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WO2025151018A1 - Anticorps bispécifique se liant de manière spécifique à c-kit et vegf, et son utilisation - Google Patents

Anticorps bispécifique se liant de manière spécifique à c-kit et vegf, et son utilisation

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Publication number
WO2025151018A1
WO2025151018A1 PCT/KR2025/000744 KR2025000744W WO2025151018A1 WO 2025151018 A1 WO2025151018 A1 WO 2025151018A1 KR 2025000744 W KR2025000744 W KR 2025000744W WO 2025151018 A1 WO2025151018 A1 WO 2025151018A1
Authority
WO
WIPO (PCT)
Prior art keywords
kit
vegf
antibody
cancer
bispecific antibody
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/KR2025/000744
Other languages
English (en)
Korean (ko)
Inventor
박상규
나태영
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.)
Novelty Nobility Inc
Original Assignee
Novelty Nobility Inc
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
Priority claimed from KR1020250004411A external-priority patent/KR20250111039A/ko
Application filed by Novelty Nobility Inc filed Critical Novelty Nobility Inc
Publication of WO2025151018A1 publication Critical patent/WO2025151018A1/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
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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 a bispecific antibody that specifically binds to c-Kit and VEGF, and more specifically, to a bispecific antibody that can bind to c-Kit and VEGF with high affinity and inhibit or neutralize the activity or activation of c-Kit and VEGF, a nucleic acid molecule encoding the bispecific antibody, a vector comprising the nucleic acid molecule, a host cell, a method for producing the bispecific antibody, a pharmaceutical composition for preventing or treating angiogenic diseases comprising the bispecific antibody as an active ingredient, a method for preventing or treating angiogenic diseases using the bispecific antibody, and a kit for detecting c-Kit and/or VEGF or diagnosing angiogenic diseases comprising the bispecific antibody.
  • Angiogenesis is the process by which new capillaries are formed from existing microvessels. Normally, it occurs only during embryonic development, wound healing, and cyclical changes in the female reproductive system, and rarely occurs under other normal conditions. However, if angiogenesis is not regulated autonomously and continues to grow pathologically, it causes various diseases.
  • hypoxia when hypoxia occurs, the expression of c-KIT in endothelial cells increases, and the production of new blood vessels by SCF (stem cell factor) in endothelial cells increases.
  • SCF stem cell factor
  • This pathological angiogenesis is a major cause of blindness in various eye diseases such as wet macular degeneration and diabetic retinopathy.
  • hypoxia occurs, the expression of HIF-1 ⁇ (hypoxia inducible factor-1 ⁇ ) increases along with SCF and c-KIT, and at this time, SCF stabilizes HIF-1 ⁇ to prolong hypoxia.
  • the inventors of the present invention developed a bispecific antibody that specifically binds to c-Kit and VEGF, and confirmed that the antibody can act as a therapeutic agent for angiogenic diseases, particularly macular degeneration, thereby completing the present invention.
  • Patent Document 0001 Republic of Korea Publication Patent No. 10-2020-0040407
  • the purpose of the present invention is to provide a bispecific antibody that specifically binds to c-Kit and VEGF.
  • Another object of the present invention is to provide a method for producing a bispecific antibody that specifically binds to c-Kit and VEGF using the host cell.
  • Another object of the present invention is to provide a method for preventing or treating angiogenic diseases using a bispecific antibody that specifically binds to the aforementioned c-Kit and VEGF.
  • the present invention provides a bispecific antibody that specifically binds to c-Kit and VEGF, comprising the following (a) and (b): (a) a first antigen binding site that specifically binds to c-Kit, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 4, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17; and (b) a second antigen binding site that specifically binds to VEGF, wherein the soluble extracellular domain of VEGF and the Fc domain of IgG are fuse
  • the second antigen binding site may include the amino acid sequence of SEQ ID NO: 21.
  • the second antigen binding site may be linked to the heavy chain C-terminus or N-terminus of the first antigen binding site by a peptide linker; or the second antigen binding site may be linked to the light chain C-terminus of the first antigen binding site by a peptide linker.
  • the bispecific antibody may be a tetravalent antibody.
  • the present invention also provides a nucleic acid molecule encoding a bispecific antibody that specifically binds to the aforementioned c-Kit and VEGF.
  • the present invention provides a recombinant vector comprising the above-described nucleic acid molecule and a transformant comprising the recombinant vector.
  • the present invention provides a pharmaceutical composition for preventing or treating angiogenic diseases, comprising a bispecific antibody that specifically binds to the aforementioned c-Kit and VEGF as an active ingredient.
  • Binding affinity refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen).
  • the affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd), which is the ratio of the dissociation rate constant and the association rate constant (koff and kon, respectively).
  • Kd dissociation constant
  • equivalent affinities can include different rate constants, as long as the ratio of the rate constants remains the same.
  • the bispecific antibody of the present invention has a dissociation constant (KD) of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g., less than or equal to 10 -7 M, for example, from 10 -7 M to 10 -13 M, for example, from 10 -9 M to 10 -13 M).
  • KD dissociation constant
  • high affinity of an antibody refers to an antibody having a KD of 10 -9 M or less, more preferably 10 -10 M or less, for its target antigen.
  • bispecific antibody comprising a first antigen binding site that specifically binds c-Kit and a second antigen binding site that specifically binds VEGF
  • bispecific antibody that specifically binds c-Kit and VEGF and "bispecific antigen-binding molecule specific for c-Kit and VEGF” are used interchangeably herein and refer to a bispecific antibody capable of binding c-Kit and VEGF with sufficient affinity to render the antibody useful as a diagnostic and/or therapeutic agent in targeting c-Kit and VEGF.
  • c-Kit used in the present invention belongs to class III of receptor tyrosine kinase (RTK) and is also known as a receptor for SCF.
  • RTK receptor tyrosine kinase
  • c-Kit one of the targets of the above angiogenesis inhibitors, belongs to class III of receptor tyrosine kinase (RTK) and is a receptor for SCF (Stem Cell Factor), which plays an important role in haematopoiesis.
