WO2013133253A1 - 抗がん剤 - Google Patents
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- WO2013133253A1 WO2013133253A1 PCT/JP2013/055927 JP2013055927W WO2013133253A1 WO 2013133253 A1 WO2013133253 A1 WO 2013133253A1 JP 2013055927 W JP2013055927 W JP 2013055927W WO 2013133253 A1 WO2013133253 A1 WO 2013133253A1
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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
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
- C07K16/3023—Lung
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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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
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- 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
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/515—Complete light chain, i.e. VL + CL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
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- 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
Definitions
- the present invention relates to an anticancer agent containing a human antibody ⁇ -type light chain that is cytotoxic against cancer cells, particularly lung cancer cells.
- the antibody is composed of a heavy chain (H chain) and a light chain (L chain).
- the heavy chain and the light chain are composed of a variable region (VR) and a constant region (CR), and the variable region has a hypervariable region (CDR: Complementarity Determining Region).
- the light chains of antibodies are classified into ⁇ and ⁇ types.
- Antibody enzymes have high molecular recognition ability and enzyme activity of antibodies, and are expected to be applied in many fields such as medical, chemical and food industries.
- an antibody enzyme that is highly specific to a target molecule and that can exhibit a disorder to the target molecule by enzyme activity is expected to be an excellent anticancer agent with few side effects.
- the main object of the present invention is to provide an anticancer agent comprising a human antibody light chain that exhibits cytotoxicity against cancer cells, particularly lung cancer cells, as an active ingredient.
- the present inventors obtained a novel human-type antibody light chain from peripheral blood obtained from a volunteer hyperimmunized multiple times using a rabies virus vaccine, and surprisingly, these were examined.
- the present inventors have found that some of the obtained human antibody ⁇ -type light chains have high cytotoxicity against cancer cells, particularly lung cancer cells, and have completed the invention.
- the anticancer agent according to the present invention is (1)
- the variable region is the amino acid sequence of SEQ ID NO: 1, an amino acid sequence in which one or several amino acids are substituted, added or deleted in the amino acid sequence, or an amino acid having 95% or more homology with the amino acid sequence A human antibody kappa light chain consisting of a polypeptide represented by the sequence and being a dimer
- the variable region is the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence, or an amino acid having 95% or more homology with the amino acid sequence A human antibody kappa light chain consisting of a polypeptide represented by the sequence and being a dimer
- the variable region is the amino acid sequence of SEQ ID NO: 9, an amino acid sequence in which one or several amino acids are substituted, added or deleted in the amino acid sequence, or an amino acid having 95% or more homology with the amino acid sequence A human antibody kappa light chain consisting of a poly
- a human antibody ⁇ -type light chain consisting of a polypeptide represented by a homologous amino acid sequence, which is a monomer, (7)
- the variable region is the amino acid sequence from 1 to 112 of SEQ ID NO: 40, the amino acid sequence in which one or several amino acids are substituted, added or deleted in the amino acid sequence, or 95% or more of the amino acid sequence It consists of a polypeptide represented by a homologous amino acid sequence and is a dimeric human antibody ⁇ -type light chain, or (8) the amino acid sequence from 1 to 107 in SEQ ID NO: 41, or the amino acid sequence
- a human antibody ⁇ type that is a dimer consisting of an amino acid sequence in which one or several amino acids are substituted, added, or deleted, or a polypeptide represented by an amino acid sequence having 95% or more homology with the amino acid sequence It contains a light chain.
- an anticancer agent having high cytotoxicity against cancer cells, particularly lung cancer cells can be provided. Since the anticancer agent of the present invention contains an antibody enzyme as an active ingredient, it has high specificity for cancer cells. Furthermore, since the amino acid sequence of the antibody enzyme is completely human, there are no problems such as allergies to humans. For this reason, the anticancer agent of the present invention is very useful as a breakthrough new drug with high activity and a sample for its development.
- FIG. 6B is a continuation of FIG. 6B and shows the amino acid sequence of the wild-type human antibody kappa light chain. It is a figure which shows the amino acid sequence of a wild type human antibody (kappa) light chain.
- FIG. 6D is a continuation of FIG. 6D and shows the amino acid sequence of the wild-type human antibody kappa light chain.
- FIG. 6B is a continuation of FIG.
- FIG. 6E shows the amino acid sequence of the wild-type human antibody kappa light chain. It is a figure which shows the amino acid sequence of a wild type human antibody (kappa) light chain.
- FIG. 6B is a continuation of FIG. 6G and shows the amino acid sequence of the wild-type human antibody kappa light chain. It is a figure which shows the amino acid sequence of a wild type human antibody (kappa) light chain. It is a figure which shows the result of the in-vivo assay. It is a figure which shows the result of a safety test (toxicity test).
- the present invention provides an anticancer agent comprising a human antibody ⁇ type light chain having cytotoxicity against cancer cells.
- the “human antibody kappa light chain” refers to a kappa light chain of a human-derived immunoglobulin (light chain).
- the “anticancer agent” means an agent having an activity of killing cancer cells or suppressing or inhibiting proliferation.
- cytotoxicity means the property of giving death or functional disorder to cells.
- the human antibody ⁇ -type light chain (hereinafter sometimes referred to as “human antibody ⁇ -type light chain according to the present invention”), which is an active ingredient of the anticancer agent according to the present invention, is specifically the following (1 ) To (8).
- the variable region is the amino acid sequence of SEQ ID NO: 1, an amino acid sequence in which one or several amino acids are substituted, added or deleted in the amino acid sequence, or an amino acid having 95% or more homology with the amino acid sequence
- a human antibody kappa light chain consisting of a polypeptide represented by a sequence and being a dimer.
- variable region is the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence, or an amino acid having 95% or more homology with the amino acid sequence A human antibody kappa light chain consisting of a polypeptide represented by a sequence and being a dimer.
- the variable region is the amino acid sequence of SEQ ID NO: 9, an amino acid sequence in which one or several amino acids are substituted, added or deleted in the amino acid sequence, or an amino acid having 95% or more homology with the amino acid sequence A human antibody kappa light chain consisting of a polypeptide represented by a sequence and being a dimer.
- variable region is the amino acid sequence of SEQ ID NO: 13, an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence, or an amino acid having 95% or more homology with the amino acid sequence A human antibody kappa light chain consisting of a polypeptide represented by a sequence and being a dimer.
