(19)
(11)EP 2 837 387 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
29.07.2020 Bulletin 2020/31

(21)Application number: 13758278.9

(22)Date of filing:  05.03.2013
(51)International Patent Classification (IPC): 
A61K 39/395(2006.01)
A61P 35/00(2006.01)
C12N 15/09(2006.01)
C07K 16/30(2006.01)
A61P 11/00(2006.01)
C07K 16/18(2006.01)
C07K 16/10(2006.01)
A61K 39/00(2006.01)
(86)International application number:
PCT/JP2013/055927
(87)International publication number:
WO 2013/133253 (12.09.2013 Gazette  2013/37)

(54)

ANTICANCER AGENT

KREBSMITTEL

AGENT ANTICANCÉREUX


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 08.03.2012 JP 2012052334

(43)Date of publication of application:
18.02.2015 Bulletin 2015/08

(73)Proprietor: Japan Science and Technology Agency
Kawaguchi-shi, Saitama 332-0012 (JP)

(72)Inventors:
  • UDA, Taizo
    Oita-shi Oita 870-0045 (JP)
  • HIFUMI, Emi
    Oita-shi Oita 870-1103 (JP)

(74)Representative: Plasseraud IP 
66, rue de la Chaussée d'Antin
75440 Paris Cedex 09
75440 Paris Cedex 09 (FR)


(56)References cited: : 
WO-A1-2008/029807
WO-A1-2011/102517
JP-A- 2006 197 930
JP-A- 2011 526 480
WO-A1-2011/102517
JP-A- 2005 508 176
JP-A- 2006 501 856
  
      
    Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


    Description

    TECHNICAL FIELD



    [0001] The present invention relates to an anticancer agent containing a human antibody κ-type light chain that demonstrates cytotoxicity against cancer cells and particularly lung cancer cells.

    [0002] The present application claims priority on the basis of Japanese Patent Application No. 2012-52334, filed in Japan on March 8, 2012.

    BACKGROUND ART



    [0003] Antibodies are composed of heavy chains (H chains) and light chains (L chains). The heavy chains and light chains are composed of a variable region (VR) and a constant region (CR), and the variable region has a complementarity determining region (CDR). Moreover, antibody light chains are classified into κ chains and λ chains.

    [0004] In recent years, attention has been focused on antibodies having an enzyme-like activity, namely, antibody enzymes. Since antibody enzymes have both the ability of antibodies to accurately recognize molecules and the activity of enzymes, they are expected to be applied in numerous fields, including medicine, the chemical industry and the food industry. In particular, since antibody enzymes exhibit high specificity for a target molecule and are able to impair target molecules due to their enzyme activity, they are expected to serve as superior anticancer agents that demonstrate few adverse side effects.

    [0005] The inventors of the present invention have heretofore conducted various innovative researches on antibody enzymes (see, for example, Patent Document 1). Antibody enzymes having complete human sequences have conventionally been unable to be obtained with the exception of the Bence-Jones Protein (BJP) obtained from multiple myeloma patients. Since there are few multiple myeloma patients and only a small amount of BJP that has enzyme activity, it was difficult to acquire a human antibody enzyme. However, since human antibody enzymes are predicted to demonstrate few adverse side effects when administered to a human body, pharmaceutical companies both at home and overseas are awaiting the development of a useful human antibody enzyme.

    Prior Art Documents


    Patent Documents



    [0006] Patent Document 1: Japanese Unexamined Patent Application, First Publication No. 2006-197930
    The international patent application published under reference WO2011/102517 discloses antiviral agents comprising an antibody κ-type light chain against rabies virus and which have particular enzymatic activities.

    DISCLOSURE OF THE INVENTION


    Problems to be Solved by the Invention



    [0007] An object of the present invention is to provide an anticancer agent that has for an active ingredient thereof a human antibody light chain that demonstrates cytotoxicity against cancer cells and particularly against lung cancer cells.

    Means for Solving the Problems



    [0008] The inventors of the present invention acquired a novel human antibody light chain from peripheral blood obtained from volunteers hyperimmunized over a plurality of times using a rabies vaccine virus, and as a result of studying those volunteers, surprisingly found that several of the resulting human antibody κ-type light chains demonstrated a high degree of cytotoxicity against cancer cells and particularly lung cancer cells, thereby leading to completion of the present invention.

    [0009] Namely, the anticancer agent according to the present invention is characterized in that it contains:
    1. (1) a human antibody κ-type light chain in the form of a dimer in which the variable region is composed of a polypeptide represented by the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having homology of 95% or more with the amino acid sequence of SEQ ID NO: 9 and having the same CDRs as the amino acid sequence of SEQ ID NO: 9;
    2. (2) a human antibody κ-type light chain in the form of a monomer in which the variable region is composed of a polypeptide represented by the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having homology of 95% or more with the amino acid sequence of SEQ ID NO: 19 and having the same CDRs as the amino acid sequence of SEQ ID NO: 19;

    Effects of the Invention



    [0010] According to the present invention, an anticancer agent can be provided that is highly cytotoxic against cancer cells and particularly lung cancer cells. Since the anticancer agent of the present invention has an antibody enzyme for the active ingredient thereof, it is highly specific for cancer cells. Moreover, since the amino acid sequence of the antibody enzyme is completely human, it is free of problems such as allergies with respect to humans. Consequently, the anticancer agent of the present invention is extremely useful as a highly active, innovative and novel pharmaceutical and as a test piece for the development thereof.

    BRIEF DESCRIPTION OF THE DRAWINGS



    [0011] 

    FIG. 1 is a diagram indicating amino acid sequences of wild type human antibody κ-type light chains.

    FIG. 2(a) schematically indicates a cDNA design for obtaining a monomer human antibody light chain, while FIG. 2(b) schematically indicates the compositions of a human antibody light chain prior to introduction of a mutation and a human antibody light chain following introduction of a mutation.

    FIG. 3 is a diagram indicating the results of purifying a polypeptide of a clone #1, with FIG. 3(a) indicating the results of newly carrying out primary purification of the polypeptide of clone #1, and FIG. 3(b) indicating the results of newly carrying out secondary purification of the polypeptide of clone #1.

    FIG. 4 is a graph indicating the results of investigating the cytotoxicity of various clones against cancer cells.

    FIG. 5 is a graph indicating the results of investigating the cytotoxicity of various clones against cancer cells.

    FIG. 6A is a diagram indicating the amino acid sequences of wild type human antibody κ-type light chains.

    FIG. 6B is a diagram indicating the amino acid sequences of wild type human antibody κ-type light chains.

    FIG. 6C is a diagram indicating the amino acid sequences of wild type human antibody κ-type light chains continuing from FIG. 6B.

    FIG. 6D is a diagram indicating the amino acid sequences of wild type human antibody κ-type light chains.

    FIG. 6E is a diagram indicating the amino acid sequences of wild type human antibody κ-type light chains continuing from FIG. 6D.

    FIG. 6F is a diagram indicating the amino acid sequences of wild type human antibody κ-type light chains continuing from FIG. 6E.

    FIG. 6G is a diagram indicating the amino acid sequences of wild type human antibody κ-type light chains.

    FIG. 6H is a diagram indicating the amino acid sequences of wild type human antibody κ-type light chains continuing from FIG. 6G.

    FIG. 6I is a diagram indicating the amino acid sequence of a wild type human antibody κ-type light chain.

    FIG. 7 is a diagram indicating the results of an in vivo assay.