  • RTK receptor tyrosine kinase
  • SCF Stem Cell Factor
  • VEGF antagonists include anti-VEGF antibodies (e.g., bevacizumab [AVASTIN®]), anti-VEGF receptor antibodies (e.g., anti-VEGFR1 antibodies, anti-VEGFR2 antibodies, etc.), and VEGF receptor-based chimeric molecules (also referred to herein as "VEGF-traps").
  • anti-VEGF antibodies e.g., bevacizumab [AVASTIN®]
  • anti-VEGF receptor antibodies e.g., anti-VEGFR1 antibodies, anti-VEGFR2 antibodies, etc.
  • VEGF receptor-based chimeric molecules also referred to herein as "VEGF-traps”
  • VEGF antagonist and "antibody comprising an antigen binding site that binds VEGF” refer to an antibody capable of binding VEGF, particularly a VEGF polypeptide, with sufficient affinity to render the antagonist or antibody useful as a diagnostic and/or therapeutic agent in the targeting of VEGF.
  • the extent of binding of the VEGF antagonist or antibody to an unrelated, non-VEGF protein is less than about 10% of the binding of the antagonist or antibody to VEGF, as measured, for example, by radioimmunoassay (RIA) or flow cytometry (FACS) or by surface plasmon resonance analysis using a biosensor system such as the Biacore® system.
  • an antibody that binds to human VEGF has a KD value for binding affinity for human VEGF of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 10 nM, ⁇ 1 nM, ⁇ 0.1 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g., less than or equal to 10 -8 M, for example, from 10 -8 M to 10 -13 M, for example, from 10 -9 M to 10 -13 M).
  • mouse antibody is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from mouse germline immunoglobulin sequences.
  • the mouse antibodies of the present disclosure may comprise amino acid residues that are not encoded by a mouse germline immunoglobulin sequence (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by in vivo complementary somatic mutagenesis).
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody e.g. a non-human antibody, refers to an antibody that has undergone humanization.
  • Other forms of “humanized antibodies” encompassed by the invention are those in which the constant region has been further modified or altered from the constant region of the original antibody to produce the properties according to the invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding.
  • Fc domain or "Fc region” is used herein to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region.
  • the term includes native sequence Fc regions and variant Fc regions.
  • Preferred peptide linkers include Gly, Glu, Asn, Lys, Ser, and Pro residues. Other neutral amino acids, such as Thr and Ala, can also be included in the linker sequence.
  • the linker sequence may consist of 1-50 amino acid residues, preferably 10-20 amino acid residues.
  • a suitable non-immunogenic linker peptide may comprise, but is not limited to, 5 to 10 consecutive glycines (G).
  • Percent (%) amino acid sequence identity to a reference polypeptide sequence is defined as the percent of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be accomplished in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA program package.
  • polypeptide is intended to encompass the singular “polypeptide” as well as the plural “polypeptides”, and refers to a molecule comprising monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds).
  • polypeptide refers to any chain or chains of two or more amino acids, and does not refer to a particular length of the product.
  • polypeptide peptide, dipeptide, tripeptide, oligopeptide, "protein", “amino acid chain”, or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of “polypeptide”, and the term “polypeptide” may be used in place of, or interchangeably with, any of these terms.
  • polypeptide is also intended to refer to the product of post-expression modification of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids.
  • the polynucleotide or nucleic acid may be or include regulatory elements such as a promoter, a ribosome binding site, or a transcription terminator.
  • isolated as used herein also refers to the nucleic acid or peptide being substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • isolated is also used herein to refer to a cell or polypeptide being separated from other cellular proteins or tissues. An isolated polypeptide is meant to include both a purified polypeptide and a recombinant polypeptide.
  • host cell refers to cells into which exogenous nucleic acid has been introduced (including the progeny of such cells).
  • Host cells include “transformants” and “transformed cells”, including the primary transformed cell and progeny derived therefrom, regardless of the number of passages.
  • the progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as that selected or selected for in the originally transformed cell are included herein.
  • a host cell is any type of cell system that can be used to produce an antibody or bispecific antibody of the invention.
  • the first antigen binding site may include a heavy chain variable region comprising an amino acid sequence having 90% or greater sequence homology to the amino acid sequence of SEQ ID NO: 27 and a light chain variable region comprising an amino acid sequence having 90% or greater sequence homology to the amino acid sequence of SEQ ID NO: 28.
  • the antibody in which the second antigen binding site is bound to the heavy chain C-terminus of the first antigen binding site by a peptide linker is 'candidate antibody A', 'candidate antibody B', 'candidate antibody B-1', and 'candidate antibody C'
  • the antibody in which the second antigen binding site is bound to the heavy chain N-terminus of the first antigen binding site by a peptide linker is 'candidate antibody E'
  • the antibody in which the second antigen binding site is bound to the light chain C-terminus of the first antigen binding site by a peptide linker is 'candidate antibody D'.
  • the bispecific antibodies of the present invention may have their Fc domains modified to reduce Fc receptor binding and/or effector function.
  • one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to produce an Fc region variant.
  • the Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.
  • the Fc domain of the bispecific antibody comprising a first antigen binding site that specifically binds c-Kit and a second antigen binding site that specifically binds VEGF can comprise one or more amino acid substitutions that reduce binding to an Fc receptor, particularly an Fc ⁇ receptor.
  • the Fc domain is of the human IgG1 subclass comprising an Fc variant comprising amino acid substitutions L234A and L235E; or an Fc variant comprising amino acid substitutions L234A, L235A I253A, H310A, P329G and H435A.
  • the Fc domain confers favorable pharmacodynamic properties to the bispecific antibody of the present invention, including a long serum half-life, which contributes to good accumulation in target tissues and a favorable tissue-to-blood distribution ratio. However, at the same time, it may lead to undesirable targeting of the bispecific antibody of the present invention to cells expressing Fc receptors rather than to desired antigen-bearing cells.
  • the Fc domain of the bispecific antibody according to one embodiment of the present invention exhibits a reduced binding affinity for Fc receptors and/or a reduced effector function compared to a native IgG Fc domain, particularly an IgG1 Fc domain.
  • the Fc domain may be a wild-type IgG1 Fc domain or a variant thereof.