- the variable region is the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence, or an amino acid having 95% or more homology with the amino acid sequence A human antibody kappa light chain consisting of a polypeptide represented by a sequence and being a monomer.
- the variable region is the 1st to 113th amino acid sequence of SEQ ID NO: 38, the amino acid sequence in which one or several amino acids are substituted, added or deleted, or the amino acid sequence and 95% or more of the amino acid sequence.
- a human antibody ⁇ -type light chain consisting of a polypeptide represented by a homologous amino acid sequence and being a monomer.
- the variable region is the amino acid sequence from 1 to 112 of SEQ ID NO: 40, the amino acid sequence in which one or several amino acids are substituted, added or deleted in the amino acid sequence, or 95% or more of the amino acid sequence
- a human antibody kappa light chain consisting of a polypeptide represented by an amino acid sequence having homology and being a dimer.
- variable region is the 1st to 107th amino acid sequence of SEQ ID NO: 41, the amino acid sequence in which one or several amino acids are substituted, added, or deleted, or 95% or more of the amino acid sequence.
- a human antibody kappa light chain consisting of a polypeptide represented by an amino acid sequence having homology and being a dimer.
- the human antibody kappa light chain which is a dimer and whose variable region consists of a polypeptide represented by the amino acid sequence of SEQ ID NO: 1 may be referred to as human antibody kappa light chain (# 1).
- the human antibody ⁇ -type light chain (# 1) can be obtained by adding a known human antibody constant region to the variable region described above.
- the full-length amino acid sequence is represented by SEQ ID NO: 2.
- CDR1 in the human antibody kappa type light chain (# 1) is positions 24 to 39 in the amino acid sequences of SEQ ID NOs: 1 and 2
- CDR2 is positions 55 to 61 in the amino acid sequences of SEQ ID NOs: 1 and 2.
- CDR3 is positions 94 to 102 in the amino acid sequences of SEQ ID NOs: 1 and 2.
- cysteine for forming a disulfide bond is present and forms a dimer.
- the human antibody ⁇ type light chain (# 1) has a cysteine for forming a disulfide bond with other light chains, like the wild type.
- the cysteine is the 220th cysteine in the amino acid sequence of SEQ ID NO: 2.
- the human antibody ⁇ -type light chain (# 1) has cytotoxicity against cancer cells, particularly lung cancer cells, as shown in Examples below. For this reason, it is suitable as an active ingredient of an anticancer agent.
- an anticancer agent for the anticancer activity of human antibody ⁇ type light chain (# 1), high molecular recognition ability for the target molecule is important.
- the active center is in the variable region.
- the human antibody kappa light chain according to the present invention forms a dimer, and the variable region is an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 1, or It may consist of a polypeptide represented by an amino acid sequence having 95% or more homology with the amino acid sequence.
- the polypeptide may be referred to as a variant of a human antibody ⁇ type light chain (# 1).
- the variant of the human antibody ⁇ type light chain (# 1) is an amino acid sequence in which one or several amino acids other than the 220th cysteine are substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 2, or the amino acid It may consist of a polypeptide represented by an amino acid sequence having 95% or more homology with the sequence.
- the human antibody kappa light chain (# 1) mutant used as the human antibody kappa light chain according to the present invention is a dimer having an anticancer activity in the same manner as the human antibody kappa light chain (# 1). It is. Therefore, in the variant of human antibody ⁇ type light chain (# 1), CDR1, CDR2, and CDR3 are identical (conserved) to the amino acid sequence of SEQ ID NO: 1 or 2, and the amino acid of SEQ ID NO: 2 A cysteine corresponding to the 220th cysteine in the sequence is also conserved.
- the variant of the human antibody ⁇ -type light chain (# 1) is a variant in which amino acids in regions other than CDR1, CDR2, and CDR3 are mutated, and amino acids in other regions of the variable region are mutated. It is preferable that
- the human antibody kappa light chain which is a dimer and whose variable region consists of a polypeptide represented by the amino acid sequence of SEQ ID NO: 9 is sometimes referred to as human antibody kappa light chain (# 4).
- the human antibody ⁇ -type light chain (# 4) can be obtained by adding a known human antibody constant region to the variable region described above.
- the full-length amino acid sequence is represented by SEQ ID NO: 10.
- CDR1 in the human antibody kappa light chain (# 4) is positions 24 to 40 in the amino acid sequences of SEQ ID NOs: 9 and 10
- CDR2 is positions 56 to 62 in the amino acid sequences of SEQ ID NOs: 9 and 10.
- CDR3 is positions 95 to 102 in the amino acid sequences of SEQ ID NOs: 9 and 10.
- a cysteine for forming a disulfide bond with another light chain is the 220th cysteine in the amino acid sequence of SEQ ID NO: 10.
- the human antibody ⁇ -type light chain (# 4) has cytotoxicity against cancer cells, particularly lung cancer cells, as shown in Examples below. For this reason, it is suitable as an active ingredient of an anticancer agent.
- the human antibody ⁇ -type light chain according to the present invention forms a dimer and the variable region is an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 9, or It may consist of a polypeptide represented by an amino acid sequence having 95% or more homology with the amino acid sequence.
- the polypeptide may be referred to as a variant of a human antibody ⁇ type light chain (# 4).
- the variant of human antibody kappa light chain (# 4) is an amino acid sequence in which one or several amino acids other than the 220th cysteine are substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 10, or the amino acid It may consist of a polypeptide represented by an amino acid sequence having 95% or more homology with the sequence.
- the human antibody ⁇ -type light chain (# 4) mutant used as the human antibody ⁇ -type light chain according to the present invention is a dimer having an anticancer activity in the same manner as the human antibody ⁇ -type light chain (# 4). It is. Therefore, in the variant of the human antibody ⁇ -type light chain (# 4), CDR1, CDR2, and CDR3 are identical (conserved) to the amino acid sequence of SEQ ID NO: 9 or 10, and the amino acid of SEQ ID NO: 10 A cysteine corresponding to the 220th cysteine in the sequence is also conserved.
- the variant of the human antibody ⁇ -type light chain (# 4) is a variant in which amino acids in regions other than CDR1, CDR2, and CDR3 are mutated, and amino acids in other regions of the variable region are mutated. It is preferable that
- the human antibody kappa light chain which is a dimer and whose variable region consists of a polypeptide represented by the amino acid sequence of SEQ ID NO: 13 may be referred to as human antibody kappa light chain (# 7).
