Cytosol-penetrating antibodies with highly efficient cytosol penetrating ability and uses thereof

Abstract

The present invention relates to: an antibody constant region in which an endosomal escape motif having activity under endosomal slightly acidic conditions is introduced into the constant region of an antibody to impart the ability of an immunoglobulin G (IgG) antibody to actively translocate from outside a cell into the cytoplasm; an antibody comprising same; a method for producing same; and use thereof. More specifically, the present invention provides a high-efficiency cytosol-penetrating antibody (cytotransmab) having an improved endosomal escape ability by introducing an endosomal escape motif into a CH3 domain, which is a heavy chain constant region of an IgG antibody, and / or a CL domain, which is a light chain constant region thereof, and relates to a pharmaceutical composition for the treatment, prevention or diagnosis of cancer, comprising the antibody.

Description

Cytoplasmic penetrating antibody having high-efficiency cytoplasmic penetrating ability and use thereof

[0001] The present invention relates to an antibody constant region in which an endosomal escape motif is introduced into the constant region of an antibody to impart the ability of an immunoglobulin G (IgG) antibody to be actively located inside the cytoplasm from outside a cell, an antibody comprising the same, a method for producing the same, and a use thereof. More specifically, the present invention relates to a highly efficient cytosol-penetrating antibody (cytotransmab) having an enhanced endosomal escape ability by introducing an endosomal escape motif into the CH3 domain, which is a heavy chain constant region, and / or the CL domain, which is a light chain constant region, of an IgG antibody, and to a pharmaceutical composition for treating, preventing, or diagnosing cancer comprising the antibody.

[0002]

[0003] An antibody is an immunoprotein that binds to an antigen and interferes with its action or removes it. There are five types of antibodies: immunoglobulin (Ig) M, IgD, IgG, IgA, and IgE. A complete immunoglobulin G (IgG) antibody forms a very stable Y-shaped structure (molecular weight, 150 kDa) made of two heavy chain (50 kDa) proteins and two light chain (25 kDa) proteins. The heavy and light chains of antibodies are divided into a variable region whose amino acid sequence differs from antibody to antibody and a constant region whose amino acid sequence is the same. The heavy chain constant region contains CH1, H (hinge), CH2, and CH3 domains. Each domain consists of two β-sheets, which are connected by disulfide bonds.

[0004] The two variable regions of the heavy and light chains combine to form an antigen binding site, one on each of the Y-shaped arms. The part that can bind to the antigen is called Fab (antibody binding fragment), and the part that cannot bind to the antigen is called Fc (crystallizable fragment). Fab and Fc are connected by a flexible hinge region.

[0005] Within the heavy chain variable region (VH) and light chain variable region (VL) of antibodies, there are regions whose amino acid sequences differ significantly from antibody to antibody. These regions are called complementarity determining regions (CDRs) because they constitute the site that binds to the antigen. Looking at the three-dimensional structure of antibodies, these CDRs form loops on the surface of the antibody, and below the loops are framework regions (FRs) that structurally support them. There are three loop structures in each of the VH and VL, and these six loop structures join together to make direct contact with the antigen.

[0006] The CH2-CH3 portion of the heavy chain constant region (Fc) of antibodies contains a binding site for the neonatal Fc receptor (FcRn), which prolongs the serum half-life of antibodies. Furthermore, the hinge-CH2 portion contains binding sites for FcR (Fc receptor) and complement, mediating antibody-dependent cellular cytotoxicity and complement-dependent cellular cytotoxicity.

[0007] Numerous therapeutic antibodies have been developed that target proteins secreted into the cell membrane or the extracellular environment, thanks to their high specificity and affinity for target proteins. All antibodies approved for therapeutic use to date specifically target extracellular or membrane proteins. In contrast, conventional antibodies are large and hydrophilic, making them unable to cross the hydrophobic lipid bilayer-bound plasma membrane or endosomal membrane, preventing them from directly penetrating living cells. Consequently, conventional antibodies face limitations in targeting various disease-related proteins within cells.

[0008] Antibodies targeting membrane receptor proteins can enter cells through the endosomal pathway after binding to the membrane receptor protein through receptor-mediated endocytosis. This process first reaches the early endosome and then goes through several routes. 1) Most antibodies go from the early endosome to the late endosome and then to the lysosome, where they are completely destroyed by acidic conditions and proteolytic enzymes. 2) Some antibodies can bind to FcRn under acidic conditions in the early endosome and go back out of the cell through the recycling endosome pathway. As endosomes mature in the above endosomal pathway, their interior gradually becomes acidic by the proton pump. Early endosomes are known to have a pH of 5.5-6.5, late endosomes have a pH of 4.5-5.5, and lysosomes have a pH of 3.5-4.5. Many protein hydrolytic enzymes are activated in the acidic environment inside lysosomes, so proteins internalized from the outside are destroyed inside lysosomes.

[0009] Antibodies that bind strongly to target membrane proteins are mostly destroyed via the endosomal-lysosomal pathway. Therefore, for an antibody to be located in the cytoplasm, it must first dissociate from the target membrane protein within an early or late endosome before entering the lysosome during its transport along the endosomal pathway following receptor-mediated internalization. This, in turn, leads to membrane destabilization and collapse, allowing the antibody to escape from the endosome into the cytoplasm—a process known as endosomal escape.

[0010] Several naturally occurring toxins and cell-penetrating peptides (CPPs) studied to date are known to penetrate living cells. However, the mechanisms by which these substances enter the cytoplasm remain unclear. There are three main hypotheses regarding their cytoplasmic penetration mechanisms.

[0011] The first hypothesis is the pore formation model, which involves the formation of pores in the cell membrane. In this hypothesis, substances such as cationic amphiphilic peptides bind to the negatively charged cell membrane, causing internal stress, inner membrane contraction, or membrane destabilization, resulting in the formation of a barrel-stave pore or toroidal channel. These pores allow intraendosome material to enter the cytoplasm. The barrel-stave pore model is often used to explain the endosomal escape mechanism of amphipathic α-helical cell-penetrating peptides. The hydrophobic domain of the amphipathic cell-penetrating peptide interacts directly with the hydrophobic hydrocarbon core of the cell membrane, while the hydrophilic domain is exposed to the outside, forming a barrel-stave pore. A toroidal channel is a doughnut-shaped pore formed when a cytoplasmic penetrant interacts with the hydrophilic head region of the cell membrane, structurally distorting the lipid bilayer. The difference between a barrel-stave pore and a toroidal channel lies in whether the hydrophobic hydrocarbon core of the phospholipid bilayer of the cell membrane is exposed. This can vary depending on the composition of the cell-penetrating peptide, its secondary structure, and whether it forms multimers.

[0012] The second hypothesis is the vesicle budding and collapse model, which proposes that cell-penetrating peptides bind to the endosomal membrane, causing it to bend (curvature) to form small vesicles in the endosome. These vesicles then collapse immediately after budding, releasing the contents within the vesicles into the cytoplasm. For vesicles to form in the endosomal membrane, the endosomal membrane must be structurally distorted. At this point, cell-penetrating peptides bind to the endosomal membrane and destabilize it, lowering the energy barrier for vesicle formation. Ultimately, the budding vesicles are unstable due to their small size, causing them to collapse immediately, releasing the contents within the vesicles into the cytoplasm.

[0013] A third hypothesis posits that endosomes rupture due to the proton sponge effect, releasing their contents into the cytoplasm. After cellular internalization, endosomes actively transport protons into the lumen of the endosome via the V-ATPase proton pump, thereby acidifying the interior of the endosome. Cytosolic penetrants, which have a strong buffering effect, intercept the protons entering the endosome, preventing further acidification. The proton pump then transports even more protons into the lumen of the endosome to lower the pH. To maintain the charge balance within the endosome, chloride ions (Cl-) also enter the lumen, increasing the ion concentration within the endosome. Water also enters the lumen to maintain osmotic pressure. This causes the endosome to swell, and this process repeats until it eventually ruptures. In this hypothesis, the efficiency of endosomal escape is determined by how well the cytoplasmic penetrant can bind to the protons flowing into the endosome, i.e., how excellent the buffering effect of the cytoplasmic penetrant is, so it is a hypothesis that is mainly explained by non-biological macromolecules that can bind many protons, such as cationic polymers, rather than proteins including peptides.

[0014] Among the above endosomal escape hypotheses, the two hypotheses, excluding the proton sponge effect, share the common premise that the cytoplasmic penetrant must first bind to the cell membrane, then locally concentrate on the cell membrane surface to reach a critical concentration, and that membrane destabilization occurs at this stage. Furthermore, these endosomal escape hypotheses assume that the endosomal escape process involves changes in the internal environment of the endosome and the endosomal membrane, and that the cytoplasmic penetrant lowers the high energy barrier required for this process. Therefore, the efficiency of the endosomal escape of the cytoplasmic penetrant depends on how effectively it can lower this energy barrier.

[0015] Various methods have been attempted to deliver antibodies into the cytosol or nucleus of living cells. One method involves genetically fusion or chemical conjugation of a cell-penetrating peptide (CPP) to a full-length IgG antibody or a fragment thereof, such as a single-chain variable fragment (scFv), to create an IgG-CPP or antibody fragment-CPP fusion protein (Bioconjugate Chem. 2019, 30:4:1028-1041).

[0016] Representative CPPs such as TAT, Penetratin, and Polyarginine peptide have been reported to be able to directly pass through the plasma membrane or to be located in the cytoplasm through the endosomal escape pathway after internalization, and to be able to deliver biopolymers such as proteins and DNA into cells. In the case of antibody-CPP fusion proteins in which the above CPPs are fused to antibodies by genetic or chemical methods, there is a technology in which TAT or Polyarginine is directly fused to antibodies, or CPPs are fused to Protein A or Protein G, which bind with high affinity to the Fc region of antibodies, and then fused to antibodies. Another example is a technology in which pH-dependent cell penetration ability is imparted to membrane-lytic spider venom peptide (M-lycotoxin), and then the peptide is treated together with an antibody to deliver the antibody into cells (US Patent No. US11179471B2).

[0017] However, the technology of directly fusing CPPs to antibodies has the problem that the linker between the protein and CPP can affect the cytoplasmic penetration ability of the CPP. In addition, the problems of the CPP itself are the limitations of the antibody-CPP fusion protein technologies. CPPs have low stability, a short half-life in the body, and no cell selectivity, so there is a potential toxicity problem due to non-specific cell penetration. In the case of viral or synthetic CPPs, there is also a problem of potential immunogenicity. In addition, there is a limitation that a significant number of CPPs are unable to escape from the endosome after internalization and are trapped within the endosome (endosomal entrapment), so only about 0.1-2% of the internalized peptides are located in the cytoplasm (Nat Commun. 2021, 12(1):3721).

[0018]

[0019] Another method is antibody-nanoparticle conjugates, which entrap antibodies in polymeric nanoparticles (NPs) or incorporate them within polymeric nanoparticles. For example, a technology for intracellular delivery exists by incorporating antibodies into mesoporous silica nanoparticles (MSNs) (Nanotechnology, 2012, 23(8):085101). While these antibody-nanoparticle conjugates are easy to synthesize and chemically optimize, their cytoplasmic delivery efficiency is low due to endosomal entrapment, similar to cell-penetrating peptides. Furthermore, when antibodies are directly bound to nanoparticles, they remain bound to nanoparticle residues after release into the cytoplasm, potentially interfering with antibody function.

[0020] In addition, there are antibody-liposome complexes that deliver antibodies into living cells using liposomes (BioTechniques, 2008, 44(7S):vii-xi). Liposomes have the advantages of excellent biocompatibility and biodegradability, but they have a low efficiency in accommodating water-soluble molecules in the liposome lumen, and because they often have a positive charge, they have the disadvantage of non-specific binding to the cell surface, similar to CPPs, resulting in low specificity for target cells. In addition, there is the problem of a short half-life because they can be removed by macrophages and other cells through opsonization, in which other serum proteins are adsorbed in the blood.

[0021] Another method reported was an antibody-nucleic acid fusion protein covalently fused to an antibody with a single-stranded phosphorothioate oligonucleotide (JCI Insight, 2019, 25;4(14):e127474). Unlike DNA nucleic acids covalently linked by phosphodiester bonds, phosphorothioated DNA oligonucleotides are linked by a phosphorothioate bond with sulfur in the oxygen position. Although the precise cytoplasmic penetration mechanism of the antibody-phosphorothioated DNA oligonucleotide fusion protein is unclear, it is presumed that the phosphorothioated DNA oligonucleotide moiety binds to the lipid bilayer of endosomes, inducing endosomal escape.

[0022] It has been reported that anti-DNA antibodies derived from patients with autoimmune diseases exhibit cytoplasmic penetration ability in the form of complete IgG without partial fusion that confers external cell penetration ability. In particular, the mouse anti-DNA 3D8 scFv, IgG antibody is internalized into cells through binding of a specific sequence in the light chain variable region (VL) to heparan sulfate proteoglycans on the cell surface, and in the endosomal environment, the amino acid residues in the light chain variable region (VL) CDR1 and CDR3 function as endosomal escape motifs, allowing penetration into the cytoplasm through interaction with the endosomal lipid bilayer (European Patent No. EP2279471B2, Cellular and Molecular Life Sciences, 2009, 66(11-12):1985-97). In addition, the endosomal escape motif of the light chain variable region (VL) of the above mouse antibody was transferred to the human antibody heavy chain variable region (VH) and light chain variable region (VL) to produce a humanized antibody, and cytosol-penetrating ability in the form of a full IgG was reported (Korean Patent No. KR10-1602870, mAbs, 2014, 6(6):1402-1414; Biochem Biophys Res Commun, 2015, 467(4):771-777). An antibody having an endosomal escape motif in the light chain variable region (VL) and / or heavy chain variable region (VH) that induces the cytosol-penetrating ability of such a full IgG antibody was reported to be a cytosol-penetrating antibody (cytotransmab).

[0023] A cytoplasmic penetrating antibody showing cytoplasmic penetrating ability in the form of the IgG was reported by fusing a cyclic peptide that binds to integrin αvβ3 / αvβ5 or epithelial cell adhesion molecule (EpCAM) receptors overexpressed on the surface of tumor cells, or a monobody that binds to EGFR (Epidermal growth factor receptor) to the light chain N-terminus of the cytotransmab antibody showing cytoplasmic penetrating ability in the form of the IgG (Biochem Biophys Res Commun, 2018, 503:2510-2516; Biochem Biophys Res Commun, 2021, 573:35-41, (Domestic Patent Application No. 10-2023-0181933). The antibody binds to tumor cell overexpressed receptors such as integrin αvβ3 / αvβ5 or EpCAM or EGFR, thereby causing receptor-mediated internalization. After entering the cell through endocytosis, it is separated from the tumor cell overexpressed receptor in the endosome-specific environment (acidic, reducing conditions) and has the ability to specifically penetrate the cytoplasm of tumor cells through an endosomal escape mechanism. The integrin αvβ3 / αvβ5-specific cytoplasmic penetrating antibody was named inCT (Science Advances, 2020, 6(3):eaay2174), the EpCAM-specific cytoplasmic penetrating antibody was named epCT (Biochem Biophys Res Commun, 2018, 503:2510-2516), and the EGFR-specific cytoplasmic penetrating antibody was named ERCT (Domestic Patent Application No. 10-2023-08181933) (Biochem Biophys Res Commun, 2018, 503:2510-2516).The inCT cytoplasmic penetrating antibody has the endosomal escape motifs 92WYW94 (Kabat numbering) in the CDR3 region of the VL and 96WYW98 (Kabat numbering) in the CDR3 region of the VH. The inCT(AAA) antibody, in which all WYW residues of the above endosomal escape motifs were substituted with alanine (Ala), did not exhibit cytoplasmic penetration ability (Science Advances, 2020, 6(3):eaay2174; Biochem Biophys Res Commun, 2018, 503:2510-2516). The cytoplasmic penetrating antibody (cytotransmab) was shown to have a pore formation mechanism in which the aromatic residues of the antibody CDR3 region are exposed to the surface through a pH-dependent structural change in the early endosome, allowing them to interact with the endosomal membrane and form toroidal pores due to destabilization of the endosomal membrane (Journal of Controlled Release, 2016, 235:165-175).

[0024] Based on the cytoplasmic penetrating antibody described above, cell / tissue-specific cytosol-penetrating interfering antibody (iMab) technology has been reported as an antibody technology for directly targeting target proteins inside the cytoplasm outside the cell (Korean Patent No. KR10-1602870, KR10-2000000, KR10-2091195, KR10-2163305, Nature Communications, 2017. 8:15090; Science Advances, 2020, 6(3):eaay2174). The cell-penetrating interfering antibody is a form in which the VH portion of the full IgG form targets the target protein inside the cytoplasm, the VL portion has the ability to escape from the endosomes, and a cyclic peptide specific for integrin αvβ3 / αvβ5 is fused to the N-terminus of the VL.

[0025] Cell-penetrating interfering antibodies (iMabs) are full-length immunoglobulin antibodies that specifically bind to cell-surface membrane protein receptors overexpressed in tumor cells or tissues, are internalized, and then localize to the cytoplasm via endosomal escape, where they bind to target proteins in the cytoplasm. For example, a cell-penetrating interfering antibody that specifically binds to activated RAS bound to GTP (RAS·GTP) in the cytoplasm and inhibits the activity of tumor-derived mutant RAS has been reported (Korean Patent No. KR10-2163305; Science Advances, 2020, 6(3):eaay2174; Nature Communications, 2017, 8:15090).

[0026] As described above, various derivative technologies have been studied based on cytoplasmic penetrating antibody technology. However, existing cytoplasmic penetrating antibodies (cytotransmasbs) have endosomal escape motifs limited to the CDR3 of the light chain variable region (VL) and heavy chain variable region (VH), which lead to low endosomal escape efficiency (approximately 13%) and a limited amount of antibody reaching the cytoplasm. Furthermore, when these cytoplasmic penetrating antibodies were applied as cell-penetrating interference antibodies, they failed to exhibit a strong effect due to the small amount reaching the cytoplasm. Furthermore, existing cytoplasmic penetrating antibodies have endosomal escape motifs in the CDR3 of the light chain variable region (VL) and heavy chain variable region (VH). Therefore, to achieve targeting ability against a target protein in the cytoplasm, only one region, either VH or VL, must be utilized, which limits their versatility.

[0027] Each variable domain (VH, VL) and constant domain (CL, CH1, CH2, CH3) that constitutes an antibody belongs to an immunoglobulin domain. An immunoglobulin domain is a type of protein domain that is formed by a two-layer sandwich structure of antiparallel β-sheets consisting of seven to nine β-strands. An important characteristic of an immunoglobulin domain is that the overall structure and function are maintained without significant damage when an appropriate mutation is introduced or the amino acid length is changed in the loop region connecting each β-strand. In addition, many proteins that have an immunoglobulin domain are involved in protein-protein interactions or protein-ligand interactions.

[0028] The most representative example is the CDR of the variable region (VH, VL) of an antibody. The variable region of an antibody is composed of a total of 9 β-strands, which are called strands A, B, C, C`, C``, D, E, F, and G, respectively, and there are AB, BC, CC`, C`C``, C``D, DE, EF, and FG loops connecting each β-strand. Among these, CDR1, 2, and 3 exist in the BC, C`C``, and FG loops, respectively. CDRs have a very high sequence and length diversity, and thus can have binding ability to numerous target proteins.

[0029] The constant region of an antibody is composed of seven β-strands, called strands A, B, C, D, E, F, and G, respectively, and AB, BC, CD, DE, EF, and FG loops connect each β-strand. Since the constant region of an antibody shows a high structural similarity to the variable region, the loops of the antibody constant region can also be endowed with binding ability to target proteins, just like CDRs.

[0030] For example, there is an Fcab (Fc fragment with antigen-binding) that has been modified to have antigen-binding ability for HER2 (human epidermal growth factor receptor 2) or VEGF (vascular endothelial growth factor receptor) in the AB and EF loops of the CH3 domain of the heavy chain constant region (Protein Eng Des Sel, 2017, 30(9):567-581). Through the Fcab technology that confers binding ability for target proteins to the AB and EF loops of the above antibody constant region, it can be confirmed that the AB and EF loops of the antibody constant region are regions that can withstand various mutation introductions and length extensions, i.e., are regions with potential for engineering.

[0031] The domains that make up antibodies are all immunoglobulin domains and are structurally very similar to each other. In particular, the 3D structure of the CL domain is very similar to that of the CH3 domain, with an RMSD (root mean square deviation) value of 1.12 Å. Therefore, by introducing mutations in the CH3 domain into structurally corresponding positions in the CL domain, the functions conferred on the CH3 domain can be conferred on the CL domain, thereby enhancing its function.

[0032] In addition, not only at the single domain level, but also the CH1-CL pair has an overall very similar shape to the CH3-CH3 pair, and the RMSD value of the CH1-CL pair tertiary structure to the CH3-CH3 pair tertiary structure is approximately 1.612 Å. At this time, the CH1-CL pair has a wider area of ​​the interacting interface of 947.2 Å, while the CH3-CH3 pair has a wider area of ​​1127.4 Å, so the CH3-CH3 pair is structurally more stable. Referring to this, there is a FabCH3 technology that replaces the CH1-CL pair with the CH3-CH3 pair, and the FabCH3 antibody has improved thermal stability while maintaining the target protein targeting ability through VH and VL.

[0033] There is also FabCab technology, which combines Fcab technology and FabCH3 technology. Specifically, a bispecific antibody that simultaneously targets HER2 and VEGF exists by replacing the CH1-CL pair in an IgG antibody that targets HER2 through the VH and VL domains with a CH3-CH3 pair that targets VEGF (Biochem Biophys Rep, 2021, 2021;26:100959). Through the FabCab technology, a desired function can be added by replacing the CH1-CL pair of an IgG antibody with a CH3-CH3 pair with a separate function. In the case of the FabCab technology, the CH3 domain that replaces the CH1 and CL domains may bind to the CH3 domain of the original antibody heavy chain constant region Fc region, which may form undesirable oligomers. To prevent this, heterodimer heavy chain constant region technology (heterodimer heterodimeric Fc) can be applied to the CH3-CH3 pair that replaces the CH1-CL pair.

[0034] Heterodimer heterodimeric Fc technology was introduced as a technology for developing bispecific antibodies. This technology induces mutations in the CH3 domains of two different Ig heavy chains through genetic engineering, inducing the two Ig heavy chains to form a heterodimer. Antibodies existing in the natural world exist as homodimers, assembled by two heavy chains with identical amino acid sequences and two light chains with identical sequences. This homodimer formation is induced through interactions between the last domains of the antibody constant region (Fc) (CH3 domains in the case of IgG), followed by the formation of disulfide bonds between the hinge regions, forming a homodimer between the heavy chains. At this time, by using the heterodimer heavy chain constant region technology, it is possible to promote heterodimer formation rather than homodimer formation by introducing mutations in amino acids that contribute to homodimer formation in the CH3 domain. For example, there is the EW-RVT technology that promotes heterodimer constant region (heterodimeric Fc) pair protein formation by introducing K360E, K409W mutations in the CH3-A domain and E347R, D399V, F405T mutations in the CH3-B domain in the CH3 domain of a human IgG1 antibody (Korean Patent No. KR10-1522954, amino acid positions are EU numbering). When the CH1-CL pair is replaced with a CH3 (heterodimeric CH3) pair to which the EW-RVT mutations have been introduced, heterodimer formation between the light chain and the heavy chain is promoted, allowing for the expression of a stable FabCab antibody.

[0035] Diphtheria toxins (DT toxin, PE toxin) derived from Pseudomonas aeruginosa inhibit protein synthesis by inactivating cytoplasmic eEF2, and are known to exhibit potent cytotoxicity, capable of killing cells even with a single molecule. Due to these properties, various studies have been conducted to develop bacterial toxins into therapeutic proteins. However, expressing these toxins in mammalian cells, which causes host cell death during the expression process, remains a significant challenge, making the expression of recombinant proteins fused with the toxins challenging.

[0036] Split intein technology can be utilized as a strategy to address this (PNAS, 2019, 166(44):22164-22172). Split inteins are inteins that are transcribed and translated by two independent genes to perform trans-splicing. Trans-splicing occurs when N-intein (Intein(N), fused to the C-terminus of the N-extein) and C-intein (Intein(C), fused to the N-terminus of the C-extein) are coexpressed. The two split intein fragments associate to restore activation and promote the linkage of the N-extein and the C-extein. This process does not require enzymes or cofactors, and the structure of the expressed protein that folds properly is sufficient.

[0037] This technology allows for the expression of antibodies fused with N-inteins in mammalian cells and for the expression of toxins fused with C-inteins in E. coli, and then the two fragments can be mixed in vitro to purify the toxin-fused antibodies.

[0038] Under this technical background, the inventors of the present application attempted to develop a highly efficient cytoplasmic penetrating antibody by introducing an endosomal escape structural motif different from that of existing cytoplasmic penetrating antibodies into the constant region of an IgG antibody. That is, previously reported cytoplasmic penetrating antibodies had endosomal escape structural motifs in the VL and VH, resulting in low endosomal escape ability. However, by introducing an endosomal escape structural motif different from that of the heavy chain constant region and / or the light chain constant region of an IgG antibody, an antibody with highly efficient endosomal escape ability was designed, constructed, and evaluated, thereby developing a completely new, highly efficient cytoplasmic penetrating antibody with highly efficient endosomal escape ability.

[0039] Specifically, the inventors of the present application developed a novel, highly efficient IgG cytoplasmic penetrating antibody (cytotransmab) by constructing an IgG antibody that introduced an endosomal escape structural motif into the CH3 domain of the heavy chain constant region of an IgG antibody. That is, it was quantitatively confirmed that the cytoplasmic penetrating antibody has improved cytoplasmic penetrating ability compared to existing cytoplasmic penetrating antibodies such as inCT and epCT65 through endosomal escape structural motifs in the existing VH and VL regions.

[0040] In addition, the inventors of the present application developed a novel IgG cytoplasmic penetrating antibody by constructing an IgG antibody in which an endosomal escape structural motif was introduced into the light chain constant region CL domain of the IgG antibody.

[0041] In addition, the inventors of the present application developed a novel, highly efficient IgG cytoplasmic penetrating antibody by constructing an IgG antibody in which an endosomal escape structural motif is endowed to the constant regions, CH3 domain and CL domain.

[0042] Moreover, the inventors of the present application developed a novel highly efficient IgG cytoplasmic penetrating antibody by constructing an IgG antibody in which the CL-CH1 pair was replaced with a CH3-CH3 pair having an endosomal escape structural motif.

[0043] By identifying and evaluating the cytoplasmic penetration ability and biophysical and chemical properties of the above-mentioned high-efficiency IgG cytoplasmic penetrating antibodies, a novel cytoplasmic penetrating antibody (cytotransmab) with high-efficiency endosomal escape ability and potential for development as a therapeutic antibody was developed.

[0044] In addition, the inventors of the present application confirmed that a high-efficiency cytoplasmic penetrating antibody in which an endosomal escape structural motif was introduced into the IgG antibody constant region did not have a significant difference in the antibody's inherent FcRn binding properties compared to a wild-type IgG antibody.

[0045] In addition, the inventors of the present application confirmed that when constructing an IgG antibody having a VH targeting activated RAS bound to α-Tubulin or GTP inserted into the heavy chain variable region (VH) of the novel high-efficiency cytoplasmic penetrating antibody, the antibody actively penetrates into the cytoplasm without a cell permeabilization process using a detergent or the like, and an anti-α-Tubulin cytoplasmic penetrating antibody and an anti-RAS cytoplasmic penetrating antibody that can target α-Tubulin or activated RAS in the cytoplasm more effectively than existing cytoplasmic penetrating antibodies were produced.

[0046] Furthermore, the inventors of the present invention confirmed that by fusing DT toxin to the novel high-efficiency cytoplasmic penetrating antibody using split-intein technology, the antibody actively penetrates the cytoplasm without treatment with detergents or the like during the cell penetration process, thereby producing a cytoplasmic penetrating antibody-based immunotoxin capable of effectively killing target cancer cells both in vitro and in vivo. Through this, the versatility and expandability of the technology were demonstrated, and the present invention was completed.

[0047] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0048]

[0049] Summary of the invention

[0050] The inventors of the present application sought to overcome the problem of low endosomal escape efficiency of existing IgG cytoplasmic penetrating antibodies due to their endosomal escape motifs being limited to only the CDR3 of the light chain variable region (VL) and heavy chain variable region (VH), and to develop a high-efficiency cytoplasmic penetrating antibody comprising an antibody constant region that introduces a new endosomal escape motif into the constant regions of the antibody.

[0051] An object of the present invention is to provide a cytoplasmic penetrating antibody or fragment thereof comprising an endosomal escape structural motif in a constant region.

[0052] The purpose of the present invention is to provide a polynucleotide encoding the cytoplasmic penetrating antibody or a fragment thereof.

[0053] The purpose of the present invention is to provide a recombinant expression vector comprising the above polynucleotide.

[0054] The purpose of the present invention is to provide an isolated host cell containing the recombinant expression vector.

[0055] The purpose of the present invention is to provide a composition for delivery of an active substance into the cytoplasm, comprising the cytoplasmic penetrating antibody or a fragment thereof.

[0056]

[0057] Solution to the problem

[0058] The present invention provides an antibody constant domain having cytoplasmic penetration ability, a highly efficient cytoplasmic penetrating antibody comprising the same, and a method for producing the same, by introducing an endosomal escape structural motif that induces endosomal escape into an IgG antibody heavy chain constant region CH3 domain and / or a light chain constant region CL domain.

[0059] The present invention relates to a cytoplasmic penetrating antibody or fragment thereof comprising an endosomal escape structural motif in the heavy chain constant region and / or the light chain constant region.

[0060] The present invention relates to a polynucleotide encoding the cytoplasmic penetrating antibody or a fragment thereof.

[0061] The present invention relates to a recombinant expression vector comprising the above polynucleotide.

[0062] The present invention relates to an isolated host cell comprising the recombinant expression vector.

[0063] The present invention relates to a composition for delivery of an active substance into the cytoplasm, comprising the cytoplasmic penetrating antibody or a fragment thereof.

