Anti-human transferrin receptor antibody with improved endosomal escape efficiency, and multispecific antibody and pharmaceutical composition using same

Abstract

The present invention relates to: an anti-human transferrin receptor antibody with improved endosomal escape efficiency; and a multispecific antibody and a pharmaceutical composition that use the anti-human transferrin receptor antibody. The present invention can provide an antibody having excellent endosomal escape efficiency by optimizing the interaction between the anti-human transferrin receptor antibody and the sugar chain region of the transferrin receptor. Accordingly, by using the anti-human transferrin receptor antibody of the present invention, a substance to be delivered can be more efficiently delivered into cells, and in particular, an active substance can enter cancer cells as well as be efficiently delivered through effective endosomal escape of the active substance. In addition, by using the anti-human transferrin receptor antibody of the present invention in a multispecific antibody, an antibody-drug conjugate, or an antibody-oligonucleotide conjugate, substances for treating cancer, brain diseases, or cell signaling-related diseases can be efficiently delivered into cells.

Description

Anti-human transferrin receptor antibody with improved endosomal escape efficiency, and multispecific antibody and pharmaceutical composition using the same

[0001] The present invention relates to an anti-human transferrin receptor antibody with improved endosomal escape efficiency, and a multispecific antibody and pharmaceutical composition using the same, and more particularly, to an anti-human transferrin receptor antibody with improved endosomal escape efficiency, enabling more effective delivery of a delivery substance into a cell, and a multispecific antibody and pharmaceutical composition using the same.

[0002] To effectively control disease-related targets within cells, precise delivery of therapeutic agents into the target cell, particularly the cytoplasm, is crucial. However, oligonucleotides and protein-based therapeutics, as polymers that have difficulty spontaneously crossing cell membranes, are limited in their ability to efficiently enter cells on their own. Therefore, these therapeutics are typically combined with antibodies, transport peptides, liposomes, or nanoparticles targeting cell surface receptors to induce intracellular uptake.

[0003] Most of these strategies utilize the endocytosis pathway to drive therapeutics into endosomes. However, the problem with this approach is that therapeutics trapped within endosomes are often degraded or released back into the cytoplasm, preventing their release into the cytoplasm. This results in an extremely low rate of reaching the target within the cytoplasm. Therefore, effective cytoplasmic delivery requires a method that enables both selective entry into target cells and effective escape from endosomes.

[0004] Meanwhile, the transferrin receptor (TfR), a receptor that mediates iron uptake, is known to be overexpressed in rapidly proliferating cancer cells and brain cells that require high metabolic activity. The transferrin receptor accepts iron-bound transferrin from the cell membrane and transports it into the cell via endosomes. Once the iron is released, the receptor is recycled. Based on these physiological characteristics, antibodies that bind to the transferrin receptor can be used to deliver therapeutic agents or polymeric drugs into target cells via the endosomal pathway.

[0005] However, the wild-type anti-transferrin receptor antibodies known to date have a low escape efficiency within endosomes, limiting the rate at which therapeutic agents reach the cytoplasm. This significantly hinders therapeutic efficacy and limits their practicality as intracellular therapeutic delivery platforms.

[0006] In this regard, Korean Patent No. 10-2527941 discloses a technology for passing through the blood-brain barrier or delivering drugs to specific tissues using an anti-transferrin receptor antibody, but the technology does not present specific technical means or improvement plans for effective escape from endosomes to the cytoplasm after the drug or antibody enters the cell.

[0007] In this situation, the inventors of the present invention discovered that by introducing a mutation into an anti-transferrin receptor antibody, it is possible to selectively enter cells through the transferrin receptor while improving the efficiency of endosomal escape, thereby effectively delivering a substance such as a therapeutic agent into cells, and thus completed the present invention.

[0008] An object of the present invention is to provide an anti-human transferrin receptor antibody (anti-hTfR) with improved endosomal escape efficiency.

[0009] Another object of the present invention is to provide a multispecific antibody produced using the above anti-human transferrin receptor antibody.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating one or more diseases selected from cancer, brain diseases, and cell signaling-related diseases, comprising the anti-human transferrin receptor antibody or multispecific antibody.

[0011] In order to achieve the above object, the present invention provides an anti-human transferrin receptor antibody comprising a heavy chain variable region (VH) consisting of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; and a light chain variable region (VL) consisting of an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, wherein at least one residue of serine (S) and tyrosine (Y) in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 is independently substituted with asparagine (N) or histidine (H).

[0012] In the present invention, one or more residues of S28 and Y96 in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 may be independently substituted with asparagine (N) or histidine (H).

[0013] In the present invention, the substitution may include one or more mutations of S28N and Y96H in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

[0014] In the present invention, one or more drug compounds may be conjugated to the anti-human transferrin receptor antibody.

[0015] In the present invention, the drug compound may be at least one selected from the group consisting of siRNA, miRNA, shRNA, growth inhibitors, toxins, radioactive isotopes, and nanoparticles.

[0016] In the present invention, the binding domain of the anti-human transferrin receptor antibody may be one.

[0017]

[0018] In addition, the present invention provides a multispecific antibody comprising at least one first binding domain that binds to human transferrin receptor (hTfR) and at least one second binding domain that binds to a target molecule, wherein the first binding domain comprises a heavy chain variable region (VH) consisting of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; and a light chain variable region (VL) consisting of an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, wherein at least one residue of serine (S) and tyrosine (Y) in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 is independently substituted with asparagine (N) or histidine (H).

[0019] In the present invention, the first binding domain may be in a form selected from the group consisting of Fab, scFv, di-scFv, dsFv, and (dsFv)2.

[0020] In the present invention, one or more drug compounds may be conjugated to the multispecific antibody.

[0021] In the present invention, the drug compound may be at least one selected from the group consisting of siRNA, miRNA, shRNA, growth inhibitors, toxins, radioactive isotopes, and nanoparticles.

[0022]

[0023] The present invention also provides a pharmaceutical composition for preventing or treating one or more diseases selected from the group consisting of cancer, brain disease, and cell signaling-related diseases, comprising the anti-human transferrin receptor antibody.

[0024] In the present invention, the cancer may be one or more diseases selected from the group consisting of pancreatic cancer, liver cancer, stomach cancer, blood cancer, bone marrow cancer, brain cancer, lung cancer, and skin cancer.

[0025] In the present invention, the brain disease may be one or more diseases selected from the group consisting of Parkinson's disease, Alzheimer's disease, traumatic brain injury, stroke, Huntington's disease, amyotrophic lateral sclerosis, spinal cord injury, alcoholic encephalopathy, alcoholic dementia, and Wernicke-Korsakoff's syndrome.

[0026] In the present invention, the cell signaling-related disease may be one or more diseases selected from the group consisting of diabetes, inflammatory diseases, immune diseases, and diabetic dementia.

[0027] The present invention optimizes the interaction between an anti-human transferrin receptor antibody and the sugar chain of the transferrin receptor, thereby providing an antibody with superior endosomal escape efficiency. Accordingly, the anti-human transferrin receptor antibody of the present invention enables more effective intracellular delivery of a delivery agent. Specifically, it enables effective delivery of the delivery agent by simultaneously allowing the delivery agent to enter cancer cells and effectively escaping from the endosomes.

[0028] In addition, when the anti-human transferrin receptor antibody of the present invention is used in a multispecific antibody, antibody-drug conjugate, or antibody-oligonucleotide conjugate, a substance for treating cancer, brain disease, or cell signaling-related disease can be efficiently delivered into cells.

[0029] Figure 1 schematically illustrates the structure of a multispecific antibody according to one embodiment of the present invention.

[0030] Figure 2a shows the results of comparing the VH sequence of chimeric antibody 128.1 and a humanized VH candidate sequence according to one embodiment of the present invention.

[0031] Figure 2b shows the results of comparing the VL sequence of chimeric antibody 128.1 and a humanized VL candidate sequence according to one embodiment of the present invention.

[0032] Figure 3 shows the SDS PAGE results for a humanized anti-human transferrin receptor antibody according to one embodiment of the present invention.

[0033] Figure 4a shows the structure of the Fab of a humanized anti-human transferrin receptor antibody according to one embodiment of the present invention.

[0034] Figure 4b shows the results of comparing the structures of pre-humanization and post-humanization antibodies according to one embodiment of the present invention.

[0035] Figure 5 shows the results of comparing the binding affinity of a humanized anti-human transferrin receptor antibody to a transferrin receptor according to one embodiment of the present invention with that of an antibody prior to humanization.

[0036] Figure 6a shows a 2D image of a human transferrin receptor and antibody complex according to one embodiment of the present invention.

[0037] Figure 6b shows a 3D image of a human transferrin receptor and antibody complex according to one embodiment of the present invention.

[0038] Figure 6c shows the structure of a complex of a transferrin receptor and an antibody according to one embodiment of the present invention superimposed on the structure of a previously identified transferrin receptor.

[0039] FIG. 6d is a visual representation of the peripheral structure of S28 (Ser28) present in the interaction site between the anti-transferrin receptor antibody variable region and the transferrin receptor sugar chain according to one embodiment of the present invention.

[0040] Figure 7 shows the modeling results for the sugar chain portion of a human transferrin receptor and antibody complex according to one embodiment of the present invention.

[0041] Figure 8 illustrates a schematic process of a split luciferase assay for measuring the endosomal escape efficiency of an anti-human transferrin receptor antibody according to one embodiment of the present invention.

[0042] Figure 9 shows the SDS-PAGE results of an anti-human transferrin receptor antibody bound to HiBiT according to one embodiment of the present invention.

[0043] Figure 10 shows the results of measuring the intracellular transmission signal of an anti-human transferrin receptor antibody according to one embodiment of the present invention.

[0044] Figure 11a shows the results of measuring the relative activity of an anti-human transferrin receptor antibody according to one embodiment of the present invention.