- the human antibody kappa light chain (# 7) can be obtained by adding a known human antibody constant region to the variable region described above, and in one embodiment, the full-length amino acid sequence is shown in SEQ ID NO: 14. .
- CDR1 in the human antibody kappa light chain (# 7) is positions 24 to 39 in the amino acid sequences of SEQ ID NOs: 13 and 14, and CDR2 is positions 55 to 61 in the amino acid sequences of SEQ ID NOs: 13 and 14.
- CDR3 is positions 94 to 101 in the amino acid sequences of SEQ ID NOs: 13 and 14.
- a cysteine for forming a disulfide bond with another light chain is the 219th cysteine in the amino acid sequence of SEQ ID NO: 14.
- the human antibody ⁇ -type light chain (# 7) has cytotoxicity against cancer cells, particularly lung cancer cells, as shown in Examples below. For this reason, it is suitable as an active ingredient of an anticancer agent.
- the human antibody ⁇ -type light chain according to the present invention forms a dimer, and the variable region has an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 13, or It may consist of a polypeptide represented by an amino acid sequence having 95% or more homology with the amino acid sequence.
- the polypeptide may be referred to as a variant of a human antibody ⁇ type light chain (# 7).
- the variant of the human antibody ⁇ -type light chain (# 7) is an amino acid sequence in which one or several amino acids other than the 219th cysteine are substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 14, or the amino acid It may consist of a polypeptide represented by an amino acid sequence having 95% or more homology with the sequence.
- the human antibody kappa light chain (# 7) mutant used as the human antibody kappa light chain according to the present invention is a dimer having an anticancer activity in the same manner as the human antibody kappa light chain (# 7). It is. Therefore, in the variant of the human antibody ⁇ type light chain (# 7), CDR1, CDR2, and CDR3 are identical (conserved) to the amino acid sequence of SEQ ID NO: 13 or 14, and the amino acid of SEQ ID NO: 14 A cysteine corresponding to the 219th cysteine in the sequence is also conserved.
- the variant of the human antibody ⁇ -type light chain (# 7) is a variant in which amino acids in regions other than CDR1, CDR2, and CDR3 are mutated and amino acids in other regions of the variable region are mutated. It is preferable that
- the variable region is composed of a polypeptide represented by the amino acid sequence of SEQ ID NO: 19, and the monomeric human antibody ⁇ -type light chain is sometimes referred to as a human antibody ⁇ -type light chain (22F6_monomer).
- the human antibody ⁇ -type light chain (22F6_monomer) may be obtained by adding a known human antibody constant region to the variable region described above.
- the full-length amino acid sequence is represented by SEQ ID NO: 20. This is shown in the amino acid sequence in which the 219th cysteine in the amino acid sequence is deleted or replaced with another amino acid (eg, alanine).
- CDR1 in the human antibody kappa light chain is positions 24 to 39 in the amino acid sequences of SEQ ID NOs: 19 and 20, and CDR2 is positions 55 to 61 in the amino acid sequences of SEQ ID NOs: 19 and 20.
- CDR3 is positions 94 to 101 in the amino acid sequences of SEQ ID NOs: 19 and 20.
- the human antibody ⁇ -type light chain (22F6_monomer) has cytotoxicity against cancer cells, particularly lung cancer cells, as shown in Examples below. For this reason, it is suitable as an active ingredient of an anticancer agent.
- the human antibody ⁇ -type light chain according to the present invention is a monomer, and the variable region is an amino acid sequence in which one or several amino acids are substituted, added, or deleted in the amino acid sequence of SEQ ID NO: 20, or the amino acid sequence And a polypeptide represented by an amino acid sequence having a homology of 95% or more.
- the polypeptide may be referred to as a variant of a human antibody kappa light chain (22F6_monomer).
- a variant of the human antibody ⁇ -type light chain (22F6_monomer) is the amino acid sequence of SEQ ID NO: 20, wherein the 219th cysteine is deleted or substituted with another amino acid, and 1 or other than the 219th amino acid It may consist of an amino acid sequence in which several amino acids are substituted, added or deleted, or a polypeptide represented by an amino acid sequence having 95% or more homology with the amino acid sequence.
- the variant of the human antibody ⁇ type light chain (22F6_monomer) used as the human antibody ⁇ type light chain according to the present invention has an anticancer activity in the same manner as the human antibody ⁇ type light chain (22F6_monomer). It has a monomer. For this reason, in the variant of human antibody kappa light chain (22F6_monomer), CDR1, CDR2, and CDR3 are identical (conserved) to the amino acid sequence of SEQ ID NO: 19 or 20, and SEQ ID NO: 20 The cysteine corresponding to the 219th cysteine in the amino acid sequence is deleted or substituted with another amino acid.
- amino acids in regions other than CDR1, CDR2, and CDR3 are mutated, and amino acids in other regions of the variable region are mutated. It is preferable that
- human antibody kappa light chain according to the present invention may contain an additional polypeptide.
- additional polypeptide include epitope-tagged polypeptides such as His tag, Myc, and Flag.
- Those skilled in the art can easily mutate one or several amino acids out of amino acid residues constituting a polypeptide or add an epitope-labeled polypeptide or the like using well-known techniques. For example, according to a known point mutation introduction method, an arbitrary base of a polynucleotide encoding a polypeptide can be mutated. In addition, a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of a polynucleotide encoding a polypeptide.
- Human antibody ⁇ -type light chains according to the present invention are naturally purified products, products of chemical synthesis procedures, and prokaryotic or eukaryotic hosts (eg, bacterial cells, yeast cells, higher plant cells, insect cells, and mammals). Products produced by recombinant technology from animal cells). Depending on the host used in the recombinant production procedure, the human antibody kappa light chain according to the present invention may be glycosylated or non-glycosylated. Furthermore, the human antibody kappa light chain according to the present invention may also contain an initiating modified methionine residue in some cases as a result of a host-mediated process.
- prokaryotic or eukaryotic hosts eg, bacterial cells, yeast cells, higher plant cells, insect cells, and mammals. Products produced by recombinant technology from animal cells).
- the human antibody kappa light chain according to the present invention may be glycosylated or non-glycosylated.
- the human antibody ⁇ -type light chain according to the present invention may be any polypeptide in which amino acids are peptide-bonded, but is not limited thereto, and may be a complex polypeptide containing a structure other than a polypeptide. Good.
