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WO2025230358A1 - Anticorps anti-récepteur de la transferrine humaine présentant une efficacité d'échappement endosomal améliorée, et anticorps multispécifique et composition pharmaceutique l'utilisant - Google Patents

Anticorps anti-récepteur de la transferrine humaine présentant une efficacité d'échappement endosomal améliorée, et anticorps multispécifique et composition pharmaceutique l'utilisant

Info

Publication number
WO2025230358A1
WO2025230358A1 PCT/KR2025/006009 KR2025006009W WO2025230358A1 WO 2025230358 A1 WO2025230358 A1 WO 2025230358A1 KR 2025006009 W KR2025006009 W KR 2025006009W WO 2025230358 A1 WO2025230358 A1 WO 2025230358A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
transferrin receptor
seq
human transferrin
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/KR2025/006009
Other languages
English (en)
Korean (ko)
Inventor
류성언
김명빈
이혜림
강지안
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Signalbio Co Ltd
Industry University Cooperation Foundation IUCF HYU
Original Assignee
Signalbio Co Ltd
Industry University Cooperation Foundation IUCF HYU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Signalbio Co Ltd, Industry University Cooperation Foundation IUCF HYU filed Critical Signalbio Co Ltd
Publication of WO2025230358A1 publication Critical patent/WO2025230358A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • 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.
  • 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.
  • 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.
  • the wild-type anti-transferrin receptor antibodies known to date have low endosome escape efficiency, limiting the rate at which therapeutic agents reach the cytoplasm. This significantly hinders therapeutic efficacy and limits their practicality as intracellular therapeutic delivery platforms.
  • 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.
  • 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.
  • An object of the present invention is to provide an anti-human transferrin receptor antibody (anti-hTfR) with improved endosomal escape efficiency.
  • Another object of the present invention is to provide a multispecific antibody produced using the above anti-human transferrin receptor antibody.
  • 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.
  • 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).
  • VH heavy chain variable region
  • VL light chain variable region consisting of an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4
  • 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).
  • 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).
  • 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.
  • the drug compound may be at least one selected from the group consisting of siRNA, miRNA, shRNA, growth inhibitors, toxins, radioactive isotopes, and nanoparticles.
  • 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).
  • VH heavy chain variable region
  • VL light chain variable region
  • the first binding domain may be in a form selected from the group consisting of Fab, scFv, di-scFv, dsFv, and (dsFv) 2 .
  • one or more drug compounds may be conjugated to the multispecific antibody.
  • the drug compound may be at least one selected from the group consisting of siRNA, miRNA, shRNA, growth inhibitors, toxins, radioactive isotopes, and nanoparticles.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 1 schematically illustrates the structure of a multispecific antibody according to one embodiment of the present invention.
  • 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.
  • 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.
  • Figure 3 shows the SDS PAGE results for a humanized anti-human transferrin receptor antibody according to one embodiment of the present invention.
  • 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.
  • Figure 6a shows a 2D image of a human transferrin receptor and antibody complex according to one embodiment of the present invention.
  • Figure 6b shows a 3D image of a human transferrin receptor and antibody complex according to one embodiment of the present invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Figure 11b shows the normalized endosomal escape efficiency of an anti-human transferrin receptor antibody according to one embodiment of the present invention.
  • Figure 12a shows the results of measuring the survival rate of K562 cells according to one embodiment of the present invention.
  • Figure 12b shows the results of measuring the survival rate of HEK293 cells according to one embodiment of the present invention.
  • Figure 13a illustrates the structure of an anti-transferrin receptor antibody-oligonucleotide conjugate according to one embodiment of the present invention.
  • 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.
  • 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.
  • 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.
  • the present invention relates to an anti-transferrin receptor antibody with improved endosomal escape efficiency.
  • an anti-transferrin receptor antibody refers to an antibody that binds to the transferrin receptor (TfR).
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • mutation means a substitution, insertion, and/or deletion of an amino acid residue.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the anti-human transferrin receptor antibody with improved endosomal escape efficiency 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.
  • VH heavy chain variable region
  • VL light chain variable region
  • 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).
  • the numbering in the amino acid sequence of the variable region of the above chimeric antibody 128.1 follows the Kabat numbering.
  • WT wild-type 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.
  • the anti-human transferrin receptor antibody with improved endosomal escape efficiency may be a humanized antibody comprising an amino acid sequence in which the variable region sequence of the chimeric antibody 128.1 is humanized.
  • 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).
  • CDR complementarity determining region
  • FR homologous human acceptor framework region
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the binding domain of the anti-transferrin receptor antibody of the present invention may be single.
