WO2020149613A1 - Antibody having improved cytosol-penetrating ability - Google Patents

Abstract

The present invention relates to an antibody having improved cytosol-penetrating ability, and a cytosol-penetrating antibody comprising an amino acid mutation on at least one selected from the group consisting of CDR1, FR, and CDR3 in a light-chain variable region, or an antigen-binding fragment thereof. The antibody was adopted using a screening method capable of evaluating the antibody per se for ability to penetrate to the cytosol, without purification after acellular protein synthesis and was found to improve in endosomal escape ability and retain an antigen targeting ability.

Description

Antibodies with enhanced cellular permeability

The present invention relates to an antibody with improved cytoplasmic permeability, and to a cytoplasmic permeable antibody or antigen-binding fragment thereof. After cell-free protein synthesis, it was adopted as a screening method capable of evaluating cytoplasmic permeability without purification, and it was confirmed that the endosomal escape ability was improved and the antigen targeting ability was maintained.

Due to the high specificity and strong binding affinity for specific ligands, therapeutic antibodies are rapidly replacing chemical drugs and have become the center of treatment in the treatment of cancer and autoimmune diseases. Target specific binding of an antibody relieves or reverses symptoms through a variety of mechanisms including receptor blockade, complement dependent cytotoxicity, antibody dependent cytotoxicity and regulation of T cell function.

However, since most antibody therapeutics developed to date cannot target cell membranes, they only target surface exposed or secreted molecules, so small molecule drugs are currently the only option to access intracellular targets. However, in order to take advantage of the antibody therapy compared to the existing chemical drugs as described above, development of a method of delivering the antibody through the cell membrane is being promoted.

The present inventors have developed a light chain variable region (VL) that exhibits cytoplasmic penetration ability, and the cytoplasmic penetration antibody TMab4 in the form of a complete IgG introduced therein, unlike the general properties of these antibodies (Choi et al., 2014). In addition, a heavy chain variable region targeting the active Ras, which is a major tumor inducer inside the cytoplasm, and a light chain variable region showing cytoplasmic penetration ability are constructed in IgG form to develop the cytoplasmic penetration antibody RT11 targeting the active Ras inside the cytoplasm. (Shin et al., 2017).

The light chain variable region of the cytoplasmic infiltrating antibody detects the acidic pH of the endosome, thereby inducing structural changes, thereby confirming the ability to escape the endosome, and the endosome escape ability is manipulated by manipulating the sequence of the ring region where structural changes occur. An improved TMab4-WYW (TMab4-3) antibody was developed (Kim et al., 2016). Finally, the RT11-3 antibody was constructed by combining the heavy chain variable region targeting the active Ras, which is the main tumor inducer inside the cytoplasm, and the light chain variable region with improved endosomal escape ability.

Through the experiment, the sequence considered to be involved in the escape of the endosome among the sequences of the light chain variable region was confirmed, but the sequence not predicted by the present inventors is also likely to be involved in the escape of the endosome. By comparing the cytoplasmic penetration ability, it was expected that a sequence with improved cytoplasmic delivery efficiency could be obtained. However, screening and expression of various cell-penetrating antibody variants for this requires numerous processes such as gene cloning, recombinant expression and purification, and there is a limitation in that a complicated process of further performing functional analysis is required. For this reason, up to now, limited variants of about 10 were constructed to evaluate cytoplasmic penetration ability.

On the other hand, as a technique for permeation of the cell membrane of an antibody, a method of using a complete immunoglobulin type antibody to permeate the cell membrane and positioning it in the cytoplasm is disclosed in Korean Patent No. 1602870. As an experimental method for confirming the degree of location in the cytoplasm, Korean Patent No. 1790669 discloses an improved split green fluorescent protein complementary system and a technique for its use. In addition, Korean Patent Registration No. 1667023 discloses a technique for easily analyzing the inflow of an antibody produced into a cytoplasm using a cell-free protein synthesis method, but a technique for an antibody having improved cytoplasmic permeability of the present invention Has not been disclosed.

Summary of the invention

The present invention has been derived by the above-mentioned needs, and the present invention has completed the present invention by providing an antibody with improved cytoplasmic permeability, and confirming that the antibody has improved permeability to the cytoplasm.

In order to achieve the above object, the present invention is an antibody with enhanced cytoplasmic permeability, comprising an amino acid variation in at least one selected from the group consisting of CDR1, FR and CDR3 among the light chain variable regions of SEQ ID NO: 2, or a cytoplasmic penetration antibody or Antigen-binding fragments are provided.

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

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

The present invention relates to a conjugate in which the cytoplasmic penetration antibody or antigen-binding fragment thereof and a bioactive molecule are fused.

The present invention is a method for placing a cytoplasmic penetration antibody or an antigen-binding fragment thereof through the cell membrane and placing it in the cytoplasm, wherein the antibody has amino acid mutations in one or more selected from the group consisting of CDR1, FR and CDR3 among the light chain variable regions of SEQ ID NO:2. How to include.

The present invention is a light chain variable region (VL) having a cytoplasmic penetration ability, which penetrates the cell membrane and induces it to be located in the cytoplasm, wherein at least one amino acid mutation selected from the group consisting of CDR1, FR and CDR3 among the light chain variable regions of SEQ ID NO:2 It relates to a light chain variable region (VL) comprising a.

1 is a confocal microscopy image confirming the cytoplasmic-infiltrated RT11-3 scFv antibody. After soluble RT11-3 scFv antibody synthesized by fusion and in situ cleavage was purified with Ni-NTA agarose beads, the purified RT11-3scFv antibody was treated to reporter cells at concentrations of 0.25, 0.5, 0.75 and 1.0 μM. And cultured in a 5% (v/v) CO ₂ incubator at 37° C. for 12 hours, fixed the cultured reporter cells with 4% paraformaldehyde for 15 minutes, and analyzed GFP fluorescence of reporter cells, Cell nuclei were stained with Hoechst 33342.

2 is a result of the cytotoxicity test for each component of the reaction mixture for reporter cells. To investigate the cytotoxicity of the major components in the reaction mixture for cell-free protein synthesis, it is the result of analyzing the cell morphology using a microscope after treating each component at the same concentration as the standard reaction mixture diluted with DMEM. 1×10 ⁵ reporter cells treated with 1.2 ml of the diluted mixture were confirmed after incubation with a 5% (v/v) CO ₂ incubator at 37° C. for 12 hours.

3 is a result of confirming cytotoxicity by diluting a cell-free protein-synthesized RT11-3 scFv antibody in various ratios (1, 2, 4 and 8 times) with DMEM. These are the results of micrograph (A) and flow cytometry (B) observed to optimize the dilution ratio with minimal effect on cell morphology and cell viability.

4 is a result confirming the effect of the ubiquitin sequence and the amount of soluble protein of RT11-3 scFv. (A) is a fusion of the nucleotide sequence of ubiquitin (UCE1) to the RT11-3 scFv gene, and the amount of protein synthesized as a cell-free protein in a standard reaction mixture was confirmed by TCA-precipitated radioactivity. Ubiquitin (UCE1) was synthesized using a gene without a sequence, UCE1-RT11-3 was synthesized using a gene fused with a ubiquitin sequence, the empty rod is the total protein synthesis amount, and the filled rod is the amount of soluble protein. to be. (B) is a result of comparing the expression level of the ubiquitin-fusion, using a standard S12 extract and a S12 extract rich in UBP1, confirmed by TCA-precipitated radioactivity measurement. The western blot result at the top confirms the cleavage of the ubiquitin tag. (C) After expressing UCE1 fused RT11-3 scFv in the presence of UBP1 S12 extract, the reaction mixture was diluted 4-fold in DMEM medium, and reporter cells (1×10 ⁴ ) were diluted with 400 μl of the reaction mixture. Cultured in the middle. After fixing the treated reporter cells with 4% paraformaldehyde, GFP fluorescence was confirmed under confocal microscopy.