- examples of the “structure other than the polypeptide” include sugar chains and isoprenoid groups, but are not particularly limited.
- the human antibody ⁇ -type light chain according to the present invention is used in the technical field such as a recombinant expression system and a cell-free expression system using a vector containing a polynucleotide encoding the human antibody ⁇ -type light chain (polypeptide). It can be produced using a known expression system.
- a polynucleotide encoding the human antibody ⁇ -type light chain according to the present invention is incorporated into a recombinant expression vector, and then introduced into a host capable of expression by a known method.
- a method of purifying a polypeptide obtained by being translated in () can be employed.
- the recombinant expression vector may or may not be a plasmid, as long as the target polynucleotide can be introduced into the host.
- the expression vector when the foreign polynucleotide is introduced into the host, the expression vector preferably incorporates a promoter that functions in the host so as to express the foreign polynucleotide.
- the method for purifying a recombinantly produced polypeptide differs depending on the host used and the nature of the polypeptide, but the target polypeptide can be purified relatively easily by using a tag or the like.
- the polynucleotide encoding the human antibody ⁇ -type light chain according to the present invention contains components necessary for the translation and synthesis of proteins such as ribosome and t-RNA. It is preferred to purify the synthesized polypeptide by adding it to the solution and incubating at an appropriate temperature.
- Cell-free protein synthesis systems include systems that use wheat germ extract, systems that use rabbit reticulocyte extract, systems that use E. coli S30 extract, and systems that use cell component extracts obtained from plant devolatilized protoplasts Is mentioned.
- eukaryotic genes are selected for translation of eukaryotic genes, ie, systems using wheat germ extract or systems using rabbit reticulocyte extract. In view of the origin of the gene (prokaryotes / eukaryotes) and the intended use of the protein after synthesis, it may be selected from the above synthesis system. As these synthesis systems, various commercially available kits can be used.
- a cell component extract obtained from plant evacuated protoplasts is preferred because it can be used as a cell-free protein synthesis solution retaining an intracellular membrane component, and therefore does not require the addition of a microsomal membrane.
- intracellular membrane component refers to an organelle composed of a lipid membrane present in the cytoplasm (ie, cells such as the endoplasmic reticulum, Golgi apparatus, mitochondria, chloroplast, vacuole and the like). Inner granules in general) are intended.
- the endoplasmic reticulum and the Golgi apparatus play an important role in post-translational modification of proteins, and are essential cellular components for the maturation of membrane proteins and secreted proteins.
- the human antibody ⁇ -type light chain synthesized by the host expression system or the cell-free protein synthesis system is purified.
- the step of purifying the human antibody kappa type light chain is prepared by preparing a cell extract from cells or tissues by a well-known method (for example, a method of centrifuging cells and tissues and then collecting a soluble fraction by centrifugation).
- Well-known methods for example, ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, And the step of purification by lectin chromatography
- HPLC high performance liquid chromatography
- the human antibody kappa light chain according to the present invention can also be purified from cells or tissues that naturally express the human antibody kappa light chain.
- antibodies or oligonucleotides can be used to identify cells or tissues that naturally express the human antibody kappa light chain according to the present invention.
- Purification of the human antibody kappa light chain from cells or tissues can be performed in the same manner as in the case of purifying the human antibody kappa light chain synthesized using a host expression system or the like.
- human antibody ⁇ -type light chain according to the present invention can also be chemically synthesized.
- the method of chemical synthesis is not particularly limited, and any method used for chemically synthesizing polypeptides may be used.
- the anticancer agent according to the present invention contains the human antibody ⁇ type light chain according to the present invention as an active ingredient.
- the human antibody ⁇ -type light chain according to the present invention exhibits cytotoxicity against cancer cells.
- the human antibody ⁇ -type light chain according to the present invention is not used for cancer cells. Specific molecules or structures on the surface are specifically recognized and bound, and at the same time, some of the components of cancer cells are decomposed by their own enzyme activity, resulting in impaired function of cancer cells and inhibition of growth. It is speculated that cell death may be induced.
- the anticancer agent according to the present invention can be administered by direct injection for use with humans or animals.
- the anticancer agent according to the present invention may also be formulated for parenteral administration, mucosal administration, intramuscular administration, intravenous administration, subcutaneous administration, intraocular administration or transdermal administration.
- the protein contained in the composition comprises a dose of 0.01-30 mg / kg body weight, preferably 0.1-10 mg / kg body weight, more preferably 0.1-1 mg / kg body weight. It can be administered in doses.
- the anticancer agent according to the present invention may contain a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof) in addition to the human antibody ⁇ -type light chain according to the present invention.
- Pharmaceutically acceptable carriers or excipients for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Ltd. (A. R. Gennaro, 1985).
- the choice of pharmaceutically usable carrier, excipient or diluent can be readily selected by one skilled in the art according to the intended route of administration and standard pharmaceutical practice.
- the anticancer agent according to the present invention may further contain any appropriate binder, lubricant, suspending agent, coating agent or solubilizer.
- composition / formulation requirements may vary.
- an anticancer agent according to the present invention may be formulated for delivery via a minipump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation, or parenterally (wherein The anticancer agents according to the present invention are formulated as injectable forms for delivery, for example, by intravenous, intramuscular or subcutaneous routes).
- the formulation can be designed to be delivered by both routes.
- the anticancer agent according to the present invention has particularly high cytotoxicity against lung cancer cells. Therefore, the anticancer agent according to the present invention is also preferably in a form that can be efficiently delivered from the nose, bronchi, etc. to lung cells, such as nasal spray or aerosol for inhalation.
- the anticancer agent according to the present invention when used for administration in vivo, various anti-cancer agents for improving the in vivo stability (blood half-life) of the human antibody ⁇ type light chain which is an active ingredient are used.
- Techniques can be used. For example, it is known that when a neonal Fc receptor (FcRn) binds to Fc, the blood half-life of an antibody such as IgG is prolonged (for example, Loopenian, DC et al., Nat Rev Immunol vol. 7 715-725 (2007)), the C-terminus of the human antibody ⁇ -type light chain according to the present invention can be modified so as to have a binding activity to FcRn.
- the human antibody kappa light chain according to the present invention can be dimerized and PEG (polyethylene glycol) can be added.
- the anticancer agent according to the present invention can be made into a kit together with, for example, instructions on the mode of administration.