  • the anti-transferrin receptor antibody may be in a form in which one of the two binding domains present in the antibody is deleted.
  • 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.
  • cancer cells can be effectively killed, thereby exhibiting a cancer treatment effect.
  • the present invention also provides a multispecific antibody using the anti-human transferrin receptor antibody.
  • 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.
  • 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.
  • hTfR human transferrin receptor
  • binding domain is interpreted as a concept encompassing antibody-derived proteins, biological proteins, and artificially designed interacting proteins.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the first binding domain can be a Fab form including VH-CH1 and VL-CL, a fragment thereof, or a recombinant form.
  • the first binding domain can be in the form of Fab, scFv, di-scFv, dsFv, (dsFv) 2 , etc.
  • 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.
  • 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
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the sdAb form refers to a single-domain variable fragment, and refers to one variable region domain.
  • 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.
  • the di-scFv form refers to a recombinant domain in which two scFvs are connected by a linker.
  • the linker may be any linker known in the art, and may be a peptide composed of 5 to 20 amino acids.
  • 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.
  • the linker may be (GGGGX) n , where X is preferably A or S, and n is preferably a natural number from 1 to 4.
  • 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.
  • (dsFv) 2 form refers to a recombinant domain in which two dsFvs are connected by a linker.
  • 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.
  • 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.
  • the first binding domain and the second binding domain may have a form linked to each chain of the Fc region.
  • 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.
  • 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.
  • 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.
  • 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.”
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a recombinant variant can be formed in the Fc region to form a multispecific antibody.
  • the dimer 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.
  • the above knob-into-hole technology is designed to form only heterodimers between the heavy chains of antibody fragments.
  • the knob is designed to have a side chain protruding toward the opposite chain and is inserted into the hole of the opposite domain.
  • the heavy chains cannot homodimerize due to side chain collisions, and only heterodimerization is possible.
  • one of the two chains constituting the Fc region may have a knob structure and the other may have a hole structure, and in this case, the chain in which the knob or hole is formed is referred to as Fc-knob or Fc-hole, respectively.
  • 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).
  • a large amino acid selected from the group consisting of tryptophan (W), arginine (R), phenylalanine (F), and tyrosine (Y).
  • W tryptophan
  • R arginine
  • F phenylalanine
  • Y tyrosine
  • the Fc-knob can be formed by forming a T366W mutation in the sequence 221 to 447 of the IgG1-Fc heavy chain.
  • 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).
  • a small amino acid selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V).
  • the Fc-hole can be formed by substituting T366S, L368A, and Y407V in the sequence 221 to 447 of the IgG1-Fc heavy chain. A mutation may have formed.
  • FIG. 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.
  • 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.
  • 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.
  • 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.
  • cancer cells can be effectively killed, thereby exhibiting a cancer treatment effect.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • VL and VH candidate sequences of the humanized anti-human transferrin receptor antibody produced as above are as follows.
  • 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.
  • VH sequence SEQ ID NO: 1
  • VL sequence SEQ ID NO: 2
  • 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.
  • the cloned antibody protein was expressed in expiCHO-S TM (Thermo Fisher Scientific). Cell culture was performed in a humidified CO 2 incubator using 125 mL Erlenmeyer cell culture flasks. ExpiFectamine TM CHO/plasmid DNA complexes were prepared and used to transfect plasmids encoding whole antibodies and Fab fragments. Ten days after transfection, the cell culture fluid was collected and the protein was purified using a Hitrap Talon column (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, and the right side shows the results under non-reduced conditions.
  • 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 ⁇ . The results of diffraction data processing are shown in Table 1 below.
  • the tertiary structure of the Fab fragment of the humanized anti-human transferrin receptor antibody was determined using the Pymol program.
  • 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.
  • RMSD C ⁇ root mean square deviation
  • Fig. 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.
  • the transferrin receptor binding affinity of the manufactured antibody was measured using an enzyme-linked immunosorbent assay (ELISA).
  • Human transferrin receptor (TfR) (2.5 ⁇ g/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.
  • HRP horseradish peroxidase
  • AB frontier horseradish peroxidase-conjugated anti-human IgG antibody
  • 50 ⁇ L of TMB solution was added and incubated at 37°C for 20 minutes.
  • 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.
  • 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.
  • 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.
  • Human transferrin receptor protein with human IgG Fc and His-tag was expressed in expiCHO-STM (Thermo Fisher Scientific). Transient transfection was performed by mixing the plasmid encoding the recombinant protein with ExpiFectamineTM 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.