Figure 5 shows the process of manufacturing the mutation location and mutation library RT11-3 scFv. The wild-type residues indicated in black in (A) were designed to be replaced by amino acids in red, and the primers shown in (B) were used to construct a library through a combination of PCR-amplified DNA fragments.

6 is a cell expression and screening results of the mutant RT11-3 scFv. The PCR-amplified mutant gene construct is expressed in a reaction mixture for cell-free protein synthesis, diluted 4 times in DMEM and added to reporter cells for 12 hours after incubation, the reporter cells are washed with PBS, and in 100 μl PBS. It is a result of measuring GFP fluorescence (excitation wavelength: 485 nm/emission wavelength: 528 nm).

7 is a result of amino acid sequence analysis of the selected variant. Wild type (WT) and selected variant RT11-3 scFv (#1-60, #5-10, #6-32 and 6-91).

8 is a result of evaluating the cytoplasmic permeability of the adopted variant clones. After expressing and purifying the selected 4 clones from a 5 ml cell-free protein synthesis reaction, the reporter cells (1×10 ⁴ cells/confocal dish) were diluted 4 times in DMEM and then purified in the same amount (1.5 μM). Treated with antibodies. After fixing with 4% paraformaldehyde for 15 minutes, GFP fluorescence of reporter cells was confirmed. The nucleus was stained with Hoechst 33342 dye.

9 is a result of performing a non-specific cell surface-binding ELISA in the pgsD-677 cell line that does not express HSPG, in order to confirm the non-specific binding ability of the anti-Ras?GTP cytoplasmic penetration antibody containing the light chain variable region with improved endosomal escape ability .

10(A) is a quantitative comparison of the number of cells obtained with trypan blue of pH7.4 and pH5.5 by an anti-Ras?GTP cytoplasmic penetration antibody comprising a light chain variable region with improved endosomal escape ability It is a graph.

Figure 10 (B) is a result of observation with a confocal microscope using calcein (calcein) for the migration to the cytoplasm according to the anti-Ras?GTP cytoplasmic penetration antibody containing a light chain variable region with improved endosomal escape ability .

Figure 10 (C) is a result of confirming the GFP fluorescence by confocal microscopy by complementary binding of the improved split green fluorescent protein of the GFP11-SBP2 fused cytoplasmic penetration antibody wild type and endosome escape enhancing mutant.

Figure 11 (A) is to confirm the specific binding of the GppNHp-associated KRasG12D of anti-Ras?GTP cytoplasmic penetration antibodies containing the light chain variable region with improved endosomal escape ability, 10 or 100nM of KRasG12D?GppNHp and 100nM It is the result of analyzing the binding capacity of KRasG12D?GDP by ELISA.

Figure 11 (B) is a result of confirming the ability to inhibit non-adherent cell growth in human colon cancer cell lines of anti-Ras?GTP cytoplasmic infiltrating antibodies comprising a light chain variable region with improved endosomal escape ability by spheroid proliferation method.

Detailed description and specific embodiments of the invention

The present invention relates to a cytoplasmic penetration antibody or antigen-binding fragment thereof, comprising an amino acid variation in at least one selected from the group consisting of CDR1, FR and CDR3 among the light chain variable regions of SEQ ID NO: 2.

The target of the antibody is preferably an antibody characterized by KRas, but is not limited thereto.

In one embodiment, the cytoplasmic penetration antibody or antigen-binding fragment thereof may include an amino acid substitution selected from the group consisting of the light chain variable region of SEQ ID NO: 2:

Amino acid A at position 34 is replaced by D or E;

Amino acid Y at position 36 is replaced by F;

Amino acid L at position 46 is substituted with K, M, I or R;

89th Q is replaced by E, M, L, I or N;

91st Y is substituted with T, M, F, I or K; And

96th Y is replaced by T, W, F, I or K.

In one embodiment, the cytoplasmic penetration antibody or antigen-binding fragment thereof may include a light chain variable region selected from the group consisting of SEQ ID NOs: 28 to 31. In some cases, the heavy chain variable region of SEQ ID NO: 1 may be further included.

An “Fv” fragment is an antibody fragment that contains a complete antibody recognition and binding site. This region consists of a dimer in which one heavy chain variable domain and one light chain variable domain are tightly virtually covalently associated with, for example, scFv.

The antibody may be in the form of, for example, scFv. The scFv includes the VH and VL domains of the antibody, and may further include a polypeptide linker between the VH domain and the VL domain, which enables to form a desired structure for antigen binding.

“Variable region” refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of complementarity determining regions (CDRs; ie CDR1, CDR2, and CDR3), and framework regions (FR). VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

“Complementarity determining regions” (CDR; ie CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable region, which are necessary for antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2 and CDR3.

“Framework region” (FR) is a variable region residue other than the CDR residues. Each variable region typically has four FRs identified as FR1, FR2, FR3 and FR4.

The antibody or antigen-binding fragment thereof according to the present invention is preferably adopted by the following screening method, but is not limited thereto.

(1) synthesizing a wild type cytoplasmic permeable target antibody by a cell-free protein synthesis method;

(2) diluting the cell-free protein synthesis reaction solution containing the target antibody synthesized in step (1) without purification, to determine a cytotoxicity and cytoplasmic permeable concentration;

(3) PCR amplifying a gene sequence capable of producing a variant cytoplasmic permeable antibody, wherein the sequence of the light chain variable region of the target antibody of step (1) is mutated;

(4) using the PCR amplification product obtained in step (3) as a synthetic template to produce mutant cytoplasmic permeable antibody by cell-free protein synthesis;

(5) treating the variant cytoplasmic permeable antibodies produced in step (4) to reporter cells at a concentration determined in step (2), and selecting the variant cytoplasmic permeability antibody with enhanced cell permeability; And

(6) identifying the affinity for the target antigen of the variant cytoplasmic permeable antibody with enhanced permeability of the cells selected in step (5); a method for screening for a permeable enhanced cytoplasmic permeable antibody.

In step (4), the PCR amplification products are sequentially sequenced from 5'to 3'in the order of promoter, fusion partner, 6×His tag, heavy chain region, linker 1, light chain region, linker 2, GFP ₁₁ , SBP2 and terminator sequence. The linked gene is preferred, but is not limited to this, and can be modified as necessary.

The target antibody is preferably a RT11-3 single chain varible fragment (SCFv) antibody or a variant thereof including the cytoplasmic permeable light chain variable region, but is not limited thereto.

The reporter cells are characterized in cells expressing the GFP _{1-10, 1-10} expressing GFP in the cytoplasm of the reporter cells is characterized in that color development of GFP fluorescence in combination with GFP ₁₁ connected to the penetration antibody. In addition, the reporter cells are preferably HeLa cells, but are not limited thereto.

The fusion partner may be used as long as it is a sequence capable of increasing the expression of downstream genes and the solubility and activity of the expressed antibody, and is preferably a ubiquitin sequence having a ubiquitin or nucleotide sequence variation, but is not limited thereto.

