Patent Grant
8268592
1. Field of the Invention
The present invention relates to a monoclonal antibody which specifically recognizes a native three-dimensional structure of an extracellular region of system ASC amino acid transporter 2 (hereinafter, referred to as “ASCT2”) and binds to the extracellular region, or an antibody fragment thereof; a hybridoma which produces the antibody; a DNA which encodes the antibody; a vector which contains the DNA; a transformant obtainable by introducing the vector; a process for producing an antibody or an antibody fragment thereof using the hybridoma or the transformant; and a therapeutic agent using the antibody or the antibody fragment thereof, and a diagnostic agent using the antibody or the antibody fragment thereof.
2. Brief Description of the Background Art
An ASCT2 polypeptide is a 10-times transmembrane protein consisting of 541 amino acids in full-length form, and functions as a transporter which transports neutral amino acids through a cell membrane depending on a sodium ion. The amino acid transporter is categorized into several systems, based on functional characteristics including substrate specificity and the like. ASCT2 belongs to System ASC and has functions of intracellular uptake of neutral amino acids such as L-alanine, L-serine, L-threonine, L-cysteine, and L-glutamine depending on a sodium ion (Non-Patent Document 1).
Further, ASCT2 is a viral cell-surface receptor which is shared by type D simian retrovirus and three type C viruses [feline endogenous virus (RD114), baboon endogenous virus, and avian reticuloendotheliosis virus] (Non-Patent Documents 2 and 3).
ASCT2 is also known as sodium-dependent neutral amino acid transporter type 2, adipocyte amino acid transporter, AAAT, neutral amino acid transporter B, ATB, amino acid transporter B0, ATB0, ATBO, baboon M7 virus receptor, M7V1, M7VS1, insulin-activated amino acid transporter, FLJ31068, RD114 virus receptor, RD114/simian type D retrovirus receptor, RDR, RDRC, or R16 (Non-patent Documents 1 and 3).
Furthermore, as another name, ASCT2 is known as Solute carrier family 1 (neutral amino acid transport), member 5, Solute carrier family 1 member 5, or SLC1A5 and belongs to SLC1 family.
With respect to the relationship between the development of cancer and the expression of ASCT2 or SLC1A5, it has been found that expression levels of three SLC1A5, SLC7A5 and SLC38A5 are significantly increased in cancerous tissues by studies using expressed sequence tag (EST) database (Non-Patent Document 4). Further, the expression of SLC1A5 is not recognized in a normal liver, but is recognized in clinical tissues of hepatocellular carcinoma or hepatoblastoma (Non-Patent Document 5). In addition, it has been found that the expression of SLC1A5 is higher in clinical tissues of poorly-differentiated astrocytoma or glioblastoma multiforme than that of normal tissues (Non-Patent Document 6).
Further, the expression of ASCT2 is detected in clinical tissues of colorectal cancer and prostate cancer by immunohistological staining or Western blotting, and patients who highly express ASCT2 have a poor outcome (Non-Patent Documents 7 and 8).
Furthermore, ASCT2 is responsible for the uptake of glutamine in several cell lines of colorectal cancer, liver cancer, breast cancer, astrocytoma and neuroblastoma (Non-Patent Documents 9 to 12), and the proliferation of cells is suppressed by competitively inhibiting intracellular uptake of glutamine using an alanine-serine-threonine mixture which is a substrate for ASCT2 (Non-Patent Document 9).
As regards an antibody which binds to ASCT2, a polyclonal antibody against partial peptides of the intracellular N-terminal or C-terminal of human ASCT2 is known (Non-Patent Documents 13, 14 and 15). Since all of these are antibodies which recognize an intracellular portion of ASCT2, they cannot bind to ASCT2 which is expressed on the cell membrane of cells. It is known that when an antibody binds to an antigen protein expressed on the cell membrane, a cellular cytotoxicity due to an effector activity of the antibody, such as antibody-dependent cellular cytotoxicity (hereinafter, referred to as “ADCC activity”) or complement-dependent cytotoxicity (hereinafter, referred to as “CDC activity”) is induced within the living body (Non-Patent Document 16). However, since these antibodies cannot bind to ASCT2 expressed on the cell membrane of cells, these cellular cytotoxicities due to the effector activity cannot be expected. Further, since all of these antibodies are antibodies which recognize an intracellular portion of ASCT2, they cannot inhibit intracellular uptake of amino acids by ASCT2 which expresses in living cells.
In addition, polyclonal antibodies which recognize human ASCT2 are commercially available from LifeSpan BioSciences (Catalog Numbers: LS-C16840 and LC-C31887), Avia Systems Biology (Catalog Number: ARP42247_T100), Santa Cruz Biotechnology (Catalog Numbers: sc-50698 and sc-50701), or Millipore (Catalog Number: AB5468).
However, it is not known a monoclonal antibody which specifically recognizes a native three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region.
An object of the present invention is to provide a monoclonal antibody which specifically recognizes a native three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region, or an antibody fragment thereof; a hybridoma which produces the antibody; a DNA which encodes the antibody; a vector which contains the DNA; a transformant obtainable by introducing the vector; a process for producing an antibody or an antibody fragment thereof using the hybridoma or the transformant; and a therapeutic agent using the antibody or the antibody fragment thereof, and a diagnostic agent using the antibody or the antibody fragment thereof.
The present invention can provide a monoclonal antibody which specifically recognizes a native three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region, or an antibody fragment thereof; a hybridoma which produces the antibody; a DNA which encodes the antibody; a vector which contains the DNA; a transformant obtainable by introducing the vector; a process for producing an antibody or an antibody fragment thereof using the hybridoma or the transformant; and a therapeutic agent using the antibody or the antibody fragment thereof, and a diagnostic agent using the antibody or the antibody fragment thereof.
The present invention relates to the following (1) to (44):
[1] A monoclonal antibody which specifically recognizes a native three-dimensional structure of an extracellular region of system ASC amino acid transporter 2 (hereinafter, referred to as “ASCT2”) and binds to the extracellular region, or an antibody fragment thereof;
[2] The monoclonal antibody or the antibody fragment thereof described in [1], which inhibits intracellular uptake of an amino acid through ASCT2;
[3] The monoclonal antibody or the antibody fragment thereof described in [1] or [2], which has cellular cytotoxicity;
[4] The monoclonal antibody or the antibody fragment thereof described in [3], in which the cellular cytotoxicity is an antibody-dependent cellular cytotoxicity (ADCC) activity or complement-dependent cytotoxicity (CDC) activity;
[5] The monoclonal antibody or the antibody fragment thereof described in any one of [1] to [4], which binds to human ASCT2 and does not bind to mouse ASCT2;
[6] The monoclonal antibody or the antibody fragment thereof described in [5], which binds to at least EL2 region of human ASCT2;
[7] The monoclonal antibody or the antibody fragment thereof described in [6], which binds to at least any one of amino acids selected from at positions 154, 159 to 160, 163 to 171, 173 to 174, 177, 188, 204 to 205, 207, 210 to 212, and 214 to 223 in the amino acid sequence represented by SEQ ID NO:2;
[8] The antibody or the antibody fragment thereof described in any one of [1] to [7], in which the monoclonal antibody is a monoclonal antibody which binds to an epitope which is recognized by a monoclonal antibody produced from any one of hybridomas selected from a hybridoma KM4008 (FERM BP-10962) and a hybridoma KM4012 (FERM BP-10963);
[9] The antibody or the antibody fragment thereof described in any one of [1] to [8], in which the monoclonal antibody is a monoclonal antibody produced from any one of hybridomas selected from a hybridoma KM4008 (FERM BP-10962) and a hybridoma KM4012 (FERM BP-10963);
[10] The antibody fragment described in any one of [1] to [9], in which the antibody fragment is an antibody fragment selected from Fab, Fab′, F(ab′)2, a single chain antibody (scFv), a dimerized V region (diabody), a disulfide stabilized V region (dsFv) and a CDR-containing peptide.
[11] The antibody or the antibody fragment thereof described in any one of [1] to [10], in which the monoclonal antibody is a recombinant antibody;
[12] The recombinant antibody or the antibody fragment thereof described in [11], in which the recombinant antibody is a recombinant antibody selected from a human chimeric antibody, a humanized antibody and a human antibody;
[13] The recombinant antibody or the antibody fragment thereof described in [11], which comprises complementarity determining regions (hereinafter, referred to as “CDRs”) of a heavy chain variable region (hereinafter, referred to as “VH”) and a light chain variable region (hereinafter, referred to as “VL”) of the monoclonal antibody described in any one of [1] to [9];
[14] The recombinant antibody or the antibody fragment thereof described in [13], in which CDR1, CDR2 and CDR3 of VH of the antibody comprise the amino acid sequences represented by SEQ ID NOs:26, 27 and 28, respectively;
[15] The recombinant antibody or the antibody fragment thereof described in [13], in which CDR1, CDR2 and CDR3 of VL of the antibody comprise the amino acid sequences represented by SEQ ID NOs:29, 30 and 31, respectively;
[16] The recombinant antibody or the antibody fragment thereof described in [13], in which CDR1, CDR2 and CDR3 of VH of the antibody comprise the amino acid sequences represented by SEQ ID NOs:26, 27 and 28, respectively, and CDR1, CDR2 and CDR3 of VL of the antibody comprise the amino acid sequences represented by SEQ ID NOs:29, 30 and 31, respectively;
[17] The recombinant antibody or the antibody fragment thereof described in [13], in which CDR1, CDR2 and CDR3 of VH of the antibody comprise the amino acid sequences represented by SEQ ID NOs:32, 33 and 34, respectively;
[18] The recombinant antibody or the antibody fragment thereof described in [13], in which CDR1, CDR2 and CDR3 of VL of the antibody comprise the amino acid sequences represented by SEQ ID NOs:35, 36 and 37, respectively;
[19] The recombinant antibody or the antibody fragment thereof described in [13], in which CDR1, CDR2 and CDR3 of VH of the antibody comprise the amino acid sequences represented by SEQ ID NOs:32, 33 and 34, respectively, and CDR1, CDR2 and CDR3 of VL of the antibody comprise the amino acid sequences represented by SEQ ID NOs:35, 36 and 37, respectively;
[20] The recombinant antibody or the antibody fragment thereof described in [13], in which CDR1, CDR2 and CDR3 of VH of the antibody comprise the amino acid sequences represented by SEQ ID NOs:49, 50 and 51, respectively,
[21] The recombinant antibody or the antibody fragment thereof described in [13], in which CDR1, CDR2 and CDR3 of VL of the antibody comprise the amino acid sequences represented by SEQ ID NOs:52, 53 and 54, respectively;
[22] The recombinant antibody or the antibody fragment thereof described in [13], in which CDR1, CDR2 and CDR3 of VH of the antibody comprise the amino acid sequences represented by SEQ ID NOs:49, 50 and 51, respectively, and CDR1, CDR2 and CDR3 of VL of the antibody comprise the amino acid sequences represented by SEQ ID NOs:52, 53 and 54, respectively;
[23] The human chimeric antibody or the antibody fragment thereof described in [12], which comprises VH and VL of the monoclonal antibody described in any one of [1] to [8];
[24] The human chimeric antibody or the antibody fragment thereof described in [23], which is selected from a human chimeric antibody in which VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:19 and VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:21, a human chimeric antibody in which VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:23 and VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:25, and a human chimeric antibody in which VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:46 and VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:48;
[25] The humanized antibody or the antibody fragment thereof described in [12], in which VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:71, or an amino acid sequence in which at least one amino acid residue selected from Val at position 2, Ser at position 9, Val at position 20, Ser at position 30, Arg at position 38, Glu at position 46, Leu at position 86, Val at position 93, Tyr at position 95 and Val at position 116 in the amino acid sequence represented by SEQ ID NO:71 is substituted with other amino acid residue, and/or in which VL of the antibody comprises an amino acid sequence represented by SEQ ID NO:72, or an amino acid sequence in which at least one amino acid residue selected from Pro at position 8, Val at position 15, Gln at position 38, Ala at position 43, Pro at position 44, Phe at position 71 and Tyr at position 87 in the amino acid sequence represented by SEQ ID NO:72 is substituted with other amino acid residue;
[26] The humanized antibody or the antibody fragment thereof described in the above [25], in which VH of the antibody comprises an amino acid sequence in which at least one modification selected from modifications for substituting Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu is introduced in the amino acid sequence represented by SEQ ID NO:71, and VL of the antibody comprises an amino acid sequence in which at least one modification selected from modifications for substituting Pro at position 8 with Thr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe is introduced in the amino acid sequence represented by SEQ ID NO:72;
[27] The humanized antibody or the antibody fragment thereof described in [25], in which a humanized antibody is selected from a humanized antibody in which VH of the antibody comprises an amino acid sequence represented by SEQ ID NO:76 and/or VL of the antibody comprises an amino acid sequence represented by SEQ ID NO:72, a humanized antibody in which VH of the antibody comprises an amino acid sequence represented by SEQ ID NO:78 and/or VL of the antibody comprises an amino acid sequence represented by SEQ ID NO:72, a humanized antibody in which VH of the antibody comprises an amino acid sequence represented by SEQ ID NO:80 and/or VL of the antibody comprises an amino acid sequence represented by SEQ ID NO:72, a humanized antibody in which VH of the antibody comprises an amino acid sequence represented by SEQ ID NO:82 and/or VL of the antibody comprises an amino acid sequence represented by SEQ ID NO:72, a humanized antibody in which VH of the antibody comprises an amino acid sequence represented by SEQ ID NO:71 and/or VL of the antibody comprises an amino acid sequence represented by SEQ ID NO:84, a humanized antibody in which VH of the antibody comprises an amino acid sequence represented by SEQ ID NO:76 and/or VL of the antibody comprises an amino acid sequence represented by SEQ ID NO:84, a humanized antibody in which VH of the antibody comprises an amino acid sequence represented by SEQ ID NO:78 and/or VL of the antibody comprises an amino acid sequence represented by SEQ ID NO:84, a humanized antibody in which VH of the antibody comprises an amino acid sequence represented by SEQ ID NO:80 and/or VL of the antibody comprises an amino acid sequence represented by SEQ ID NO:84, and a humanized antibody in which VH of the antibody comprises an amino acid sequence represented by SEQ ID NO:82 and/or VL of the antibody comprises an amino acid sequence represented by SEQ ID NO:84;
[28] A hybridoma which produces the monoclonal antibody described in any one of [1] to [27];
[29] The hybridoma described in [28], in which the hybridoma is a hybridoma KM4008 (FERM BP-10962) or a hybridoma KM4012 (FERM BP-10963)];
[30] A DNA which encodes the antibody or the antibody fragment thereof described in any one of [1] to [29];
[31] A recombinant vector which comprises the DNA described in [30];
[32] A transformant obtainable by introducing the recombinant vector described in [31] into a host cell;
[33] A process for producing the antibody or the antibody fragment thereof described in any one of [1] to [27], comprising culturing the hybridoma described in [28] or [29] or the transformant described in [32] in a medium to form and accumulate the antibody or the antibody fragment thereof described in any one of [1] to [27] in the culture and collecting the antibody or the antibody fragment thereof from the culture;
[34] A therapeutic agent for ASCT2 related disease comprising the antibody or the antibody fragment thereof described in any one of [1] to [27] as an active ingredient;
[35] The therapeutic agent described in [34], in which the disease relating to ASCT2 is cancer;
[36] The therapeutic agent described in [35], in which the cancer is blood cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer or prostate cancer;
[37] A method for immunologically detecting or measuring ASCT2, using the antibody or the antibody fragment thereof described in any one of [1] to [27];
[38] The method described in [37], in which the method for immunologically measuring is an immunoprecipitation method;
[39] A method for immunologically detecting or measuring a cell which expresses ASCT2, using the antibody or the antibody fragment thereof described in any one of [1] to [27];
[40] The method described in [39], in which the method for immunologically detecting is a fluorescent cell staining method;
[41] A reagent for immunologically detecting or measuring ASCT2 using antibody or the antibody fragment thereof described in any one of [1] to [27];
[42] A diagnostic agent for ASCT2 related disease using the antibody or the antibody fragment thereof described in any one of [1] to [27];
[43] The diagnostic agent described in [42], in which the disease relating to ASCT2 is cancer; and
[44] The diagnostic agent described in [43], in which the cancer is blood cancer, esophageal cancer, gastric cancer, colorectal cancer, liver cancer or prostate cancer.