    FIG. 8 is a diagram indicating the results of safety studies (toxicity studies).


    BEST MODE FOR CARRYING OUT THE INVENTION



    [0012] The present invention provides an anticancer agent containing a human antibody κ-type light chain that demonstrates cytotoxicity against cancer cells. In the description of the present application, a "human antibody κ-type light chain" refers to a κ-type light chain of human-derived immunoglobulin.

    [0013] In the description of the present application, an "anticancer agent" refers to a pharmaceutical agent having an activity that eradicates cancer cells or suppresses or inhibits the proliferation thereof.

    [0014] In addition, in the description of the present application, "cytotoxicity" refers to a property that induces cell death or causes functional impairment in cells.

    [0015] More specifically, the active ingredient of the anticancer agent according to the present invention in the form of a human antibody κ-type light chain (to also be referred to as the "human antibody κ-type light chain according to the present invention") is any of those described in (1) or (2) below.
    1. (1) A human antibody κ-type light chain in the form of a dimer in which the variable region is composed of a polypeptide represented by the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having homology of 95% or more with the amino acid sequence of SEQ ID NO: 9 and having the same CDRs as the amino acid sequence of SEQ ID NO: 9.
    2. (2) A human antibody κ-type light chain in the form of a monomer in which the variable region is composed of a polypeptide represented by the amino acid sequence of SEQ ID NO: 19, or an amino acid sequence having homology of 95% or more with that amino acid sequence of SEQ ID NO: 19 and having the same CDRs as the amino acid sequence of SEQ ID NO: 19.


    [0016] The human antibody κ-type light chain in the form of a dimer in which the variable region is composed of a polypeptide represented by the amino acid sequence of SEQ ID NO: 1 may also be referred to as human antibody κ-type light chain (#1). The human antibody κ-type light chain (#1) can have a known human antibody constant region added to the aforementioned variable region, and in one aspect, the entire length of the amino acid sequence is as shown in SEQ ID NO: 2. CDR1 in the human antibody κ-type light chain (#1) consists of the 24th to 39th amino acids in the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, CDR2 consists of the 55th to 61st amino acids in the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, and CDR3 consists of the 94th to 102nd amino acids in the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2.

    [0017] A cysteine residue for forming a disulfide bond is present in a wild type antibody κ-type light chain that results in the formation of a dimer. The human antibody κ-type light chain (#1) also has a cysteine residue for forming a disulfide bond with another light chain in the same manner as the wild type. For example, in the case the human antibody κ-type light chain (#1) is composed of a polypeptide represented by the amino acid sequence of SEQ ID NO: 2, then the cysteine residue is the cysteine residue at position 220 in the amino acid sequence of SEQ ID NO: 2.

    [0018] As will be subsequently indicated in the examples, the human antibody κ-type light chain (#1) demonstrates cytotoxicity against cancer cells and particularly lung cancer cells. Consequently, it is preferable for use as an active ingredient of an anticancer agent. Since the ability to accurately recognize a target molecule is important for the human antibody κ-type light chain (#1) to demonstrate anticancer activity, the active center of the anticancer activity of the human antibody κ-type light chain (#1) is in the variable region.

    [0019] Being able to easily modify several amino acids among amino acid residues composing a polypeptide without having a significant effect on the structure or function of the polypeptide is commonly known in the art. Moreover, in addition to artificial modification, mutants are also commonly known to exist in naturally-occurring proteins that do not cause a significant change in the structure or function of that protein. Furthermore, in the description of the present application, the substitution, addition or deletion of one or a plurality of amino acids in a specific amino acid sequence X is referred to as mutation.

    [0020] The human antibody κ-type light chain may form a dimer in which the variable region is composed of a polypeptide represented by an amino acid sequence in which one or a plurality of amino acids in the amino acid sequence of SEQ ID NO: 1 have been substituted, added or deleted, or an amino acid sequence having homology of 95% or more with that amino acid sequence. This polypeptide may also be referred to as a mutant of the human antibody κ-type light chain (#1). A mutant of the human antibody κ-type light chain (#1) may also be composed of a polypeptide represented by an amino acid sequence in which one or a plurality of amino acids other than the cysteine at position 220 in the amino acid sequence of SEQ ID NO: 2 have been substituted, added or deleted, or an amino acid sequence having homology of 95% or more with that amino acid sequence.

    [0021] A mutant of the human antibody κ-type light chain (#1) used as the human antibody κ-type light chain according to the present disclosure is a dimer having an anticancer action in the same manner as the human antibody κ-type light chain (#1) . Consequently, CDR1, CDR2 and CDR3 of a mutant of the human antibody κ-type light chain (#1) are identical to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 (are preserved therein), and the cysteine corresponding to cysteine at position 220 in the amino acid sequence of SEQ ID NO: 2 is also preserved. In other words, a mutant of the human antibody κ-type light chain (#1) is preferably such that amino acids in regions other than CDR1, CDR2 and CDR3 are mutated and amino acids in other regions of the variable region are mutated.

    [0022] A human antibody κ-type light chain in the form of a dimer in which the variable region is composed of a polypeptide represented by the amino acid sequence of SEQ ID NO: 9 may also be referred to as human antibody κ-type light chain (#4). The human antibody κ-type light chain (#4) can have a known human antibody constant region added to the aforementioned variable region, and in one embodiment, the entire length of the amino acid sequence is as shown in SEQ ID NO: 10. CDR1 in the human antibody κ-type light chain (#4) consists of the 24th to 40th amino acids in the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10, CDR2 consists of the 56th to 62nd amino acids in the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10, and CDR3 consists of the 95th to 102nd amino acids in the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 10. In addition, a cysteine residue for forming a disulfide bond with another light chain is the cysteine residue at position 220 in the amino acid sequence of SEQ ID NO: 10.

    [0023] As will be subsequently indicated in the examples, the human antibody κ-type light chain (#4) demonstrates cytotoxicity against cancer cells and particularly lung cancer cells. Consequently, it is preferable for use as an active ingredient of an anticancer agent.

    [0024] The human antibody κ-type light chain according to the present invention may form a dimer in which the variable region is composed of a polypeptide represented by an amino acid sequence of SEQ ID NO: 9, or an amino acid sequence having homology of 95% or more with the amino acid sequence of SEQ ID NO: 9. This polypeptide may also be referred to as a mutant of the human antibody κ-type light chain (#4). A mutant of the human antibody κ-type light chain (#4) may also be composed of a polypeptide represented by an amino acid sequence of SEQ ID NO: 10, or an amino acid sequence having homology of 95% or more with the amino acid sequence of SEQ ID NO: 10.

    [0025] A mutant of the human antibody κ-type light chain (#4) used as the human antibody κ-type light chain according to the present invention is a dimer having an anticancer action in the same manner as the human antibody κ-type light chain (#4). Consequently, CDR1, CDR2 and CDR3 of a mutant of the human antibody κ-type light chain (#4) are identical to the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 10 (are preserved therein), and the cysteine corresponding to cysteine at position 220 in the amino acid sequence of SEQ ID NO: 10 is also preserved. In other words, a mutant of the human antibody κ-type light chain (#4) is such that amino acids in regions other than CDR1, CDR2 and CDR3 are mutated and amino acids in other regions of the variable region are mutated.