The present invention relates to a conjugate in which a cytoplasmic penetration antibody or an antigen-binding fragment thereof and a bioactive molecule are fused. In addition, the present invention relates to a composition for bioactive molecule delivery in the cytoplasm comprising a cytoplasmic penetration antibody or antigen-binding fragment thereof.

The bioactive molecule may be a form fused or bound to an antibody, for example, one or more selected from the group consisting of peptides, proteins, toxins, antibodies, antibody fragments, RNA, siRNA, DNA, small molecule drugs, nanoparticles and liposomes However, it is not limited thereto.

The proteins include antibodies, antibody fragments, immunoglobulins, peptides, enzymes, growth factors, cytokines, transcription factors, toxins, antigenic peptides, hormones, transport proteins, motor function proteins, receptors, signals ( signaling protein, storage protein, membrane protein, transmembrane protein, internal protein, external protein, secreted protein, viral protein, sugar protein, truncated protein, protein complex, or chemically modified Protein and the like.

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

The siRNA (Small interfering RNA) is an RNA interference material having a small size composed of dsRNA, and serves to decompose by binding to an mRNA having a target sequence, and to treat a target mRNA as a therapeutic agent for a disease or experimentally It is used extensively herein, with the activity of inhibiting the expression of proteins that are degraded and translated by the target mRNA.

The DNA or deoxyribonucleic acid is a type of nucleic acid, and has two types of a monosaccharide, deoxyribose, with a phosphate group bound to a backbone chain, purine, and pyrimidine. It is composed of nucleobases and is a substance that stores genetic information of cells.

The small molecule drug has a molecular weight of less than about 1000 Daltons and is widely used herein to refer to an organic compound, an inorganic compound, or an organometallic compound having activity as a therapeutic agent for a disease. Small molecule drugs used in the present invention include oligopeptides and other biomolecules having molecular weights less than about 1000 Daltons.

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

The liposome consists of one or more lipid bilayer membranes surrounding the aqueous inner compartment, which are capable of associating themselves. Liposomes can be characterized by membrane type and size. Small unilamellar vesicles (SUVs) have a single film and may have a diameter of 20 nm to 50 nm. Large unilamellar vesicles (LUV) can have a diameter of 50 nm or more. Oligolamellar large vesicles and multilamellar large vesicles have multiple, generally concentric, membrane layers and may be 100 nm or more in diameter. Liposomes with multiple non-concentric membranes, ie several small vesicles contained within larger vesicles, are called multivesicular vesicles.

The "fusion" or "binding" is to integrate two molecules with different or identical functions or structures, and any physical, chemical or biological method capable of binding the tumor-penetrating peptide to the protein, small molecule drug, nanoparticle or liposome. It may be a fusion by. The fusion may preferably be by a linker peptide, and the linker peptide may relay fusion with the active material at various positions of the antibody light chain variable region, antibody, or fragment thereof of the present invention.

The present invention is a method for placing a cytoplasmic penetration antibody or an antigen-binding fragment thereof through the cell membrane and placing it in the cytoplasm, wherein the antibody has amino acid mutations in one or more selected from the group consisting of CDR1, FR and CDR3 among the light chain variable regions of SEQ ID NO:2. How to include.

The present invention is a light chain variable region (VL) having a cytoplasmic penetration ability, which penetrates the cell membrane and induces it to be located in the cytoplasm, wherein at least one amino acid mutation selected from the group consisting of CDR1, FR and CDR3 among the light chain variable regions of SEQ ID NO:2 It relates to a light chain variable region (VL) comprising a.

The antibody may include an amino acid substitution selected from the group consisting of light chain variable region of SEQ ID NO: 2 (position is according to Kabat numbering).

Amino acid A at position 34 is replaced by D or E;

Amino acid Y at position 36 is replaced by F;

Amino acid L at position 46 is substituted with K, M, I or R;

89th Q is replaced by E, M, L, I or N;

91st Y is substituted with T, M, F, I or K; And

96th Y is replaced by T, W, F, I or K.

Specifically, the antibody may include a light chain variable region selected from the group consisting of SEQ ID NOs: 28 to 31.

The present invention provides a pharmaceutical composition for the prevention or treatment of cancer, comprising the cytoplasmic penetration antibody or antigen-binding fragment thereof and an active substance in the cytoplasm delivered thereby.

By using the active material, it is possible to impart to the antibody the characteristics of remaining in the cytoplasm by penetrating into the cell without affecting the antigen's high specificity and high affinity. Through this, it is present in the cytoplasm, and a wide flat surface between the protein and the protein Through this, it is possible to expect a high effect in treatment and diagnosis related to tumor- and disease-related factors constituting a structure-composite interaction.

While it is possible to selectively inhibit KRas mutant, which is a major drug resistance-related factor of various existing tumor therapeutic agents, effective anticancer activity can be expected through concurrent treatment with existing therapeutic agents.

The cancer 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, melanoma of the skin or eye, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine adenocarcinoma, Parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocyte lymphoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver tumor, breast cancer, colon cancer, colon cancer, Endometrial or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulva cancer, thyroid cancer, liver cancer and head and neck cancer may be selected from the group consisting of.

When the composition is prepared as a pharmaceutical composition for preventing or treating cancer, the composition may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the composition is commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, fine Crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like, but is not limited thereto. The pharmaceutical composition may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The pharmaceutical composition for the prevention or treatment of cancer may be administered orally or parenterally. In the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration may be administered. When administered orally, proteins or peptides are digested, so oral compositions must be formulated to coat the active agent or to protect it from degradation in the stomach. In addition, the composition can be administered by any device capable of transporting the active substance to the target cell.

Suitable dosages of the pharmaceutical composition for the prevention or treatment of cancer are factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response sensitivity. It can be variously prescribed by. The preferred dosage of the composition is in the range of 0.001-100 mg/kg on an adult basis. The term “pharmaceutically effective amount” means an amount sufficient to prevent or treat cancer, or to prevent or treat diseases caused by angiogenesis.

The composition may be prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and/or excipient, or by incorporating it into a multi-dose container, according to a method easily carried out by those skilled in the art. At this time, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be in the form of ex-agent, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer. In addition, the composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Meanwhile, since the composition includes an antibody or an antigen-binding fragment, it may be formulated as an immune liposome. Liposomes comprising antibodies can be prepared according to methods well known in the art. The immune liposome is a lipid composition comprising phosphatidylcholine, cholesterol and polyethylene glycol-derivatized phosphatidylethanolamine, and may be prepared by reverse phase evaporation. For example, Fab' fragments of an antibody can be conjugated to liposomes through a disulfide-replacement reaction. Chemotherapeutic agents, such as doxorubicin, may additionally be included in the liposome.

The present invention provides a composition for diagnosing cancer comprising the cytoplasmic penetration antibody or an antigen-binding fragment thereof and an active substance in the cytoplasm delivered thereby.

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

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

The molecular imaging phosphor refers to all materials that generate fluorescence, and it is preferable to emit red or near-infrared fluorescence, and a phosphor having a high quantum yield is more preferable, but is not limited thereto. .

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

The phosphor is preferably fluorescein, BODYPY, tetramethyl rhodamine, Alexa, cyanine, allopicocyanine or derivatives thereof. Does not work.

The fluorescent protein is preferably, but not limited to, Dronepa protein, fluorescent chromogenic gene (EGFP), red fluorescent protein (DsRFP), cyanine phosphor that exhibits near-infrared fluorescence, or other fluorescent protein. .

