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WO2010074218A1 - Anticorps anti-humain de midkine - Google Patents

Anticorps anti-humain de midkine Download PDF

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Publication number
WO2010074218A1
WO2010074218A1 PCT/JP2009/071567 JP2009071567W WO2010074218A1 WO 2010074218 A1 WO2010074218 A1 WO 2010074218A1 JP 2009071567 W JP2009071567 W JP 2009071567W WO 2010074218 A1 WO2010074218 A1 WO 2010074218A1
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Prior art keywords
amino acid
antibody
seq
acid sequence
mdk
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Japanese (ja)
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賢蔵 高田
りゅう 三浦
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Evec Inc
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Evec Inc
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to a human monoclonal antibody that binds to human midkine (hereinafter sometimes referred to as MDK) or an antigen-binding fragment thereof, and a pharmaceutical composition containing the antibody or fragment.
  • MDK human midkine
  • MDK Midkine
  • cytokines having actions such as cytoprotective action / growth action, promotion of migration of inflammatory cells such as macrophages and neutrophils, and suppression of apoptosis.
  • MDK is mainly composed of two domains, the N-terminal side and the C-terminal side, and has three disulfide bonds (SS bonds) on the N-terminal side and two on the C-terminal side.
  • SS bonds disulfide bonds
  • PTN heparin-binding growth factor pleiotrophin
  • PTN has about 50% identity with MDK.
  • PTN has the ability to cause neurite outgrowth and growth promotion, cell transformation or angiogenesis.
  • MDK is most strongly expressed in metaphase embryos, and its expression in adults is limited, but it is characterized by being strongly expressed in the processes of carcinogenesis, inflammation, and repair.
  • Non-patent Document 2 shows that adhesion after surgery is greatly reduced in knockout mice lacking MDK.
  • MDK is involved in inflammatory diseases such as various autoimmune diseases, rheumatoid arthritis, multiple sclerosis, postoperative adhesion, inflammatory bowel disease, psoriasis, asthma, and neutrophil dysfunction. It is known.
  • C Furthermore, it is known that the endometriosis also has a higher MDK level than healthy women (Non-patent Document 3).
  • MDK has an intimal thickening action, it is effective for vascular occlusion diseases such as restenosis after vascular reconstruction, cardiac coronary vasoocclusive disease, cerebrovascular occlusion disease, arteriosclerosis, cerebral infarction and the like.
  • Drugs containing MDK inhibitors are disclosed in a plurality of patent publications (Patent Documents 1, 2, 3 and 4).
  • Patent Documents 1, 2, 3 and 4 disclose a plurality of patent publications.
  • Non-patent Document 4 antibody expression against the antibody drug used was frequently observed (Non-patent Document 4), and in recent years, human antibodies have attracted attention.
  • Non-patent Document 4 Non-patent Document 4
  • human antibodies or antibody-binding fragments that specifically bind to MDK and sufficiently inhibit its biological activity are therapeutic strategies or preventive strategies for various diseases caused by MDK. It is considered useful.
  • a human anti-MDK monoclonal antibody having higher affinity is desired.
  • it is desired to provide a pharmaceutical composition comprising a monoclonal antibody having excellent affinity and neutralizing ability for MDK that causes various disease states and an antigen-binding fragment thereof.
  • a human monoclonal antibody that is not recognized as a foreign substance by the human immune system an antibody against an antibody drug is not produced
  • the present inventors have succeeded in obtaining a human monoclonal antibody having high affinity for MDK, and the antibody exhibits the biological activity of MDK. After confirming neutralization, the present invention was completed.
  • the present invention relates to an anti-MDK monoclonal antibody or a binding fragment thereof, a DNA (polynucleotide) encoding the antibody or binding fragment, a vector containing the DNA, and a host cell containing the vector described below. And so on.
  • a monoclonal antibody that specifically binds to human-derived midkine (MDK) and can neutralize its biological activity (a) from the group consisting of the amino acid sequences of SEQ ID NOs: 7, 8, and 9, and amino acid sequences having deletion, substitution, insertion and / or addition mutations of one to several amino acid residues in those amino acid sequences Any one or more amino acid sequences of selected light chain CDRs 1, 2 and 3, and (b) the amino acid sequences of SEQ ID NOs: 10, 11 and 12, and 1 to several amino acid residues in those amino acid sequences
  • An anti-human MDK monoclonal antibody comprising one or more amino acid sequences of heavy chain CDRs 1, 2 and 3 selected from the group consisting of amino acid sequences having deletion, substitution, insertion and / or addition mutations of An antigen-binding fragment thereof.
  • LCVR light chain variable region
  • HCVR heavy chain variable region
  • the antibody or antigen thereof according to (1) above which comprises a light chain (L chain) represented by the amino acid sequence of SEQ ID NO: 2 and a heavy chain (H chain) represented by the amino acid sequence of SEQ ID NO: 4. Binding fragment.
  • a disease involving midkine comprising the antibody or antigen-binding fragment thereof according to any one of (1) to (12) above and a pharmaceutically acceptable carrier.
  • Pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of (1) to (12) above and a pharmaceutically acceptable carrier.
  • Pharmaceutical composition comprising the disease involving MDK is an autoimmune disease, an inflammatory disease, cancer, endometriosis or a vascular occlusive disease.