[0045] Figure 11b shows the normalized endosomal escape efficiency of an anti-human transferrin receptor antibody according to one embodiment of the present invention.

[0046] Figure 12a shows the results of measuring the survival rate of K562 cells according to one embodiment of the present invention.

[0047] Figure 12b shows the results of measuring the survival rate of HEK293 cells according to one embodiment of the present invention.

[0048] Figure 13a illustrates the structure of an anti-transferrin receptor antibody-oligonucleotide conjugate according to one embodiment of the present invention.

[0049] Figure 13b shows the results of measuring the expression level of DUSP28 mRNA in cancer cells treated with an anti-transferrin receptor antibody-oligonucleotide conjugate according to one embodiment of the present invention.

[0050] Figure 13c shows the results of inhibition of cancer cell colony formation by treatment with an anti-transferrin receptor antibody-oligonucleotide conjugate according to one embodiment of the present invention.

[0051] Figure 13d shows the number of cancer cell colonies following treatment with an anti-transferrin receptor antibody-oligonucleotide conjugate according to one embodiment of the present invention.

[0052] Hereinafter, specific implementations of the present invention will be described in more detail. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0053]

[0054] The present invention relates to an anti-transferrin receptor antibody with improved endosomal escape efficiency.

[0055] An anti-transferrin receptor antibody (anti-TfR antibody) refers to an antibody that binds to the transferrin receptor (TfR). Specifically, the term "anti-transferrin receptor antibody" used in the present invention may be an antibody that undergoes endocytosis when bound to the human transferrin receptor (human TfR, hTfR), and may have an amino acid sequence derived from chimeric antibody 128.1.

[0056] In the present invention, the term "antibody" includes a molecule derived from immunoglobulin (Ig) that immunologically has reactivity with a specific antigen(s), and the immunoglobulin may be IgG, IgA, IgE, IgD or IgM such as IgG1, IgG2, IgG3, IgG4. In addition, the term "antibody" includes both polyclonal antibodies and monoclonal antibodies, and is meant to include forms produced by genetic engineering such as chimeric antibodies (e.g., humanized murine antibodies), humanized antibodies and heterologous antibodies (e.g., bispecific antibodies, multispecific antibodies).

[0057] The anti-human transferrin receptor antibody of the present invention is an antibody protein comprising a variable region, and its form can be produced by changing it according to the purpose. The anti-human transferrin receptor antibody of the present invention can be a whole antibody having both Fab and Fc regions, an antibody fragment, or a recombinant antibody thereof. For example, the antibody fragment and recombinant antibody can be in the form of Fab, scFv, di-scFv, dsFv, (dsFv)2, etc., or a form in which these are linked to an Fc region.

[0058] In the present invention, the term "mutation" means a substitution, insertion, and / or deletion of an amino acid residue. Preferably, a mutation in the present invention includes a substitution of an amino acid residue. The substitution of an amino acid residue is indicated by the amino acid residue present in the parent wild-type protein, the number of the amino acid residue, and the order of the substituted amino acid residue.

[0059] In the present invention, by confirming various antibody mutations and the structure of receptor-antibody complexes using cryo-electron microscopy and modeling structures, it was discovered that anti-transferrin receptor antibodies bind to and interact with the sugar chain region of the transferrin receptor, thereby inducing a structural change of the transferrin receptor, and that the regulation of the interaction between the antibody and the sugar chain region of the receptor is important for optimizing the efficiency of endosomal escape.

[0060] In particular, it was found that the amino acid residues of the anti-transferrin receptor antibody that interact with the sugar chain region of the transferrin receptor are located adjacent to the CDR grafting region for humanization of the antibody, and thus, in order to optimize the endosomal escape efficiency of the humanized anti-transferrin receptor antibody, both CDR grafting for antibody humanization and control of antibody-receptor sugar chain region interactions must be considered.

[0061] In addition, we found that the affinity of anti-transferrin receptor antibodies and transferrin receptors can vary depending on the pH change of the endosome, and that the efficiency of endosomal escape can be increased by introducing amino acid mutations that can optimize the affinity of anti-transferrin receptors and transferrin receptors depending on the pH change.

[0062] Specifically, anti-transferrin receptor antibodies enter cells via receptor-mediated endocytosis in the form of endosomes, which become acidic pH environments. Under these acidic pH conditions, amino acid mutations that alter the affinity of anti-transferrin receptor antibodies to the transferrin receptor can be introduced to optimize the affinity between the anti-transferrin receptor antibodies and the transferrin receptor, thereby increasing the efficiency of endosomal escape.

[0063] In particular, when a specific amino acid residue is substituted with histidine, histidine has a positive charge under acidic conditions, and this positive charge can affect the affinity of anti-transferrin receptor antibodies and transferrin receptors, thereby increasing the efficiency of endosomal escape.

[0064] Accordingly, the present invention provides an anti-transferrin receptor antibody exhibiting excellent endosomal escape efficiency by introducing a mutation into an amino acid residue that interacts with the sugar chain portion of the transferrin receptor in the anti-transferrin receptor antibody, or by introducing a mutation into an amino acid residue that can affect the affinity of the anti-transferrin receptor antibody and the transferrin receptor under acidic pH conditions.

[0065]

[0066] The anti-human transferrin receptor antibody with improved endosomal escape efficiency according to the present invention comprises an amino acid sequence based on the heavy chain variable region (VH) and / or the light chain variable region (VL) of chimeric antibody 128.1, which is reported to bind to the human transferrin receptor and cause endocytosis.

[0067] In the present invention, the term "chimeric antibody" refers to an antibody in which the variable region is derived from a non-human species and the constant region is derived from a different species (e.g., human).

[0068] The numbering in the amino acid sequence of the variable region of the above chimeric antibody 128.1 follows the Kabat numbering.

[0069] The wild-type (WT) sequences of the heavy chain variable region (VH) and light chain variable region (VL) of the above chimeric antibody 128.1 can be represented by sequence numbers 1 and 2 below, respectively.

[0070]

[0071] [Sequence number 1] Chimeric128.1 VH

[0072] EVQLQQSGPELVKPGASMKISCKASGYSFTGYTMNWVKQSHGENLEWIGRINPHNGGTDYNQKFKDKAPLTVDKSSNTAYMELLSLTSEDSAVYYCARGYYYYSLDYWGQGTSVTVSS

[0073]

[0074] [Sequence number 2] Chimeric128.1 VL

[0075] QIVLTQSPAIMSASPGEKVTTMTCSASSSIDYIHWYQQKSGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEPEDAATYYCHQRNSYPWTFGGGTRLEIR

[0076]

[0077] In addition, the anti-human transferrin receptor antibody with improved endosomal escape efficiency according to the present invention may be a humanized antibody comprising an amino acid sequence in which the variable region sequence of the chimeric antibody 128.1 is humanized.

[0078] In the present invention, the term "humanized antibody" refers to a non-human antibody that has been genetically engineered to include a non-human variable domain that has been modified to have a high level of sequence homology to human antibody constant and variable domains. The humanized antibody can be produced by grafting the complementarity determining region (CDR) of a non-human antibody onto a homologous human acceptor framework region (FR).

[0079] In the present invention, a humanized anti-human transferrin receptor antibody was manufactured by comparing the amino acid sequences of the VH and VL regions of the existing anti-human transferrin receptor antibody 128.1 with the VH and VL of human antibodies registered in the NCBI database to find a structurally similar human antibody framework sequence and performing CDR grafting.

[0080] The sequence of the heavy chain variable region of the humanized anti-human transferrin receptor antibody manufactured through this can be represented by SEQ ID NO: 3 below, and the sequence of the light chain variable region can be represented by SEQ ID NO: 4 below.

[0081]

[0082] [Sequence number 3] Humanized 128.1 VH

[0083] EVKLQQSGPELVKPGASVKMSCKASGYSFTGYTMNWVKQKPGQGLEWIGRINPHNGGTDYNQKFKDKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGYYYYSLDYWGQGTTLTVSS

[0084]

[0085] [Sequence number 4] Humanized 128.1 VL

[0086] QIVLTQSPAIMSASPGEKVTTMTCSASSSIDYIHWYQQKPGTSPKKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRNSYPWTFGGGTKLEIK

[0087]

[0088] The anti-human transferrin receptor antibody according to the present invention comprises a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0089] In addition, the heavy chain variable region and the light chain variable region may each include an amino acid sequence that is at least 90% identical, preferably 95% identical, and more preferably 98% identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, respectively.

[0090] In the present invention, it was discovered that anti-human transferrin receptor antibody 128.1, which is reported to bind to the transferrin receptor and cause endocytosis, interacts with the sugar chain region of the receptor when binding to the transferrin receptor, and this interaction changes the structure of the transferrin receptor. It was also discovered that the endosomal escape efficiency of the anti-human transferrin receptor antibody can be improved by introducing a mutation in the amino acid sequence related to this structural change.

[0091] In addition, we found that introducing mutations in specific amino acid residues in the acidic pH environment inside the endosome can improve the efficiency of endosomal escape by changing the affinity of anti-transferrin receptor antibodies and transferrin receptors in the acidic pH environment.

[0092] Specifically, the anti-human transferrin receptor antibody according to the present invention may have a structure in which at least one residue of serine (S) and tyrosine (Y) in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 is independently substituted with asparagine (N) or histidine (H).

[0093] Preferably, at least one residue among S28 and Y96 in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 may be independently substituted with asparagine (N) or histidine (H), and more preferably, at least one mutation among S28N and Y96H in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 may be included.

[0094] In one embodiment of the present invention, by modeling the structure of the receptor-antibody complex and confirming it with an ultra-low temperature microscope, it was confirmed that the S28 residue in the amino acid sequence of SEQ ID NO. 1 or SEQ ID NO. 3 affects the interaction between the sugar chain region of the transferrin receptor and the transferrin receptor antibody, thereby improving the endosomal escape efficiency of the transferrin receptor antibody.