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

The present invention relates to a nucleic acid encoding the cytoplasmic penetration antibody or antigen-binding fragment thereof.

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

The polynucleotide includes not only the nucleotide sequence encoding the light chain variable region (VL) and heavy chain variable region (VH) with improved endosomal escape ability described above, but also a complementary sequence to the sequence. The complementary sequence includes not only perfectly complementary sequences, but also substantially complementary sequences.

The nucleic acid can be modified. Such modifications include addition, deletion or non-conservative substitutions or conservative substitutions of nucleotides. The nucleic acid encoding the amino acid sequence is also interpreted to include a nucleotide sequence showing substantial identity to the nucleotide sequence. The substantial identity is at least 80% homology when aligning the nucleotide sequence with any other sequence to the maximum correspondence and analyzing the aligned sequence using an algorithm commonly used in the art. It may be a sequence that exhibits at least 90% homology or at least 95% homology.

The DNA encoding the antibody is readily separated or synthesized using conventional procedures (eg, by using an oligonucleotide probe capable of specifically binding DNA encoding the heavy and light chains of the antibody).

The vector may be a vector system that simultaneously expresses the light and heavy chains in one vector, or a system that expresses each in separate vectors. In the latter case, both vectors can be introduced into the host cell through co-transfomation and targeted transformation.

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

In the recombinant vector, the light chain variable region, heavy chain variable region and/or linker between them provided in the present invention may be operably linked to a promoter. The term “operatively linked” refers to a functional bond between a nucleotide expression control sequence (eg, promoter sequence) and another nucleotide sequence. Thus, the regulatory sequence can thereby regulate the transcription and/or translation of the other nucleotide sequence.

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

The recombinant vector can be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vector used is an expression vector, and a prokaryotic cell is a host, a strong promoter capable of progressing transcription (eg, pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.) , It usually contains a ribosome binding site for initiation of translation and a transcription/detox termination sequence. When eukaryotic cells are used as hosts, the origin of replication operating in eukaryotic cells included in the vector includes f1 origin of replication, SV40 origin of replication, pMB1 origin of replication, adeno origin of replication, AAV origin of replication, CMV origin of replication, and BBV origin of replication. Including, but not limited to. In addition, a promoter derived from the genome of a mammalian cell (eg, a metallothionine promoter) or a promoter derived from a mammalian virus (eg, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, Cytomegalovirus (CMV) promoter and HSK's tk promoter) can be used and generally have a polyadenylation sequence as the transcription termination sequence.

A host cell transformed with the recombinant vector can be provided.

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

For insertion of the recombinant vector into a host cell, an insertion method well known in the art can be used. For the transport method, for example, when the host cell is a prokaryotic cell, a CaCl2 method or an electroporation method can be used, and when the host cell is a eukaryotic cell, a micro-injection method, calcium phosphate precipitation method, electroporation method, liposome -Mediated transfection and gene bombardment may be used, but is not limited thereto.

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

Hereinafter, the present invention will be described in more detail using examples. It is obvious to those skilled in the art that these examples are only intended to illustrate the present invention more specifically and that the scope of the present invention is not limited by them.

[Materials and methods]

1. Materials

HeLa cells were purchased from the American Type Culture Collection (ATCC) and supplemented with 10% fetal bovine serum (GE Healthcare, Logan, UT, USA) and 1% antibiotic-antibacterial (Thermo Fisher Scientific, Waltham, MA, USA). Cultured in one DMEM (Life Technologies, Grand Island, NY, USA).

ATP, GTP, UTP, CTP, Creatine Phosphate and Creatine Kinase were purchased from Roche Applied Science, Indianapolis, IN, USA.

L-[U- ¹⁴ C] leucine was purchased from Perkin Elmer (Waltham, MA, USA). All other chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification.

The S12 extract was prepared from E. coli strain BL21Star (DE3) (Thermo Fisher Scientific) containing plasmid pTUM4 according to a method known in the art.

2. Construction of modified RT11-3 scFv gene

The VL and VH sequences of antibodies that are inherent in the cytoplasm and bind to KRas·GTP were assembled in the scFv format with (G ₄ S) ₃ linkers between the two chains (RT11-3 scFv).

Modeling of the three-dimensional structure of the mutant RT11-3 scFvs was performed using the ABodyBuilder algorithm (http://opig.stats.ox.ac.uk/webapps/sabpred), and the three-dimensional structure image of the antibody was PyMol It was shown using the program (Schrodinger, Cambridge, MA, USA).

The sequences of RT11 VH and hT4-3 VL constituting RT11-3 scFv are shown in Table 1.

Using the primers disclosed in Table 2, among the amino acid residues constituting RT11-3 scFv, six amino acid residues located in FR2, CDR1 and CDR3 were mutated.

Each of the PCR techniques using the primer sequences disclosed in Table 2 was mutated, and a combination of modified genes was prepared by combining by overlap-extension PCR.

Then, the PCR library was cloned into plasmid pK7 (pK7-RT11-3Lib) and used to transform the E. coli DH5α strain. The individual variant RT11-3 gene was amplified by colony PCR and used as a template in cell-free protein synthesis. According to the experiment, to enhance soluble expression, a ubiquitin sequence was inserted upstream of the modified RT11-3 scFv gene.

3. Cell-free protein synthesis of modified RT11-3 scFvs

The standard reaction mixture for cell-free protein synthesis of RT11-3 scFv is 57 mM HEPES-KOH (pH 8.2); 1.2 mM ATP; 0.85 mM each of GTP, UTP and CTP; 80 mM ammonium acetate; 34 μg/ml 1-5-formyl-5,6,7,8-tetrahydro folic acid (folic acid); 1.0 amino acid each of 20 amino acids; 2% PEG (8,000); 3.2 U/ml creatine kinase; 67 mM creatine phosphate; 0.01 mM L-[U- ¹⁴ C] leucine (11.1 GBq/mmol); 27% (v/v) S12 extract; And 50 ng/ml of PCR amplified DNA component.

On the other hand, for in situ cleavage of ubiquitin during expression of a structure having an N-terminal ubiquitin sequence, a reaction mixture was prepared using a S12 extract rich in UBP1 instead of a standard S12 extract, and the cell-free protein synthesis reaction was performed at 30°C for 1 hour. For a while.

Quantification of scFv was quantified by measuring the TCA insoluble radioactivity level using a Tri-Carb 2810TR liquid scintillation counter. The amount of soluble antibody was determined by measuring the level of TCA insoluble radioactivity in the supernatant after centrifugation (13,000 x g, 10 minutes). The size of the cell-free protein synthesized scFv was confirmed by electrophoresis on 16% tricine gel, followed by Coomassie blue staining or Western blotting.

4. Purification of cell-free protein synthesized RT11-3 scFv

After the cell-free protein synthesis reaction, the reaction mixture was centrifuged at 13,000×g for 10 minutes, and 5 ml of the supernatant was equilibrated with PBS, and then mixed with 400 μl of the Ni-NTA slurry. After washing 3 times with 2 ml of wash buffer (50 mM NaH2PO4, 300 mM NaCl and 10 mM imidazole), the resin-bound scFv was dissolved in 250 μl of elution buffer (50 mM NaH ₂ PO ₄ , 300 mM NaCl and 100 mM) (Imidazole). The eluted solution was desalted and concentrated using a centrifugal ultrafiltration device (Merck Millipore, Burlington, MA, USA) equipped with a barrier membrane of 10,000 molecular weight. 500 μl of eluent was added and centrifuged at 14,000×g for 10 minutes. The concentrated protein (50 μl) was diluted with 450 μl of PBS and centrifuged again. This process was repeated 5 times. After the final centrifugation step, desalted, concentrated scFv was recovered and diluted with PBS to the desired concentration.