  • the Fc domain exhibits less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5%, of the binding affinity for an Fc receptor relative to a native IgG1 Fc domain (or the bispecific antigen binding molecule of the present invention comprising a native IgG1 Fc domain), and/or less than 50%, preferably less than 20%, more preferably less than 10% and most preferably less than 5%, of the effector function relative to a native IgG1 Fc domain.
  • the Fc domain does not substantially bind to and/or induce effector function of an Fc receptor.
  • the Fc receptor is an Fc ⁇ receptor.
  • the Fc domain can be engineered to have reduced binding affinity and/or reduced effector function for an Fc receptor, compared to a non-engineered Fc domain (wild-type Fc).
  • the Fc domain of the bispecific antibody of the present invention can comprise one or more amino acid mutations that reduce the binding affinity and/or effector function of the Fc domain for an Fc receptor.
  • the same one or more amino acid mutations are present in each of the two subunits of the Fc domain. The amino acid mutations can reduce the binding affinity of the Fc domain for an Fc receptor.
  • the amino acid mutations can reduce the binding affinity of the Fc domain for an Fc receptor by at least 2-fold, at least 5-fold, or at least 10-fold, or can eliminate binding affinity such that the Fc domain does not bind to an Fc receptor at all.
  • the bispecific antibody of the invention comprising an engineered Fc domain may exhibit a binding affinity for an Fc receptor of less than 20%, particularly less than 10%, more particularly less than 5%, compared to a bispecific antibody of the invention comprising a non-engineered Fc domain.
  • the Fc receptor is an Fc ⁇ receptor.
  • the Fc receptor is a human Fc receptor.
  • the Fc receptor is an activating human Fc ⁇ receptor, more particularly human Fc ⁇ RIIIa, Fc ⁇ RI or Fc ⁇ RIIa, preferably human Fc ⁇ RIIIa.
  • the binding affinity for complement components particularly binding affinity for C1q, may also be reduced.
  • the binding affinity for neonatal Fc receptor (FcRn) may be reduced. Substantially similar binding to FcRn, i.e. preservation of the binding affinity of the Fc domain for the receptor, is achieved when the Fc domain exhibits a binding affinity for FcRn that is greater than about 70% of the binding affinity of the non-engineered form of the Fc domain.
  • the Fc domain, or a bispecific antibody of the invention comprising the Fc domain can exhibit such affinities of greater than about 80% and even greater than about 90%.
  • the Fc domain of the bispecific antigen binding molecule of the invention is engineered to have reduced effector function relative to the non-engineered Fc domain.
  • an exemplary bispecific antibody having a wild-type Fc domain may be, but is not limited to, candidate antibody A or candidate antibody D.
  • the heavy chain of the first antigen-binding portion of candidate antibody A or candidate antibody D having a wild-type Fc domain may comprise or consist of the amino acid sequence of SEQ ID NO: 29, and the light chain of the first antigen-binding portion may comprise or consist of the amino acid sequence of SEQ ID NO: 30.
  • amino acid sequences of the above sequence numbers 29 and 30 are as follows.
  • the Fc domain of the bispecific antibody according to the invention may comprise amino acid substitutions as defined above.
  • an exemplary bispecific antibody having LALAPG introduced into the Fc domain may be, but is not limited to, candidate antibody B or candidate antibody E.
  • the heavy chain of candidate antibody B or candidate antibody E having LALAPG introduced into the Fc domain may comprise or consist of the amino acid sequence of SEQ ID NO: 31, and the light chain of the first antigen-binding site may comprise or consist of the amino acid sequence of SEQ ID NO: 30.
  • amino acid sequence of the above sequence number 31 is as follows.
  • amino acid sequence of the above sequence number 32 is as follows.
  • amino acid sequence of the above sequence number 33 is as follows.
  • Binding to an Fc receptor can be readily measured by surface plasmon resonance (SPR) using standard equipment, such as, for example, an ELISA or a BIAcore instrument (GE Healthcare), or the Fc receptor itself can be obtained by recombinant expression.
  • SPR surface plasmon resonance
  • the binding affinity of an Fc domain, or of a cell-activating bispecific antibody comprising an Fc domain, for an Fc receptor can be assessed using cell lines known to express particular Fc receptors, such as the human NK cell-expressed Fc ⁇ IIIa receptor.
  • the effector functions of the Fc domain, or of a bispecific antibody of the invention comprising an Fc domain can be measured by methods known in the art.
  • Faricimab brand name: Vabysmo
  • a commercially available treatment for macular degeneration is a 2(1+1) antibody that binds Ang-2 and VEGF CrossMab.
  • two types of 2(1+1)valent antibodies [Fab-VEGFR-Fc(K-H) and Fab-VEGFR-Fc(H-K)] were produced by linking the first antigen binding site and the second antigen binding site to anti-c-Kit antibody and VEGFR using the "knob-into-hole" technology, and one type of 2(1+1)valent antibody in which c-Kit scFv and VEGFR trap were linked with a peptide linker (G4S or G8) was produced, and their purity, stability (HMW and LMW production), thermostability, and binding affinity to c-Kit and VEGF were evaluated.
  • these antibodies showed endotoxin detection, low stability (HMW and LMW production), low thermostability,
  • the antibody in the form of Fab-VEGFR-Fc(K-H) produced above was named a “comparative antibody” and was used for comparison of efficacy with the bispecific antibody according to the present invention.
  • the "knob-into-hole” technique is described, for example, in U.S. Pat. No. 5,731,168; U.S. Pat. No. 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) and Carter, J Immunol Meth 248, 7-15 (2001).
  • the method involves introducing a knob ("knob") into the contact surface of a first polypeptide and introducing a corresponding cavity ("hole”) into the contact surface of a second polypeptide such that the knob is positioned within the cavity to promote heterodimer formation and prevent homodimer formation.
  • the bispecific antibody may have a change in the amino acid sequence of the antibody in order to improve its binding affinity and/or other biological properties.
  • modifications include, for example, deletion, insertion, and/or substitution of amino acid sequence residues of the antibody.
  • Such amino acid mutations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of the amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes.
  • arginine, lysine, and histidine alanine, glycine, and serine
  • phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.
  • the bispecific antibodies of the present invention and the nucleic acid molecules encoding them are interpreted to also include sequences showing substantial identity with the sequences listed in the sequence listing.