    [0026] A human antibody κ-type light chain in the form of a dimer in which the variable region is composed of a polypeptide represented by the amino acid sequence of SEQ ID NO: 13 may also be referred to as human antibody κ-type light chain (#7). The human antibody κ-type light chain (#7) can have a known human antibody constant region added to the aforementioned variable region, and in one aspect, the entire length of the amino acid sequence is as shown in SEQ ID NO: 14. CDR1 in the human antibody κ-type light chain (#7) consists of the 24th to 39th amino acids in the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14, CDR2 consists of the 55th to 61st amino acids in the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14, and CDR3 consists of the 94th to 101st amino acids in the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 14. In addition, a cysteine residue for forming a disulfide bond with another light chain is the cysteine residue at position 219 in the amino acid sequence of SEQ ID NO: 14.

    [0027] As will be subsequently indicated in the examples, the human antibody κ-type light chain (#7) demonstrates cytotoxicity against cancer cells and particularly lung cancer cells. Consequently, it is preferable for use as an active ingredient of an anticancer agent.

    [0028] The human antibody κ-type light chain may form a dimer in which the variable region is composed of a polypeptide represented by an amino acid sequence in which one or a plurality of amino acids in the amino acid sequence of SEQ ID NO: 13 have been substituted, added or deleted, or an amino acid sequence having homology of 95% or more with that amino acid sequence. This polypeptide may also be referred to as a mutant of the human antibody κ-type light chain (#7). A mutant of the human antibody κ-type light chain (#7) may also be composed of a polypeptide represented by an amino acid sequence in which one or a plurality of amino acids other than the cysteine at position 219 in the amino acid sequence of SEQ ID NO: 14 have been substituted, added or deleted, or an amino acid sequence having homology of 95% or more with that amino acid sequence.

    [0029] A mutant of the human antibody κ-type light chain (#7) used as the human antibody κ-type light chain according to the present disclosure is a dimer having an anticancer action in the same manner as the human antibody κ-type light chain (#7) . Consequently, CDR1, CDR2 and CDR3 of a mutant of the human antibody κ-type light chain (#7) are identical to the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14 (are preserved therein), and the cysteine corresponding to cysteine at position 219 in the amino acid sequence of SEQ ID NO: 14 is also preserved. In other words, a mutant of the human antibody κ-type light chain (#7) is preferably such that amino acids in regions other than CDR1, CDR2 and CDR3 are mutated and amino acids in other regions of the variable region are mutated.

    [0030] A human antibody κ-type light chain in the form of a monomer in which the variable region is composed of a polypeptide represented by the amino acid sequence of SEQ ID NO: 19 may also be referred to as human antibody κ-type light chain (22F6_monomer). The human antibody κ-type light chain (22F6_monomer) can have a known human antibody constant region added to the aforementioned variable region, and in one embodiment, the entire length of the amino acid sequence is represented by an amino acid sequence in which the 219th cysteine in the amino acid sequence of SEQ ID NO: 20 has been deleted or substituted with another amino acid (such as alanine) . CDR1 in the human antibody κ-type light chain (22F6_monomer) consists of the 24th to 39th amino acids in the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 20, CDR2 consists of the 55th to 61st amino acids in the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 20, and CDR3 consists of the 94th to 101st amino acids in the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO: 20.

    [0031] As will be subsequently indicated in the examples, the human antibody κ-type light chain (22F6_monomer) demonstrates cytotoxicity against cancer cells and particularly lung cancer cells. Consequently, it is preferable for use as an active ingredient of an anticancer agent.

    [0032] The human antibody κ-type light chain according to the present invention may be a monomer in which the variable region is composed of a polypeptide represented by an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having homology of 95% or more with the amino acid sequence of SEQ ID NO: 20. This polypeptide may also be referred to as a mutant of the human antibody κ-type light chain (22F6_monomer). A mutant of the human antibody κ-type light chain (22F6_monomer) may also be composed of a polypeptide represented by an amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having homology of 95% or more with the amino acid sequence of SEQ ID NO: 20.

    [0033] A mutant of the human antibody κ-type light chain (22F6_monomer) used as the human antibody κ-type light chain according to the present invention is a monomer having an anticancer action in the same manner as the human antibody κ-type light chain (22F6_monomer). Consequently, CDR1, CDR2 and CDR3 of a mutant of the human antibody κ-type light chain (22F6_monomer) are identical to the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 20 (are preserved therein), and the cysteine corresponding to cysteine at position 219 in the amino acid sequence of SEQ ID NO: 20 is deleted or substituted with another amino acid. In other words, a mutant of the human antibody κ-type light chain (22F6_monomer) is such that amino acids in regions other than CDR1, CDR2 and CDR3 are mutated and amino acids in other regions of the variable region are mutated.

    [0034] In addition, the human antibody κ-type light chain according to the present invention may also contain an additional polypeptide. Typical examples of additional polypeptides include epitope-tagged polypeptides such as those tagged with His tag, Myc or Flag.

    [0035]  A person with ordinary skill in the art is able to easily mutate one or a plurality of amino acids among amino acid residues that compose a polypeptide or add an epitope-tagged polypeptide using a known technology. For example, an arbitrary base of a polynucleotide that encodes a polypeptide can be mutated in accordance with a known point mutagenesis method. In addition, a primer corresponding to an arbitrary site of a polynucleotide that encodes a polypeptide can be designed to create a deletion mutant or an addition mutant.

    [0036] The human antibody κ-type light chain according to the present invention includes a naturally-occurring purification product, a product obtained by a chemical synthesis procedure, and a product produced by recombination technology from a prokaryotic host or eukaryotic host (including bacterial cells, yeast cells, higher plant cells, insect cells and mammalian cells). The human antibody κ-type light chain may or may not be glycosylated depending on the host used in the recombinant production procedure. Moreover, the human antibody κ-type light chain according to the present invention can contain a modified initiating methionine group in several cases as a result of a host intervention process.

    [0037] Although the human antibody κ-type light chain according to the present invention may be a polypeptide in which amino acids are linked by peptide bonds, it is not limited thereto, and the polypeptide may also be a composite polypeptide containing a structure other than that of a polypeptide. As used in the present description, although examples of a "structure other than that of a polypeptide" include sugar chains and isoprenoid groups, there are no particular limitations thereon.

    [0038] The human antibody κ-type light chain according to the present invention can be produced using an expression system known in the art, such as a recombination expression system or a cell-free expression system, by using a vector containing a polynucleotide encoding the human antibody κ-type light chain (polypeptide).

    [0039] In the case of using a recombination expression system, a method can be employed having the steps of, for example, incorporating a polynucleotide encoding the human antibody κ-type light chain according to the present invention into a recombination expression vector followed by introducing into a host enabling expression thereof according to a known method, translating within the host (transformant) and purifying the resulting polypeptide. The recombination expression vector may or may not be a plasmid, and is only required to enable the target polynucleotide to be introduced into the host.

    [0040] In the case of introducing an exogenous polynucleotide into a host in this manner, a promoter that functions in the host so as to express exogenous polynucleotides is preferably incorporated into the expression vector. Although the method used to purify the recombinantly produced polypeptide varies according to the properties of the host and polypeptide used, a target polypeptide can be purified comparatively easily using a tag and the like.

    [0041] In the case of using a cell-free expression system (cell-free protein synthesis system), a polynucleotide encoding the human antibody κ-type light chain according to the present invention is preferably added to a solution containing components such as ribosomes or t-RNA required for protein translation and synthesis followed by incubating at a suitable temperature and purifying the synthesized polypeptide.