  • MDK is involved, comprising administering to a mammal an antibody or antigen-binding fragment thereof according to any of (1) to (12) above in an effective amount for treating a disease involving MDK. How to treat the disease.
  • the disease involving MDK is an autoimmune disease, an inflammatory disease, cancer, endometriosis or a vaso-occlusive disease.
  • the anti-MDK antibody or antigen-binding fragment thereof according to the present invention specifically binds to MDK that causes various diseases, loses (neutralizes) its biological activity, and is a conventional anti-MDK (against MDK). Higher neutralization ability can be exhibited than the MDK antibody.
  • a human monoclonal antibody has no immunogenicity and no immune reaction.
  • the human monoclonal antibodies of the present invention are more suitable for human administration than chimeric or humanized antibodies.
  • the anti-MDK antibody or antigen-binding fragment thereof according to the present invention is useful as a preventive or therapeutic agent for diseases involving MDK.
  • diseases involving MDK include autoimmune diseases, inflammatory diseases, cancer, endometriosis, and vascular occlusive diseases.
  • the present invention has a high affinity for an antigen, which is characterized by a dissociation constant of 1 ⁇ 10 ⁇ 9 M or less, which has been difficult to realize with conventional antibody drugs.
  • a particularly preferred pharmaceutical composition comprising a human monoclonal antibody according to the invention is advantageous in that it is effective in very small amounts.
  • Antibody according to the present invention or antigen-binding fragment thereof The first aspect of the present invention is as follows: (1) an anti-human MDK monoclonal antibody capable of specifically binding to human-derived midkine (MDK) and neutralizing its biological activity Or an antigen-binding fragment thereof.
  • This antibody or antigen-binding fragment thereof typically contains the following (a) and (b).
  • the term “antibody” is four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. It shall refer to an immunoglobulin molecule consisting of things.
  • the monoclonal antibody in the present invention also consists of immunoglobulin molecules each containing two light chains (L chains) and heavy chains (H chains).
  • Each heavy chain is composed of a heavy chain variable region (sometimes referred to as “HCVR” or “V H ”) and a heavy chain constant region (the heavy chain constant region is composed of three domains, “C H 1”). , “C H 2” and “C H 3” (generic name: C H )).
  • Each L chain consists of an L chain variable region (sometimes referred to as “LCVR” or “V L ”) and an L chain constant region (the L chain constant region is a single domain, referred to as “C L ”). May be).
  • HCVR and LCVR are particularly important in that they are involved in antibody binding specificity. Since antibodies interact with target antigens primarily through LCVR and HCVR amino acid residues, the amino acid sequences within the variable region are more different between individual antibodies than sequences outside the variable region. Furthermore, HCVR and LCVR can be further subdivided into a region called a framework region (FR) and a hypervariable region called a complementarity determining region (CDR) that are kept more constant between various antibodies. HCVR and LCVR are each composed of 3 CDRs and 4 FRs, which are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus.
  • FR framework region
  • CDR complementarity determining region
  • antibody fragment refers to a fragment of one or more antibodies capable of specifically binding to an antigen (eg, MDK).
  • the fragment includes a peptide having a minimum amino acid sequence that specifically binds to an antigen.
  • antigen-binding fragment also included in the “antigen-binding fragment” are single-chain antibodies (scFV), bispecific antigens, multispecific antigens and the like having variable regions and complementarity determining regions of antibodies that specifically bind to antigens.
  • scFV single-chain antibodies
  • bispecific antigens bispecific antigens
  • multispecific antigens multispecific antigens and the like having variable regions and complementarity determining regions of antibodies that specifically bind to antigens.
  • antibody or antigen-binding fragment is also simply referred to as “antibody”.
  • an “antibody capable of neutralizing the biological activity of MDK” is intended to refer to an antibody that inhibits the biological activity of MDK by binding to MDK.
  • Bioactivity of MDK includes cytoprotection / growth activity, migration promoting activity of inflammatory cells such as macrophages and neutrophils, apoptosis inhibiting activity, osteoclast differentiation promoting activity, intimal thickening promoting activity, Endometrial differentiation promoting activity is known.
  • the terms “inhibitory effect”, “inhibition”, “suppression”, “can inhibit” and the like refer to biological activity attributable to an antigen (MDK) of about 5-100%, preferably 10 -100%, more preferably 20-100%, more preferably 30-100%, more preferably 40-100%, more preferably 50-100%, more preferably 60-100%, more preferably 70-100. %, More preferably 80 to 100%.
  • MDK antigen
  • variable region sequence derived from a specific naturally occurring antibody or the sequence of the CDR portion is a different antibody having different properties.
  • an expression vector such that it is contained in an invariant region or framework sequence derived from it, a recombinant antibody that mimics the properties of a particular naturally occurring antibody can be expressed. Therefore, it is not necessary to obtain the entire sequence of a particular antibody when recreating an intact recombinant antibody having binding properties similar to those of the original antibody.
  • the antibody heavy and light chain variable region sequence or CDR portion sequences may be sufficient for this purpose.
  • SEQ ID NOs: 7, 8, and 9 are amino acid sequences corresponding to light chain CDR1, CDR2, and CDR3, respectively.