The present invention relates to a monoclonal antibody which specifically recognizes a native three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region, or an antibody fragment thereof.
The species of ASCT2 of the present invention is not particularly limited. An example of the species may preferably be a mammal, specifically a human.
The amino acid sequence information of ASCT2 is available from a known database such as NCBI (www.ncbi.nlm.nih.gov/). Examples include human ASCT2 (NCBI Accession No. NP—005619) represented by SEQ ID NO:2, mouse ASCT2 (NCBI Accession No. NP—033227) represented by SEQ ID NO:86, and the like.
ASCT2 of the present invention includes a polypeptide comprising the amino acid sequence represented by SEQ ID NO:2 or an amino acid sequence wherein one or more amino acid residue(s) is/are deleted, substituted and/or added in the amino acid sequence represented by SEQ ID NO:2 and having a function of ASCT2. ASCT2 of the present invention also includes a polypeptide having 60% or more homology, preferably 80% or more homology, more preferably 90% or more homology, further preferably 95% or more homology to the amino acid sequence represented by SEQ ID NO:2 and having a function of ASCT2.
In the present invention, the polypeptide comprising an amino acid sequence wherein one or more amino acid residue(s) is/are deleted, substituted and/or added in the amino acid sequence represented by SEQ ID NO:2 can be obtained by site-specific mutagenesis described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci., USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985), etc. For example, it can be obtained by introducing a site-specific mutation into DNA having the nucleotide sequence encoding a polypeptide having the amino acid sequence represented by SEQ ID NO:2. The number of amino acid residues which are deleted, substituted and/or added is not specifically limited. The suitable number is 1 to dozens, preferably 1 to 20, more preferably 1 to several, further preferably 1 to 5.
Examples of the gene encoding ASCT2 include the nucleotide sequence represented by SEQ ID NO:1. Examples of the gene encoding ASCT2 also include a gene containing a DNA comprising a nucleotide sequence having deletion, substitution or addition of one or more nucleotides in the nucleotide sequence represented by SEQ ID NO:1 and encoding a polypeptide having the function of ASCT2; a gene containing a DNA comprising a nucleotide sequence having at least 60% or higher homology, preferably 80% or higher homology, and more preferably 95% or higher homology with the nucleotide sequence represented by SEQ ID NO:1 and encoding a polypeptide having the function of ASCT2; a gene comprising a DNA which hybridizes with a DNA having the nucleotide sequence represented by SEQ ID NO:1 under stringent conditions and containing a DNA that encodes a polypeptide having the function of ASCT2, and the like.
In the present invention, the DNA which hybridizes under stringent conditions refers to a DNA which is obtained by colony hybridization, plaque hybridization, Southern hybridization, DNA microarray, or the like using, a DNA having the nucleotide sequence represented by SEQ ID NO:1 as a probe. A specific example of such DNA is a hybridized colony- or plaque derived DNA which can be identified by performing hybridization at 65° C. in the presence of 0.7 to 1.0 mol/l sodium chloride using a filter or slide glass with the PCR product or oligo DNA having immobilized thereon, and then washing the filter or slide glass at 65° C. with a 0.1 to 2-fold concentration SSC solution (1-fold concentration SSC solution: 150 mmol/l sodium chloride and 15 mmol/l sodium citrate). Hybridization can be carried out according to the methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Lab. Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997); DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995). Specifically, the DNA capable of hybridization includes DNA having at least 60% or more homology, preferably 80% or more homology, more preferably 90% or more homology, and most preferably 95% or more homology to the nucleotide sequence represented by SEQ ID NO:1.
In the nucleotide sequence of the gene encoding a protein of a eukaryote, genetic polymorphism is often recognized. The ASCT2 gene used in the present invention also includes a gene in which small modifications are generated in the nucleotide sequence by such polymorphism.
The number of the homology described in the present invention may be a number calculated by using a homology search program known by the skilled person, unless otherwise indicated. Regarding the nucleotide sequence, the number may be calculated by BLAST [J. Mol. Biol., 215, 403 (1990)] with a default parameter or the like, and regarding the amino acid sequence, the number may be calculated by using BLAST2 [Nucleic Acids Res., 25, 3389 (1997); Genome Res., 7, 649 (1997); www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.html] with a default parameter or the like.
As the default parameter, G (cost to open gap) is 5 for the nucleotide sequence and 11 for the amino acid sequence; —E (cost to extend gap) is 2 for the nucleotide sequence and 1 for the amino acid sequence; —q (penalty for nucleotide mismatch) is −3; —r (reward for nucleotide match) is 1; —e (expect value) is 10; —W (wordsize) is 11 residues for the nucleotide sequence and 3 residues for the amino acid sequence; —y (dropoff (X) for blast extensions in bits) is 20 for blastn and 7 for a program other than blastn; —X (X dropoff value for gapped alignment in bits) is 15; and —Z (final X dropoff value for gapped alignment in bits) is 50 for blastn and 25 for a program other than blastn (wwvv.ncbi.nlm.nih.gov/blast/html/blastcgihelp.html).
A polypeptide comprising a partial sequence of the amino acid sequence represented by SEQ ID NO:2 can be prepared by methods known to those skilled in the art, for example, by partially deleting a DNA encoding the amino acid sequence represented by SEQ ID NO:2, and culturing a transformant into which an expression vector containing the partially deleted DNA is introduced. Based on the thus constructed polypeptide or DNA, a polypeptide in which one or more amino acids are deleted, substituted and/or added in a partial sequence of the amino acid sequence represented by SEQ ID NO:2 can also be obtained, in accordance with the same method as described above. Further, the polypeptide comprising a partial sequence of the amino acid sequence represented by SEQ ID NO:2, or the polypeptide in which one or more amino acids are deleted, substituted and/or added in a partial sequence of the amino acid sequence represented by SEQ ID NO:2 can also be produced by a chemical synthesis method such as a fluorenylmethyloxycarbonyl (Fmoc) method, a t-butyloxycarbonyl (tBoc) method, or the like.
Examples of the extracellular region of ASCT2 in the present invention include a region predicted by a known transmembrane region prediction programs SOSUI (sosui. proteome.bio.tuat. ac.jp/sosuiframe0. html), TMEMM ver. 2 (www. cbs.dtu.dk/services/TMH1VINI-2.0/), ExPAsy Proteomics Server (Ca.expasy.org/), or the like, based on the amino acid sequence of the polypeptide represented by SEQ ID NO:2. Specifically, examples include five regions, at positions 74 to 98, at positions 154 to 224, at positions 287 to 305, at positions 357 to 376 and at positions 420 to 425 which are the extracellular region predicted by ExPASy Proteomics Server. Alternatively, examples include five regions, at positions 65 to 88, at positions 152 to 224, at positions 288 to 306, at positions 361 to 380 and at positions 447 to 451 which are the extracellular regions predicted in the literature [J. Biol. Chemistry, 271, 18657 (1996)] or [J. Virology, 73, 4470 (1999)]. In the present invention, the five extracellular regions are indicated as an EL1 region, an EL2 region, an EL3 region, an EL4 region and an EL5 region sequentially from the N-terminal side. For example, the EL2 region in an amino acid sequence of the ASCT2 polypeptide represented by SEQ ID NO:2 corresponds to positions 154 to 224 or positions 152 to 224.
The monoclonal antibody of the present invention binds to an extracellular region of ASCT2, preferably any one of the above-mentioned EL1 to EL5 regions, and more preferably the EL2 region.
The native three-dimensional structure of the extracellular region of ASCT2 in the present invention may be any structure, so long as it has a three-dimensional structure equivalent to a three-dimensional structure which can be formed in a native state by the extracellular region of ASCT2 having the amino acid sequence represented by SEQ ID NO:2.
Whether the antibody or the antibody fragment thereof of the present invention specifically recognizes a native three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region can be confirmed by radioimmunoassay using a solid-phase sandwich method or the like, or by a known immunological detection method for ASCT2-expressing cells using such as enzyme immunoassay (ELISA) method or the like, preferably a method capable of investigating a binding of an antibody to a particular antigen and a cell expressing the particular antigen, such as a fluorescent cell staining method. For example, specific examples include a fluorescent antibody staining method using an FMAT8100HTS System (manufactured by Applied Biosystems) [Cancer Immunol. Immunother., 36, 373 (1993)] or the like, a fluorescent cell staining method using flow cytometry, surface plasmon resonance using a Biacore System (manufactured by GE Healthcare) or the like, or other methods. Furthermore, a known immunological detection methods [Monoclonal Antibodies—Principles and practice, Third edition, Academic Press (1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Experimental Manual, Kodan-sha Scientific (1987)] and the like can be combined to confirm the binding.
In the present invention, the cell expressing ASCT2 may be any cell, so long as it expresses ASCT2, and examples include a cell which is naturally present in the human body, a cell line established from the cell which is naturally present in the human body, a cell obtained by gene recombination technique and the like.
The cell which is naturally present in the human body includes a cell expressing the polypeptide in the body of a cancer patient, such as a cell expressing the ASCT2 among tumor cells obtained by biopsy or the like.
Example of a cell line established from the cell which is naturally present in the human body include a cell line expressing ASCT2, among cell lines prepared by establishing the ASCT2-expressing cells obtained from the above cancer patients, and examples include the multiple myeloma cell line KMS-11 [Human Science Research Resources Bank (HSRRB) Accession No. JCRB1179], RPMI 8226 (HSRRB Accession No. JCRB0034) and the like.
Specific examples of the cell obtained by gene recombination techniques may include an ASCT2-expressing cell obtained by introducing an expression vector comprising an ASCT2-encoding cDNA into an insect cell, an animal cell or the like, and others.
The monoclonal antibody of the present invention includes an antibody produced by a hybridoma and a recombinant antibody produced by a transformant transformed with an expression vector containing a gene encoding an antibody.
The hybridoma can be prepared, for example, by preparing the above cell expressing ASCT2 as an antigen, inducing an antibody-producing cell having antigen specificity from an animal immunized with the antigen, and fusing the antigen-producing cell with a myeloma cell. The anti-ASCT2 antibody can be obtained by culturing the hybridoma or administering the hybridoma cell into an animal to cause ascites tumor in the animal and separating and purifying the culture or the ascites.
The animal immunized with an antigen may be any animal, so long as a hybridoma can be prepared, and mouse, rat, hamster, rabbit or the like is suitably used. Also, the antibody of the present invention includes an antibody produced by a hybridoma obtained by fusion of the cell having antibody-producing activity can be obtained from such an animal and immune in vitro with a myeloma cell.
Specific examples of the monoclonal antibody produced by the hybridoma of the present invention may include an antibody KM3998 produced by a hybridoma KM3998, an antibody KM4000 produced by a hybridoma KM4000, an antibody KM4001 produced by a hybridoma KM4001, an antibody KM4008 produced by a hybridoma KM4008, an antibody KM4012 produced by a hybridoma KM4012, an antibody KM4018 produced by a hybridoma KM4018, and the like. The hybridomas KM4008 and KM4012 have been deposited to National Institute of Advanced Industrial Science and Technology under the Budapest Treaty, on May 1, 2008, as FERM BP-10962 and FERM BP-10963, respectively.
Further, the monoclonal antibody of the present invention also includes a monoclonal antibody that binds to an epitope to which an antibody produced from the above-mentioned hybridomas binds.
The recombinant antibody of the present invention includes an antibody produced by gene recombination, such as a human chimeric antibody, a humanized antibody, a human antibody and an antibody fragment thereof. Among the recombinant antibodies, one having character of a common monoclonal antibody, low immunogenicity and prolonged half-life in blood is preferable as a therapeutic agent.
Examples of the recombinant antibody include a recombinant antibody modified the above mentioned monoclonal antibody by using recombinant technologies.
Examples of the recombinant antibody of the present invention include a recombinant antibody in which Complementarity Determining Region (hereinafter referred to as CDR) 1, CDR2 and CDR3 of a heavy chain variable region (hereinafter, referred to as VH) of the antibody comprise the amino acid sequences represented by SEQ ID NOs:26, 27 and 28, respectively, and/or CDR1, CDR2 and CDR3 of a light chain variable region (hereinafter, referred to as VL) of the antibody comprise the amino acid sequences represented by SEQ ID NOs:29, 30 and 31, respectively; a recombinant antibody in which CDR1, CDR2 and CDR3 of VH of the antibody comprise the amino acid sequences represented by SEQ ID NOs:32, 33 and 34, respectively, and/or CDR1, CDR2 and CDR3 of VL of the antibody comprise the amino acid sequences represented by SEQ ID NOs:35, 36 and 37, respectively; a recombinant antibody in which CDR1, CDR2 and CDR3 of VH of the antibody comprise the amino acid sequences represented by SEQ ID NOs:49, 50 and 51, respectively, and/or CDR1, CDR2 and CDR3 of VL of the antibody comprise the amino acid sequences represented by SEQ ID NOs:52, 53 and 54, respectively; and the like.
The human chimeric antibody is an antibody comprising VH and VL of an antibody of a non-human animal, and a heavy chain constant region (hereinafter referred to as CH) and a light chain constant region (hereinafter referred to as CL) of a human antibody. Specifically, the human chimeric antibody of the present invention can be produced by obtaining cDNAs encoding VH and VL from a hybridoma which produces a monoclonal antibody which specifically recognizes a native three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region, inserting each of them into an expression vector for animal cell comprising DNAs encoding CH and CL of human antibody to thereby construct a vector for expression of human chimeric antibody, and then introducing the vector into an animal cell to express the antibody.
As the CH of the human chimeric antibody, any CH can be used, so long as it belongs to human immunoglobulin (hereinafter referred to as “hIg”), and those belonging to the hIgG class are preferred, and any one of the subclasses belonging to the hIgG class, such as hIgG1, hIgG2, hIgG3 and hIgG4, can be used. As the CL of the human chimeric antibody, any CL can be used, so long as it belongs to the hIg class, and those belonging to κ class or λ class can be used.
Examples of the human chimeric antibody of the present invention include a human chimeric antibody in which VH of the antibody comprises the amino acid sequences represented by SEQ ID NO:19, and VL of the antibody comprises the amino acid sequences represented by SEQ ID NO:21; a human chimeric antibody in which VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:23, and VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:25; a human chimeric antibody in which VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:46, and VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:48; and the like. Specific examples include human chimeric antibodies cKM4008, cKM4012, cKM4018 and the like.
A humanized antibody is an antibody in which amino acid sequences of CDRs of VH and VL of an antibody derived from a non-human animal are grafted into appropriate positions of VH and VL of a human antibody, and is also called a human CDR-grafted antibody, a reshaped-antibody or the like. The humanized antibody of the present invention can be produced by constructing cDNAs encoding an antibody variable region (hereinafter referred to as “V region”) in which the amino acid sequences of CDRs of VH and VL of an antibody derived from a non-human animal produced by a hybridoma which produces a monoclonal antibody which specifically recognizes three-dimensional structure of ASCT2 and binds to the extracellular region are grafted into frameworks (hereinafter referred to as “FR”) of VH and VL of a suitable human antibody, inserting each of them into a vector for expression of animal cell comprising genes encoding CH and CL of a human antibody to thereby construct a vector for expression of humanized antibody, and introducing it into an animal cell to thereby express and produce the humanized antibody.
As the amino acid sequences of FRs of VH and VL of a humanized antibody, any amino acid sequences can be used, so long as they are amino acid sequences of FRs of VH and VL, respectively, derived from a human antibody. Examples include amino acid sequences of FRs of VH and VL of human antibodies registered in database such as Protein Data Bank, common amino acid sequences of each sub group of FRs of VH and VL of human antibodies described in, for example, Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991), and the like.
As the CH of the humanized antibody, any CH can be used, so long as it belongs to the Mg class, and those of the hIgG class are preferred and any one of the subclasses belonging to the hIgG class, such as hIgG1, hIgG2, hIgG3 and hIgG4 can be used. As the CL of the humanized antibody, any CL can be used, so long as it belongs to the Mg class, and those belonging to the κ class or λ class can be used.