    [0042] Examples of cell-free protein synthesis systems include systems using wheat germ extract, systems using rabbit reticulocyte extract, systems using E. coli S30 extract and systems using cell component extracts obtained from plant devacuolated protoplasts. In general, although eukaryotic cell systems, namely, systems using wheat germ extract or systems using rabbit reticulocyte extract, are selected for translation of eukaryotic genes, the aforementioned synthesis system is selected in consideration of such factors as the origin of the gene to be translated (prokaryotic or eukaryotic) or the purpose for which the protein is to be used following synthesis. Various commercially available kits can be used for these synthesis systems.

    [0043] Furthermore, since various viral gene products frequently express activity by going through a complex biochemical reaction involving the cytomembrane, such as the endoplasmic reticulum or Golgi bodies, following translation, it is necessary to add cytomembrane components (such as microsomal membrane) in order to reproduce the various biochemical reactions in vitro. Cell component extracts obtained from plant devacuolated protoplasts are preferable since they can be used as a cell-free protein synthesis liquid that retains cytomembrane components, thereby eliminating the need to add microsomal membrane.

    [0044] As used in the present description, "cytomembrane components" are intended to refer to cell organelles composed of lipid membrane present in the cytomembrane (namely, all types of intracellular granules such as endoplasmic reticulum, Golgi bodies, mitochondria, chloroplast and vacuoles). In particular, since endoplasmic reticulum and Golgi bodies fulfill an important role in post-translation modification of proteins, they are essential cell components for maturation of membrane proteins and secretory proteins.

    [0045] Human antibody κ-type light chain synthesized with a host expression system or a cell-free protein synthesis system is preferably purified. Although a step for purifying human antibody κ-type light chain is preferably a step in which a cell extract is prepared from cells or tissue using a known method (such as a method in which the cells or tissue is homogenized, followed by centrifuging and recovering the soluble fraction), followed by purifying the human antibody κ-type light chain from this cell extract using a known method (such as ammonium sulfate precipitation or ethanol precipitation, acid extraction, anionic or cationic chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography or lectin chromatography), it is not limited thereto. High-performance liquid chromatography (HPLC) is most preferably used for purification.

    [0046] In addition, the human antibody κ-type light chain according to the present invention can also be purified from cells or tissues that express the human antibody κ-type light chain in nature. For example, cells or tissues that express the human antibody κ-type light chain according to the present invention in nature can be identified using an antibody or an oligonucleotide. Purification of a human antibody κ-type light chain from cells or tissue can also be carried out in the same manner as in the case of purifying a human antibody κ-type light chain synthesized using a host expression system and the like.

    [0047] In addition, the human antibody κ-type light chain according to the present invention can also be chemically synthesized. There are no particular limitations on the chemical synthesis method, and may be carried out by any method used when chemically synthesizing polypeptides.

    [0048] The anticancer agent according to the present invention has the human antibody κ-type light chain according to the present invention as an active ingredient thereof. Although the mechanism of action by which the human antibody κ-type light chain according to the present invention demonstrates cytotoxicity against cancer cells has not been completely determined, it is presumed that, as a result of the human antibody κ-type light chain according to the present invention specifically recognizing and binding to a specific molecule or structure on the surface of cancer cells simultaneous to decomposing a portion of the components of cancer cells by utilizing its own enzyme activity, the function of the cancer cells is impaired, proliferation thereof is inhibited or cell death is induced.

    [0049] The anticancer agent according to the present invention can be injected or administered directly for use in humans or animals. The anticancer agent according to the present invention can also be formulated for parenteral administration, mucosal administration, intramuscular administration, intravenous administration, subcutaneous administration, intraocular administration or transcutaneous administration. Typically, protein contained in a composition can be administered at a dose of 0.01 mg/kg to 30 mg/kg of body weight, preferably at 0.1 mg/kg to 10 mg/kg of body weight, and even more preferably at 0.1 mg/kg to 1 mg/kg of body weight.

    [0050] The anticancer agent according to the present invention can also contain a pharmaceutically acceptable carrier, diluent or vehicle (including combinations thereof) in addition to the human antibody κ-type light chain according to the present invention. Pharmaceutically acceptable carriers or vehicles for therapeutic use are commonly known in the field of pharmacy, and are described in, for example, Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro, ed., 1985). Pharmaceutically usable carriers, vehicles or diluents can be suitably selected by a person with ordinary skill in the art in accordance with the intended administration route and standard pharmaceutical practices. In addition, the anticancer agent according to the present invention can further contain an arbitrary suitable binder, lubricant, suspension agent, coating agent or solubilizing agent.

    [0051] Conditions required for composition and/or formulation can vary depending on the use of different delivery systems. As an example thereof, the anticancer agent according to the present invention can be formulated so as to be delivered using a minipump, by a mucosal route in the form of, for example, a nasal spray or aerosol for inhalation, or for parenteral delivery (here, the anticancer agent according to the present invention is formulated in an injectable form for delivery via, for example, an intravenous route, an intramuscular route or a subcutaneous route). Alternatively, the formula can be designed so as to be delivered by both routes. For example, the anticancer agent according to the present invention demonstrates a high level of cytotoxicity against lung cancer cells in particular. Consequently, the anticancer agent according to the present invention is preferably in the form of a nasal spray or aerosol for inhalation that enables it to be efficiently delivered to pneumocytes from the nose or bronchi.

    [0052] In addition, in the case of using the anticancer agent according to the present invention in an application in which it is administered into the body, various technologies can be used for improving the stability (half-life in blood) of the active ingredient in the form of the human antibody κ-type light chain in the body. For example, the half-life in the blood of antibodies such as IgG is known to be prolonged if neonatal Fc receptor (FcRn) is bound to the Fc region (see, for example, Roopenian , D. C., et al., Nat. Rev. Immunol., Vol. 7, 715-725 (2007)), and the C-terminal of the human antibody κ-type light chain according to the present invention can be modified so as to have binding activity with FcRn. In addition, the human antibody κ-type light chain according to the present invention can be in the form of a dimer, and polyethylene glycol (PEG) can be added as well.

    [0053] The anticancer agent according to the present invention can be incorporated in a kit, for example, together with instructions and the like on the form in which it is to be administered. The kit can also contain various other pharmaceuticals that can be used with the anticancer agent according to the present invention.

    [0054] In addition, since the anticancer agent according to the present invention has for the active ingredient thereof an antibody κ-type light chain that is highly effective in recognizing a target molecule, it does not demonstrate cytotoxicity against cancer cells in which the target molecule of the antibody light chain is not present on the cell surface thereof. Consequently, the anticancer agent of the present invention is expected to be useful in distinguishing types of cancer.

    Examples



    [0055] Although the following provides a more detailed explanation of the present invention through examples thereof, the present invention is not limited by these examples.

    [Example 1]


    (1. Preparation of Human Peripheral Blood cDNA)



    [0056] Lymphocytes were isolated using Ficoll-paque from peripheral blood acquired from volunteers hyperimmunized over a plurality of times using rabies virus vaccine. Total RNA was obtained from roughly 3.0 × 107 isolated lymphocytes using an RNA extraction kit (Stratagene Corp.). The target cDNA (cDNA library) was then prepared by reverse transcribing the total RNA with the ThermoScript RT-PCT System (Invitrogen Inc.) using oligo(dT) as primer.