[0064] The present invention relates to a highly efficient cytoplasmic penetrating antibody or Fc fragment thereof, which introduces an endosomal escape structural motif through amino acid mutation in a specific region of the CH3 domain of the heavy chain constant region of an IgG antibody, so that an IgG antibody internalized into a cell in response to a change in the slightly acidic environment of an endosome interacts with the endosomal membrane of a lipid bilayer to induce membrane destabilization and collapse of the endosomal membrane, thereby allowing the antibody to escape from the endosome to the cytoplasm, and a method for producing the same.

[0065] In addition, the present invention relates to a highly efficient cytoplasmic penetrating antibody or Fab fragment thereof, which introduces an endosomal escape structural motif through mutation of an amino acid in a specific region of an IgG antibody light chain constant region CL domain, thereby inducing destabilization and collapse of the endosomal membrane through interaction with the endosomal membrane of a lipid bilayer in response to a change in the weakly acidic environment of the endosome, thereby allowing the antibody to escape from the endosome to the cytoplasm, and a method for producing the same.

[0066] In addition, the present invention relates to a highly efficient cytoplasmic penetrating antibody having a heavy chain constant region CH3 domain and a light chain constant region CL domain having the endosomal escape structural motif designed above, and a method for producing the same.

[0067] In addition, the present invention relates to a highly efficient cytoplasmic penetrating antibody comprising an antibody in which the CL-CH1 pair position of an IgG antibody is replaced with a heterodimeric CH3 pair having the endosomal escape structural motif designed above.

[0068] In addition, the present invention relates to an endosomal escape structural motif through amino acid substitution at a specific position and a combination thereof, which improves an IgG antibody comprising a heavy chain constant region CH3 domain and / or a light chain constant region CL domain having an endosomal escape structural motif, while maintaining the unique physicochemical properties of the antibody and not causing non-specific binding properties.

[0069] The present invention relates to a specific amino acid composition and position for providing an endosomal escape structural motif in the CH3 domain and / or CL domain, which are constant regions of an antibody.

[0070] The present invention relates to a cytoplasmic penetrating antibody or Fab fragment thereof having a high-efficiency endosomal escape ability by replacing the CL-CH1 pair of an existing IgG antibody with a heterodimeric CH3-CH3 pair having the endosomal escape structural motif, and a method for producing the same.

[0071] The present invention relates to a specific amino acid composition and position for replacing the CL-CH1 pair of an antibody with a heterodimeric CH3-CH3 pair having the endosomal escape structural motif.

[0072] In addition, the present invention relates to an antibody-drug conjugate comprising the high-efficiency cytoplasmic penetrating antibody or an Fc fragment or Fab fragment thereof, or a composition for intracytoplasmic delivery of an active substance. Specifically, the present invention relates to a cytoplasmic penetrating antibody fusion protein that can deliver the loaded payload into the cytoplasm very efficiently when various payloads (e.g., proteins, fusion proteins, enzymes, toxins, siRNA, small molecule compounds, peptides, DNA, mRNA, etc.) are fused to the high-efficiency cytoplasmic penetrating antibody, and the cytoplasmic penetrating antibody fusion protein that can expect the payload's effect.

[0073] In addition, the present invention relates to a cytosol-penetrating interfering antibody with endosomal escape ability, which has the ability to target a target protein in the cytoplasm in the CDR regions of the heavy chain variable region (VH) and light chain variable region (VL) of the high-efficiency cytoplasm-penetrating antibody and has the property of modulating the activity of the target protein, and a method for producing the same.

[0074] The present invention also provides polynucleotide and amino acid sequences encoding any of the above cytoplasmic penetrating antibodies or Fc fragments thereof.

[0075]

[0076] Figure 1 is a schematic diagram of a cytoplasmic penetrating antibody in the full IgG form with an endosomal escape structural motif in the CH3 domain.

[0077] Figure 2 shows the results of modeling the Fc tertiary structure of a full IgG CH3 domain variant having an endosomal escape structural motif using Alphafold2, and analyzing the intramolecular interaction patterns of the antibodies using Pymol molecular visualizer.

[0078] Figure 3 is a schematic diagram of the expected endosomal escape mechanism of a cytoplasmic penetrating antibody in the full IgG form having an endosomal escape structural motif in the antibody constant region CH3 domain.

[0079] Figure 4 is a schematic diagram illustrating the process by which GFP fluorescence is observed by complementary binding of improved split green fluorescent protein when a cytoplasmic penetrating antibody is located in the cytoplasm.

[0080] Figure 5 shows the results of analysis using 12% SDS-PAGE under reducing or non-reducing conditions and size exclusion chromatography after purifying the CH3 domain variants of Figure 2.

[0081] Figure 6 shows the evaluation of non-specific binding through ELISA at concentrations of 100 nM and 500 nM for the CH3 domain variants of Figure 2.

[0082] Figure 7 shows the evaluation of non-specific binding to cell membranes through flow cytometry at concentrations of 500 nM and 1 μM for the CH3 domain variants of Figure 2.

[0083] FIG. 8 shows the construction of cell lines in which the SA-GFP1-10 reporter protein was transduced into MDA-MB-231 and SW480 cells, which are cell lines expressing integrin αvβ3 and αvβ5, to evaluate the cytoplasmic penetration ability of the CH3 domain variants of FIG. 2 through the complementary binding system of the improved split green fluorescent protein, and the expression level of the SA-GFP1-10 reporter protein was evaluated through Western blotting.

[0084] Figures 9a and 9b show the cytoplasmic penetration ability evaluated at a concentration of 500 nM by live cell imaging in MDA-MB-231 and SW480 cell lines through the complementary binding system of the improved split green fluorescent protein for the CH3 domain variants of Figure 2.

[0085] Figure 10 shows the evaluation of the concentration-dependent cytoplasmic penetration ability of the CH3 domain variants of Figure 2 through a complementary binding system of the improved split green fluorescent protein in MDA-MB-231 cells by live cell imaging. Figure 11 shows the evaluation of the pH-dependent interaction of the CH3 domain variants with the cell membrane in Ramos cells, an endosomal membrane model cell line, to indirectly confirm the endosomal escape mechanism of the CH3 domain variants of Figure 2.

[0086] Figure 12 is a schematic diagram of a fully IgG cytoplasmic penetrating antibody having an endosomal escape structural motif in the CL domain.

[0087] Figure 13 shows the results of modeling the Fab tertiary structure of a full IgG CL domain variant having an endosomal escape structural motif using Alphafold2, and analyzing the intramolecular interaction patterns of each antibody using Pymol molecular visualizer.

[0088] Figure 14 shows the results of analysis using 12% SDS-PAGE under reducing or non-reducing conditions and size exclusion chromatography after purifying the CL domain variants of Figure 13.

[0089] Figure 15 shows the cytoplasmic penetration ability evaluated at a concentration of 100 nM or 500 nM by live cell imaging through a complementary binding system of improved split green fluorescent protein for the CL domain variants of Figure 13.

[0090] Figure 16 is a schematic diagram of a cytoplasmic penetrating antibody in the full IgG form having an endosomal escape structural motif in the heavy chain constant region CH3 domain and the light chain constant region CL domain.

[0091] Figure 17 shows the results of analysis using 12% SDS-PAGE under reducing or non-reducing conditions and size exclusion chromatography after purifying the CH3 and CL domain variants of Figure 16.

[0092] Figure 18 shows the evaluation of non-specific binding at concentrations of 100 nM and 500 nM for the CH3 and CL domain variants of Figure 16 through ELISA.

[0093] Figure 19 shows the evaluation of non-specific binding to cell membranes by flow cytometry at concentrations of 500 nM and 1 μM for the CH3 and CL domain variants of Figure 16.

[0094] Figure 20 shows the cytoplasmic penetration ability evaluated at a concentration of 100 nM or 500 nM by live cell imaging through a complementary binding system of an improved split green fluorescent protein for a variant without non-specific binding among the CH3 and CL domain variants of Figure 16.

[0095] Figure 21 shows the results of confocal microscopy using a complementary binding system of an improved split green fluorescent protein to confirm the cytoplasmic penetration ability of the cytoplasmic penetrating antibody in2C41 according to the concentration.

[0096] Figure 22 is a schematic diagram illustrating the process of bioluminescence by complementary binding of a split nanoluciferase luminescent protein when a cytoplasmic penetrating antibody is located in the cytoplasm.

[0097] Figure 23 is a schematic diagram showing the calculation formula for quantifying the endosomal escape efficiency in the complementary binding system of the split nanoluciferase luminescent protein of Figure 22, along with the experimental process.

[0098] Figure 24 shows the results of purification of cytoplasmic penetrating antibodies having an endosomal escape structural motif in the constant region fused to HiBiT, analyzed by 12% SDS-PAGE under reducing or non-reducing conditions, and analyzed using size exclusion chromatography.

[0099] Figure 25 shows the quantification of endosomal escape efficiency at a concentration of 500 nM for cytoplasmic penetrating antibodies having an endosomal escape structural motif in the constant region fused to HiBiT via a complementary binding system of a split nanoluciferase luminescent protein.

[0100] Figure 26 shows a pulse-chase experiment observed using a confocal microscope to confirm the transport process of the cytoplasmic penetrating antibody in2C41 that has entered the cell.

[0101] Figure 27 shows the movement of cytoplasmic antibody in2C41 into the cytoplasm in the presence or absence of a pharmacological inhibitor, observed using a confocal microscope using calcein.

[0102] Figure 28 shows a flow cytometry analysis of the binding of cytoplasmic penetrating antibody in2C41 to the cell membrane according to pH.

[0103] Figure 29 shows the results of live cell imaging in Ramos cells to evaluate whether trypan blue, which has no membrane permeability, can be obtained through cell membrane perforation by the cytoplasmic penetrating antibody in2C41 according to pH and whether the resulting cell membrane perforation is a transient and reversible phenomenon.

[0104] Figure 30 shows a confocal microscope observation of whether the cytoplasmic penetrating antibody in2C41 enters the cell through the cell membrane according to pH.

[0105] Figure 31 shows the thermal stability of cytoplasmic penetrating antibodies (in2C11, in2C21, in2C41) having an endosomal escape structural motif in the antibody constant region, evaluated by differential scanning calorimetry.

[0106] Figure 32 shows the pH-dependent affinity analysis for the heavy chain constant region receptor FcRn using BLI for in2C41, which has an endosomal escape structural motif in the heavy chain constant region CH3 domain and the light chain constant region CL domain.

[0107] Figure 33 shows the pharmacokinetics of cytoplasmic penetrating antibody in2C41 in BALB / c nude mice.

[0108] Figure 34 is a schematic diagram of an in2C7N cytoplasmic penetrating antibody with enhanced endosomal escape ability through a FabCab domain in which the CH1-CL pair of a full IgG antibody is replaced with a CH3-CH3 pair having an endosomal escape structural motif.

[0109] Figure 35 shows the Fab structure of a FabCab domain variant in which the CL-CH1 pair of a full IgG antibody is replaced with a CH3-CH3 pair having an endosomal escape structural motif, modeled using Alphafold2, and the intramolecular interaction patterns of the antibody are analyzed using the Pymol molecular visualizer.

[0110] Figure 36 shows the results of analyzing the FabCab domain variants of Figure 35 by 12% SDS-PAGE under reducing or non-reducing conditions and using size exclusion chromatography after purifying them.

[0111] Figure 37 shows the evaluation of nonspecific binding at concentrations of 100 nM and 500 nM for the cytoplasmic penetrating antibody in2C71, which substitutes the CL-CH1 pair of a full IgG antibody with a CH3-CH3 pair having an endosomal escape structural motif, by ELISA.

[0112] Figure 38 shows the evaluation of non-specific binding to the cell membrane by flow cytometry at concentrations of 500 nM and 1 μM for the cytoplasmic penetrating antibody in2C71, which substitutes the CL-CH1 pair of a full IgG antibody with a CH3-CH3 pair having an endosomal escape structural motif.

[0113] Figure 39 shows the endosomal escape ability evaluated at a concentration of 500 nM by live cell imaging through a complementary binding system of an improved split green fluorescent protein to a cytoplasmic penetrating antibody in2C71 in which the CH1-CL pair of a full IgG antibody is replaced with a CH3-CH3 pair having an endosomal escape structural motif.

[0114] Figure 40 shows the pH-dependent interaction of cytoplasmic penetrating antibodies with the cell membrane in Ramos cells, an endosomal membrane model cell line, to indirectly confirm the endosomal escape mechanism of cytoplasmic penetrating antibodies (in2C41 and in2C71) having an endosomal escape structural motif in the constant region.

[0115] Figure 41 is a schematic diagram illustrating the construction of a full IgG form of an anti-α-Tubulin cytoplasmic penetrating antibody (inTu41) to confirm the activity of a cytoplasmic penetrating antibody (in2C41) having an endosomal escape structural motif in the constant region.

[0116] Figure 42 shows the results of 12% SDS-PAGE analysis under reducing or non-reducing conditions and size exclusion chromatography after purification of a full IgG form of anti-α-Tubulin cytoplasmic penetrating antibody (inTu41).

[0117] Figure 43 shows the results of a confocal microscope examination of the overlap between a full IgG form of anti-α-Tubulin cytoplasmic penetrating antibody (inTu41) and α-Tubulin, a cytoskeletal protein located in the cytoplasm.

[0118] Figure 44 is a schematic diagram illustrating the construction of a full IgG form of an anti-RAS cytoplasmic penetrating antibody (inRas41) to confirm the activity of a cytoplasmic penetrating antibody (in2C41) having an endosomal escape structural motif in the constant region.

[0119] Figure 45 shows the results of 12% SDS-PAGE analysis under reducing or non-reducing conditions and size exclusion chromatography after purification of an anti-RAS cytoplasmic penetrating antibody (inRas41) in the form of a full IgG.

[0120] Figure 46 shows the results of confocal microscopy to confirm whether there is overlap between the anti-RAS cytoplasmic penetrating antibody (inRas41) in the form of a full IgG and intracellular RAS in SW480 (KRASG12V) cells.

[0121] Figure 47 is a schematic diagram illustrating the construction of an immunotoxin (ER2C41-DTA) that targets the cancer cell overexpressed receptor EGFR and fuses a bacterial toxin (DT toxin) to a cytoplasmic penetrating antibody (ER2C41) having an endosomal escape structural motif in its constant region, and a schematic diagram showing the splicing process.

[0122] Figure 48 shows the results of 12% SDS-PAGE analysis under reducing or non-reducing conditions and size exclusion chromatography analysis to confirm whether the toxin was well fused to the antibody after fusing the bacterial toxin through splicing.

[0123] Figure 49 is a schematic diagram depicting the process by which an immunotoxin endowed with cytoplasmic penetration ability through an endosomal escape structural motif in the constant region exhibits toxicity toward target cells.

[0124] Figure 50 shows the results of evaluating cytotoxicity in cell lines showing different EGFR expression levels to evaluate the target cell-specific cytotoxicity of a cytoplasmic penetrating antibody-based immunotoxin (ER2C41-DTA) targeting the cancer cell overexpressed receptor EGFR.

[0125] Figure 51 shows the results of evaluating the efficacy of a cytoplasmic antibody-based immunotoxin (ER2C41-DTA) targeting the overexpressed receptor EGFR in cancer cells in a mouse xenograft model using the EGFR-overexpressing cell line A431, and measuring the tumor weight after the end of treatment.

[0126] Figure 52 shows the results of evaluating the survival rate and weight gain / loss of mice during treatment in the mouse xenograft model of Figure 51 and evaluating serum AST and ALT levels after the end of treatment.

[0127]

[0128] Specific details for carrying out the invention

[0129] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0130] The present invention is to develop a highly efficient cytosol-penetrating antibody (cytotransmab) that introduces an endosomal escape motif to impart endosomal escape ability to the heavy chain constant region and / or the light chain constant region of an immunoglobulin G (IgG) antibody, thereby allowing an IgG antibody containing the same to actively localize from outside the cell to the cytoplasm inside the cell.

[0131] The “cytosol-penetrating antibody with high efficient cytosol-penetrating ability in antibody constant region” according to the present invention means an antibody that binds to membrane protein receptors overexpressed in target cells such as tumor tissues in the form of immunoglobulin, is internalized, and then is located in the cytoplasm through endosomal escape.

[0132] Specifically, the present invention aims to develop a cytoplasmic penetrating antibody that is located in the cytoplasm by introducing amino acid mutations that induce interaction with the cell membrane into the constant region of an antibody as in SEQ ID NOS: 2 to 12 to impart an endosomal escape structural motif to each of the antibody constant region CH3 and CL domains, thereby inducing an increase in interaction with the endosomal membrane and membrane destabilization through a change in the charge state of amino acids in the constant region in the mildly acidic environment of the endosomes after internalization into a living cell (endocytosis), and ultimately causing the collapse of the endosomal membrane.

[0133] The present invention more specifically completed the present invention by improving the antibody constant region to impart cytoplasmic penetration ability to the antibody constant region, identifying the biophysical and chemical properties of the mutants, and confirming and evaluating their improved cytoplasmic penetration ability compared to existing cytoplasmic penetration antibodies in a living cell-based assay system.

[0134] In the present invention, “endosomal escape” may mean that an antibody located in an endosome escapes the endosome in an endosome-specific environment through cell internalization and is located in the cytoplasm.

[0135] In the present invention, the “endosome escape structural motif” refers to a primary structure including a specific amino acid sequence having a structural feature that induces endosomal escape and a tertiary structure formed thereby. It can be used interchangeably with “endosome escape ability motif” or “endosome escape ability possessing motif.” An antibody including a constant region including an “endosome escape structural motif” and a substance targeting a target cell or tissue-specific antigen is capable of “target cell / tissue-specific cytoplasmic penetration,” and a “cytoplasmic penetrating antibody” refers to an antibody that has penetrated into a cell by cell internalization and has escaped from the endosome into the cytoplasm, and can be used interchangeably with “an antibody having cytoplasmic penetration ability.”

[0136] The present invention relates to an antibody in the form of a complete immunoglobulin having an endosomal escape structural motif in the antibody constant region.

[0137]

[0138] The logic of amino acid selection for the endosomal escape structural motif in the antibody constant region.

[0139] The above endosomal escape structural motif utilizes three amino acid residues (arginine (R), tryptophan (W), and glutamic acid (E)) to enable the antibody to induce endosomal membrane destabilization through interaction with the endosomal membrane specific to the mildly acidic environment of the endosomes after cell internalization.

[0140] Arginine (R) and tryptophan (W) are amino acids known to contribute most significantly to cell membrane interaction and cell membrane destabilization in many cell penetrating peptide studies. Arginine can contribute to the initial binding with the phospholipid head group of the cell membrane and membrane destabilization, and tryptophan can destabilize the cell membrane by being located at the interface between the hydrophilic and hydrophobic portions of the cell membrane. In particular, when a cation-π interaction is formed between arginine and tryptophan, tryptophan can enhance the interaction of arginine with the membrane. In one embodiment, the endosomal escape structural motif of the antibody constant region may include two or more arginine and tryptophan, and may be located at a close distance such that a cation-π interaction is formed between them.

[0141] Specifically, during cytoplasmic penetration, the positive amino acid arginine participates in the initial binding to the cell membrane through electrostatic interaction with the negatively charged cell membrane surface. The amine group of lysine (K), another positive amino acid, can form hydrogen bonds with one phosphate group per molecule, whereas the guanidinium group of arginine can form bidentate hydrogen bonds with two or more phosphate groups per molecule. Through this, arginine can induce the formation of negative Gaussian curvature in the cell membrane, destabilizing lipid packing. Furthermore, the guanidinium group of arginine has a larger dipole moment than the amine group of lysine. Therefore, unlike lysine, arginine can maintain a positive charge even in hydrophobic environments, allowing it to attract water molecules and phospholipid hydrophilic heads into the hydrocarbon core of the cell membrane via hydrogen bonding. When arginine attracts water molecules or phospholipid hydrophilic heads within the hydrophobic phospholipid bilayer, it can significantly destabilize or structurally distort the cell membrane, potentially leading to temporary and / or localized membrane disruption.

[0142] Tryptophan (W) is the most hydrophobic amino acid. Furthermore, it can form hydrogen bonds through the nitrogen in its side chain, indole, and interact through its π-electron system. Due to its complex properties of both strong hydrophobicity and electrostatic interactions, it can destabilize the cell membrane by forming a positive Gaussian curvature at the interface between hydrophilic and hydrophobic environments, increasing lateral pressure, or reducing the density of the membrane lipid packing.

[0143] However, arginine or tryptophan alone faces a significant energy barrier when crossing the hydrophobic core of the lipid bilayer. To overcome this, we used a strategy of placing arginine and tryptophan within a distance of 3.5–6 Å to promote the cation-π interaction induced by the arginine-tryptophan pair (RW pair). Tryptophan can exert a synergistic effect with arginine in terms of membrane destabilization. When the cation-π interaction is formed between tryptophan and arginine, tryptophan can help arginine form an ion pair with the hydrophilic head of the membrane phospholipid. In addition, the cation-π interaction between tryptophan and arginine can partially cover the positive charge of arginine, thereby lowering the energy barrier required for hydrophilic arginine to penetrate the membrane interior. Because the cation-π interaction between tryptophan and arginine can be maintained within the hydrocarbon core, it can facilitate deeper insertion of the hydrophilic arginine into the membrane's hydrocarbon core when interacting with the cell membrane. This allows tryptophan to facilitate arginine's initial membrane binding and membrane destabilization processes, promoting more efficient membrane pore formation and ultimately enhancing the antibody's ability to escape from the endosomes.

[0144] At this time, since all cells have a negative surface charge and a hydrophobic interior of the cell membrane, the endosomal escape structural motif can be composed of arginine and tryptophan to induce interaction with the cell membrane, but at the same time, it can increase the non-specific binding of the antibody to the cell membrane. Non-specific binding can cause problems such as toxic effects due to indiscriminate interaction with cells in vivo and reduced half-life in vivo. In addition, since tryptophan can lower protein solubility and induce aggregate formation, the endosomal escape structural motif must be designed to interact with the cell membrane specifically in the endosomal environment and must be designed so that there are no disadvantages in terms of developability.

[0145] To prevent nonspecific interactions of the arginine-tryptophan pair (RW pair) with the cell membrane and ensure selective interactions with the endosomal membrane, we placed a cluster of Glu residues (so-called E patches) near the RW pair to take advantage of the slightly acidic endosomal lumen (pH 5–6). The carboxyl side chain of Glu has a low pK a (4.3), but clustering the Glu residues reduces this pK a can rise.

[0146] Endosomes are known to be slightly acidic, with a pH of 5.5-6.5 in the early endosome and a pH of 4.5-5.5 in the late endosome, due to the internalization of the cell by vacuolar ATPase. Therefore, by introducing amino acids whose charge state can change depending on pH into the endosomal escape structural motif, the endosomal escape structural motif can have different physicochemical properties when exposed to the slightly acidic pH of the endosomes than when exposed to the neutral pH. Through this pH-dependent endosomal escape structural motif, it is possible to induce interaction with the cell membrane specifically in the endosomal environment and suppress nonspecific binding in the neutral pH environment of the body circulation.

[0147] Aspartic acid (D) and glutamic acid (E) are negatively charged amino acids at neutral pH, but as the pH decreases, the carboxylic acid (COO-) on the amino acid side chain can be protonated. This increases their hydrophobicity and allows them to form hydrophobic interactions with surrounding hydrophobic substances, making them pH-dependent amino acids. The pK of aspartic acid and glutamic acid a The pK values ​​are around 3.8 and 4.2, respectively, indicating that glutamic acid is closer to the endosomal slightly acidic environment (pH 4.5-6.5) than aspartic acid. a have

[0148] At this time, the pK of glutamic acid a (approximately 4.3) is still lower than the slightly acidic environment of the endosomes (pH 4.5-6.5), so most of the carboxylic acids in the side chain remain in a negatively charged state and the rate of hydrogenation is low. As a strategy to overcome this, glutamic acid is placed in multiple adjacent positions in the tertiary structure to increase the pK of glutamic acid. acan increase. When there is a functional group with the same charge in the adjacent position, glutamic acid becomes prone to losing charge as the equilibrium shifts toward losing charge to reduce charge repulsion. That is, when glutamic acid is densely packed in adjacent tertiary structure positions, the side chain carboxyl group of glutamic acid becomes hydrogenated and loses charge, which can reduce charge repulsion and become a more stable state. Through this, the pK of the side chain carboxylic acid of glutamic acid a can increase, ultimately making glutamic acid more easily hydrogenated even under slightly acidic conditions.

[0149] Therefore, we designed an endosomal escape motif with pH dependence by including a glutamic acid patch, which densely packs glutamic acid in adjacent positions of a specific tertiary structure, in the endosomal escape motif. The glutamic acid patch of the endosomal escape motif causes electrostatic repulsion with the negatively charged cell surface under physiological conditions of neutral pH. However, in the endosomal environment of slightly acidic pH, glutamic acid is hydrogenated and loses its charge, so it does not repel from the cell surface, and can induce cell membrane destabilization together with arginine and tryptophan through hydrophobic interactions with the hydrocarbon chains of phospholipids. In addition, glutamic acid is known to contribute most to improving protein solubility, and the introduction of glutamic acid can be expected to have the effect of offsetting the deterioration of physical properties caused by the introduction of tryptophan and arginine.

[0150] Therefore, the above endosomal escape structural motif includes an amino acid composition of arginine, tryptophan and glutamic acid so that the antibody can induce endosomal membrane destabilization through interaction with the endosomal membrane specific to the mildly acidic environment of the endosomes after cell internalization.

[0151]

[0152] The logic of amino acid positioning in the endosomal escape structural motif of antibody constant regions

[0153] The endosomal escape structural motif having an amino acid composition of arginine, tryptophan, and glutamic acid was designed to have a position of the endosomal escape structural motif by considering the primary, secondary, and tertiary structures of the antibody constant region so that the antibody can induce pH-dependent interaction with the endosomal membrane and endosomal membrane destabilization through the endosomal escape structural motif of the constant region.

[0154] Specifically, endosomal escape structural motifs were introduced into the CH3 of the antibody constant region, the AB and EF loops of the CL domain, and the C-terminal loop. The AB, EF loops, and the C-terminal loop of the constant region are all located at the lower end of each domain, and the lower end of each domain interacts with the lower end of the opposite domain to form a duplex (CL-CH1 or CH3-CH3 pair). Therefore, when endosomal escape structural motifs are introduced into the AB, EF loops, and the C-terminal loop of the constant region, an avidity effect can be expected because two endosomal escape motifs are located in close proximity due to the characteristics of the duplex.

[0155] Each amino acid position of the endosomal escape structural motif having an amino acid composition of arginine, tryptophan, and glutamic acid was selected based on the following three criteria, taking into account the interactions between amino acids in the AB, EF loop, and C-terminal loop tertiary structures of the CH3 or CL domain and the change in charge state of each amino acid.

[0156]

[0157] 1) Identification of surface-exposed amino acid positions

[0158] All amino acids constituting the endosomal escape structural motifs of the lower AB, EF, and C-terminal loops of the antibody constant region were intended to be exposed on the surface of the tertiary structure to facilitate interaction with the cell membrane and charge state changes depending on pH. To this end, surface-exposed amino acid positions in the CH3 and CL domains were identified from the tertiary model structures of the Fc and Fab regions using the GETAREA web server (https: / curie.utmb.edu / getarea.html).

[0159] At this time, GETAREA calculates the Ratio (%) of the solvent accessible surface area (SASA) based on the 3D structure to identify surface exposed amino acid positions, non-surface exposed amino acid positions, and buried amino acid positions. At this time, a “surface exposed amino acid position” refers to a state where the Ratio (%) is 50 or more. In addition, a “non-surface exposed amino acid position” refers to a state where the Ratio (%) is 50 or less, and a “buried amino acid position” refers to a state where the Ratio (%) is 20 or less.

[0160] The 3D structure (PDB file) of the wild-type Fc region or Fab region was input into the GETAREA web server, and the SASA Ratio (%) was obtained for the AB, EF, and C-terminal loops of each domain. Based on the surface-exposed amino acid position information, the surface-exposed amino acid positions in the AB, EF, and C-terminal loops were selected.

[0161]

[0162] 2) Selection of a position that can induce a cation-π interaction between arginine (R) and tryptophan (W) in the endosomal escape structural motif.

[0163] In the endosomal escape structural motif, when arginine (R) and tryptophan (W) form a cation-π interaction, the binding to the cell membrane is strengthened, and the cell membrane can be efficiently destabilized after binding. Therefore, among the surface-exposed amino acid positions in the AB, EF, and C-terminal loops, positions where the distance between the Cα of two amino acids or the distance between the side chains is within 6 Å were selected so that arginine and tryptophan form a cation-π interaction pair. In addition, when the arginine-tryptophan pair is placed in a position where the amino acid side chains face the same direction in the AB loop and EF loop where an α-helix exists, the cation-π interaction by arginine and tryptophan can stabilize the α-helix. Therefore, by introducing an arginine-tryptophan pair at a surface-exposed amino acid position in the α-helix portion of the AB loop and EF loop, the interaction with the cell membrane was strengthened while providing structural stability.

[0164] At this time, since the steric hindrance formed by the introduction of a large number of large amino acids, such as tryptophan, into a narrow area can affect protein folding, the amino acids around the positions of arginine and tryptophan were replaced with small amino acids, such as alanine (A), to prevent potential steric hindrance.

[0165]

[0166] 3) Positioning of the glutamic acid (E) patch in the endosomal escape structural motif

[0167] To induce endosomal pH-specific interactions with the endosomal mildly acidic membrane, glutamic acid (E) patches were introduced at surface-exposed amino acid positions in the AB, EF, and C-terminal loops.

[0168] At this time, the distance between the side chains of the glutamic acid patch can increase due to electrostatic repulsion between the side chains when each glutamic acid that composes it is crowded in adjacent positions. Therefore, the glutamic acid patch is introduced at a position where the distance between the amino acid side chains as well as the distance between the amino acid Cα is as close as possible, so that even if the distance between the side chains becomes somewhat far, the glutamic acids have an electrostatic effect on each other, and the pK a It was made so that it could rise.

[0169] Additionally, the glutamic acid patch was positioned around the arginine-tryptophan pair. This is because, when the glutamic acid patch is located around the arginine-tryptophan pair, at neutral pH, the glutamic acid patch can suppress nonspecific binding that may occur due to arginine-tryptophan, and at the slightly acidic pH of the endosome, the glutamic acid patch is hydrogenated to facilitate the interaction of arginine-tryptophan with the cell membrane.