Specific examples of the humanized antibody of the present invention include a humanized antibody wherein VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:71, or an amino acid sequence in which at least one amino acid residue selected from Val at position 2, Ser at position 9, Val at position 20, Ser at position 30, Arg at position 38, Glu at position 46, Leu at position 86, Val at position 93, Tyr at position 95 and Val at position 116 in the amino acid sequence represented by SEQ ID NO:71 is substituted with other amino acid residue(s), and/or wherein VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:72, or an amino acid sequence in which at least one amino acid residue selected from Pro at position 8, Val at position 15, Gln at position 38, Ala at position 43, Pro at position 44, Phe at position 71 and Tyr at position 87 in the amino acid sequence represented by SEQ ID NO:72 is substituted with other amino acid residue(s); and the like. In this connection, the number of modifications which are introduced is not limited.
Examples of the amino acid sequence of VH of the antibody include preferably:
a humanized antibody comprising an amino acid sequence in which Ser at position 9, Val at position 20, Arg at position 38, Glu at position 46, Val at position 93, Tyr at position 95 and Val at position 116 in the amino acid sequence represented by SEQ ID NO:71 are substituted with other amino acid residues,
a humanized antibody comprising an amino acid sequence in which Val at position 20, Glu at position 46, Tyr at position 95 and Val at position 116 in amino acid sequence represented by SEQ ID NO:71 are substituted with other amino acid residues, or
a humanized antibody comprising an amino acid sequence in which Glu at position 46, and Tyr at position 95 in the amino acid sequence represented by SEQ ID NO:71 are substituted with other amino acid residues.
The amino acid sequence of VH of the antibody obtained by the above amino acid modifications may include, for example, an amino acid sequence in which at least one modification selected from amino acid modifications for substituting Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu is introduced in the amino acid sequence represented by SEQ ID NO:71.
Specific example of the amino acid sequence of VH in which ten modifications are introduced include an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced in the amino acid sequence represented by SEQ ID NO:71.
Specific examples of the amino acid sequence of VH in which nine modifications are introduced in the amino acid sequence represented by SEQ ID NO:71 include the following amino acid sequences:
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, and Val at position 116 with Leu are introduced, and
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced.
Specific examples of the amino acid sequence of VH in which eight modifications are introduced in the amino acid sequence represented by SEQ ID NO:71 include the following amino acid sequences:
an amino acid sequence in which substitutions of Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Val at position 20 with Ile, Ser at position 30 with Thr, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Val at position 20 Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Ser at position 30 with Thr, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Ser at position 30 with Thr, Arg at position 38 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Arg at position 38 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Glu at position 46 with Lys, Leu at position 86 with Val, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Leu at position 86 with Val, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Val at position 93 with Thr, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, and Tyr at position 95 with Phe are introduced, and
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, and Val at position 93 with Thr are introduced.
Specific examples of the amino acid sequence of VH in which seven modifications are introduced in the amino acid sequence represented by SEQ ID NO:71 include the following amino acid sequences:
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Glu at position 46 with Lys, Leu at position 86 with Val, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Val at position 20 with Ile, Ser at position 30 with Thr, Glu at position 46 with Lys, Leu at position 86 with Val, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Val at position 20 with Ile, Ser at position 30 with Thr, Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, Val at position 20 with Ile, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Glu at position 46 with Lys, Leu at position 86 with Val, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Ser at position 30 with Thr, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced, and the like.
Specific examples of the amino acid sequence of VH in which six modifications are introduced in the amino acid sequence represented by SEQ ID NO:71 include the following amino acid sequences:
an amino acid sequence in which substitutions of Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Arg at position 38 with Lys, Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Arg at position 38 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Val at position 93 with Thr, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced, and the like.
Specific examples of the amino acid sequence of VH in which five modifications are introduced in the amino acid sequence represented by SEQ ID NO:71 include the following amino acid sequences:
an amino acid sequence in which substitutions of Val at position 2 with Ile, Val at position 20 with Ile, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Ser at position 30 with Thr, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Glu at position 46 with Lys, Leu at position 86 with Val, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced, and the like.
Specific examples of the amino acid sequence of VH in which four modifications are introduced in the amino acid sequence represented by SEQ ID NO:71 include the following amino acid sequences:
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, Glu at position 46 with Lys, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Arg at position 38 with Lys, Glu at position 46 with Lys, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Glu at position 46 with Lys, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Arg at position 38 with Lys, Glu at position 46 with Lys, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Glu at position 46 with Lys, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Arg at position 38 with Lys, Glu at position 46 with Lys, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Arg at position 38 with Lys, Glu at position 46 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Glu at position 46 with Lys, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced, and the like.
Specific examples of the amino acid sequence of VH in which three modifications are introduced in the amino acid sequence represented by SEQ ID NO:71 include the following amino acid sequences:
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, and Arg at position 38 with Lys are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, and Glu at position 46 with Lys are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, and Val at position 93 with Thr are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 20 with Ile, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Arg at position 38 with Lys, and Glu at position 46 with Lys are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Arg at position 38 with Lys, and Val at position 93 with Thr are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Arg at position 38 with Lys, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Arg at position 38 with Lys, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Glu at position 46 with Lys, and Val at position 93 with Thr are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Glu at position 46 with Lys, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Glu at position 46 with Lys, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Val at position 93 with Thr, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Arg at position 38 with Lys, and Glu at position 46 with Lys are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Arg at position 38 with Lys, and Val at position 93 with Thr are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Arg at position 38 with Lys, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Arg at position 38 with Lys, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Glu at position 46 with Lys, and Val at position 93 with Thr are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Glu at position 46 with Lys, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Glu at position 46 with Lys, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Val at position 93 with Thr, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Arg at position 38 with Lys, Glu at position 46 with Lys, and Val at position 93 with Thr are introduced,
an amino acid sequence in which substitutions of Arg at position 38 with Lys, Glu at position 46 with Lys, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Arg at position 38 with Lys, Glu at position 46 with Lys, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Arg at position 38 with Lys, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Arg at position 38 with Lys, Val at position 93 with Thr, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Arg at position 38 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Glu at position 46 with Lys, Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Glu at position 46 with Lys, Val at position 93 with Thr, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Glu at position 46 with Lys, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced, and the like.
Specific examples of the amino acid sequence of VH in which two modifications are introduced in the amino acid sequence represented by SEQ ID NO:71 include the following amino acid sequences:
an amino acid sequence in which substitutions of Val at position 2 with Ile, and Ser at position 9 with Pro are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, and Val at position 20 with Ile are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, and Ser at position 30 with Thr are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, and Arg at position 38 with Lys are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, and Glu at position 46 with Lys are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, and Leu at position 86 with Val are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, and Val at position 93 with Thr are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 2 with Ile, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, and Val at position 20 with Ile are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, and Ser at position 30 with Thr are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, and Arg at position 38 with Lys are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, and Glu at position 46 with Lys are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, and Leu at position 86 with Val are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, and Val at position 93 with Thr are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Ser at position 9 with Pro, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, and Ser at position 30 with Thr are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, and Arg at position 38 with Lys are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, and Glu at position 46 with Lys are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, and Leu at position 86 with Val are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, and Val at position 93 with Thr are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 20 with Ile, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Ser at position 30 with Thr, and Arg at position 38 with Lys are introduced,
an amino acid sequence in which substitutions of Ser at position 30 with Thr, and Glu at position 46 with Lys are introduced,
an amino acid sequence in which substitutions of Ser at position 30 with Thr, and Leu at position 86 with Val are introduced,
an amino acid sequence in which substitutions of Ser at position 30 with Thr, and Val at position 93 with Thr are introduced,
an amino acid sequence in which substitutions of Ser at position 30 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Ser at position 30 with Thr, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Arg at position 38 with Lys, and Glu at position 46 with Lys are introduced,
an amino acid sequence in which substitutions of Arg at position 38 with Lys, and Leu at position 86 with Val are introduced,
an amino acid sequence in which substitutions of Arg at position 38 with Lys, and Val at position 93 with Thr are introduced,
an amino acid sequence in which substitutions of Arg at position 38 with Lys, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Arg at position 38 with Lys, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Glu at position 46 with Lys, and Leu at position 86 with Val are introduced,
an amino acid sequence in which substitutions of Glu at position 46 with Lys, and Val at position 93 with Thr are introduced,
an amino acid sequence in which substitutions of Glu at position 46 with Lys, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Glu at position 46 with Lys, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Leu at position 86 with Val, and Val at position 93 with Thr are introduced,
an amino acid sequence in which substitutions of Leu at position 86 with Val, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Leu at position 86 with Val, and Val at position 116 with Leu are introduced,
an amino acid sequence in which substitutions of Val at position 93 with Thr, and Tyr at position 95 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 93 with Thr, and Val at position 116 with Leu are introduced, and
an amino acid sequence in which substitutions of Tyr at position 95 with Phe, and Val at position 116 with Leu are introduced.
Specific examples of the amino acid sequence of VH in which one modification is introduced in the amino acid sequence represented by SEQ ID NO:71 include the following amino acid sequences:
an amino acid sequence in which a substitution of Val at position 2 with Ile is introduced,
an amino acid sequence in which a substitution of Ser at position 9 with Pro is introduced,
an amino acid sequence in which a substitution of Val at position 20 with Ile is introduced,
an amino acid sequence in which a substitution of Ser at position 30 with Thr is introduced,
an amino acid sequence in which a substitution of Arg at position 38 with Lys is introduced,
an amino acid sequence in which a substitution of Glu at position 46 with Lys is introduced,
an amino acid sequence in which a substitution of Leu at position 86 with Val is introduced,
an amino acid sequence in which a substitution of Val at position 93 with Thr is introduced,
an amino acid sequence in which a substitution of Tyr at position 95 with Phe is introduced, and
an amino acid sequence in which a substitution of Val at position 116 with Leu is introduced.
With regard to VL of the antibody, preferable examples include
a humanized antibody comprising an amino acid sequence in which Val at position 15, Ala at position 43, Pro at position 44, Phe at position 71, and Tyr at position 87 in the amino acid sequence represented by SEQ ID NO:72 are substituted with other amino acid residues,
a humanized antibody comprising an amino acid sequence in which Val at position 15, Phe at position 71 and Tyr at position 87 in amino acid sequence represented by SEQ ID NO:72 are substituted with other amino acid residues, and the like.
The amino acid sequence of VL of the antibody obtained by the above amino acid modifications include, for example, an amino acid sequence in which at least one modification selected from amino acid modifications for substituting Pro at position 8 with Thr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe is introduced in the amino acid sequence represented by SEQ ID NO:72.
Specific examples of the amino acid sequence of VL in which seven modifications are introduced include an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced in the amino acid sequence represented by SEQ ID NO:72.
Specific examples of the amino acid sequence of VL in which six modifications are introduced in the amino acid sequence represented by SEQ ID NO:72 include the following amino acid sequences:
an amino acid sequence in which substitutions of Val at position 15 with Leu, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Ala at position 43 with Thr, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Gln at position 38 with Arg, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala at position 43 with Thr, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro at position 44 with Val, and Tyr at position 87 with Phe are introduced, and
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro at position 44 with Val, and Phe at position 71 with Tyr are introduced.
Specific examples of the amino acid sequence of VL in which five modifications are introduced in the amino acid sequence represented by SEQ ID NO:72 include the following amino acid sequences:
an amino acid sequence in which substitutions of Gln at position 38 with Arg, Ala at position 43 with Thr, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, Ala at position 43 with Thr, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, Gln at position 38 with Arg, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, Gln at position 38 with Arg, Ala at position 43 with Thr, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro at position 44 with Val, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro at position 44 with Val, and Phe at position 71 with Tyr are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Ma at position 43 with Thr, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Gln at position 38 with Arg, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Gln at position 38 with Arg, Ala at position 43 with Thr, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro at position 44 with Val, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro at position 44 with Val, and Phe at position 71 with Tyr are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Ala at position 43 with Thr, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Ala at position 43 with Thr, Pro at position 44 with Val, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Ala at position 43 with Thr, Pro at position 44 with Val, and Phe at position 71 with Tyr are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Gln at position 38 with Arg, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Gln at position 38 with Arg, Pro at position 44 with Val, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Gln at position 38 with Arg, Pro at position 44 with Val, and Phe at position 71 with Tyr are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala at position 43 with Thr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala at position 43 with Thr, and Phe at position 71 with Tyr are introduced, and
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala at position 43 with Thr, and Pro at position 44 with Val are introduced.
Specific examples of the amino acid sequence of VL in which four modifications are introduced in the amino acid sequence represented by SEQ ID NO:72 include the following amino acid sequences:
an amino acid sequence in which substitutions of Val at position 15 with Leu, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, Ala at position 43 with Thr, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, Gln at position 38 with Arg, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced, and the like.
Specific examples of the amino acid sequence of VL in which three modifications are introduced in the amino acid sequence represented by SEQ ID NO:72 may include the following amino acid sequences:
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, and Phe at position 71 with Tyr are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Val at position 15 with Leu, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, Gln at position 38 with Arg, and Phe at position 71 with Tyr are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, Gln at position 38 with Arg, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, Ala at position 43 with Thr, and Phe at position 71 with Tyr are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, Ala at position 43 with Thr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, Pro at position 44 with Val, and Phe at position 71 with Tyr are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, Pro at position 44 with Val, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Gln at position 38 with Arg, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Ala at position 43 with Thr, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced, and the like.
Specific examples of the amino acid sequence of VL in which two modifications are introduced in the amino acid sequence represented by SEQ ID NO:72 include the following amino acid sequences:
an amino acid sequence in which substitutions of Pro at position 8 with Thr, and Val at position 15 with Leu are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, and Gln at position 38 with Arg are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, and Ala at position 43 with Thr are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, and Pro at position 44 with Val are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, and Phe at position 71 with Tyr are introduced,
an amino acid sequence in which substitutions of Pro at position 8 with Thr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, and Gln at position 38 with Arg are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, and Ala at position 43 with Thr are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, and Pro at position 44 with Val are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, and Phe at position 71 with Tyr are introduced,
an amino acid sequence in which substitutions of Val at position 15 with Leu, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Gln at position 38 with Arg, and Ala at position 43 with Thr are introduced,
an amino acid sequence in which substitutions of Gln at position 38 with Arg, and Pro at position 44 with Val are introduced,
an amino acid sequence in which substitutions of Gln at position 38 with Arg, and Phe at position 71 with Tyr are introduced,
an amino acid sequence in which substitutions of Gln at position 38 with Arg, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Ala at position 43 with Thr, and Pro at position 44 with Val are introduced,
an amino acid sequence in which substitutions of Ala at position 43 with Thr, and Phe at position 71 with Tyr are introduced,
an amino acid sequence in which substitutions of Ala at position 43 with Thr, and Tyr at position 87 with Phe are introduced,
an amino acid sequence in which substitutions of Pro at position 44 with Val, and Phe at position 71 with Tyr are introduced,
an amino acid sequence in which substitutions of Pro at position 44 with Val, and Tyr at position 87 with Phe are introduced, and
an amino acid sequence in which substitutions of Phe at position 71 with Tyr, and Tyr at position 87 with Phe are introduced.
Specific examples of the amino acid sequence of VL in which one modification is introduced in the amino acid sequence represented by SEQ ID NO:72 may include the following amino acid sequences:
an amino acid sequence in which a substitution of Pro at position 8 with Thr is introduced,
an amino acid sequence in which a substitution of Val at position 15 with Leu is introduced,
an amino acid sequence in which a substitution of Gln at position 38 with Arg is introduced,
an amino acid sequence in which a substitution of Ala at position 43 with Thr is introduced,
an amino acid sequence in which a substitution of Pro at position 44 with Val is introduced,
an amino acid sequence in which a substitution of Phe at position 71 with Tyr is introduced, and
an amino acid sequence in which a substitution of Tyr at position 87 with Phe is introduced.