  • SEQ ID NOs: 10, 11 and 12 are amino acid sequences corresponding to CDR1, CDR2 and CDR3 of the heavy chain, respectively.
  • preferred antibodies of the invention include all SEQ ID NOs: 7-12 (light chain CDR1, CDR2 and CDR3, and heavy chain CDR1, CDR2 and CDR3).
  • the CDR sequence has 1 to several (specifically, 1 to 9, 1 to 8, 1 to 7, in the amino acid sequence of SEQ ID NOs: 7 to 12, An amino acid sequence having a deletion, substitution, insertion and / or addition mutation of 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1) amino acid residues There may be.
  • the amino acid sequence other than the CDR is not particularly limited, and so-called CDR-grafted antibodies in which the amino acid sequence other than the CDR is derived from other antibodies, particularly other types of antibodies, are also included in the antibody of the present invention.
  • humanized antibodies in which amino acid sequences other than CDRs are also derived from humans are preferred, but one to several amino acid residues (specific numbers are the same as described above) in the framework region (FR) as necessary. There may be deletion, substitution, insertion and / or addition mutations.
  • a known method can be used as a method for producing a humanized antibody (Riechmann L, et al., Reshaping human antibodies for therapy. Nature, 332: 323-327, 1988). In the present invention, of course, fully human antibodies are preferred.
  • deletion, substitution, insertion and / or addition of one or more amino acid residues in the amino acid sequence of the protein of the present invention means that one or more amino acid residues in one and a plurality of amino acid sequences in the same sequence It means that there are amino acid residue deletion, substitution, insertion and / or addition, and two or more of deletion, substitution, insertion and addition may occur simultaneously. Examples of amino acid residues that can be substituted with each other are shown below. Amino acid residues contained in the same group can be substituted for each other.
  • Group A leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine;
  • Group B aspartic acid, glutamic acid, isoaspartic acid , Isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid;
  • group C asparagine, glutamine;
  • group D lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid;
  • group E Proline, 3-hydroxyproline, 4-hydroxyproline;
  • Group F serine, threonine, homoserine;
  • Group G phenylalanine, tyrosine.
  • more preferred antibodies are: (a) the amino acid sequence of SEQ ID NO: 5; 1 to several (specifically, 1 to 9, 1 to 8, 1 7-7, 1-6, 1-5, 1-4, 1-3, 1-2, or 1) amino acid residue deletion, substitution, insertion and / or addition mutation An amino acid sequence having an identity of 95% or more (preferably 96% or more, 97% or more, 98% or more, 99% or more or 99.5% or more) with the amino acid sequence of SEQ ID NO: 5 The light chain variable region (LCVR) shown, and (b) the absence of one to several amino acid residues (specific numbers are the same as described above) in the amino acid sequence of SEQ ID NO: 6; An amino acid sequence having a deletion, substitution, insertion and / or addition mutation; or SEQ ID NO: : 6 amino acid sequence 95% or more (specific percentages as above) containing the heavy chain variable region represented by amino acid sequence identity with (HCVR).
  • LCVR light chain variable region
  • more preferred antibodies are (a) a light chain variable region (LCVR) represented by the amino acid sequence of SEQ ID NO: 5 and (b) a heavy chain variable region (HCVR) represented by the amino acid sequence of SEQ ID NO: 6. ).
  • further preferred antibodies are: (a) amino acid sequence of SEQ ID NO: 2; deletion of 1 to several amino acid residues (specific numbers are the same as above) in the amino acid sequence of SEQ ID NO: 2.
  • amino acid sequence having a substitution, insertion and / or addition mutation or a light chain represented by an amino acid sequence having 95% or more identity (specific% is the same as above) with the amino acid sequence of SEQ ID NO: 2 ( L chain), and (b) amino acid sequence of SEQ ID NO: 4; deletion, substitution, insertion of one to several amino acid residues (specific numbers are the same as above) in the amino acid sequence of SEQ ID NO: 4 And / or an amino acid sequence having an additional mutation; or a heavy chain (H chain) represented by an amino acid sequence having 95% or more identity (specific% is the same as above) with the amino acid sequence of SEQ ID NO: 4 contains.
  • the most preferable antibody in the present invention is a fully human monoclonal antibody comprising a light chain (L chain) represented by the amino acid sequence of SEQ ID NO: 2 and a heavy chain (H chain) represented by the amino acid sequence of SEQ ID NO: 4. It is.
  • a preferred antibody class (subclass) in the present invention is IgG1 ( ⁇ ).
  • anti-MDK antibody or antigen-binding fragment thereof specifically binds to MDK causing various diseases, neutralizes its biological activity, and is more neutralized than conventional anti-MDK antibodies. It has the function.
  • specifically binds means to recognize and bind to a predetermined antigen.
  • the dissociation constant (Kd value) with MDK (particularly human MDK) in the antibody of the present invention is preferably 1 ⁇ 10 ⁇ 7 M or less, more preferably 1 ⁇ 10 ⁇ 8 M or less, and even more preferably 1 ⁇ 10 ⁇ 9 M or less, more preferably 9 ⁇ 10 ⁇ 10 M or less, more preferably 8 ⁇ 10 ⁇ 10 M or less, more preferably 7 ⁇ 10 ⁇ 10 M or less, more preferably 6.5 ⁇ 10 ⁇ 10 M or less, More preferably 6 ⁇ 10 ⁇ 10 M or less, further preferably 5.5 ⁇ 10 ⁇ 10 M or less, still more preferably 5 ⁇ 10 ⁇ 10 M or less, and most preferably 4.5 ⁇ 10 ⁇ 10 M or less. .