5. Analysis of cytoplasmic penetration efficiency of cell-free protein synthesized scFv

The cytoplasmic penetration efficiency of RT11-3 scFv was evaluated by split-GFP complementation assay. The cytoplasmic-penetration efficiency of a cell-free protein synthesized RT11-3 scFv variant was measured using a HeLa cell line expressing a fusion structure of streptavidin and GFP _1-10 fragment [HeLa (SA-GFP _1-10 )].

RT11-3 scFv antibody contains a streptavidin-binding peptide (SBP2) sequence linked to the GFP ₁₁ fragment, and thus, when the antibody is delivered to the cytoplasm, through the interaction between streptavidin and SBP2 , From the fragmented GFP _1-10 and GFP ₁₁ fragments fused to show GFP fluorescence, cytoplasmic penetration of the RT11-3 scFv antibody was confirmed.

HeLa-SA-GFP _1-10 cells were inoculated in 96-well plates at a density of 1×10 ⁴ cells per well in 100 μl of DMEM and cultured at 37° C. for 5 hours at 5% (v/v) CO ₂ condition. . After washing with PBS, reporter cells were treated with 50 μl of cell-free protein synthesis RT11-3 scFv for 12 hours. Thereafter, the reporter cells were washed twice with PBS, and resuspended in 100 μl of PBS, and then GFP fluorescence (excitation wavelength: 485 nm / emission wavelength: 528 nm) was measured. Whether or not the cytoplasmic delivery of RT11-3 scFv was confirmed through confocal microscopy image analysis of reporter cells.

6. Cell Morphology and Viability Analysis

Flow cytometry (FACS Canto, BD Biosciences, East Rutherford, NJ, USA) was performed to determine if the reaction mixture for cell-free protein synthesis affects the morphology and viability of reporter cells.

3×10 ⁵ reporter cells were treated with 1.2 ml of reaction mixture diluted in various ratios for 12 hours, and then analyzed by flow cytometry. Cell morphology was determined based on forward scattering and lateral scattering light.

7. Conversion of RT11-3 scFv to IgG

Heavy chain variable region (RT11 VH: SEQ ID NO: X) and heavy chain constant region of an antibody in which DNA encoding the secretion signal peptide is fused at the 5'end to construct a heavy chain expression vector for production in the form of a complete IgG monoclonal antibody DNAs encoding heavy chains containing (CH1-hinge-CH2-CH3) were cloned into NotD/HindIII into pcDNA3.4 (Invitrogen) vector, respectively. In addition, in order to construct a vector expressing a light chain, a cytoplasmic penetration light chain variable region (hT4-3, #1-60, #5-10, #6-32, fused with DNA encoding a secretory signal peptide at the 5'end) 6-91) and the DNA encoding the light chain containing the light chain constant region (CL) were cloned into NotD/HindIII into the pcDNA3.4 vector, respectively.

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

8. Cell-Based Enzyme-Linked Immunosorbent (ELISA) Analysis to Confirm Nonspecific Binding Capacity of Mutant RT11-3 IgG

The experiment was conducted to confirm the change in the non-specific binding capacity of the antibody according to the manipulation of the light chain variable region sequence. After culturing the HeLa (HSPG+) and pgsD-677 (HSPG-) cell lines in a 96-well plate so that the cells were completely filled at the bottom of the well, the cells were washed three times with a washing buffer (HBSS buffer, 50MM HEPES). Then, PBS, RT11-3, #1-60, #5-10, #6-32, 6-91, cetuximab 100 were added to a blocking buffer (HBSS buffer, 50MM HEPES, 1% BSA). , 50, 25, 12.5, 6.25 and 3.125ng/ml diluted and incubated for 2 hours at 4 ℃. After washing three times with washing buffer, HRP-conjugated anti-human mAb is conjugated with a labeled antibody. Absorbance was quantified at 450 nm by reacting with TMB ELISA solution.

9. Analysis of cytoplasmic penetration ability of mutant RT11-3 IgG antibody

Three assays were performed to analyze the cytoplasmic penetration ability of the cytoplasmic penetration antibody.

(1) Trypan Blue Assay

In a 24-well plate, a cover slip was placed and 200 μl of 0.01% poly-L-lysine solution was added to the floating cell to attach Ramos to the plate, and reacted at 25° C. for 20 minutes. After washing with PBS, 5×10 ⁴ Ramos cells per well were put in 0.5 ml of medium containing 10% FBS and cultured at 37° C. for 30 minutes. Cell adhesion was confirmed and the cytoplasmic pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4), the initial endosomal pH pH 5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5) 200 μl, 0.5 μM and 1 μM RT11-3, #1-60, #5-10, #6-32, and 6-91 were added and incubated at 37°C for 2 hours. Then, after carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl per well was dispensed and observed under a microscope.

(2) Calcein Assay

A cover slip was placed in a 24-well plate, and 2.5×10 ⁴ HeLa cells per well were put into 0.5 ml of a medium containing 10% FBS and cultured at 5% CO ₂ and 37° C. for 12 hours. After confirming cell adhesion, RT11-3, #1-60, #5-10, #6-32, 6-91 0.1, 0.25, 0.5 μM were cultured at 37° C. for 6 hours. After 4 hours, the wells containing the antibody were treated with 150 μM calcein and incubated at 37° C. for 2 hours. After removing the medium and washing with PBS, proteins attached to the cell surface were removed with a weakly acidic solution (200 mM glycine, 150 mM NaCl pH 2.5). After PBS washing, cells were fixed for 10 minutes at 25°C after adding 4% paraformaldehyde. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope.

(3) Split-GFP Compensation Assay

A cover slip was placed in a 24-well plate, and 2.5×10 ⁴ HeLa cells per well were put in 0.5 ml of a medium containing 10% FBS and cultured at 5% CO ₂ and 37° C. for 12 hours. After confirming cell adhesion, 200 μl of RT11-3, #1-60, #5-10, #6-32 and 6-91 IgG were treated and incubated for 6 hours. Thereafter, the reporter cells were washed with PBS and a weakly acidic solution, and then the cells were fixed. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope.

10. Enzyme-linked immunosorbent assay for mutant RT11-3 IgG antigen (ELISA)

The experiment was conducted to confirm the functional change of the heavy chain variable region according to the change of the light chain variable region. TMab4-3, which is a cytoplasmic penetration antibody containing TMab4 VH without antigenic targeting ability and cytoplasmic penetration light chain variable region, was used as a control, and an anti-Ras?GTP cytoplasmic penetration antibody RT11-3 containing a light chain variable region with improved cytoplasmic penetration ability, After #1-60, #5-10, #6-32 and 6-91 were combined at a concentration of 5 μg/ml in 96-well EIA/RIA plates for 1 hour at room temperature, 0.1% TBST (12 mM Tris, It was washed three times for 10 minutes with pH 7.4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween20, 5 mM MgCl ₂ ). Then, 4% TBSB (12mM Tris, pH7.4, 137mM NaCl, 2.7mM KCl, 4% BSA, 10mM MgCl ₂ ) was combined for 1 hour and washed 3 times with 0.1% TBST for 10 minutes. KRas protein bound with GppNHp was diluted with concentrations of 100nM and 10nM, and KRas protein bound with GDP was diluted with 4% TBSB at a concentration of 100nM for 1 hour at room temperature, washed 3 times for 10 minutes with 0.1% TBST. . HRP-conjugated anti-his mAb was conjugated with a labeled antibody. Absorbance was quantified at 450 nm by reacting with TMB ELISA solution.