In addition, specific examples of the humanized antibody of the present invention include a humanized antibody wherein VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:71 and/or VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:72, a humanized antibody wherein VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:76 and/or VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:72, a humanized antibody wherein VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:78 and/or VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:72, a humanized antibody wherein VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:80 and/or VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:72, a humanized antibody wherein VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:82 and/or VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:72, a humanized antibody wherein VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:71 and/or VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:84, a humanized antibody wherein VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:76 and/or VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:84, a humanized antibody wherein VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:78 and/or VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:84, a humanized antibody wherein VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:80 and/or VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:84, a humanized antibody wherein VH of the antibody comprises the amino acid sequence represented by SEQ ID NO:82 and/or VL of the antibody comprises the amino acid sequence represented by SEQ ID NO:84 and the like.
A human antibody is originally an antibody naturally existing in the human body, and it also includes an antibody obtained from a human antibody phage library or a human antibody-producing transgenic animal, which is prepared based on the recent advanced techniques in genetic engineering, cell engineering and developmental engineering.
The antibody existing in the human body can be prepared, for example by isolating a human peripheral blood lymphocyte, immortalizing it by infecting with EB virus or the like, and then cloning it to thereby obtain lymphocytes capable of producing the antibody, culturing the lymphocytes thus obtained, and purifying the antibody from the supernatant of the culture.
The human antibody phage library is a library in which antibody fragments such as Fab and scFv are expressed on the phage surface by inserting a gene encoding an antibody prepared from a human B cell into a phage gene. A phage expressing an antibody fragment having the desired antigen binding activity can be recovered from the library, using its activity to bind to an antigen-immobilized substrate as the index. The antibody fragment can be converted further into a human antibody molecule comprising two full H chains and two full L chains by genetic engineering techniques.
A human antibody-producing transgenic animal is an animal in which a human antibody gene is integrated into cells. Specifically, a human antibody-producing transgenic animal can be prepared by introducing a gene encoding a human antibody into a mouse ES cell, grafting the ES cell into an early stage embryo of other mouse and then developing it. A human antibody is prepared from the human antibody-producing transgenic non-human animal by obtaining a human antibody-producing hybridoma by a hybridoma preparation method usually carried out in non-human mammals, culturing the obtained hybridoma and forming and accumulating the human antibody in the supernatant of the culture.
In the amino acid sequence constituting the above antibody or antibody fragment, an antibody or antibody fragment thereof in which one or more amino acids are deleted, substituted, inserted or added, having activity similar to the above antibody or antibody fragment is also included in the antibody or antibody fragment of the present invention.
The number of amino acids which are deleted, substituted, inserted and/or added is one or more, and is not specifically limited, but it is within the range where deletion, substitution or addition is possible by known methods such as the site-directed mutagenesis described in Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory Press (1989); Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997); Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982); Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985); Proc. Natl. Acad. Sci. USA, 82, 488 (1985) or the like. For example, the number is 1 to dozens, preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5.
The expression “one or more amino acid residue(s) is/are deleted, substituted, inserted and/or added” in the amino acid sequence of the above antibody means the followings. That is, it means there is deletion, substitution, insertion or addition of one or plural amino acids at optional positions in the same sequence and in one or plural amino acid sequences. Also, the deletion, substitution, insertion or addition may occur at the same time and the amino acid which is substituted, inserted or added may be either a natural type or a non-natural type. The natural type amino acid includes L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.
Preferable examples of mutually substitutable amino acids are shown below. The amino acids in the same group are mutually substitutable.
Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine
Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid
Group C: asparagine, glutamine
Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid
Group E: proline, 3-hydroxyproline, 4-hydroxyproline
Group F: serine, threonine, homoserine
Group G: phenylalanine, tyrosine
The antibody fragment of the present invention includes Fab, F(ab′)2, Fab′, scFv, diabody, dsFv, a peptide comprising CDR and the like.
An Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen binding activity, in which about a half of the N-terminal side of H chain and the entire L chain, among fragments obtained by treating an IgG antibody molecule with a protease, papain (cleaved at an amino acid residue at position 224 of the H chain), are bound together through a disulfide bond. The Fab of the present invention can be produced by treating a monoclonal antibody which specifically recognizes three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region with papain. Also, the Fab can be produced by inserting DNA encoding Fab of the antibody into an expression vector for prokaryote or an expression vector for eukaryote, and introducing the vector into a prokaryote or eukaryote to express the Fab.
An F(ab′)2 is an antibody fragment having a molecular weight of about 100,000 and having antigen binding activity and comprising two Fab regions which are bound in the hinge position obtained by digesting the lower part of two disulfide bonds in the hinge region of IgG with an enzyme, pepsin. The F(ab′)2 of the present invention can be produced by treating a monoclonal antibody which specifically recognizes three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region with pepsin. Also, the F(ab′)2 can be produced by binding Fab′ described below via a thioether bond or a disulfide bond.
An Fab′ is an antibody fragment having a molecular weight of about 50,000 and antigen binding activity, which is obtained by cleaving a disulfide bond at the hinge region of the above F(ab′)2. The Fab′ of the present invention can be produced by F(ab′)2 which specifically recognizes three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region with a reducing agent, such as dithiothreitol. Also, the Fab′ can be produced by inserting DNA encoding Fab′ fragment of the antibody into an expression vector for prokaryote or an expression vector for eukaryote, and introducing the vector into a prokaryote or eukaryote to express the Fab′.
An scFv is a VH-P-VL or VL-P-VH polypeptide in which one chain VH and one chain VL are linked using an appropriate peptide linker (hereinafter referred to as “P”) and is an antibody fragment having antigen binding activity. The scFv of the present invention can be produced by obtaining cDNAs encoding VH and VL of a monoclonal antibody which specifically recognizes three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region, constructing DNA encoding scFv, inserting the DNA into an expression vector for prokaryote or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote to express the scFv.
A diabody is an antibody fragment wherein scFv is dimerized, is an antibody fragment having divalent antigen binding activity. In the divalent antigen binding activity, two antigens may be the same or different. The diabody of the present invention can be produced by obtaining cDNAs encoding VII and VL of a monoclonal antibody which specifically recognizes three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region, constructing DNA encoding scFv so that the length of the amino acid sequence of the peptide linker is 8 or less residues, inserting the DNA into an expression vector for prokaryote or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote to express the diabody.
A dsFv is obtained by binding polypeptides in which one amino acid residue of each of VH and VL is substituted with a cysteine residue via a disulfide bond between the cysteine residues. The amino acid residue to be substituted with a cysteine residue can be selected based on a three-dimensional structure estimation of the antibody in accordance with a known methods [Protein Engineering, 7, 697 (1994)]. The dsFv of the present invention can be produced by obtaining cDNAs encoding VH and VL of a monoclonal antibody which specifically recognizes three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region, constructing DNA encoding dsFv, inserting the DNA into an expression vector for prokaryote or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote to express the dsFv.
A peptide comprising CDR is constituted by including one or more regions of CDRs of VH or VL. Peptide comprising plural CDRs can be bound directly or via an appropriate peptide linker. The peptide comprising CDR of the present invention can be produced by constructing DNA encoding CDRs of VH and VL of a monoclonal antibody which specifically recognizes three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region, inserting the DNA into an expression vector for prokaryote or an expression vector for eukaryote, and then introducing the expression vector into a prokaryote or eukaryote to express the peptide. The peptide comprising CDR can also be produced by a chemical synthesis method such as Fmoc method or tBoc method.
The monoclonal antibody of the present invention includes an antibody conjugate in which a monoclonal antibody or an antibody fragment thereof which specifically recognizes a three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region is chemically or genetically bound to a radioisotope, an agent having a low molecular weight, an agent having a high molecular weight, a protein, a therapeutic antibody or the like.
The antibody conjugate of the present invention can be produced by chemically conjugating a radioisotope, an agent having a low molecular weight, an agent having a high molecular weight, a protein, a therapeutic antibody or the like to the N-terminal side or C-terminal side of an H chain or an L chain of the monoclonal antibody or the antibody fragment thereof, an appropriate substituent or side chain of the antibody or the antibody fragment, a sugar chain in the antibody or the antibody fragment or the like, which specifically recognizes a three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region in the present invention [Antibody Engineering Handbook, published by Chijin Shokan (1994)].
Also, the antibody conjugate can be genetically produced by linking a DNA encoding the monoclonal antibody or the antibody fragment thereof which specifically recognizes three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region in the present invention to other DNA encoding a protein or a therapeutic antibody to be conjugated, inserting the DNA into a vector for expression, and introducing the expression vector into an appropriate host cell.
The radioisotope includes 131I, 125I, 90Y, 64Cu, 199Tc, 77Lu, 211At and the like. The radioisotope can directly be conjugated with the antibody by Chloramine-T method or the like. Also, a substance chelating the radioisotope can be conjugated with the antibody. The chelating agent includes 1-isothiocyanatobenzyl-3-methyldiethylene-triaminepentaacetic acid (MX-DTPA) and the like.
The agent having a low molecular weight includes an anti-tumor agent such as an alkylating agent, a nitrosourea agent, a metabolism antagonist, an antibiotic substance, an alkaloid derived from a plant, a topoisomerase inhibitor, an agent for hormonotherapy, a hormone antagonist, an aromatase inhibitor, a P glycoprotein inhibitor, a platinum complex derivative, an M-phase inhibitor and a kinase inhibitor [Rinsho Syuyo-gaku (Clinical Oncology), Gan to Kagaguryoho-Sha (1996)], a steroid agent such as hydrocortisone and prednisone, a nonsteroidal agent such as aspirin and indomethacin, immune-regulating agent such as aurothiomalate, penicillamine, immuno-suppressing agent such as cyclophosphamide and azathioprine, anti-inflammatory agent such as anti-histamine agent, for example, chlorpheniramine maleate and clemastine [Ensho to Kouensho-Ryoho (Inflammation and Anti-inflammation Therapy), Ishiyaku Shuppann (1982)] and the like. Examples of the antitumor agent include amifostine (Ethyol), cisplatin, dacarbazine (DTIC), dactinomycin, mecloretamin (nitrogen mustard), streptozocin, cyclophosphamide, iphosphamide, carmustine (BCNU), lomustine (CCNU), doxorubicin (adriamycin), epirubicin, gemcitabine (Gemsal), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, fluorouracil, vinblastine, vincristine, bleomycin, daunomycin, peplomycin, estramustine, paclitaxel (Taxol), docetaxel (Taxotea), aldesleukin, asparaginase, busulfan, carboplatin, oxaliplatin, nedaplatin, cladribine, camptothecin, 10-hydroxy-7-ethylcamptothecin (SN38), floxuridine, fludarabine, hydroxyurea, iphosphamide, idarubicin, mesna, irinotecan (CPT-11), nogitecan, mitoxantrone, topotecan, leuprolide, megestrol, melfalan, mercaptopurine, hydroxycarbamide, plicamycin, mitotane, pegasparagase, pentostatin, pipobroman, streptozocin, tamoxifen, goserelin, leuprorelin, flutamide, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil, hydrocortisone, prednisolone, methylprednisolone, vindesine, nimustine, semustine, capecitabine, Tomudex, azacytidine, UFT, oxaliplatin, gefitinib (Iressa), imatinib (STI 571), elrotinib, FMS-like tyrosine kinase 3 (Flt3) inhibitor, vascular endothelial growth factor receptor (VEGFR) inhibitor, fibroblast growth factor receptor (FGFR) inhibitor, epidermal growth factor receptor (EGFR) inhibitor such as Iressa and Tarceva, radicicol, 17-allylamino-17-demethoxygeldanamycin, rapamycin, amsacrine, all-trans-retinoic acid, thalidomide, lenalidomide, anastrozole, fadrozole, letrozole, exemestane, gold thiomalate, D-penicillamine, bucillamine, azathioprine, mizoribine, cyclosporine, rapamycin, hydrocortisone, bexarotene (Targretin), tamoxifen, dexamethasone, progestin substances, estrogen substances, anastrozole (Arimidex), Leuplin, aspirin, indomethacin, celecoxib, azathioprine, penicillamine, gold thiomalate, chlorpheniramine maleate, chlorpheniramine, clemastine, tretinoin, bexarotene, arsenic, voltezomib, allopurinol, calicheamicin, ibritumomab tiuxetan, Targretin, ozogamine, clarithromycin, leucovorin, ifosfamide, ketoconazole, aminoglutethimide, suramin, methotrexate, maytansinoid and derivatives thereof.
The method for conjugating the agent having low molecular weight with the antibody includes a method in which the agent and an amino group of the antibody are conjugated through glutaraldehyde, a method in which an amino group of the agent and a carboxyl group of the antibody are conjugated through water-soluble carbodiimide, and the like.
The agent having a high molecular weight includes polyethylene glycol (hereinafter referred to as “PEG”), albumin, dextran, polyoxyethylene, styrene-maleic acid copolymer, polyvinylpyrrolidone, pyran copolymer, hydroxypropylmethacrylamide, and the like. By binding these compounds having a high molecular weight to an antibody or antibody fragment, the following effects are expected: (1) improvement of stability against various chemical, physical or biological factors, (2) remarkable prolongation of half life in blood, (3) disappearance of immunogenicity, suppression of antibody production, and the like [Bioconjugate Drug, Hirokawa Shoten (1993)]. For example, the method for binding. PEG to an antibody includes a method in which an antibody is allowed to react with a PEG-modifying reagent [Bioconjugate Drug, Hirokawa Shoten (1993)]. The PEG-modifying reagent includes a modifying agent of ε-amino group of lysine (Japanese Published Unexamined Patent Application No. 178926/86), a modifying agent of a carboxyl group of aspartic acid and glutamic acid (Japanese Published Unexamined Patent Application No. 23587/81), a modifying agent of a guanidino group of arginine (Japanese Published Unexamined Patent Application No. 117920/90) and the like.
The immunostimulator may be any natural products known as immunoadjuvants. Examples of an agent enhancing immunogen include β(1→3)glucan (lentinan, schizophyllan), α-galactosylceramide (KRN7000) and the like.
The protein includes a cytokine or a growth factor which activates a immunocompetent cell, such as NK cell, macrophage or neutrophil, a toxic protein, and the like.
Examples of the cytokine or the growth factor include interferon (hereinafter referred to as “INF”)-α, INF-β, INF-γ, interleukin (hereinafter referred to as “IL”)-2, IL-12, IL-15, IL-18, IL-21, IL-23, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), macrophage-colony stimulating factor (M-CSF) and the like. The toxic protein includes ricin, diphtheria toxin, ONTAK and the like, and also includes a toxic protein wherein mutation is introduced into a protein in order to control the toxicity.
The therapeutic antibody includes an antibody against an antigen in which apoptosis is induced by binding of the antibody, an antibody against an antigen participating in formation of pathologic state of tumor, an antibody which regulates immunological function and an antibody relating to angiogenesis in the pathologic part.
The antigen in which apoptosis is induced by binding of the antibody includes cluster of differentiation (hereinafter “CD”) 19, CD20, CD21, CD22, CD23, CD24, CD37, CD53, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD79b, CD80 (B7.1), CD81, CD82, CD83, CDw84, CD85, CD86 (B7.2), human leukocyte antigen (HLA)-Class II, epidermal growth factor receptor (EGFR) and the like.
The antigen participating in formation of pathologic state of tumor or the antigen for the antibody which regulates immunological function includes CD4, CD40, CD40 ligand, B7 family molecule (CD80, CD86, CD274, B7-DC, B7-H2, B7-H3, B7-H4), ligand of B7 family molecule (CD28, CTLA-4, ICOS, PD-1, BTLA), OX-40, OX-40 ligand, CD137, tumor necrosis factor (TNF) receptor family molecule (DR4, DR5, TNFR1, TNFR2), TNF-related apoptosis-inducing ligand receptor (TRAIL) family molecule, receptor family of TRAIL family molecule (TRAIL-R1, TRAIL-R2, TRAIL-R3, TRAIL-R4), receptor activator of nuclear factor kappa B ligand (RANK), RANK ligand, CD25, folic acid receptor 4, cytokine [IL-1α, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, transforming growth factor (TGF) β, TNFα, etc.], receptors of these cytokines, chemokine (SLC, ELC, I-309, TARC, MDC, CTACK, etc.) and receptors of these chemokines.