    (2. Acquisition of Human Antibody κ-type Light Chain Genes)



    [0057] PCR reactions were carried out in two stages using the cDNA acquired in step 1 above as template and using primers for amplifying antibody light chain gene having a Vκ gene belonging to subgroup II to obtain roughly 750 bp PCR products (κ-type light chain genes belonging to subgroup II). These PCR products were cloned and subjected to sequence analysis, and the Vκ gene in each germline gene was estimated by a homology search. As a result, all of the resulting 18 clones belonged to subgroup II. Among these, nine clones, namely, clone #1 (germline genotype: A18b), clone #2 (germline genotype: A3/A19), clone #4 (germline genotype: 011/01), clone #7 (germline genotype: A3/A19), clone #8 (germline genotype: A18b), clone #9 (germline genotype: A18b), clone #11 (germline genotype: A18b), clone #13 (germline genotype: A3/A19) and clone #14 (germline genotype: A3/A19) were used in subsequent experimentation.

    (3. Expression of Human Antibody κ-type Light Chains)



    [0058] Each of the clones acquired in step 2 above was respectively introduced into a plasmid vector having an His tag sequence site followed by introducing the plasmid vector into Escherichia coli to produce transformants. When each transformant was cultured and subjected to induction of expression with IPTG, the protein expressed in the E. coli was able to be identified as a human antibody light chain by SDS-PAGE analysis and Western blotting using anti-human(Fab')2 antibody. The resulting human antibody light chains had M (initiating methionine) on the N-terminal and LEHHHHHH (SEQ ID NO: 23) derived from the plasmid vector on the C-terminal.

    (4. Preparation of Human Peripheral Blood cDNA)



    [0059] Subjects were hyperimmunized over a plurality of times using rabies virus vaccine followed by measurement of serum neutralizing activity. Peripheral blood was collected from the donor subject having the highest level of serum neutralizing activity (7.21 U), and lymphocytes were isolated from the peripheral blood using Ficoll-paque. Total RNA was then obtained from roughly 3.0 × 107 isolated lymphocytes using an RNA extraction kit (Stratagene Corp.). cDNA to be used as template was prepared in a PCR reaction to be subsequently described by reverse transcribing the total RNA with the ThermoScript RT-PCR System (Invitrogen Inc.) using oligo(dT) as primer.

    (5. Acquisition of Human Antibody κ-type Light Chain Genes)



    [0060] A PCR reaction was carried out using a primer set for comprehensively amplifying human antibody light chain gene and using the cDNA acquired in step 4 above as template to obtain a roughly 660 bp PCR product. This PCR product was purified and inserted into the E. coli expression vector pET101/D-TOPO® (Invitrogen Inc.) to construct an LCA library. Furthermore, protein in which an His tag was added to the C-terminal of the protein encoded by the PCR product was expressed from an expression vector in which the PCR product was inserted in the pET101/D-TOPO vector. PCR reactions were carried out using the cDNA of this LCA library as template and using primers for amplifying human antibody light chain gene having a Vκ gene belonging to subgroup II to obtain roughly 660 bp PCR products. These PCR products were cloned and subjected to sequence analysis and their amino acid sequences and light chain variable and constant regions were estimated using analytical software (Genetix® Ver. 8) followed by estimation of the Vκ gene in each germline gene. Among these clones, two clones, namely, clone 22F6 (germline genotype: A3/A19) and clone 23D4 (germline genotype: A3/A19) were used in subsequent experimentation. The resulting human antibody light chains had M (initiating methionine) on the N-terminal and LEHHHHHH (SEQ ID NO: 23) derived from the plasmid vector on the C-terminal.

    [0061] As a result of sequencing each clone, the total length of the human antibody light chain pertaining to clone #1 (human antibody light chain (#1_WT)) was the base sequence indicated in SEQ ID NO: 27, the total length of the human antibody light chain pertaining to clone #8 (human antibody light chain (#8_WT)) was the base sequence indicated in SEQ ID NO: 28, the total length of the human antibody light chain pertaining to clone #9 (human antibody light chain (#9_WT)) was the base sequence indicated in SEQ ID NO: 29, the total length of the human antibody light chain pertaining to clone #11 (human antibody light chain (#11_WT)) was the base sequence indicated in SEQ ID NO: 30, the total length of the human antibody light chain pertaining to clone #4 (human antibody light chain (#4_WT)) was the base sequence indicated in SEQ ID NO: 31, the total length of the human antibody light chain pertaining to clone #2 (human antibody light chain (#2_WT)) was the base sequence indicated in SEQ ID NO: 32, the total length of the human antibody light chain pertaining to clone #7 (human antibody light chain (#7_WT)) was the base sequence indicated in SEQ ID NO: 33, the total length of the human antibody light chain pertaining to clone #13 (human antibody light chain (#13_WT)) was the base sequence indicated in SEQ ID NO: 34, the total length of the human antibody light chain pertaining to clone #14 (human antibody light chain (#14_WT)) was the base sequence indicated in SEQ ID NO: 35, the total length of the human antibody light chain pertaining to clone 22F6 (human antibody light chain (22F6_WT)) was the base sequence indicated in SEQ ID NO: 36, and the total length of the human antibody light chain pertaining to clone 23D4 (human antibody light chain (23D4_WT)) was the base sequence indicated in SEQ ID NO: 37.

    [0062] The amino acid sequences estimated from each of the base sequences are shown in FIG. 1. In addition, the locations of the variable regions, constant regions and CDR1 to CDR3 are also shown. The human antibody light chain pertaining to clone #1 (human antibody light chain (#1_WT)) was the amino acid sequence shown in SEQ ID NO: 2, the human antibody light chain pertaining to clone #8 (human antibody light chain (#8_WT)) was the amino acid sequence shown in SEQ ID NO: 4, the human antibody light chain pertaining to clone #9 (human antibody light chain (#9_WT)) was the amino acid sequence shown in SEQ ID NO: 6, the human antibody light chain pertaining to clone #11 (human antibody light chain (#11_WT)) was the amino acid sequence shown in SEQ ID NO: 8, the human antibody light chain pertaining to clone #4 (human antibody light chain (#4_WT)) was the amino acid sequence shown in SEQ ID NO: 10, the human antibody light chain pertaining to clone #2 (human antibody light chain (#2_WT)) was the amino acid sequence shown in SEQ ID NO: 12, the human antibody light chain pertaining to clone #7 (human antibody light chain (#7_WT)) was the amino acid sequence shown in SEQ ID NO: 14, the human antibody light chain pertaining to clone #13 (human antibody light chain (#13_WT)) was the amino acid sequence shown in SEQ ID NO: 16, the human antibody light chain pertaining to clone #14 (human antibody light chain (#14_WT)) was the amino acid sequence shown in SEQ ID NO: 18, the human antibody light chain pertaining to clone 22F6 (human antibody light chain (22F6_WT)) was the amino acid sequence shown in SEQ ID NO: 20, and the human antibody light chain pertaining to clone 23D4 (human antibody light chain (23D4_WT)) was the amino acid sequence shown in SEQ ID NO: 22.

    [0063] Furthermore, the wild type human antibody light chains used in the present example were polypeptides in which methionine was added to the N-terminal of each amino acid sequence shown in FIG. 1 and LEHHHHHH (SEQ ID NO: 23) derived from the plasmid vector was added to the C-terminal.