[0095] Meanwhile, the binding domain of the anti-transferrin receptor antibody of the present invention may be single. Specifically, the anti-transferrin receptor antibody may be in a form in which one of the two binding domains present in the antibody is deleted.

[0096] According to the present invention, an anti-human transferrin receptor antibody having improved endosomal escape efficiency can be provided by regulating the interaction between the sugar chain portion of the transferrin receptor and the transferrin receptor antibody through the mutation, or by changing the affinity of the transferrin receptor and the transferrin receptor antibody in an acidic pH environment. The anti-human transferrin receptor antibody of the present invention can be used in a multispecific antibody, antibody-drug conjugate, or antibody-oligonucleotide conjugate to facilitate the endosomal escape process within cells expressing the transferrin receptor on the surface, thereby enabling more effective delivery of drugs, therapeutic agents, oligonucleotides, and the like into the cells.

[0097] In particular, in one embodiment of the present invention, it was confirmed that by combining siRNA capable of suppressing the expression of a specific protein in cancer cells with an anti-human transferrin receptor antibody that has introduced a mutation and delivering it into cancer cells, cancer cells can be effectively killed, thereby exhibiting a cancer treatment effect.

[0098]

[0099] Accordingly, the present invention also provides a multispecific antibody using the anti-human transferrin receptor antibody.

[0100] In the present invention, the term "multispecific antibody" means an antibody capable of binding to two or more different antigens or receptors, such as a bispecific antibody, a trispecific antibody, etc., and includes a form produced by genetic engineering.

[0101] In the present invention, the multispecific antibody may comprise one or more first binding domains that bind to the human transferrin receptor (hTfR) and one or more second binding domains that bind to a target molecule. Furthermore, the multispecific antibody according to the present invention may further comprise one or more binding domains (multiple binding domains) that are different from the first and second binding domains. In this case, in addition to the targets of the first and second binding domains, the antibody may bind to other target molecules, suggesting various therapeutic strategies.

[0102] In the present invention, the term "binding domain" is interpreted as a concept encompassing antibody-derived proteins, biological proteins, and artificially designed interacting proteins. For example, the second binding domain and the multiple binding domains may each independently include, in addition to antibody-derived proteins, biological proteins or artificially designed interacting proteins.

[0103] For example, the multispecific antibody of the present invention may include a first binding domain that binds to a human transferrin receptor, a second binding domain that binds to a target molecule, and an Fc region, in which case the first binding domain and the second binding domain may have a structure in which they are linked to the Fc region. Alternatively, a fusion protein produced to have multispecificity using a linker other than an Fc region-derived protein may also be included in the category of the multispecific antibody of the present invention.

[0104] Specifically, the multispecific antibody of the present invention may comprise a variable region sequence of an anti-human transferrin receptor antibody of the present invention in one arm (a first binding domain) and a therapeutic protein capable of binding to a target expressing a transferrin receptor in the other arm (a second binding domain). Accordingly, the multispecific antibody of the present invention binds to the human transferrin receptor through the first binding domain and moves into cells through transcytosis, thereby effectively delivering the therapeutic protein of the second binding domain into the cells.

[0105] In the present invention, the first binding domain is a region including a heavy chain variable region (VH) and a light chain variable region (VL), and its form can be manufactured by changing it according to the purpose. In the present invention, the first binding domain can be a Fab form including VH-CH1 and VL-CL, a fragment thereof, or a recombinant form. For example, the first binding domain can be in the form of Fab, scFv, di-scFv, dsFv, (dsFv)2, etc.

[0106] In addition, the multispecific antibody of the present invention may be in a form in which the second binding domain is deleted, and specifically, the multispecific antibody may be in a form in which a binding domain (first binding domain) is present on one arm, but only an Fc region to which a binding domain is not attached is present on the other arm.

[0107] In the multispecific antibody of the present invention, the heavy chain variable region (VH) of the first binding domain comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, and the light chain variable region (VL) comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, wherein at least one residue of serine (S) and tyrosine (Y) in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 is independently substituted with asparagine (N) or histidine (H). Accordingly, the first binding domain can effectively induce transcytosis or endocytosis through the human transferrin receptor to transport the multispecific antibody into cells and facilitate endosomal escape.

[0108] In the multispecific antibody of the present invention, the second binding domain may comprise a protein that binds to a target molecule, such as an antigen-binding region of an antibody that binds to a target molecule, or a therapeutic protein. In one embodiment of the present invention, the second binding domain may bind to a target molecule to promote neuronal growth.

[0109] In the second binding domain, the antigen-binding region of the antibody that binds to the target molecule may refer to a portion of the antibody that specifically binds to part or all of the antigen (target molecule) and includes a region complementary to part or all of the antigen. The form of the antigen-binding region is not particularly limited, and may be in the form of not only a Fab form but also sdAb, scFv, di-scFv, dsFv, (dsFv)2, etc.

[0110] The Fab form includes the CH1 domain of the variable region (VH) and constant region (CH) of the heavy chain, and the variable region (VL) and constant region (CL) of the light chain, and a disulfide bond is formed between the CH1 and CL. In addition, the sdAb form refers to a single-domain variable fragment, and refers to one variable region domain.

[0111] Meanwhile, the scFv form refers to a single-chain variable fragment in which variable regions are connected, and refers to a recombinant domain in which VH and VL regions are connected by a peptide linker. In addition, the di-scFv form refers to a recombinant domain in which two scFvs are connected by a linker. The linker may be a linker known in the art, and may be a peptide composed of 5 to 20 amino acids. Preferably, the linker may be composed of one or more amino acids selected from the group consisting of G, A, S, P, E, T, D, and K. For example, the linker may be (GGGGX) n It can be, and it is preferable that X is A or S, and it is preferable that n is a natural number from 1 to 4.

[0112] In addition, the dsFv form is similar to scFv in that the variable regions are connected as disulfide-linked variable fragments, but it refers to a recombinant domain in which the VH and VL regions are connected by a disulfide bond rather than a linker. The (dsFv)2 form refers to a recombinant domain in which two dsFvs are connected by a linker.

[0113] The target molecule of the second binding domain may be a variety of cellular functional proteins, such as dual specificity protein phosphatase, RAF kinase, MAP kinase, and K-ras, which are intracellular proteins that act in the cell signaling process.

[0114]

[0115] In the present invention, the first binding domain and the second binding domain may have a form linked to each chain of the Fc region.

[0116] In the present invention, the term "Fc region" refers to a C-terminal region including the CH2 and CH3 domains (or CH2, CH3, and CH4 domains) among the heavy chain constant regions of an immunoglobulin, and is used to encompass a wild-type Fc region and variants thereof. The immunoglobulin that serves as the parent of the Fc region may be IgG1, IgG2, IgG3, or IgG4, and preferably IgG1.

[0117] In the present invention, the Fc region may refer to a region extending from residue 221 of a human IgG1 heavy chain to the C-terminus, or a region further including a hinge in the region. The numbering of amino acid residues in the Fc region follows the EU numbering, which defines the numbering of residues within a human immunoglobulin heavy chain.

[0118] In the present invention, the term "wild-type Fc region" includes an amino acid sequence that matches the amino acid sequence of the Fc region of an immunoglobulin found in nature.

[0119] In the present invention, the term "Fc region variant" refers to one that includes one or more amino acid residues that differ from the wild-type Fc region, and may be abbreviated as "Fc variant." In the present invention, the Fc variant may have a homology of about 80% or more, preferably about 90% or more, with the parent wild-type Fc region sequence.

[0120] In the present invention, each chain of the Fc region may include heavy chain sequences 221 to 447 of IgG1. The heavy chain sequences 221 to 447 of IgG1 may be represented by the amino acid sequence of SEQ ID NO: 5 below.

[0121]

[0122] [SEQ ID NO: 5] IgG1 221-447

[0123] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0124]

[0125] In the present invention, each binding domain and Fc region may be linked by 0 to 20 amino acid residues. That is, the binding domain and Fc region may be directly linked or linked via a linker consisting of 1 to 20 amino acids. In this case, each binding domain may be linked to an amino acid located at the N-terminus, C-terminus, or between them of the Fc region, and preferably, may be linked to the N-terminus.

[0126] For example, the first binding domain may be linked to one to three of the four possible binding sites, including the two N-terminals and the two C-terminals of the dimer of the Fc region, and the second binding domain may be linked to one or more of the remaining sites. Alternatively, it is also possible to produce and use fusion proteins having various orientations in which the first binding domain is linked to the second binding domain via a linker.

[0127] In the present invention, a recombinant variant can be formed in the Fc region to form a multispecific antibody.

[0128] For example, when the first binding domain and the second binding domain are attached to the two chains of the Fc region dimer, the dimer can be formed by introducing a recombinant variant. The recombinant variant for forming the dimer can be formed using the knob-into-hole technology.

[0129] The above knob-into-hole technology is designed to form only heterodimers between the heavy chains of antibody fragments. Here, the knob is designed to have a side chain protruding toward the opposite chain and is inserted into the hole of the opposite domain. As a result, the heavy chains cannot homodimerize due to side chain collisions, and only heterodimerization is possible. In the present invention, one of the two chains constituting the Fc region may have a knob structure and the other may have a hole structure. In this case, the chain in which the knob or hole is formed is referred to as Fc-knob or Fc-hole, respectively.

[0130] The above Fc-knob can be formed by substituting one or more amino acids in a chain constituting the Fc region with a large amino acid selected from the group consisting of tryptophan (W), arginine (R), phenylalanine (F), and tyrosine (Y). For example, the Fc-knob can be formed by forming a T366W mutation in the sequence 221 to 447 of the IgG1-Fc heavy chain.