The antigen for the antibody which inhibits angiogenesis in the pathologic part includes vascular endothelial growth factor (VEGF), angiopoietin, fibroblast growth factor (FGF), EGF, platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), erythropoietin (EPO), TGFβ, IL-8, Ephilin, SDF-1, receptors thereof and the like.
A fusion antibody with a protein or therapeutic antibody can be produced by linking a cDNA encoding a monoclonal antibody or antibody fragment to a cDNA encoding the protein, constructing a DNA encoding the fusion antibody, inserting the DNA into an expression vector for prokaryote or eukaryote, and then introducing the expression vector into a prokaryote or eukaryote to express the fusion antibody.
In the case where the above antibody conjugate is used for the detection method, method for quantitative determination, detection reagent, reagent for quantitative determination or diagnostic agent in the present invention, examples of the agent to which a monoclonal antibody or an antibody fragment thereof which specifically recognizes a three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region is bound includes a label used in routine immunological detecting or measuring method. The label includes enzymes such as alkaline phosphatase, peroxidase and luciferase, luminescent materials such as acridinium ester and lophine, fluorescent materials such as fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (RITC), and the like.
Furthermore, the present invention includes a monoclonal antibody which inhibits intracellular uptake of amino acids by ASCT2, and an antibody fragment thereof.
Examples of the method of evaluating an inhibitory activity of the antibody or antibody fragment thereof of the present invention on intracellular uptake of amino acids by ASCT2, include a method in which an antibody or an antibody fragment thereof is allowed to react with a normal or cancer cell expressing ASCT2, and then is evaluated the inhibition of glutamine-dependent proliferation using a viable cell counting reagent, or the like [J. Surgical Research, 90, 149 (2000)], a method in which an antibody or an antibody fragment thereof is allowed to react with a normal or cancer cell expressing ASCT2, and then is evaluated the inhibition of uptake of amino acids such as radioactive material-labeled alanine using appropriate equipment such as scintillation counter [J. Biol. Chem., 271, 14883 (1996)], or the like.
In addition, the present invention includes a monoclonal antibody and an antibody fragment thereof having a cellular cytotoxicity such as a complement-dependent cytotoxicity (CDC) activity, or an antibody-dependent cellular cytotoxicity (ADCC) activity.
The CDC activity or ADCC activity of the antibody or the antibody fragment thereof of the present invention against the antigen-positive cell line can be evaluated by known assay methods [Cancer Immunol. Immunother, 36, 373 (1993)].
Furthermore, the present invention includes a monoclonal antibody having an apoptosis-inducing activity, and an antibody fragment thereof.
Moreover, the present invention includes a monoclonal antibody which does not bind to mouse ASCT2, but also binds to human ASCT2, and an antibody fragment thereof.
Further, the present invention includes a monoclonal antibody which binds to at least the EL2 region of human ASCT2, and an antibody fragment thereof, for example, a monoclonal antibody which binds to at least any one of amino acids at positions 154, 159 to 160, 163 to 171, 173 to 174, 177, 188, 204 to 205, 207, 210 to 212, and 214 to 223, in the amino acid sequence represented by SEQ ID NO:2, and an antibody fragment thereof.
The binding specificity of the antibody or antibody fragment thereof of the present invention can be evaluated by a known epitope analysis method, for example by measuring the binding activity to human/mouse chimeric ASCT2 in which an appropriate site is replaced with a sequence of mouse ASCT2, based on the amino acid sequence information.
Also, the present invention relates to a therapeutic agent for a disease relating to ASCT2 comprising the monoclonal antibody or antibody fragment which specifically recognizes three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region as an active ingredient.
The disease relating to ASCT2 is not limited, so long as it is a disease relating to a cell expressing ASCT2, such as cancer.
The cancer includes blood cancer, breast cancer, uterine cancer, colorectal cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, renal cancer, rectal cancer, thyroid cancer, uterine cervix cancer, small intestinal cancer, prostate cancer and pancreatic cancer. Preferable examples of the cancer include blood cancer, esophageal cancer, stomach cancer, colorectal cancer, liver cancer and prostate cancer. Examples of blood cancer include myeloid leukemia, lymphoid leukemia, multiple myeloma, Hodgkin's lymphoma and non-Hodgkin's lymphoma.
The therapeutic agent in the present invention includes a therapeutic agent comprising the above monoclonal antibody or antibody fragment of the present invention as an active ingredient.
The therapeutic agent comprising the antibody or antibody fragment thereof, or conjugate thereof of the present invention may comprise only the antibody or antibody fragment thereof, or conjugate thereof as an active ingredient. It is generally preferred that the therapeutic agent is prepared as a pharmaceutical preparation produced by an appropriate method well known in the technical field of pharmaceutics, and by mixing it with one or more pharmaceutically acceptable carriers.
It is preferred to administer the therapeutic agent by the route that is most effective for the treatment. Examples include oral administration and parenteral administration, such as buccal, tracheal, rectal, subcutaneous, intramuscular or intravenous administration and intravenous administration is preferred.
The therapeutic agent may be in the form of spray, capsules, tablets, powder, granules, syrup, emulsion, suppository, injection, ointment, tape, and the like.
Although the dose or the frequency of administration varies depending on the objective therapeutic effect, administration method, treating period, age, body weight and the like, it is usually 10 μg/kg to 8 mg/kg per day and per adult.
Further, the present invention relates to a method for immunologically detecting or measuring ASCT2, a reagent for immunologically detecting or measuring ASCT2, a method for immunologically detecting or measuring a cell expressing ASCT2, and a diagnostic agent for diagnosing a disease relating to ASCT2, comprising a monoclonal antibody which specifically recognizes a native three-dimensional structure of an extracellular region of ASCT2 and binds to the extracellular region, or an antibody fragment thereof as an active ingredient.
In the present invention, the method for detecting or measuring the amount of ASCT2 may be any known method. For example, it includes an immunological detecting or measuring method.
The immunological detecting or measuring method is a method in which an antibody amount or an antigen amount is detected or determined using a labeled antigen or antibody. Examples of the immunological detecting or measuring method include radioactive substance-labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, Western blotting method, physico-chemical method and the like.
The above disease relating to ASCT2 can be diagnosed by detecting or measuring a cell expressing ASCT2 by using the monoclinal antibody or antibody fragment of the present invention.
For the detection of the cell expressing ASCT2, known immunological detection methods can be used, and an immunoprecipitation method, a fluorescent cell staining method, an immune tissue staining method and the like are preferably used. Also, a fluorescent antibody staining method using FMAT 8100 HTS system (Applied Biosystem) and the like can be used.
In the present invention, the living body sample to be used for detecting or measuring ASCT2 is not particularly limited, so long as it has a possibility of containing a cell expressing ASCT2, such as tissue cells, blood, blood plasma, serum, pancreatic fluid, urine, fecal matter, tissue fluid or culture fluid.
The diagnostic agent containing the monoclonal antibody or antibody fragment thereof, or conjugate thereof may further contain a reagent for carrying out an antigen-antibody reaction or a reagent for detection of the reaction depending on the desired diagnostic method. The reagent for carrying out the antigen-antibody reaction includes a buffer, a salt, and the like. The reagent for detection includes a reagent generally used for the immunological detecting or measuring method, such as labeled secondary antibody which recognizes the monoclonal antibody, antibody fragment thereof or conjugates thereof and substrate corresponding to the labeling.
The present invention can provide a monoclonal antibody which specifically recognizes a native three-dimensional structure of an extracellular region of system ASC amino acid transporter 2 (hereinafter referred to as ASCT2) and binds to the extracellular region, or an antibody fragment thereof; a hybridoma which produces the antibody; a DNA which encodes the antibody; a vector which contains the DNA; a transformant obtainable by introducing the vector; a process for producing an antibody or an antibody fragment thereof using the hybridoma or the transformant; and a therapeutic agent using the antibody or the antibody fragment thereof, and a diagnostic agent using the antibody or the antibody fragment thereof.
A process for producing the antibody of the present invention, a method for treating the disease and a method for diagnosing the disease are specifically described below.
1. Preparation Method of Monoclonal Antibody
(1) Preparation of Antigen
ASCT2 or a cell expressing ASCT2 as an antigen can be obtained by introducing an expression vector comprising cDNA encoding a full length of ASCT2 or a partial length thereof is introduced into Escherichia coli, yeast, an insect cell, an animal cell or the like. In addition, ASCT2 can be purified from various human tumor cell lines, human tissue and the like which express a large amount of ASCT2. The tumor cell line and the tissue can be allowed to use as antigens. Furthermore, a synthetic peptide having a partial sequence of the ASCT2 can be prepared by a chemical synthesis method such as Fmoc method or tBoc method and used as an antigen.
ASCT2 used in the present invention can be produced, for example, by expressing a DNA encoding ASCT2 in a host cell using a method described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) or the like according to the following method.
Firstly, a recombinant vector is prepared by inserting a full length cDNA comprising the region encoding ASCT2 into downstream of a promoter of an appropriate expression vector. At this time, if necessary, a DNA fragment having an appropriate length containing a region encoding the polypeptide based on the full length cDNA, and the DNA fragment may be used instead of the above full length cDNA. Next, a transformant producing ASCT2 can be obtained by introducing the recombinant vector into a host cell suitable for the expression vector.
The expression vector may be any one, so long as it can replicate autonomously in the host cell to be used or it can be integrated into a chromosome comprising an appropriate promoter at such a position that the DNA encoding the polypeptide can be transcribed.
The host cell may be any one, so long as it can express the objective gene. Examples include a microorganism which belongs to the genera Escherichia, such as Escherichia coli, yeast, an insect cell, an animal cell and the like.
When a prokaryote such as Escherichia coli is used as the host cell, it is preferred that the recombinant vector used in the present invention is autonomously replicable in the prokaryote and comprising a promoter, a ribosome binding sequence, the DNA encoding ASCT2 and a transcription termination sequence. The recombinant vector is not necessary to have a transcription termination sequence, but a transcription termination sequence is preferably set just below the structural gene. The recombinant vector may further comprise a gene regulating the promoter.
Also, the above recombinant vector is preferably a plasmid in which the space between Shine-Dalgarno sequence, which is the ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 nucleotides).
Furthermore, the nucleotide sequence of the DNA encoding ASCT2 can be substituted with another base so as to be a suitable codon for expressing in a host cell, thereby improve the productivity of the objective ASCT2.
Any expression vector can be used, so long as it can perform in the host cell to be used. Examples of the expression vector includes pBTrp2, pBTac1, pBTac2 (all manufactured by Roche Diagnostics), pKK233-2 (manufactured by Pharmacia), pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN), pKYP10 (Japanese Published Unexamined Patent Application No. 110600/83), pKYP200 [Agricultural Biological Chemistry, 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)], pBluescript II SK(−) (manufactured by Stratagene), pTrs30 [prepared from Escherichia coli JM109/pTrS30 (FERM BP-5407)], pTrs32 [prepared from Escherichia coli JM109/pTrS32 (FERM BP-5408)], pGHA2 [prepared from Escherichia coli IGHA2 (FERM BP-400), Japanese Published Unexamined Patent Application No. 221091/85], pGKA2 [prepared from Escherichia coli IGKA2 (FERM BP-6798), Japanese Published Unexamined Patent Application No. 221091/85], pTerm2 (U.S. Pat. No. 4,686,191, U.S. Pat. No. 4,939,094, U.S. Pat. No. 5,160,735), pSupex, pUB110, pTP5, pC194, pEG400 [J. Bacteriol., 172, 2392 (1990)], pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen), pME18SFL3 and the like.
Any promoter can be used, so long as it can function in the host cell to be used. Examples include promoters derived from Escherichia coli, phage and the like, such as trp promoter (Ptrp), lac promoter, PL promoter, PR promoter and T7 promoter. Also, artificially designed and modified promoters, such as a promoter in which two Ptrp are linked in tandem, tac promoter, lacT7 promoter and letI promoter, can be used.
Examples of host cell include Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W3110, Escherichia colt NY49, Escherichia coli DH5α and the like.
Any introduction method of the recombinant vector can be used, so long as it is a method for introducing DNA into the host cell, and examples include a method using a calcium ion described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982) and Molecular & General Genetics, 168, 111 (1979) and the like.
When an animal cell is used as the host cell, any expression vector can be used, so long as it can function in the animal cell. Examples include pcDNAI, pcDM8 (manufactured by Funakoshi), pAGE107 [Japanese Published Unexamined Patent Application No. 22979/91; Cytotechnology, 3, 133 (1990)], pAS3-3 (Japanese Published Unexamined Patent Application No. 227075/90), pCDM8 [Nature, 329, 840, (1987)], pcDNAI/Amp (manufactured by Invitrogen), pcDNA3.1 (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochemistry, 101, 1307 (1987)], pAGE210, pME18SFL3, pKANTEX93 (WO 97/10354) and the like.
Any promoter can be used, so long as it can function in an animal cell. Examples include a promoter of immediate early (IE) gene of cytomegalovirus (CMV), SV40 early promoter, a promoter of retrovirus, a metallothionein promoter, a heat shock promoter, SRα promoter, Molony murine leukemia virus promoter or enhancer, and the like. Also, the enhancer of the IE gene of human CMV can be used together with the promoter.
The host cell includes human Namalwa cell, monkey COS cell, Chinese hamster ovary (CHO) cell, HST5637 (Japanese Published Unexamined Patent Application No. 299/88) and the like.
Any introduction method of the recombinant vector can be used, so long as it is a method for introducing DNA into an animal cell, and examples include electroporation [Cytotechnology, 3, 133 (1990)], the calcium phosphate method (Japanese Published Unexamined Patent Application No. 227075/90), the lipofection method [Proc. Natl. Acad. Sci. USA, M, 7413 (1987)], and the like.
ASCT2 can be produced by culturing the transformant derived from a microorganism, an animal cell or the like having a recombinant vector comprising the DNA encoding ASCT2 in a medium to form and accumulate ASCT2 in the culture, and recovering it from the culture. The method for culturing the transformant in the medium is carried out according to the usual method used in culturing of hosts.
When ASCT2 is expressed in a cell derived from eukaryote, ASCT2 to which sugars or sugar chains bind can be obtained.
When a microorganism transformed with a recombinant vector containing an inducible promoter is cultured, an inducer can be added to the medium, if necessary. For example, isopropyl-β-D-thiogalactopyranoside or the like can be added to the medium when a microorganism transformed with a recombinant vector using lac promoter is cultured; or indoleacrylic acid or the like can be added thereto when a microorganism transformed with a recombinant vector using trp promoter is cultured.
When a transformant obtained using an animal cell as the host cell is cultured, the medium includes generally used RPMI 1640 medium [The Journal of the American Medical Association, 199, 519 (1967)], Eagle's MEM medium [Science, 122, 501 (1952)], Dulbecco's modified MEM medium [Virology, 8, 396 (1959)] and 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)], Iscoove's modified Dulbecco's medium (IMDM), the media to which fetal calf serum, etc. is added, and the like. The culturing is carried out generally at a pH of 6 to 8 and 30 to 40° C. for 1 to 7 days in the presence of 5% CO2. If necessary, an antibiotic such as kanamycin or penicillin can be added to the medium during the culturing.
Regarding the expression method of the gene encoding ASCT2, in addition to direct expression, secretory production, fusion protein expression and the like can be carried out according to the method described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989).
The process for producing ASCT2 includes a method of intracellular expression in a host cell, a method of extracellular secretion from a host cell, a method of producing on a host cell membrane outer envelope, and the like. The appropriate method can be selected by changing the host cell used and the structure of the ASCT2 produced.
When the ASCT2 is produced in a host cell or on a host cell membrane outer envelope, ASCT2 can be positively secreted extracellularly in accordance with the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], the methods described in Japanese Published Unexamined Patent Application No. 336963/93 and WO 94/23021, and the like.
Also, the production amount of ASCT2 can be increased in accordance with the method described in Japanese Published Unexamined Patent Application No. 227075/90 utilizing a gene amplification system using a dihydrofolate reductase gene.
The resulting ASCT2 can be isolated and purified, for example, as follows.