  • the substantial identity means a sequence showing at least 60% identity, more preferably 70% identity, even more preferably 80% identity, and most preferably at least 90% identity when the sequences of the present invention and any other sequences are aligned to the greatest extent possible and the aligned sequences are analyzed using an algorithm commonly used in the art.
  • the bispecific antibody may comprise a peptide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the above defined amino acid sequence.
  • the second aspect of the present invention relates to a nucleic acid molecule encoding the aforementioned bispecific antibody or fragment thereof according to the present invention, a vector comprising the nucleic acid molecule, and a host cell comprising the vector.
  • the nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 27 and 28, respectively may include or consist of the nucleotide sequences of SEQ ID NOs: 60 and 61, in that order, but are not limited thereto.
  • the nucleotide sequences encoding the amino acid sequences of SEQ ID NOs: 29, 30, 31, 32 and 33, respectively may include or consist of the nucleotide sequences of SEQ ID NOs: 62, 63, 64, 65 and 66, in that order, but are not limited thereto.
  • nucleotide sequences of the above sequence numbers 62, 63, 64, 65 and 66 are as follows.
  • the nucleic acid molecule can comprise a nucleic acid molecule having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide sequence defined above.
  • the vector includes a plasmid vector; a phagemid vector; a cosmid vector; and a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector, and is preferably a plasmid vector.
  • the vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression.
  • the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.
  • the vector of the present invention is an expression vector and uses a prokaryotic cell as a host, it generally includes a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL ⁇ promoter, pR ⁇ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence.
  • a strong promoter capable of driving transcription e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL ⁇ promoter, pR ⁇ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.
  • E. coli e.g., BL21, HB101, DH5 ⁇ , etc.
  • coli tryptophan biosynthetic pathway (Yanofsky, C. (1984), J. Bacteriol., 158:1018-1024) and the left-hand promoter of phage ⁇ (pL ⁇ promoter, Herskowitz, I. and Hagen, D. (1980), Ann. Rev. Genet., 14:399-445) can be used as regulatory regions.
  • the vector that can be used in the present invention can be produced by manipulating plasmids (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series (pET28a, pET21a, etc.), and pUC19, etc.), phagemids (e.g., pComb3X), phages (e.g., ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ z1, and M13, etc.), or viruses (e.g., SV40, etc.) that are frequently used in the art.
  • plasmids e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR32
  • the expression vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selectable marker, for example, a resistance gene for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.
  • an antibiotic resistance gene commonly used in the art as a selectable marker, for example, a resistance gene for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.
  • the vector expressing the bispecific antibody of the present invention may be a vector system in which the first antigen binding site and the second antigen binding site are expressed in a single vector in a form in which they are linked by a peptide linker, or a system in which the first antigen binding site and the second antigen binding site are expressed in separate vectors.
  • transfection refers to introducing a desired gene into a host cell using the recombinant vector of the present invention, and is used in the same meaning as “transformation”. Therefore, “transfection” and/or “transformation” into a host cell includes any method for introducing a nucleic acid into an organism, cell, tissue, or organ, and can be performed by selecting a standard technique suitable for the host cell as known in the art.
  • Such methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, stirring using silicon carbide fibers, Agrobacterium-mediated transformation, PEG, dextran sulfate, lipofectamine, and desiccation/inhibition-mediated transformation methods.
  • the culture of the transformant for the antibody can be carried out according to the appropriate medium and culture conditions known in the art. This culture process can be easily adjusted and used by those skilled in the art according to the selected strain. Cell culture is divided into suspension culture and attachment culture according to the cell growth method, and batch, fed-batch, and continuous culture methods according to the culture method. The medium used for culture must appropriately satisfy the requirements of the specific strain.
  • the medium used for culturing animal cells contains various carbon sources, nitrogen sources, and trace element components.
  • carbon sources that can be used include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose, fats such as soybean oil, sunflower oil, castor oil, and coconut oil, fatty acids such as palmitic acid, stearic acid, and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid.
  • These carbon sources can be used alone or in combination.
  • nitrogen sources examples include organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn steep liquor (CSL), and soybean meal, and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. These nitrogen sources can be used alone or in combination.
  • the above-mentioned badge may contain, as personnel, potassium dihydrogen phosphate, potassium dihydrogen phosphate and the corresponding sodium-containing salt. In addition, it may contain a metal salt such as magnesium sulfate or iron sulfate. In addition, it may contain amino acids, vitamins, and suitable precursors, etc.
  • compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be added to the culture in an appropriate manner to adjust the pH of the culture.
  • an antifoaming agent such as fatty acid polyglycol ester can be used to suppress bubble formation.
  • oxygen or an oxygen-containing gas e.g., air is injected into the culture.
  • the temperature of the culture is usually 20°C to 45°C, preferably 25°C to 40°C.
  • the antibody obtained by culturing the transformant can be used in an unpurified state, and can be further purified to a high purity using various conventional methods, such as dialysis, salt precipitation, and chromatography, and then used.
  • various conventional methods such as dialysis, salt precipitation, and chromatography
  • the method using chromatography is the most commonly used, and the type and order of the column can be selected from ion exchange chromatography, size exclusion chromatography, and affinity chromatography depending on the characteristics of the antibody, the culturing method, etc.
  • the present invention provides a c-Kit and VEGF bispecific antibody according to the first aspect for use in preventing or treating angiogenic diseases and a pharmaceutical composition for preventing or treating angiogenic diseases comprising the same as an active ingredient.
  • the present invention provides the use of a c-Kit and VEGF bispecific antibody according to the first aspect for the manufacture of a medicament for the prevention or treatment of angiogenic diseases.
  • the above angiogenic disease refers to a disease related to the occurrence or progression of angiogenesis. If it is a disease that can be treated with the bispecific antibody according to the present invention, it may be included in the scope of angiogenic disease.
  • the above angiogenic disease may be selected from the group consisting of cancer, leukemia, ocular vascular disease, rheumatoid arthritis, psoriasis, chronic wound, chronic inflammation, hemangioma, angiofibroma, vascular malformation, arteriosclerosis, vascular adhesion, vasculitis, pyogenic granuloma, bullous disease, pulmonary hypertension, asthma, nasal polyp, infectious disease, inflammatory bowel disease, periodontal disease, peritoneal adhesion, endometrium, uterine bleeding, ovarian cyst, osteomyelitis, osteophyte, sepsis, and autoimmune disease, but is not limited thereto.