11.Assessment of mutant RT11-3 IgG's ability to inhibit tumor cell growth

Changes in tumor cell growth inhibitory ability according to the improvement of cytoplasmic penetration ability of anti-Ras?GTP cytoplasmic penetration antibody were analyzed. 1×10 ³ human colorectal cancer cell lines SW480 and Colo320DM per well were diluted in 50 µl medium containing 1% FBS per well in a low-attchatment 96-well plate, and after 12-18 hours, spheroid formation was confirmed. . Then, 50 μl of the medium containing 1% FBS was incubated for 48 hours at 37° C. and 5% CO ₂ conditions with 0.5 and 2 μM antibodies. Thereafter, the antibody was further treated twice, and cultured for 48 hours. Finally, after incubation for a total of 144 hours, 50 μl of CellTiterGlo (Promega) was added to quantify luminescence.

Example 1. Cytoplasmic penetration of cell-free protein synthesized RT11-3 scFv

As a platform expressing RT11-3 scFv, a cell-free protein synthesis system derived from E. coli extract was used. The RT11-3 scFv was produced at a concentration of 6.4 μM (221 μg/ml) in an initial experiment using a standard reaction mixture, and about 25% of the synthetic protein was included in the soluble fraction. RT11-3 scFv synthesized in 5 ml reaction was purified using Ni-NTA agarose resin. As described in [Materials and Methods], after desalting and concentration, about 1.8 nmole (62 μg) of purified protein was obtained in 0.3 ml of phosphate buffered saline (PBS: 6.0 μM).

The purified RT11-3 scFv antibody was treated in a medium containing HeLa-SA-GFP1-10 reporter cells, and then confirmed through confocal microscopy image analysis. The cytoplasmic penetration of the cell-free protein-synthesized RT11-3 scFv antibody was confirmed from the GFP fluorescence intensity increasing in proportion to the amount of protein added to the medium (FIG. 1 ).

Example 2. Establishing cytoplasmic-penetration assay

Engineering cytoplasmic infiltrating antibodies involves the expression and screening of many gene structures, and is very difficult if each variant must be purified for functional analysis. Therefore, it was investigated whether the cell-free protein-synthesized RT11-3 scFv can be directly used for cell-based functional analysis without a separate purification process.

After the cell-free protein synthesis of RT11-3 scFv, 400 μl of the reaction mixture was added to the reporter cells without purification. As a result, the reporter cells to which the cell-free protein compound was added were found to die. After incubation for 12 hours, the cells died and decomposed. Subsequently, it was confirmed that the cytotoxicity appeared to be caused by the components of the S12 extract derived from E. coli. Reporter cells were hardly affected when cultured separately from the rest of the main reactants (salt solution, PEG, creatine phosphate and amino acids) other than E. coli derived S12 extract added during cell-free protein synthesis (Fig. 2).

In addition, by diluting the cell-free protein synthesis reaction mixture, the cell viability of reporter cells was analyzed by diluting the cell-free protein synthesis reaction mixture by concentration in DMEM (Dulbecco's Modified Eagle's Medium) in order to confirm that the above-mentioned cytotoxicity is alleviated. .

As a result of confocal microscopy image analysis, the reporter cells were not significantly affected when the reaction mixture was diluted 4 times or more (FIG. 3A), and similarly, in the flow cytometry, the proportion of living cells was diluted 4 times or more for 12 hours. It was confirmed that cells were alive without being affected by incubation with (FIG. 3B).

Recently, a ubiquitin (hereinafter referred to as'UCE1') base sequence has been developed that stimulates the translation of a fusion protein much more effectively than a wild-type ubiquitin sequence. Can be improved up to. When fused to the gene encoding RT11-3 of the present invention, as a result of acellular protein synthesis of RT11-3 scFv fused with UCE1, soluble RT11-3 scFv of about 2.8 μM was produced (FIG. 4A ).

In addition, an antibody having a wild-type RT11-3 scFv sequence was produced using S12 extract containing UBP1 (ubiquitin carboxyl-terminal hydrolase 1), and UBP1 contained in the S12 extract cleaves the UCE1 tag in situ during a cell-free protein synthesis reaction. Did. Western blot analysis confirmed that the concentration of the soluble protein of RT11-3 scFv in which the ubiquitin sequence was removed was maintained by maintaining cell-free protein synthesis from the extract rich in UBP1 (FIG. 4B ).

Example 3. Expression of RT11-3 modification and evaluation of cytoplasmic permeability

The mutant RT11-3 gene was mutated through the process disclosed in FIG. 5, and the mutated gene library was cloned into the pK7 plasmid (pK7RT11-3).

Individual mutant genes were PCR amplified from 950 colonies of E. coli transformed with pK7RT11-3Lib and expressed in a reaction mixture for cell-free protein synthesis in 96-well micro titer plates as described in Materials and Methods. After the plate was incubated for 1 hour, 15 μl of each reaction mixture was diluted 4 fold in DMEM and 50 μl was transferred to aspirated reporter cells. When the intracellular GFP signal was analyzed in the reporter cell, as shown in FIG. 6, the mutant clones showed various GFP fluorescence intensities, and some variant clones showed significantly stronger GFP fluorescence than the parent gene. Among them, we selected the four clones (#1-60, #5-10, #6-32 and 6-91) that showed the strongest fluorescence compared to the parent RT11-3, and the nucleotide sequence of these variants ((# 1-60 (SEQ ID NO: 24), #5-10 (SEQ ID NO: 25), #6-32 (SEQ ID NO: 26), 6-91 (SEQ ID NO: 27)) and amino acid sequence (#1-60 (SEQ ID NO: 28), #5-10 (SEQ ID NO: 29), #6-32 (SEQ ID NO: 30), 6-91 (SEQ ID NO: 31)) were analyzed (FIG. 7). The amount of cytoplasmic penetrating antibody in this assay can be influenced by the efficiency of endosomal escape as well as its expression level. According to the results of the Split-GFP complementation assay, reporter cells of the same concentration (1.5 μM) as the selected mutant scFv were purified after purification. Measurement of the intensity of GFP fluorescence in reporter cells by confocal microscopy showed that GFP fluorescence showed the strongest antibody with #6-32, and GFP fluorescence intensity in the order of antibodies with 6-91, #1-60, and #5-10. It was confirmed (Fig. 8).

Example 4. Fully IgG-type cytoplasmic penetration antibody mutation RT11-3 expression and purification including the selected light chain variable region (VL) with improved endosomal escape ability

In Examples 1 to 3, the cytoplasmic penetration antibody was constructed in scFv form to select a light chain variable region with improved endosomal escape ability. Improving the escape ability of the selected endosomes The light chain variable region was expressed and purified in animal cells as a monoclonal antibody in the form of a complete IgG to confirm the improvement of the escape ability of endosomes.