When ASCT2 is intracellularly expressed in a dissolved state, the cells after culturing are recovered by centrifugation, suspended in an aqueous buffer and then disrupted using ultrasonicator, French press, Manton Gaulin homogenizer, dynomill or the like to obtain a cell-free extract. The cell-free extract is centrifuged to obtain a supernatant, and a purified preparation can be obtained by subjecting the supernatant to a general protein isolation and purification techniques such as solvent extraction; salting out with ammonium sulfate etc.; desalting; precipitation with an organic solvent; anion exchange chromatography using a resin such as diethylaminoethyl (DEAE)-sepharose, DIAION HPA-75 (manufactured by Mitsubishi Chemical); cation exchange chromatography using a resin such as S-Sepharose FF (manufactured by Pharmacia); hydrophobic chromatography using a resin such as butyl-Sepharose or phenyl-Sepharose; gel filtration using a molecular sieve; affinity chromatography; chromatofocusing; electrophoresis such as isoelectric focusing; and the like which may be used alone or in combination.
When ASCT2 is expressed intracellularly by forming an inclusion body, the cells are recovered, disrupted and centrifuged in the same manner, and the inclusion body of ASCT2 are recovered as a precipitation fraction. The recovered inclusion body of the protein is solubilized with a protein denaturing agent. The protein is made into a normal three-dimensional structure by diluting or dialyzing the solubilized solution, and then a purified preparation of ASCT2 is obtained by the same isolation purification method as above.
When ASCT2 or the derivative such as a glycosylated product is secreted extracellularly, ASCT2 or the derivative such as a glycosylated product can be recovered from the culture supernatant. That is, the culture is treated by a method such as centrifugation in the same manner as above to obtain a culture supernatant, a purified preparation of ASCT2 can be obtained from the culture supernatant by the same isolation purification method as above.
Also, ASCT2 used in the present invention can be produced by a chemical synthesis method, such as Fmoc method or tBoc method. Also, it can be chemically synthesized using a peptide synthesizer manufactured by Advanced ChemTech, Perkin-Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation, or the like.
(2) Immunization of Animal and Preparation of Antibody-Producing Cell for Fusion
A mouse, rat or hamster 3 to 20 weeks old is immunized with the antigen prepared in the above (1), and antibody-producing cells are collected from the spleen, lymph node or peripheral blood of the animal. Also, when the increase of a sufficient titer in the above animal is recognized due to low immunogenicity, a ASCT2 knockout mouse may by used as an animal to be immunized.
The immunization is carried out by administering the antigen to the animal through subcutaneous, intravenous or intraperitoneal injection together with an appropriate adjuvant (for example, complete Freund's adjuvant, combination of aluminum hydroxide gel with pertussis vaccine, or the like). When the antigen is a partial peptide, a conjugate is produced with a carrier protein such as BSA (bovine serum albumin), KLH (keyhole limpet hemocyanin) or the like, which is used as the antigen.
The administration of the antigen is carried out 5 to 10 times every one week or every two weeks after the first administration. On the 3rd to 7th day after each administration, a blood sample is collected from the fundus of the eye, the reactivity of the serum with the antigen is tested, for example, by enzyme immunoassay [Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988)] or the like. An animal showing a sufficient antibody titer in their sera against the antigen used for the immunization is used as the supply source of antibody-producing cells for fusion.
Three to seven days after final administration of the antigen, tissue containing the antibody-producing cells such as the spleen from the immunized animal is excised to collect the antibody-producing cells. When the spleen cells are used, the spleen is cut out and loosened, followed by centrifuged. Then, antibody-producing cells for fusion are obtained by removing erythrocytes.
(3) Preparation of Myeloma Cell
An established cell line obtained from mouse is used as myeloma cells. Examples include 8-azaguanine-resistant mouse (derived from BALB/c) myeloma cell line P3-X63Ag8-U1 (P3-U1) [Current Topics in Microbiology and Immunology, 18, 1 (1978)], P3-NS1/1-Ag41 (NS-1) [European J. Immunology, 6, 511 (1976)], SP2/0-Ag14 (SP-2) [Nature, 276, 269 (1978)], P3-X63-Ag8653 (653) [J. Immunology, 123, 1548 (1979)], P3-X63-Ag8 (X63) [Nature, 256, 495 (1975)] and the like.
The myeloma cells are subcultured in a normal medium [a medium in which glutamine, 2-mercaptoethanol, gentamicin, FBS and 8-azaguanine are added to RPMI-1640 medium] and they are subcultured in the normal medium 3 or 4 days before cell fusion to ensure the cell number of 2×107 or more on the day for fusion.
(4) Cell Fusion and Preparation of Hybridoma for Producing Monoclonal Antibody
The antibody-producing cells for fusion obtained by the above (2) and myeloma cells obtained by the above (3) were sufficiently washed with a minimum essential medium (MEM) or PBS (1.83 g of disodium hydrogen phosphate, 0.21 g of potassium dihydrogen phosphate, 7.65 g of sodium chloride, 1 liter of distilled water, pH 7.2) and mixed to give a ratio of the antibody-producing cells:the myeloma cells=5 to 10:1, followed by centrifugation. Then, the supernatant is discarded. The precipitated cell group is sufficiently loosened. After loosening the precipitated cell, the mixture of polyethylene glycol-1000 (PEG-1000), MEM and dimethylsulfoxide is added to the cell under stirring at 37° C. In addition, 1 to 2 mL of MEM medium is added several times every one or two minutes, and MEM is added to give a total amount of 50 mL. After centrifugation, the supernatant is discarded. After the cells are gently loosen, the cells are gently suspended in HAT medium [a medium in which hypoxanthine, thymidine and aminopterin is added to the normal medium]. The suspension is cultured in a 5% CO2 incubator for 7 to 14 days at 37° C.
After the culturing, a portion of the culture supernatant is sampled and a hybridoma which is reactive to an antigen containing ASCT2 and is not reactive to an antigen not containing ASCT2 is selected by binding assay as described below. Then, cloning is carried out twice by a limiting dilution method [Firstly, HT medium (HAT medium from which aminopterin is removed) is used, and secondly, the normal medium is used], and a hybridoma which shows a stably high antibody titer is selected as the monoclonal antibody-producing hybridoma.
(5) Preparation of Purified Monoclonal Antibody
The hybridoma cells producing a monoclonal antibody obtained by the above (4) are administered by intraperitoneal injection into 8- to 10-week-old mice or nude mice treated with 0.5 mL of pristane (2,6,10,14-tetramethylpentadecane (pristane) is intraperitoneally administered, followed by feeding for 2 weeks). The hybridoma develops ascites tumor in 10 to 21 days. The ascitic fluid is collected from the mice, centrifuged to remove solids, subjected to salting out with 40 to 50% ammonium sulfate and then precipitated by caprylic acid, passed through a DEAE-Sepharose column, a protein A column or a gel filtration column to collect an IgG or IgM fraction as a purified monoclonal antibody.
Furthermore, a monoclonal antibody-producing hybridoma obtained by the above (4) is cultured in RPMI1640 medium containing 10% FBS or the like and the supernatant is removed by centrifugation. The precipitated cells are suspended in Hybridoma SFM medium containing 5% DIGO GF21 and cultured for 3 to 7 days. The purified monoclonal antibody can be obtained by centrifusing the obtained cell suspension, followed by purifying the resulting supernatant with Protein A column or Protein G column to collect the IgG fractions.
The subclass of the antibody can be determined using a subclass typing kit by enzyme immunoassay. The amount of the protein can be determined by the Lowry method or from the absorbance at 280 nm.
(6) Selection of Monoclonal Antibody
Selection of monoclonal antibody is carried out by the following binding assay using an enzyme immunoassay method and inhibition assay of intracellular uptake of amino acids.
(6-a) Binding Assay
As the antigen, a gene-introduced cell or a recombinant protein obtained by introducing an expression vector containing a cDNA encoding ASCT2 obtained in (1) into Escherichia coli, yeast, an insect cell, an animal cell or the like, or a purified polypeptide or partial peptide obtained from a human tissue is used. When the antigen is a partial peptide, a conjugate is prepared with a carrier protein such as BSA or KLH and is used.
After making these antigens into a solid layer by dispensing in a 96-well plate, a substance to be tested such as serum, a culture supernatant of a hybridoma or a purified monoclonal antibody is dispensed therein as the primary antibody and allowed to react. After thoroughly washing with PBS, PBS-Tween, an anti-immunoglobulin antibody labeled with biotin, an enzyme, a chemiluminescent material, a radiation compound or the like is dispensed therein as the secondary antibody and allowed to react. After thoroughly washing with PBS-Tween, the reaction is carried out in response to the label of the secondary antibody to select a monoclonal antibody which specifically reacts with the antigen.
(6-b) Inhibition Assay of Intracellular Uptake of Amino Acids
As an assay cell, a gene-introduced cell in which the expression vector comprising cDNA encoding ASCT2 is introduced into a cell such as an animal cell obtained in the above (1), or a normal or cancer cell expressing ASCT2 can be used.
The activity of the monoclonal antibody or antibody fragment thereof of the present invention to inhibit intracellular uptake of amino acids through ASCT2 is evaluated using a method comprising reacting a monoclonal antibody or an antibody fragment thereof with a normal or cancer cell expressing ASCT2, and then evaluating the inhibition of glutamine-dependent proliferation using a viable cell counting reagent, or the like [J. Surgical Research, 90, 149 (2000)], a method comprising reacting a monoclonal antibody or an antibody fragment thereof with a normal or cancer cell expressing ASCT2 and then evaluating the inhibition of uptake of amino acids such as radioactive material-labeled alanine using appropriate equipment such as scintillation counter [J. Biol. Chem., 271, 14883 (1996)] or the like.
2. Preparation of Recombinant Antibody
As production examples of recombinant antibodies, processes for producing a human chimeric antibody and a humanized antibody are shown below.
(1) Construction of Vector for Expression of Recombinant Antibody
A vector for expression of recombinant antibody is an expression vector for animal cell into which DNAs encoding CH and CL of a human antibody have been inserted, and is constructed by cloning each of DNAs encoding CH and CL of a human antibody into an expression vector for animal cell.
The C region of a human antibody may be CH and CL of any human antibody. Examples include CH belonging to γ1 subclass, CL belonging to κ class, and the like. As the DNAs encoding CH and CL of a human antibody, the cDNA may be generally used and a chromosomal DNA comprising an exon and an intron can be also used. As the expression vector for animal cell, any expression vector can be used, so long as a gene encoding the C region of a human antibody can be inserted thereinto and expressed therein. Examples include pAGE107 [Cytotechnol., 3, 133 (1990)], pAGE103 [J. Biochem., 101, 1307 (1987)], pHSG274 [Gene, 27, 223 (1984)], pKCR [Proc. Natl. Acad. Sci. USA, 78, 1527 (1981)], pSG1bd2-4 [Cytotechnol., 4, 173 (1990)], pSE1UK1Sed1-3 [Cytotechnol., 13, 79 (1993)] and the like. Examples of a promoter and enhancer used for an expression vector for animal cell include an SV40 early promoter [J. Biochem., 101, 1307 (1987)], a Moloney mouse leukemia virus LTR [Biochem. Biophys. Res. Commun., 149, 960 (1987)], an immunoglobulin H chain promoter [Cell, 41, 479 (1985)] and enhancer [Cell, 33, 717 (1983)] and the like.
The vector for expression of recombinant antibody may be either of a type in which a gene encoding an antibody H chain and a gene encoding an antibody L chain exist on separate vectors or of a type in which both genes exist on the same vector (tandem type). In respect of easiness of construction of a vector for expression of recombinant antibody, easiness of introduction into animal cells, and balance between the expression amounts of antibody H and L chains in animal cells, a tandem type of the vector for expression of recombinant antibody is more preferred [J. Immunol. Methods, 167, 271 (1994)]. Examples of the tandem type of the vector for expression of recombinant antibody include pKANTEX93 (WO 97/10354), pEE18 [Hybridoma, 17, 559 (1998)], and the like.
(2) Obtaining of cDNA Encoding V Region of Antibody Derived from Non-Human Animal and Analysis of Amino Acid Sequence
cDNAs encoding VH and VL of non-human antibody and analysis of amino acid sequence are obtained as follows.
mRNA is extracted from hybridoma cells producing an antibody derived from a non-human animal to synthesize cDNA. The synthesized cDNA is cloned into a vector such as a phage or a plasmid, to prepare a cDNA library. Each of a recombinant phage or recombinant plasmid containing cDNA encoding VH or VL is isolated from the library using DNA encoding a part of the C region or V region of an mouse antibody as the probe. The full length of the nucleotide sequences of VH and VL of mouse antibody of interest on the recombinant phage or recombinant plasmid are determined, and the full length of the amino acid sequences of VH and VL are deduced from the nucleotide sequences, respectively.
Examples of the non-human animal for preparing a hybridoma cell which produces a non-human antibody include mouse, rat, hamster, rabbit, or the like. Any animals can be used so long as a hybridoma cell can be produced therefrom.
Examples of the method for preparing total RNA from a hybridoma cell include a guanidine thiocyanate-cesium trifluoroacetate method [Methods in Enzymol., 154, 3 (1987)] using a kit such as RNA easy kit (manufactured by Qiagen) and the like.
Examples of the method for preparing mRNA from total RNA include an oligo (dT) immobilized cellulose column method [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)], a method using a kit such as Oligo-dT30<Super>mRNA Purification Kit (manufactured by Takara Bio) and the like. Also, examples of a kit for preparing mRNA from a hybridoma cell include Fast Track mRNA Isolation Kit (manufactured by Invitrogen), Quick Prep mRNA Purification Kit (manufactured by Pharmacia) and the like.
Examples of the method for synthesizing cDNA and preparing a cDNA library include known methods [Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Lab. Press (1989); Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)]; a method using a kit such as Super Script Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured by GIBCO BRL), ZAP-cDNA Kit (manufactured by Stratagene), etc.; and the like.
The vector into which the synthesized cDNA using mRNA extracted from a hybridoma cell as the template is inserted for preparing a cDNA library may be any vector, so long as the cDNA can be inserted. Examples include ZAP Express [Strategies, 5, 58 (1992)], pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989)], λzapII (manufactured by Stratagene), λgt10 and λgt11 [DNA Cloning: A Practical Approach, I, 49 (1985)], Lambda BlueMid (manufactured by Clontech), λExCell and pT7T3 18U (manufactured by Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)], pUC18 [Gene, 33, 103 (1985)], and the like.
Any Escherichia coli for introducing the cDNA library constructed by a phage or plasmid vector may be used, so long as the cDNA library can be introduced, expressed and maintained. Examples include XL1-Blue MRF' [Strategies, 5, 81 (1992)], C600 [Genetics, 39, 440 (1954)], Y1088 and Y1090 [Science, 222: 778 (1983)], NM522 [J. Mol. Biol., 166, 1 (1983)], K802 [J. Mol. Biol., 16, 118 (1966)], JM105 [Gene, 38, 275 (1985)], and the like.
A colony hybridization or plaque hybridization method using an isotope- or fluorescence-labeled probe may be used for selecting cDNA clones encoding VH and VL of a non-human antibody or the like from the cDNA library [Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989)]. Also, the cDNAs encoding VH and VL can be prepared through polymerase chain reaction (hereinafter referred to as “PCR”; Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989); Current Protocols in Molecular Biology, Supplement 1, John Wiley & Sons (1987-1997)) by preparing primers and using cDNA prepared from mRNA or a cDNA library as the template.
The nucleotide sequence of the cDNA can be determined by digesting the cDNA selected with appropriate restriction enzymes and the like, cloning the fragments into a plasmid such as pBluescript SK(−) (manufactured by Stratagene), carrying out the reaction by a usually used nucleotide analyzing method such as analyzing the sequence using an automatic nucleotide sequence analyzer such as A.L.F. DNA sequencer (manufactured by Pharmacia) after the dideoxy method [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)].
Whether the obtained cDNAs encode the full amino acid sequences of VL and VL of the antibody containing a secretory signal sequence can be confirmed by estimating the full length of the amino acid sequences of VH and VL from the determined nucleotide sequence and comparing them with the full length of the amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. The length of the secretory signal sequence and N-terminal amino acid sequence can be deduced by comparing the full length of the amino acid sequences of VH and VL of the antibody comprising a secretory signal sequence with full length of the amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)], and the subgroup to which they belong can also be known. Furthermore, the amino acid sequence of each of CDRs of VH and VL can be found by comparing the obtained amino acid sequences with amino acid sequences of VH and VL of known antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)].