    (6. Production of Monomer Human Antibody Light Chains)



    [0064] The human antibody κ-type light chains of the clones acquired in steps 2 and 5 above formed dimers due to the formation of disulfide (S-S) bonds by cysteine on the C-terminal. Then, cDNA was designed so as to form only monomer human antibody enzymes by introducing a mutation in which the cysteine involved in S-S bond formation (cysteine on the C-terminal of the amino acid sequences of FIG. 1) is substituted with alanine. The details of this design with respect to the human antibody light chain having LEHHHHHH derived from the plasmid vector on the C-terminal thereof (#1_WT) are shown in FIG. 2. As shown in FIG. 2(a), TGT encoding cysteine at position 220 in the full-length human antibody enzyme gene is substituted with GCT. As a result, as shown in FIG.2(b), although a dimer is formed in the original amino acid sequence due to the presence of cysteine at position 220, S-S bonds are not formed in the substituted amino acid sequence as a result of substituting alanine at positon 220, thereby resulting in a monomer.

    [0065] More specifically, TGT encoding the aforementioned cysteine in the wild-type full-length human antibody enzyme gene was substituted with GCTCTCGAGCACCACCACCACCACCACTGA (SEQ ID NO: 26) that encodes ALEHHHHHH (SEQ ID NO: 25) (having a stop codon) . In other words, the monomer human antibody light chain used in the present example was a polypeptide in which methionine was added to the N-terminal of each amino acid sequence shown in FIG. 1 and ALEHHHHHH was added to the C-terminal instead of cysteine. Furthermore, among those mutants obtained in this manner, in which the cysteine involved in S-S bonding was substituted with alanine, the mutant of human antibody light chain (#1_WT) is referred to as the human antibody light chain(#1_C220A), the mutant of human antibody light chain (#8_WT) is referred to as the human antibody light chain(#8_C220A), the mutant of human antibody light chain (#9_WT) is referred to as the human antibody light chain(#9_C220A), the mutant of human antibody light chain (#11_WT) is referred to as the human antibody light chain(#11_C220A), the mutant of human antibody light chain (#4_WT) is referred to as the human antibody light chain(#4_C220A), the mutant of human antibody light chain (#2_WT) is referred to as the human antibody light chain(#2_C220A), the mutant of human antibody light chain (#7_WT) is referred to as the human antibody light chain(#7_C220A), the mutant of human antibody light chain (#13_WT) is referred to as the human antibody light chain(#13_C220A), the mutant of human antibody light chain (#14_WT) is referred to as the human antibody light chain(#14_C220A), the mutant of human antibody light chain (22F6_WT) is referred to as the human antibody light chain (22F6_C220A) and the mutant of human antibody light chain (23D4_WT) is referred to as the human antibody light chain(23D4_C220A).

    (7. Purification of Human Antibody Light Chains)



    [0066] Each of the human antibody light chains was subjected to primary purification and secondary purification in the manner described below. FIG. 3(a) is a diagram indicating the results of Ni-NTA column chromatography and SDS-PAGE analysis during primary purification of human antibody light chain (#1_WT) and human antibody light chain (#1_C220A) . FIG. 3(b) is a diagram indicating the results of cation exchange chromatography and SDS-PAGE analysis during secondary purification.

    [0067] 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 and 0.005% Tween 20) was passed through the column after applying the sample until all the flow-through fraction had passed through the column. As indicated by the broken line in the graph on the left side, the concentration of imidazole was increased gradually from 40 mM to 300 mM to elute a component bound to the gel. An Ni-NTA agarose column (diameter: 1 cm, 2 ml) was used for the column and the flow rate was maintained at 0.1 mL/min throughout purification. As shown on the right side of FIG. 3(a), a target band of roughly 31 kDa was detected in fractions 30 to 37. These samples were combined and subjected to the secondary purification indicated below.

    [0068] As shown on the left side of FIG. 3(b), buffer A (50 mM sodium acetate (pH 5.4), 0.2 M NaCl and 0.005% Tween 20) was passed through the column after applying the sample until all the flow-through fraction had passed through the column. As indicated by the broken line in the graph on the left side, the concentration of NaCl was increased gradually from 0.2 M to 0.4 M to elute a component bound to the gel. The SP5PW column (Tosho Corp.) was used for the column and the flow rate was maintained at 0.1 ml/min throughout purification. Components contained in the sample prior to purification, the region "a" surrounded by broken lines in the graph (fraction numbers 10 to 15) and the region "c" surrounded by broken lines in the graph (fraction numbers 25 to 30) were analyzed by SDS-PAGE. As shown on the right side of FIG. 3(b), a target band of roughly 31 kDa was detected in regions "a" and "c" in the reduced sample. In addition, in the unreduced sample, a roughly 31 kDa band was detected only in region "a" while a roughly 51 kDa band was detected only in region "c". As has been described above, the monomer of the antibody light chain is roughly 31 kDa and the dimer is roughly 51 kDa. Sample a is the monomer fraction of the antibody light chain while sample c is the dimer fraction of the antibody light chain.

    [0069] The other clones also contained dimers and monomers in the expression products of the wild-type human antibody light chains in the same manner as clone (#1), dimers were purified by two-stage purification utilizing Ni-NTA column chromatography and cation exchange chromatography, monomers were contained in the expression products of mutants in which cysteine involved in S-S bonding had been mutated to alanine, and the monomers were purified by the same two-stage purification.

    (8. Cytotoxicity against Cancer Cells)



    [0070] A test was conducted of the cytotoxicity of various human antibody κ-type light chains against cancer cells. Human alveolar adenocarcinoma cell line A549 purchased from ATCC was used for the cancer cells, and the cells were cultured in accordance with routine methods using F-12K medium containing 10% fetal calf serum (FCS).

    [0071]  First, after thawing and recovering frozen A549 cells, 100 µl aliquots of the cells were disseminated in a 96-well plate to a concentration of 5 × 103 cells/well. After culturing for 24 hours at 37°C and removing the medium added to the 96-well plate by decantation, each human antibody κ-type light chain adjusted to a concentration of about 1 mg/mL was added in 100 µL aliquots to each well. 10 µl aliquots of WST-1 reagent (Roche Diagnostics GmbH) were added to each well at 24 hours and 48 hours after adding the human antibody κ-type light chains (48 hours and 72 hours after disseminating the cells), followed by measurement of absorbance of the formasan pigment formed (Abs 450 nm) 1, 1.5 and 2 hours later. Cell viability was determined in each well based on the resulting absorbance results using a value of 100% for cell viability in a well to which a human antibody κ-type light chain was not added (N.C.) followed by evaluation of cytotoxicity of the added human antibody κ-type light chains.

    [0072] Cell viability at 24 hours and 48 hours after adding human antibody κ-type light chain is shown in FIG. 4, FIG. 5, Table 1 and Table 2 for each of the human antibody κ-type light chains . The results for the clones having a germline genotype of A18b or 011/o1 are shown in FIG. 4 and Table 1, and the results for the clones having a germline genotype of A3/A19 are shown in FIG. 5 and Table 2. In addition, Tables 1 and 2 also indicate the concentrations of the human antibody κ-type light chains in the wells.
    [Table 1]
    CloneConcentration in well (µM)Cell viability (%)
    After 24 hrAfter 48 hr
    #1_WT 44 60 60
    #1 C220A 40 72 75
    #8 WT 29 79 81
    #8 C220A 38 96 93
    #9 WT 20 78 75
    #9 C220A 40 86 84
    #11 WT 28 73 80
    #11 C220A 44 95 94
    #4 WT 20 50 54
    #4 C220A 44 96 93
    [Table 2]
    CloneConcentration in well (µM)Cell viability (%)
    After 24 hrAfter 48 hr
    #2 WT 27 81 86
    #2 C220A 40 94 95
    #7 WT 35 49 53
    #7 C220A 40 92 93
    #13 WT 40 101 101
    #13 C220A 56 97 81
    #14 WT 32 100 94
    #14 C220A 40 89 88
    22F6 WT 62 80 92
    22F6 C220A 32 65 64
    23D4 WT 28 83 85
    23D4 C220A 44 96 92


    [0073] As a result, the four clones consisting of clone (#1_WT), clone (#4_WT), clone (#7_WT) and clone (22F6_C220A) demonstrated cytotoxicity on the order of 40% to 50% against A549 cells. Other clones were observed to hardly demonstrate any cytotoxicity against A549 cells.