  • the Kd value of an anti-MDK antibody when used for the treatment of human diseases, it is advantageous in terms of safety and medical costs to reduce the dose of the antibody drug. Therefore, the smaller the Kd value of an anti-MDK antibody is, the more preferable it is. 1 ⁇ 10 ⁇ 9 M or less, especially 4.5 ⁇ 10 ⁇ 10 M or less, which suppresses the dose compared with existing antibody drugs. This is desirable.
  • a known method can be used to measure the dissociation constant between the antibody and MDK. For example, it can be measured by a protein interaction analyzer such as BIACORE 3000 (registered trademark) using an anti-MDK antibody immobilized on a chip.
  • the neutralizing ability of the anti-MDK antibody for example, Wilms tumor-derived cultured cell line G-401 (Developmental Biol., 159, 392-402 (1993)) known to proliferate in an autocrine MDK-dependent manner. Can be used to investigate. That is, the neutralizing activity of the anti-MDK antibody can be measured by adding an anti-MDK antibody to the culture system of the G-401 cell line and measuring its growth inhibitory effect.
  • the anti-MDK antibody or antigen-binding fragment thereof according to the present invention preferably has about 1 ⁇ g / mL (about 7 nM) as a neutralizing activity for G-401 cells proliferating in an autocrine MDK-dependent manner.
  • it has a cell growth inhibition rate of about 50% with respect to negative subjects (for example, human IgG1). More preferably, it has a cell growth inhibition rate of about 50% at about 0.1 ⁇ g / mL (about 0.7 nM) or less. Even more preferably, it has a growth inhibition rate of about 50% or more at 12 ng / mL (about 0.08 nM).
  • the antibody of the present invention is a full-length antibody or an antigen-binding fragment thereof, or SEQ ID NOs: 5 and 6 indicating a variable region, or amino acids of SEQ ID NOs: 7 to 12 indicating a complementarity determining region (CDR).
  • CDR complementarity determining region
  • heavy and light chain leader sequences are cleaved during protein maturation and do not contribute to the properties of the final antibody, but to add missing sequences, cloned cDNA sequences are ligated.
  • it can be combined with synthetic oligonucleotides by PCR amplification.
  • the entire variable region can be synthesized as a set of short, overlapping oligonucleotides and combined in a PCR amplification method to create a fully artificial variable region clone.
  • DNA according to the present invention is a DNA encoding an anti-MDK monoclonal antibody or antigen-binding fragment thereof capable of specifically binding to MDK and neutralizing its biological activity, comprising SEQ ID NOs: 2 and 4-12.
  • the isolated DNA is also the present invention as long as it has high identity with the DNA. include.
  • High stringency conditions are, for example, 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide at 50 ° C. (e.g., J. Sambrook et al Molecular Cloning, A Laboratory Manual 2 nd ed., Cold Spring Harbor Laboratory Press (1989), especially section 11.45 “Conditions for Hybridization of Oligonucleotide Probes”). Under these conditions, it can be expected that a polynucleotide (eg, DNA) having high identity can be efficiently obtained as the temperature is increased.
  • factors affecting the stringency of hybridization include multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration, and those skilled in the art will select these factors as appropriate. It is possible to achieve similar stringency.
  • DNA that hybridizes under highly stringent conditions include DNA encoding the amino acid sequence of SEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11 or 12, for example, 70% or more 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more of DNA having identity.
  • the identity of the base sequence can be determined using the above-described identity search algorithm (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc Natl Acad Sci USA 90: 5873, 1993) .
  • the DNA preferred in the present invention is DNA encoding the amino acid sequences of SEQ ID NOs: 5 and 6, and more preferably DNA encoding the amino acid sequences of SEQ ID NOs: 2 and 4.
  • the present invention also relates to a vector incorporating the above DNA, a host cell introduced with the vector, and an antibody production method using these.
  • the antibody of the present invention can also be prepared as a recombinant human antibody using a known method (see Nature, 312: 643, 1984, Nature, 321: 522, 1986, etc.).
  • the antibody of the present invention can be produced by culturing host cells into which the vector of the present invention has been introduced, and purifying the produced antibody from the culture supernatant or the like.
  • an expression vector for animal cells containing a gene encoding human antibody C H and / or human antibody C L prepared from the same cell or another human cell with cDNA encoding V H and V L. It can be produced by constructing a human antibody expression vector by inserting each vector, introducing it into an animal cell and expressing it.
  • the vector into which the nucleic acid encoding the V H or V L of the antibody of the present invention is not necessarily limited, but a vector or a high expression vector that is generally used for expression of a protein gene and the like and that is suitable for expression of an antibody gene is preferred. . Suitable examples include vectors containing the EF promoter and / or CMV enhancer.
  • expression vectors usually incorporating a nucleic acid encoding V H or V L are prepared and cotransfected into a host cell, but they may be incorporated into a single expression vector.