  • the ocular vascular disease may be selected from the group consisting of diabetic retinopathy, macular degeneration, age-related macular degeneration, glaucoma, glaucomatous retinitis pigmentosa, choroidal neovascularization, retinopathy of prematurity, corneal dystrophy, and retinal detachment, but is not limited thereto.
  • the pharmaceutical composition according to the present invention may contain the bispecific antibody alone, or may additionally contain one or more pharmaceutically acceptable carriers, excipients or diluents.
  • the pharmaceutical composition of the present invention can be administered to mammals, including humans, by any method.
  • it can be administered orally or parenterally.
  • parenteral administration method it can be intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration, but is not limited thereto.
  • the pharmaceutical composition of the present invention can be prepared in an injectable formulation and administered by lightly pricking the skin with a 30-gauge thin injection needle, or by directly applying it to the skin.
  • excipients may include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, and maltitol; starches including corn starch, wheat starch, rice starch, and potato starch; cellulosics including cellulose, methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; and fillers such as gelatin and polyvinylpyrrolidone.
  • cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, if desired.
  • the pharmaceutical composition of the present invention may further include an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifier, and a preservative.
  • an anticoagulant e.g., a sulfate, a sulfate, a sulfate, a sulfate, a sulfate, a sulfate, a sulfate, a sulfate, a sulfate, a sulfate, a ointments for external use, oils, moisturizers, gels, aerosols and nasal inhalers by methods known in the art. These formulations are described in a generally known reference in all pharmaceutical chemistry (Remington's Pharmaceutical Science, 15th Edition, 1975 Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour).
  • the total effective amount of the pharmaceutical composition of the present invention can be administered to a patient as a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time.
  • the pharmaceutical composition of the present invention can have different contents of the effective ingredient depending on the degree of symptoms of the disease.
  • the daily dosage of the pharmaceutical composition of the present invention can be 0.0001 to 100 mg/kg.
  • the dosage of the pharmaceutical composition of the present invention takes into account various factors such as the administration route and the number of treatments as well as the patient's age, weight, health condition, sex, severity of the disease, diet, and excretion rate, and a person having ordinary knowledge in the art will be able to determine an appropriate effective dosage for the patient.
  • the pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effect of the present invention.
  • composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents.
  • composition of the present invention and other therapeutic agents may be administered simultaneously, separately, or sequentially.
  • the other therapeutic agent may be a substance already known to have a therapeutic or improving effect on neovascular diseases, and includes all other anticancer therapies other than drug therapy, such as radiation therapy.
  • the bispecific antibody and the other therapeutic agent contained in the composition of the present invention may be formulated separately in separate containers or may be formulated together in the same container.
  • the term "subject” includes, but is not limited to, any animal (e.g., human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig, or rodent). This term does not indicate a particular age or gender. Thus, it is intended to include female/female or male/male, adult/adult and newborn subjects, as well as fetuses.
  • a patient refers to a subject suffering from a disease or disorder. The term patient includes human and veterinary subjects.
  • the fifth aspect of the present invention relates to a kit for detecting c-Kit and/or VEGF comprising the above-described bispecific antibody and a method for detecting c-Kit and/or VEGF using the above-described bispecific antibody.
  • the kit of the present invention can be manufactured to be suitable for various immunoassays or immunostaining.
  • the immunoassays or immunostaining include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), immunofluorescence, Western blotting, immunohistochemistry staining, flow cytometry, immunocytochemistry, radioimmunoassay (RIA), immunoprecipitation assay, radioimmunoassay (RIA), and protein chips.
  • Tools or reagents used in immunological analysis may include suitable carriers or supports, labels capable of generating detectable signals, solubilizers, detergents, and stabilizers.
  • Suitable carriers may include, but are not limited to, a substrate capable of measuring enzyme activity when the label is an enzyme, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, a chromogenic substrate, and a reaction terminator.
  • the bispecific antibodies against c-Kit and VEGF included in the kit of the present invention can preferably be immobilized on a suitable carrier or support using various methods as disclosed in the literature, examples of suitable carriers or supports include PBS, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluoropolymer, agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethyl cellulose, polyacrylamide, polysterene, gabbro, filter paper, ion exchange resin, plastic film, plastic tube, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, metal, glass, glass beads, or magnetic particles.
  • Other solid substrates include cell culture plates, ELISA plates, tubes and polymeric membranes.
  • the support may have any possible shape, for example spherical (bead), cylindrical (inside of a test tube or well), planar (sheet
  • Labels capable of generating a detectable signal enable qualitative or quantitative measurement of the formation of an antigen-antibody complex
  • examples of such labels include enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes.
  • Enzymes that can be used include ⁇ -glucuronidase, ⁇ -D-glucosidase, urease, peroxidase (such as horseradish peroxidase), alkaline phosphatase, acetylcholinesterase, glycose oxidase, hexokinase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, invertase, and luciferase.
  • Fluorescent substances that can be used include fluorescein, isothiocyanate, rhodamine, phycoerythelin, phycocyanin, allophycocyanin, and fluorsine isothiocyanate.
  • Ligands include biotin derivatives, etc.
  • luminescent substances include acridinium ester, luciferin, etc.
  • Microparticles include colloidal gold, colored latex, etc.
  • redox molecules include ferrocene, ruthenium complexes, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, hydroquinone, etc.
  • Radioisotopes include 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re, etc. However, in addition to those exemplified above , any one that can be used in an immunological analysis method can be used.
  • an enzyme chromogenic substrate for example, when horseradish peroxidase (HRP) is selected as an enzyme label, a solution containing 3-amino-9-ethylcarbazole, 5-aminosalicylic acid, 4-chloro-1-naphthol, o-phenylenediamine, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), 3,3-diaminobenzidine, 3,3',5,5'-tetramethylbenzidine, o-dianisidine, or 3,3-dimethoxybenzidine can be used as a substrate.