- the kit can also contain various drugs that can be used in combination with the anticancer agent according to the present invention.
- the anticancer agent according to the present invention uses an antibody ⁇ -type light chain with high target molecule recognition ability as an active ingredient, cancer cells in which the target molecule of the antibody ⁇ -type light chain does not exist on the cell surface Does not exert cytotoxicity. For this reason, the anticancer agent of the present invention is expected to be useful for identifying the type of cancer.
- Example 1 (1. Preparation of human peripheral blood cDNA) Lymphocytes were isolated using Ficoll-paque from peripheral blood obtained from multiple hyperimmunized volunteers using a rabies virus vaccine. Using RNA extraction kit (Stratagene), total RNA was obtained from about 3.0 ⁇ 10 7 separated lymphocytes. The target cDNA (cDNA library) was prepared by reverse transcription of total RNA using ThermoScript RT-PCR System (Invitrogen) and oligo (dT) as a primer.
- ThermoScript RT-PCR System Invitrogen
- oligo oligo
- step 2 (2. Acquisition of human antibody kappa light chain gene) 1 above.
- a two-step PCR reaction is performed using a primer for amplifying an antibody light chain gene having a V ⁇ gene belonging to subgroup II, and a PCR product of about 750 bp ( ⁇ belonging to subgroup II) Type light chain gene).
- PCR products were cloned, sequence analysis was performed, and V ⁇ genes in each germline gene were estimated by homology search. As a result, all 18 clones obtained belonged to subgroup II.
- clone # 1 (germline genotype: A18b), clone # 2 (germline genotype: A3 / A19), clone # 4 (germline genotype: O11 / o1), clone # 7 ( Germline genotype: A3 / A19), clone # 8 (germline genotype: A18b), clone # 9 (germline genotype: A18b), clone # 11 (germline genotype: A18b), Nine clones of clone # 13 (germline genotype: A3 / A19) and clone # 14 (germline genotype: A3 / A19) were used in the subsequent experiments.
- RNA extraction kit (Stratagene)
- total RNA was obtained from about 3.0 ⁇ 10 7 separated lymphocytes.
- ThermoScript RT-PCR System (Invitrogen)
- the total RNA was reverse transcribed using oligo (dT) as a primer, thereby preparing cDNA as a template in the PCR reaction described below.
- PCR reaction was performed using a primer set for comprehensively amplifying the human antibody light chain gene to obtain a PCR product of about 660 bp.
- This PCR product was purified and inserted into an E. coli expression vector pET101 / D-TOPO vector (registered trademark, Invitrogen) to construct an LCA library. From an expression vector in which a PCR product is inserted into a pET101 / D-TOPO vector, a protein in which a His tag is added to the C terminus of the protein encoded by the PCR product is expressed.
- a PCR reaction was performed using primers for amplifying a human antibody light chain gene having a V ⁇ gene belonging to subgroup II to obtain a PCR product of about 660 bp.
- These PCR products are cloned, sequence analysis is performed, and amino acid sequences and light chain variable and constant regions are estimated using analysis software (GENETIX (registered trademark) Ver. 8).
- the V ⁇ gene in (germine line) was estimated.
- two clones, clone 22F6 (germline genotype: A3 / A19) and clone 23D4 (germline genotype: A3 / A19) were used in subsequent experiments.
- the obtained human antibody light chain had M (starting methionine) at the N-terminus and LEHHHHHH (SEQ ID NO: 23) derived from a plasmid vector at the C-terminus.
- the full length of the human antibody light chain (human antibody light chain (# 1_WT)) related to clone # 1 is the base sequence shown in SEQ ID NO: 27, and the human type related to clone # 8
- the total length of the antibody light chain (human antibody light chain (# 8_WT)) is the base sequence shown in SEQ ID NO: 28, and the human antibody light chain (human antibody light chain (# 9_WT)) according to clone # 9 Is the base sequence shown in SEQ ID NO: 29, and the full length of the human antibody light chain (human antibody light chain (# 11_WT)) according to clone # 11 is the base sequence shown in SEQ ID NO: 30
- the full length of the human antibody light chain (human antibody light chain (# 4_WT)) according to clone # 4 is the base sequence shown in SEQ ID NO: 31, and the human antibody light chain (human type according to clone # 2) Total length of antibody light chain (# 2_WT
- the full length of the human antibody light chain according to 13 is the base sequence represented by SEQ ID NO: 34
- the human antibody light chain according to clone # 14 (human antibody light chain)
- the total length of (# 14_WT)) is the base sequence shown in SEQ ID NO: 35
- the full length of the human antibody light chain (human antibody light chain (22F6_WT)) related to clone 22F6 is the base sequence shown in SEQ ID NO: 36
- the full length of the human antibody light chain (human antibody light chain (23D4_WT)) related to clone 23D4 was the base sequence shown in SEQ ID NO: 37.
- the amino acid sequence deduced from each base sequence is shown in FIG. FIG. 1 also shows the positions of the variable region, constant region, and CDR1-3.
- the human antibody light chain (human antibody light chain (# 1_WT)) according to clone # 1 has the amino acid sequence shown in SEQ ID NO: 2, and the human antibody light chain (human antibody light chain (human antibody light chain ( # 8_WT)) is the amino acid sequence shown in SEQ ID NO: 4, and the human antibody light chain (human antibody light chain (# 9_WT)) according to clone # 9 is the amino acid sequence shown in SEQ ID NO: 6,
- the human antibody light chain according to # 11 (human antibody light chain (# 11_WT)) has the amino acid sequence shown in SEQ ID NO: 8
- the human antibody light chain according to clone # 4 human antibody light chain (# 4_WT) is the amino acid sequence shown in SEQ ID NO: 10
- the chain (human antibody light chain (22F6_WT)) is the amino acid sequence shown in SEQ ID NO: 20, and the human antibody light chain related to clone 23D4 (human antibody light chain (23D4_WT)) is the amino acid shown in SEQ ID NO: 22. It was an array.
- the wild-type human antibody light chain used in this example is a polymorphism in which methionine is added to the N-terminus of each amino acid sequence shown in FIG. 1 and LEHHHHHH (SEQ ID NO: 23) derived from a plasmid vector is added to the C-terminus. It was a peptide.