[0131] The above Fc-hole can be formed by substituting one or more amino acids in the chain constituting the Fc region with a small amino acid selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). For example, the Fc-hole can be formed by mutations T366S, L368A, and Y407V in the sequence 221 to 447 of the IgG1-Fc heavy chain.

[0132] Figure 1 schematically illustrates the structure of a multispecific antibody according to an exemplary embodiment of the present invention. The multispecific antibody of the present invention may have a structure in which a first binding domain in the form of a Fab is linked to an Fc-hole, and a second binding domain including a therapeutic protein that binds to a target molecule is linked to an Fc-knob.

[0133] In one embodiment of the present invention, when the first binding domain and the second binding domain are attached to the N-terminus and C-terminus (or vice versa) of each Fc chain, it is possible to produce a multispecific antibody using an Fc homodimer. In this case, it can be called an Fc homodimer multispecific antibody.

[0134] The multispecific antibody of the present invention can readily enter cells expressing the human transferrin receptor through its domain that binds to the human transferrin receptor and can easily cross the blood-brain barrier. This allows the domain that binds to the target molecule and exerts a therapeutic effect to be effectively delivered into the cell, resulting in excellent therapeutic effects for diseases associated with the transferrin receptor.

[0135] Specifically, since the mutant antibody according to the present invention is capable of effective endocytosis into cells expressing the transferrin receptor, by using this, a therapeutic agent targeting a disease target inside cancer cells expressing the transferrin receptor can be effectively delivered into the cancer cells, and thus can be usefully used in the development of cancer therapeutic agents. In addition, since the transferrin receptor is closely connected to various cell signaling pathways, the mutant antibody according to the present invention can also be utilized in the treatment or prevention of diseases related to cell signaling abnormalities, such as diabetes, inflammatory diseases, immune diseases, and diabetic dementia.

[0136]

[0137] The anti-human transferrin receptor antibody or multispecific antibody of the present invention may be conjugated with a drug compound or oligonucleotide to form an antibody-drug conjugate (ADC) or an antibody-oligonucleotide conjugate (AOC).

[0138] The above antibody-drug conjugate or antibody-oligonucleotide conjugate is intended to deliver a low-molecular-weight drug compound or oligonucleotide into a cell by utilizing endocytosis of an anti-human transferrin receptor antibody via a human transferrin receptor. When a drug compound or oligonucleotide is attached to the anti-human transferrin receptor antibody of the present invention or a multispecific antibody using the same, effective drug or oligonucleotide delivery into a cell expressing the transferrin receptor is possible, and the delivery efficiency of a drug or oligonucleotide that has difficulty passing through the blood-brain barrier can be improved. In addition, for the treatment of cancer, neuronal cell disease, or cell signaling-related disease, the efficiency of endosomal escape for transporting the drug or oligonucleotide into the cytoplasm of a cell is improved, enabling effective delivery of the drug or oligonucleotide.

[0139] In the present invention, the drug or oligonucleotide may be linked to the C-terminus and / or N-terminus of each antibody chain, as well as to one or more amino acid residues within the chain. The drug or oligonucleotide may be conjugated using a suitable linker. The drug or oligonucleotide may be any drug compound, growth inhibitor, toxin, radioisotope, miRNA, siRNA, shRNA, nanoparticle, etc. known in the art.

[0140] In particular, in one embodiment of the present invention, it was confirmed that by combining siRNA capable of suppressing the expression of a specific protein in cancer cells with an anti-human transferrin receptor antibody that has introduced a mutation and delivering it into cancer cells, cancer cells can be effectively killed, thereby exhibiting a cancer treatment effect.

[0141]

[0142] The present invention also relates to a pharmaceutical composition comprising the anti-human transferrin receptor antibody of the present invention or a multispecific antibody using the same.

[0143] The pharmaceutical composition may be used for the treatment of cancer, and the cancer may include one or more selected from the group consisting of pancreatic cancer, liver cancer, stomach cancer, blood cancer, bone marrow cancer, brain cancer, lung cancer, and skin cancer. In these cancer diseases, transferrin receptors are overexpressed on the surface of cancer cells for iron metabolism and cell proliferation, and thus, the anti-human transferrin receptor antibody of the present invention or a multispecific antibody using the same can effectively deliver a therapeutic agent into the cancer cells.

[0144] In one embodiment of the present invention, when cancer cells expressing transferrin receptors were treated with the anti-human transferrin receptor antibody of the present invention, it was confirmed that the antibody selectively entered the cancer cells, thereby enabling effective delivery of therapeutic substances into the cancer cells.

[0145] In addition, the pharmaceutical composition may be used for treating a brain disease, and the brain disease may include at least one selected from the group consisting of Parkinson's disease, Alzheimer's disease, traumatic brain injury, stroke, Huntington's disease, amyotrophic lateral sclerosis, spinal cord injury, alcoholic encephalopathy, alcoholic dementia, and Wernicke-Korsakoff's syndrome.

[0146] For example, when a multispecific antibody manufactured by fusing with a protein that promotes nerve cell growth according to one embodiment of the present invention is used, it can be effectively used for the prevention or treatment of brain diseases such as Parkinson's disease, Alzheimer's disease, and traumatic brain injury.

[0147] The above pharmaceutical composition may be used for treating a disease related to cell signaling associated with a transferrin receptor, and the disease related to cell signaling may include at least one selected from the group consisting of diabetes, inflammatory diseases, immune diseases, and diabetic dementia.

[0148] In these cell signaling-related diseases, transferrin receptors are overexpressed on the cell surface due to DNA synthesis, cell growth, immune response, energy metabolism, redox reactions, etc., and therefore, by using the anti-human transferrin receptor antibody of the present invention, a therapeutic substance for the disease can be effectively delivered into the cells.

[0149] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the antibody of the present invention.

[0150] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are 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 of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0151] The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0152] The pharmaceutical composition of the present invention may be formulated in the form of a sterile injection solution, a lyophilized formulation, a pre-filled syringe solution, an oral formulation, a topical preparation, or a suppository, according to conventional methods. Since proteins or peptides are digested upon oral administration, oral compositions may be formulated to coat the active agent or protect it from degradation in the stomach.

[0153] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0154] The pharmaceutical composition of the present invention may further comprise at least one other therapeutic or diagnostic agent. For example, it may further comprise interferon, anti-S protein monoclonal antibody, anti-S protein polyclonal antibody, nucleoside analog, DNA polymerase inhibitor, or siRNA agent.

[0155] The appropriate dosage of the pharmaceutical composition of the present invention may 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 daily dosage of the pharmaceutical composition of the present invention may be 0.001 to 100 mg / kg.

[0156] The present invention also relates to a method for preventing or treating one or more diseases selected from the group consisting of cancer, brain diseases, and cell signaling-related diseases, wherein the method may include a step of administering a pharmaceutical composition comprising the anti-human transferrin receptor antibody of the present invention or a multispecific antibody using the same.

[0157] In the present invention, the subject of administration may be a subject, specifically a subject requiring an anti-human transferrin receptor antibody or a multispecific antibody using the same, and the subject may be an animal, and typically a mammal.

[0158]

[0159] Example

[0160]

[0161] The present invention is described in more detail through the following examples. However, these examples are intended to illustrate some experimental methods and configurations of the present invention, and the scope of the present invention is not limited to these examples.

[0162]

[0163] Experimental Example 1: Analysis of amino acid residues involved in the interaction between the sugar chain region of the transferrin receptor and the transferrin receptor antibody.

[0164]

[0165] 1-1. Determination of candidate sequences of humanized anti-human transferrin receptor antibodies

[0166]

[0167] CDR grafting:

[0168] To humanize antibodies, the amino acid sequences of the existing anti-human transferrin receptor antibody 128.1 VH and VL were compared with the VH and VL sequences of human antibodies registered in the NCBI database to identify framework sequences suitable for producing humanized anti-human transferrin receptor antibodies. For CDR grafting, the CDR and FR regions of the 128.1 antibody sequence were separated using the AbYsis program according to methods such as Kabat and IMGT, and the same process was performed for human antibody candidates. The amino acid sequences of the CDR regions isolated from the 128.1 antibody and the FR sequences isolated from the human antibody candidates were recombined according to methods such as Kabat and IMGT to produce the VL and VH candidate sequences of one humanized antibody.

[0169] The VL and VH candidate sequences of the humanized anti-human transferrin receptor antibody produced as above are as follows.

[0170]

[0171] Humanization 128.1 VL Candidate Sequence_1:

[0172] QIVLTQSPAIMSASPGEKVTTMTCSASSSIDYIHWYQQKPGTSPKKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRNSYPWTFGGGTKLEIK

[0173]

[0174] Humanization 128.1 VL Candidate Sequence_2:

[0175] QIVSTQSPAIMSASPGEKVTTMTCSASSSIDYIHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRNSYPWTFGGGTKLEIK

[0176]

[0177] Humanization 128.1 VL Candidate Sequence_3:

[0178] QIVLTQSPAIMSASPGEKVTTMTCSASSSIDYMQWYQQKPGTSPKKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRNSYPWTFGGGTKLEIK

[0179]

[0180] Humanization 128.1 VL Candidate Sequence_4:

[0181] QIVSTQSPAIMSASPGEKVTMTCSASSSIDYMQWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRNSYPWTFGGGTKLEIK

[0182]

[0183] Humanization 128.1 VH candidate sequence_1:

[0184] EVKLQQSGPELVKPGASVKMSCKASGYSFTGYTMNWVKQKPGQGLEWIGRINPHNGGTDYNQKFKDKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGYYYYSLDYWGQGTTLTVSS

[0185]

[0186] Humanization 128.1 VH candidate sequence_2:

[0187] EVKLQQSGPELVKPGASVKMSCKASGYTFTGYTMNWVKQKPGQGLEWIGRINPHNGGTDYNQKFKDKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGYYYYSLDYWGQGTTLTVSSAST