Moreover, the novelty of the full length of the amino acid sequence of VH and VL can be examined by carrying out a homology search with sequences in any database, for example, SWISS-PROT, PIR-Protein or the like using the obtained full length of the amino acid sequences of VH and VL, for example, according to the BLAST method [J. Mol. Biol., 215, 403 (1990)] or the like.
(3) Construction of Vector for Expression of Human Chimeric Antibody
cDNAs encoding VH and VL of antibody of non-human animal are respectively cloned in the upstream of genes encoding CH or CL of human antibody of vector for expression of recombinant antibody mentioned in the above (1) to thereby construct a vector for expression of human chimeric antibody. For example, in order to ligate cDNA comprising a nucleotide sequence of 3′-terminal of VH or VL of antibody of non-human animal and a nucleotide sequence of 5′-terminal of CH or CL of human antibody, each cDNA encoding VH and VL of antibody of non-human animal is prepared so as to encodes appropriate amino acids encoded by a nucleotide sequence of a linkage portion and designed to have an appropriate recognition sequence of a restriction enzyme. The obtained cDNAs encoding VH and VL of antibody are respectively cloned so that each of them is expressed in an appropriate form in the upstream of gene encoding CH or CL of human antibody of the vector for expression of humanized antibody mentioned in the above (1) to construct a vector for expression of human chimeric antibody.
In addition, cDNA encoding VH or VL of non-human animal is amplified by PCR using a synthetic DNA having a recognition sequence of an appropriate restriction enzyme at both ends and each of them is cloned to the vector for expression of recombinant antibody obtained in the above (1).
(4) Construction of cDNA Encoding V Region of Humanized Antibody
cDNAs encoding VH or VL of a humanized antibody can be obtained as follows. First, amino acid sequences of framework region (hereinafter referred to as “FR”) in VH or VL of a human antibody to which amino acid sequences of CDRs in VH or VL of an antibody derived from a non-human animal antibody are transplanted are respectively selected. Any amino acid sequences of FR of a human antibody can be used, so long as they are derived from human. Examples include amino acid sequences of FRs of human antibodies registered in database such as Protein Data Bank or the like, and amino acid sequences common to subgroups of FRs of human antibodies [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)], and the like. In order to inhibit the decrease in the binding activity of the antibody, amino acid sequences having high homology (at least 60% or more) with the amino acid sequence of FR in VH or VL of the original antibody is selected.
Then, amino acid sequences of CDRs of the original antibody are grafted to the selected amino acid sequence of FR in VH or VL of the human antibody, respectively, to design each amino acid sequence of VH or VL of a humanized antibody. The designed amino acid sequences are converted to DNA sequences by considering the frequency of codon usage found in nucleotide sequences of genes of antibodies [Sequence of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)], and the DNA sequence encoding the amino acid sequence of VH or VL of a humanized antibody is designed.
Based on the designed nucleotide sequences, several synthetic DNAs having a length of about 100 nucleotides are synthesized, and PCR is carried out using them. In this case, it is preferred that 6 synthetic DNAs per each of the H chain and the L chain are designed in view of the reaction efficiency of PCR and the lengths of DNAs which can be synthesized.
Furthermore, the cDNA encoding VH or VL of a humanized antibody can be easily cloned into the vector for expression of humanized antibody constructed in (1) by introducing the recognition sequence of an appropriate restriction enzyme to the 5′ terminal of the synthetic DNAs existing on the both ends.
After the PCR, an amplified product is cloned into a plasmid such as pBluescript SK (−) (manufactured by Stratagene) or the like, and the nucleotide sequence is determined according to a method similar to the method described in (2) to obtain a plasmid having a DNA sequence encoding the amino acid sequence of VH or VL of a desired humanized antibody.
(5) Modification of Amino Acid Sequence of V Region of Humanized Antibody
It is known that when a humanized antibody is produced by simply grafting only CDRs in VH and VL of an antibody derived from a non-human animal into FRs of VH and VL of a human antibody, its antigen binding activity is lower than that of the original antibody derived from a non-human animal [BIO/TECHNOLOGY, 9, 266 (1991)]. In humanized antibodies, among the amino acid sequences of FRs in VH and VL of a human antibody, an amino acid residue which directly relates to binding to an antigen, an amino acid residue which interacts with an amino acid residue in CDR, and an amino acid residue which maintains the three-dimensional structure of an antibody and indirectly relates to binding to an antigen is identified and modified to an amino acid residue which is found in the original non-humahumanized antibody to thereby increase the antigen binding activity which has been decreased.
In order to identify the amino acid residues relating to the antigen binding activity in FR, the three-dimensional structure of an antibody is constructed and analyzed by X-ray crystallography [J. Mol. Biol., 112, 535 (1977)], computer-modeling [Protein Engineering, 7, 1501 (1994)] or the like. In addition, various attempts must be currently be necessary, for example, several modified antibodies of each antibody are produced and the correlation between each of the modified antibodies and its antibody binding activity is examined.
The modification of the amino acid sequence of FR in VH and VL of a human antibody can be accomplished using various synthetic DNA for modification according to PCR as described in (4). With regard to the amplified product obtained by the PCR, the nucleotide sequence is determined according to the method as described in (2) so that whether the objective modification has been carried out is confirmed.
(6) Construction of Vector for Expression of Humanized Antibody
A vector for expression of humanized antibody can be constructed by cloning each cDNA encoding VH or VL of a constructed recombinant antibody into upstream of each gene encoding CH or CL of the human antibody in the vector for expression of recombinant antibody as described in (1).
For example, when recognizing sequences of an appropriate restriction enzymes are introduced to the 5′-terminal of synthetic DNAs positioned at both ends among synthetic DNAs used in the construction of VH or VL of the humanized antibody in (4) and (5), cloning can be carried out so that they are expressed in an appropriate form in the upstream of each gene encoding CH or CL of the human antibody in the vector for expression of humanized antibody as described in the above (1).
(7) Transient Expression of Recombinant Antibody
In order to efficiently evaluate the antigen binding activity of various humanized antibodies produced, the recombinant antibodies can be expressed transiently using the vector for expression of recombinant antibody as described in (3) and (6) or the modified expression vector thereof.
Any cell can be used as a host cell, so long as the host cell can express a recombinant antibody. Generally, COS-7 cell (ATCC CRL1651) is used [Methods in Nucleic Acids Res., CRC Press, 283 (1991)]. Examples of the method for introducing the expression vector into COS-7 cell include a DEAE-dextran method [Methods in Nucleic Acids Res., CRC Press, 283 (1991)], a lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], and the like.
After introduction of the expression vector, the expression amount and antigen binding activity of the recombinant antibody in the culture supernatant can be determined by the enzyme immunoassay [Monoclonal Antibodies—Principles and practice, Third edition, Academic Press (1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988), Monoclonal Antibody Experiment Manual, Kodansha Scientific (1987)] and the like.
(8) Obtaining Transformant which Stably Expresses Recombinant Antibody and Preparation of Recombinant Antibody
A transformant which stably expresses a recombinant antibody can be obtained by introducing the vector for expression of recombinant antibody described in (3) and (6) into an appropriate host cell.
Examples of the method for introducing the expression vector into a host cell include electroporation [Japanese Published Unexamined Patent Application No. 257891/90, Cytotechnology, 3, 133 (1990)] and the like.
As the host cell into which a vector for expression of a recombinant antibody is introduced, any cell can be used, so long as it is a host cell which can produce the recombinant antibody. Examples include mouse SP2/0-Ag14 cell (ATCC CRL1581), mouse P3X63-Ag8.653 cell (ATCC CRL1580), CHO cell in which a dihydrofolate reductase gene (hereinafter referred to as “dhfr”) is defective [Proc. Natl. Acad. Sci. USA., 77, 4216 (1980)], lection resistance-acquired Lec13 [Somatic Cell and Molecular genetics, 12, 55 (1986)], CHO cell in which α1,6-fucosyltransaferse gene is defected (WO 2005/35586, WO 02/31140), rat YB2/3HL.P2.G11.16Ag.20 cell (ATCC CRL1662), and the like.
In addition, host cells in which activity of a protein such as an enzyme relating to synthesis of an intracellular sugar nucleotide, GDP-fucose, a protein such as an enzyme relating to the modification of a sugar chain in which 1-position of fucose is bound to 6-position of N-acetylglucosamine in the reducing end through α-bond in a complex type N-glycoside-linked sugar chain, or a protein relating to transport of an intracellular sugar nucleotide, GDP-fucose, to the Golgi body are introduced is decreased or deleted, preferably CHO cell in which α-1,6-fucosyltransferase gene is defected as described in WO05/35586, WO02/31140 or the like, can also be used.
After introduction of the expression vector, transformants which express a recombinant antibody stably are selected by culturing in a medium for animal cell culture containing an agent such as G418 sulfate (hereinafter referred to as “G418”) or the like (Japanese Published Unexamined Patent Application No. 257891/90).
Examples of the medium for animal cell culture include RPMI1640 medium (manufactured by Invitrogen), GIT medium (manufactured by Nihon Pharmaceutical), EX-CELL301 medium (manufactured by JRH), IMDM medium (manufactured by Invitrogen), Hybridoma-SFM medium (manufactured by Invitrogen), media obtained by adding various additives such as fetal calf serum (hereinafter referred to as “FCS”) to these media, and the like. The recombinant antibody can be produced and accumulated in a culture supernatant by culturing the selected transformants in a medium. The expression amount and antigen binding activity of the recombinant antibody in the culture supernatant can be measured by ELISA or the like. Also, in the transformant, the expression amount of the recombinant antibody can be increased by using DHFR amplification system or the like according to the method disclosed in Japanese Published Unexamined Patent Application No. 257891/90.
The recombinant antibody can be purified from the culture supernatant of the transformant by using a protein A column [Monoclonal Antibodies—Principles and practice, Third edition, Academic Press (1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988)]. For example, the recombinant antibody can be purified by a combination of gel filtration, ion-exchange chromatography, ultrafiltration and the like. The molecular weight of the H chain or the L chain of the purified recombinant antibody or the antibody molecule as a whole is determined by polyacrylamide gel electrophoresis (hereinafter referred to as “SDS-PAGE”) [Nature, 227, 680 (1970)], Western blotting [Monoclonal Antibodies—Principles and practice, Third edition, Academic Press (1996), Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988)], and the like.
3. Activity Evaluation of the Monoclonal Antibody or Antibody Fragment
The activity of the purified monoclonal antibody or antibody fragment of the present invention can be evaluated in the following manner.
The binding activity to ASCT2-expressing cell is evaluated by the binding assay described in the above 1 (6a). Furthermore, it can be measured by fluorescent antibody technique [Cancer Immunol. Immunother., 36, 373 (1993)], a surface plasmon resonance method using such as BIAcore system or the like.
The inhibitory activity on intracellular uptake of amino acids by ASCT2 can be evaluated by the method described in the above 1-(6b).
In addition, CDC activity or ADCC activity against an antigen positive cell line is evaluated by a known method [Cancer Immunol. Immunother., 36, 373 (1993)].
4. Method for Treating Disease Using the Anti-ASCT2 Monoclonal Antibody or Antibody Fragment of the Present Invention
A monoclonal antibody which specifically recognizes a native three-dimensional structure of ASCT2 and binds to the extracellular region, or an antibody fragment thereof, of the present invention can be used for treating a disease relating to ASCT2.
The therapeutic agent comprising the monoclonal antibody or antibody fragment of the present invention or derivatives thereof may be only the antibody or antibody fragment or derivatives thereof as an active ingredient, and is preferably supplied as a pharmaceutical preparation produced by an appropriate method well known in the technical field of pharmaceutics, by mixing it with one or more pharmaceutically acceptable carriers.
Examples of a route of administration include oral administration and parenteral administration, such as buccal, tracheal, rectal, subcutaneous, intramuscular or intravenous administration. Examples of the dosage form includes sprays, capsules, tablets, powder, granules, syrups, emulsions, suppositories, injections, ointments, tapes and the like.
The pharmaceutical preparation suitable for oral administration includes emulsions, syrups, capsules, tablets, powders, granules and the like.
Liquid preparations such as emulsions and syrups can be produced using, as additives, water; sugars such as sucrose, sorbitol and fructose; glycols such as polyethylene glycol and propylene glycol; oils such as sesame oil, olive oil and soybean oil; antiseptics such as p-hydroxybenzoic acid esters; flavors such as strawberry flavor and peppermint; and the like.
Capsules, tablets, powders, granules and the like can be produced using, as additives, excipients such as lactose, glucose, sucrose and mannitol; disintegrating agents such as starch and sodium alginate; lubricants such as magnesium stearate and talc; binders such as polyvinyl alcohol, hydroxypropylcellulose and gelatin; surfactants such as fatty acid ester; plasticizers such as glycerin; and the like.
The pharmaceutical preparation suitable for parenteral administration includes injections, suppositories, sprays and the like.
Injections can be prepared using a carrier such as a salt solution, a glucose solution or a mixture of both thereof.
Suppositories can be prepared using a carrier such as cacao butter, hydrogenated fat or carboxylic acid.
Sprays can be prepared using the antibody or antibody fragment as such or using it together with a carrier which does not stimulate the buccal or airway mucous membrane of the patient and can facilitate absorption of the compound by dispersing it as fine particles. The carrier includes lactose, glycerol and the like. It is possible to produce pharmaceutical preparations such as aerosols and dry powders.
In addition, the components exemplified as additives for oral preparations can also be added to the parenteral preparations.
5. Method for Diagnosing Disease Using the Anti-ASCT2 Monoclonal Antibody or Antibody Fragment of the Present Invention
A disease relating to ASCT2 can be diagnosed by detecting or determining ASCT2 or a cell expressing ASCT2 using the monoclonal antibody or antibody fragment of the present invention.
A diagnosis of cancer, one of the diseases relating to ASCT2, can be carried out by, for example, the detection or measurement of ASCT2 as follows.
Firstly, on the living body samples collected from two or more of the living bodies of healthy persons, the expressed amount of ASCT2 in the living body samples of healthy persons is confirmed by carrying out detection or measurement of ASCT2 by the following immunological means using the monoclonal antibody or antibody fragment of the present invention or derivatives thereof. By examining the amount of ASCT2 also in the living body samples of the person to be tested in the same manner, the amount is compared with the amount in healthy persons. When the amount of the polypeptide in the person to be tested is increased in comparison with the healthy persons, it can be diagnosed that cancer is positive.
An immunological method is a method in which an antibody amount or an antigen amount is detected or determined using a labeled antigen or antibody. Examples of the immunological method include radioactive substance-labeled immunoantibody method, enzyme immunoassay, fluorescent immunoassay, luminescent immunoassay, Western blotting method, physico-chemical means and the like.
Examples of the radioactive substance-labeled immunoantibody method include a method, in which the antibody or antibody fragment of the present invention is allowed to react with an antigen or a cell expressing an antigen, then anti-immunoglobulin antibody subjected to radioactive labeling, a binding fragment thereof or the like is allowed to react therewith, followed by determination using a scintillation counter or the like.
Examples of the enzyme immunoassay include a method, in which the antibody or antibody fragment of the present invention is allowed to react with an antigen or a cell expressing an antigen or the like, then an anti-immunoglobulin antibody or an binding fragment thereof subjected to antibody labeling is allowed to react therewith and the colored pigment is measured by a spectrophotometer, and, for example, sandwich ELISA may be used. As a label used in the enzyme immunoassay, any known enzyme label [Enzyme Immunoassay, published by Igaku Shoin (1987)] can be used. Examples include alkaline phosphatase labeling, peroxidase labeling, luciferase labeling, biotin labeling and the like.