- the human antibody kappa light chain of the clone obtained in (1) forms a dimer because a cysteine at the C-terminus forms a disulfide (SS) bond. Therefore, by introducing a mutation that replaces the cysteine involved in the SS bond (C-terminal cysteine in the amino acid sequence of FIG. 1) with alanine, so that only the monomeric human antibody enzyme is formed. Designed. Details of this design for the human antibody light chain (# 1_WT) with the plasmid vector-derived LEHHHHHH added to the C-terminus are shown in FIG. As shown in FIG.
- the TGT encoding the 220th cysteine in the full-length human antibody enzyme gene was replaced with GCT.
- a dimer is formed because the 220th amino acid is cysteine in the original amino acid sequence, but the 220th amino acid is alanine in the amino acid after substitution.
- the SS bond is not formed and becomes a monomer.
- TGT encoding the cysteine in the gene of the wild-type full-length human antibody enzyme was replaced with GCTCTCGAGCACCACCACCACCACCACCACTACTGA (SEQ ID NO: 26) encoding ALEHHHHHH (SEQ ID NO: 25) (+ stop codon). That is, the monomeric human antibody light chain used in this example has methionine added to the N-terminus of each amino acid sequence shown in FIG. 1, and ALEHHHHHH added in place of the C-terminal cysteine. It was a polypeptide.
- the human antibody light chain (# 1_WT) mutant is human antibody light chain (# 1_C220A), human antibody.
- a variant of the light chain (# 8_WT) is a human antibody light chain (# 8_C220A)
- a variant of the human antibody light chain (# 9_WT) is a human antibody light chain (# 9_C220A)
- a human antibody light chain (# 11_WT) is a human antibody light chain (# 11_C220A)
- a human antibody light chain (# 4_WT) is a human antibody light chain (# 4_C220A)
- a human antibody light chain (# 2_WT) is a mutation.
- Human antibody light chain (# 2_C220A), human antibody light chain (# 7_WT) variant is human antibody light chain (# 7_C220A), and human antibody light chain (# 13_WT) variant is human.
- Antibody light chain (# 3_C220A), human antibody light chain (# 14_WT) variant is human antibody light chain (# 14_C220A), human antibody light chain (22F6_WT) variant is human antibody light chain (22F6_C220A), human antibody The variant of the light chain (23D4_WT) is referred to as the human antibody light chain (23D4_C220A), respectively.
- FIG. 3 (a) shows the results of Ni-NTA column chromatogram and SDS-PAGE analysis in the primary purification of human antibody light chain (# 1_WT) and human antibody light chain (# 1_C220A).
- FIG. 3 (b) is a diagram showing the results of cation exchange chromatogram and SDS-PAGE analysis in secondary purification. As shown on the left side of FIG. 3 (a), buffer A (25 mM Tris-HCl (pH 8.0), 0.25 M NaCl, 40 mM imidazole, 0.005% Tween 20) was run.
- buffer A 25 mM Tris-HCl (pH 8.0), 0.25 M NaCl, 40 mM imidazole, 0.005% Tween 20
- the concentration of imidazole was gradually increased from 40 mM to 300 mM to elute the components bound to the gel.
- a Ni-NTA agarose column (diameter 1 cm, 2 ml) was used as the column, and the flow rate was maintained at 0.1 mL / min throughout the purification.
- the target band of about 31 kDa was detected in fraction numbers 30 to 37. The samples were combined into one and subjected to the following secondary purification.
- buffer A 50 mM sodium acetate (pH 5.4), 0.2 M NaCl, 0.005% until the flow-through fraction runs out after sample application. Of Tween 20). Then, as indicated by the broken line in the left graph, the NaCl concentration was gradually increased from 0.2M to 0.4M to elute the components bound to the gel. SP5PW (TOSHO) was used as the column and the flow rate was maintained at 0.1 ml / min throughout the purification. Included in sample before purification, region “a” (fraction numbers 10 to 15) surrounded by broken line in graph, and region “c” (fraction numbers 25 to 30) surrounded by broken line in graph Components were analyzed by SDS-PAGE.
- a target band of about 31 kDa was detected in a and c in the reduced sample.
- a band of about 31 kDa was detected only in a, and a band of about 51 kDa was detected only in c.
- the antibody light chain monomer is about 31 kDa and the dimer is about 51 kDa.
- sample a is the monomeric fraction of the antibody light chain and sample c is the dimer fraction of the antibody light chain.
- the expression product of the wild-type human antibody light chain contains a dimer and a monomer.
- the dimer was purified by two-step purification by ion exchange chromatogram, and a monomer was contained in the expression product of the mutant in which cysteine contributing to SS bond was mutated to alanine.
- the monomer was purified by step purification.
- frozen A549 cells were thawed and recovered, and then seeded at 100 ⁇ L each in a 96-well plate at 5 ⁇ 10 3 cells / well. After culturing at 37 ° C. for 24 hours, the medium added to the 96-well plate was removed by decantation, and then 100 ⁇ L of each human antibody ⁇ type light chain prepared to about 1 mg / mL was added to each well. . 10 ⁇ L of WST-1 reagent (Roche) was added to each well 24 hours and 48 hours after addition of human antibody ⁇ type light chain (48 hours and 72 hours after seeding the cells). After each addition, the absorbance (Abs 450 nm) of the produced formazan dye was measured after 1, 1.5, and 2 hours.
- the cell viability of each well (NC) to which no human antibody kappa light chain was added was defined as 100%, the cell viability of each well was determined, and the added human antibody kappa Cytotoxicity of type light chain was evaluated.
- FIG. 4 and Table 1 show the results of the clones having the germline genotype A18b or O11 / o1
- FIG. 5 and Table 2 show the results of the clones having the germline genotype A3 / A19.
- Tables 1 and 2 also show the concentration of the human antibody kappa light chain in the well.
- clone (# 1_WT), clone (# 4_WT), clone (# 7_WT), and clone (22F6_C220A) showed cytotoxicity of about 40 to 50% on A549 cells. For other clones, almost no cytotoxicity against A549 cells was observed.
- the clone (# 4_WT) and the clone (# 7_WT) showed particularly strong cytotoxicity.
- clone (# 7_WT) showed that the number of cells in the well hardly changed before the addition of human antibody kappa light chain (0 hour) and after the addition of human antibody kappa light chain (48 hours).
- the clone (# 1_H31Y C220A) showed high cytotoxicity against A549 cells, MOLT-4 cells, and ES-2 cells.