[0188]

[0189] Humanization 128.1 VH candidate sequence_3:

[0190] EVKLQQSGPELVKPGASVKMSCKASGYSFTGYTMNWVKQKPGQGLEWIGRINPHNGGTDYNQKFKDKATTLTVDKSSSTAYMELSSLTSEDSAVYYCARGYYYYSLDYWGQGTTLTVSS

[0191]

[0192] Humanization 128.1 VH candidate sequence_4:

[0193] EVKLQQSGPELVKPGASVKMSCKASGYTFTGYTMNWVKQKPGQGLEWIGRINPHNGGTDYNQKFKDKATTLTVDKSSSTAYMELSSLTSEDSAVYYCARGYYYYSLDYWGQGTTLTVSS

[0194]

[0195] Humanization 128.1 VH candidate sequence_5:

[0196] EVKLQQSGPELVKPGASVKMSCKASGYSFTGYTMHWVKQKPGQGLEWIGYINPHNGGTNYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGYYYYSLDYWGQGTTLTVSS

[0197]

[0198] Humanization 128.1 VH candidate sequence_6:

[0199] EVKLQQSGPELVKPGASVKMSCKASGYSFTGYTMHWVKQKPGQGLEWIGYINPHNGGTNYNEKFKGKATLTVDKSSSTAYMELSSLTSEDSAVYYCARGYYYYSLDYWGQGTTLTVSS

[0200]

[0201] Antibody prediction model creation and amino acid optimization:

[0202] The generated sequence was compared with the VH sequence (SEQ ID NO: 1) and VL sequence (SEQ ID NO: 2) of chimeric antibody 128.1 to identify elements likely to cause instability through humanization. The amino acids corresponding to the Vernier zone were identified using the Biophi program, and the changes occurring in the corresponding amino acids due to humanization were examined. A humanization predicted structural model file was generated using the AlphaFold program. Based on the generated model file, amino acids likely to cause structural instability were identified, and these were reflected in the design of the humanized sequence.

[0203] The results of comparing the VH sequence (SEQ ID NO: 1) and VL sequence (SEQ ID NO: 2) of the above chimeric antibody 128.1 with the humanized VH and VL candidate sequences are shown in Figures 2a and 2b.

[0204] Through this process, among the candidate sequences, humanized 128.1 VH candidate sequence_1 (SEQ ID NO: 3) and VL candidate sequence_1 (SEQ ID NO: 4), which are sequences that are judged to be able to effectively bind to the human transferrin receptor and have the lowest instability through humanization, were used to produce humanized anti-human transferrin receptor antibodies.

[0205]

[0206] 1-2. Preparation and structural analysis of humanized anti-human transferrin receptor antibodies

[0207]

[0208] Gene cloning of humanized anti-human transferrin receptor antibodies:

[0209] Gene fragments encoding the VH and VL sequences determined in 1-1 above were synthesized. The heavy and light chain variable regions were amplified by polymerase chain reaction (PCR). The variable regions were inserted into the pcDNA 3.1 / myc-His A plasmid vector (Invitrogen), which has the human IgG CH1 or CH1-Fc region and His-tag in the heavy chain and the human kappa chain CL in the light chain.

[0210]

[0211] Protein expression and purification:

[0212] The antibody protein cloned from the above gene was expressed as expiCHO-S TM (Thermo Fisher Scientific). Cell culture was performed in a humidified CO2 incubator using 125 mL Erlenmeyer cell culture flasks. To transfect plasmids encoding whole antibodies and Fab fragments, ExpiFectamine TM CHO / plasmid DNA complexes were prepared and used. Cell cultures were collected 10 days after transfection, and proteins were purified using Hitrap Talon columns (Cytiva). SDS-PAGE was performed on the purified Fab fragments, and the results are shown in Figure 3. Figure 3 shows the results under reduced conditions, while the right side shows the results under non-reduced conditions.

[0213]

[0214] Crystallization and structural elucidation of a humanized anti-human transferrin receptor antibody:

[0215] Fab fragment crystals of a humanized anti-human transferrin receptor antibody were grown at 18°C ​​by the sitting drop vapor diffusion method. X-ray diffraction data were obtained by protecting the crystals with a solution containing 0.2 M ammonium sulfate, 0.1 M sodium acetate trihydrate (pH 4.6), 24% polyethylene glycol 6,000, and 20% ethylene glycol. X-ray diffraction experiments were performed at Pohang Accelerator Beamline 7A, and the crystals diffracted to a resolution of 1.6 Å. The results of diffraction data processing are shown in Table 1 below.

[0216] Additionally, the tertiary structure of the antibody was identified and refined based on the molecular substitution method using the PHENIX program, and the results are shown in Table 2 below.

[0217]

[0218]

[0219]

[0220]

[0221]

[0222] Structural analysis of humanized anti-human transferrin receptor antibodies:

[0223] To determine how antibody humanization affected the structure of the antibody, we performed an analysis of the antibody structure.

[0224] The tertiary structure of the Fab fragment of the humanized anti-human transferrin receptor antibody was determined using the Pymol program.

[0225] Additionally, the structures of the pre-humanized and post-humanized antibodies were compared using the same program, and only the VH and VL regions were used in the analysis to prevent the influence of the flexibility between the VH and VL regions of the antibody and the CH1 and CL1 regions on the analysis.

[0226] Furthermore, the average value of the Cα root mean square deviation (RMSD) was calculated using the same program to determine the degree of difference between the two antibody structures.

[0227] The structure of the Fab of the humanized anti-human transferrin receptor antibody is shown in Figure 4a, and the results of comparing the structures of the antibody before and after humanization are shown in Figure 4b.

[0228] As can be seen in Figure 4b, the structure of the pre-humanized and post-humanized antibodies was confirmed by overlapping them, and it was confirmed that the CDR regions were in a form that matched each other, indicating that the structural similarity of the CDR regions was high.

[0229] In addition, the average value of Cα root mean square deviation (RMSD) was calculated to be 0.58Å, confirming that the structural difference between the two antibodies was small.

[0230] Through this, it was possible to predict that the manufactured humanized anti-human transferrin receptor antibody could effectively bind to the transferrin receptor like the pre-humanized antibody.

[0231]

[0232] Affinity measurement of humanized anti-human transferrin receptor antibodies to the transferrin receptor:

[0233] To confirm that the manufactured humanized anti-human transferrin receptor antibody can effectively bind to the transferrin receptor, the transferrin receptor binding affinity of the manufactured antibody was measured using an enzyme-linked immunosorbent assay (ELISA).

[0234] Human transferrin receptor (TfR) (2.5 μg / ml) was coated onto a 96-well half-area plate (Corning) overnight at 4°C. The humanized anti-human transferrin receptor antibody and pre-humanized antibody prepared above were diluted 5-fold from 50 nM to 0.64 pM. Dilutions were made using 5% skimmed milk-containing PBS (pH 7.5, same as the blocking buffer). The plate was washed three times with PBS (pH 7.5), blocked with blocking buffer at room temperature for 2 hours, and then washed several more times. Various concentrations of anti-TfR antibody were added to the wells to induce binding for 2 hours. Unbound antibodies were then washed twice with PBST and twice with PBS.

[0235] Horseradish peroxidase (HRP)-conjugated anti-human IgG antibody (AB frontier) was added and incubated for 2 hours. After an antibody washing step, 50 μL of TMB solution was added and incubated at 37°C for 20 minutes. Finally, an equal volume of stop solution was added, mixed, and the absorbance was measured at 450 nm using an EMax microplate reader (Molecular Devices). All steps except the TMB incubation were performed at room temperature.

[0236] The binding affinity of the humanized anti-transferrin receptor antibody manufactured above to the transferrin receptor was measured and the results compared with the antibody before humanization are shown in Figure 5.

[0237] As can be seen in Fig. 5, the pre-humanization antibody and the post-humanization antibody showed similar binding affinity, confirming that the humanized anti-transferrin receptor antibody of the present invention effectively binds to the transferrin receptor.

[0238]

[0239] 1-3. Determination of amino acid residues for introducing mutations for the production of humanized anti-human transferrin receptor antibody mutants.

[0240]

[0241] Gene cloning:

[0242] A gene fragment encoding human transferrin receptor protein (using the gene sequence encoding amino acids 121 to 760 of the human transferrin receptor protein) was amplified by PCR. The gene was inserted into the pcDNA 3.1 / myc-His A plasmid vector (Invitrogen) containing the human IgG Fc region gene using restriction enzymes and T4 ligase, and a His-tag was linked to the C-terminus of the expressed protein. The HRV-3C protease recognition sequence was inserted between the human IgG Fc and the transferrin receptor.

[0243]

[0244] Protein expression and purification:

[0245] Human transferrin receptor protein with human IgG Fc and His-tag was expressed in expiCHO-S™ (Thermo Fisher Scientific). Transient transfection was performed by mixing the plasmid encoding the recombinant protein with ExpiFectamine™ CHO reagent. After transfection, cells were cultured in a CO2 incubator for approximately 10 days, and the cell culture was separated by centrifugation. The culture was incubated with HRV-3C protease to cleave the link between the transferrin receptor and human IgG Fc, and the transferrin receptor protein was purified using Ni-NTA resin (QIAGEN). The purified transferrin receptor protein was mixed with an anti-human transferrin receptor antibody Fab fragment and subjected to size-exclusion chromatography.

[0246]

[0247] Structural elucidation of a complex of humanized anti-human transferrin receptor antibody and transferrin receptor by cryo-electron microscopy:

[0248] The complex of human transferrin receptor protein and anti-human transferrin receptor antibody Fab fragment prepared as described above was loaded onto a glow-discharged Quantifoil R1.2 / 1.3 grid and then processed using a Vitrobot Mark IV (FEI) device to prepare a sample.