Specifically, in order to construct a heavy chain expression vector for producing in the form of a complete IgG monoclonal antibody, a heavy chain variable region of an antibody in which DNA encoding the secretion signal peptide is fused at the 5'end (RT11 VH: SEQ ID NO: 1) DNA encoding the heavy chain containing the heavy chain constant region (CH1-hinge-CH2-CH3) was cloned into NotD/HindIII in pcDNA3.4 (Invitrogen) vector, respectively. In addition, in order to construct a vector expressing a light chain, a cytoplasmic penetration light chain variable region (hT4-3, #1-60, #5-10, #6-32, fused with DNA encoding a secretory signal peptide at the 5'end) 6-91) and the DNA encoding the light chain containing the light chain constant region (CL) were cloned into NotD/HindIII into pcDNA3.4 (Invitrogen) vector, respectively.

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 Polyethylenimine (PEI) (Polyscience). Upon 200 ml transfection in a shake flask (Corning), HEK293-F cells were seeded in 100 ml of medium at a density of 2×10 ⁶ cells/ml, and cultured at 150 rpm, 8% CO ₂ . For the production of each monoclonal antibody, the appropriate heavy and light chain plasmids were diluted in 10 ml of FreeStyle 293 expression medium (Invitrogen) to 125 μg of heavy chain and 125 μg of light chain to a total of 250 μg (2.5 μg/ml), 750 μg (7.5 μg/ml) of PEI Was mixed with diluted 10 ml of medium and reacted at room temperature for 10 minutes. Thereafter, the reacted mixed medium was put into cells seeded with 100 ml previously, and then cultured at 150 rpm and 8% CO2 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 taken with reference to the standard protocol. Antibodies were applied to Protein A Sepharose column (GE healthcare) and washed with PBS (pH 7.4). After eluting the antibody at pH 3.0 with 0.1M glycine buffer, the sample was immediately neutralized with 1M Tris buffer. The eluted antibody fraction was concentrated by exchanging buffer with PBS (pH 7.4) through a dialysis method. The purified protein was quantified using absorbance and absorption coefficient at a wavelength of 280 nm.

Example 5. Confirmation of non-specific binding of the cytoplasmic penetration antibody in the form of a complete IgG comprising a light chain variable region (VL) with improved endosome escape capability

Anti-Ras?GTP cytoplasmic penetration using the existing hT4-3 light chain, wherein the non-specific binding of the complete IgG form of the cytoplasmic penetration antibody comprising the light chain variable region (VL) enhanced in the selected endosomes escape ability constructed in Example 4 above It was observed using a cell-based ELISA for comparison with the antibody (RT11-3).

Specifically, the HeLa (HSPG+), pgsD-677 (HSGP-) cell line was incubated in a 96-well plate so that the cells were completely filled at the bottom of the well, and then washed three times with a washing buffer (HBSS buffer, 50 mM HEPES). Then, PBS, RT11-3, #1-60, #5-10, #6-32, #6-91, cetuximab were added to blocking buffer (HBSS buffer, 50mM HEPES, 1% BSA) 100, 50, 25 , 12.5, 6.25 and diluted to a concentration of 3.125ng/ml, and incubated at 4°C for 2 hours. After washing three times with washing buffer, HRP-conjugated anti-human mAb was conjugated with a labeled antibody. Absorbance was quantified by reacting with TMB ELISA solution. The highest non-specific cell surface binding capacity was measured in #6-32 antibody, and the other antibodies confirmed the non-specific cell surface binding capacity almost similar to that of wild type RT11-3 (FIG. 9).

Example 6 Evaluation of Endosomes Escape Ability of Cytoplasmic Infiltration Antibodies in Complete IgG Form Containing Enhanced Light Chain Variable Region (VL)

In Example 5, a light chain variable region with improved endosomal escape ability was selected in the form of scFv, and the endosomes escape ability improvement of the complete IgG form containing this light chain variable region was improved.

(1) Trypan Blue Assay

In a 24-well plate, 200 µl of a 0.01% poly-L-lysine solution was added to attach a cover slip and attach the floating cell Ramos to the plate, and reacted at 25° C. for 20 minutes. After washing with PBS, 5×10 ⁴ Ramos cells per well were put into 0.5 ml of a medium containing 10% FBS and cultured at 37° C. for 30 minutes. Check the cell adhesion and the pH 7.4 buffer (HBSS (Welgene), 50 mM HEPES pH 7.4), which is the cytoplasmic pH, pH 5.5 buffer (HBSS (Welgene), 50 mM MES pH 5.5), which is the initial endosomal pH, RT11-3, # 200 μl 1-60, #5-10, #6-32, #6-91 0.5 μM and 1 μM were added and cultured for 2 hours at 37°C. After carefully washing with PBS, 10 μl of trypan blue was mixed with 190 μl of PBS, and 200 μl per well was dispensed and observed under a microscope. The #1-60, #5-10, and #6-32 antibodies except for the #6-91 antibody showed improved trypan blue results compared to RT11-3 (FIG. 10(A)).

(2) Calcein Fluorescence Assay

A cover slip was placed in a 24-well plate, and 2.5×10 ⁴ HeLa cells per well were put into 0.5 ml of a medium containing 10% FBS and cultured at 5% CO 2 at 37° C. for 12 hours. After confirming cell adhesion, RT11-3, #1-60, #5-10, #6-32, #6-91 0.1, 0.25, 0.5 μM were cultured at 37° C. for 6 hours. After 4 hours, 150 μM of calcein was treated in the wells containing the antibody and cultured at 37° C. for 2 hours. After removing the medium and washing with PBS, proteins attached to the cell surface were removed with a weakly acidic solution (200 mM glycine, 150 mM NaCl pH 2.5). After PBS washing, 4% paraformaldehyde was added and cells were fixed for 10 minutes at 25 degrees. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. In cells treated with #1-60, #5-10, and #6-32 antibodies, green calcein fluorescence spreading in the cytoplasm was more clearly observed than those treated with RT11-3. On the other hand, in cells treated with #6-91 antibody, green calcein fluorescence spreading in the cytoplasm was observed similarly to RT11-3 (Fig. 10(B)).

Accordingly, it was confirmed that even when the light chain variable region with improved endosomes escape ability selected in the form of scFv was newly constructed and expressed and purified with an endosomes escape ability enhancement cytoplasmic penetration antibody in full IgG form, it was confirmed that the endosomes escape ability was improved compared to the wild type.

Example 7. Cytoplasmic penetration through complementary binding of an improved split green fluorescent protein Confirmation of the cytoplasmic location of a monoclonal antibody

Improved endosomal escape ability An improved split green fluorescent protein complementation system (Registration No.: 10-1790669) was used to confirm the improvement of the cytoplasmic position of the cytoplasmic penetration antibody. To this end, RT11-3-GFP11-SBP2, #1-60-GFP11-SBP2, #5-10-GFP11-SBP2, #6-32-GFP11- fused with GFP11-SBP2 peptide to the heavy chain C-terminus of RT11 SBP2, and #6-91-GFP11-SBP2 were constructed.

Specifically, in the same manner as in Example 2, when the transformed HeLa cell line stably expressing SA-GFP1-10 was prepared to stabilize the cells, RT11-3-GFP11-SBP2, #1-60-GFP11-SBP2, # 5-10-GFP11-SBP2, #6-32-GFP11-SBP2, #6-91-GFP11-SBP2 0.2, 0.4, and 0.8 μM were cultured at 37° C. for 6 hours. After washing with PBS and a weakly acidic solution, the cells were fixed. The nuclei were stained (blue fluorescence) using Hoechst 33342 and observed with a confocal microscope. In cells treated with #1-60, #5-10, and #6-32 antibodies, improved GFP fluorescence was observed compared to cells treated with RT11-3. On the other hand, GFP fluorescence similar to RT11-3 was observed in cells treated with #6-91 antibody. Therefore, it was confirmed that the amount of cytoplasmic penetration antibody located in the cytoplasm by escaping the endosome was improved as a result (FIG. 10(C)).