  • the host cell into which the expression vector is introduced is not necessarily limited, but a cell that is generally used for the expression of protein genes and the like and is particularly suitable for the expression of antibody genes is preferred. Examples include bacteria (such as E. coli), actinomycetes, yeast, insect cells (such as SF9), and mammalian cells (such as COS-1, CHO, and myeloma cells).
  • a recombinant animal cell line that stably produces the antibody for example, a CHO cell line is generally used.
  • Known methods can be used to generate, clone, and amplify and screen such recombinant cell lines (eg, Omasa T .: J. Biosci. Bioeng., 94, 600-605). , See 2002 etc.).
  • the present invention includes an antigen-binding fragment of the antibody of the present invention in addition to an antibody composed of two heavy chains and two light chains.
  • the antigen-binding fragment include Fab (fragment of antigen binding), Fab ′, and F (ab ′) 2.
  • Fab fragment of antigen binding
  • Fab ′ fragment of antigen binding
  • F (ab ′) 2 a single chain antibody
  • peptides containing active fragments of antibodies include peptides containing CDRs.
  • These can be produced by a known method such as a method of treating the antibody of the present invention with an appropriate proteolytic enzyme or a gene recombination technique. Purification of the antibody can be performed using a known purification means such as salting out, gel filtration, ion exchange chromatography or affinity chromatography.
  • scFv fragmentd by artificially shuffling V H and V L genes using phage display antibody technology that utilizes genetic engineering technology to express recombinant antibodies (recombinant antibodies) on the phage surface.
  • a single antibody can be expressed as a phage fusion protein by expressing a single chain fragment of variable region) antibody.
  • This technique is highly evaluated as a humanized antibody production technique that can avoid immunity and is replaced with a cell fusion method. Any specific antibody or antigen-binding fragment thereof produced using this technique with reference to the amino acid sequences of SEQ ID NOs: 2 and 4 to 12 in the present specification belongs to the technical scope of the present invention.
  • an antibody obtained by applying the Potentgent technology which has been developed in recent years, to significantly improve the ADCC activity of an antibody by modifying the sugar chain portion of the antibody (reference: Niwa R. , et al, Clin. Cancer Res., 10, 6248-6255 (2004)) or an antibody obtained by applying the Complement technology for improving CDC activity to the antibody of the present invention (reference: Kanda S., et al, Glycobiology, 17, 104-118 (2007)) also belongs to the technical scope of the present invention.
  • polyclonal antibodies and monoclonal antibodies are usually obtained using laboratory animals such as mice, rabbits, goats, etc., but the antibodies thus obtained were used. Since it has a sequence characteristic to animal species, if it is administered to humans as it is, it is recognized as a foreign substance by the human immune system, and a human anti-animal antibody response may occur (that is, an antibody of an antibody is produced).
  • the anti-MDK monoclonal antibody or antigen-binding fragment thereof according to the present invention can be obtained from blood-derived antibody-producing cells such as healthy persons, and is a fully human antibody. Even if this fully human antibody is administered to the human body as an antibody drug, it is considered that it has no immunogenicity and no immune reaction is observed.
  • the anti-MDK monoclonal antibody of the present invention has higher neutralizing ability than the conventional anti-MDK monoclonal antibody, the same therapeutic effect can be expected with a smaller dose.
  • the present invention prevents or treats a disease involving midkine (MDK), comprising the antibody or antigen-binding portion thereof and a pharmaceutically acceptable carrier.
  • MDK midkine
  • a pharmaceutical composition is provided.
  • a disease caused by MDK is a disease in which inhibition of MDK activity is predicted to reduce the symptoms and / or progression of the disease.
  • the term “disease associated with MDK” means that the subject suffering from the disease has MDK causing the disease pathology or exacerbating the disease It is intended to include diseases and other diseases that have been shown to be or are believed to be Diseases involving MDK include, for example, autoimmune diseases and inflammatory diseases ((2004) Biochem. Biophys. Res. Commun. 317, 108-113 .; (2004) Arthritis Rheum. 50, 1420-1429 .; (2008 ) PNAS 105, 3915-3920), cancer (Muramatsu T., (2002), J. Biochem., 132 (359-371); Takei, Y. et al, (2001) Cancer Res.
  • pharmaceutically acceptable carrier includes any and all physiologically compatible solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like.
  • pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof.
  • the composition preferably contains a pH adjuster or isotonic agent such as sugar, polyalcohol such as mannitol or sorbitol, or sodium chloride.
  • Pharmaceutically acceptable carriers can additionally contain minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, buffering agents, stabilizing agents and the like that increase the preservability or effectiveness of the antibody or antibody portion.
  • compositions of the present invention can be made into various dosage forms.
  • Such compositions include, for example, liquids, semi-solids such as solutions (eg, injectable and infusible solutions) and dispersions, suspensions, tablets, capsules, troches, pills, powders, liposomes, suppositories, etc.
  • Solid dosage forms are included. The preferred form depends on the intended mode of administration and therapeutic application. Generally preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used to passively immunize humans with other antibodies.
  • Preferred dosage forms are parenteral (eg, intravenous, subcutaneous, intraperitoneal, intramuscular).
  • the antibody is administered by intravenous infusion or intravenous injection.