[0249] Image data were acquired by photographing grids constructed using a 200-kV Glacios electron microscope equipped with a Falcon 4. The data were processed using the CryoSPARC program, and through iterative 2D classification and non-uniform refinement, a 4.39-Å electron density map was ultimately generated.

[0250] Figure 6a is an image of a complex of transferrin receptor and antibody obtained through 2D classification, and Figure 6b shows a 3D image of the complex finally generated.

[0251] Furthermore, based on the electron density map, tertiary structure model refinement was performed using the Coot and PHENIX programs. Table 3 presents the statistical results of data collection and processing using cryo-electron microscopy for the transferrin receptor and antibody complex, and Table 4 presents the results of structural refinement using cryo-electron microscopy for the complex.

[0252]

[0253]

[0254]

[0255]

[0256]

[0257] In addition, in order to specifically confirm the interaction between the sugar chain portion of the transferrin receptor and the antibody within the complex of the transferrin receptor and the antibody, the structure of the complex of the transferrin receptor and the antibody and the previously identified transferrin receptor were superimposed and compared using the secondary structure matching superpose method, and the results are shown in Figure 6c.

[0258] Furthermore, based on the structural analysis results of the complex, the surrounding structure of S28 (Ser28) present in the interaction site between the antibody variable region and the transferrin receptor sugar chain was visualized and shown in Figure 6d. Specifically, Figure 6d shows a sphere based on Cα of S28.

[0259] As can be confirmed in Figures 6a, 6b, and 6c, it was found that antibody binding affected the entire structure of the transferrin receptor, and in particular, in Figure 6c, it was confirmed that when the antibody bound to the transferrin receptor, the structure of the transferrin receptor became wider than before antibody binding.

[0260] Additionally, as can be confirmed in Fig. 6d, it was found that a specific amino acid residue, such as S28, in the anti-human transferrin receptor antibody interacts with the sugar chain region of the transferrin receptor.

[0261]

[0262] Structural elucidation through modeling of humanized anti-human transferrin receptor antibodies and transferrin receptor complexes:

[0263] To further investigate the effect of anti-transferrin receptor antibodies on the sugar chain region of the transferrin receptor and the relationship between the structural changes of the transferrin receptor and the sugar chain region that was not clearly observed in the cryo-microscope, additional modeling was performed, and the results are shown in Figure 7.

[0264] As can be seen in Figure 7, it was confirmed that specific amino acid residues of the anti-transferrin receptor antibody interact with the sugar chain region of the transferrin receptor.

[0265]

[0266] Determination of residues to be mutated that affect interactions with the sugar chain region of the transferrin receptor:

[0267] Based on the results of the structural analysis of the above human transferrin receptor antibody and the antibody-transferrin receptor complex, the positions of amino acid residues for mutation introduction were selected, which are expected to improve the endosomal escape efficiency without affecting CDR grafting during the humanization process of the antibody while interacting with the sugar chain region of the transferrin receptor, and as a result, the S28 residue of the amino acid sequence of SEQ ID NO. 3 was selected as the residue for mutation introduction.

[0268]

[0269] Experimental Example 2: Evaluation of Endosomal Escape Efficiency by Introducing Mutations

[0270]

[0271] In the above Experimental Example 1, when a mutation is introduced into the S28 residue of the amino acid sequence of SEQ ID NO. 3, which is an amino acid residue that affects the interaction between the sugar chain region of the transferrin receptor and the transferrin receptor antibody, whether the endosomal escape efficiency is improved, and an amino acid residue that is expected to affect the affinity of the anti-transferrin receptor antibody and the transferrin receptor is introduced to change the affinity of the transferrin receptor and the transferrin receptor antibody in the acidic pH environment within the endosome, an endosomal escape efficiency evaluation experiment according to the introduction of a mutation was conducted to select an amino acid residue that can improve the endosomal escape efficiency.

[0272]

[0273] System design for measuring the endosomal escape efficiency of anti-transferrin receptor antibodies:

[0274] A luminescence measurement system was designed to measure the endosomal escape efficiency of anti-transferrin receptor antibodies by measuring luminescence emitted within cells.

[0275] Split luciferase used for luminescence measurement is composed of a large BiT protein (LgBiT, 17.9 kDa) and a high-affinity complementary peptide (HiBiT, 1.3 kDa). LgBiT and HiBiT do not produce a luminescence signal when present alone, and a luminescence signal is generated only when HiBiT binds with high affinity to the LgBiT protein. Based on this characteristic, in this experimental example, the cytosolic delivery was evaluated by quantitatively measuring the signals of cells expressing LgBiT and HiBiT-labeled antibodies using the Split luciferase assay. Since LgBiT can be partially secreted into the extracellular space, to prevent this, LgBiT was designed to be located in the cytoplasm by fusing with beta-actin. A rough outline of the split luciferase assay used in this experimental example is shown in Figure 8.

[0276] Additionally, the amino acid sequences of ahTfR (anti-human transferrin receptor antibody)_LC_HiBiT-EEP, ahTfR_LC, HiBiT, EEP, and LgBiT-actin used in this experimental example are shown below.

[0277]

[0278] ahTfR_LC_HiBiT-EEP sequence

[0279] METPAQLLFLLLLWLPDTTGDIQIVLTQSPAIMSASPGEKVTMTCSASSSIDYIHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRNSYPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECGGGTGGGVSGWRLFKKISGGGGWWG

[0280]

[0281] ahTfR_LC 서열

[0282] METPAQLLFLLLLWLPDTTGDIQIVLTQSPAIMSASPGEKVTMTCSASSSIDYIHWYQQKPGTSPKRWIYDTSKLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRNSYPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0283]

[0284] HiBiT 서열

[0285] VSGWRLFKKIS

[0286]

[0287] EEP 서열

[0288] GWWG

[0289]

[0290] LgBiT-actin 서열

[0291] MVFTLEDFVGDWEQTAAYNLDQVLEQGGVSSLLQNLAVSVTPIQRIVRSGENALKIDIHVIIPYEGLSADQMAQIEEVFKVVYPVDDHHFKVILPYGTLVIDGVTPNMLNYFGRPYEGIAVFDGKKITVTGTLWNGN KIIDERLITPDGSMLFRVTINSLEGGSGGSMDDDIAALVVDNGSGMCKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYVGDEAQSKRGILTLKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEA PLNPKANREKMTQIMFETFNTPAMYVAIQAVLSLYASGRTTGIVMDSGDGVTHTVPIYEGYALPHAILRLDLAGRDLTDYLMKILTERGYSFTTTAEREIVRDIKEKLCYVALDFEQEMATAASSSSLEKSYELPDG QVITIGNERFRCPEALFQPSFLGMESCGIHETTFNSIMKCDVDIRKDLYANTVLSGGTTMYPGIADRMQKEITALAPSTMKIKIIAPPERKYSVWIGGSILASLSTFQQMWISKQEYDESGPSIVHRKCFTGHHHHHH

[0292]

[0293] Construction of protein expression plasmids:

[0294] To produce antibodies that can be used in the split luciferase assay, protein expression plasmids were constructed.

[0295] Anti-transferrin receptor antibody was prepared in a similar manner to the method in Experimental Example 1, but LgBiT was replaced with pcDNA TM 3.1 / Myc-His A vector was subcloned, and β-actin DNA was ordered from Addgene and fused with LgBiT to prevent secretion of LgBiT.

[0296] Luciferase LgBiT-HiBiT is a pcDNA containing the light chain of an anti-human transferrin receptor antibody. TM3.1 / Myc-His A vector was subcloned, and a portion of LgBiT was deleted using the [QuikChange Site-Directed Mutagenesis kit (Stratagene)] protocol.

[0297] Afterwards, the endosomal escape peptide (EEP) was inserted via PCR, and mutations were introduced using the [QuikChange Site-Directed Mutagenesis kit (Stratagene)] protocol for anti-human transferrin receptor antibodies to enhance endosomal escape.

[0298] Specifically, two protein expression plasmids with amino acid mutations were prepared by introducing a mutation into the S28 residue of the amino acid sequence of SEQ ID NO: 3, which is the amino acid residue to which a mutation was decided in Experimental Example 1.

[0299] In addition, a total of 15 protein expression plasmids were additionally prepared by introducing mutations at 15 amino acid residue positions in the amino acid sequence of sequence number 3 that are expected to affect the affinity of the anti-transferrin receptor antibody and the transferrin receptor based on the charge characteristics of the amino acid residues in an acidic pH environment.

[0300]

[0301] Expression and purification of antibodies:

[0302] Using the plasmid prepared as described above, anti-human transferrin receptor antibody was expressed in ExpiCHO cells.

[0303] Cells were cultured in ExpiCHO expression medium (Thermo Fisher Scientific) and maintained at 37°C, 120 rpm, and 8% CO2. Subcultures were performed when the cell density reached approximately 4 to 6 x 106 cells / mL, and the cells were diluted to 3 to 4 x 106 cells / mL for transfection. Transfections were performed using the ExpiCHO expression system kit (Thermo Fisher Scientific), and the final cell density was 6 x 106 cells / mL. ExpiCHO expression medium preheated to 37°C was used as the medium.

[0304] For transfection, plasmid DNA and ExpiFectamine CHO reagent at concentrations of 0.5–1.0 μg / mL were diluted in chilled OptiPRO medium and incubated for 1–5 minutes. The transfection complex (ExpiFectamine CHO / plasmid DNA mixture) was then slowly added to ExpiCHO cells in a 125 mL Erlenmeyer flask fitted with a filter cap.

[0305] After 18 to 22 hours of transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were added to the flask, and the flask was transferred to an incubator at 32°C, 120 rpm, and 5% CO2.

[0306] After transfection, ExpiCHO cells were cultured for 10 to 12 days and centrifuged at 6000 rpm for 20 min at 4°C. The supernatant was filtered through a 0.45 μm nitrocellulose mixed ester membrane filter from Advantec and HiTrap TM TALON TM Purification was performed using a column (Cytiva).