- clone (# 7 RLI) and clone (C51) showed high cytotoxicity against MOLT-4 cells.
- clone (# 4) showed high cytotoxicity against ES-2 cells.
- the amino acid sequence of the clone (# 1_H31Y C220A) is SEQ ID NO: 38
- the amino acid sequence of the clone (# 7 VL (I)) is SEQ ID NO: 39
- the amino acid sequence of the clone (# 7 RLI) is SEQ ID NO: 40
- the amino acid sequence of clone (C51) is SEQ ID NO: 41
- the amino acid sequence of clone (C87) is SEQ ID NO: 42
- the amino acid sequence of clone (# 7 EI) is SEQ ID NO: 43
- the present invention can be used in the fields of development of new anticancer agents and cancer treatment.
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Abstract
Description
本願は、2012年3月8日に、日本に出願された特願2012-52334号に基づき優先権を主張し、その内容をここに援用する。
(1)可変領域が配列番号1のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖、
(2)可変領域が配列番号7のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖、
(3)可変領域が配列番号9のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖、
(4)可変領域が配列番号13のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖、
(5)可変領域が配列番号19のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、単量体であるヒト抗体κ型軽鎖、
(6)可変領域が配列番号38の1~113番目のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、単量体であるヒト抗体κ型軽鎖、
(7)可変領域が配列番号40の1~112番目のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖、又は
(8)可変領域が配列番号41の1~107番目のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖
を含有することを特徴とする。
また、本願明細書において、「細胞傷害性」とは、細胞に対して死又は機能障害を与える性質を意味する。
(1)可変領域が配列番号1のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖。
(2)可変領域が配列番号7のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖。
(3)可変領域が配列番号9のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖。
(4)可変領域が配列番号13のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖。
(5)可変領域が配列番号19のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、単量体であるヒト抗体κ型軽鎖。
(6)可変領域が配列番号38の1~113番目のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、単量体であるヒト抗体κ型軽鎖。
(7)可変領域が配列番号40の1~112番目のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖。
(8)可変領域が配列番号41の1~107番目のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖。
(1.ヒト末梢血cDNAの調製)
狂犬病ウイルスのワクチンを用いて、複数回にわたって過剰免疫されたボランティアから得られた末梢血から、Ficoll-paqueを用いてリンパ球を分離した。RNA extraction kit(Stratagene)を用いて、分離した約3.0×107個のリンパ球からトータルRNAを得た。TheromoScript RT-PCR System(Invitrogen)を用い、oligo(dT)をプライマーとして、トータルRNAを逆転写することにより、目的とするcDNA(cDNAライブラリ-)を調製した。
前記1.で取得したcDNAを鋳型として、サブグループIIに属するVκ遺伝子を有する抗体軽鎖遺伝子を増幅するためのプライマーを用いた2段階のPCR反応を行い、約750bpのPCR産物(サブグループIIに属するκ型軽鎖遺伝子)を得た。これらのPCR産物をクローニングし、シーケンス解析を行い、相同性検索により、それぞれの生殖細胞系列遺伝子(germline gene)におけるVκ遺伝子を推定した。この結果、得られた18クローンすべてがサブグループIIに属していた。このうち、クローン#1(生殖細胞系列遺伝子型:A18b)、クローン#2(生殖細胞系列遺伝子型:A3/A19)、クローン#4(生殖細胞系列遺伝子型:O11/o1)、クローン#7(生殖細胞系列遺伝子型:A3/A19)、クローン#8(生殖細胞系列遺伝子型:A18b)、クローン#9(生殖細胞系列遺伝子型:A18b)、クローン#11(生殖細胞系列遺伝子型:A18b)、クローン#13(生殖細胞系列遺伝子型:A3/A19)、及びクローン#14(生殖細胞系列遺伝子型:A3/A19)の9クローンを以降の実験に用いた。
前記2.で取得した各クローンをそれぞれ、Hisタグ配列サイトを有するプラスミドベクターに導入し、当該プラスミドベクターを大腸菌に導入して形質転換体を作製した。各形質転換体を培養し、IPTGによる発現誘導を行ったところ、SDS-PAGE分析及び抗ヒト型(Fab’)2抗体を用いたウエスタンブロッティングにより、大腸菌において発現したタンパク質がヒト型抗体軽鎖であることを同定することができた。得られたヒト型抗体軽鎖は、N末端にM(開始メチオニン)を、C末端にプラスミドベクター由来のLEHHHHHH(配列番号23)を有していた。
狂犬病ウイルスのワクチンを用いて、複数回にわたって被験者を過剰免疫し、血清の中和活性を測定した。血清の中和活性が最も高かった(7.2IU)被験者をドナーとして末梢血を採取し、Ficoll-paqueを用いて当該末梢血からリンパ球を分離した。RNA extraction kit(Stratagene)を用いて、分離した約3.0×107個のリンパ球からトータルRNAを得た。TheromoScript RT-PCR System(Invitrogen)を用い、oligo(dT)をプライマーとして、トータルRNAを逆転写することにより、後述するPCR反応において鋳型とするcDNAを調製した。