  • the antibody is administered by intramuscular or subcutaneous injection.
  • the antibodies and antibody fragments of the present invention can be incorporated into pharmaceutical compositions suitable for parenteral administration.
  • the antibody or antibody portion is preferably prepared as an injectable preparation containing 0.1 to 250 mg / mL of antibody when a single type is used.
  • an injectable preparation containing 0.001 to 100 mg / mL of antibody.
  • the mixing ratio of the plurality of types of antibodies can be set as appropriate.
  • the injectable preparation can be constituted by dissolving the active ingredient in a liquid or freeze-drying the active ingredient in a flint or amber vial, ampoule or prefilled syringe.
  • the buffer can be L-histidine (1-50 mM) at pH 5.0-7.0 (optimally pH 6.0), and 5-10 mM L-histidine, optimally.
  • Other suitable buffering agents include, but are not limited to, sodium succinate, sodium citrate, sodium phosphate, or potassium phosphate.
  • Sodium chloride can be used to change the osmotic pressure of a 0-300 mM concentration solution (150 mM, optimal for liquid dosage forms).
  • the lyophilized dosage form can contain a cryoprotectant, primarily 0-10% (optimally 0.5-5.0%) sucrose.
  • suitable cryoprotectants include mannitol, trehalose and lactose.
  • the lyophilized dosage form can contain a bulking agent, primarily 1-10% mannitol (2-4% when optimal).
  • stabilizers primarily 1-50 mM (optimally 5-10 mM) L-methionine can be used.
  • stabilizers include glycine, arginine, polysorbate 80, and the like, and in the case of polysorbate 80 can include 0-0.05% (optimally 0.005-0.01%).
  • surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactant.
  • the pharmaceutical composition generally must be sterile or stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration.
  • a sterile injectable solution is made by mixing the required amount of active compound (ie, antibody or antibody portion) in a suitable solvent with one or a combination of the above components, if necessary, followed by filter sterilization. It can be prepared by doing.
  • dispersions are prepared by mixing the active compound with a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • a sterile powder formulation for the preparation of a sterile injectable solution the preferred method of preparation is freeze vacuum drying and spray drying of the sterile filtered solution as previously described, thereby adding to the active ingredient powder, A composition containing any other desired ingredients is obtained.
  • the proper fluidity of the solution can be achieved, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. Can be maintained.
  • Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts or gelatin.
  • an antibody or antibody portion of the invention is formulated together with one or more other therapeutic agents useful for treating a disease caused by MDK, or such other therapeutic agent and Administer at the same time.
  • an anti-MDK antibody or antibody portion of the invention can be combined with one or more other antibodies that bind to other targets (eg, antibodies that bind to other cytokines or antibodies that bind to cell surface molecules). It can be formulated or co-administered with such other antibodies.
  • one or more antibodies of the present invention can be used in combination of two or more of the aforementioned therapeutic agents. Such combination therapy can advantageously utilize lower doses of the administered therapeutic agent, thus avoiding the toxicities or complications that may be associated with various single therapies.
  • the anti-MDK monoclonal antibody and the antigen-binding fragment thereof according to the present invention are obtained by isolating a cell clone that produces the antibody from blood of a healthy person through various steps, and obtaining antibody-positive cells from the obtained antibody-producing cell library.
  • the antibody obtained from the antibody-positive cell supernatant after the selection step can be obtained by affinity purification.
  • the method of recovering monoclonal antibody from cells induced to proliferate can be carried out by well-known methods commonly used in the production of monoclonal antibodies.
  • Lymphocytes that produce antibodies that bind to MDK are selected from the antibody-producing cell library, and the antibodies are removed. That is, a cell population (clone) that produces an antibody that binds to MDK is selected from the antibody-producing cell library by a limiting dilution method.
  • ELISA For detection of the fraction that binds to MDK, it is preferable to employ ELISA using MDK and a labeled mouse anti-human IgG antibody.
  • a cell population (clone) that produces only the target antibody can be obtained.
  • Affinity purification using Protein A or G To purify anti-MDK antibodies, selected cells can be grown in roller bottles, 2 liter spinner flasks, or other culture systems. The supernatant can be filtered and concentrated, and then the protein can be purified by affinity chromatography such as Protein A or Protein G-Sepharose (Piscataway, NJ, Pharmacia). The buffer solution can be exchanged into PBS and the concentration can be determined by OD 280 or preferably by nephelometer analysis. The isotype can be determined by a method specific to the isotype antigen.
  • the anti-MDK antibody thus obtained is a fully human antibody prepared from B lymphocytes sensitized in the human body, the possibility of an immune reaction to the antibody is low.
  • Another feature of the production of antibody-producing cell clones is the use of an EB virus that has the activity of inducing proliferation by infecting B lymphocytes.
  • the advantage of the EB virus method is that a natural antibody produced in the human body can be produced and an antibody with high affinity can be obtained.
  • human antibodies against MDK have been found to be about 10-100 times more affinity than antibodies made by artificially immunizing mice.
  • a population of B lymphocytes proliferated by EB virus infection becomes a library of antibody-producing cells.
  • a specific antibody-producing cell clone can be isolated from this library to obtain a human antibody.