[0307] The column was regenerated with 0.2 M EDTA (pH 7.5), 0.05 M cobalt(II) chloride, 0.3 M NaCl and equilibrated with equilibration buffer (0.04 M Tris-HCl (pH 7.5), 0.5 M NaCl). The supernatant was combined with the column, washed with 120 mL of wash buffer (0.04 M Tris-HCl (pH 7.5), 0.5 M NaCl), and then washed once more with 70 mL of second wash buffer (0.04 M Tris-HCl (pH 7.5), 0.2 M NaCl, 0.025 M imidazole).

[0308] After that, HiTrap TM TALON TM The antibody bound to the column resin was eluted using elution buffer (0.04 M Tris-HCl (pH 7.5), 0.2 M NaCl, 0.5 M imidazole). The eluted buffer was HiTrap TM Imidazole was removed by exchanging with PBS using a desalting column (Cytiva). Finally, the purified antibody was confirmed by SDS-PAGE and stored at -80°C.

[0309] The SDS-PAGE results of the manufactured antibody are shown in Figure 9.

[0310]

[0311] Cell culture:

[0312] To measure the endosomal escape efficiency of anti-human transferrin receptor antibodies, SK-BR-3 cells overexpressing transferrin receptor were cultured in RPMI 1640 medium (L-glutamine, sodium bicarbonate) supplemented with 10% FBS and 100-fold concentration of antibiotic-antimycotic.

[0313] Cells were cultured at 37°C under 5% CO2 conditions and subcultured every 3 to 4 days. When the cell density reached 70 to 80%, the culture medium was removed, and 2 mL of Trypsin-EDTA solution preheated to 37°C was treated and reacted for 3 to 5 minutes. After trypsin treatment was completed, the cells were collected and centrifuged at 1,000 rpm for 5 minutes. After removing the supernatant, the cells were washed with 2 mL of DPBS (Dulbecco's Phosphate-Buffered Saline, WELGENE) preheated to 37°C, and centrifuged again at 1,000 rpm for 5 minutes. Afterwards, DPBS was removed, and preheated medium was added. Centrifuged cells of an appropriate density were transferred to a cell culture dish containing 10 mL of preheated medium and cultured.

[0314]

[0315] Analysis of endosomal escape efficiency using luminescence measurements:

[0316] The above-prepared SK-BR-3 cells were seeded at 5 x 10 per well in a 96-well cell culture plate one day before the experiment. 5 Cells were seeded at a cell density of 10 μg / mL. Transfection of a DNA plasmid containing LgBiT-actin was performed using Lipofectamine 3000 transfection reagent (Invitrogen). After 48 hours, cells expressing LgBiT-actin protein were washed, and the cells were treated with different concentrations of the anti-human TfR-HiBiT-EEP antibody prepared above.

[0317] Cells were incubated for 6 hours at 37°C and 5% CO2, then washed twice with medium. NanoGlo Live Cell Substrate (Promega) was then added, and 2030 VICTOR TMThe luminescence signal was measured using X2 Multilabel Reader (PerkinElmer), and the results of the intracellular transduction signal normalized to the WT of the manufactured antibodies are shown in Figure 10.

[0318] The mutations introduced into the antibodies manufactured above are shown in Table 5 below based on the amino acid sequence of sequence number 3.

[0319]

[0320] Sample number Introduction mutation 1Y98H2S28N3G26H4T30H5Y32H6S28H and Y96H7S28H8T30H and Y96H9S28H and Y97H10G26E and S28H11F29H12G31H13Y99H14Y96H15Y97H16N55H17N58H

[0321]

[0322] As shown in Fig. 10, the endosomal escape efficiency of antibodies with introduced mutations was measured, and it was confirmed that the mutant antibody with introduced S28N (αTfR A in Fig. 10) and the mutant antibody with introduced Y96H (αTfR B in Fig. 10) showed a much higher intracellular delivery signal than the WT, thereby confirming that the mutant antibodies have very excellent endosomal escape efficiency.

[0323] The VH sequences of the mutant antibody introducing S28N (αTfR A in Figure 10) and the mutant antibody introducing Y96H (αTfR B in Figure 10) are shown below, and the introduced mutant amino acids are underlined.

[0324]

[0325] [SEQ ID NO: 6] Humanized 128.1 VH S28N mutant sequence

[0326] EVKLQQSGPELVKPGASVKMSCKASGYNFTGYTMNWVKQKPGQGLEWIGRINPHNGGTDYNQKFKDKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGYYYYSLDYWGQGTTLTVSS

[0327]

[0328] [SEQ ID NO: 7] Humanized 128.1 VH Y96H mutant sequence

[0329] EVKLQQSGPELVKPGASVKMSCKASGYSFTGYTMNWVKQKPGQGLEWIGRINPHNGGTDYNQKFKDKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGHYYYSLDYWGQGTTLTVSS

[0330]

[0331] Experimental Example 3: Precise Measurement of the Relative Endosomal Escape Efficiency of Mutant Antibodies to WT

[0332]

[0333] An experiment was conducted to more closely measure the relative endosomal escape efficiency of the two mutant antibodies confirmed in Experimental Example 2 above compared to the WT.

[0334] As in Experimental Example 2, when the HiBiT peptide is fused with another protein, such as an anti-human transferrin antibody, the luminescence activity itself may be affected, which may result in differences in luminescence activity between anti-human transferrin mutant antibodies.

[0335] To correct for this, the relative activity of anti-human transferrin-HiBiT-EEP antibodies was measured using cell lysates expressing LgBiT-actin, and the endosomal escape efficiencies of the mutant antibodies introducing S28N, the mutant antibodies introducing Y96H, and the WT antibody were normalized and compared based on this.

[0336] To prepare cell lysate samples, SK-BR-3 cells cultured in Experimental Example 2 were seeded into 96-well cell culture plates one day before transfection, and one day later, transfection was performed with the LgBiT-actin plasmid using Lipofectamine 3000 transfection reagent. The cells were cultured for two days, lysed using RIPA buffer, and the prepared cell lysate samples were reacted with purified anti-human transferrin-HiBiT-EEP antibodies for 15 minutes. The relative activity measurements of the mutant antibodies introducing S28N (A) and the mutant antibodies introducing Y96H (B) measured in this way against the WT are shown in Figure 11a.

[0337] In addition, based on the results shown in Fig. 11a, the endosomal escape efficiency of the mutant antibody introducing S28N (A), the mutant antibody introducing Y96H (B), and the WT was normalized and shown in Fig. 11b.

[0338] As can be seen in Figure 11b, even when the endosomal escape efficiency was normalized based on the relative activity measurement results of the three antibodies, it was found that the mutant antibody introducing S28N (A) and the mutant antibody introducing Y96H (B) still showed much better endosomal escape efficiency than the WT.

[0339]

[0340] Experimental Example 4: Evaluation of intracancer cell delivery efficiency

[0341]

[0342] Preparation of anti-human transferrin receptor antibody-avidin fusion protein:

[0343] A mutant antibody was prepared in the same manner as in Experimental Example 2, but to fuse the anti-human transferrin receptor antibody with avidin, avidin was replaced with pcDNA. TM3.1 / Myc-His A vector was subcloned. The heavy chain of the anti-human transferrin receptor antibody was inserted into pcDNA containing avidin. TM 3.1 / Myc-His A vector was subcloned to produce an anti-human transferrin receptor antibody-avidin fusion protein. Since the anti-human transferrin receptor antibody-avidin fusion protein is known to exhibit apoptotic activity in cancer cells, the delivery efficiency of the anti-human transferrin receptor antibody into cancer cells can be evaluated by evaluating the survival rate of cancer cells treated with the fusion protein.

[0344]

[0345] Cell culture:

[0346] To measure the delivery efficiency of the anti-human transferrin receptor antibody-avidin fusion protein manufactured above into cancer cells, K562 cells overexpressing the transferrin receptor and HEK293 cells expressing the receptor at a low level were cultured.

[0347] Cells were cultured in the same manner as in Experimental Example 2, but HEK293 cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium, 4500 mg / L D-glucose, L-glutamine, 110 mg / L sodium pyruvate, sodium bicarbonate) supplemented with 10% FBS and 100-fold concentration of antibiotic-antimycotic, and K562 cells were cultured in RPMI 1640 medium (L-glutamine, sodium bicarbonate) supplemented with 10% FBS and 100-fold concentration of antibiotic-antimycotic.

[0348]

[0349] Measurement of intra-cancer cell delivery efficiency:

[0350] HEK293 cells were seeded into 96-well cell culture plates one day prior to the experiment at a density of 3 x 10³ per well. After overnight culture, the cells were treated with different concentrations of anti-human transferrin receptor antibody-avidin fusion protein and incubated for 48 hours at 37°C in a 5% CO2 atmosphere. EZ-Cytox (Dogene Bio) was added to each well, and the absorbance was measured at 450 nm.

[0351] Additionally, K562 cells were seeded at a cell density of 5 x 10³ per well in a 96-well cell culture plate and treated with different concentrations of anti-human transferrin receptor antibody-avidin fusion protein. Subsequently, the cells were cultured for 72 hours at 37°C and 5% CO2, and EZ-Cytox was added to each well and incubated at 37°C and 5% CO2.

[0352] Absorbance was measured at 450 nm using an EMax microplate reader. Cell-free medium was used as a blank sample, and cells not treated with anti-human transferrin receptor antibody-avidin fusion protein were used as a control. Cell viability was calculated according to the following formula.

[0353]

[0354] [Formula 1]

[0355] Survival rate (%) = [(absorbance test - Absorbance blank ) / (absorbance control - Absorbance blank )] x 100

[0356]

[0357] The results of cancer cell survival rates measured as above are shown in Figures 12a and 12b. Figures 12a and 12b show the survival rates of K562 cells and HEK293 cells, respectively.

