anti-RAS in the form of a complete IgG containing the skeleton iMab of GTP Combined Confirmation of maintenance of specific binding with K-RAS

(1) Affinity measurement in the combined form of GTP and GDP of the K-RAS mutant K-RAS G12D

The GTP-bound form of the target molecule K-RAS G12D and the GDP-bound form were bound to a 96-well EIA/RIA plate (COSTAR Corning) at 37°C for 1 hour, followed by 0.1% PBST (0.1% Tween20, pH 7.4, Wash three times for 10 minutes with 137 mM NaCl, 12 mM phosphate, 2.7 mM KCl) (SIGMA). Combined with 5% PBSS (5% Skim milk, pH 7.4, 137 mM NaCl, 12 mM phosphate, 2.7 mM KCl) (SIGMA) for 1 hour, and then washed 3 times with 0.1% PBST for 10 minutes. Subsequently, IgG-type monoclonal antibodies RT11-WYW, RT11-Neci, RT11-Nimo, RT11-Pani, RT11-Pert, RT11-Lumr, RT11-Emib were combined and washed 3 times for 10 minutes with 0.1% PBST. It binds with an anti-human antibody (alkaline phosphatase-conjugated anti-human mAb) (SIGMA) conjugated with goat-derived AP as a labeling antibody. By reacting with pNPP (p-nitrophenyl palmitate) (Sigma), absorbance at 405 nm was quantified. As a result of affinity analysis for the K-RAS mutation, the complete IgG form of anti-RAS iMab containing a monoclonal antibody skeleton with five improved endosomes, except for RT11-Nimo, showed a difference in affinity with RT11-WYW. , All clones did not bind to the GDP-bound K-RAS used as a negative control (FIG. 22A)

(2) Preparation of anti-RAS iMab in the form of complete IgG containing a monoclonal antibody skeleton with improved endosomes escape ability by fusion with RGD10 peptide

Anti-RAS iMab in the form of complete IgG containing a monoclonal antibody skeleton with improved endosomes escape ability has been removed from the ability to penetrate cells, so it is specific to integrin αvβ3, which is overexpressed in neovascular cells and various tumors to enable cell penetration. The RGD10 peptide having a light chain was fused genetically using two linkers of GGGGS at the N-terminus. In the case of RGD10 peptide, it has similar affinity to RGD4C peptide, but only one disulfide bond by two cysteines exists, and genetic engineering fusion is possible. In addition, based on the expression yield, endosomes escape ability, affinity analysis experiment results for Ras, RGD10 peptide was fused to the N-terminus of the light chain variable region of excellent candidate antibodies RT11-Pani and RT11-Neci (Fig. 22b ).

Then, 2x104 cells per well were diluted in 0.5ml of each of the SW480 cell lines in a 24-well plate and incubated for 12 hours, 37 degrees, 5% CO2, and then RT11-i-WYW, RT11-i-Neci, RT11- 1 μM of i-Pani was treated and cultured at 37 degrees for 12 hours. Thereafter, the antibody and the nucleus were labeled under the same conditions as in Example 2, and the Ras-labeled antibody was reacted at 37°C for 1 hour, followed by staining with a secondary antibody and observed with a confocal microscope. As a result, the green fluorescent RT11-i-WYW, RT11-i-Neci, and RT11-i-Pani overlapped with the red fluorescent activated RAS. As a result of the above experiment, it was confirmed that the anti-RAS iMab in the form of a complete IgG containing the activated RAS in the cell and the monoclonal antibody skeleton with improved endosomes escape ability was specifically bound (FIG. 22C).

Experimental Example 6. Improved a little escape motif introduced into the heavy chain variable region and the improved performance endosomes escape the endosome given of a monoclonal antibody having a light chain variable region framework full IgG form of the cellular penetration antibody comprising an endo Confirmation of improvement in escape ability .

(1) Preparation of complete IgG-type cytoplasmic infiltrating antibody

A schematic diagram illustrating the construction of a fully IgG-type cytoplasmic infiltrating antibody including a heavy chain variable region introduced with an improved endosomes escape motif and a light chain variable region having a monoclonal antibody skeleton with improved endosomes escape capability is shown in FIG.23a. Using the same method as in Example 1, an animal encoded with a light chain including a heavy chain including an improved endosomes escape motif introduced heavy chain variable region and a light chain variable region having a monoclonal antibody skeleton imparting improved endosomes escape ability The expression vector was transiently transfected into HEK293F protein-expressing cells at the same time. Thereafter, purification of the complete IgG-type cytoplasmic penetration antibody was performed in the same manner as in Example 1.