  • HRP horseradish peroxidase
  • alkaline phosphatase when alkaline phosphatase is selected as an enzyme label, a solution containing 5-bromo-4-chloro-3-indolyl phosphate, nitroblue tetrazolium, or p-nitrophenyl phosphate can be used as a substrate.
  • ⁇ -D-galactosidase when ⁇ -D-galactosidase is selected as an enzyme label, a solution containing o-nitrophenyl- ⁇ -D-galactoside or 5-bromo-4-chloro-3-indole- ⁇ -D-galactopyranoside can be used as a substrate.
  • various enzymes and enzyme chromogenic substrates known in the art can be used.
  • a method for detecting c-Kit and/or VEGF according to one embodiment of the present invention may detect c-Kit and/or VEGF proteins in a biological sample or a mammal including a human.
  • the method for detecting c-Kit and/or VEGF according to the present invention may include a step of detecting a c-Kit and/or VEGF antigen-antibody complex using the above-described bispecific antibody.
  • the sample is a biological sample, and may include, but is not limited to, tissue, cell, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph node, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells, and bacterial expression systems.
  • the biological sample may be isolated from a mammal, including a human, having a disease in which c-Kit and/or VEGF is overexpressed or a disease related to c-Kit and/or VEGF, or may be isolated from an animal model that overexpresses c-Kit and/or VEGF protein or an animal model of a disease related to c-Kit and/or VEGF, but is not limited thereto.
  • These biological samples can be reacted with the bispecific antibody of the present invention, with or without manipulation, to confirm the presence or absence of c-Kit and VEGF proteins.
  • the mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans, non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).
  • domestic animals e.g., cattle, sheep, cats, dogs, and horses
  • primates e.g., humans, non-human primates such as monkeys
  • rabbits e.g., mice and rats.
  • the colony was cultured and DNA was obtained using a Mini-prep (Qiagen, 27106) kit.
  • the obtained DNA was digested with the same restriction enzyme, and the insertion of the gene into the vector was confirmed by the size of the cleaved band through electrophoresis, and then sequencing (commissioned by Bioneer) was used for final analysis, and a vector expressing anti-c-Kit antibody was obtained.
  • ExpiFectamine CHO transfection kit Gibco, A29129
  • Optipro SFM Gibco, 12309019
  • DNA transfection was performed at a concentration of 1 ⁇ g/mL.
  • supplements were added on the first day and the culture conditions were changed to 5% CO2 , 32°C, and 120 rpm. Supplements were then added again on the fifth day and cultured for 10 to 12 days to complete production.
  • the steady-state affinity method was used to calculate the binding affinity (KD).
  • the steady-state affinity method is not suitable, so the binding rate constant ( ) and the dissociation rate constant ( ) was calculated, and the binding affinity (KD) was calculated as the ratio of the dissociation rate and the association rate. If the binding reaction signal was too low or non-specific binding occurred, the binding affinity (KD) could not be accurately determined, so only the binding was determined.
  • ADCC antibody-dependent cellular cytotoxicity
  • cytotoxicity was confirmed to occur in the group treated with the wild-type Fc, and no cytotoxicity was observed in the groups treated with the mutants LALAPG, LAGA, and LALE, respectively.
  • the mutants LALAPG, LAGA, and LALE were confirmed to still have toxicity.
  • Trastuzumab was used as a positive control. Therefore, it was confirmed that the LALAPG, LAGA, and LALE Fc mutants significantly reduced ADCC compared to the wild-type Fc, and these three Fc mutants were the optimal mutants.
  • bispecific antibodies linked by the G8 peptide linker and each introducing LALAPG and LALE Fc mutations were named 'candidate antibody B' (LALAPG) and 'candidate antibody C' (LALE), respectively, and the structure and sequence information of these bispecific antibodies are shown in Figures 3a to 3b and Tables 9 to 12, respectively.
  • a bispecific antibody was produced by additionally introducing I253A, H310A, and H435A mutations into the above candidate antibody B, and this was named 'candidate antibody B-1'.
  • the binding affinity of candidate antibody B-1 to human FcRn was evaluated by SPR analysis. As confirmed in Table 13, no significant interaction between antibody B-1 and human FcRn was observed at pH 6.0, and no KD value was measured at pH 7.4.
  • Candidate Antibody A Candidate Antibody B
  • Candidate Antibody B-1 Candidate Antibody
  • Candidate Antibody C
  • Candidate Antibody D
  • Candidate antibody E Molecular form of bispecific antibodies IgG(H)-VEGFR IgG(H)-VEGFR IgG(H)-VEGFR IgG(H)-VEGFR VEGFR- IgG(L) VEGFR- IgG(H) valence 2+2 2+2 2+2 2+2 2+2 2+2 2+2 Fc Engineering Wild type LALAPG LALAPG-AAA LALE Wild type LALAPG Linker type G5 G8 G8 G8 G5 G8
  • Each antibody was diluted to 1 ⁇ g/ml in DPBS and prepared as a two-fold serial dilution in the dilution plate. After blocking, the solution was removed from the assay plate, and the plate was washed three times with 300 ⁇ l/well of 0.05% PBST buffer. Using a multichannel pipette, 100 ⁇ l of antibody was transferred from the dilution plate to the assay plate, the plate was sealed with a film, and incubated for 1 h at 25 ⁇ 2°C without shaking.
  • the plate was washed three times with 300 ⁇ l/well of 0.05% PBST buffer, and 100 ⁇ l of the diluted secondary antibody solution (80 ng/mL; final secondary antibody) was added to each well using a multichannel pipette.
  • the plate was sealed with a film and incubated for 1 h at 25 ⁇ 2°C without shaking. After incubation with secondary antibodies, the solution was removed from the assay plate.
  • the plates were washed three times with 300 ⁇ l/well of 0.05% PBST buffer, and 100 ⁇ l of TMB substrate reagent solution was added to each well using a multichannel pipette.
  • the plates were covered with a film and incubated at 25 ⁇ 2°C for 5 min. The plates were read within 10 min after adding 100 ⁇ l of 1 N sulfuric acid to the wells. Plates were read at absorbance at 450 nm using a SPECTROstar NaNo.