[0358] As can be seen in Figures 12a and 12b, in K562 cells overexpressing the transferrin receptor, cell viability decreased as the concentration of the mutant antibody introducing S28N (A) and the mutant antibody introducing Y96H (B) increased, and it was found that a lower cell viability was shown compared to the case in which no mutation was introduced.

[0359] In contrast, in HEK293 cells expressing low levels of transferrin receptor, no apoptosis-inducing effect was observed following treatment with anti-human transferrin receptor antibody-avidin fusion protein.

[0360] Through this, it was found that the manufactured anti-human transferrin receptor antibody-avidin fusion protein exhibited a specific apoptosis-inducing effect on K562 cells overexpressing the transferrin receptor, and that a better apoptosis-inducing effect was exhibited when a mutation was introduced.

[0361]

[0362] Experimental Example 5: Confirming the effectiveness of cancer treatment through intracellular siRNA delivery.

[0363]

[0364] In order to measure the cancer cell killing effect of the anti-transferrin receptor antibody introducing the mutation of the present invention upon delivery of siRNA into cancer cells, an anti-transferrin receptor antibody-siRNA conjugate, which is an antibody-oligonucleotide conjugate (AOC), was prepared using siRNA that can specifically bind to DUSP28, and the cancer cell killing effect was evaluated.

[0365] DUSP28 (Dual Specificity Phosphatase 28) is an enzyme that regulates the phosphorylation status of proteins overexpressed in cancer cells, and suppressing the expression of the enzyme with siRNA can result in a cancer cell death effect.

[0366]

[0367] Preparation of anti-transferrin receptor antibody-siRNA conjugates with introduced mutations:

[0368] An anti-transferrin receptor antibody-siRNA conjugate was prepared using an anti-transferrin receptor antibody with S28N introduced among the anti-transferrin receptor antibodies manufactured in Experimental Example 2. The siRNA that specifically binds to DUSP28 was manufactured by Bioneer.

[0369] siRNA specifically binding to DUSP28 and anti-transferrin receptor antibodies were linked via disulfide bonds. To this end, specific residues in the anti-transferrin receptor antibody were mutated to cysteine ​​using the [QuikChange Site-Directed Mutagenesis kit] protocol.

[0370] siRNA was modified with the 5'-thiol modifier C6 SS and 2'-O-methylation was performed to improve serum and intracellular stability. The disulfide bonds of the thiol-modified oligonucleotides were reduced to the active sulfhydryl form using DTT.

[0371] The siRNA solution was treated with DTT and reacted at room temperature for 30 minutes, and excess DTT and unwanted thiol fragments were removed using ethyl acetate to prevent oxidative dimerization prior to disulfide bond formation.

[0372] Afterwards, the reduced siRNA was mixed with an anti-transferrin receptor antibody at an equimolar concentration, diamide was added, and the mixture was reacted at room temperature for 1 hour to form a disulfide bond, and then separated on a 1.5% agarose gel containing ethidium bromide.

[0373] The structure of the manufactured anti-transferrin receptor antibody-siRNA conjugate is shown in Figure 13a.

[0374]

[0375] Confirmation of decreased mRNA expression by anti-transferrin receptor antibody-siRNA treatment with introduced mutations:

[0376] AsPC-1 cells were seeded at 1 x 105 per well in a 24-well cell culture plate (SPL) and cultured overnight at 37°C and 5% CO2. When the cells reached 60-80% confluence, the existing medium was removed and replaced with RPMI-1640 medium containing 1% FBS, and the cells were cultured.

[0377] The cultured cells were treated with anti-transferrin receptor antibody-siRNA conjugates at various concentrations for 4 days, and RNA extraction and RT-PCR were performed.

[0378] Transfection of cells was performed using [Lipofectamine RNAiMAX (Invitrogen)]. After 48 hours, the culture medium was removed, and 200 μL of prewarmed Trypsin-EDTA was treated for 34 minutes. The cells were then harvested in microcentrifuge tubes (SPL) and centrifuged at 2300 g and 4°C for 10 minutes. After removing the supernatant, RNA was extracted using [Accuprep Universal RNA Extraction Kit (Bioneer)]. For normalization, RNA was quantified using OPTIZEN NanoQ Lite and then diluted to the same concentration.

[0379] To assess the relative mRNA levels of target genes, [One Step PrimeScript TMReverse transcription polymerase chain reaction (RT-PCR) was performed using a RT-PCR Kit (Takara Bio Inc.). Primers for human DUSP28 and β-actin were purchased from Bionics, and the primer sequences are presented in Table 6 below.

[0380]

[0381] Gene primer sequence DUSP28 forward 5'-CCTTCCAGATGGTGAAGAGC-3'reverse 5'-GGTGAATGTGGGTGACACTG-3'β-actin forward 5'-AGAGCCTCGCCTTTGCCGATC-3'reverse 5'-CTGGGCCTCGTCGCCCACATA-3'

[0382]

[0383] The results of mRNA expression levels measured as described above are shown in Fig. 13b. As can be confirmed in Fig. 13b, the DUSP28 mRNA expression level was found to be reduced in cancer cells treated with anti-transferrin receptor antibody-siRNA.

[0384] Through this, it was confirmed that the anti-transferrin receptor antibody introducing the mutation of the present invention can efficiently deliver siRNA into cancer cells.

[0385]

[0386] Analysis of cancer cell colony formation inhibition by anti-transferrin receptor antibody-siRNA conjugate treatment

[0387] AsPC-1 cells were seeded at 800 cells per well in a 24-well culture plate and cultured overnight. The following day, the cells were treated with various concentrations of antibody-siRNA conjugates and cultured for 10 days. After 10 days, the remaining medium was removed and washed twice with DPBS. After removing the DPBS, the cells were fixed with a mixture of methanol and acetic acid and incubated for 5 minutes. After fixation, the fixative solution was removed, and the cells were stained with a 0.5% crystal violet solution for 15 minutes.

[0388] Afterwards, the plate was washed with tap water, and colony formation images were captured using a Cloneselect Imager (Molecular Devices), and the results are shown in Figure 13c. In addition, the average number of cancer cell colonies was calculated and is shown in Figure 13d.

[0389] As can be seen in Figures 13c and 13d, the formation of cancer cell colonies was inhibited when the anti-transferrin receptor antibody-siRNA conjugate was treated.

[0390] Through this, it was confirmed that the anti-transferrin receptor antibody introducing the mutation of the present invention can efficiently deliver siRNA into cancer cells.

[0391]

[0392] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical concept or essential features. In this regard, it should be understood that the embodiments described above are illustrative in all respects and are not restrictive.

Claims

1. An anti-human transferrin receptor antibody comprising a heavy chain variable region (VH) consisting of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; and a light chain variable region (VL) consisting of an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, An anti-human transferrin receptor antibody, wherein at least one of serine (S) and tyrosine (Y) residues in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 is independently substituted with asparagine (N) or histidine (H).

2. In paragraph 1, An anti-human transferrin receptor antibody, wherein at least one of residues S28 and Y96 in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 is independently substituted with asparagine (N) or histidine (H).

3. In paragraph 1, An anti-human transferrin receptor antibody, wherein the substitution comprises at least one mutation of S28N and Y96H in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

3.

4. In paragraph 1, An anti-human transferrin receptor antibody, wherein one or more drug compounds are conjugated to the antibody.

5. In paragraph 4, An anti-human transferrin receptor antibody, wherein the drug compound is at least one selected from the group consisting of siRNA, miRNA, shRNA, growth inhibitors, toxins, radioactive isotopes, and nanoparticles.

6. In paragraph 1, An anti-human transferrin receptor antibody having one binding domain of the above anti-human transferrin receptor antibody.

7. A multispecific antibody comprising at least one first binding domain that binds to human transferrin receptor (hTfR) and at least one second binding domain that binds to a target molecule, The first binding domain comprises a heavy chain variable region (VH) consisting of an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3; and a light chain variable region (VL) consisting of an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; A multispecific antibody, wherein at least one of serine (S) and tyrosine (Y) residues in the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 is independently substituted with asparagine (N) or histidine (H).

8. In paragraph 7, A multispecific antibody, wherein the first binding domain is in a form selected from the group consisting of Fab, scFv, di-scFv, dsFv and (dsFv)2.

9. In paragraph 7, A multispecific antibody wherein the first binding domain and the second binding domain are linked to an Fc region.

10. In paragraph 7, A multispecific antibody, wherein one or more drug compounds are conjugated to the antibody.

11. In paragraph 10, A multispecific antibody, wherein the drug compound is at least one selected from the group consisting of siRNA, miRNA, shRNA, growth inhibitors, toxins, radioactive isotopes, and nanoparticles.

12. A pharmaceutical composition for preventing or treating one or more diseases selected from the group consisting of cancer, brain disease, and cell signaling-related diseases, comprising an anti-human transferrin receptor antibody according to any one of claims 1 to 6.

13. In paragraph 12, A pharmaceutical composition, wherein the cancer is at least one disease selected from the group consisting of pancreatic cancer, liver cancer, stomach cancer, blood cancer, bone marrow cancer, brain cancer, lung cancer, and skin cancer.

14. In paragraph 12, A pharmaceutical composition, wherein the brain disease is at least one disease selected from the group consisting of Parkinson's disease, Alzheimer's disease, traumatic brain injury, stroke, Huntington's disease, amyotrophic lateral sclerosis, spinal cord injury, alcoholic encephalopathy, alcoholic dementia, and Wernicke-Korsakoff's syndrome.

15. In paragraph 12, A pharmaceutical composition, wherein the cell signaling-related disease is at least one disease selected from the group consisting of diabetes, inflammatory disease, immune disease, and diabetic dementia.

Images