  • the antibody or antigen-binding fragment thereof of the present invention also includes nucleic acids, vectors and host cells for expressing the recombinant antibody of the present invention or antibody-binding portions thereof.
  • SEQ ID NO: 1 Represents the anti-human midkine antibody (EV3032) light chain nucleotide sequence.
  • SEQ ID NO: 2 Anti-human midkine antibody (EV3032) represents the light chain amino acid sequence.
  • SEQ ID NO: 3 Anti-human midkine antibody (EV3032) represents the heavy chain nucleotide sequence.
  • SEQ ID NO: 4 Anti-human midkine antibody (EV3032) represents the heavy chain amino acid sequence.
  • SEQ ID NO: 5 This represents the amino acid sequence of the anti-human midkine antibody (EV3032) L chain variable region.
  • SEQ ID NO: 6 Anti-human midkine antibody (EV3032) represents the heavy chain variable region amino acid sequence.
  • SEQ ID NO: 7 Represents the anti-human midkine antibody (EV3032) L chain CDR1 amino acid sequence.
  • SEQ ID NO: 8 Represents the anti-human midkine antibody (EV3032) L chain CDR2 amino acid sequence.
  • SEQ ID NO: 9 Represents the anti-human midkine antibody (EV3032) L chain CDR3 amino acid sequence.
  • SEQ ID NO: 10 Anti-human midkine antibody (EV3032) represents the heavy chain CDR1 amino acid sequence.
  • SEQ ID NO: 11 Anti-human midkine antibody (EV3032) represents the heavy chain CDR2 amino acid sequence.
  • SEQ ID NO: 12 Anti-human midkine antibody (EV3032) represents the heavy chain CDR3 amino acid sequence.
  • SEQ ID NO: 13 This represents the nucleotide sequence of IgG1 H chain FR4.
  • SEQ ID NO: 14 IgG1 H chain The nucleotide sequence of KpnI site-introduced FR4.
  • SEQ ID NO: 15 EV3011 represents the nucleotide sequence of H chain FR4.
  • SEQ ID NO: 16 EV3011 represents the nucleotide sequence of H4 KpnI site-introduced FR4.
  • lymphocytes were isolated from the peripheral blood of healthy individuals, and after EBV infection, they were seeded in 96-well plates. After culturing for about 3 weeks, the culture supernatant was collected, and whether or not anti-MDK antibody was produced was confirmed by ELISA (primary screening). ELISA was performed using a 96 well plate coated with MDK. The cell population confirmed to produce anti-MDK antibody was diluted and seeded in a new 96-well plate. After 3 weeks of culture, secondary screening for anti-MDK antibody was performed.
  • the resulting antibody positive cell population was further diluted and seeded in a new 96 well plate.
  • the proliferating wells were screened for anti-MDK antibody by ELISA. Furthermore, by repeating this cloning operation, a cell clone producing the target antibody was obtained.
  • a KpnI site (GGTACC) was introduced into FR4 of the IgG1 H chain, but the amino acid residue was not converted.
  • the expression plasmid was cleaved at the HindIII site derived from the expression vector located on the 5 ′ side of the IgG1 H chain DNA and the newly introduced KpnI site to remove the leader sequence and variable region of the IgG1 H chain. Further, a DNA fragment encoding from the leader sequence to FR4 was amplified by PCR using the IgG3 MDK antibody H chain cDNA as a template. A HindIII site was added to the 5′-end primer used for PCR, and a KpnI site was added to the 3′-end primer.
  • IgG1 H chain FR4 (5'TGGGGCCAGGGgACCACGGTCACCGTCTCCTCA 3 '; SEQ ID NO: 13)
  • IgG1 H chain KpnI site introduction FR4 (5'TGGGGCCAGGGtACCACGGTCACCGTCTCCTCA 3 '; SEQ ID NO: 14)
  • EV3011 H chain FR4 (5'TGGGGCAAAGGgACCACGGTCACCGTCTCCTCA 3 '; SEQ ID NO: 15) EV3011 H chain KpnI site introduction FR4: (5'TGGGGCAAAGGtACCACGGTCACCGTCTCCTCA 3 '; SEQ ID NO: 16)
  • the obtained PCR product was digested with HindIII and KpnI and incorporated into the HindIII-KpnI site of the expression vector. It was confirmed by sequencing that the constructed expression plasmid encoded DNA of the full-length IgG1 H chain in which the leader sequence of the anti-MDK antibody H chain and the variable region and the constant region of IgG1 H chain were linked.
  • the anti-MDK antibody IgG1 H chain expression plasmid and the expression plasmid inserted with the L chain cDNA were simultaneously introduced into 293T cells, and the ELISA method confirmed that the antibody in the culture supernatant was a human IgG1 antibody and bound to MDK. confirmed.
  • the resulting anti-MDKIgG1 antibody (EV3032) expression plasmid was introduced into CHO cells.
  • the same method as in the case of 293T cells described above was employed.
  • a CHO cell clone that constantly expresses the antibody was obtained.
  • CHO cells stably expressing the antibody were cultured in a serum-free medium, and the culture supernatant was collected.
  • This culture supernatant was added to a Protein A column, and a purified antibody was obtained by affinity purification.