    [0074] Among these four clones, clone (#4_WT)and clone (#7_WT) demonstrated particularly potent cytotoxicity. Among these, clone (#7_WT), namely, human antibody κ-type light chain (#7), was suggested to have an effect that suppresses proliferation of A549 cells since there were hardly any changes in the number of cells in the wells between prior to addition of the human antibody κ-type light chain (0 hours) and after addition of the human antibody κ-type light chain (48 hours).

    [0075] In addition, on the basis of the results for the clones used in this test, potent cytotoxicity was suggested to be present in dimers since it was observed that dimers (WT) have a tendency to demonstrate more potent cytotoxicity than monomers.

    [0076] In addition, cytotoxicity of human antibody κ-type light chain against various cell lines was evaluated in the same manner as described above while also including other clones. Those results are shown in Table 3.
    [Table 3]
    Cell typeCloneCell viability (%)
    24 hr after addition48 hr after addition
    A549 #1 H31Y C220A 72 53
    #7 VL(I) 77 82
    #7 RLI 74 90
    C51 78 87
    C87 75 65
    MOLT-4 #1 H31Y C220A 57.1 60.2
    #4 wt 73.8 90.6
    #7 EI 87 80.2
    #7 TR 93.2 74.1
    #7 RLI 55 73
    #7 VL 77.3 84.2
    S13 75.3 108
    S21 78.2 91.3
    S38 77.3 85.5
    C51 59.4 63
    ES-2 #1 H31Y C220A 59.9 72.7
    #4 83.9 93.3
    #7 wt 98.3 98.6
    #7 RLI 100 94.8
    #10 79.9 92.4
    #11 57 78.8
    22F6 63.1 89
    22F6 C220A 53.2 67.4
    C51 71.2 70.7
    C67 69.7 76.1
    C82 62.2 72.7
    C88 78.1 76.6
    BxPC #4 58 63.3
    #7 G 88.3 71.9
    #7 EI 80.5 69.9
    #7 RLI 87.9 77.3
    #7 VL 77.7 75.9
    #13 120.4 67.6
    #14 116.6 69.3
    22F6 115.2 65.5
    B-16 #7 wt 85 92


    [0077] As a result, clone (#1_H31Y C220A) demonstrated a high level of cytotoxicity against A549 cells, MOLT-4 cells and ES-2 cells. In addition, clone (#7 RLI) and clone (C51) demonstrated a high level of cytotoxicity against MOLT-4 cells. Moreover, clone (#4) demonstrated a high level of cytotoxicity against ES-2 cells.

    [0078] Furthermore, the amino acid sequence of clone (#1_H31Y C220A) is shown in SEQ ID NO: 38, the amino acid sequence of clone (#7 VL(I)) is shown in SEQ ID NO: 39, the amino acid sequence of clone (#7 RLI) is shown in SEQ ID NO: 40, the amino acid sequence of clone (C51) is shown in SEQ ID NO: 41, the amino acid sequence of clone (C87) is shown in SEQ ID NO: 42, the amino acid sequence of clone (#7 EI) is shown in SEQ ID NO: 43, the amino acid sequence of clone (#7 TR) is shown in SEQ ID NO: 44, the amino acid sequence of clone (#7 VL) is shown in SEQ ID NO: 45, the amino acid sequence of clone (S13) is shown in SEQ ID NO: 46, the amino acid sequence of clone (S21) is shown in SEQ ID NO: 47, the amino acid sequence of clone (S38) is shown in SEQ ID NO: 48, the amino acid sequence of clone (#10) is shown in SEQ ID NO: 49, the amino acid sequence of clone (C67) is shown in SEQ ID NO: 50, the amino acid sequence of clone (C82) is shown in SEQ ID NO: 51, the amino acid sequence of clone (C88) is shown in SEQ ID NO: 52, and the amino acid sequence of clone (#7 G) is shown in SEQ ID NO: 53.

    [0079] In addition, as shown in FIGs. 7 and 8, the anticancer agent containing the human antibody κ-type light chain of the present application did not demonstrate any toxicity in animal studies .

    INDUSTRIAL APPLICABILITY



    [0080] The present invention allows the development of a novel anticancer agent and the use thereof in the field of cancer treatment.

    SEQUENCE LISTING



    [0081] 

    <110> Japan Science and Technology Agency

    <120> Anticancer agent

    <130> PC16746

    <150> JP2012-052334
    <151> 2012-03-08

    <160> 53

    <170> PatentIn version 3.1

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    Claims

    1. A pharmaceutical composition for use in the treatment of lung cancer by eradicating cancer cells or suppressing or inhibiting a proliferation of the cancer cells, comprising:

    (1) a human antibody κ-type light chain in the form of a dimer in which a variable region is composed of a polypeptide represented by
    an amino acid sequence of SEQ ID NO: 9, or
    an amino acid sequence having an identity of 95% or more with the amino acid sequence of SEQ ID NO: 9 and having the same CDRs as the amino acid sequence of SEQ ID NO: 9; or

    (2) a human antibody κ-type light chain in the form of a monomer in which a variable region is composed of a polypeptide represented by
    an amino acid sequence of SEQ ID NO: 19, or
    an amino acid sequence having an identity of 95% or more with the amino acid sequence of SEQ ID NO: 19 and having the same CDRs as the amino acid sequence of SEQ ID NO: 19;

    wherein the CDRs in the amino acid sequence of SEQ ID NO: 9 consist of the 24th to 40th amino acid residues, the 56th to 62nd amino acid residues, and the 95th to 102nd amino acid residues; and
    wherein the CDRs in the amino acid sequence of SEQ ID NO: 19 consist of the 24th to 39th amino acid residues, the 55th to 61st amino acid residues, and the 94th to 101st amino acid residues.
     
    2. A pharmaceutical composition for use according to claim 1, wherein

    the human antibody κ-type light chain of (1) above is a human antibody κ-type light chain in the form of a dimer composed of a polypeptide represented by
    an amino acid sequence of SEQ ID NO: 10, or
    an amino acid sequence having an identity of 95% or more with the amino acid sequence of SEQ ID NO: 10 and having the same CDRs as the amino acid sequence of SEQ ID NO: 10; or

    the human antibody κ-type light chain of (2) above is a human antibody κ-type light chain in the form of a monomer composed of a polypeptide represented by an amino acid sequence in which cysteine at position 219 has been deleted or substituted by an amino acid other than cysteine in the amino acid sequence of SEQ ID NO: 20, or an amino acid sequence having an identity of 95% or more with this amino acid sequence and having the same CDRs as the amino acid sequence of SEQ ID NO: 20;

    wherein the CDRs in the amino acid sequence of SEQ ID NO: 10 consist of the 24th to 40th amino acid residues, the 56th to 62nd amino acid residues, and the 95th to 102nd amino acid residues; and
    wherein the CDRs in the amino acid sequence of SEQ ID NO: 20 consist of the 24th to 39th amino acid residues, the 55th to 61st amino acid residues, and the 94th to 101st amino acid residues.
     