  • a running buffer was prepared by diluting 1X from 20X HBS-EP+ buffer with deionized water, and a regeneration solution (10 mM NaOH) was prepared by diluting 10 N NaOH to 10 mM with deionized water. Immobilization and binding assays were performed at 25 °C using 1X HBS-EP+ buffer as a running buffer.
  • c-Kit protein was directly immobilized on the sensor chip surface via primary amine coupling.
  • the sensor chip surfaces of the sample and reference flow cells were activated with freshly mixed EDC/NHS mixture for 420 s.
  • c-Kit protein was diluted in 10 mM sodium acetate solution and injected into the sample flow cell to achieve an appropriate immobilization level, and the reference flow cell was set as blank.
  • the remaining active coupling sites were blocked with ethanolamine for 420 s.
  • the capture molecule protein A
  • the amine coupling process was the same as for c-Kit analysis, except that protein A was immobilized on both channels. Then, diluted antibodies or Eylea were injected only into the sample flow cell, and captured by pre-immobilized protein A using affinity for the Fc region.
  • Binding assays were performed at a flow rate of 30 uL/min. All analytes were serially diluted in running buffer, and all samples were prepared on ice. Analyte samples were injected during the binding step, followed by running buffer for separation. The sensor chip surface was regenerated at each cycle by injecting a regeneration solution. For the c-Kit assay, 10 mM NaOH solution was used as the regeneration solution, and for the VEGF assay, 10 mM glycine, pH 1.5 solution was used. The analyte concentrations and running configurations are shown in Tables 20 and 21.
  • Running configuration Assay type Multi-cycle reaction rate Start cycle 2 times, using running buffer Blank cycle 2 cycles per analyte, after concentration series Bonding time c-Kit analysis: 240 seconds, VEGF165 analysis: 120 seconds Harry time c-Kit analysis: 480 seconds, VEGF165 analysis: 240 seconds Flow rate 30 uL/min
  • PBS (1X) buffer to be used as a negative control was prepared by diluting PBS (20X) buffer, and the stock solution of each test sample was diluted to a concentration of 1 mg/mL using PBS (1X) buffer so that the total amount of test sample for each reaction was 5 ug.
  • Protein Thermal ShiftTM Dye (1000X) was freshly diluted to 8X using PBS (1X) buffer.
  • the reaction components described in Table 22 below were added to each well of the MicroAmp® Optical 8-tube strip.
  • the tubes were sealed with MicroAmp® Optical 8-Cap Strips. Samples were mixed using a vortex mixer and then centrifuged. Each reaction was prepared in quadruplicate. Fluorescence readings were monitored using a real-time PCR instrument, StepOnePlus® Real-Time PCR System, and control software. Protein melting reactions were performed by setting the experimental characteristics as described in Table 23 below.
  • Tm Melting temperature
  • Results were statistically analyzed using one-way ANOVA and Tukey's multiple comparisons using GraphPad Prism 10 software.
  • the six bispecific antibodies in Table 16 above were frozen at -80°C for 16 hours and then thawed at 4°C for 2 hours. This was performed as 1 cycle, and the cycle was repeated 5 times. Afterwards, the antibody stock solution was diluted to 1 mg/mL using the formulation buffer, and SEC-HPLC analysis was performed under the conditions of Table 25, and the results are shown in Tables 26 to 31.
  • Human umbilical vein endothelial cells (HUVEC; Lonza_C2519A) cultured in EBM medium containing 0.5% FBS were pretreated with the indicated concentrations of each constructed bispecific antibody for 2 h. The cells were additionally exposed to 100 mM CoCl 2 (hypoxia) or 100 ng/ml LPS overnight. Endothelial permeability was determined by measuring the passage of FITC-dextran on HUVEC monolayers. To measure fluorescein isothiocyanate-dextran permeability, excitation was performed at 480 nm and fluorescence was detected at 520 nm.
  • the vascular permeability of candidate antibodies B and C was compared with that of Eylea alone, anti-c-Kit antibody alone, or Fariximab-treated groups, and the vascular permeability of candidate antibodies C and E was compared with that of anti-c-Kit antibody alone or comparator antibody-treated groups.
  • the comparator antibody is a 2(1+1) antibody in the form of Fab-VEGFR-Fc(K-H) that links anti-c-Kit antibody IgG (knob) and Eylea (VEGFR trap (hole)) using knob-into-hole technology.
  • the dexamethasone-treated group was used as a positive control.
  • candidate antibody B showed a significantly superior vascular permeability inhibition effect compared to the combination treatment group of Aylea and anti-c-Kit antibodies and the paricimab treatment group
  • candidate antibodies C and E showed a significantly superior vascular permeability inhibition effect compared to the comparative antibody
  • candidate antibody C showed a significantly superior vascular permeability inhibition effect compared to the combination treatment group of Aylea and anti-c-Kit antibodies, and showed a vascular permeability inhibition effect similar to the paricimab treatment group.
  • candidate antibodies B, C and E were observed to significantly inhibit the effect on paracellular permeability of HUVECs in vitro , suggesting that the bispecific candidate antibodies according to the present invention inhibit hypoxia- or LPS-induced venous endothelial barrier disruption.
  • Membranes were blocked with SuperBlock blocking buffer in TBST for 1 hour. Membranes were incubated overnight at 4°C with the following primary antibodies: pho-cKit (1:1000), pho-AKT (1:1000), pho-Erk (1:3000), cKit (1:1000), AKT (1:1000), Erk (1:3000), ⁇ -actin (1:10000). Each of the primary antibodies was diluted in SuperBlock blocking buffer dissolved in TBST. The membranes were washed with TBST for 10 min, repeated three times.
  • the membranes were incubated for 1 h at room temperature with the following secondary antibodies: mouse-anti-goat IgG-HRP (1:5000), mouse anti-rabbit IgG-HRP (1:5000), goat anti-mouse IgG-HRP (1:5000).
  • the above secondary antibodies were used by diluting each with 5% skim milk dissolved in TBST.
  • the membrane was washed with TBST for 10 minutes, and this was repeated three times.
  • the membrane was detected using ECL buffer.
  • the signal transduction inhibitory effects of candidate antibodies B and B-1 were compared with the anti-c-Kit antibody treatment group, and the signal transduction inhibitory effects of candidate antibodies C and E were compared with the comparative antibody treatment group.