  • 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.
  • FEI Vitrobot Mark IV
  • 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- ⁇ electron density map was ultimately generated.
  • 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.
  • Table 3 presents the statistical results of data collection and processing using cryo-electron microscopy for the transferrin receptor and antibody complex
  • Table 4 presents the results of structural refinement using cryo-electron microscopy for the complex.
  • 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.
  • 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.
  • a luminescence measurement system was designed to measure the endosomal escape efficiency of anti-transferrin receptor antibodies by measuring luminescence emitted within cells.
  • 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.
  • LgBiT 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.
  • ahTfR anti-human transferrin receptor antibody
  • An anti-transferrin receptor antibody was prepared in a similar manner to that in Experimental Example 1, but LgBiT was subcloned into the pcDNA TM 3.1/Myc-His A vector, and ⁇ -actin DNA was ordered from Addgene and fused with LgBiT to prevent secretion of LgBiT.
  • Luciferase LgBiT-HiBiT was subcloned into pcDNA TM 3.1/Myc-His A vector containing the light chain of anti-human transferrin receptor antibody, and a portion of LgBiT was deleted using the [QuikChange Site-Directed Mutagenesis kit (Stratagene)] protocol.
  • 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.
  • 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.
  • 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.
  • anti-human transferrin receptor antibody was expressed in ExpiCHO cells.
  • 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.
  • plasmid DNA and ExpiFectamine CHO reagent 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.
  • 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 .
  • 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 ⁇ m nitrocellulose mixed ester membrane filter (Advantec) and purified using a HiTrap TM TALON TM column (Cytiva).
  • 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).
  • the antibody bound to the HiTrap TM TALON TM column resin was eluted using elution buffer (0.04 M Tris-HCl (pH 7.5), 0.2 M NaCl, 0.5 M imidazole).
  • elution buffer 0.4 M Tris-HCl (pH 7.5), 0.2 M NaCl, 0.5 M imidazole.
  • the eluted buffer was exchanged with PBS using a HiTrap TM Desalting column (Cytiva) to remove imidazole.
  • the purified antibody was confirmed by SDS-PAGE and stored at -80°C.
  • 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.
  • 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.
  • DPBS Dynamiconitrate
  • the prepared SK-BR-3 cells were seeded into 96-well cell culture plates one day before the experiment at a density of 5 x 10 5 cells per well.
  • Transfection of the DNA plasmid containing LgBiT-actin was performed using Lipofectamine 3000 transfection reagent (Invitrogen). After 48 hours, the cells expressing LgBiT-actin protein were washed, and the cells were treated with the prepared anti-human TfR-HiBiT-EEP antibody at different concentrations.
  • the endosomal escape efficiency of antibodies with introduced mutations was measured, and it was confirmed that the mutant antibody with introduced S28N ( ⁇ TfR A in Fig. 10) and the mutant antibody with introduced Y96H ( ⁇ TfR B in Fig. 10) showed a much higher intracellular delivery signal than the WT, and through this, it was confirmed that the mutant antibody had a very excellent endosomal escape efficiency.
  • VH sequences of the mutant antibody introducing S28N ( ⁇ TfR A in Figure 10) and the mutant antibody introducing Y96H ( ⁇ TfR B in Figure 10) are shown below, and the introduced mutant amino acids are underlined.
  • the luminescence activity itself may be affected, which may result in differences in luminescence activity between anti-human transferrin mutant antibodies.
  • 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 2 days and 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.
  • a mutant antibody was prepared in the same manner as in Experimental Example 2, but avidin was subcloned into the pcDNA TM 3.1/Myc-His A vector to fuse the anti-human transferrin receptor antibody with avidin.
  • the heavy chain of the anti-human transferrin receptor antibody was subcloned into the pcDNA TM 3.1/Myc-His A vector into which avidin had been inserted to prepare 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 when treated with the fusion protein.
  • K562 cells overexpressing the transferrin receptor and HEK293 cells expressing the receptor at a low level were cultured.
  • HEK293 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.
  • DMEM Dulbecco's Modified Eagle's Medium, 4500 mg/L D-glucose, L-glutamine, 110 mg/L sodium pyruvate, sodium bicarbonate
  • RPMI 1640 medium L-glutamine, sodium bicarbonate
  • HEK293 cells were seeded into 96-well cell culture plates one day prior to the experiment at a density of 3 x 103 cells 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 under 5% CO2 conditions. EZ-Cytox (Dogene Bio) was added to each well, and the absorbance was measured at 450 nm.