前記4.で取得したcDNAを鋳型として、ヒト抗体軽鎖遺伝子を網羅的に増幅するためのプライマーセットを用いてPCR反応を行い、約660bpのPCR産物を得た。このPCR産物を精製し、大腸菌発現ベクターpET101/D-TOPOベクター(登録商標、Invitrogen)に挿入し、LCAライブラリを構築した。なお、pET101/D-TOPOベクターにPCR産物を挿入した発現ベクターからは、当該PCR産物がコードするタンパク質のC末端にHisタグを付加させたタンパク質が発現される。このLCAライブラリのcDNAを鋳型として、サブグループIIに属するVκ遺伝子を有するヒト抗体軽鎖遺伝子を増幅するためのプライマーを用いてPCR反応を行い、約660bpのPCR産物を得た。これらのPCR産物をクローニングし、シーケンス解析を行い、解析ソフトウェア(GENETIX(登録商標) Ver.8)を用いて、アミノ酸配列ならびに軽鎖の可変領域および定常領域を推定し、それぞれの生殖細胞系列遺伝子(germline gene)におけるVκ遺伝子を推定した。これらのクローンの中から、クローン22F6(生殖細胞系列遺伝子型:A3/A19)及びクローン23D4(生殖細胞系列遺伝子型:A3/A19)の2クローンを以降の実験に用いた。得られたヒト型抗体軽鎖は、N末端にM(開始メチオニン)を、C末端にプラスミドベクター由来のLEHHHHHH(配列番号23)を有していた。
前記2.及び5.で取得したクローンのヒト抗体κ型軽鎖は、C末端にあるシステインによりジスルフィド(S-S)結合が形成されるため、二量体を形成している。そこで、S-S結合に関与するシステイン(図1のアミノ酸配列のC末端のシステイン)をアラニンに置換する変異を導入して、単量体のヒト型抗体酵素のみが形成されるように、cDNAを設計した。C末端にプラスミドベクター由来のLEHHHHHHを付加したヒト型抗体軽鎖(#1_WT)に対するこの設計の詳細を図2に示す。図2の(a)に示すように、全長のヒト型抗体酵素の遺伝子における220番目のシステインをコードするTGTを、GCTに置換した。これによって、図2の(b)に示すように、元のアミノ酸配列では220番目がシステインであるために二量体が形成されるが、置換後のアミノ酸では220番目がアラニンであるために、S-S結合が形成されず、単量体となる。
各ヒト型抗体軽鎖を以下の様に一次精製および二次精製した。図3の(a)は、ヒト型抗体軽鎖(#1_WT)及びヒト型抗体軽鎖(#1_C220A)の一次精製におけるNi-NTAカラムクロマトグラムおよびSDS-PAGE分析の結果を示す図である。図3の(b)は、二次精製における陽イオン交換クロマトグラムおよびSDS-PAGE分析の結果を示す図である。
図3の(a)左段に示すように、サンプルのアプライ後に素通り画分が流れ切るまで、緩衝液A(25mMのTris-HCl(pH8.0)、0.25MのNaCl、40mMのイミダゾール、0.005%のTween20)を流した。そして、左段のグラフにおける破線に示されるように、イミダゾールの濃度を40mMから300mMまで漸次的に上昇させて、ゲルと結合した成分を溶出させた。カラムとしてNi-NTAアガロースカラム(直径1cm、2ml)を用い、精製を通して流速を0.1mL/分に維持した。図3の(a)の右段に示すように、目的とする約31kDaのバンドが、フラクション番号30~37に検出された。このサンプルを1つにまとめて、次の二次精製を行った。
各種ヒト抗体κ型軽鎖のがん細胞に対する細胞障害性について試験した。がん細胞は、ATCCより購入したA549(ヒト肺胞上皮がん細胞株)を用い、10%FCS(ウシ胎児血清)含有F-12K培地を用いる通常の方法で培養した。
当該4クローンのうち、クローン(#4_WT)及びクローン(#7_WT)は特に強い細胞傷害性を示した。中でもクローン(#7_WT)は、ヒト抗体κ型軽鎖添加前(0時間)とヒト抗体κ型軽鎖添加後(48時間)において、ウェル内の細胞数がほとんど変化しなかったことから、クローン(#7_WT)、すなわちヒト抗体κ型軽鎖(#7)は、A549細胞の増殖を抑制する効果があることが示唆された。
また、今回用いたクローンの結果から、二量体(WT)のほうが単量体よりも細胞傷害性が強い傾向が観察されたことから、二量体に強い細胞傷害性が存在することも示唆された。
また、他のクローンを含めたものについても、各種細胞株に対し、上記と同様にヒト抗体κ型軽鎖の細胞障害性を評価した。その結果を表3に示す。
なお、クローン(#1_H31Y C220A)のアミノ酸配列は配列番号38に、クローン(#7 VL(I))のアミノ酸配列は配列番号39に、クローン(#7 RLI)のアミノ酸配列は配列番号40に、クローン(C51)のアミノ酸配列は配列番号41に、クローン(C87)のアミノ酸配列は配列番号42に、クローン(#7 EI)のアミノ酸配列は配列番号43に、クローン(#7 TR)のアミノ酸配列は配列番号44に、クローン(#7 VL)のアミノ酸配列は配列番号45に、クローン(S13)のアミノ酸配列は配列番号46に、クローン(S21)のアミノ酸配列は配列番号47に、クローン(S38)のアミノ酸配列は配列番号48に、クローン(#10)のアミノ酸配列は配列番号49に、クローン(C67)のアミノ酸配列は配列番号50に、クローン(C82)のアミノ酸配列は配列番号51に、クローン(C88)のアミノ酸配列は配列番号52に、クローン(#7 G)のアミノ酸配列は配列番号53に、それぞれ記載されている。
また、図7、図8に示す通り、本願のヒト抗体κ型軽鎖を含有する抗がん剤は、動物実験において毒性を全く示さなかった。
Claims (2)
- (1)可変領域が配列番号1のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖、
(2)可変領域が配列番号7のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖、
(3)可変領域が配列番号9のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖、
(4)可変領域が配列番号13のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖、
(5)可変領域が配列番号19のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、単量体であるヒト抗体κ型軽鎖、
(6)可変領域が配列番号38の1~113番目のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、単量体であるヒト抗体κ型軽鎖、
(7)可変領域が配列番号40の1~112番目のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖、又は
(8)可変領域が配列番号41の1~107番目のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖
を含有することを特徴とする抗がん剤。 - 前記(1)のヒト抗体κ型軽鎖が、配列番号2のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖であり、
前記(2)のヒト抗体κ型軽鎖が、配列番号8のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖であり、
前記(3)のヒト抗体κ型軽鎖が、配列番号10のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖であり、
前記(4)のヒト抗体κ型軽鎖が、配列番号14のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖であり、
前記(5)のヒト抗体κ型軽鎖が、配列番号20のアミノ酸配列中の219番目のシステインが削除又はシステイン以外のアミノ酸に置換されているアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、単量体であるヒト抗体κ型軽鎖であり、
前記(6)のヒト抗体κ型軽鎖が、配列番号38のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、単量体であるヒト抗体κ型軽鎖であり、
前記(7)のヒト抗体κ型軽鎖が、配列番号40のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖であり、又は
前記(8)のヒト抗体κ型軽鎖が、配列番号41のアミノ酸配列、又は当該アミノ酸配列において1若しくは数個のアミノ酸が置換、付加、若しくは欠失したアミノ酸配列、又は当該アミノ酸配列と95%以上の相同性を有するアミノ酸配列によって示されるポリペプチドからなり、二量体であるヒト抗体κ型軽鎖である、
請求項1に記載の抗がん剤。
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