(2) Endosome escape ability check

*Improved endosomes escape motif introduced heavy chain variable region and a light chain variable region with a monoclonal antibody skeleton that gives improved endosomes escape ability, using calcein to move the cytoplasmic infiltrating antibody in the form of complete IgG into the cytoplasm. Observation with a confocal microscope and calcein fluorescence in the confocal micrograph was quantified. As a result, in the cells treated with TMab4-HW1-ep41 at each concentration, the intensity of green calcein fluorescence spreading in the cytoplasm was stronger than that of the cells treated with TMab4-ep41 (FIG. 23B).

Cells that obtained trypan blue according to pH by a fully IgG-type cytoplasmic penetrating antibody including a heavy chain variable region introduced with an improved endosomes escape motif and a light chain variable region having a monoclonal antibody skeleton that gave improved endosomes escape ability. The number of were compared quantitatively. As a result, in the case of TMab4-HW1-ep41, it was observed that the trypan blue acquisition increased in a concentration-dependent manner compared to TMab4-ep41 (FIG. 23C).

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

So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an illustrative point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the above description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Endosomal escape motif enabling antibody to escape from endosomes and uses thereof <130> 241 <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 Pro 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 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 Pro 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 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 Pro 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 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 Pro 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 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 Pro 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 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 Pro 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 Pro 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 Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Val Ser 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 Pro 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 Phe 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 Pro 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 Pro 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 (13)

The antibody is located in CDR3 (Complementarity determining region-3) of the antibody. After the antibody is intracellularly introduced into the cell by the endocytosis ability of the antibody itself, endosomal escape including the following amino acid sequence for imparting endosomal elimination ability A light chain variable region or heavy chain variable region comprising an endosomal escape motif.
-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 selected from the group consisting of Tryptophan (W).
The method according to claim 1,
Wherein the endosomal cleavage structure motif X1-X2-X3 comprises a WWH, WYW, YWW, WYH or YWH amino acid.
The method according to claim 1,
The endosomal escape sequence motif X1-X2-X3 is derived from the N-terminus of the light chain variable region, the 92,93, and 94 amino acids, the N-terminus of the heavy chain variable region, the 96,97 and 98 amino acids, and the N-terminus of the light chain variable region 92, 93 and 94 amino acids and the 96, 97 and 98 amino acids from the N terminus of the heavy chain variable region.
(Provided that the amino acid position is according to the Kabat number)
The method according to claim 1,
(SEQ ID NO: 1), QQYWYWMYT (SEQ ID NO: 2), QQYYWWMYT (SEQ ID NO: 3), QQYWYHMYT (SEQ ID NO: 4), QQYYWHMYT (SEQ ID NO: 5), GWYWMDL (SEQ ID NO: 6), GWYWFDL ), GWYWGFDL (SEQ ID NO: 8) and YWYWMDL (SEQ ID NO: 9).
A light chain variable region according to any one of claims 1 to 4 or a heavy chain variable region or a light chain variable region according to any one of claims 1 to 4 and a heavy chain variable region An endothelium-derived antibody or fragment thereof.
6. The method of claim 5,
Wherein the antibody is a mouse, chimeric, human, or humanized antibody.
6. The method of claim 5,
Wherein the antibody is an IgG, IgM, IgA, IgD or IgE antibody or fragment thereof.
6. The method of claim 5,
Wherein the antibody is located in the cytoplasm by intrusion of endosomes after intracellularization, or an antibody or fragment thereof having enhanced endosomal elimination capability.
6. The method of claim 5,
Wherein said antibody targets cytoplasm, nucleus, mitochondria, endoplasmic reticulum, or intracellular polymer, or an endo-escape enhancing antibody or fragment thereof.
An antibody or fragment thereof having enhanced endo-escape ability according to claim 5, and
A biologically active molecule selected from the group consisting of peptides, proteins, small molecule drugs, nanoparticles and liposomes fused to said antibody.
11. The method of claim 10,
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, Wherein the cancer is selected from the group consisting of endometrium or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, mucin cancer, thyroid cancer, liver cancer and head and neck cancer.
An antibody or fragment thereof having enhanced endo-escape ability according to claim 5, and
A biologically active molecule selected from the group consisting of peptides, proteins, small molecule drugs, nanoparticles and liposomes fused to said antibody.
13. The method of claim 12,
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, Endometrium or uterine cancer, salivary gland cancer, renal cancer, liver cancer, prostate cancer, mucin cancer, thyroid cancer, liver cancer, and head and neck cancer.

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