  • 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. Afterwards, they were cultured for 72 hours under conditions of 37°C and 5% CO 2 , and EZ-Cytox was added to each well and incubated under conditions of 37°C and 5% CO 2 .
  • Figures 12a and 12b show the survival rates of K562 cells and HEK293 cells, respectively.
  • 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.
  • 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.
  • AOC antibody-oligonucleotide conjugate
  • 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.
  • 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.
  • siRNA specifically binding to DUSP28 and anti-transferrin receptor antibodies were linked via disulfide bonds.
  • specific residues in the anti-transferrin receptor antibody were mutated to cysteine using the [QuikChange Site-Directed Mutagenesis kit] protocol.
  • siRNA was modified with the 5'-thiol modifier C6 S-S 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.
  • 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.
  • 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.
  • 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 under 5% CO 2 conditions. 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.
  • SPL 24-well cell culture plate
  • 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.
  • RT-PCR reverse transcription polymerase chain reaction
  • Fig. 13b 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.
  • the anti-transferrin receptor antibody introducing the mutation of the present invention can efficiently deliver siRNA into cancer cells.
  • 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.
  • the anti-transferrin receptor antibody introducing the mutation of the present invention can efficiently deliver siRNA into cancer cells.

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Abstract

La présente invention concerne : un anticorps anti-récepteur de la transferrine humaine présentant une efficacité d'échappement endosomal améliorée ; et un anticorps multispécifique et une composition pharmaceutique qui utilisent l'anticorps anti-récepteur de la transferrine humaine. La présente invention peut fournir un anticorps ayant une excellente efficacité d'échappement endosomal par optimisation de l'interaction entre l'anticorps anti-récepteur de transferrine humaine et la région de chaîne glucidique du récepteur de transferrine. L'anticorps anti-récepteur de transferrine humaine de la présente invention permet ainsi d'administrer plus efficacement une substance à administrer dans des cellules, et en particulier, de faire entrer dans des cellules cancéreuses une substance active qui peut être efficacement administrée par échappement endosomal efficace. De plus, en utilisant l'anticorps anti-récepteur de transferrine humaine de la présente invention dans un anticorps multispécifique, un conjugué anticorps-médicament, ou un conjugué anticorps-oligonucléotide, des substances pour le traitement du cancer, de maladies cérébrales ou de maladies associées à la signalisation cellulaire peuvent être efficacement administrés dans des cellules.
PCT/KR2025/006009 2024-05-03 2025-05-02 Anticorps anti-récepteur de la transferrine humaine présentant une efficacité d'échappement endosomal améliorée, et anticorps multispécifique et composition pharmaceutique l'utilisant Pending WO2025230358A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102052571B1 (ko) * 2010-11-30 2019-12-05 제넨테크, 인크. 저친화도 혈액-뇌 장벽 수용체 항체 및 그의 용도
KR20210035021A (ko) * 2016-12-14 2021-03-31 리간달 인코포레이티드 핵산 및 단백질 페이로드 전달을 위한 방법 및 조성물
WO2021205358A1 (fr) * 2020-04-08 2021-10-14 Janssen Biotech, Inc. Compositions et méthodes d'administration à la barrière hémato-encéphalique
KR20230016147A (ko) * 2021-07-15 2023-02-01 한양대학교 산학협력단 뇌혈관장벽 통과 효율이 향상된 항 인간 트랜스페린 수용체 항체, 및 이를 이용한 다중특이적 항체 및 약학 조성물
US11834510B2 (en) * 2018-12-21 2023-12-05 Avidity Biosciences, Inc. Anti-transferrin receptor antibodies and uses thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102052571B1 (ko) * 2010-11-30 2019-12-05 제넨테크, 인크. 저친화도 혈액-뇌 장벽 수용체 항체 및 그의 용도
KR20210035021A (ko) * 2016-12-14 2021-03-31 리간달 인코포레이티드 핵산 및 단백질 페이로드 전달을 위한 방법 및 조성물
US11834510B2 (en) * 2018-12-21 2023-12-05 Avidity Biosciences, Inc. Anti-transferrin receptor antibodies and uses thereof
WO2021205358A1 (fr) * 2020-04-08 2021-10-14 Janssen Biotech, Inc. Compositions et méthodes d'administration à la barrière hémato-encéphalique
KR20230016147A (ko) * 2021-07-15 2023-02-01 한양대학교 산학협력단 뇌혈관장벽 통과 효율이 향상된 항 인간 트랜스페린 수용체 항체, 및 이를 이용한 다중특이적 항체 및 약학 조성물

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