[0170] At this time, if a positive amino acid such as lysine or arginine is at a distance and angle that can form a salt bridge with the glutamic acid, the equilibrium shifts to maintain a negative charge because glutamic acid has a negative charge and it is more stable to form a salt bridge with the positive amino acid. In other words, when glutamic acid forms a salt bridge with a surrounding positive amino acid, the pK of the side chain carboxylic acid of glutamic acid a can be lowered, and as a result, hydrogenation of glutamic acid may become difficult in a slightly acidic pH environment. The pK of glutamic acid depends on the surrounding amino acid environment. aTaking the changes into account, glutamic acid was introduced at a position among the surface-exposed amino acid positions of the AB, EF, and C-terminal loops of the constant region that does not form salt bridges with surrounding arginine or lysine.

[0171] Additionally, for the same reason as above, if lysine or arginine, which can form a salt bridge with glutamic acid, exists at a position adjacent to glutamic acid, these lysine or arginine can be replaced with asparagine (N) or glutamine (Q), which are amino acids of similar size but no charge.

[0172] For the glutamic acid patch introduced with the above logic, the pK of the glutamic acid side chain was obtained using the PROPKA 3.1 web server (https: / biolib.com / bio-utils / propka / ). a Predict pK a It was confirmed that the pK of the charged amino acid side chain was increased. PROPKA 3.1 is based on the tertiary structure. a Therefore, the three-dimensional structure (PDB file) of the wild-type Fc region or Fab region and the three-dimensional model structure (PDB file) of the Fc region or Fab region of the variant are input to the PROPKA 3.1 web server, and the pK of the glutamic acid side chain is predicted through the glutamic acid patch. a It was confirmed that the increase was

[0173] Therefore, the glutamic acid patch was introduced at an amino acid position that is close to the distance between amino acids Cα, is around the arginine-tryptophan pair, and does not form a salt bridge with the surrounding positive amino acids among the surface-exposed amino acid positions of the constant regions AB, EF, and the C-terminal loop.

[0174]

[0175] The present invention provides the amino acid composition and position of the endosomal escape structural motif of an antibody constant region designed based on the above logic.

[0176] The above endosomal escape structural motif may be characterized by being included in at least one selected from the group consisting of a CH3 domain of a heavy chain constant region and a CL domain of a light chain constant region.

[0177] In one aspect, the present invention may be characterized in that the endosomal escape structural motif is located in the AB, EF loop and C-terminus of the heavy chain constant region CH3 domain of sequence number 1.

[0178]

[0179] The N-terminal position of the sequence of sequence number 1 is 351, the C-terminal position is 447, and it contains 97aa.

[0180] It comprises a CH3 domain having an endosomal escape structural motif consisting of the following amino acid sequence at the AB, EF loop and C-terminal position. The amino acid residue numbers below follow the EU numbering.

[0181] AB loop: 353 PS-XXEXXKXX 362

[0182] In the above AB loop, X may be selected from the group consisting of arginine (R), tryptophan (W), glutamic acid (E), alanine (A), asparagine (N), or glutamine (Q).

[0183] EF loop: 411 TV-XXX-RW-XX-GNVF 423

[0184] In the above EF loop, X may be selected from the group consisting of arginine (R), tryptophan (W), glutamic acid (E), alanine (A), asparagine (N), or glutamine (Q).

[0185] C-terminal: 439 X-SLS-X-SPGK 447

[0186] At the C-terminus, X may be selected from the group consisting of arginine (R), tryptophan (W), glutamic acid (E), alanine (A), asparagine (N), or glutamine (Q).

[0187] In one aspect, the present invention may be characterized in that the endosomal escape structural motif is located in the AB, EF loop and C-terminus of the light chain constant region CL domain of SEQ ID NO: 17.

[0188]

[0189] In the sequence of sequence number 17, the N-terminal position is 118, the C-terminal position is 217, and it contains 97aa.

[0190] It comprises a light chain constant region having a CL domain having an endosomal escape structural motif consisting of the following amino acid sequence at the AB, CD, EF loop and C-terminal position. The amino acid residue numbers below follow the EU numbering.

[0191] AB loop: 120 PS-XXQXXSXX 129

[0192] In the above AB loop, X can be selected from the group consisting of arginine (R), tryptophan (W), glutamic acid (E), alanine (A), asparagine (N), or glutamine (Q).

[0193] EF loop: 180 TL-XXX-DY-XX-HKVY 192

[0194] In the above EF loop, X can be selected from the group consisting of arginine (R), tryptophan (W), glutamic acid (E), alanine (A), asparagine (N), or glutamine (Q).

[0195] C-terminal: 207 X-SFN-X-GEC 214

[0196] At the C-terminus, X may be selected from the group consisting of arginine (R), tryptophan (W), glutamic acid (E), alanine (A), asparagine (N), or glutamine (Q).

[0197] The present invention relates to a cytoplasmic penetrating antibody, or an Fc fragment thereof, or a Fab fragment thereof, comprising a heavy chain constant region having an endosomal escape structural motif capable of escaping from an endosome to the cytoplasm. According to one embodiment, in FIG. 9, it was confirmed that the endosomal escape structural motif introduced into the CH3 domain of the heavy chain constant region of in2C11 was approximately 2.2 times improved compared to the conventional cytoplasmic penetrating antibody inCT.

[0198] The above endosomal escape structural motif can be introduced at a corresponding position in sequence and / or structure in other isotypes (IgA, IgE, IgD, IgM) and subclasses (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2) of immunoglobulins when there is high sequence homology and / or structural homology.

[0199] The heavy chain constant region CH3 domain having the above endosomal escape structural motif may comprise one or more sequences selected from the group consisting of SEQ ID NOs: 2 to 12.

[0200] The heavy chain constant region CH3 domain having the endosomal escape structural motif may comprise a sequence having at least 80% homology, for example 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology, with a sequence of a heavy chain constant region selected from the group consisting of SEQ ID NOs: 2 to 12.

[0201] The light chain constant region CL domain having the above endosomal escape structural motif may comprise one or more sequences selected from the group consisting of SEQ ID NOs: 18 and 19.

[0202] The light chain constant region CL domain having the above endosomal escape structural motif can comprise a sequence having at least 80% homology, for example 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology, with a sequence of a light chain constant region selected from the group consisting of SEQ ID NOs: 18, 19.

[0203] In the present invention, the cytoplasmic penetrating antibody or fragment thereof may be characterized by comprising the following structure:

[0204] (1) A CH1-CL pair comprising a heavy chain constant region CH1 domain and a light chain constant region CL domain constant region containing an endosomal escape structural motif;

[0205] (2) a constant region CH3-CH3 pair comprising an endosomal escape structural motif in the heavy chain constant region CH3 domain; and / or

[0206] (3) A constant region CH3-CH3 pair in which the constant region CH1-CL pair is replaced with the constant region CH3-CH3 pair of (2), and the constant region CH3-CH3 pair includes an endosomal escape structural motif in the heavy chain constant region CH3 domain. The heavy chain constant region CH1 and light chain constant region CL pairs are replaced with a CH3-CH3 pair including an endosomal escape structural motif, and the endosomal escape structural motif is included in six CH3 domains, and the CH3-CH3 pair replacing the CH1-CL pair may be a heterodimer.

[0207] In the present invention, a FabCab format antibody is constructed in which the constant region CH1-CL pair is replaced with a CH3-CH3 pair containing an endosomal escape structural motif, thereby introducing an endosomal escape structural motif into a total of six CH3 domains, thereby enhancing cytoplasmic penetration ability. At this time, in order to prevent the CH3 domain replacing the CH1-CL pair from inappropriately assembling with the CH3 domain of the Fc region to form an oligomer, a heterodimer heavy chain constant region (heterodimer heterodimeric Fc) technology may be introduced. Specifically, the CH3 domain replacing the CH1 domain may include Q347R, D399V, F405T (EU numbering) mutations, and the CH3 domain replacing the CL domain may include K360E, K409W (EU numbering) mutations.

[0208] The constant region heterodimer CH3 pair having the above endosomal escape structural motif may comprise one or more sequences selected from the group consisting of SEQ ID NOs: 23 to 26. The antibody comprising the CH3-CH3 pair replacing the CH1-CL pair may be characterized by comprising one or more sequences selected from the group consisting of SEQ ID NOs: 23 to 26.

[0209] The constant region heterodimer CH3 pair having the above endosomal escape structural motif can comprise a sequence having at least 80% homology, for example 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology, with a sequence of a constant region selected from the group consisting of SEQ ID NOs: 23 to 26.

[0210] According to one embodiment of the present invention, a highly efficient cytoplasmic penetrating antibody having an endosomal escape structural motif in the antibody constant region CH3 domain and / or CL domain comprises a substance that specifically binds to a cell surface membrane protein receptor overexpressed in a target cell / tissue.

[0211] The high-efficiency cytoplasmic penetrating antibody having an endosomal escape structural motif in the constant region CH3 domain and / or CL domain is a cytoplasmic penetrating antibody by constant region that specifically binds to a membrane protein receptor on the cell surface overexpressed in a target cell / tissue, is internalized (endocytosis), and then is located in the cytoplasm of the cell by endosomal escape ability.

[0212] The high-efficiency cytoplasmic penetrating antibody having an endosomal escape structural motif in the CH3 domain and / or CL domain may specifically recognize one or more selected from among target cell / tissue-specific antigens, for example, epithelial cellular adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), and integrin αvβ3 / αvβ5 overexpressed in tumor cells.

[0213] The above tumor cell line may overexpress at least one selected from EpCAM, EGFR, and integrin αvβ3 / αvβ5.

[0214] The substance targeting the target cell or tissue-specific antigen may be, for example, a ligand, oligopeptide, antibody or fragment thereof, or aptamer capable of specifically binding to the antigen, but is not limited thereto.

[0215] According to one embodiment of the present invention, the cytoplasmic penetrating antibody according to the present invention is characterized by being fused with a cyclic peptide or monobody targeting a receptor overexpressed in tumor tissue for tumor tissue-specific cytoplasmic penetration, wherein the cyclic peptide may be a cyclic peptide in4 (DGVRQCRGDCFDGPL) targeting integrin αvβ3 / αvβ5 (Science Advances, 2020, 6(3):eaay2174). The monobody may be a monobody ER (VSDVPRDLEVVAATPTSLLISWDSGRGS YQYYRITYGETGGNSPVQEFHVPGPHHTATISGLKPGVDYTITVYAVTDHKPHADGPHTYHESPISINYRTEIDKPSQ) targeting EGFR (Domestic Patent Application No. 10-2023-0181933).

[0216] The above-mentioned circular peptide may be directly bound to the N- or C-terminus of the antibody or may be bound via a linker. The linker may be a peptide having various sequences and lengths, and may be a GGGGS, (GGGGS)2, (GGGGS)4, ASTKGP, or ASTKGPSVFPLAP sequence linker that provides structural flexibility without being cleaved by proteolytic enzymes.

[0217] The present invention is to develop a highly efficient IgG cytoplasmic penetrating antibody by introducing an endosomal escape motif into the heavy chain constant region and / or the light chain constant region of an IgG antibody.

[0218] In order to achieve the above purpose, the heavy chain constant region CH3 domain of the high-efficiency cytoplasmic penetrating antibody according to the present invention is composed of an AB loop including an amino acid sequence of the following general formula 1, an EF loop including an amino acid sequence of the following general formula 2, and a C-terminal loop including an amino acid sequence of the following general formula 3.

[0219] In addition, the light chain constant region CL domain of the high-efficiency cytoplasmic penetrating antibody according to the present invention is composed of an AB loop comprising an amino acid sequence of the following general formula 4, an EF loop comprising an amino acid sequence of the following general formula 5, and a C-terminal loop comprising an amino acid sequence of the following general formula 6. The amino acid residue numbers below follow the EU numbering.

[0220] In addition, the heterodimeric CH3 pair of the high-efficiency cytoplasmic penetrating antibody according to the present invention is composed of an AB loop comprising an amino acid sequence of the following general formula 1, an EF loop comprising an amino acid sequence of the following general formula 2, and a C-terminal loop comprising an amino acid sequence of the following general formula 3. The amino acid residue numbers below follow the EU numbering.

[0221] [General Formula 1]

[0222] 353 PS-XXEXXKXX 362

[0223] In the above general formula 1, X can be R, W, E, A, N or Q.

[0224] [General Formula 2]

[0225] 411 TV-XXX-RW-XX-GNVF 423

[0226] In the above general formula 2, X can be R, W, E, A, N or Q.

[0227] [General Formula 3]

[0228] 439 X-SLS-X-SPGK 447

[0229] In the above general formula 3, X can be R, W, E, A, N or Q.

[0230] [General Formula 4]

[0231] 120 PS-XXQXXSXX 129

[0232] In the above general formula 4, X can be R, W, E, A, N or Q.

[0233] [General Formula 5]

[0234] 180 TL-XXX-DY-XX-HKVY 192

[0235] In the above general formula 5, X can be R, W, E, A, N or Q.

[0236] [General Formula 6]

[0237] 207 X-SFN-X-GEC 214

[0238] In the above general formula 6, X can be R, W, E, A, N or Q.

[0239] The fragments of the present invention include Fc, Fab, F(ab'), F(ab')2, and Fv, etc. 'Fc' is a fragment obtained when an immunoglobulin (Ig) molecule is decomposed with papain, and is a region in which the variable region (VL) and constant region (CL) of the light chain and the variable region (VH) and heavy chain constant region 1 (CH1) of the heavy chain are removed.

[0240] Among antibody fragments, Fab has a single antigen-binding site, consisting of the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1) of the heavy chain. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the CH1 domain of the heavy chain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds.

[0241] Specifically, a complete immunoglobulin antibody or Fc fragment thereof comprising a CH3 domain selected from the amino acid sequences of SEQ ID NOs: 2 to 12 is provided.

[0242] Also provided is a complete immunoglobulin antibody or Fab fragment thereof comprising a CL domain selected from the amino acid sequence of SEQ ID NO: 18 or 19.

[0243] Also provided is a complete immunoglobulin antibody comprising a CH3 domain selected from the amino acid sequences of SEQ ID NOs: 2 to 12 and a CL domain selected from the amino acid sequences of SEQ ID NOs: 18 or 19.

[0244] Also provided is a complete immunoglobulin antibody or Fab fragment thereof comprising a CH3 domain selected from the amino acid sequences of SEQ ID NOs: 2 to 12 and a heterodimeric CH3 pair having the amino acid sequences of SEQ ID NOs: 23 to 26.

[0245] The present invention also provides a highly efficient cytoplasmic penetrating antibody specific for target cells / tissues that specifically binds to a membrane protein receptor on the cell surface overexpressed in a living target cell or tissue, is internalized into the cell (endocytosis), and then is located in the cytoplasm of the cell through the endosomal escape ability via the endosomal escape structural motif of the antibody constant region.

[0246] The present invention provides a nucleic acid encoding the antibody, its Fc fragment or Fab fragment.

[0247] The present invention also provides an expression vector comprising the nucleic acid.

[0248] The present invention also provides a recombinant cell transformed with the expression vector.

[0249] The present invention also provides an Fc having an endosomal escape structural motif introduced into a specific region of a CH3 domain of a heavy chain constant region of an existing IgG antibody through amino acid mutation, a cytoplasmic penetrating antibody comprising the Fc, and a method for producing the same, comprising the following steps: (1) a step of selecting an amino acid position at which an endosomal escape structural motif is to be introduced among the lower loops (AB, EF, C-terminal end loop) of the CH3 domain of the antibody heavy chain constant region; (2) a step of preparing a heavy chain expression vector of a cytoplasmic penetrating antibody by cloning a heavy chain nucleic acid including a CH3 domain having an endosomal escape structural motif at the selected amino acid position; (3) a step of preparing a light chain expression vector by cloning a wild-type light chain nucleic acid; (4) a step of simultaneously transforming an animal cell for protein expression with the prepared heavy chain and light chain expression vectors to express a cytoplasmic penetrating antibody in the form of a complete immunoglobulin; and (5) a step of purifying and recovering the expressed cytoplasmic penetrating antibody.

[0250] The present invention also provides a Fab having an endosomal escape structural motif introduced into a specific region of a light chain constant region CL domain of an existing IgG antibody through amino acid mutation, a cytoplasmic penetrating antibody comprising the Fab, and a method for producing the same, comprising the following steps: (1) a step of selecting an amino acid position at which an endosomal escape structural motif is to be introduced among the lower loops (AB, EF, C-terminal end loop) of the antibody light chain constant region CL domain; (2) a step of preparing a cytoplasmic penetrating antibody light chain expression vector by cloning a light chain nucleic acid including a CL domain having an endosomal escape structural motif at the selected amino acid position; (3) a step of preparing a heavy chain expression vector by cloning a wild-type IgG heavy chain nucleic acid; (4) a step of simultaneously transforming an animal cell for protein expression with the prepared heavy chain and light chain expression vectors to express a cytoplasmic penetrating antibody in the form of a complete immunoglobulin; and (5) a step of purifying and recovering the expressed cytoplasmic penetrating antibody.

[0251] The present invention also provides a highly efficient cytoplasmic penetrating antibody having a heavy chain constant region CH3 domain and a light chain constant region CL domain having an endosomal escape structural motif as designed above, and a method for producing the same, comprising the following steps: (1) preparing a heavy chain expression vector by cloning a heavy chain nucleic acid including a CH3 domain having an endosomal escape structural motif; (2) preparing a light chain expression vector of a cytoplasmic penetrating antibody by cloning a light chain nucleic acid including a CL domain having an endosomal escape structural motif; (3) simultaneously transforming the prepared heavy chain and light chain expression vectors into an animal cell for protein expression to express a cytoplasmic penetrating antibody in the form of a complete immunoglobulin; and (4) purifying and recovering the expressed cytoplasmic penetrating antibody.

[0252] The present invention also provides a method for producing a cytoplasmic penetrating antibody having a high-efficiency endosomal escape ability by replacing the CL-CH1 pair of an existing IgG antibody with a heterodimeric CH3 pair having the endosomal escape structural motif, including the following steps.

[0253] Specifically, to promote heterodimer formation of the heterodimer CH3 pair replacing the CL-CH1 pair, the EW-RVT mutation among the heterodimer heavy chain constant region (heterodimer heterodimeric Fc) technologies can be utilized. The CH3 (EW) replacing CL has K360E, K409W mutations in CH3 for heterodimer CH3 pair formation, and the CH3 (EW) domain is replaced from position 109 (EU numbering) of the light chain, and 445Pro, 446Gly, and 447Lys are substituted with Gly, Glu, and Cys, respectively, for disulfide bond formation with the heavy chain. CH3 (RVT) replacing CH1 has Q347R, D399V, F405T mutations for heterodimeric CH3 pairing in CH3, and positions 119 (EU numbering) of the heavy chain are replaced with CH3 (RVT), and 445Pro, 446Gly, and 447Lys are substituted with Gly, Glu, and Cys, respectively, for disulfide bond formation with the light chain. The positions of the above amino acids all follow the EU numbering.

[0254] (1) A step for preparing a heavy chain expression vector of a cytoplasmic penetrating antibody, cloning a heavy chain nucleic acid comprising a heavy chain constant region (CH3(RVT)-hinge-CH2-CH3) and a heavy chain variable region (VH), in which CH1 is replaced with CH3-RVT having an endosomal escape structural motif and a heterodimer CH3 formation mutation; (2) A step for preparing a light chain expression vector of a cytoplasmic penetrating antibody, cloning a light chain nucleic acid comprising a light chain constant region (CH3(EW)) and a light chain variable region (VL), in which CL is replaced with CH3-EW having an endosomal escape structural motif and a heterodimer formation mutation; (3) A step for expressing a cytoplasmic penetrating antibody in the form of a complete immunoglobulin by co-transfecting an animal cell for protein expression with the prepared heavy chain and light chain expression vectors; and (4) a step for purifying and recovering the expressed cytoplasmic penetrating antibody.

[0255] The present invention also provides a method for producing a target cell / tissue-specific cytoplasmic penetrating antibody having an endosomal escape structural motif in a CH3 domain and / or a CL domain, and GFP11-SBP2 or HiBiT fused to the heavy chain C-terminus, comprising the following steps: (1) producing a heavy chain expression vector of a cytoplasmic penetrating antibody fused with GFP11-SBP2 or HiBiT, by cloning a nucleic acid in which a heavy chain including a CH3 domain having an endosomal escape structural motif and GFP11-SBP2 or HiBiT is linked to the C-terminus of the heavy chain; (2) producing a light chain expression vector by cloning a nucleic acid in which a light chain including a CL domain or a wild-type CL domain having an endosomal escape structural motif and a circular peptide targeting a target cell / tissue-specific overexpressed receptor are fused to the N-terminus of the light chain; (3) a step of simultaneously transforming the heavy and light chain expression vectors manufactured above into animal cells for protein expression to express a cytoplasmic penetrating antibody in the form of a complete immunoglobulin; and (4) a step of purifying and recovering the expressed cytoplasmic penetrating antibody.

[0256] The present invention also provides a method for producing a target cell / tissue-specific cytoplasmic penetrating antibody having an endosomal escape structural motif in a CH3 domain and / or a CL domain and having a targeting ability for alpha tubulin (α-Tubulin), a cytoplasmic scaffolding protein, in a heavy chain variable region (VH), the method comprising the following steps: (1) producing an anti-α-Tubulin heavy chain expression vector comprising a CH3 domain having an endosomal escape structural motif and a heavy chain variable region (VH) having an α-Tubulin targeting ability; (2) producing a light chain expression vector by cloning a nucleic acid in which a light chain comprising a CL domain having an endosomal escape structural motif and a circular peptide targeting a target cell / tissue-specific overexpressed receptor are fused to the N-terminus of the light chain; (3) A step of simultaneously transforming the heavy and light chain expression vectors manufactured above into animal cells for protein expression to express an anti-α-Tubulin cytoplasmic penetrating antibody in the form of a complete immunoglobulin. And (4) a step of purifying and recovering the expressed anti-α-Tubulin cytoplasmic penetrating antibody.

[0257] The present invention also provides a method for producing a target cell / tissue-specific cell-penetrating interference antibody having an endosomal escape structural motif in a CH3 domain and / or a CL domain and having a targeting ability against activated RAS, which is one of cytoplasmic proteins, in a heavy chain variable region (VH), comprising the following steps: (1) producing an anti-RAS heavy chain expression vector having an endosomal escape structural motif, which includes a CH3 domain having an endosomal escape structural motif and a heavy chain variable region (VH) having a targeting ability against activated RAS; (2) producing a light chain expression vector by cloning a nucleic acid in which a light chain including a CL domain having an endosomal escape structural motif and a circular peptide targeting a target cell / tissue-specific overexpressed receptor are fused to the N-terminus of the light chain; (3) simultaneously transforming the produced heavy chain and light chain expression vectors into an animal cell for protein expression to express an anti-RAS cell-penetrating interference antibody in the form of a complete immunoglobulin; And (4) a step of purifying and recovering the above-mentioned anti-RAS cell penetration interference antibody.

[0258] The present invention also provides a method for producing an antibody in which a specific functional protein is fused through trans-splicing with a protein having an endosomal escape structural motif in a CH3 domain and / or a CL domain, and an N-intein fused to a light chain constant region (CL) and a C-intein, the method comprising the following steps: (1) producing a heavy chain expression vector including a CH3 domain having an endosomal escape structural motif; (2) producing a light chain expression vector including a CL domain having an endosomal escape structural motif and a light chain in which a circular peptide targeting a target cell / tissue-specific overexpressed receptor is fused to the N-terminus of the light chain and an N-intein is fused to the C-terminus of the light chain; (3) producing a functional protein expression vector in which a C-intein is fused to the N-terminus; (4) simultaneously transforming the produced heavy chain and light chain expression vectors into an animal cell for protein expression to express a cytoplasmic penetrating antibody in the form of a complete immunoglobulin; (5) A step of transforming the functional protein expression vector fused with the manufactured C-intein into E. coli for protein expression to express the functional protein; (6) A step of purifying and recovering the expressed cytoplasmic penetrating antibody and functional protein; (7) A step of trans-splicing the purified cytoplasmic penetrating antibody and functional protein to fuse them and purifying and recovering them.

[0259] The present invention also provides a conjugate comprising the antibody, its Fc fragment or Fab fragment, and a bioactive molecule linked to the cytoplasmic penetrating antibody.

[0260] The present invention also provides a bispecific or multispecific antibody comprising the antibody, an Fc fragment or Fab fragment thereof, and the cytoplasmic penetrating antibody.

[0261] The cytoplasmic penetrating antibody according to the present invention may be IgG, IgM, IgA, IgD or IgE, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA1, IgA2, or IgD type, and most preferably, it may be a monoclonal antibody of the complete IgG type. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types and has subclasses of gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1) and alpha2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0262] The term "heavy chain" refers to both a full-length heavy chain and fragments thereof, comprising a variable domain VH and three constant domains CH1, CH2 and CH3, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. In addition, the term "light chain" as used herein refers to both a full-length light chain and fragments thereof, comprising a variable domain VL and a constant domain CL, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.

[0263] Monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFV), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFV) and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of the above antibodies, are included, but are not limited thereto.

[0264] “Interaction” refers to interactions between molecules, such as those detectable using, for example, a yeast two-hybrid assay. The term interaction is intended to encompass “binding” interactions between two molecules.

[0265] A monoclonal antibody is an antibody obtained from a substantially homogeneous population of antibodies, i.e., identical except for possible naturally occurring mutations that may occur in trace amounts within the individual antibodies. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to conventional (polyclonal) antibodies, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0266] For example, monoclonal antibodies useful in the present invention can be produced by hybridoma methods, or can be produced using recombinant DNA methods in bacterial, eukaryotic, or plant cells. Furthermore, monoclonal antibodies can be isolated from phage antibody libraries.

[0267] The term "affinity" refers to the ability of an antibody to specifically recognize and bind to a specific site of an antigen. High affinity, along with the specificity of an antibody for an antigen, is an important factor in the immune response. The affinity constant (K D ) can be determined using biolayer interferometry (BLI), for example, Octet Qke. Biolayer interferometry data can be used to fit a 1:1 Langmuir coupling model (simultaneously k on ,k off ) and rate constant k off / k on Affinity constant (K) calculated from the ratio of D ) is obtained.

[0268] Considering the mutations having the above-described biological equivalent activity, the antibody of the present invention or the nucleic acid molecule encoding the same is interpreted to also include a sequence showing substantial identity with the sequence described in the sequence number. The above-described substantial identity means a sequence showing at least 90% homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, or 99% or more homology when the sequence of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0269] Alignment methods for sequence comparison are well known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is available from NBCI and other sources and can be used online in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx. BLAST is accessible at www.ncbi.nlm.nih.gov / BLAST / . Instructions for comparing sequence homology using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0270] Based on this, the antibody or antigen-binding fragment thereof of the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology to the specified sequences or the entirety of the sequences described in the specification. Such homology may be determined by sequence comparison and / or alignment using methods known in the art. For example, the percent sequence homology of the nucleic acids or proteins of the present invention may be determined using sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection.

[0271] The term "nucleic acid" encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides, the basic structural units of nucleic acids, include not only natural nucleotides but also analogues with modified sugar or base moieties. The sequence of the nucleic acid encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, non-conservative substitutions, or conservative substitutions of nucleotides.

[0272] The DNA encoding the antibody can be readily isolated or synthesized using conventional molecular biological techniques (e.g., by using an oligonucleotide probe that can specifically bind to the DNA encoding the antibody and its heavy and light chains), and the nucleic acid can be isolated and inserted into a replicable vector for further cloning (DNA amplification) or further expression. Based on this, the present invention relates to a recombinant expression vector comprising the nucleic acid from another aspect.

[0273] As used herein, the term "vector" refers to a means for expressing a target gene in a host cell, and includes viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. Components of a vector typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more antibiotic resistance marker genes, an enhancer element, a promoter, and a transcription terminator sequence. A nucleic acid encoding an antibody is operably linked to a promoter and a transcription terminator sequence, etc. "Operably linked" means a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription regulator binding sites) and another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence. The above vector contains antibiotic resistance genes commonly used in the art as selectable markers, for example, resistance genes for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0274] In another aspect, the present invention relates to an isolated host cell transfected with the recombinant expression vector. The host cell used to produce the antibody of the present invention may be a prokaryotic cell, yeast cell, or higher eukaryotic cell, but is not limited thereto.

[0275] Prokaryotic host cells can be used, such as strains of the genus Bacillus, such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis and Staphylococcus (e.g., Staphylococcus carnosus).

[0276] However, examples of host cell lines useful as animal cells may include, but are not limited to, MDA-MB-231, A549, A431, HT29, HCC827, SK-MEL-2, SW620, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0277] The host cells described above can be cultured in various media. Any commercially available medium can be used as a culture medium. Any other essential supplements known to those skilled in the art may be included at appropriate concentrations. Culture conditions, such as temperature and pH, are already used with the host cells selected for expression and will be readily apparent to those skilled in the art.

[0278] The above recovery can be performed by removing impurities, for example, by centrifugation or ultrafiltration, and the resultant product can be purified, for example, using affinity chromatography. Additional purification techniques, for example, anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc., can be used.

[0279] The present invention also provides a composition for intracytoplasmic delivery of an active substance comprising the cytoplasmic penetrating antibody, its Fc fragment, or Fab fragment.

[0280] The above active substance may be in a form fused or bound to an antibody, and the active substance may be one or more selected from the group consisting of, for example, peptides, proteins, toxins, antibodies, antibody fragments, RNA, siRNA, DNA, small molecule drugs, nanoparticles, and liposomes, but is not limited thereto.

[0281] The protein may be an antibody, an antibody fragment, an immunoglobulin, a peptide, an enzyme, a growth factor, a cytokine, a transcription factor, a toxin, an antigenic peptide, a hormone, a transport protein, a motor protein, a receptor, a signaling protein, a storage protein, a membrane protein, a transmembrane protein, an internal protein, an external protein, a secreted protein, a viral protein, a glycoprotein, a truncated protein, a protein complex, or a chemically modified protein.