Example 8. Evaluation of tumor cell growth inhibitory ability of anti-Ras?GTP cytoplasmic penetration antibody comprising light chain variable region with improved endosomal escape ability

TMab4-3, which is a cytoplasmic penetration antibody containing TMab4 VH without an antigen targeting ability and an endosomal escape light chain variable region, was used as a control, and an anti-Ras?GTP cytoplasmic penetration antibody RT11- comprising an endosomal escape ability enhanced light chain variable region 3, #1-60, #5-10, #6-32, #6-91 were combined at a concentration of 5 μg/ml in 96-well EIA/RIA plates for 1 hour at room temperature, and then 0.1% TBST (12 mM) Tris, pH 7.4, 137mM NaCl, 2.7mM KCl, 0.1% Tween20, 5mM MgCl ₂ ) were washed 3 times for 10 minutes. Then, 4% TBSB (12mM Tris, pH 7.4, 137mM NaCl, 2.7mM KCl, 4% BSA, 10mM MgCl ₂ ) was combined for 1 hour and washed 3 times with 0.1% TBST for 10 minutes. KRas protein bound with GppNHp was diluted with various concentrations of 100nM and 10nM, and KRas protein bound with GDP was diluted with 4% TBSB at 100nM concentration for 1 hour at room temperature and then washed 3 times for 10 minutes with 0.1% TBST. . HRP-conjugated anti-his mAb was conjugated with a labeled antibody. 450nm absorbance was quantified by reacting with TMB ELISA solution. Antibodies #1-60, #6-32, and #6-91, except for #5-10 antibodies, show KRasG12D binding similar to RT11-3, and the Ras targeting ability of heavy chain variable regions is affected by changes in light chain variable regions. It was confirmed that it was hardly received (Fig. 11(A)).

In addition, 1×10 ³ of the above cell lines were diluted in 50 µl of a medium containing 1% FBS per well in a low-attchatment 96-well plate, and after 12-18 hours, spheroid formation was confirmed. Subsequently, 50 μl of the medium containing 1% FBS was incubated for 48 hours at 37° C. and 5% CO ₂ conditions with 0.5 and 2 μM antibodies. Thereafter, the antibody was further treated twice to incubate for 48 hours. Finally, after incubation for a total of 144 hours, 50 μl of CellTiterGlo (Promega) was added to quantify luminescence.

As a result, as shown in FIG. 11(B), when compared with the existing RT11-3, the #1-60 and #6-32 antibodies, which have improved endosomal escape ability and maintain antigen targeting ability, are Ras mutant cell line specific cells. It was confirmed that the growth inhibitory effect was improved.

The present invention relates to an antibody having improved cytoplasmic permeability, and is an antibody having a mutant sequence introduced into a light chain variable region. The antibody according to the present invention has an effect of improving endosomal escape ability and maintaining antigen targeting ability.

Electronic file attached.

Claims (17)

1. A cytoplasmic penetration antibody or antigen-binding fragment thereof, comprising an amino acid variation in at least one selected from the group consisting of CDR1, FR and CDR3 among the light chain variable regions of SEQ ID NO: 2.
2. The cytoplasmic penetration antibody or antigen-binding fragment thereof according to claim 1, wherein the target of the antibody is KRas.
3. According to claim 1, wherein the light chain variable region of SEQ ID NO: 2 comprises an amino acid substitution selected from the group consisting of (position according to Kabat numbering), cytoplasmic penetration antibody or antigen-binding fragment thereof:

   Amino acid A at position 34 is replaced by D or E;

   Amino acid Y at position 36 is replaced by F;

   Amino acid L at position 46 is substituted with K, M, I or R;

   89th Q is replaced by E, M, L, I or N;

   91st Y is substituted with T, M, F, I or K; And

   96th Y is replaced by T, W, F, I or K.
4. According to claim 1, comprising a light chain variable region selected from the group consisting of SEQ ID NO: 28 to 31, cytoplasmic penetration antibody or antigen-binding fragment thereof.
5. According to claim 1, wherein the antibody is a cytoplasmic penetration antibody or antigen-binding fragment thereof, characterized in that the scFv form.
6. According to claim 1, Further comprising a heavy chain variable region of SEQ ID NO: 1, the cytoplasmic penetration antibody or antigen-binding fragment thereof.
7. A nucleic acid encoding the cytoplasmic penetration antibody of any one of claims 1 to 6 or an antigen-binding fragment thereof.
8. A composition for delivery of a bioactive molecule in the cytoplasm comprising the cytoplasmic penetration antibody of any one of claims 1 to 6 or an antigen-binding fragment thereof.
9. The composition of claim 8, wherein the active material is at least one selected from the group consisting of peptides, proteins, toxins, antibodies, antibody fragments, RNA, siRNA, DNA, small molecule drugs, nanoparticles, and liposomes.
10. A conjugate in which the cytoplasmic penetrating antibody of any one of claims 1 to 6 or an antigen-binding fragment thereof and a bioactive molecule are fused.
11. The conjugate of claim 10, wherein the bioactive molecule is selected from the group consisting of peptides, proteins, small molecule drugs, nanoparticles, and liposomes.
12. A method of placing a cytoplasmic penetration antibody or antigen-binding fragment thereof through the cell membrane and placing it in the cytoplasm, wherein the antibody comprises an amino acid variation in one or more selected from the group consisting of CDR1, FR and CDR3 among the light chain variable regions of SEQ ID NO:2 .
13. The method of claim 12, wherein the antibody comprises an amino acid substitution selected from the group consisting of the light chain variable region of SEQ ID NO: 2 (position is according to Kabat numbering):

    Amino acid A at position 34 is replaced by D or E;

    Amino acid Y at position 36 is replaced by F;

    Amino acid L at position 46 is substituted with K, M, I or R;

    89th Q is replaced by E, M, L, I or N;

    91st Y is substituted with T, M, F, I or K; And

    96th Y is replaced by T, W, F, I or K.
14. The method of claim 12, comprising a light chain variable region selected from the group consisting of SEQ ID NOs: 28 to 31.
15. A light chain variable region (VL) having a cytoplasmic penetration ability that penetrates into a cell membrane and induces its location in the cytoplasm, wherein the light chain variable region of SEQ ID NO: 2 comprises an amino acid variation in one or more selected from the group consisting of CDR1, FR and CDR3 Light chain variable region (VL).
16. The light chain variable region (VL) according to claim 15, wherein the antibody comprises an amino acid substitution selected from the group consisting of light chain variable region of SEQ ID NO: 2 (position is according to Kabat numbering):

    Amino acid A at position 34 is replaced by D or E;

    Amino acid Y at position 36 is replaced by F;

    Amino acid L at position 46 is substituted with K, M, I or R;

    89th Q is replaced by E, M, L, I or N;

    91st Y is substituted with T, M, F, I or K; And

    96th Y is replaced by T, W, F, I or K.
17. The light chain variable region (VL) according to claim 15, comprising a light chain variable region selected from the group consisting of SEQ ID NOs: 28 to 31.

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