  • Column is HiTrap rProteinA FF (Amersham) prepacked columns were used, and the purification conditions were those recommended by the column manufacturer.
  • MDK binding of the antibody was confirmed by ELISA.
  • an antibody H chain of about 50 kDa and an antibody L chain of about 25 kDa were confirmed by SDS-PAGE.
  • Affinity Analysis of Anti-MDK Antibody was performed by surface plasmon resonance (SPR) using BIA Core System (registered trademark). The method used was an antibody capture method using a ProteinG-immobilized sensor chip. The method followed the manufacturer's recommended protocol (see the BIACORE, Biocore J instruction manual). The dissociation constant (KD (M)) for MDK was 4.5 ⁇ 10 ⁇ 10 M, confirming that it has a very high affinity for MDK.
  • G401 Wilms tumor-derived cultured cell line G401 is known to proliferate in an autocrine MDK-dependent manner (Developmental Biol., 159, 392-402 (1993)) . Therefore, whether or not the anti-MDK antibody (EV3032) inhibits MDK proliferation activity was examined using G401 cells.
  • G401 cells suspended in 10% FCS-containing McCoy's 5A medium were seeded in a 96-well plate at a concentration of 2,000 cells / well and cultured overnight. On the next day, the medium was removed, and a serum-free medium containing antibodies (OPTI-MEM, Invitrogen) was added.
  • the antibody was diluted 2-fold in the range of 7.8-1000 ng / ml, and human IgG1 antibody was used as a control antibody.
  • the cell count kit (WST-1 assay, doujin) was used to evaluate the viability of G401 cells by the intensity of color development (A450 / ref.595). If the antibody has neutralizing activity, it can neutralize MDK that G401 cells autocrine and suppress the growth of G401 cells.
  • the results are shown in FIG. IC 50 was determined by standard methods. IC 50 was 12 ng / ml, confirming that it has a very high neutralizing ability.
  • the anti-MDK antibody or antigen-binding fragment thereof according to the present invention is useful in a small amount as a preventive or therapeutic agent for pathological conditions caused by MDK, such as cancer and inflammatory diseases. Conceivable.
  • the anti-midkine antibody of the present invention and a pharmaceutical composition containing the antibody can be used for prevention or treatment of autoimmune diseases, inflammatory diseases, cancer, endometriosis, vascular occlusive diseases and the like.

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Abstract

Cette invention se rapporte à un anticorps monoclonal humain qui présente une excellente affinité vis-à-vis de la Midkine (parfois désignée sous le nom de « MDK ») et une excellente activité de neutralisation de celle-ci, qui induit divers états pathologiques ; à une composition pharmaceutique qui contient l'anticorps ; et à d'autres choses. Cette invention se rapporte de manière spécifique à un anticorps monoclonal humain capable de se lier à une MDK humaine, ou à un fragment de liaison à l'antigène de celle-ci ; à une composition pharmaceutique qui contient l'anticorps ou le fragment ; et à d'autres choses. La composition pharmaceutique peut être utilisée pour la prévention ou le traitement de maladies auto-immunes, de maladies inflammatoires, de cancers, d'endométrioses, de maladies occlusives vasculaires et similaires.
PCT/JP2009/071567 2008-12-25 2009-12-25 Anticorps anti-humain de midkine Ceased WO2010074218A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014021339A1 (fr) * 2012-07-30 2014-02-06 国立大学法人名古屋大学 Anticorps monoclonal dirigé contre la protéine midkine d'origine humaine
EP2686016A4 (fr) * 2011-03-14 2014-09-03 Cellmid Ltd Anticorps de reconnaissance de domaine n-terminal de midkine

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999003493A1 (fr) * 1997-07-14 1999-01-28 Meiji Milk Products Co., Ltd. Medicaments contenant midkine en tant principe actif ou inhibiteurs desdits medicaments
JP2002085058A (ja) * 2000-09-07 2002-03-26 Meiji Milk Prod Co Ltd ヒトｍｋに対するモノクローナル抗体
WO2008059616A1 (fr) * 2006-11-14 2008-05-22 Medical Therapies Limited Anticorps reconnaissant le domaine c de la midkine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999003493A1 (fr) * 1997-07-14 1999-01-28 Meiji Milk Products Co., Ltd. Medicaments contenant midkine en tant principe actif ou inhibiteurs desdits medicaments
JP2002085058A (ja) * 2000-09-07 2002-03-26 Meiji Milk Prod Co Ltd ヒトｍｋに対するモノクローナル抗体
WO2008059616A1 (fr) * 2006-11-14 2008-05-22 Medical Therapies Limited Anticorps reconnaissant le domaine c de la midkine

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2686016A4 (fr) * 2011-03-14 2014-09-03 Cellmid Ltd Anticorps de reconnaissance de domaine n-terminal de midkine
WO2014021339A1 (fr) * 2012-07-30 2014-02-06 国立大学法人名古屋大学 Anticorps monoclonal dirigé contre la protéine midkine d'origine humaine
JPWO2014021339A1 (ja) * 2012-07-30 2016-07-21 国立大学法人名古屋大学 ヒトミッドカインに対するモノクローナル抗体
US9840552B2 (en) 2012-07-30 2017-12-12 National University Corporation Nagoya University Monoclonal antibody against human midkine

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