[0282] The above RNA or ribonucleic acid is a type of nucleic acid that forms a chain structure of nucleotides based on ribose, a type of pentose sugar, and has a structure in which a single helix is ​​long and coiled, and is created by transcribing a portion of DNA. In one specific embodiment, the RNA may be selected from rRNA, mRNA, tRNA, miRNA, snRNA, snoRNA, and aRNA, but is not limited thereto.

[0283] The above siRNA (small interfering RNA) is an RNA interference agent with a small size composed of dsRNA, which acts to bind to and degrade mRNA having a target sequence, and is widely used in the present invention by utilizing the activity of degrading the target mRNA and inhibiting the expression of a protein translated by the target mRNA as a therapeutic agent for diseases or experimentally.

[0284] The above DNA or deoxyribonucleic acid is a type of nucleic acid, and is composed of a backbone chain in which a phosphate group is bonded to the monosaccharide deoxyribose and a nucleobase having two types of purines and pyrimidines, and is a substance that stores the genetic information of cells.

[0285] The term "small molecule drug" is broadly used herein to refer to an organic, inorganic, or organometallic compound having a molecular weight of less than about 1000 daltons and having activity as a therapeutic agent for diseases. The small molecule drug used in the present invention includes oligopeptides and other biomolecules having a molecular weight of less than about 1000 daltons.

[0286] The above nanoparticle refers to a particle composed of materials having a diameter of 1 to 1000 nm, and the nanoparticle may be a metal / metal core-shell composite composed of a metal nanoparticle, a metal nanoparticle core, and a metal shell surrounding the core, a metal / non-metal core-shell composed of a metal nanoparticle core and a non-metal shell surrounding the core, or a non-metal / metal core-shell composite composed of a non-metal nanoparticle core and a metal shell surrounding the core. According to one specific example, the metal may be selected from, but is not limited to, gold, silver, copper, aluminum, nickel, palladium, platinum, ferromagnetic iron, and oxides thereof, and the non-metal may be selected from, but is not limited to, silica, polystyrene, latex, and acrylate series materials.

[0287] The liposomes are composed of one or more lipid bilayer membranes surrounding an aqueous internal compartment capable of self-assembly. Liposomes can be characterized by their membrane type and size. Small unilamellar vesicles (SUVs) have a single membrane and can have a diameter of 20 nm to 50 nm. Large unilamellar vesicles (LUVs) can have a diameter greater than 50 nm. Oligolamellar and multilamellar large vesicles have multiple, usually concentric, membrane layers and can have a diameter greater than 100 nm. Liposomes with multiple non-concentric membranes, i.e., multiple smaller vesicles contained within a larger vesicle, are called multivesicular vesicles.

[0288] The above "fusion" or "binding" refers to the integration of two molecules with different or identical functions or structures, and may be a fusion by any physical, chemical, or biological method that allows the tumor-penetrating peptide to be bound to the protein, small molecule drug, nanoparticle, or liposome. The fusion may preferably be by a linker peptide, which may mediate fusion with the active agent at various positions of the antibody light chain variable region, antibody, or fragment thereof of the present invention.

[0289] A pharmaceutical composition for preventing or treating cancer is provided, comprising the cytoplasmic penetrating antibody or fragment thereof and an intracellular active substance delivered thereby.

[0290] The cancer may be selected from the group consisting of 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, cutaneous or intraocular melanoma, rectal cancer, anal cancer, esophageal cancer, small intestine cancer, endocrine cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, chronic or acute leukemia, lymphocytic lymphoma, hepatocellular carcinoma, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, large intestine 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.

[0291] When manufactured as a pharmaceutical composition for the prevention or treatment of cancer, the composition may include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the composition is one commonly used in formulation, and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0292] The appropriate dosage of a pharmaceutical composition for the prevention or treatment of cancer can be prescribed in various ways depending on factors such as the formulation method, administration method, patient age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. The preferred dosage of the composition is within the range of 0.001-100 mg / kg for adults. The term "pharmaceutically effective amount" refers to an amount sufficient to prevent or treat cancer or to prevent or treat diseases caused by angiogenesis.

[0293] The composition may be prepared in a unit dose form or may be placed in a multi-dose container by formulating the composition using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person skilled in the art. In this case, 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 an extract, powder, granule, tablet or capsule, and may additionally include a dispersing agent or a stabilizer. In addition, the composition may be administered as an individual therapeutic agent or in combination with another therapeutic agent, and may be administered sequentially or simultaneously with conventional therapeutic agents. Meanwhile, since the composition includes an antibody or an antigen-binding fragment, it may be formulated as an immunoliposome. Liposomes containing antibodies may be prepared according to methods widely known in the art. The immunoliposome may be a lipid composition containing phosphatidylcholine, cholesterol and polyethylene glycol-derivatized phosphatidylethanolamine, and may be prepared by a reverse phase evaporation method. For example, the Fab' fragment of an antibody can be conjugated to liposomes via a disulfide-exchange reaction. Chemotherapeutic agents, such as doxorubicin, can be additionally incorporated into the liposomes.

[0294] The present invention provides a composition for diagnosing cancer comprising the cytoplasmic penetrating antibody or a fragment thereof and an intracellular active substance delivered thereby.

[0295] The above "diagnosis" refers to confirming the presence or characteristics of a pathophysiological condition. In the present invention, diagnosis refers to confirming the presence or absence of cancer and its progression.

[0296] The above antibodies in the form of complete immunoglobulins and fragments thereof can be combined with a fluorophore for molecular imaging to diagnose cancer through imaging.

[0297] The above-mentioned fluorescent material for molecular imaging refers to any material that generates fluorescence, and it is preferable that it emits red or near-infrared fluorescence, and a fluorescent material with a high quantum yield is more preferable, but is not limited thereto.

[0298] The above-described molecular imaging fluorophore is preferably, but not limited to, a fluorophore, fluorescent protein or other imaging material that can bind to a tumor-penetrating peptide that specifically binds to the antibody in the form of the complete immunoglobulin and its fragments.

[0299] The fluorescent material is preferably, but not limited to, fluorescein, BODYPY, tetramethylrhodamine, alexa, cyanine, allophycocyanine or derivatives thereof.

[0300] The fluorescent protein is preferably, but not limited to, a dronpa protein, an epifluorescent gene (EGFP), a red fluorescent protein (DsRFP), a cyanine fluorophore exhibiting near-infrared fluorescence, Cy5.5, or other fluorescent proteins.

[0301] Other imaging materials include, but are not limited to, iron oxide and radioactive isotopes, and can be applied to imaging equipment such as MR and PET.

[0302]

[0303] Example

[0304] The present invention will be described in more detail below through examples. However, these examples are intended to exemplify the present invention and the scope of the present invention is not limited to these examples.

[0305]

[0306] Example 1. Design of an antibody (in2C1N) having an endosomal escape structural motif in the heavy chain constant region CH3 domain.

[0307] To develop a cytoplasmic penetrating antibody having an endosomal escape structural motif in the CH3 domain, an endosomal escape structural motif was designed to induce interaction with the endosomal membrane in the AB loop, EF loop, and C-terminal loop of the CH3 domain of a representative IgG1 antibody, and 11 CH3 domain variants were constructed as shown in Table 1 below.

[0308] The existing inCT cytoplasmic penetrating antibody targets the CDR3 region of VL. 92 WYW 94 (Amino acid residue numbers are Kabat numbering) and CDR3 region of VH 96 WYW 98 (Kabat numbering) as an endosomal escape structural motif. In addition, the inCT(AAA) antibody, in which all of the above endosomal escape structural motifs, WYW, were replaced with alanine (Ala), did not show cytoplasmic penetration ability.

[0309] Therefore, we constructed CH3 domain variants based on the inCT(AAA) antibody, which has no cytoplasmic penetration ability, and aimed to evaluate whether they have cytoplasmic penetration ability only through the endosomal escape structural motif of the CH3 domain. That is, we constructed CH3 domain variants based on inCT(AAA) through a heavy chain including the heavy chain variable region (VH) of SEQ ID NO: 14 and the heavy chain constant region (CH1-hinge-CH2-CH3) having the CH3 domain variants of SEQ ID NOs: 2 to 12, and a light chain including the light chain variable region (VL) of SEQ ID NO: 16 and the light chain constant region (CL) of SEQ ID NO: 17, and aimed to confirm the endosomal escape ability dependent on the endosomal escape structural motif of the CH3 domain variant.

[0310] At this time, the cytoplasmic penetrating antibody with a new endosomal escape structural motif introduced into the constant region was named in2C, and the CH3 domain variant with an endosomal escape structural motif introduced into CH3 was named in2C1N. (For example, the CH3 domain variant with the 1st endosomal escape structural motif introduced into the CH3 domain is in2C11, and the CH3 domain variant with the 2nd endosomal escape structural motif introduced into the CH3 domain is in2C12.) In addition, antibodies with only arginine and tryptophan mutations without glutamic acid patch mutations in the endosomal escape structural motifs of in2C11 and in2C12 were named in2C11, respectively. W / O E , in2C12 W / O E was named (W / OE means that the glutamate patch was removed from the endosomal escape structural motif).

[0311] Additionally, the endosomal escape structural motif of sequence number 13 ( 96 WYW 98 ) and a heavy chain comprising a heavy chain constant region (CH1-hinge-CH2-CH3) having a wild-type IgG1 CH3 domain of SEQ ID NO: 1, and an endosomal escape structural motif of SEQ ID NO: 15 ( 92 WYW 94 ) and a light chain including a wild-type light chain constant region (CL) of SEQ ID NO: 17 were used as a control for the physical property evaluation and cytoplasmic penetration ability evaluation by co-expressing and purifying the inCT cytoplasmic penetrating antibody.

[0312] Along with this, the endosomal escape structure motif of sequence number 14 ( 96 WYW 98 ) comprising a heavy chain variable region (VH) in which alanine (Ala) is substituted and a heavy chain constant region (CH1-hinge-CH2-CH3) having a wild-type IgG1 CH3 domain of SEQ ID NO: 1, and an endosomal escape structural motif of SEQ ID NO: 16 ( 92 WYW 94) was co-expressed and purified with an inCT(AAA) antibody having no cytoplasmic penetration ability through a light chain comprising a light chain variable region (VL) substituted with alanine (Ala) and a wild-type light chain constant region (CL) of SEQ ID NO: 17, and used as a control for the evaluation of physical properties or cytoplasmic penetration ability.

[0313] The endosomal escape structural motif of the CH3 domain variant was introduced into the surface-exposed amino acid positions of the AB, EF, and C-terminal loops of the CH3 domain to facilitate interaction with the endosomal membrane and hydrogenation according to changes in the pH environment. To this end, the 3D structure (PDB file) of the wild-type IgG1 antibody Fc region was entered into the GETAREA web server (https: / curie.utmb.edu / getarea.html), and the solvent accessible surface area (SASA) ratio (%) was used to identify the surface-exposed amino acid positions in the AB, EF, and C-terminal loops of the CH3 domain on the web, as shown in Table 3 below.

[0314] CH3 domain mutants were designed by introducing arginine, tryptophan, and glutamic acid mutations constituting the endosomal escape motif at surface-exposed amino acid positions of the CH3 domain AB, EF, and C-terminal loop.

[0315] The amino acid sequence of the IgG1 wild-type CH3 domain and the amino acid sequences of 11 CH3 domain variants possessing the endosomal escape structural motif are shown in Table 1 below.

[0316]

[0317] Figure 1 is a schematic diagram of a cytoplasmic penetrating antibody that introduces an endosomal escape structural motif into the AB, EF loop, and C-terminal loop of the CH3 domain.

[0318] Figure 2 shows the 3D structure of the Fc region containing 11 CH3 domain variants that introduced the endosomal escape structural motif of the amino acid sequence into the AB, EF and C-terminal loops of the CH3 domain, modeled using Alphafold2, and the intramolecular interaction patterns analyzed using the Pymol molecular visualizer.

[0319] To analyze the difference between the tertiary structure of the wild-type CH3 domain and the tertiary structure of the CH3 domain mutant, the RMSD (Root Mean Square Deviation) of the CH3 domain mutant duplex of FIG. 2 with respect to the wild-type CH3 domain duplex was calculated, and is shown in Table 2 below.

[0320]

[0321] Through Figure 2, it was confirmed that the tertiary structure of the CH3 domain mutant was similar to the overall shape and protein folding state of the tertiary structure of the wild-type CH3 domain, and as shown in Table 2, it was confirmed that the structure was maintained very similarly to the wild-type CH3 domain with an RMSD value of 0.2 Å or less. However, although the RMSD value of in2C17 increased slightly to 2.24 Å, the overall folding state of the antibody was maintained similarly.

[0322] In addition, the 3D structures of CH3 domain variants modeled through Alphafold2 in Fig. 2 were input into GETAREA to obtain the solvent accessible surface area ratio (%), which is specified in Table 3 below. Through this, it was confirmed that the degree of solvent exposure of each amino acid did not significantly change even when the endosomal escape structural motif was introduced into the AB, EF, and C-terminal loops of the CH3 domain. However, there were also cases where the solvent accessible surface area ratio decreased from about 50% to about 30% or less, such as Q418W and K439W.

[0323] The following paragraphs detail the endosomal escape structural motifs of CH3 domain variants.

[0324]

[0325]

[0326] The endosomal escape structural motif of in2C11 of sequence number 4 consists of two tryptophan mutations (R355W, Q418W), two arginine mutations (L358R, L443R), one alanine mutation (K414A), and three glutamic acid mutations (N361E, Q362E, D413E) in the AB loop, the EF loop, and the C-terminal loop.

[0327] For arginine and tryptophan mutations, L358R and R355W interact with a cation-π at a distance of 4.1 Å, as shown in Fig. 2b, and L443R and Q418W interact with a cation-π at a distance of 4.1 Å. At this time, K414 was substituted with a smaller alanine (K414A) to prevent steric hindrance between Q418W and W417.

[0328] In the case of glutamic acid mutations, three glutamic acids are densely packed in adjacent positions in a patch shape, and the pK of the carboxyl group of the glutamic acid side chain is a It was intended to increase the pK of the side chain carboxyl group of glutamic acid. a is around 4.2, and in the slightly acidic pH environment of endosomes (4.5 - 6.5), the rate of dehydrogenation is much higher than the rate of hydrogenation, so a large number of glutamic acids maintain negative charges in the endosome. However, when negative charges are concentrated, hydrogenation and charge loss are energetically preferred because it can reduce charge repulsion between them, so pK a It increases and can promote hydrogenation under slightly acidic conditions.

[0329] Specifically, the distance between the Cα of D413 and the Cα of Q362 is 4.5 Å, and the distance between the Cα of Q362 and the Cα of N361 is 3.8 Å. If the three amino acids above are replaced with glutamic acid, Q362E located in the middle will be surrounded by amino acids with the same negative charge within 5 Å from the Cα. The 3D model structure of in2C11 with the glutamic acid patches of the three amino acids (N361E, Q362E, D413E) introduced was input into the PROPKA 3.1 web server (https: / biolib.com / bio-utils / propka / ) to obtain the pK of glutamic acid. a When predicted, the pK of Q362E as shown in Fig. 3d a The original pK is 5.02 a It was confirmed that the pK increased by about 0.8 from 4.2. According to the Henderson-Hasselbalch equation, this is the original pK based on the initial endosomal slightly acidic environment of pH 5.5. a When the pK is 4.2, the ratio of hydrogenated and dehydrogenated glutamic acid is approximately 1:20, while the pK a pK of 5.0, which is 0.5 higher a In this case, the ratio is approximately 1:3, which means that the proportion of glutamic acid being hydrogenated has increased by more than 6 times. In the late endosome, which is slightly acidic, the ratio is approximately 2:1, which means that the proportion of glutamic acid being hydrogenated has increased even more. Therefore, the pK through glutamic acid patches that densely cluster glutamic acid mutations in adjacent locations a We confirmed that the increase in endosome hydrogenation rate could result in target cell-specific cytoplasmic penetration, which could lead to receptor-mediated cellular internalization.

[0330] Therefore, in2C11 was designed to confer cytoplasmic penetration ability and pH dependency by densely clustering four pairs of arginine and tryptophan that form cation-π interactions based on the CH3 dimer at the lower part of the CH3 domain of the antibody Fc region and introducing glutamic acid patches located around the periphery on both sides of the CH3 domain of the antibody Fc region.

[0331]

[0332] in2C11 of sequence number 2 W / O E is a mutant in which the glutamic acid patch is removed from in2C11 and only arginine and tryptophan mutations are introduced, as shown in Fig. 2c. This mutant was constructed to verify the function of the glutamic acid patch introduced into in2C11, namely, whether it can actually prevent nonspecific binding and deterioration of physical properties due to arginine and tryptophan, and whether it can impart pH dependence.

[0333]

[0334] The endosomal escape structural motif of in2C13 of SEQ ID NO: 6 consists of two tryptophan mutations (R355W, Q418W), two arginine mutations (L358R, L443R), one alanine mutation (K414A), and five glutamic acid mutations (N361E, Q362E, D413E, S415E, Q419E) in the AB loop, EF loop, and C-terminal loop. These mutations were constructed to enhance the effect of the glutamic acid patch by introducing two additional glutamic acids (S415E, Q419E) on the side of the α-helix present in the EF loop in in2C11. The 3D model structure of in2C13 with a glutamic acid patch consisting of five glutamic acids (N361E, Q362E, D413E, S415E, Q419E) was input into the PROPKA 3.1 web server to obtain the pK of glutamic acid. a When predicted, the pK of Q362E as shown in Fig. 2f a pK of 5.03, S415E apK at the position where it is surrounded by negative charges of 5.05 a It was confirmed that the value increased by approximately 0.8.

[0335]

[0336] The endosomal escape structural motif of in2C12 of SEQ ID NO: 5 consists of three tryptophan mutations (R355W, S415W, Q418W), three arginine mutations (L358R, Q419R, L443R), one alanine mutation (K414A), and three glutamic acid mutations (N361E, Q362E, D413E) in the AB loop, EF loop, and C-terminal loop. This mutation was constructed to strengthen the interaction with the cell membrane by introducing an additional arginine-tryptophan pair (S415W, Q419R) on the side of the α-helix in the EF loop in in2C11, thereby introducing arginine and tryptophan in a wider region than in2C11. As shown in Fig. 2d, the pK of Q362E due to the cation-π interaction and glutamic acid patch introduced in in2C11 a As the rise was maintained, it was confirmed that the additionally introduced S415W and Q419R formed cation-π interactions at a distance of 4.9 Å.

[0337] Therefore, in2C12 was designed to confer cytoplasmic penetration ability and pH dependency by densely clustering six pairs of arginine and tryptophan that form cation-π interactions based on the CH3 dimer at the lower part of the CH3 domain of the antibody Fc region and introducing glutamic acid patches located around the periphery on both sides of the CH3 domain of the antibody Fc region.

[0338]

[0339] in2C12 of sequence number 3 W / O Eis a mutant in which the glutamic acid patch is removed from in2C12 and only arginine and tryptophan mutations are introduced, as shown in Fig. 2e. This mutant was constructed to verify the function of the glutamic acid patch introduced into in2C12, namely, whether it can actually prevent nonspecific binding and deterioration of physical properties caused by arginine and tryptophan, and whether it can impart pH dependence.

[0340]

[0341] The endosomal escape structural motif of in2C14 of sequence number 7 consists of four tryptophan mutations (R355W, D413W, Q418W, Q419W), three arginine mutations (L358R, S415R, L443R), one alanine mutation (K414A), and two glutamic acid mutations (N361E, Q362E) in the AB loop, EF loop, and C-terminal loop. In in2C12, the arrangement of arginine and tryptophan was changed differently, and D413E of in2C11 was substituted with tryptophan (D413W) and S415W was substituted with arginine (S415R), forming an arginine-tryptophan pair of S415R and D413W, thereby inducing interaction with the cell membrane. As shown in Figure 2g, it was confirmed that D413W and S415R formed a cation-π interaction at a distance of 5.4 Å, and the pK of Q362E was a pK as 5.05 a The rise was maintained.

[0342] Therefore, in2C14 was designed to confer cytoplasmic penetration ability and pH dependency by densely clustering six pairs of arginine and tryptophan that form cation-π interactions based on the CH3 dimer at the lower part of the CH3 domain of the antibody Fc region and introducing glutamic acid patches located around the periphery on both sides of the CH3 domain of the antibody Fc region.

[0343]

[0344] in2C15 of sequence number 8 and in2C16 of sequence number 9 are mutants that form an endosomal escape structural motif with the same logic as in2C11, but with different positions of arginine, tryptophan, and glutamic acid.

[0345] The endosomal escape structural motif of in2C15 consists of two tryptophan mutations (L358W, T359W), one arginine mutation (D356R), one glutamine mutation (K414Q), and four glutamic acid mutations (N361E, D413E, S415E, Q418E) in the AB loop, EF loop, and C-terminal loop. As shown in Fig. 2h, the cation-π interaction formed between R355W and L358R in in2C11 was changed to a cation-π interaction between R355 and L358W. In addition, the 3D model structure of in2C15, in which a glutamic acid patch consisting of four glutamic acids (N361E, D413E, S415E, Q418E) was introduced, was entered into the PROPKA 3.1 web server, and the pK of glutamic acid was calculated. a When predicted, pK of D413E as in Fig. 3h a The original pK is 4.7 a It increased by about 0.5 from 4.2. In addition, to prevent glutamic acid from forming a salt bridge with lysine (K414) and lowering the pka of glutamic acid, glutamine, which has a side chain length similar to glutamic acid but no charge (K414Q), was introduced to remove the positive charge.

[0346] The endosomal escape structural motif of in2C16 consists of two tryptophan mutations (D356W, L358W), one arginine mutation (T359R), one glutamine mutation (K414Q), and four glutamic acid mutations (N361E, D413E, S415E, Q418E) in the AB loop, EF loop, and C-terminal loop. As shown in Fig. 2i, a cation-π interaction is formed between R355 and D356W, and a cation-π interaction is formed between T359R and D356W. The glutamic acid patch is identical to that of in2C15, except that K414Q was introduced to remove the positive charge at position 414 to prevent glutamic acid from forming a salt bridge with lysine (K414).

[0347] Therefore, in2C15 and in2C16 were designed to densely cluster four pairs of arginine and tryptophan that form CH3 dimer-based cation-π interactions at the bottom of the CH3 domain of the antibody Fc region, and to introduce glutamic acid patches in a different form from in2C11, in2C12, in2C13, and in2C14 onto both sides of the CH3 domain of the antibody Fc region, thereby imparting cytoplasmic penetration ability and pH dependency.

[0348] in2C17 of sequence number 10 and in2C18 of sequence number 11 are mutants in which arginine, tryptophan, and glutamic acid are arranged with reference to in2C11, in2C15, and in2C16, which have no non-specific binding and excellent yield and / or endosomal escape ability.

[0349] The endosomal escape structural motif of in2C17 consists of two tryptophan mutations (L358W, K439W), two arginine mutations (D356R, T359R), one asparagine mutation (K414N), and three glutamic acid mutations (N361E, Q362E, D413E) in the AB loop, the EF loop, and the C-terminal loop.

[0350] The arginine-tryptophan pair of in2C17 maintains the R355-L358W of in2C15 and in2C16, and a new D356R-K439W was introduced at the interface between the CH3 domain duplexes, replacing the existing salt bridge with a cation-π interaction. When the wild-type CH3 domain forms a homodimer, various interactions occur at the interface between each CH3 domain. As shown in Fig. 2j, D356 of the wild-type CH3 domain and K439 of the opposite CH3 domain form a salt bridge at a distance of 4.7 Å. When D356R and K439W were introduced to change the salt bridge to a cation-π interaction, and the three-dimensional model structure was confirmed as shown in Fig. 3k, it was confirmed that a cation-π interaction was formed between D356R-K439W at a distance of 3.1 Å.

[0351] The same glutamic acid patch (N361E, Q362E, D413E) as in2C11 was introduced, and the 3D model structure of in2C17 was input into the PROPKA 3.1 web server to obtain the pK of glutamic acid. a When predicted, the pK of Q362E as in Fig. 3k a The original glutamic acid pK is 5.16 a It was confirmed that it increased by about 0.9 from 4.2. In addition, glutamine (K414Q), which was introduced to prevent glutamic acid from forming a salt bridge with lysine (K414), was changed to asparagine (N), which is smaller in size.

[0352] The endosomal escape structural motif of in2C18 of SEQ ID NO: 11 consists of three tryptophan mutations (R355W, L358W, K439W), two arginine mutations (D356R, T359R), one asparagine mutation (K414N), and three glutamic acid mutations (N361E, Q362E, D413E) in the AB loop, EF loop, and C-terminal loop. D356R-K439W forms a cation-π interaction at a distance of 3.3 Å. The pK of glutamic acid was determined using the PROPKA 3.1 web server in the same manner as above. a When predicted, the pK of Q362E as in Fig. 2k a The original glutamic acid pK is 5.03 a It was confirmed that it increased by about 0.8 from 4.2.

[0353] The endosomal escape structural motif of in2C19 of SEQ ID NO: 12 consists of three tryptophan mutations (L358W, Q418W, K439W), three arginine mutations (D356R, T359R, L443R), one alanine mutation (K414A), and three glutamic acid mutations (N361E, Q362E, D413E) in the AB loop, EF loop, and C-terminal loop. in2C19 is a mutant that combines in2C11 and in2C17. As shown in Fig. 2l, R355-T359R formed a cation-π interaction at 5.5 Å, and introduction of L443R-Q418W of in2C11 induced a cation-π interaction at a distance of 4.6 Å between the two amino acids. At this time, lysine was substituted with alanine (K414A) to prevent steric hindrance due to Q418W. In addition, D356R-K439W of in2C17 was introduced to induce a cation-π interaction at a distance of 3.3 Å between the two amino acids. The glutamic acid patch is identical to in2C11, and the pK of glutamic acid was determined using the PROPKA 3.1 web server in the same manner as above. aWhen predicted, Fig. 2l and pK of this Q362E a It was confirmed that it rose to 5.02.

[0354] Therefore, in2C19 was designed to confer cytoplasmic penetration ability and pH dependency by densely clustering six pairs of arginine and tryptophan that form CH3 dimer cation-π interactions at the bottom of the CH3 domain of the antibody Fc region and introducing glutamic acid patches identical to those of in2C11 onto both sides of the CH3 domain of the antibody Fc region.

[0355]

[0356] Figure 3 is a schematic diagram showing the expected mechanism by which a cytoplasmic penetrating antibody having an endosomal escape structural motif in the CH3 domain is internalized into a cell and then located in the cytoplasm, and the design strategy of the endosomal escape structural motif to impart pH dependence to the cytoplasmic penetrating antibody.

[0357] The cytoplasmic penetrating antibody is specifically internalized into target cells overexpressing the integrin αvβ3 / αvβ5 receptor through the in4 circular peptide specific for the integrin αvβ3 / αvβ5 receptor fused to the light chain N-terminus. After internalization, the cytoplasmic penetrating antibody is detached from the receptor under the mildly acidic and reducing conditions of the endosome, and interacts with the endosomal membrane through the arginine and tryptophan of the endosomal escape structural motif introduced into the CH3 domain and / or the CL domain, destabilizing the membrane, resulting in the collapse of the endosomal membrane and endosomal escape from the endosome to the cytoplasm. Therefore, the cytoplasmic penetrating antibody does not go to the Golgi or lysosomes.

[0358] At this time, the glutamic acid patch around arginine and tryptophan in the endosomal escape structural motif inhibits the interaction with the cell membrane because it causes electrostatic repulsion with the negatively charged cell membrane in the physiological environment of pH 7.4. However, in the late endosomal environment where the pH is lowered to 4.5 - 5.5, the carboxyl group of the glutamic acid side chain (COO - ) is hydrogenated and loses its negative charge, thereby reducing electrostatic repulsion with the cell membrane, and strengthening the hydrophobic interaction between the hydrogenated glutamic acid and the hydrophobic tail region of the cell membrane phospholipid, thereby promoting interaction with the cell membrane.

[0359]

[0360] Example 2. Evaluation of cytoplasmic localization and cytoplasmic penetrability of cytoplasmic penetrable antibodies through complementary binding of improved split green fluorescent protein.

[0361] To confirm that the constructed CH3 domain variants are located in the cytoplasm and to quantitatively compare and evaluate the cytoplasmic penetration ability between the variants, an improved split green fluorescent protein complementary binding system (Korean Patent No. KR10-1522954, Biochem Biophys Res Commun, 2015, 467(4):771-777) was used. Figure 4 is a schematic diagram depicting the process in which GFP fluorescence is observed due to complementary binding of the improved split green fluorescent protein when the cytoplasmic penetrating antibody is located in the cytoplasm. The observation of GFP fluorescence proves that the cytoplasmic penetrating antibody is located in the cytoplasm, and the cytoplasmic penetration ability of each variant can be evaluated through quantitative analysis of GFP fluorescence. Therefore, all cytoplasmic penetrating antibodies in the form of full IgG with cytoplasmic penetration ability in the CH3 domain were constructed and evaluated in a form in which the GFP11-SBP2 fragment was fused to the heavy chain C-terminus.

[0362]

[0363] Example 3. Expression and purification of a cytoplasmic penetrating antibody (in2C1N-GFP11-SBP2) having an endosomal escape structural motif in the CH3 domain, fused with a circular peptide targeting a membrane protein receptor overexpressed on the surface of tumor cells and GFP11-SBP2.

[0364] To identify biophysicochemical properties of CH3 domain mutants and evaluate cytoplasmic penetration ability through the complementary binding system of improved split green fluorescent protein, we constructed in2C1N-GFP11-SBP2, a CH3 domain mutant in which GFP11-SBP2 is fused to the C-terminus of the antibody heavy chain.

[0365] At this time, a circular peptide (in4, DGVRQCRGDCFDGPL) (Science Advances, 2020, 6(3):eaay2174) targeting integrin αvβ3 / αvβ5, a type of membrane protein receptor overexpressed on the surface of tumor cells / tissues, was fused to the light chain N-terminus of the cytoplasmic penetrating antibody to induce cell internalization through tumor cell / tissue-specific receptors.