    Ansprüche

    1. Eine pharmazeutische Zusammensetzung zur Verwendung bei der Behandlung von Lungenkrebs durch die Vernichtung von Krebszellen oder die Unterdrückung oder Inhibition einer Proliferation von Krebszellen, umfassend:

    (1) eine Human-Antikörper-

    -Typ-Leichtkette in der Form eines Dimers, in welcher eine variable Region zusammengesetzt ist aus einem Polypeptide, das dargestellt wird durch

    eine Aminosäuresequenz nach SEQ ID Nr. 9 oder

    eine Aminosäuresequenz mit mindestens 95%iger Sequenzidentität mit der Aminosäuresequenz gemäß SEQ ID Nr. 9 oder denselben CDRs wie die Aminosäuresequenz gemäß SEQ ID Nr. 9 oder

    (2) eine Human-Antikörper-

    -Typ-Leichtkette in der Form eines Monomers, in welcher eine variable Region zusammengesetzt ist aus einem Polypeptid, das dargestellt wird durch

    eine Aminosäuresequenz gemäß SEQ ID Nr. 19 oder

    eine Aminosäuresequenz mit mindestens 95%iger Sequenzidentität mit der Aminosäuresequenz gemäß SEQ ID Nr. 19 und mit denselben CDRs wie die Aminosäuresequenz gemäß SEQ ID Nr. 19;

    wobei die CDRs in Aminosäuresequenz gemäß SEQ ID Nr. 9 aus den 24. bis 40. Aminosäure-Resten, den 56. bis 62. Aminosäure-Resten und den 95. bis 102. Aminosäure-Resten bestehen und
    wobei die CDRs in der Aminosäuresequenz gemäß SEQ ID Nr. 19 aus den 24. bis 39. Aminosäure-Resten, den 55. bis 61. Aminosäure-Resten und den 94. bis 101. Aminosäure-Resten bestehen.
     
    2. Eine pharmazeutische Zusammensetzung zur Verwendung nach Anspruch 1, wobei die Human-Antikörper-

    -Typ-Leichtkette von (1) oben eine Human-Antikörper-

    -Typ-Leichtkette in der Form eines Dimers, das zusammengesetzt ist aus einem Polypeptid, das dargestellt wird durch

    eine Aminosäuresequenz gemäß SEQ ID Nr. 10 oder

    eine Aminosäuresequenz mit mindestens 95%iger Sequenzidentität mit der Aminosäuresequenz gemäß SEQ ID Nr. 10 und mit denselben CDRs wie die Aminosäuresequenz gemäß SEQ ID Nr. 10 oder

    die Human-Antikörper-

    -Typ-Leichtkette von (2) oben eine Human- Antikörper-

    -Typ-Leichtkette in der Form eines Monomers ist, das aus einem Polypeptid zusammengesetzt, das durch eine Aminosäuresequenz dargestellt wird, in der das Cystein an der Position 219 entfernt oder substituiert worden ist durch eine andere Aminosäure als das Cystein von SEQ ID Nr. 20 oder einer Aminosäuresequenz mit einer Sequenzidentität von mindestens 95% mit dieser Aminosäuresequenz und mit denselben CDRs wie die Aminosäuresequenz gemäß SEQ ID Nr. 20;

    wobei die CDRs in der Aminosäuresequenz gemäß SEQ ID Nr. 10 aus den 24. bis 40. Aminosäure-Resten, den 56. bis 62. Aminosäure-Resten und den 95. bis 102. Aminosäure-Resten bestehen; und
    wobei die CDRS in der Aminosäuresequenz von SEQ ID Nr. 20 aus den 24. bis 39. Aminosäure-Resten, den 55. bis 61. Aminosäure-Resten und den 94. bis 101. Aminosäure-Resten bestehen.
     


    Revendications

    1. Composition pharmaceutique pour son utilisation dans le traitement du cancer du poumon par éradication des cellules cancéreuses ou suppression ou inhibition de la prolifération des cellules cancéreuses, comprenant :

    (1) une chaîne légère de type κ d'anticorps humain sous la forme d'un dimère dans lequel une région variable est composée d'un polypeptide représenté par
    une séquence d'acides aminés de SEQ ID N° : 9, ou
    une séquence d'acides aminés ayant une identité de 95 % ou plus avec la séquence d'acides aminés de SEQ ID N° : 9 et ayant les mêmes CDR que la séquence d'acides aminés de SEQ ID N° : 9 ; ou

    (2) une chaîne légère de type κ d'anticorps humain sous la forme d'un monomère dans lequel une région variable est composée d'un polypeptide représenté par une séquence d'acides aminés de SEQ ID N° : 19, ou
    une séquence d'acides aminés ayant une identité de 95 % ou plus avec la séquence d'acides aminés de SEQ ID N° : 19 et ayant les mêmes CDR que la séquence d'acides aminés de SEQ ID N° : 19 ;
    dans laquelle les CDR dans la séquence d'acides aminés de SEQ ID N° : 9 sont constituées des 24e à 40e résidus d'acides aminés, des 56e à 62e résidus d'acides aminés, et des 95e à 102e résidus d'acides aminés ; et
    dans laquelle les CDR dans la séquence d'acides aminés de SEQ ID N° : 19 sont constituées des 24e à 39e résidus d'acides aminés, des 55e à 61e résidus d'acides aminés, et des 94e à 101e résidus d'acides aminés.


     
    2. Composition pharmaceutique pour son utilisation selon la revendication 1, dans laquelle
    la chaîne légère de type κ d'anticorps humain de (1) ci-dessus est une chaîne légère de type κ d'anticorps humain sous la forme d'un dimère composé d'un polypeptide représenté par
    une séquence d'acides aminés de SEQ ID N° : 10, ou
    une séquence d'acides aminés ayant une identité de 95 % ou plus avec la séquence d'acides aminés de SEQ ID N° : 10 et ayant les mêmes CDR que la séquence d'acides aminés de SEQ ID N° : 10 ; ou
    la chaîne légère de type κ d'anticorps humain de (2) ci-dessus est une chaîne légère de type κ d'anticorps humain sous la forme d'un monomère composé d'un polypeptide représenté par une séquence d'acides aminés dans laquelle la cystéine au niveau de la position 219 a été supprimée ou substituée par un acide aminé autre que la cystéine dans la séquence d'acides aminés de SEQ ID N° : 20, ou une séquence d'acides aminés ayant une identité de 95 % ou plus avec cette séquence d'acides aminés et ayant les mêmes CDR que la séquence d'acides aminés de SEQ ID N° : 20 ;
    dans laquelle les CDR dans la séquence d'acides aminés de SEQ ID N° : 10 sont constituées des 24e à 40e résidus d'acides aminés, des 56e à 62e résidus d'acides aminés, et des 95e à 102e résidus d'acides aminés ; et
    dans laquelle les CDR dans la séquence d'acides aminés de SEQ ID N° : 20 sont constituées des 24e à 39e résidus d'acides aminés, des 55e à 61e résidus d'acides aminés, et des 94e à 101e résidus d'acides aminés.
     




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    Cited references

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