  • the comparative antibody was the same as that used in Example 6-1.
  • candidate antibodies B, B-1, C, and E were observed to effectively inhibit SCF-dependent c-Kit signaling in HRMEC or LAD2.
  • HRMEC or ARPE-19 (ATCC: CRL-2302) cells cultured in medium containing 0.5% FBS were pretreated with each bispecific antibody for 6 h. The cells were additionally exposed to 1% O 2 hypoxia or LPS for 12 h. Cell supernatants were harvested and quantitative ELISA for VEGF, Ang-2, IL-6, IL-8, and MCP-1 was performed.
  • the cytokine inhibitory effect of candidate antibody A was compared with the Ilia treatment group, the cytokine inhibitory effects of candidate antibodies B and C were compared with the Ilia treatment group, anti-c-Kit antibody treatment group, and fariximab treatment group (dexamethasone treatment group was used as a positive control group), and the cytokine inhibitory effects of candidate antibodies C and E were compared with the comparative antibody treatment group.
  • the comparative antibody is the same as that used in Example 6-1.
  • candidate antibodies A, B, C, and E dose-dependently reduced the levels of cytokines induced by hypoxia or LPS in HRMEC and ARPE19 cells in vitro
  • candidate antibodies B and C showed significantly superior cytokine inhibitory effects compared to the combined treatment group of Aylea and anti-c-Kit antibodies and the Fariximab treatment group.
  • LCNV were generated on experimental day 0 (D0) in 6- to 8-week-old male C57BL/6 mice, recapitulating the subretinal neovascularization characteristic of human wet age-related macular degeneration (wAMD).
  • D1 One day after laser administration (D1), each bispecific antibody was administered via intravitreal injection.
  • Lesion patency was assessed by quantitative fluorescein angiography (qFA) on days 7 and 13 (D7 and D13) post-laser administration.
  • Animals were anesthetized with an IP injection of ketamine/xylazine, and 10% sodium fluorescein was administered via IP injection at a dose of 10 ⁇ L/g body weight.
  • Fluorescent fundus images were captured for one eye of each animal with a Micron IV imaging system during lesion filling with fluorescein, and again 2 min later. Fluorescence intensity of one lesion per eye was quantified via the integrated density function using ImageJ software. The difference in integrated density between the two hours post-injection was reported as a readout of lesion leakage.
  • candidate antibody C showed a much better effect on vascular permeability than the combined treatment with Ilia and anti-c-Kit antibodies.
  • candidate antibody C showed a statistically significant reduction in CNV lesion area compared to the combined treatment with Ilia and anti-c-Kit antibody.
  • each dual-specific antibody was administered into the vitreous of mice at different concentrations (0.5, 1, or 2 ⁇ g/ ⁇ L per eye) immediately after LCNV was induced in Example 7-1, and qFA and optical coherence tomography (OCT) were performed 10 days (D10) after laser administration.
  • OCT optical coherence tomography
  • the vehicle-treated negative control group was administered the formulation buffer
  • the reference drug-treated positive control group was administered aflibercept at 2 or 20 ⁇ g/ ⁇ L per eye.
  • CTF Corrected Total Fluorescence
  • FFA fundus fluorescein angiography
  • the candidate antibody A administration group showed a significant decrease in CTF value compared to the vehicle-treated negative control group, and there was no statistically significant difference according to the dosage.
  • the candidate antibody A administration group showed a significant decrease in CNV lesion volume compared to the vehicle-treated negative control group.

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Abstract

La présente invention concerne un anticorps bispécifique se liant de manière spécifique à c-kit et VEGF et, plus particulièrement : un anticorps bispécifique capable d'inhiber ou de neutraliser l'activité ou l'activation de c-kit et de VEGF par liaison à c-kit et VEGF avec une affinité élevée ; une molécule d'acide nucléique codant pour l'anticorps bispécifique ; un vecteur comprenant la molécule d'acide nucléique ; un procédé de préparation de l'anticorps bispécifique ; une composition pharmaceutique pour prévenir ou traiter des maladies angiogéniques, la composition comprenant l'anticorps bispécifique en tant que principe actif ; une méthode de prévention ou de traitement de maladies angiogéniques à l'aide de l'anticorps bispécifique ; et un kit c-kit et/ou VEGF ou kit de diagnostic de maladie angiogénique comprenant l'anticorps bispécifique.
PCT/KR2025/000744 2024-01-12 2025-01-13 Anticorps bispécifique se liant de manière spécifique à c-kit et vegf, et son utilisation Pending WO2025151018A1 (fr)

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KR1020250004411A KR20250111039A (ko) 2024-01-12 2025-01-10 c-Kit 및 VEGF에 특이적으로 결합하는 이중특이적 항체 및 이의 용도
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WO2015063187A1 (fr) * 2013-10-30 2015-05-07 Sergej Michailovic Kiprijanov Protéines multivalentes de liaison à l'antigène
KR20200040407A (ko) * 2018-10-10 2020-04-20 주식회사 노벨티노빌리티 신규 항-c-KIT 항체
WO2022253314A1 (fr) * 2021-06-04 2022-12-08 信达生物制药(苏州)有限公司 Molécule de liaison bispécifique se liant au vegf et à l'ang2 et son utilisation

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KR20110008086A (ko) * 2008-04-11 2011-01-25 메리맥 파마슈티컬즈, 인크. 인간 혈청 알부민 링커 및 그 콘쥬게이트
JP2012525149A (ja) * 2009-04-27 2012-10-22 オンコメッド ファーマシューティカルズ インコーポレイテッド ヘテロ多量体分子を作製するための方法
WO2015063187A1 (fr) * 2013-10-30 2015-05-07 Sergej Michailovic Kiprijanov Protéines multivalentes de liaison à l'antigène
KR20200040407A (ko) * 2018-10-10 2020-04-20 주식회사 노벨티노빌리티 신규 항-c-KIT 항체
WO2022253314A1 (fr) * 2021-06-04 2022-12-08 信达生物制药(苏州)有限公司 Molécule de liaison bispécifique se liant au vegf et à l'ang2 et son utilisation

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