[0366] Specifically, to construct a heavy chain expression vector for producing a monoclonal antibody in the form of a full IgG, DNA encoding a heavy chain variable region (VH, SEQ ID NO: 14) of an antibody fused with a DNA encoding a secretory signal peptide at the 5' end and a heavy chain constant region of CH1-hinge-CH2-CH3 having a CH3 domain variant (SEQ ID NO: 2 to 12) were each cloned into the pcDNA3.4 vector using NotI / HindIII. At this time, to construct a cytoplasmic penetrating antibody fused with GFP11-SBP2, GFP11-(G4S)3-SBP2 was fused to the heavy chain C-terminus using a linker of three GGGS units. In addition, to construct a vector expressing a light chain, DNA encoding a light chain, including an antibody light chain variable region (VL, SEQ ID NO: 16) and a light chain constant region (CL, SEQ ID NO: 17) fused to a DNA encoding a secretory signal peptide at the 5' end, was cloned into the pcDNA3.4 vector using NotI / HindIII. The variable region sequences of inCT and inCT(AAA) of SEQ ID NOs: 13 to 16 are specified in Table 4 below.

[0367] The cytoplasmic penetrating antibody was expressed and purified by transient transfection of a heavy chain expression vector including a VH of SEQ ID NO: 14 and a CH3 domain variant of SEQ ID NOs: 2 to 12 and a common light chain expression vector including a VL of SEQ ID NO: 16 and a light chain constant region sequence of SEQ ID NO: 17.

[0368]

[0369] In shake flasks, HEK293-F cells (Invitrogen) growing in suspension in serum-free FreeStyle 293 expression medium (Invitrogen) were transfected with a mixture of the above plasmids and polyethylenimine (PEI) (Polyscience, 24765-1). 2.0 × 10 HEK293-F cells were transfected in 200 mL shake flasks (Corning). 6 Cells were seeded in 100 mL of medium at a density of 10 cells / mL and cultured at 130 rpm, 8% CO2, 37°C. To produce antibodies, the appropriate heavy and light chain plasmids were diluted to 125 μg of heavy chain and 125 μg of light chain in 10 mL FreeStyle 293 expression medium (total 250 μg (2.5 μg / mL)) and filtered, mixed with 10 mL of medium in which 750 μg (7.5 μg / mL) of PEI was diluted, and reacted at room temperature for 10 minutes. After that, the reacted mixed medium was added to the cells seeded in 100 mL previously and cultured at 130 rpm, 8% CO2, 37°C for 4 hours, and then the remaining 100 mL of FreeStyle 293 expression medium was added and cultured for 6 days.

[0370] Proteins were purified from cell culture supernatants using a standard protocol. Antibodies were applied to a Protein A Sepharose column (GE Healthcare) and washed with PBS (pH 7.4). The antibody was eluted at pH 3.0 with 0.1 M glycine buffer, and the sample was immediately neutralized with 1 M Tris buffer (pH 9.0). The eluted antibody fraction was concentrated by dialysis into histidine buffer (25 mM histidine, 150 mM NaCl, pH 6.5). After purification, the protein concentration was quantified using a BCA protein assay kit (Pierce, 23225).

[0371]

[0372] The production yields of CH3 domain mutants are shown in Table 5 above, and although there are cases where the yield is reduced by about 55%, such as in2C18-GFP11-SBP2, compared to the wild type inCT(AAA)-GFP11-SBP2, it was confirmed that all CH3 domain mutants had a satisfactory production yield of more than 30 mg per liter.

[0373] Hereinafter, a cytoplasmic penetrating antibody (in2C1N-GFP11-SBP2) having an endosomal escape structural motif in the CH3 domain, fused to a prototypical peptide targeting a membrane protein receptor overexpressed on the surface of tumor cells and GFP11-SBP2, is described as in2C1N. For example, in2C11-GFP11-SBP2 is in2C11.

[0374]

[0375] Example 4. Physical property evaluation of a cytoplasmic penetrating antibody (in2C1N) having an endosomal escape structural motif in the heavy chain constant region CH3 domain.

[0376] Figure 5 shows the results of analysis of 11 antibodies having an endosomal escape structural motif in the CH3 domain by 12% SDS-PAGE under reducing or non-reducing conditions and size-exclusion chromatography after purification together with the control antibody inCT or inCT(AAA). Specifically, all 11 CH3 domain variants were confirmed to have a molecular weight of approximately 162 to 163 kDa under non-reducing conditions, like the control antibody, and showed a molecular weight of approximately 55 kDa for the heavy chain (approximately 50 kDa for the antibody heavy chain and approximately 5 kDa for the GFP11-SBP2 fragment) and approximately 26.5 kDa for the light chain (approximately 25 kDa for the antibody light chain and 1.5 kDa for the in4 circular peptide) under reducing conditions. This demonstrates that the purified in4 circular peptide and the cytoplasmic penetrating antibody fused with GFP11-SBP2 exist as a single entity in solution and do not form dimers or oligomers through unnatural disulfide bonds.

[0377] The lower graph in Figure 5 shows the mAU values ​​at 280 nm for 11 antibodies with an endosomal escape structural motif in the CH3 domain, using size exclusion chromatography (Superdex 200 increased 10 / 300GL, Cytiva). Specifically, 20 μL of the 11 CH3 domain variants and the control inCT (AAA) and / or Trastuzumab were prepared at a concentration of 1 mg / mL and placed in the insert to prepare the sample. PBS (10 mM Na2HPO4, 137 mM NaCl, 2.7 mM KCl, 1.8 mM KH2PO4, pH 7.4) buffer was flowed at a flow rate of 0.75 mL / min, and the pressure and mAU values ​​at 280 nm were allowed to stabilize before preparation. Afterwards, 10 μL of the prepared sample was loaded at the same flow rate, and the mAU values ​​at 280 nm were measured for 40 minutes. in2C12 W / O E, except for in2C12 and in2C14, the remaining eight variants showed a single peak without oligomerization and tailing, similar to the control antibody inCT (AAA) or Trastuzumab. This confirms that in2C12 W / O E , it was confirmed that the remaining eight mutants, excluding in2C12 and in2C14, had similar properties to the control wild-type antibody.

[0378]

[0379] Example 5. Evaluation of non-specific binding of a cytoplasmic penetrating antibody (in2C1N) having an endosomal escape structural motif in the heavy chain constant region CH3 domain.

[0380] Figure 6 shows the results of ELISA performed on four antigens, dsDNA, Insulin, Keyhole limpet hemocyanin (KLH), and Cardiolipin, which can confirm nonspecific binding of antibodies, to confirm nonspecific binding of cytoplasmic penetrating antibodies having an endosomal escape structural motif in the CH3 domain.

[0381] Specifically, 50 μL of four antigens, 1 μg / mL dsDNA (Sigma, D4522), 5 μg / mL Insulin (Sigma, I9278), 5 μg / mL Keyhole limpet hemocyanin (Sigma, H8283), and 10 μg / mL Cardiolipin (Sigma, C0563) diluted in PBS (pH 7.4), were dispensed per well of a 96-well EIA / RIA plate and allowed to adsorb to the plate surface for 1 hour at room temperature. Afterwards, 100 μL of PBS-T solution (0.1% Tween 20 in PBS) was dispensed per well of the plate and shaken off, repeating this process three times to wash the plate. Then, 50 μL of blocking solution (2% skim milk in PBS-T) was dispensed per well to block the non-adsorbed surface and allowed to adsorb for 1 hour at room temperature. Afterwards, 100 μL per well was dispensed and washed three times with PBS-T solution, and 50 μL of each antibody diluted to a concentration of 100 nM or 500 nM in blocking solution was dispensed to each well and treated for 1 hour at room temperature. RT11-i antibody is an antibody with many positively charged amino acids in CDR-L1 and has non-specific binding to various antigens including HSPG, so it was used as a positive control for the experiment. InCT (AAA) of the wild-type IgG1 CH3 domain was used as a negative control. Afterwards, 100 μL per well was dispensed and washed three times with PBS-T solution, and 50 μL of HRP-conjugated anti-human monoclonal antibody (Invitrogen, 31413) diluted 1:8,000 in blocking solution was dispensed to each well and treated for 1 hour at room temperature.Finally, 100 μL per well was dispensed with PBS-T solution, and the process of shaking off the solution was repeated three times to wash the well. Then, 25 μL of TMB ELISA solution (Thermo Scientific™, 34028), which provides a substrate for HRP, was dispensed and reacted for 2 minutes. After 2 minutes, 25 μL of 2 N sulfuric acid solution (SAMCHUN, S2129) was dispensed to stop the reaction, and the absorbance was measured at 450 nm.

[0382] In the above experiment, it is determined that a non-specific binding is present when the absorbance at 450 nm is 0.05 or higher for at least one of the four antigens at a concentration of 100 nM. The experimental results show that in2C11 for dsDNA antigen +W / O E , in2C12 W / O E , in2C12, and in2C14 showed nonspecific binding at 100 nM.

[0383] At this time, in2C11 W / O E and in2C12 W / O E are mutants in which the glutamic acid patch was removed and only arginine and tryptophan mutations were introduced to confirm the function of the glutamic acid patch in in2C11 and in2C12, respectively. When the non-specific binding to dsDNA was evaluated in Fig. 6, in2C11 without the glutamic acid patch W / O E and in2C12 W / O E showed high nonspecific binding to dsDNA, but in2C11 and in2C12 with glutamic acid patches showed significantly reduced nonspecific binding to dsDNA. This confirmed that the glutamic acid patch actually reduces nonspecific binding that can be induced by arginine-tryptophan. Consequently, in2C11 W / O E and in2C12 W / O E , in2C12, and in2C14 were confirmed to have non-specific binding to the above four antigens, and the remaining seven mutants were selected as candidate substances.

[0384]

[0385] Figure 7 shows the results of flow cytometry analysis performed to confirm the nonspecific binding to the cell membrane of a cytoplasmic penetrating antibody having an endosomal escape structural motif in the CH3 domain. Specifically, it was confirmed whether nonspecific binding to the cell membrane was shown in two cell lines (K-562, CHO-K1) that do not express integrin αvβ3 / αvβ5. The two cell lines were cultured in RPMI medium containing 10% FBS and 1% ABAM at 5% CO2 and 37°C, and 1 × 10 per sample. 5 The cells were prepared in PBS-F solution (pH 7.4 PBS, 1% BSA). Each cell line was treated with CH3 domain variants at concentrations of 500 nM and 1 μM, along with the wild-type negative control inCT or inCT(AAA) and the positive control RT11-i, at 4°C for 1 hour. After washing with PBS-F solution, the cells were treated with Alexa Fluor 647-conjugated anti-human IgG (H+L) antibody (Invitrogen, A21445) at 4°C for 30 minutes. After washing with PBS-F solution, the cells were analyzed using a flow cytometer (FACSCalibur, BD). Experimental results in in2C11 W / O E , in2C12 W / O E , in2C12, in2C13, and in2C14 were confirmed to exhibit significantly more non-specific binding to the cell surfaces of K-562 and CHO-K1 than the wild-type negative control, and therefore, the six mutants excluding these were selected as candidates.

[0386]

[0387] Example 6. Construction of a reporter cell line for evaluating the cytoplasmic penetration ability of a cytoplasmic penetrating antibody through complementary binding of an improved split green fluorescent protein.

[0388] An assay system was established to quantitatively evaluate the cytoplasmic penetration ability of cytoplasmic penetrating antibodies using complementary binding of improved split green fluorescent proteins. Specifically, transfected MDA-MB-231 and SW480 cell lines stably expressing SA-GFP1-10 were established using MDA-MB-231 and SW480, which express integrin αvβ3 / αvβ5. First, 8 × 10 per well were seeded in 6-well plates for lentivirus particle production. 5 HEK293T cells were cultured in DMEM medium containing 10% FBS and 1% ABAM for 16 hours at 37°C in 5% CO2. After 16 hours, the medium was replaced with 2 mL of Opti-MEM medium. Transfection was performed with lentiviral packaging plasmids (pMDLg / Prre (Addgene, 12251), pRSV-Rev (Addgene, 12253), pMD2.G (Addgene, 12259)) and lentiviral transfer plasmid (pLJM-SA-GFP1-10) using Lipofectamine™ 3000 transfection reagent (Invitrogen, L3000001) according to the manufacturer's suggested method. After 6 hours, the medium was replaced with 2 mL of DMEM, and the cells were cultured for 48 hours to produce viral particles. After obtaining the supernatant from each well, centrifugation was performed at 500 g for 10 minutes at room temperature to remove cell debris, the supernatant was filtered through a 0.45 μm filter, and virus particles were concentrated using a Lenti-X concentrator (TAKARA, 631231).

[0389] To transduce MDA-MB-231 cells using lentiviral particles, 8 × 10 per well in a 6-well plate 5MDA-MB-231 or SW480 cells were cultured in 2 mL of RPMI medium containing 10% FBS and 1% ABAM for 16 h at 5% CO2, 37°C. To improve transduction efficiency, polybrene infection / transfection reagent (Sigma, TR-1003) was diluted to 12 μg / mL in RPMI medium, and the concentrated virus particles were diluted in this medium and infected into MDA-MB-231 or SW480 cells. The 6-well plates were centrifuged at 1,200 g for 60 min at 30°C and cultured at 5% CO2, 37°C. After 6 h, the medium was replaced with RPMI medium containing 10% FBS and 1% ABAM, and cultured for 48 h. Afterwards, cells from each well were obtained and cultured in RPMI medium containing 1 μg / mL puromycin and 10% FBS to select only cells containing the target plasmid. A single cell line stably expressing SA-GFP1-10 was selected from the constructed cell line through single cell selection.

[0390] Figure 8 shows the results of Western blotting to confirm the intracellular SA-GFP1-10 expression of selected single cell lines MDA-MB-231-SA-GFP1-10 and SW480-SA-GFP1-10 cells. To obtain cell lysates, cells were lysed by adding lysis buffer (10 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% SDS, 1 mM EDTA, Inhibitor cocktail (Sigma, 78440)) and boiling at 95 °C for 20 minutes, and the protein concentration in the lysate was quantified using a BCA protein assay kit (Pierce, 23225). 15 μg of each cell lysate was loaded, and the SDS-PAGE gel was transferred to a PVDF membrane and treated with blocking solution (4% BSA in 0.1% TBS-T) at room temperature for 1 hour. Afterwards, anti-flag M2 mouse monoclonal antibody (Sigma, F1804), which recognizes the flag tag fused to SA-GFP1-10, was diluted 1:1,000 in blocking solution as the primary antibody and treated at room temperature for 3 hours. As the primary antibody to confirm β-actin, anti-β-actin monoclonal antibody (Santa Cruz, 69879) was diluted 1:1,000 in blocking solution and treated at room temperature for 2 hours. After washing twice with TBS-T solution (0.1% Tween 20 in TBS) for 10 minutes each, anti-mouse IgG-HRP conjugated polyclonal antibody (Sigma, A9044) was diluted 1:4,000 in 0.1% TBS-T solution and treated at room temperature for 1 hour. After washing twice with 0.1% TBS-T solution for 10 minutes each, the target band was detected using West-Q Femto Clean ECL solution (GenDEPOT, W3680-010).Analysis was performed using ImageQuant LAS4000 mini (GE Healthcare). Target bands were detected in SW480-SA-GFP1-10 single cells (lane 1) and MDA-MB-231-SA-GFP1-10 single cells (lane 3), while no bands were detected in the wild-type SW480 (lane 2) and MDA-MB-231 (lane 4) cells, which were negative controls. This confirmed that the SA-GFP1-10 fragment protein was expressed in the constructed and selected single cell lines, and the cytoplasmic penetration ability of the CH3 domain mutants was evaluated using two cell lines, MDA-MB-231-SA-GFP1-10 and SW480-SA-GFP1-10.

[0391]

[0392] Example 7. Evaluation of cytoplasmic penetration ability of a cytoplasmic penetrating antibody (in2C1N) having an endosomal escape structural motif in the heavy chain constant region CH3 domain.

[0393] Figure 9 shows the results of observing and quantifying GFP fluorescence by complementary binding of improved split green fluorescent protein using a live cell imaging device, Lionheart Fx (Biotek), to evaluate the cytoplasmic penetration ability of a cytoplasmic penetrating antibody (in2C1N) having an endosomal escape structural motif in the CH3 domain.

[0394] Specifically, transfected MDA-MB-231-SA-GFP1-10 or SW480-SA-GFP1-10 cell lines stably expressing SA-GFP1-10 were cultured in RPMI medium containing 10% FBS and 1% ABAM. 2 × 10 per well in a 96-well black plate (Greiner, 655090). 3 dog (MDA-MB-231) or 4 × 10 3Dog (SW480) cells were aliquoted at 100 μL each and cultured for 48 hours under 5% CO2, 37°C conditions. After removing the supernatant, 500 nM concentrations of positive control inCT, negative control inCT (AAA), and CH3 domain variants were treated at 37°C for 12 hours. Afterwards, to reduce fluorescence signal interference due to FBS and phenol red contained in the existing medium, all medium in each well was removed, and 50 μL of FluoroBrite™ DMEM (Gibco, A1896701), a medium suitable for fluorescence imaging, was dispensed per well to replace the medium. Live cell images were analyzed in real time through the bright field channel and GFP channel of Lionheart Fx. The intracellular fluorescence intensity by CH3 domain mutants was quantified as the Mean GFP value, which is the sum of the total intracellular fluorescence intensity in the image divided by the total number of cells, using the Gen5 image analysis program (Biotek), and expressed as a relative value to the fluorescence intensity quantitative value of the inCT treatment group, which is the positive control group.

[0395] Figure 9a shows the results of evaluating the cytoplasmic penetration ability of six mutants, in2C11, in2C15, in2C16, in2C17, in2C18, and in2C19, compared with inCT at a concentration of 500 nM using MDA-MB-231-SA-GFP1-10 cells. The quantitative results showed that in2C11 and in2C19 showed approximately 2.2- and 2.4-fold enhanced cell-based fluorescence intensities compared to inCT, respectively, and exhibited superior cytoplasmic penetration ability compared to other mutants.

[0396] Figure 9b shows the results of the cytoplasmic penetration ability evaluation system in the above MDA-MB-231-SA-GFP1-10 cells, which were evaluated under the same conditions as Figure 11a using another cell line, SW480-SA-GFP1-10 cells, to show that the results are not cell line dependent. The quantitative results show that in2C11 and in2C19 showed cell-based fluorescence intensities that were approximately 2.2-fold and 2.4-fold enhanced, respectively, compared to inCT, and exhibited superior cytoplasmic penetration ability compared to other mutants. Since in2C11 and in2C19 showed the best endosomal escape ability in both cell lines, these two mutants were selected as lead substances.

[0397]

[0398] Example 8. Evaluation of concentration-dependent cytoplasmic penetration ability of cytoplasmic penetrating antibodies having an endosomal escape structural motif in the heavy chain constant region CH3 domain.

[0399] Figure 10 shows the results of observing GFP fluorescence by complementary binding of improved split green fluorescent protein in a concentration-dependent manner for two types of lead antibodies in2C11 and in2C19 selected through Example 7 among cytoplasmic penetrating antibody CH3 domain variants having an endosomal escape structural motif in the CH3 domain using Lionheart Fx (Biotek).

[0400] Specifically, 2 × 10 per well in a 96 well black plate (Greiner, 655090) under the same conditions as in Example 7. 3 dog (MDA-MB-231) or 4 × 10 3Dog (SW480) cells were aliquoted at 100 μL each and cultured for 48 hours under 5% CO2, 37°C conditions. After removing the supernatant, the positive control inCT, the negative control inCT (AAA), and two CH3 domain mutants were treated at concentrations of 500 nM, 100 nM, and 10 nM at 37°C for 12 hours. After removing all the medium in each well, 50 μL of FluoroBrite™ DMEM was dispensed per well to replace the medium, and the images of each well were analyzed in real time using the Bright field channel and GFP channel of Lionheart Fx while the cells were alive. As in Figure 9, the cell-based fluorescence intensity by the mutants was plotted as a relative value to the fluorescence intensity quantitative value of the positive control inCT 500 nM treatment group.

[0401] Through this, we confirmed the concentration-dependent cytoplasmic penetration ability of two types of leading antibodies, in2C11 and in2C19. At 10 nM, the cellular fluorescence intensity due to inCT and most of the mutants was very weak, making it difficult to distinguish the difference, but the fluorescence signals could be compared starting from a concentration of 100 nM, and in2C11 and in2C19 showed a higher level of fluorescence intensity at a concentration of 100 nM than the inCT 500 nM treatment group. Through this, we confirmed the concentration-dependent cytoplasmic penetration ability of two types of leading antibodies, in2C11 and in2C19.

[0402]

[0403] Example 9. Evaluation of pH-dependent interaction of a cytoplasmic penetrating antibody having an endosomal escape structural motif in the heavy chain constant region CH3 domain with the cell membrane.

[0404] For the cytoplasmic penetrating antibody of the present invention to ultimately reside in the cytoplasm after internalization, it must escape from the endosomes. Previous studies have shown that the endosomal escape ability of the existing cytoplasmic penetrating antibody inCT is due to pH-sensitive interactions between the membrane-binding motif located in CDR3 and residues in the framework region (Journal of Controlled Release, 2016, 235:165-175).

[0405] Since CH3 mutants harbor mutations in the CH3 domain that favor endosomal, slightly acidic environment-specific membrane interactions, we attempted to indirectly elucidate the pH-dependent membrane interactions and endosomal escape mechanisms of cytoplasmically penetrating antibodies using the same experimental methods as in previous studies. We assessed trypan blue dye uptake by Ramos human Burkitt's lymphoma cells, a non-adherent cell line lacking HSPG expression.

[0406] Figure 11 shows the results of a live cell imaging device, Lionheart Fx (Biotek), used in Ramos cells to confirm whether trypan blue without membrane permeability can be obtained by pore formation by a cytoplasmic penetrating antibody having an endosomal escape structure motif in the CH3 domain depending on pH.

[0407] Specifically, Ramos cell line was cultured in RPMI medium environment containing 10% FBS, 1% ABAM. Cell suspension containing a certain number of cells was prepared in 1 X HBSS buffer (Hank's Balanced Salt Solution (Sigma H4641), 50 mM HEPES, pH 7.4) with cytoplasmic pH of 7.4 and HBSS buffer (Hank's Balanced Salt Solution (Sigma H4641), 50 mM MES pH 5.5) with early endosomal pH of 5.5, and 5 × 10 per well in a 96-well plate (SPL, 30096). 4 Ramos cells were dispensed in 50 μL each. CH3 domain variants and control antibodies prepared at a concentration of 4 μM were treated in each well at 50 μL each, and finally, 2 μM concentration was treated in a volume of 100 μL at 37 °C for 2 hours. Trypan blue solution (0.4%, Gibco, 15250061) was added at a 2:1 ratio to stain, and the cells were centrifuged at 1,300 rpm for 3 minutes to attach to the bottom. This process was repeated twice, and carefully washed with PBS (pH 7.4). Afterwards, the trypan blue staining pattern of live Ramos cells was observed using a color bright field microscope of a live cell imaging device, Lionheart Fx (Biotek), in a PBS (pH 7.4) environment.

[0408] In Fig. 11, the number of cells that acquired trypan blue was quantitatively compared and presented in a graph. Specifically, the number of cells that acquired trypan blue among all cells was counted and presented as a percentage. The mean value (mean) was presented in the graph after counting more than 200 cells in total. It was confirmed that trypan blue was acquired in cells to which CH3 domain variants were added only under pH 5.5 conditions. In particular, in2C11 and in2C19, which have high cytoplasmic penetration ability and the same glutamic acid patch, showed a high trypan blue acquisition percentage of about 27% only at pH 5.5 (Fig. 17b). In addition, in2C11 without glutamic acid mutation W / O E In the case of , trypan blue acquisition of about 24% and 23% was observed at both pH 7.4 and pH 5.5, respectively. This confirmed that the endosomal escape structural motif of the CH3 domain according to the present invention can interact with the cell membrane specifically in a weakly acidic environment through the glutamic acid patch.

[0409]

[0410] Example 10. Design of a cytoplasmic penetrating antibody (in2C2N) having an endosomal escape structural motif in the light chain constant region CL domain.

[0411] Among the cytoplasmic penetrating antibodies (in2C1N) having an endosomal escape structural motif in the CH3 domain in Examples 1 to 9 above, two species, in2C11 and in2C19, were selected without problems in yield, physical properties, or non-specificity, and with improved cytoplasmic penetrating ability. Thereafter, as shown in Fig. 12, the same endosomal escape structural motif was introduced into the structurally corresponding position of the CL domain, which has a high structural similarity to the CH3 domain, to impart cytoplasmic penetrating ability to the CL domain as well.

[0412] At this time, the CL domain variant in which the endosomal escape structural motif was introduced into the CL domain was named in2C2N. Specifically, the CL domain variant in2C21 was constructed by grafting the endosomal escape structural motif of the in2C11 CH3 domain onto the CL domain. In addition, the CL domain variant in2C22 was constructed by grafting the endosomal escape structural motif of the in2C19 CH3 domain onto the CL domain.

[0413] When grafting the endosomal escape structural motif of the CH3 domain, amino acids expected to interact with the cell membrane in the wild-type CH3 domain that do not exist in the CL domain were introduced into the CL domain. For example, the wild-type residue R355 of the in2C19 CH3 domain forms an arginine-tryptophan cation-π interaction of R355-L358W, and the corresponding residue in the CL domain is D122, so the D122R mutation was added to in2C22.

[0414] As shown in Fig. 12, a CL domain variant was constructed based on the inCT(AAA) antibody, which has no cytoplasmic penetration ability, to evaluate whether it has cytoplasmic penetration ability through the endosomal escape structural motif of the CL domain. That is, a CL domain variant was constructed through a heavy chain including a heavy chain variable region (VH) of SEQ ID NO: 14 and a heavy chain constant region (CH1-hinge-CH2-CH3) having a CH3 domain of SEQ ID NO: 1, and a light chain including a light chain variable region (VL) of SEQ ID NO: 16 and a light chain constant region (CL) having endosomal escape structural motifs of SEQ ID NOs: 18 and 19.

[0415] Additionally, as in Example 1, cytoplasmic penetrating antibodies inCT and inCT(AAA) were co-expressed and purified and used as controls for the evaluation of physical properties and cytoplasmic penetrating ability. The amino acid sequences of specific CL domain variants are specified in Table 6 below.

[0416]

[0417] Figure 13 shows the 3D structure of a Fab region containing a CL domain variant in which the endosomal escape structural motif of a CH3 domain variant is grafted onto the CL domain, modeled using Alphafold2, and the intramolecular interaction patterns analyzed using the Pymol molecular visualizer.

[0418] Additionally, to analyze the structural differences between each CL domain variant and the CH3 domain having the same endosomal escape structural motif, the root mean square deviation (RMSD) of the CL domain variants with respect to the CH3 domain variants having the same endosomal escape structural motif was calculated. Specifically, the wild-type CL domain was compared with the wild-type CH3 domain, the CL domain of in2C21 was compared with the CH3 domain of in2C11, and the CL domain of in2C22 was compared with the CH3 domain of in2C19.

[0419]

[0420] Through the above Table 7, it was confirmed that both in2C21 and in2C22 had similar RMSD values ​​(1.425 Å) between the wild-type CH3 domain and the wild-type CL domain, which confirmed that the structure of the CL domain was maintained when the endosomal escape structural motifs of the CH3 domain mutants were grafted to the corresponding positions of the CL domain.

[0421] When constructing an endosomal escape structural motif in the CL domain by introducing the endosomal escape structural motif of the CH3 domain variants into the corresponding position of the CL domain, smooth interaction with the cell membrane can be expected only when the endosomal escape structural motif is exposed on the antibody surface, just like the CH3 domain. Therefore, the 3D structures of the CL domain variants modeled using Alphafold2 (Figure 16) were input into GETAREA to obtain the solvent accessible surface area ratio (%), which is specified in Table 8 below. Through this, it was confirmed that, just like the CH3 domain, most of the positions where the endosomal escape structural motifs of the AB, EF, and C-terminal loops of the CL domain were introduced were exposed on the surface.

[0422]

[0423] The following paragraphs detail the endosomal escape structural motifs of the CL domain variants.

[0424]

[0425] The endosomal escape structural motif of in2C21 of SEQ ID NO: 18 consists of two tryptophan mutations (D122W, E187W), one arginine mutation (L125R), one alanine mutation (K183A), and three glutamic acid mutations (G128E, T129E, S182E) in the AB loop, the EF loop, and the C-terminal loop.

[0426] CL domains 122, 125, 128, 129, 182, 183, and 187, into which each mutation was introduced, correspond to CH3 355, 358, 361, 362, 413, 414, and 418 in the tertiary structure (Fig. 2b, Fig. 13b).

[0427] For arginine and tryptophan mutations, the mutations (R355W, L358R, Q418W) introduced in in2C11 were identically introduced into CL so that the amino acid interactions induced in2C11 appeared in the CL domain. In the case of L443R introduced in in2C11, the wild-type sequence was preserved because the corresponding position (spot 211) in the wild-type CL is already arginine. Through this, as shown in Fig. 13b, the cation-π interaction formed by R355W, L358R, Q418W, and L443R in in2C11 was also formed in in2C21 through D122W, L125R, E187W, and R211. To prevent steric hindrance caused by Q418W in in2C11, the surrounding lysine was substituted with alanine (K414A). Similarly, in in2C21, to prevent steric hindrance caused by E187W, the lysine in the structurally corresponding position was substituted with alanine (K183A). In addition, the glutamic acid patch (N361E, Q362E, D413E), which was introduced to impart pH dependence to in2C11, was introduced in the same structurally corresponding position in the CL domain (G128E, T129E, S182E). As in in2C11, in the glutamic acid patch where glutamic acid is densely packed in adjacent positions, the pKa of T129E was 5.18, and the pKa of S182E was 4.9, confirming that the pKa of glutamic acid increases.

[0428] Therefore, in2C21 mimics the endosomal escape structural motif of in2C11, and two pairs of arginine and tryptophan that form cation-π interactions are densely packed at the bottom of the antibody CL domain, and a glutamic acid patch located peripherally is introduced on the side of the antibody CL domain to impart cytoplasmic penetration ability and pH dependency.

[0429]

[0430] The endosomal escape structural motif of in2C22 of SEQ ID NO: 19 consists of four tryptophan mutations (E123W, L125W, E187W, K207W), two arginine mutations (D122R, K126R), one alanine mutation (K183A), and three glutamic acid mutations (G128E, T129E, S182E) in the AB loop, EF loop, and C-terminal loop.

[0431] CLs 122, 123, 125, 126, 128, 129, 182, 183, 187, and 207, into which each mutation was introduced, correspond to CH3 355, 356, 358, 359, 361, 362, 413, 414, 418, and 439 in the tertiary structure (Fig. 2l, Fig. 13c).

[0432] For arginine and tryptophan mutations, the mutations introduced in in2C19 (D356R, L358W, T359R, Q418W, K439W) and the sequence of wild-type CH3 (R355) were introduced into CL so that the amino acid interaction induced in2C19 would appear in the CL domain. In this case, in the case of L443R of in2C19, the wild-type sequence was preserved because the corresponding position (211) in the wild-type CL is already arginine.

[0433] Through this, the cation-π interaction formed by R355, L358W, T359R, Q418W, L443R in in2C19 was also formed in in2C22 through D122R, L125W, K126R, E187W, K207W, R211. At this time, in in2C19, D356R forms a cation-π interaction with the opposite CH3 K439W at the CH3-CH3 interface, and in in2C22, in a similar manner, E123W formed a cation-π interaction with CH1 K213 at the CL-CH1 interface. In addition, to prevent steric hindrance due to Q418W in in2C19, the surrounding lysine was substituted with alanine (K414A), and in in2C22, to prevent steric hindrance due to E187W, the lysine in the structurally corresponding position was substituted with alanine (K183A).

[0434] In addition, the glutamic acid patches (N361E, Q362E, D413E) introduced to impart pH dependence to in2C19 were introduced identically to the structurally corresponding positions in the CL domain (G128E, T129E, S182E). This is identical to the glutamic acid patch of in2C22, and it was confirmed that the pKa of T129E increased to 5.14 and the pKa of S182E increased to 4.9.

[0435]

[0436] Example 11. Expression and purification of a cytoplasmic penetrating antibody (in2C2N-GFP11-SBP2) having an endosomal escape structural motif in the light chain constant region CL domain.

[0437] To identify biophysicochemical properties of CL domain mutants and evaluate their endosomal escape ability through the complementary binding system of improved split green fluorescent protein, we constructed a CL domain mutant, in2C2N-GFP11-SBP2, in which GFP11-SBP2 was fused to the C-terminus of the antibody heavy chain. Similar to the previous CH3 domain mutants, a circular peptide (in4, DGVRQCRGDCFDGPL) targeting integrin αvβ3 / αvβ5, a type of membrane protein receptor overexpressed on the surface of tumor cells / tissues, was fused to the N-terminus of the light chain to induce cellular internalization through tumor cell / tissue-specific receptors.

[0438] Specifically, to construct a heavy chain expression vector for producing a monoclonal antibody in the form of a full IgG, the heavy chain variable region (VH, SEQ ID NO: 14) of the antibody fused with a DNA encoding a secretory signal peptide at the 5' end and the DNA encoding a heavy chain including CH1-hinge-CH2-CH3 having a wild-type CH3 domain (SEQ ID NO: 1) were each cloned into the pcDNA3.4 vector using NotI / HindIII. At this time, to construct a cytoplasmic penetrating antibody fused with GFP11-SBP2, GFP11-(G4S)3-SBP2 was fused to the heavy chain C-terminus using a linker of three GGGS units. In addition, to construct a vector expressing the light chain, DNA encoding the light chain, including the antibody light chain variable region (VL, SEQ ID NO: 16) and light chain constant region (CL, SEQ ID NO: 18 or 19) fused to DNA encoding a secretory signal peptide at the 5' end, was cloned into the pcDNA3.4 vector using NotI / HindIII.

[0439] The cytoplasmic penetrating antibody (in2C2N-GFP11-SBP2) was expressed and purified by transient transfection of a common heavy chain expression vector containing the VH of SEQ ID NO: 14 and the wild-type CH3 domain of SEQ ID NO: 1 and a light chain expression vector containing the VL of SEQ ID NO: 16 and the light chain constant region sequence of SEQ ID NO: 18 or 19.

[0440] In a shake flask, transfection was performed using a mixture of HEK293-F cells (Invitrogen) and polyethylenimine (PEI) (Polyscience, 24765-1) suspended in serum-free FreeStyle 293 expression medium (Invitrogen) using the same method as in Example 3. In addition, purification and protein quantification of the CL variants were also performed in the same manner as in Example 3.

[0441]

[0442] The production yields of specific CL domain mutants are shown in Table 9 above. in2C22-GFP11-SBP2 had a very low yield, and in2C21-GFP11-SBP2 showed a yield of about 40% less than that of the wild-type inCT(AAA)-GFP11-SBP2, but showed a respectable yield of more than 30 mg per liter.

[0443] Hereinafter, a cytoplasmic penetrating antibody (in2C2N-GFP11-SBP2) fused with a prototypical peptide targeting a membrane protein receptor overexpressed on the surface of tumor cells and GFP11-SBP2 is described as in2C2N. For example, in2C21-GFP11-SBP2 is in2C21.

[0444]

[0445] Example 12. Physical properties evaluation of a cytoplasmic penetrating antibody (in2C2N) having an endosomal escape structural motif in the light chain constant region CL domain.

[0446] Figure 14 shows the physical properties of two types of in2C21 and in2C22, purified together with the control antibody inCT (AAA), through 12% SDS-PAGE and size exclusion chromatography under reducing or non-reducing conditions in the same manner as in Example 4. Specifically, it was confirmed through SDS-PAGE that both types of in2C21 and in2C22 had a molecular weight of about 162 to 163 kDa under non-reducing conditions like the control antibody, and showed a molecular weight of about 55 kDa for the heavy chain (about 50 kDa for the antibody heavy chain and about 5 kDa for the GFP11-SBP2 fragment) and about 26.5 kDa for the light chain (about 25 kDa for the antibody light chain and 1.5 kDa for the in4 circular peptide) under reducing conditions. This demonstrates that the purified in4 circular peptide and the cytoplasmic penetrating antibody fused with GFP11-SBP2 exist as a single entity in solution and do not form dimers or oligomers through unnatural disulfide bonds. Furthermore, when the mAU value at 280 nm was measured using size exclusion chromatography (Superdex 200 increased 10 / 300GL, Cytiva), in2C21 and in2C22 were observed as single peaks without oligomerization or tailing, similar to the control Trastuzumab.

[0447]

[0448] Example 13. Evaluation of cytoplasmic penetration ability of a cytoplasmic penetrating antibody (in2C21) having an endosomal escape structural motif in the light chain constant region CL domain.

[0449] Figure 15 shows the results of observing and quantifying GFP fluorescence by complementary binding of improved split green fluorescent protein using a live cell imaging device, Lionheart Fx (Biotek), to evaluate the cytoplasmic penetration ability of the CL domain mutant in2C21.

[0450] Specifically, in the transfected MDA-MB-231-SA-GFP1-10 cell line stably expressing SA-GFP1-10, antibody treatment was performed at concentrations of 100 nM and 500 nM in the same manner as in Example 7, and then the cell images were analyzed in real time using the Bright field channel and GFP channel of Lionheart Fx in a living cell state. The intracellular fluorescence intensity by CH3 or CL domain variants was quantified using the Gen5 image analysis program (Biotek) in the same manner as in Example 7 and presented in a graph. The experimental results showed that in2C21 showed a cell-based fluorescence intensity that was approximately 1.0 times that of inCT, i.e., the same level as the positive control. in2C21 showed a lower cytoplasmic penetration ability than in2C11, which is thought to be because the CH3 domain variant has an avidity effect as a CH3-CH3 dimer, whereas the CL domain variant introduced a mutation into a single molecule. Consequently, we confirmed that when the endosomal escape structural motif introduced into the CH3 domain was grafted to the corresponding position of the CL domain, the cytoplasmic penetration ability could be achieved by the CL domain. In addition, we confirmed that the cytoplasmic penetration ability was lower when the endosomal escape structural motif of the same composition and position was introduced into the CL domain singleton than when it was introduced into the CH3 domain doubleton.

[0451]

[0452] Example 14. Expression and purification of a cytoplasmic penetrating antibody (in2C4N-GFP11-SBP2) having an endosomal escape structural motif in the heavy chain constant region CH3 domain and the light chain constant region CL domain.

[0453] To design a cytoplasmic penetrating antibody having high cytoplasmic penetration ability through the CH3 and CL domains by introducing an endosomal escape structural motif into both the CH3 and CL domains, the CH3 domain variants and CL domain variants selected through the preceding Examples 1 to 13 were combined. As shown in Fig. 18, the cytoplasmic penetrating antibody having an endosomal escape structural motif introduced into the CH3 and CL domains was named in2C4N.

[0454] To characterize the biophysicochemical properties of CH3 and CL domain variants (in2C4N) and to evaluate their cytoplasmic penetration potential via the improved complementary binding system of split green fluorescent protein, in2C4N-GFP11-SBP2 was constructed by fusion of GFP11-SBP2 to the C-terminus of the antibody heavy chain. Similar to the construction of the previous CH3 domain variants or CL domain variants, a circular peptide (in4, DGVRQCRGDCFDGPL) targeting integrin αvβ3 / αvβ5, a type of membrane protein receptor overexpressed on the surface of tumor cells / tissues, was fused to the N-terminus of the light chain to induce cellular internalization via tumor cell / tissue-specific receptors.

[0455] Specifically, among the CH3 domain variants evaluated through Figures 9a and 9b of Example 7, four variants (in2C11, in2C17, in2C18, in2C19) with enhanced cytoplasmic penetration ability and two CL domain variants (in2C21, in2C22) were combined to express and purify eight CH3 and CL domain variants having endosomal escape structural motifs in both the CH3 and CL domains as full IgG monoclonal antibodies. The designation of CH3 and domain variant in2C4N is shown in Table 10 below.

[0456]

[0457] Specifically, in the case of in2C41-GFP11-SBP2, it was expressed using a heavy chain expression plasmid containing CH1-hinge-CH2-CH3 having the VH of SEQ ID NO: 14 and the CH3 of in2C11 of SEQ ID NO: 4, and a light chain expression plasmid having the VL of SEQ ID NO: 16 and the CL of SEQ ID NO: 18. In the other cases, it was expressed using a heavy chain expression vector containing each CH3 domain variant and a light chain expression vector containing each CL domain variant. Their expression in animal cells was carried out in HEK293-F cells in the same manner as in Example 3.

[0458] Thereafter, purification and protein quantification of CH3 and CL domain variants were performed in the same manner as in Example 3. The specific production yields of eight types of in2C4N-GFP11-SBP2 are shown in Table 13 above. Among the eight types of CH3 and CL domain combination antibodies, three types, in2C41, in2C42, and in2C44, showed satisfactory yields of more than 20 mg per liter.

[0459] Hereinafter, a cytoplasmic penetrating antibody (in2C4N-GFP11-SBP2) fused with a prototypical peptide targeting a membrane protein receptor overexpressed on the surface of tumor cells and GFP11-SBP2 is described as in2C4N. For example, in2C41-GFP11-SBP2 is in2C41.

[0460]

[0461] Example 15. Physical property evaluation of a cytoplasmic penetrating antibody (in2C4N) having an endosomal escape structural motif in the heavy chain constant region CH3 domain and the light chain constant region CL domain.

[0462] Figure 17 shows the results of analyzing eight CH3 and CL domain variants having endosomal escape structural motifs in the CH3 domain and CL domain using 12% SDS-PAGE under reducing or non-reducing conditions and size-exclusion chromatography after purification together with the control antibody inCT (AAA). Specifically, all eight CH3 and CL domain variants were confirmed to have a molecular weight of approximately 162 to 163 kDa under non-reducing conditions, like the control antibody, and showed a molecular weight of approximately 55 kDa for the heavy chain (approximately 50 kDa for the antibody heavy chain and approximately 5 kDa for the GFP11-SBP2 fragment) and approximately 26.5 kDa for the light chain (approximately 25 kDa for the antibody light chain and 1.5 kDa for the in4 circular peptide) under reducing conditions. In the case of in2C43, in2C45, in2C46, and in2C47, the heavy chain showed double bands rather than a single band under reducing conditions. It was confirmed that the remaining four CH3 and CL domain mutants, excluding the above four, existed as a single entity in solution and did not form duplexes or oligomers through unnatural disulfide bonds. The graph on the right side of Figure 17 shows the mAU values ​​at 280 nm measured in the same manner as in Example 4 using size exclusion chromatography (Superdex 200 increased 10 / 300GL, Cytiva) for three CH3 and CL domain mutants (in2C41, in2C42, and in2C44) that showed a good yield of more than 10 mg per liter. In the three mutants, a single peak was observed without oligomers or tailing, as in the control group Trastuzumab and inCT (AAA).

[0463] Therefore, further evaluation was conducted on the three CH3-CL domain variants (in2C41, in2C42, in2C44) with satisfactory production yields of more than 10 mg per liter [Table 10] and similar properties to the control wild-type antibody.

[0464]

[0465] Example 16. Nonspecific binding evaluation of a cytoplasmic penetrating antibody (in2C4N) having an endosomal escape structural motif in the heavy chain constant region CH3 domain and the light chain constant region CL domain.

[0466] Figure 18 shows the results of ELISA for four antigens, dsDNA, Insulin, Keyhole limpet hemocyanin (KLH), and Cardiolipin, which can confirm nonspecific binding of antibodies, to confirm nonspecific binding of three lead antibodies (in2C41, in2C42, and in2C44) having endosomal escape structural motifs in the CH3 and CL domains. The experiment was performed in the same manner as Example 5. In the experiment, nonspecific binding was determined when an absorbance of 0.05 or higher at 450 nm was shown for at least one of the four antigens at a concentration of 100 nM. The experimental results showed that in2C42, unlike the control and the other two mutants, showed nonspecific binding to the dsDNA antigen at concentrations of 100 nM and 500 nM. Therefore, the other two mutants, excluding in2C42, were selected as candidates.

[0467] Figure 19 shows the results of flow cytometry analysis to confirm non-specific binding to cell membranes for three lead antibodies (in2C41, in2C42, in2C44) having endosomal escape structural motifs in the CH3 and CL domains. The experiment was performed in the same manner as in Example 5. The experimental results confirmed that in2C42 and in2C44 showed non-specific binding to the cell surfaces of K-562 and CHO-K1 compared to the wild-type negative control. Therefore, among the mutants having endosomal escape structural motifs in the CH3 and CL domains, in2C41, which had good physical properties, was selected as the final material for the CH3-CL mutant.

[0468]

[0469] Example 17. Evaluation of cytoplasmic penetration ability of cytoplasmic penetrating antibodies having endosomal escape structural motifs in the heavy chain constant region CH3 domain and the light chain constant region CL domain.

[0470] Figure 20 shows the results of observing and quantifying GFP fluorescence by complementary binding of improved split green fluorescent protein to evaluate the cytoplasmic penetration ability of in2C41 using a live cell imaging device, Lionheart Fx (Biotek). In the same manner as in Example 7, MDA-MB-231-SA-GFP1-10 cells were treated with a cytoplasmic penetrating antibody at a concentration of 500 nM for 12 hours, and then the cell images were analyzed in real time in a living cell state using the Bright field channel and GFP channel of Lionheart Fx. The intracellular fluorescence intensity by CH3 and CH3-CL mutants was quantified using the Gen5 image analysis program (Biotek) in the same manner as in Example 7, and the fluorescence intensity was expressed in a graph.

[0471] When the cytoplasmic penetration abilities of in2C11, in2C21, and in2C41 treated with 100 nM or 500 nM concentrations in MDA-MB-231-SA-GFP1-10 cells were compared, in2C41 showed a fluorescence intensity approximately 2.6 times that of inCT, and in2C11 and in2C21 showed a fluorescence intensity approximately 2.2 times that of inCT, and in2C21 showed a fluorescence intensity approximately 1.1 times that of inCT, similar to Example 14. Through this, it was confirmed that in2C41, which has an endosomal escape structural motif in the constant region CH3 domain and CL domain, had the highest cytoplasmic penetration ability among the cytoplasmic penetration antibodies evaluated.

[0472]

[0473] Example 18. Evaluation of GFP fluorescence according to concentration-dependent cytoplasmic penetration of a cytoplasmic penetrating antibody having an endosomal escape structural motif in the heavy chain constant region CH3 domain and the light chain constant region CL domain.

[0474] Figure 21 shows the cytoplasmic penetration ability of in2C41 according to the concentration, observed by confocal microscopy using a complementary binding system of an improved split green fluorescent protein.

[0475] Specifically, MDA-MB-231-SA-GFP1-10 cells constructed in Example 6 were cultured in RPMI medium containing 10% FBS and 1% ABAM. A coverslip (Marienfeld, 01-115-20) with a radius of 12 mm was placed in a well of a 24-well plate (SPL, 30024), and 500 μL of poly-L-lysine solution (Sigma, P4707) was dispensed and coated. After washing twice with sterile water, the plate was sufficiently dried at room temperature for more than 2 hours. 1 × 10 cells were seeded per well in the prepared plate. 5MDA-MB-231-SA-GFP1-10 cells were aliquoted with 500 μL each and cultured for 24 h at 37°C in a 5% CO2 atmosphere. After removing the supernatant, the cells were treated with GFP11-SBP2-fused in2C41 and control antibodies (inCT(AAA), inCT) at various concentrations (1, 10, 50, 100, and 500 nM) at 37°C for 12 h. The supernatant was then removed, washed twice with PBS (pH 7.4), and acid-washed twice with low-pH glycine buffer (200 mM glycine, 150 mM NaCl, pH 2.5) for 30 s each. After washing twice with PBS, the cells were fixed with 4% paraformaldehyde (Thermo Scientific™, 28908) solution at room temperature for 10 min. After washing three times with PBS, nuclei were stained (blue fluorescence) for 5 minutes with Hoechst 33342 (Thermo Scientific™, 62249). After washing three more times with PBS, the cover glass in the well was removed with tweezers and mounted on a glass slide (Matsunami, HMA-APS-11) using mounting medium (Dako, S3023) for sampling. Each sample was observed using a confocal microscope (Leica, Stellaris 5).

[0476] In Fig. 21, the intensity of GFP fluorescence observed from the cytoplasm was quantified as a change in magnitude relative to the negative control, inCT (AAA), and is presented in a graph. As shown in Fig. 20, in2C41 showed a fluorescence intensity approximately 2.6 times that of inCT, and GFP fluorescence was observed even at a lower concentration of 10 nM compared to the positive control, inCT.

[0477]

[0478] Example 19. Quantitative endosomal escape efficiency evaluation of cytoplasmic penetrating antibodies via complementary binding of improved split green fluorescent protein.

[0479] Table 12 below shows the endosomal escape efficiency of cytoplasmic penetrating antibodies (inCT, in2C41) according to concentration using the complementary binding system of the improved split green fluorescent protein.

[0480]

[0481] Specifically, the cytoplasmic penetration efficiency quantification system was developed in a previous study (BBRC, 2015;467(4):771-777) using a complementary binding system of an improved split green fluorescent protein and Western blot to quantify the cytoplasmic penetration efficiency according to the antibody concentration (0.1, 0.5, 1 μM).

[0482] The cytoplasmic penetration efficiency of the two cytoplasmic penetrating antibodies increased in proportion to their concentration. In particular, the cytoplasmic penetration efficiency of the cytoplasmic penetrating antibody in2C41 developed in the present invention was confirmed to be approximately three times higher than that of the cytoplasmic penetrating antibody inCT, used as a positive control.

[0483]

[0484] Example 20. Expression and purification of HiBiT fusion antibodies utilizing the complementary binding system of split nanoluciferase luminescent proteins.

[0485] For cytoplasmic penetrating antibodies with cytoplasmic penetration ability through the endosomal escape structural motif of the constant region, the cytoplasmic penetration ability and endosomal escape efficiency were evaluated using another cytoplasmic penetration ability evaluation system, the complementary binding system of split nanoluciferase luminescent protein (Nature Communications, 2021, 12(1):3721). This was intended to be compared with the results of cytoplasmic penetration ability evaluation using the improved complementary binding system of split green fluorescent protein.

[0486] Figure 22 is a schematic diagram illustrating the process of bioluminescence emitted by complementary binding of a split nanoluciferase luminescent protein when a cytoplasmic penetrating antibody is located in the cytoplasm. By measuring the luminescence emitted through complementary binding of LgBiT, which is expressed by being fused to cytoplasmic actin, and HiBiT, which is fused to the C-terminus of the heavy chain of the cytoplasmic penetrating antibody, the endosomal escape efficiency of the cytoplasmic penetrating antibody can be quantified.

[0487] Figure 23 illustrates the process for quantifying the endosomal escape efficiency of the complementary binding system of the split nanoluciferase luminescent protein. The live signal, which is the luminescence caused by the cytosolically delivered antibody, is measured, and to exclude the luminescence caused by the extracellularly secreted LgBiT, the supernatant from each well is measured and the media signal is subtracted from the live signal to obtain the cytosolic signal (hereinafter referred to as cytosolic delivery). In addition, the association signal (hereinafter referred to as total cellular association), which is the luminescence of the entire cell caused by the cytosolically delivered antibody and the endosomal entrapped antibody through cell membrane permeabilization by treating with digitonin, is measured. Through this, the endosomal escape efficiency can be expressed as a percentage by dividing the measured cytosolic delivery value by the total cellular association value.

[0488] To utilize the above system, a cytoplasmic penetrating antibody was constructed by fusing HiBiT (VSGWRLFKKIS) to the heavy chain C-terminus, which has cytoplasmic penetrating ability through the endosomal escape structural motif of the constant region.

[0489] Specifically, to construct heavy chain expression vectors for producing inCT-HiBiT, inCT(AAA)-HiBiT, and in2C11-HiBiT, in2C21-HiBiT, and in2C41-HiBiT as monoclonal antibodies in the form of full IgG, DNA encoding the heavy chain variable region (VH, SEQ ID NO: 14) of the antibody fused with DNA encoding a secretory signal peptide at the 5'-terminus and DNA encoding the heavy chain containing CH1-hinge-CH2-CH3 having a CH3 domain (SEQ ID NO: 1 or 4) were each cloned into the pcDNA3.4 vector using NotI / HindIII. At this time, HiBiT was fused using a GSSG three-linker at the heavy chain C-terminus. In the case of the light chain, the light chain DNA including the light chain variable region (VL, SEQ ID NO: 16) and the light chain constant region (CL, SEQ ID NO: 17) constructed in Example 3 or the light chain DNA including the light chain variable region (VL, SEQ ID NO: 16) and the light chain constant region (CL, SEQ ID NO: 18) constructed in Example 12 was used.

[0490] In the case of inCT(AAA)-HiBiT, inCT-HiBiT, and in2C11-HiBiT, the heavy chain expression vector including the VH of SEQ ID NO: 14 and the CH3 domain of SEQ ID NO: 1 or 4 and the common light chain expression vector including the VL of SEQ ID NO: 16 and the CL domain of SEQ ID NO: 17 were transiently transfected together, and then expressed and purified. In the case of in2C41-HiBiT, the heavy chain expression vector including the VH of SEQ ID NO: 14 and the CH3 domain of SEQ ID NO: 4 and the common light chain expression vector including the VL of SEQ ID NO: 16 and the CL domain of SEQ ID NO: 18 were transiently transfected together, and then expressed and purified. Their expression in animal cells was performed in HEK293-F cells in the same manner as in Example 3.

[0491] Thereafter, purification and protein quantification of the cytoplasmic penetrating antibody fused with HiBiT were performed in the same manner as in Example 3, and their production yields in animal cells are shown in Table 15 below. in2C41, in which HiBiT having an endosomal escape structural motif in the constant region CH3 and CL domains was fused, showed a yield of approximately 52.5% compared to wild-type inCT(AAA)-HiBiT, but a satisfactory yield of more than 20 mg per liter was observed.

[0492]

[0493] Figure 24 shows the cytoplasmic penetrating antibodies having endosomal escape structural motifs in the constant region CH3 and / or CL domains fused to HiBiT, purified and analyzed by 12% SDS-PAGE and size exclusion chromatography under reducing or non-reducing conditions. Specifically, all three antibodies (in2C11-HiBiT, in2C21-HiBiT, in2C41-HiBiT) were confirmed to have a molecular weight of approximately 154 kDa under non-reducing conditions, and showed a molecular weight of the heavy chain of approximately 51 kDa (antibody heavy chain approximately 51 kDa and HiBiT fragment approximately 3 kDa) and a molecular weight of the light chain of approximately 26.8 kDa (antibody light chain approximately 25.3 kDa and in4 circular peptide 1.5 kDa) under reducing conditions. When the mAU value at 280 nm was measured using size exclusion chromatography (Superdex 200 increased 10 / 300GL, Cytiva) in the same manner as in Example 3, a single peak was observed for all three antibodies (in2C11-HiBiT, in2C21-HiBiT, in2C41-HiBiT) without any special oligomerization or tailing, like the control group Trastuzumab and inCT-HiBiT.

[0494] Through this, it was confirmed that the cytoplasmic penetrating antibody having an endosomal escape structural motif in the constant region fused to HiBiT had a satisfactory production yield of more than 20 mg per liter [Table 13] and similar properties to the control wild-type antibody.

[0495]

[0496] Example 21. Evaluation of endosomal escape efficiency of HiBiT fusion antibodies using a complementary binding system of split nanoluciferase luminescent proteins.

[0497] Figure 25 shows the quantification of the endosomal escape efficiency at a concentration of 500 nM in HEK293T cells through a complementary binding system of split nanoluciferase luminescent proteins for cytoplasmic penetrating antibodies (in2C11-HiBiT, in2C21-HiBiT, in2C41-HiBiT) having an endosomal escape structural motif in the constant region fused to HiBiT.

[0498] Specifically, a transfected HEK293T-LSA cell line stably expressing LgBit-SNAP-Actin (LSA) was provided by Monash University (Australia). The HEK293T-LSA cell line was cultured in a DMEM medium environment containing 10% FBS and 1% ABAM. 5 × 10 cells were seeded per well in a 96-well white plate (Greiner, 655098). 3 The cells were aliquoted at 100 μL each and cultured for 24 hours under 5% CO2, 37°C. After removing the supernatant, the positive control inCT-HiBiT, the negative control inCT(AAA)-HiBiT, and three cytoplasmic penetrating antibodies with endosomal escape structural motifs in the constant region (in2C11-HiBiT, in2C21-HiBiT, in2C41-HiBiT) at a concentration of 500 nM were treated at 37°C for 12 hours.

[0499] At this time, GFP-HiBiT and pHD118-GFP-HiBiT, which have been reported to have endosomal escape efficiency in the complementary binding system of the above-mentioned split luciferase luminescent protein, were evaluated together as another control. pHD118 is an endosomal escape peptide (EEP) reported to have pH-dependent endosomal escape ability (Nature Communications, 2021, 12(1):3721).

[0500] Afterwards, to reduce interference by phenol red contained in the existing medium, the supernatant was removed and the cells were washed three times with DMEM (Cytiva, SH30585.02, 10% FBS) that does not contain phenol red. For measuring the total cellular association signal, the wells were permeabilized by treating the cells with 75 μL of digitonin solution (Millipore, 300410-250MGCN, 0.01% w / v in DMSO) at 37 °C for 1 hour before substrate treatment. Similarly, the wells for measuring cytosolic delivery were washed three times with DMEM (Cytiva, SH30585.02, 10% FBS) that does not contain phenol red, and then 75 μL of new medium was added. For measuring the total cellular association signal after permeabilization with digitonin and for measuring cytosolic delivery (live signal and media signal), 25 μL of substrate fumarizine (Promega, N2012) was added simultaneously to the wells, and incubated for 10 minutes on an orbital shaker at room temperature. Then, luminescence was measured using a plate reader Cytation 3 (Biotek, USA) to obtain the live signal and total cellular association signal. Then, 50 μL of the supernatant from each well where the live signal was measured was carefully transferred to a new plate, and luminescence was measured to obtain the media signal. The measured values ​​were calculated as shown in Figure 25, and the quantification results of the endosomal escape efficiency are shown in Figure 25.

[0501] In HEK293-LSA cells, the endosomal escape efficiency of the conventional cytoplasmic penetrating antibody inCT was approximately 4.2%, and that of inCT (AAA), which has no cytoplasmic penetration ability, was approximately 1.9%. The endosomal escape efficiencies of in2C11 were approximately 8.8%, in2C21 approximately 5.3%, and in2C41 approximately 12.1%. These are 2.0-, 1.3-, and 2.8-fold higher than the endosomal escape efficiency of inCT, respectively. GFP and pHD118-GFP showed low endosomal escape efficiencies of 1-2%, similar to the endosomal escape efficiencies reported in the literature.

[0502] Through the above experiments, the reliability of the evaluation system was confirmed by the fact that the quantitative results of the cytoplasmic penetration ability through the complementary binding system of the improved split green fluorescent protein corresponded to the quantitative results of the endosomal escape efficiency through the complementary binding system of the split nanoluciferase. In addition, all five antibodies treated with HEK293-LSA showed similar values ​​of total cellular association, indicating that the five antibodies associated and internalized into HEK293-LSA cells at similar levels. This confirmed that the increased endosomal escape efficiency of the cytoplasmic penetrating antibodies in2C11, in2C21, and in2C41 was due to the increased cytosolic delivery of these antibodies, not to the total cellular association.

[0503]

[0504] Example 22. Identification of the transport pathway after cell internalization of a cytoplasmic penetrating antibody having an endosomal escape structural motif in the heavy chain constant region CH3 domain and the light chain constant region CL domain.

[0505] Figure 26 shows the results of a confocal microscope observation using a pulse-chase experiment to observe the intracellular transport pathway of in2C41 that has entered the cell, showing the overlap of in2C41 with several intracellular organelles (early endosomes, late endosomes / lysosomes, endoplasmic reticulum, Golgi).

[0506] Specifically, 1 × 10 per well was added to a lysine-coated plate prepared in the same manner as in Example 18. 5MDA-MB-231 cells were aliquoted in 500 μL each and cultured for 24 hours under 5% CO2, 37°C conditions. The supernatant was removed, and in2C41 was treated at a concentration of 2 μM at 37°C for 30 minutes. After washing with PBS and acid wash, the cells were cultured in an RPMI medium environment containing 10% FBS and 1% ABAM for 0, 2, 6, 12, and 18 hours at 37°C, respectively. After removing the supernatant, PBS washing and cell fixation were performed, and the cells were permeabilized by treating them with 1 X PERM buffer, which is a 1 X dilution of permeabilization buffer (10 X) (Invitrogen, 00-8333-56) in 1% BSA (1% BSA in PBS) solution, for 10 minutes at room temperature. Afterwards, blocking solution (2% BSA in PBS) was treated at room temperature for 2 hours to prevent nonspecific binding of staining antibodies. Early endosomes, endoplasmic reticulum, and Golgi were primarily labeled by treating with anti-EEA1 mouse IgG (Santa Cruz, sc-53939), anti-calnexin mouse IgG (Santa Cruz, sc-70481), and anti-58K Golgi mouse IgG (Santa Cruz, sc-58770) at 25 °C for 4 hours, respectively. Among intracellular organelles, late endosomes / lysosomes were treated with LysoTracker Red DND-99 (Invitrogen, L7528) diluted to 1 μM in the medium before the cell fixation step, and treated at 37 °C for 30 minutes, followed by the same cell fixation, cell permeabilization, and blocking processes. Afterwards, the intracellular in2C41 and intracellular organelles primarily labeled with mouse antibodies were secondarily labeled with FITC (green fluorescent substance)-linked anti-human Fc antibody (Sigma, F9512) and Alexa Fluor 555 (red fluorescent substance)-linked anti-human Fc antibody (Invitrogen, A212422), respectively. Afterwards, PBS washing, nuclear staining, sampling, and observation were performed using a confocal microscope in the same manner as in Example 18.

[0507] As shown in Fig. 26, after treatment with in2C41 for 30 min, in2C41 was found to be spread throughout the cytoplasm in cells cultured for 0 and 2 h, with some strongly overlapping with EEA1 and relatively weakly overlapping with LysoTracker. After 6 h, it was confirmed that the green fluorescence signal of in2C41 within the cytoplasm gradually decreased. In particular, in2C41 did not overlap with calnexin and 58K Golgi at any time point, confirming that in2C41 moves directly from late endosomes to the cytoplasm without additional movement to the endoplasmic reticulum and Golgi.

[0508]

[0509] Example 23. Confirmation of the effect of acidification of late endosomes on the endosomal escape of cytoplasmic penetrating antibodies having endosomal escape structural motifs in the heavy chain constant region CH3 domain and the light chain constant region CL domain.

[0510] Figure 27 shows the results of observing the cytoplasmic penetration ability of in2C41 in the presence or absence of a pharmacological inhibitor using calcein using a confocal microscope.

[0511] Specifically, 1 × 10 per well was added to a lysine-coated plate prepared in the same manner as Example 18. 5MDA-MB-231 cells were aliquoted in 500 μL each and cultured for 24 hours under 5% CO2, 37°C conditions. The pharmacological inhibitors used thereafter were bafilomycin A1, which inhibits endosome acidification by inhibiting the ATPase hydrogen pump, wortmannin, which inhibits maturation of early endosomes into late endosomes, and brefeldin A, which inhibits transport from endosomes to the endoplasmic reticulum and Golgi. Bafilomycin A1 (200 nM), wortmannin (200 nM), and brefeldin A (7 μM) were diluted in serum-free RPMI medium, respectively, and treated to the cells, followed by incubation at 37°C for 30 minutes. After removing the supernatant, the cells were washed twice with PBS, and treated with 1 μM of in2C41 and control antibodies and 200 μM of calcein (sigma, C0875), a green fluorescent substance that is not membrane-permeable, at 37°C for 12 hours. Thereafter, the cells were washed twice with PBS, fixed, sampled, and observed using a confocal microscope as in Example 18.

[0512] As shown in Figure 27, in cells treated with bafilomycin A1 and wortmannin, in2C41 was unable to escape from the endosome, and calcein was still trapped in the endosome, whereas in cells treated with brefeldin A, calcein was found to be spread throughout the cytoplasm. This confirmed that in2C41 escapes from the endosome in the acidic environment of the late endosome, and moves directly into the cytoplasm without moving to the endoplasmic reticulum and Golgi, as in Example 23.

[0513]

[0514] Example 24. Evaluation of cell membrane binding ability and cell penetration ability of a cytoplasmic penetrating antibody having an endosomal escape structural motif in the heavy chain constant region CH3 domain and the light chain constant region CL domain depending on pH.

[0515] Figure 28 shows the results of flow cytometry analysis evaluating the cell membrane binding of in2C41 and control antibodies according to pH.

[0516] Specifically, CHO-K1 cells lacking expression of integrin αvβ3 / αvβ5 were seeded at 2 × 10 per sample. 5 After preparation, the cells were washed with pH 7.4 buffer (PBS, 2% FBS, 50 mM HEPES (Sigma, H3537)) and pH 5.5 buffer (PBS, 2% FBS, 50 mM MES (Sigma, H76039)), respectively. Then, in2C41 antibody and control antibody were diluted to a concentration of 3 μM in each pH buffer and treated with the cells at 4 °C for 1 h. After washing with each pH buffer, FITC (green fluorescent substance)-conjugated anti-human Fc antibody (Sigma, F9512) was diluted in each pH buffer and treated with the cells at 4 °C for 30 min. After washing with each pH buffer, the cells were analyzed using a flow cytometer (Agilent, Novocyte).

[0517] As shown in Figure 28, it was confirmed that in2C41 specifically binds to the cell membrane under pH 5.5 conditions.

[0518] Figure 29 shows the results of confirming whether membrane-permeable trypan blue is absorbed through the formation of cell membrane perforations of in2C41 according to pH and whether the formed cell membrane perforations are temporary and reversible, using live cell imaging equipment Lionheart Fx (Biotek).

[0519] Specifically, Ramos cells were cultured with in2C41 or control antibodies at a concentration of 1 or 2 μM at 37°C for 2 hours under pH 7.4 and pH 5.5 conditions in the same manner as in Example 9. To confirm whether the cell membrane perforation formation was temporary and reversible, cells cultured with antibodies (2 μM) under pH 5.5 conditions were washed with PBS and then cultured again under pH 7.4 RPMI medium conditions for 2 hours. Afterwards, trypan blue staining was performed in the same manner as in Example 9 and observation was performed using a live cell imaging device.

[0520] The number of cells that absorbed trypan blue was quantitatively compared and presented in a graph in Fig. 29. Specifically, the number of cells that acquired trypan blue among all cells was counted and presented as a percentage. The mean value (mean) was presented in the graph after counting a total of 200 or more cells. It was confirmed that trypan blue was absorbed in proportion to the concentration of in2C41 under pH 5.5 conditions. On the other hand, in the case of cells cultured for 2 hours in a pH 7.4 medium after culture at pH 5.5, trypan blue absorption did not occur, confirming that the formation of cell membrane perforations by in2C41 is a temporary and reversible phenomenon.

[0521] Figure 30 shows confocal microscopy observations of whether in2C41 can completely pass through the cell membrane and move to the opposite side depending on pH.

[0522] Specifically, 1 × 10 per well was added to a lysine-coated plate prepared in the same manner as in Example 18. 5CHO-K1 cells were aliquoted in 500 μL portions and cultured for 24 hours under 5% CO2, 37°C conditions. In the same two buffer environments as in Figure 30, 5 μM of in2C41 and control antibodies were treated and cultured at 37°C for 2 hours. Afterwards, PBS washing, fixation, cell permeabilization, and blocking processes were performed in the same manner as in Example 19, and FITC (green fluorescence)-linked anti-human Fc antibody (sigma, F9512) was treated for 1 hour at 25°C conditions. Afterwards, the cells were washed twice with PBS and sampled on a coverglass in the same manner as in Example 18.

[0523] As shown in Figure 30, it was confirmed that in2C41 was specifically located within the cell under pH 5.5 conditions and could penetrate the membrane through the cell membrane pores created in the endosomal environment and move into the cytoplasm.

[0524]

[0525] Example 25. Evaluation of the thermostability of a cytoplasmic penetrating antibody having an endosomal escape structural motif in the heavy chain constant region CH3 domain and the light chain constant region CL domain.

[0526] Figure 31 shows the thermal stability of cytoplasmic penetrating antibodies (in2C11, in2C21, in2C41) having an endosomal escape structural motif in the antibody constant region, their parent antibody inCT (AAA), and the therapeutically approved antibody Trastuzumab, evaluated using differential scanning calorimetry.

[0527] Specifically, antibodies were prepared by diluting 1 mg / ml in histidine buffer (25 mM histidine, 150 mM NaCl, pH 6.5) and heated at a rate of 1 °C / min from 25 °C to 90 °C in a MicroCal PEAQ-DSC (Malvern Panalytical, UK) instrument. The melting temperature (Tm) of the antibody was then calculated by subtracting the value measured under the same conditions with only the buffer.

[0528] As shown in Figure 31, in2C21, which has an endosomal escape structural motif in the CL domain, has two melting points T, identical to inCT (AAA) and trastuzumab, which were used as controls. m1 (CH2 domain), T m2 (Fab, CH3 domain) was measured. In particular, T m2 The slight difference in melting point between trastuzumab and T might be due to the differences in the VH and VL domains (Ionescu et al. 2008). In contrast, cytoplasmic penetrating antibodies (in2C11, in2C41) with an endosomal escape structural motif in the CH3 domain exhibited a T m1 Additional melting point (T) at about 4°C higher than m3 , CH3 domain) was measured. This is similar to a previous study in which mutations were introduced into the CH3 domain loop region to confer binding ability to a specific antigen, and it was confirmed that the heat stability decreased by approximately 10°C due to the mutations introduced into the CH3 domain loop (Lobner et al. 2017).

[0529]

[0530] Example 26. Confirmation of maintenance of binding ability to FcRn of a cytoplasmic penetrating antibody having an endosomal escape structural motif in the heavy chain constant region CH3 domain and the light chain constant region CL domain.

[0531] Figure 32 shows the results of bio-layer interferometry using Octet Qke (Sartorius) to confirm whether a cytoplasmic penetrating antibody (in2C41) with an endosomal escape structural motif in the CH3 and CL domains retains binding ability to FcRn like wild-type IgG1 Fc.

[0532] Specifically, three types of antibodies, including cytoplasmic penetrating antibodies (in2C11, in2C41) with endosomal escape structural motifs in their constant regions and the therapeutically approved antibody Trastuzumab, were diluted to 50 μg / mL in 1X kinetics buffer (Sartorius, 18-1105) and immobilized to a Fab2G biosensor (Sartorius, 18-5125) at 1.8 nm. After stabilization in 100 mM PBS at pH 6.0 or pH 7.4 containing 0.05% Tween 20, the binding ability to recombinant human FcRn (SinoBiological, CT009-H08H) diluted to concentrations of 31.3, 62.5, 125, 250, 500, and 1,000 nM in the same buffer was measured. The measured data were fitted to a 1:1 Langmuir coupling model (simultaneously k on ,k off ) and rate constant k off / k on Affinity constant (K) calculated from the ratio of D ) was obtained.

[0533] Experimental results showed that cytoplasmic penetrating antibodies (in2C11, in2C41) with endosomal escape structural motifs in the CH3 and CL domains showed binding affinity similar to that of Trastuzumab with wild-type IgG1 Fc. The binding affinity for FcRn at pH 6.0 (K D) were similar around 20 nM, and at pH 7.4, all antibodies did not bind to FcRn, confirming that the endosomal escape structural motif introduced into the CH3 and CL domains of in2C41 did not affect FcRn binding.

[0534]

[0535] Example 27. Pharmacokinetics of cytoplasmic penetrating antibodies having endosomal escape structural motifs in the heavy chain constant region CH3 domain and the light chain constant region CL domain.

[0536] Figure 33 shows the results of evaluating the pharmacokinetics of in2C41 in BALB / c nude mice.

[0537] Specifically, BALB / c nude mice were intravenously injected with cytoplasmic penetrating antibody in2C41 and control inCT at 20 mg / kg, and mouse blood was collected at 1, 4, 12, and 24 hours; and 2, 4, and 7 days, respectively. The collected blood was centrifuged at 12,000 rpm, 10 min, and 4 °C, and only the supernatant plasma was stored at -80 °C. ELISA was then performed to analyze the concentrations of in2C41 and inCT in the plasma. Specifically, anti-human Fab antibodies (sigma) were each incubated at a concentration of 2.5 μg / ml in a 96-well half-area plate (Corning) for 1 hour at room temperature, and then washed three times with 0.1% PBST (PBS, pH 7.4, 0.1% Tween 20). After binding for 1 hour with 1% PBSB (PBS, pH 7.4, 1% BSA), the collected blood and purified antibodies (for the reference curve) were diluted in 1% PBSB and bound for 2 hours, and washed three times with 0.1% PBST. After that, HRP-conjugated anti-human IgG, Fc antibody (Sigma) was bound as a labeled antibody for 1 hour, and washed three times with 0.1% PBST. After reacting with TMB ELISA solution, the reaction was stopped with sulfuric acid solution, and the absorbance at 450 nm was quantified.

[0538] As shown in Figure 33, it was confirmed that in2C41 exhibited pharmacokinetics similar to the control antibody inCT (T1 / 2β (inCT): 92.4 ± 3.9 h, T 1 / 2 β (in2C41): 86.9 ± 3.0 h) It was confirmed that the endosomal escape structural motif introduced into the antibody constant region did not affect the half-life of the antibody.

[0539]

[0540] Example 28. Design of a cytoplasmic penetrating antibody (in2C71) having a FabCab domain in which the CH1-CL pair of the antibody is replaced with a CH3-CH3 pair and an endosomal escape structural motif in the CH3 domain.

[0541] Following the Fcab (Fc with antigen-binding constant domains) technology, which imparts antigen-binding ability to the CH3 domain, the FabCH3 technology was reported, which improves thermostability by replacing the CH1-CL pair in an antibody Fab fragment with a CH3-CH3 pair. By fusing the above two technologies, the FabCab (Fab with antigen-binding constant domains, Fab with antigen-binding ability in the constant region) technology, which imparts antigen-binding ability to the CH3 domain of FabCH3, was reported. Utilizing this, a cytoplasmic penetrating antibody was constructed in which the CH1-CL pair of an antibody Fab was replaced with a CH3-CH3 pair having an endosomal escape structural motif. At this time, the FabCab domain variant in which the CH1-CL pair in an antibody Fab fragment was replaced with a CH3-CH3 pair having an endosomal escape structural motif was named in2C7N. At this time, since light chain homodimers or heavy chain oligomers may be generated during the protein assembly process when both CH3 replacing CH1 and CH3 replacing CL are wild-type, to prevent this, a heterodimer Fc (Heterodimer heterodimeric Fc) forming mutation, EW-RVT, was introduced into the CH3 pair (Korean Patent No. KR10-1522954).

[0542] Figure 34 is a schematic diagram of a FabCab domain variant (in2C7N) with enhanced cytoplasmic penetration ability by replacing the CH1-CL pair with a heterodimer CH3 pair having an endosomal escape structural motif. In inCT(AAA)-FabCab, in which the CH1-CL pair of inCT(AAA) is replaced with an EW-RVT heterodimer CH3 pair, an endosomal escape structural motif was introduced into the heterodimer CH3 pair to construct in2C7N.

[0543]

[0544] Specifically, a FabCab domain variant was constructed by a heavy chain comprising a heavy chain constant region (CH3(RVT)-hinge-CH2-CH3) having a VH of SEQ ID NO: 14 and a CH3 domain into which an RVT mutation of SEQ ID NO: 22, 24, or 26 was introduced, and a light chain comprising a VL of SEQ ID NO: 16 and a CH3 domain into which an EW mutation of SEQ ID NO: 21, 23, or 25 was introduced. The constituent domains and amino acid sequences of the heavy and light chains of the FabCab domain variant (in2C7N) are shown in Table 16 above.

[0545] Figure 35 shows the 3D structure of a FabCab fragment, in which the CH1-CL pair of the wild-type Fab domain is replaced with a CH3-CH3 pair harboring an endosomal escape structural motif and a heterodimer-forming mutation. This structure was modeled using Alphafold2, and the intramolecular interaction patterns were analyzed using the Pymol molecular visualizer. This confirmed that the 3D structures of the FabCab domain variants (in2C71 and in2C72) are similar to the folded state of the 3D structure of the wild-type IgG1 Fc of Figure 2A.

[0546]

[0547] To analyze the structural differences between the FabCab fragments of the FabCab domain variants (in2C71 and in2C72) and the wild-type Fc region, the RMSD of the CH3 domain heterodimer of FabCab relative to the CH3 domain homodimer (CH3-CH3 pair) was calculated and is shown in Table 15 above. The RMSD value of the wild-type FabCab without the endosomal escape structural motif and the wild-type IgG1 Fc was 0.189 Å, the RMSD value of in2C71 and in2C11 was 0.172 Å, and the RMSD value of in2C72 and in2C19 was 0.141 Å. All of the RMSD values ​​were less than 0.2 Å, confirming that the structures were very similar to the wild type.

[0548] It was confirmed that the inCT(AAA) FabCab consisting of sequence numbers 21 and 22 folded in the same form as the wild-type Fc of Fig. 2a, as shown in Fig. 35a.

[0549] in2C71, consisting of sequence numbers 23 and 24, introduced the endosomal escape structural motif of the CH3 domain of in2C11 into the FabCab CH3-CH3 pair of inCT(AAA)-FabCab. The CH3-CH3 pair, like inCT(AAA)-FabCab, induces heterodimeric CH3 pair formation between the FabCab heavy and light chains through EW-RVT. The endosomal escape structural motif is the same as in2C11 and consists of two tryptophan mutations (R355W, Q418W), two arginine mutations (L358R, L443R), one alanine mutation (K414A), and three glutamic acid mutations (N361E, Q3622, D413E) in the AB loop and EF loop and the C-terminal loop. The intramolecular interaction pattern of the endosomal escape structural motif was confirmed to be the same as that in in2C11 of Fig. 2b, as shown in Fig. 35b.

[0550] in2C72, consisting of sequence numbers 25 and 26, introduced the endosomal escape structural motif of the CH3 domain of in2C19 into the FabCab CH3-CH3 pair of inCT(AAA)-FabCab. The CH3-CH3 pair, like the inCT(AAA)-FabCab, induces heterodimeric CH3 pair formation between the FabCab heavy and light chains through EW-RVT. The endosomal escape structural motif is the same as in2C19, consisting of three tryptophan mutations (L358W, Q418W, K439W), three arginine mutations (D356R, T359R, L443R), one alanine mutation (K414A), and three glutamic acid mutations (N361E, Q362E, D413E) in the AB loop, EF loop, and C-terminal loop. The intramolecular interaction pattern of the endosomal escape structural motif was confirmed to be the same as that in in2C19 of Fig. 2l, as shown in Fig. 35c.

[0551]

[0552] Example 29. Expression and purification of a cytoplasmic penetrating antibody (in2C7N-GFP11-SBP2) having a FabCab domain in which the CH1-CL pair of the antibody is replaced with a CH3-CH3 pair and an endosomal escape structural motif in the CH3 domain.

[0553] To identify the biophysical and chemical properties of in2C71, which introduced the endosomal escape motif of in2C11 into the FabCab domain, and in2C72, which introduced the endosomal escape motif of in2C19 into the FabCab domain, and to evaluate the endosomal escape ability through the complementary binding system of the improved split green fluorescent protein, in2C7N-GFP11-SBP2 was constructed with GFP11-SBP2 fused to the C-terminus of the antibody heavy chain. In addition, inCT(AAA)-FabCab -GFP11-SBP2, a wild-type FabCab format antibody without the endosomal escape motif, was constructed together and its biophysical and chemical properties were compared.

[0554] As in the previous example, a circular peptide (in4, DGVRQCRGDCFDGPL) targeting integrin αvβ3 / αvβ5, a type of membrane protein receptor overexpressed on the surface of tumor cells / tissues, was fused to the light chain N-terminus of the cytoplasmic penetrating antibody to induce cellular internalization through the tumor cell / tissue-specific receptor.

[0555] Specifically, to construct a light chain expression vector for producing inCT(AAA)-FabCab-GFP11-SBP2 as a monoclonal antibody in the form of a full IgG, the DNA encoding the light chain, including the VL of the antibody (SEQ ID NO: 16) fused to the 5'-terminus with a DNA encoding a secretory signal peptide and CH3(EW) (SEQ ID NO: 23) with an EW mutation introduced for heterodimer formation, was cloned into the pcDNA3.4 vector with NotI / HindIII. In addition, to construct a vector for expressing the heavy chain, the DNA encoding the heavy chain, including the VH of the antibody (SEQ ID NO: 14) fused to the 5'-terminus with a DNA encoding a secretory signal peptide and CH3(RVT) (SEQ ID NO: 24) with an RVT mutation introduced for heterodimer formation, was cloned into the pcDNA3.4 vector with NotI / HindIII, respectively. At this time, to construct a cytoplasmic penetrating antibody fused with GFP11-SBP2, GFP11-(G4S)3-SBP2 was fused using a GGGS three-linker at the heavy chain C-terminus.

[0556] To construct a light chain expression vector for producing in2C71-GFP11-SBP2 as a monoclonal antibody in the form of a full IgG, the DNA encoding the light chain, including the VL of the antibody (SEQ ID NO: 16) fused to the DNA encoding the secretory signal peptide at the 5' end and CH3 11(EW) (SEQ ID NO: 25) with the EW mutation for heterodimer formation and the endosomal escape structural motif introduced, was cloned into the pcDNA3.4 vector with NotI / HindIII. In addition, to construct a vector for expressing the heavy chain, the DNA encoding the heavy chain, including the VH of the antibody (SEQ ID NO: 14) fused to the DNA encoding the secretory signal peptide at the 5' end and CH3 11(RVT) (SEQ ID NO: 26) with the RVT mutation for heterodimer formation and the endosomal escape structural motif introduced, was cloned into the pcDNA3.4 vector with NotI / HindIII. Similarly, to construct a cytoplasmic penetrating antibody fused with GFP11-SBP2, GFP11-(G4S)3-SBP2 was fused to the heavy chain C-terminus using a GGGS three-linker.

[0557] To construct a light chain expression vector for producing in2C72-GFP11-SBP2 as a monoclonal antibody in the form of a full IgG, the DNA encoding the light chain, including the VL of the antibody (SEQ ID NO: 16) fused to the DNA encoding the secretory signal peptide at the 5' end and CH3 19(EW) (SEQ ID NO: 27) with the EW mutation for heterodimer formation and the introduction of the endosomal escape structural motif, was cloned into the pcDNA3.4 vector with NotI / HindIII. In addition, to construct a vector for expressing the heavy chain, the DNA encoding the heavy chain, including the VH of the antibody (SEQ ID NO: 14) fused to the DNA encoding the secretory signal peptide at the 5' end and CH3 19(RVT) (SEQ ID NO: 28) with the RVT mutation for heterodimer formation and the introduction of the endosomal escape structural motif, was cloned into the pcDNA3.4 vector with NotI / HindIII. Similarly, to construct a cytoplasmic penetrating antibody fused with GFP11-SBP2, GFP11-(G4S)3-SBP2 was fused to the heavy chain C-terminus using a GGGS three-linker.

[0558] The three cytoplasmic penetrating antibodies in the FabCab format were purified by transiently transfecting HEK293-F cells together with a light chain expression vector of VL-CH3(EW) containing the VL of SEQ ID NO: 16 and the CH3(EW) of SEQ ID NO: 21, 23, or 25 and a heavy chain expression vector of VH-CH3(RVT)-hinge-CH2-CH3 containing the VH of SEQ ID NO: 14 and the CH3(RVT) of SEQ ID NO: 22, 24, or 26. The transfection was performed using the same method as in Example 3.

[0559]

[0560] Afterwards, purification and protein quantification of the FabCab domain mutants were also performed in the same manner as in Example 3. Their production yields in animal cells are shown in Table 16 above. in2C72-GFP11-SBP2 showed a significantly low yield of 0.34 mg per liter. inCT(AAA)-FabCab-GFP11-SBP2 and in2C71, in2C72-GFP11-SBP2 showed a significantly reduced yield of about 75% compared to the wild-type inCT(AAA)-GFP11-SBP2, but showed a respectable yield of more than 10 mg per liter.

[0561] Hereinafter, a FabCab domain variant (in2C7N-GFP11-SBP2) fused with a prototypical peptide targeting a membrane protein receptor overexpressed on the surface of tumor cells and GFP11-SBP2 is described as in2C7N. For example, in2C71-GFP11-SBP2 is in2C71.

[0562]

[0563] Example 30. Physical properties evaluation of a FabCab in which the CH1-CL pair of an antibody is replaced with a CH3-CH3 pair and a cytoplasmic penetrating antibody (in2C7N) having an endosomal escape structural motif in the CH3 domain.

[0564] Figure 36 shows the physical properties of in2C71, in2C72, and inCT(AAA)-FabCab purified using 12% SDS-PAGE and size exclusion chromatography under reducing or non-reducing conditions. Specifically, all three antibodies were confirmed to have a molecular weight of approximately 166 to 167 kDa under non-reducing conditions, and showed a heavy chain molecular weight of approximately 56 kDa (antibody heavy chain approximately 51 kDa and GFP11-SBP2 fragment approximately 5 kDa) and a light chain molecular weight of approximately 26.8 kDa (antibody light chain approximately 25.3 kDa and in4 circular peptide 1.5 kDa) under reducing conditions. However, a band of approximately 50 kDa in size was observed under non-reducing conditions, which was thought to be due to the formation of some homodimers between the two VL-CH3(EW) light chains. When the mAU values ​​of the samples were measured at 280 nm using size exclusion chromatography (Superdex 200 increased 10 / 300GL, Cytiva) in the same manner as in Example 4, both in2C71 and inCT(AAA)-FabCab showed a single peak without any special oligomerization or tailing, like the control groups Trastuzumab and inCT(AAA).

[0565] Through this, it was confirmed that in2C71, which has an endosomal escape structural motif in three CH3-CH3 pairs, has a satisfactory production yield of more than 10 mg per liter [Table 16] and similar properties to the control wild-type antibody.

[0566]

[0567] Example 31. Evaluation of nonspecific binding of FabCabs in which the antibody CH1-CL dimer is replaced with a CH3-CH3 dimer and cytoplasmic penetrating antibodies having an endosomal escape structural motif in the CH3 domain.

[0568] Figure 37 shows the results of ELISA performed on four antigens, dsDNA, Insulin, Keyhole limpet hemocyanin (KLH), and Cardiolipin, which can confirm the nonspecific binding of a cytoplasmic penetrating antibody (in2C71) having an endosomal escape structural motif in three CH3-CH3 pairs, to confirm the nonspecific binding of the antibody. The experiment was performed in the same manner as in Example 5.

[0569] In the above experiment, non-specific binding was determined when an absorbance of 0.05 or higher at 450 nm was observed for at least one of the four antigens at a concentration of 100 nM. It was confirmed that in2C71 did not have any particular non-specific binding to the four antigens, similar to the negative control antibody.

[0570] Figure 38 shows the results of flow cytometry analysis performed to confirm nonspecific binding to the cell membrane of the cytoplasmic penetrating antibody in2C71 in FabCab format. The experiment was performed in the same manner as in Example 5. The results confirmed that in2C71, similar to the wild-type negative control, did not exhibit nonspecific binding to the cell surfaces of K-562 and CHO-K1.

[0571]

[0572] Example 32. Evaluation of cytoplasmic penetration ability of FabCab with CH1-CL pair replaced by CH3-CH3 pair and cytoplasmic penetration antibody (in2C71) with endosomal escape structural motif in CH3 domain.

[0573] Figure 39 shows the results of observing and quantifying GFP fluorescence resulting from complementary binding of improved split green fluorescent protein using a live cell imaging device, Lionheart Fx (Biotek), to evaluate the cytoplasmic penetration ability of cytoplasmic penetrating antibodies (in2C41 and in2C71) having endosomal escape structural motifs in their antibody constant regions. The experimental process and quantification of intracellular fluorescence intensity were performed in the same manner as in Example 7. The experimental results showed that in2C71 showed a cell-based fluorescence intensity approximately 3.0 times higher than inCT. This confirms that in2C71, which has endosomal escape structural motifs in three CH3-CH3 pairs by replacing the CH1-CL pair with a CH3-CH3 pair, has a cytoplasmic penetration ability that is approximately 3 times higher than inCT, at a level higher than in2C41 (approximately 2.5 times higher than inCT) through the reinforcing effect of multiple endosomal escape structural motifs.

[0574]

[0575] Example 33. Evaluation of pH-dependent interaction of cytoplasmic penetrating antibodies having endosomal escape structural motifs in their constant regions with the cell membrane.

[0576] Figure 40 shows the results of a live cell imaging device, Lionheart Fx (Biotek, USA), used to confirm the possibility of obtaining trypan blue without membrane permeability through pore formation by a cytoplasmic penetrating antibody having an endosomal escape structural motif in its constant region in Ramos cells according to pH. The experiment was performed in the same manner as in Example 9.

[0577] In Fig. 40, the number of cells that acquired trypan blue was quantitatively compared and presented in a graph. Specifically, the number of cells that acquired trypan blue among all cells was counted and presented as a percentage. The mean value (mean) was presented in the graph after counting a total of 200 or more cells. It was confirmed that trypan blue was acquired only under pH 5.5 conditions in cells to which a cytoplasmic penetrating antibody having an endosomal escape structural motif in the constant region was added. In particular, in2C41 and in2C71, which have high cytoplasmic penetrating ability, showed a high trypan blue acquisition percentage of approximately 50% only at pH 5.5. Through this, it was confirmed that the cytoplasmic penetrating antibodies having an endosomal escape structural motif in the constant region according to the present invention (in2C41 and in2C71) can interact with the cell membrane specifically in a slightly acidic environment through a glutamic acid mutation.

[0578]

[0579] Example 34. Design of a full IgG form anti-α-Tubulin cytoplasmic penetrating antibody (inTu41) targeting intracellular α-Tubulin.

[0580] A novel, highly efficient cytoplasmic penetrating antibody with an endosomal escape structural motif in its constant region will ultimately increase the amount of antibody located in the cytoplasm, allowing for more effective targeting of proteins located inside the cytoplasm.

[0581] Figure 41 is a schematic diagram illustrating the construction of a full IgG form of an anti-α-Tubulin cytoplasmic penetrating antibody (inTu41) to confirm the activity of in2C41 with enhanced cytoplasmic penetrating ability. The antibody variable region sequences required for the construction of the anti-α-Tubulin cytoplasmic penetrating antibody are shown in Table 17 below.

[0582] For animal cell expression of a full IgG form of an anti-α-Tubulin cytoplasmic penetrating monoclonal antibody, the DNA encoding the heavy chain having a heavy chain variable region (VH, SEQ ID NO: 27) that specifically binds to the cytoskeleton α-Tubulin and a heavy chain constant region (CH1-hinge-CH2-CH3) containing the CH3 domain of in2C41 (SEQ ID NO: 4) and a DNA coding for a secretory signal peptide fused to the 5' end was cloned into the pcDNA3.4 vector using NotI / HindIII. In addition, the DNA coding for the light chain having a light chain variable region (VL, SEQ ID NO: 16) and a CL domain of in2C21 (SEQ ID NO: 18) fused to the DNA coding for a secretory signal peptide at the 5' end was cloned into the pcDNA3.4 vector using NotI / HindIII. Afterwards, the heavy and light chain-encoded animal expression vectors were simultaneously transiently transfected into HEK293-F cells using the same method as in Example 3, and the antibodies were purified.

[0583]

[0584] In addition, inTu, which has anti-α-Tubulin VH in a wild-type antibody without cytoplasmic penetration ability, was constructed and used as a control group for the experiment, and inCT-based anti-α-Tubulin cytoplasmic penetration antibody (inTuCT), which has been reported to penetrate the cytoplasm through cell-penetrating light chain VL (SEQ ID NO: 15) and target intracellular α-Tubulin through anti-α-Tubulin VH (SEQ ID NO: 27), was constructed and used as a control group for the experiment.

[0585] For inTu construction, DNA encoding a heavy chain having a heavy chain variable region VH (SEQ ID NO: 27) that specifically binds to cytoskeletal α-Tubulin and a wild-type heavy chain constant region (CH1-hinge-CH2-CH3) fused to the 5' end of the DNA encoding a secretory signal peptide was cloned into the pcDNA3.4 vector using NotI / HindIII. In addition, DNA encoding a light chain including a light chain variable region (VL, SEQ ID NO: 16) and a wild-type light chain constant region (CL, SEQ ID NO: 17) fused to the 5' end of the DNA encoding a secretory signal peptide was cloned into the pcDNA3.4 vector using NotI / HindIII. Thereafter, the heavy chain and light chain-encoding animal expression vectors were transiently transfected simultaneously into HEK293-F cells in the same manner as in Example 3 to purify the antibody.

[0586] For inTuCT, the heavy chain of inTu and the light chain of inCT were simultaneously transiently transfected into HEK293-F cells using the same method, and the antibodies were purified.

[0587]

[0588] Protein quantification of all full IgG forms of anti-α-Tubulin antibodies was performed using the same method as in Example 3. The production yields of inTu, inTuCT, and inTu41 are shown in Table 18 above. inTuCT, which has cytoplasmic penetration ability through the light chain variable region VL, and inTu41, which has cytoplasmic penetration ability through the constant regions CH3 and CL, both showed a good yield of more than 20 mg per liter, similar to inTu.

[0589] Figure 42 is a graph showing the mAU values ​​at 280 nm measured using size exclusion chromatography (Superdex 200 increased 10 / 300GL, Cytiva) in the same manner as in Example 4 for anti-α-Tubulin antibodies in the form of full IgG and their controls. A single peak was observed for all three anti-α-Tubulin antibodies without oligomerization or tailing, as with the control Trastuzumab.

[0590]

[0591] Example 35. Confirmation of overlap between anti-α-Tubulin cytoplasmic penetrating antibody (inTu41) in full IgG form and cytoskeletal α-Tubulin.

[0592] Figure 43 shows the results of a confocal microscope examination of the overlap between a full IgG form of anti-α-Tubulin cytoplasmic penetrating antibody (inTu41) and α-Tubulin located in the cytoplasm.

[0593] Specifically, 1 × 10 per well was added to a lysine-coated plate prepared in the same manner as in Example 18. 5HeLa cells were aliquoted in 500 μL each and cultured for 24 hours under 5% CO2, 37°C conditions. After removing the supernatant, the cells were treated with anti-α-Tubulin cytoplasmic penetrating antibody (inTu41) and control antibody at a concentration of 0.1 or 1 μM at 37°C for 12 hours. Thereafter, PBS washing, acid washing, cell fixation, cell permeabilization, and blocking processes were performed in the same manner as in Example 23. Afterwards, anti-α-Tubulin mouse antibody (Santa Cruz, sc-32293) was diluted in blocking solution as the primary antibody for α-Tubulin staining and treated at 25°C for 1 hour. After washing three times with PBS, the intracellular antibody and anti-α-Tubulin mouse antibody were diluted in blocking solution with FITC (green fluorescence)-conjugated anti-human Fc antibody (sigma, F9512) and Alexa Fluor 647 (red fluorescence)-conjugated anti-mouse IgG antibody (Invitrogen, A21235), respectively, and treated at 25°C for 1 hour. Thereafter, PBS washing, nuclear staining, sampling, and observation were performed using a confocal microscope in the same manner as in Example 18.

[0594] As shown in Figure 43, in the case of inTu(AAA), which has no cytoplasmic penetration ability, a dot-shaped fluorescent signal indicating that the antibody was trapped in the endosome was observed regardless of the antibody concentration. On the other hand, in cells treated with inTu41, inTu41 labeled with green fluorescence and cytoplasmic α-Tubulin labeled with red fluorescence overlapped at both concentrations. In particular, inTuCT, used as a positive control at a concentration of 0.1 μM, was observed to be trapped in the endosome in the same manner as inTu(AAA), used as a negative control, confirming that inTu41 developed in the present invention is more effectively located in the cytoplasm than inTuCT.

[0595]

[0596] Example 36. Design of a RAS-targeting cytoplasmic penetrating antibody (inRas41) in the form of a full IgG targeting intracellular activated RAS.

[0597] In addition to the cytoskeletal protein α-Tubulin, we conducted experiments to determine whether other cytoplasmic proteins could be effectively targeted. Figure 44 is a schematic diagram illustrating the construction of a fully IgG-type RAS-targeting cytoplasmic-penetrating antibody to confirm the activity of in2C41, which has enhanced cytoplasmic penetration ability.

[0598] The antibody variable region sequences required for constructing anti-RAS cytoplasmic penetrating antibodies are shown in Table 19 above. For animal cell expression of anti-RAS cytoplasmic penetrating monoclonal antibodies in the form of full IgG, DNA encoding a secretory signal peptide was fused to the 5' end. The heavy chain having CH1-hinge-CH2-CH3, which includes RT22 VH (Science Advances, 2020, 6(3):eaay2174, SEQ ID NO: 28) that specifically binds to RAS and the CH3 domain of in2C41 (SEQ ID NO: 4), was cloned into the pcDNA3.4 vector using NotI / HindIII. In addition, DNA encoding a light chain having a light chain variable region (VL, SEQ ID NO: 30) and the CL domain of in2C41 (SEQ ID NO: 18), which was fused to the 5' end. The light chain was cloned into the pcDNA3.4 vector using NotI / HindIII. Afterwards, the heavy and light chain-encoded animal expression vectors were simultaneously transiently transfected into HEK293-F cells using the same method as in Example 3, and the antibodies were purified.

[0599] In addition, an anti-RAS cytoplasmic penetrating antibody (inRas37), which has been reported to penetrate the cytoplasm through the cell-penetrating light chain VL (SEQ ID NO: 29) and target intracellular RAS through the anti-RAS RT22 VH (SEQ ID NO: 28), was constructed and used as a positive control for intracellular RAS targeting, and inRas37(AAA) having the anti-RAS RT22 VH (SEQ ID NO: 28) in the wild-type VL (SEQ ID NO: 30) without cytoplasmic penetrating ability was constructed and used as a negative control for cytoplasmic penetrating ability.

[0600] To construct inRas37 and inRas37(AAA), DNA encoding a heavy chain having a heavy chain variable region VH (SEQ ID NO: 28) that specifically binds to RAS and a wild-type heavy chain constant region (CH1-hinge-CH2-CH3) fused to the 5' end of the DNA encoding a secretory signal peptide was cloned into the pcDNA3.4 vector with NotI / HindIII. In addition, DNA encoding a light chain comprising a light chain variable region (VL, SEQ ID NO: 29 or 30) and a wild-type light chain constant region (CL, SEQ ID NO: 17) fused to the 5' end of the DNA encoding a secretory signal peptide was cloned into the pcDNA3.4 vector with NotI / HindIII. Afterwards, HEK293-F cells were transiently transfected at the same time as in Example 3, and the antibodies were purified.

[0601] Protein quantification of all full IgG forms of anti-RAS antibodies was performed using the same method as in Example 3. The purification yields of inRas37, inRas37(AAA), and inRas41 are shown in Table 19 below. inRAS41, which has an endosomal escape structural motif in the constant regions CH3 and CL, showed a yield of 42% lower than inRAS37, but showed a satisfactory yield of approximately 27 mg per liter.

[0602]

[0603] Figure 45 shows the physical properties of anti-RAS cytoplasmic penetrating antibodies (inRas41) having an endosomal escape structural motif in the constant region and their controls, analyzed by 12% SDS-PAGE and size exclusion chromatography under reducing or non-reducing conditions after purification. Specifically, all three antibodies in Table 21 were confirmed to have a molecular weight of approximately 150 kDa under non-reducing conditions, and showed molecular weights of approximately 49 kDa for the heavy chain and approximately 26.5 kDa for the light chain (approximately 25 kDa for the antibody light chain and 1.5 kDa for the inRas4 circular peptide) under reducing conditions. When the mAU values ​​at 280 nm were measured using size exclusion chromatography (Superdex 200 increased 10 / 300GL, Cytiva) in the same manner as in Example 4, a single peak was observed for all three anti-RAS antibodies without oligomerization or tailing, as with the control Trastuzumab.

[0604]

[0605] Example 37. Confirmation of overlap between anti-RAS cytoplasmic penetrating antibody (inRas41) in full IgG form and RAS.

[0606] Figure 46 shows the results of a confocal microscope analysis to confirm whether there is overlap between a full IgG form of anti-RAS cytoplasmic penetrating antibody (inRas41) and RAS located in the cytoplasm.

[0607] Specifically, 2 × 10 per well were added to a lysine-coated plate prepared in the same manner as in Example 18. 5SW480 cells were aliquoted in 500 μL each and cultured for 24 hours under 5% CO2, 37°C conditions. After removing the supernatant, the cells were treated with anti-α-Ras cytoplasmic penetrating antibody (inRas41) and control antibody at a concentration of 0.1 or 1 μM at 37°C for 12 hours. Thereafter, PBS washing, acid washing, cell fixation, cell permeabilization, and blocking were performed in the same manner as in Example 23. Afterwards, anti-Ras rabbit antibody (Abcam, ab108602) was diluted in blocking solution as the primary antibody for RAS staining and treated at 25°C for 4 hours. After washing three times with PBS, the intracellular antibody and anti-Ras rabbit antibody were diluted in blocking solution with FITC (green fluorescence)-conjugated anti-human Fc antibody (sigma, F9512) and Alexa Fluor 555 (red fluorescence)-conjugated anti-rabbit IgG antibody (Invitrogen, A21428), respectively, and treated at 25°C for 1 hour. Afterwards, PBS washing, nuclear staining, sampling, and observation were performed in the same manner as in Example 18, using a confocal microscope.

[0608] As shown in Figure 46, in the case of inRas(AAA) which has no cytoplasmic penetration ability, a dot-shaped fluorescent signal indicating that the antibody was trapped in the endosome was observed regardless of the antibody concentration. On the other hand, in cells treated with inRas41, inRas41 labeled with green fluorescence and cytoplasmic RAS labeled with red fluorescence overlapped at both concentrations. In particular, inRas37, used as a positive control at a concentration of 0.1 μM, was observed to be trapped in the endosome in the same manner as inRas(AAA), used as a negative control, confirming that inRas41 developed in the present invention is more effectively located in the cytoplasm than inRas37.

[0609]

[0610] Example 38. Design of an immunotoxin having cytoplasmic penetration ability.

[0611] To evaluate whether an antibody possessing cytoplasmic penetrability in its constant region can deliver active proteins into cells and its potential as a therapeutic agent, a cytoplasmic antibody-based immunotoxin was designed. Figure 47 is a schematic depicting the process of fusing the catalytic domain (DTA) of diphtheria (Corynebacterium diphtheriae) to the antibody via trans-splicing. The protein sequences required for the expression of the cytoplasmic antibody-based immunotoxin are shown in Table 20 below.

[0612]

[0613] To enable splicing of toxins and antibodies ER CT, ER CT(AAA), ER After fusing the N-intein to the C-terminus of the light chain of the 2C41 antibody and the C-intein to the N-terminus of the toxin, they were expressed and purified in animal cells and E. coli, respectively, and then mixed to induce trans-splicing, and then purified again to obtain an antibody fused with the toxin.

[0614] To produce antibodies capable of toxin and trans-splicing ER CT-intein(N), ER CT(AAA)-intein(N), ER2C41-intein(N) was constructed. To this end, a monobody targeting EGFR, a type of membrane protein receptor overexpressed on the surface of tumor cells / tissues, was fused to the N-terminus of the light chain via a (G4S)4 linker, and a cysteine-deficient N-intein required for trans-splicing was fused to the C-terminus of the light chain via a (G4S)2 linker. Specifically, to construct a light chain expression vector for production as a monoclonal antibody capable of performing trans-splicing, DNA encoding a secretory signal peptide at the 5' end, DNA encoding an EGFR targeting monobody (SEQ ID NO: 31), DNA encoding a light chain including the antibody's light chain variable region (VL, SEQ ID NO: 15 or 16) and the antibody's light chain constant region (CL, SEQ ID NO: 17 or 18), and DNA encoding an N-intein (SEQ ID NO: 32) at the 3' end were fused and cloned into the pcDNA3.4 vector with NotI / HindIII. To construct a heavy chain expression vector for production as a monoclonal antibody, DNA encoding a heavy chain including the antibody heavy chain variable region (VH, SEQ ID NO: 13 or 14) and the antibody heavy chain constant region (CH1-hinge-CH2-CH3) including the antibody CH3 domain (CH3, SEQ ID NO: 1 or 4) was fused to DNA encoding a secretory signal peptide at the 5' end, and the DNA was cloned into the pcDNA3.4 vector using NotI / HindIII. Expression of these in animal cells was performed in HEK293-F cells in the same manner as in Example 3.

[0615] To produce a toxin capable of trans-splicing with antibodies, intein(C)-DTA was constructed. The cysteine-deficient C-intein was fused to the N-terminus of the catalytic domain of DT toxin via a (G4S)2 linker. Specifically, the DNA encoding the catalytic domain of DT toxin (SEQ ID NO: 34) was fused to the DNA encoding the C-intein (SEQ ID NO: 33) at the 5' end, and the DNA encoding eight histidines was fused to the 3' end, and the resulting DNA was cloned into the pET23m vector with NotI / NcoI. The vector was transformed into E. coliSoluBL21(DE3) (Genlantis), and the transformed E. coli was grown in 2X TY media until OD 600 The cells were cultured at 37°C until reaching a median of 0.6. 1 mM IPTG was then added and cultured at 20°C for 24 h. The cells were then centrifuged to obtain a cell pellet, which was resuspended in TBS (50 mM Tris, 150 mM NaCl) and then disrupted using an ultrasonicator. The cells were centrifuged again, and the supernatant was filtered through a 0.45 μm filter and purified using Ni-NTA resin (Thermo Scientific, 88221).

[0616] The purified antibody and toxin were mixed at 10 μM and 60 μM, respectively, in TBS (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 7.0) and reacted at 18°C ​​for 18 hours. After the reaction was completed, purification was performed using Protein A resin in the same manner as in Example 3, and the purified extract was repurified using Ni-NTA resin to purify only the antibody fused to the toxin. The antibody fused to the toxin ER CT-DTA, ER CT(AAA)-DTA, ER It was designated 2C41-DTA.

[0617] Figure 48 shows the results of 12% SDS-PAGE analysis of antibodies before and after toxin fusion, and the results of size exclusion chromatography analysis. After toxin fusion, a band corresponding to the toxin-fused light chain is observed at 60 kDa, and the peak in size exclusion chromatography shifts to the left after toxin fusion. This confirms that at least one toxin is fused to the purified immunotoxin.

[0618]

[0619] Example 39. Cytotoxicity evaluation of immunotoxins with cytoplasmic penetration ability.

[0620] To determine whether the cytoplasmic penetrating antibody fused with the toxin can deliver active proteins such as toxins into cells and to evaluate their potential as therapeutic proteins, cytotoxicity was evaluated. Figure 49 is a schematic diagram illustrating the cytotoxic process of an immunotoxin with cytoplasmic penetrating ability. The immunotoxin based on the cytoplasmic penetrating antibody fused with a monobody targeting the EGFR, a receptor overexpressed on cancer cells, binds to and is internalized by cells expressing EGFR, dissociates from EGFR in the mildly acidic environment of the endosomes, and then escapes the endosome via an endosomal escape structural motif and translocates into the cytoplasm. Upon reaching the cytoplasm, the catalytic domain of the DT toxin fused to the antibody ADP-ribosylates EF2, inhibiting protein synthesis and ultimately causing cell death. DT toxin must translocate to the cytoplasm to exhibit cytotoxicity; the catalytic domain of DT toxin alone cannot reach the cytoplasm. Therefore, by fusing a cytoplasmic penetrating antibody and a toxin that are only capable of penetrating the cytoplasm when exposed to the mildly acidic environment of the endosomes after target cell internalization, target cell-specific cytotoxicity can be induced.

[0621] Figure 50 shows the results of evaluating cytotoxicity against four types of cells with different expression levels of EGFR to evaluate target cell-specific cytotoxicity of an immunotoxin with cytoplasmic penetration ability. SK-MEL-2 (EGFR-), a cell line that does not express EGFR, HT29 (EGFR+), a cell line that expresses EGFR at the level of normal cells, HCC827 (EGFR++), a cell line that overexpresses EGFR, and A431 (EGFR+++), a cell line that highly overexpresses EGFR, were cultured in an RPMI medium environment containing 10% FBS and 1% ABAM. 2 × 10 per well in a 96-well plate (SPL, 30096). 3 The cells were aliquoted into 100 μL each and cultured under 5% CO2, 37°C conditions until the cell density reached 60%. After removing the supernatant, the cytoplasmic penetrating antibodies, with or without toxins, were serially diluted 5-fold starting from 10 μM in RPMI medium containing 10% FBS and 1% ABAM and treated at 37°C for 72 hours. After removing all the supernatant and washing with DPBS, 50 μL of Crystal violet solution was added to each well and reacted at room temperature for 20 minutes. After washing four times with DBPS, the absorbance was measured at 570 nm, and the absorbance value of each well was divided by the absorbance value of the well treated only with RPMI medium instead of the antibody, and this was used to calculate the cell viability.

[0622] As a result, in cell lines that do not express EGFR or express EGFR at the level of normal cells (SK-MEL-2, HT29), almost no cell death was observed even at 10 μM, whereas high cytotoxicity was confirmed in cell lines that overexpress EGFR (HCC827, A431). In addition, previously developed ER Compared to CT-based immunotoxins, ER2C41-based immunotoxin demonstrated approximately 15- to 20-fold enhanced cytotoxicity in EGFR-overexpressing cell lines. On the other hand, it was not endowed with cytoplasmic penetration ability. ER CT(AAA)-based immunotoxin and non-toxin-fused ER The 2C41 antibody was found to have very low cytotoxicity in cell lines overexpressing EGFR.

[0623]

[0624] Example 40. Evaluation of the efficacy of an immunotoxin with cytoplasmic penetration ability in mice.

[0625] To evaluate the in vivo efficacy of immunotoxins with cytoplasmic penetration ability, mouse experiments were performed. EGFR-overexpressing cell line A431 was dissociated by trypsin treatment and 1 × 10 8 A cell suspension of 10 cells / mL was prepared and mixed with an equal amount of Matrigel (Corning, 354234). 100 μL (5 × 10) was injected subcutaneously into the flank of 4-week-old female nude mice (Balb / c nude) using a 1 mL syringe. 6 cells) were injected. After 4 to 5 days, when the tumor size reached 80 to 100 mm3, the mice were randomly divided into 4 groups and (i) vehicle, (ii) ERCT-DTA 20 mg / kg, (iii) ERCT(AAA)-DTA 20 mg / kg, and (iv) ER2C41-DTA 20 mg / kg were injected into the tail vein at 3-day intervals. The tumor size and body weight were measured every 3 days, and when the tumor size in the vehicle-treated group reached 1,000 mm3, the treatment was stopped, the tumors were excised, and the masses were measured.

[0626] Figure 51 shows the results of evaluating the tumor size during 6 treatments and the tumor weight after the end of treatment. There is no cytoplasmic invasion. ERIn the group treated with CT(AAA)-DTA, there was no tumor growth inhibition effect at all, similar to the vehicle. ER CT-DTA and ER For 2C41-DTA, tumor growth inhibition effects of 45% and 96% were confirmed, respectively, and it was confirmed that this difference was similar to Example 39, and that the 2C41-based immunotoxin with improved cytoplasmic penetration ability showed a greater tumor growth inhibition effect. Figure 52 shows the results of evaluating the survival rate and body weight increase and decrease of mice during treatment, and the results of evaluating the serum ALT and AST levels after the end of treatment. No individual died or showed significant changes in body weight during the six administration periods. In addition, no significant changes were observed in the serum ALT and AST levels in any experimental group. Through this, it was confirmed that the immunotoxin based on a cytoplasmic penetrating antibody with an endosomal escape structural motif introduced into the antibody constant region exhibits a therapeutic effect on target cancer cells without side effects, and that improved cytoplasmic penetration ability can also lead to improved therapeutic effects.

[0627]

[0628] The IgG high-efficiency cytoplasmic penetrating antibody having an endosomal escape structural motif in the antibody constant region according to the present invention is actively internalized in living cells and located in the cytoplasm through the endosomal escape ability in the endosome, so that the IgG antibody can ultimately be distributed from outside the living cell to the cytoplasm without the help of a special external protein delivery system or substance.

[0629] IgG cytoplasmic penetrating antibodies with WYW endosomal escape structural motifs in previously developed VH and VL (e.g., inCT, epCT65, ERIn the case of CT, etc., there was a limitation that the effect in the cytoplasm was not strong because the amount of antibody reaching the cytoplasm was small due to the low endosomal escape ability. As a new strategy to overcome this, a cytoplasm-penetrating antibody with an endosomal escape structural motif in the antibody constant region can be located in the cytoplasm with an endosomal escape ability that is approximately 2-3 times higher than that of existing cytoplasm-penetrating antibodies. Ultimately, this significantly increases the amount of antibody distributed in the cytoplasm by the cytoplasm-penetrating antibody.

[0630] In addition, when various payloads (e.g., proteins, fusion proteins, enzymes, toxins, siRNA, small molecule compounds, peptides, DNA, mRNA, etc.) are fused to a high-efficiency cytoplasmic penetrating antibody having an endosomal escape structural motif in the antibody constant region according to the present invention, the loaded payload can be delivered into the cytoplasm very efficiently, and the effect of the payload can be expected.

[0631] In addition, the IgG high-efficiency cytoplasmic penetrating antibody having an endosomal escape structural motif in the antibody constant region according to the present invention overcomes the limitation of existing cytoplasmic penetrating antibodies that they must contain an endosomal escape structural motif in the VH / VL region, and has great versatility because it can deliver antibodies to the cytoplasm while maintaining the original function of other antibodies - targeting ability for target proteins - in the VH / VL region.

[0632] In addition, if the VH and VL of the IgG high-efficiency cytoplasmic penetrating antibody having an endosomal escape structural motif in the antibody constant region according to the present invention are made to specifically recognize a cytoplasmic target protein, it can be expected to have the effect of actively penetrating from outside the cell into the cytoplasm and inhibiting and / or activating the activity of the cytoplasmic target protein.

[0633] Moreover, the cytoplasmic penetrating antibody obtained by improving the target cell-specific antibody constant region according to the present invention has a high production yield and excellent biophysical properties, and maintains thermal stability and serum half-life similar to those of existing wild-type antibodies, making it easy to develop as a basic technology for therapeutic drugs, and unlike other cell-penetrating peptides that are non-specific, it can penetrate the cytoplasm specifically into target cells / tissues. In addition, since the constant region commonly possessed by general IgG antibodies has been improved, it has the versatility to be easily applied to other antibodies, and it can be utilized as a carrier to deliver various types of active substances into the cytoplasm of cells, and since strong cytoplasmic penetrating ability is granted only to cells that overexpress the target receptor, even if a highly toxic payload is fused, there is almost no toxicity in normal cells with low target receptor expression levels, and thus, it can be utilized in pharmaceutical compositions for the treatment and prevention of various diseases in the future.

[0634]

[0635] Electronic file attached.

Claims

1. A cytoplasmic penetrating antibody or fragment thereof comprising an endosomal escape structural motif in the heavy chain constant region and / or the light chain constant region.

2. A cytoplasmic penetrating antibody or fragment thereof, characterized in that in claim 1, the endosomal escape structural motif is included in the CH3 domain of the heavy chain constant region and / or the CL domain of the light chain constant region.

3. A cytoplasmic penetrating antibody or fragment thereof, characterized in that in the first paragraph, the endosomal escape structural motif is located in the AB, EF loop and C-terminus of the heavy chain constant region CH3 domain of sequence number 1.

4. In the third paragraph, a cytoplasmic penetrating antibody or fragment thereof, characterized in that the endosomal escape structural motif comprises the following, and the amino acid residue numbers follow the EU numbering: AB loop: 353 PS-XXEXXKXX 362 In the above AB loop, X can be selected from the group consisting of arginine (R), tryptophan (W), glutamic acid (E), alanine (A), asparagine (N), or glutamine (Q); EF loop: 411 TV-XXX-RW-XX-GNVF 423 In the above EF loop, X can be selected from the group consisting of arginine (R), tryptophan (W), glutamic acid (E), alanine (A), asparagine (N), or glutamine (Q); and C-terminal: 439 X-SLS-X-SPGK 447 At the C-terminus, X can be selected from the group consisting of arginine (R), tryptophan (W), glutamic acid (E), alanine (A), asparagine (N), or glutamine (Q).

5. A cytoplasmic penetrating antibody or fragment thereof, characterized in that in the first paragraph, the endosomal escape structural motif is located in the AB, EF loop and C-terminus of the light chain constant region CL domain of SEQ ID NO:

17.

6. In the fifth paragraph, a cytoplasmic penetrating antibody or fragment thereof, characterized in that the endosomal escape structural motif comprises the following, and the amino acid residue numbers follow the EU numbering: AB loop: 120 PS-XXQXXSXX 129 In the above AB loop, X can be selected from the group consisting of arginine (R), tryptophan (W), glutamic acid (E), alanine (A), asparagine (N), or glutamine (Q); EF loop: 180 TL-XXX-DY-XX-HKVY 192 In the above EF loop, X can be selected from the group consisting of arginine (R), tryptophan (W), glutamic acid (E), alanine (A), asparagine (N), or glutamine (Q); and C-terminal: 207 X-SFN-X-GEC 214 At the C-terminus, X can be selected from the group consisting of arginine (R), tryptophan (W), glutamic acid (E), alanine (A), asparagine (N), or glutamine (Q).

7. In the first paragraph, the endosomal escape structural motif comprises arginine (R), tryptophan (W) and glutamic acid (E), and a cytoplasmic penetrating antibody or fragment thereof characterized in that arginine (R), tryptophan (W) and glutamic acid (E) are structurally densely packed.

8. A cytoplasmic penetrating antibody or fragment thereof, characterized in that the heavy chain constant region CH3 domain including the endosomal escape structural motif in the first paragraph comprises one or more sequences selected from the group consisting of SEQ ID NOs: 2 to 12.

9. A cytoplasmic penetrating antibody or fragment thereof, characterized in that the light chain constant region CL domain including the endosomal escape structural motif in paragraph 1 comprises the sequence of SEQ ID NO: 18 or 19.

10. A cytoplasmic penetrating antibody or fragment thereof, characterized in that the endosomal escape structural motif in the first paragraph is included in IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA1, IgA2, IgD or kappa (κ) and lambda (λ) light chains.

11. In paragraph 1, a cytoplasmic penetrating antibody or fragment thereof characterized by comprising the following (1) or (2), or comprising (1) and (2): (1) A CH1-CL pair comprising a heavy chain constant region CH1 domain and a light chain constant region CL domain constant region including an endosomal escape structural motif; (2) a constant region CH3-CH3 pair comprising an endosomal escape structural motif in the heavy chain constant region CH3 domain; and / or (3) A constant region CH3-CH3 pair in which the constant region CH1-CL pair is replaced with the constant region CH3-CH3 pair of (2), and which includes an endosomal escape structural motif in the heavy chain constant region CH3 domain.

12. A cytoplasmic penetrating antibody or a fragment thereof, characterized in that the heavy chain constant region CH1 and light chain constant region CL pair in claim 11 are replaced with a CH3-CH3 pair including an endosomal escape structural motif, so that six CH3 domains include an endosomal escape structural motif, and the CH3-CH3 pair replacing the CH1-CL pair is a heterodimer.

13. A cytoplasmic penetrating antibody or fragment thereof, characterized in that the antibody comprising a CH3-CH3 pair replacing the CH1-CL pair in claim 12 comprises one or more sequences selected from the group consisting of SEQ ID NOs: 23 to 26.

14. A polynucleotide encoding a cytoplasmic penetrating antibody or a fragment thereof according to any one of claims 1 to 13.

15. A recombinant expression vector comprising the polynucleotide of clause 14.

16. An isolated host cell containing the recombinant expression vector of clause 15.

17. A composition for intracytoplasmic delivery of an active substance comprising a cytoplasmic penetrating antibody or a fragment thereof according to any one of claims 1 to 13.

18. A composition according to claim 17, characterized in that the active substance 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.

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