Sandwich ELISA is a method in which an antibody is bound to a solid phase, antigen to be detected or measured is trapped and another antibody is allowed to react with the trapped antigen. In the ELISA, two kinds of antibody which recognizes the antigen to be detected or measured or the antibody fragment thereof in which antigen recognizing site is different are prepared and one antibody or antibody fragments is previously adsorbed on a plate (such as a 96-well plate) and another antibody or antibody fragment is labeled with a fluorescent substance such as FITC, an enzyme such as peroxidase, or biotin. The plate to which the above antibody is adsorbed is allowed to react with the cell separated from living body or disrupted cell suspension thereof, tissue or disintegrated solution thereof, cultured cells, serum, pleural effusion, ascites, eye solution or the like, then allowed to react with labeled monoclonal antibody or antibody fragment and a detection reaction corresponding to the labeled substance is carried out. When an antigen concentration in the sample to be tested is measured by the method, antigen concentration in the sample to be tested can be calculated from a calibration curve prepared by a stepwise dilution of antigen of known concentration. As an antibody used for sandwich ELISA, any of a polyclonal antibody and a monoclonal antibody may be used or antibody fragments such as Fab, Fab′ and F(ab)2 may be used. As a combination of two kinds of antibodies used in sandwich ELISA, a combination of monoclonal antibodies or antibody fragments recognizing different epitopes may be used or a combination of polyclonal antibody with monoclonal antibody or antibody fragments may be used.
A fluorescent immunoassay includes a method described in the literatures [Monoclonal Antibodies—Principles and practice, Third Edition, Academic Press (1996); Manual for Monoclonal Antibody Experiments, Kodansha Scientific (1987)] and the like. As a label for the fluorescent immunoassay, any of known fluorescent labels (Fluorescent Immunoassay, Soft Science, (1983)) may be used as described already. Examples include FITC, RITC and the like.
The luminescent immunoassay can be carried out using the methods described in the literature [Bioluminescence and Chemical Luminescence, Rinsho Kensa, 42, Hirokawa Shoten (1998)] and the like. As a label used for luminescent immunoassay, any of known luminescent labels can be included. Examples include acridinium ester, lophine or the like may be used.
Western blotting is a method in which an antigen or a cell expressing an antigen is fractionated by SDS-polyacrylamide gel electrophoresis [Antibodies—A Laboratory Manual (Cold Spring Harbor Laboratory, 1988)], the gel is blotted onto PVDF membrane or nitrocellulose membrane, the membrane is allowed to react with antigen-recognizing antibody or antibody fragment, further allowed to react with an anti-mouse IgG antibody or antibody fragment which is labeled with a fluorescent substance such as FITC, an enzyme label such as peroxidase, a biotin labeling, or the like, and the label is visualized to confirm the reaction. An example thereof is described below. Cells or tissues in which a polypeptide having the amino acid sequence represented by SEQ ID NO:2 is expressed are dissolved in a solution and, under reducing conditions, 0.1 to 30 μg as a protein amount per lane is electrophoresed by an SDS-PAGE method. The electrophoresed protein is transferred to a PVDF membrane and allowed to react with PBS containing 1 to 10% of BSA (hereinafter referred to as “BSA-PBS”) at room temperature for 30 minutes for blocking. Here, the monoclonal antibody of the present invention is allowed to react therewith, washed with PBS containing 0.05 to 0.1% Tween 20 (hereinafter referred to as “Tween-PBS”) and allowed to react with goat anti-mouse IgG labeled with peroxidase at room temperature for 2 hours. It is washed with Tween-PBS and a band to which the monoclonal antibody is bound is detected using ECL Western Blotting Detection Reagents (manufactured by Amersham) or the like to thereby detect a polypeptide having the amino acid sequence represented by SEQ ID NO:2. As an antibody used for the detection in Western blotting, an antibody which can be bound to a polypeptide having no three-dimensional structure of a natural type is used.
The physicochemical method is specifically carried out by reacting ASCT2 as the antigen with the antibody or antibody fragment of the present invention to form an aggregate, and detecting this aggregate. Other examples of the physicochemical methods include a capillary method, a one-dimensional immunodiffusion method, an immunoturbidimetry, a latex immunoturbidimetry [Handbook of Clinical Test Methods, Kanehara Shuppan, (1988)] and the like. For example, in a latex immunodiffusion method, a carrier such as polystyrene latex having a particle size of about of 0.1 to 1 μm sensitized with antibody or antigen may be used and when an antigen-antibody reaction is carried out using the corresponding antigen or antibody, scattered light in the reaction solution increases while transmitted light decreases. When such a change is detected as absorbance or integral sphere turbidity, it is now possible to measure antigen concentration, etc. in the sample to be tested.
On the other hand, for the detection or measurement of the cell expressing ASCT2, known immunological detection methods can be used, and an immunoprecipitation method, a immuno cell staining method, an immune tissue staining method, a fluorescent antibody staining method and the like are preferably used.
An immunoprecipitation method is a method in which a cell expressing ASCT2 is allowed to react with the monoclonal antibody or antibody fragment of the present invention and then a carrier having specific binding ability to immunoglobulin such as protein G-Sepharose is added so that an antigen-antibody complex is precipitated. Also, the following method can be carried out. The monoclonal antibody or antibody fragment of the present invention is solid-phased on a 96-well plate for ELISA and then blocked with BSA-PBS. When the antibody is in a non-purified state such as a culture supernatant of hybridoma cell, anti-mouse immunoglobulin or rat immunoglobulin or protein A or G or the like is previously adsorbed on a 96-well plate for ELISA and blocked with BSA-PBS and a culture supernatant of hybridoma cell is dispensed thereto for binding. After BSA-PBS is discarded and the residue is sufficiently washed with PBS, reaction is carried out with a dissolved solution of cells or tissues expressing ASCT2. An immune precipitate is extracted from the well-washed plate with a sample buffer for SDS-PAGE and detected by the above-described Western blotting.
An immune cell staining method and an immune tissue staining method are methods where cells or tissues in which antigen is expressed are treated, if necessary, with a surfactant, methanol or the like to make an antibody easily permeate to the cells or tissues, then the monoclonal antibody of the present invention is allowed to react therewith, then further allowed to react with an anti-immunoglobulin antibody or binding fragment thereof subjected to fluorescent labeling such as FITC, enzyme labeling such as peroxidase or biotin labeling and the label is visualized and observed under a microscope Also, the detection can be carried out by a fluorescent antibody staining method [Monoclonal Antibodies—Principles and practice, Third Edition, Academic Press (1996), Manual for Experiments of Monoclonal Antibodies, Kodansha Scientific (1987)] in which cells are allowed to react with a fluorescence-labeled antibody and analyzed by a flow cytometer. Particularly, since the monoclonal antibody or antibody fragment of the present invention binds to an extracellular region of ASCT2, it can be preferably used for detection of a cell expressing the polypeptide maintaining a natural type three-dimensional structure by a fluorescent antibody staining method.
In addition, in the case of using FMAT8100HTS system (manufactured by Applied Biosystems) and the like among fluorescent antibody staining methods, the antigen quantity or antibody quantity can be measured without separating the formed antibody-antigen complex and the free antibody or antigen which is not concerned in the formation of the antibody-antigen complex.
The present invention is described below by Examples; however, the present invention is not limited to the following Examples.
(1) Construction of Xenografts with Subcutaneous Transplantation of a Cancer Cell Line into SCID Mice and Preparation of Tumor Mass
In accordance with the following procedure, human cancer cell lines were subcutaneously transplanted into SCID mice to thereby construct xenografts. A tumor mass was extracted and prepared from the resulting xenografts.
Cells derived from human pancreatic cancer cell lines [ASPC-1 (ATCC Accession No. CRL-1682), CaPan-1 (ATCC Accession No. HTB-79), PANC-1 (ATCC Accession No. CRL-1469)], and human colorectal cancer cell lines [Colo205 (Riken (Physicochemical Research Institute) Cell Bank No. RCB2127), HT-29 (ATCC Accession No. HTB-38), LS180 (ATCC Accession No. CCL-187), SW1116 (ATCC Accession No. CCL-233), and WiDr (ATCC Accession No. CCL-218)] were suspended in PBS to give a cell density of about 1×108 cells/mL. Into the ventral hypodermis of 4 Fox CHASE C.B-17/Icr-scidJcl mice per group (male, 5 weeks old, purchased from CLEA Japan), 100 μL/animal of each cell suspension was transplanted.
Each of the cells were suspended and subcultured in an RPMI 1640 medium (manufactured by Invitrogen) containing 10% inactivated fetal bovine serum (manufactured by Invitrogen), in a CO2 incubator at 37° C. to use for transplantation.
The resulting xenografts were named as xASPC1, xCaPan1, xPANC1, xColo205, xHT29, xLS180, xSW1116, and xWiDr, respectively.
After tumor transplantation, the diameter of tumor mass was measured day by day using vernier calipers. The animals in which the major axis of the tumor became about 1 cm were sequentially sacrificed by bleeding under anesthesia and then each tumor mass was excised. Each tumor mass was cut into 4 portions and quickly frozen using liquid nitrogen, followed by storage in a freezer at −80° C.
(2) Extraction of Total RNA and Purification of Poly A(+) RNA
From cell lines and tumor mass of xenografts prepared in the above (1), total RNA was extracted and poly A(+) RNA was purified in accordance with the following procedure.
As a cell line, blood cancer-derived cell line {KG-1 (ATCC Accession No. CCL-246), THP-1 (ATCC Accession No. TIB-202), HL-60 (ATCC Accession No. CCL-240) [all, acute myeloid leukemia (AML) derived cell line], CCRF-CEM (ATCC Accession No. CCL-119), CCRF-SB (ATCC Accession No. CCL-120), Jurkat (ATCC Accession No. TIB-152), HSB-2 (ATCC Accession No. CCL-120), HPB-ALL (Riken Cell Bank No.:RCB1935) [all, acute lymphatic leukemia (ALL) derived cell line], K-562 (ATCC Accession No. CCL-243), KU812 (ATCC Accession No. CRL-2099) [all, chronic myelocytic leukemia (CML) derived cell line], KMS-11 (HSRRB No. JCRB1179), ARH-77 (ATCC Accession No. CRL-1621), IM-9 (ATCC Accession No. CCL-159), RPMI8226 (HSRRB No. JCRB0034), U266B1 (ATCC Accession No. TIB-196), MC/CAR (ATCC Accession No. CRL-8083) [all, multiple myeloma (MM) derived cell line], HS-Sultan (ATCC Accession No. CRL-1484), Daudi (ATCC Accession No. CCL-213), Raji (ATCC Accession No. CCL-86), Ramos (ATCC Accession No. CRL-1596) [all, Burkitt's lymphoma (BL) derived cell line], U-937 (ATCC Accession No. CRL-1593.2), ML-1 (DSMZ No. ACC464) [all, histiocytic lymphoma (HS) derived cell line]}, lung cancer derived cell line [PC-14 (Riken Cell Bank No.:RCB0446), PC-7 (Immuno-Biological Laboratories Co., Ltd.; Product No.:37011), PC-9 (Immuno-Biological Laboratories Co., Ltd.; Product No. 37012), PC-1 (Immuno-Biological Laboratories Co., Ltd.; Product No. 37008) (all, non-small cell lung cancer), Lu-139 (Riken Cell Bank No.:RCB0469), NCI-H69 (ATCC Accession No. HTB-119), RERF-LC-MA (HSRRB No. JCRB0812), SBC-5 (HSRRB No. JCRB0819) (all, small-cell lung cancer)], gastric cancer derived cell line [Kato III (Riken Cell Bank No. RCB2088), MKN-74 (HSRRB No. JCRB0255), NUGC-4 (Riken Cell Bank No. RCB 1939), AZ-521 (HSRRB No. JCRB0061)], colorectal cancer derived cell line {Colo205 (Riken Cell Bank No. RCB2127), HT-29 (ATCC Accession No. HTB-38), LS174T[European Collection of Cell Cultures (ECACC) No. 87060401], LS180 (ATCC Accession No. CCL187), SW1116 (ATCC Accession No. CCL-233)}, pancreatic cancer derived cell line [ASPC-1 (ATCC Accession No. CRL-1682), BXPC-3 (ATCC Accession No. CRL-1687), CaPan-1 (ATCC Accession No. HTB-79)], malignant melanoma (melanoma) derived cell line [G-361 (ATCC Accession No. CRL-1424), HMV-1 (Riken Cell Bank No. RCB0004), SK-MEL-28 (ATCC Accession No. HTB-72)], and normal human lung fibroblast [MRC-5 (ATCC Accession No. CCL-171)] were used.
The extraction of total RNA from the cell lines was carried out as follows. In the case of adhesive cell lines, the medium was removed using an aspirator after culturing, washing with an appropriate volume of PBS was carried out and cells were recovered using a spatula made of silicone. They were sufficiently suspended and lysed by adding 1 mL of a TRIzol Reagent (manufactured by Invitrogen) per the cells corresponding to 10 cm2 of cultured area. In addition, the resulting cell lysate was passed through an 18-gauge injection needle 10 times to cleave the genomic DNA into pieces. In the case of floating cell lines, the cell culture was centrifuged at 1,500 rpm for 5 minutes using a refrigerated centrifuge (manufactured by Hitachi Koki, Himac CF15R, rotor: T11A21), the medium was removed by decantation and the cells were suspended in PBS. The cell suspension was centrifuged again at 1,500 rpm for 5 minutes using a refrigerated centrifuge (manufactured by Hitachi Koki, Himac CF15R, rotor: T11A21) and the cells were recovered. To 1×107 recovered cells, 1 mL of a TRIzol Reagent (manufactured by Invitrogen) was added and suspended sufficiently so as to lyse cells. The cell lysate was passed through an 18-gauge injection needle 10 times to cleave the genomic DNA into pieces.
Lysis of tumor mass of the xenografts prepared in (1) and extraction of total RNA were carried out as follows. Frozen tumor mass was poured into 10 mL of a TRIzol Reagent (manufactured by Invitrogen) and immediately lysed using a Polytron homogenizer (PT 2100, manufactured by Kinematica) at 30,000 rpm for 15 seconds to give cell lysates. Each of the cell lysates was centrifuged at 11,000 rpm for 10 minutes using a refrigerated centrifuge (manufactured by Hitachi Koki, Himac CF15R, rotor: T11A21) and each of the supernatants was transferred to a fresh tube carefully so as not to carry over the precipitate. To the supernatant, 2 mL of chloroform was added and vigorously stirred for 15 seconds and the mixture was allowed to stand at room temperature for 2 to 3 minutes and centrifuged at 3,000 rpm for 90 minutes at 4° C. using a refrigerated centrifuge (manufactured by Hitachi Koki, Himac CF7D2, rotor: RT3S3). Each of the supernatants was transferred to a fresh tube, and 5 mL of isopropanol was added thereto, followed by gentle mixing. The mixture was allowed to stand at room temperature for 10 minutes and centrifuged at 11,000 rpm for 10 minutes using a refrigerated centrifuge (manufactured by Hitachi Koki, Himac CF15R, rotor: T11A21) and, after removing the supernatant, 10 mL of a 75% aqueous ethanol solution was added thereto, followed by mixing and centrifugation at 11,000 rpm for 5 minutes using a refrigerated centrifuge (manufactured by Hitachi Koki, Himac CF15R, rotor: T11A21) to give a precipitate. The precipitate was dissolved in an appropriate volume of RNase-free water to prepare a total RNA sample. The concentration of the total RNA sample was measured by an absorption spectrophotometer and it was confirmed that the ratio of A260/A280 was 1.7 or more. When the ratio of A260/A280 was less than 1.7, further purification was carried out using an RNeasy kit (manufactured by Qiagen).
From 400 μg of the total RNA sample prepared as above, poly A(+) RNA was purified using a Micro Poly(A) Pure Kit (manufactured by Ambion) in accordance with the instructions attached thereto.
(3) Synthesis of cDNA
cDNA was synthesized from the poly A(+) RNA obtained in (2) or commercially available tissue-derived mRNA using a SuperScript First-Strand Synthesis System for RT-PCR (manufactured by Invitrogen).
As mRNA derived from human normal tissues (liver, lung, whole brain, heart, stomach, spleen, spinal cord, small intestine, skeletal muscle, uterus, respiratory tract, thyroid gland, thymus gland, testis, salivary gland, prostate gland, placenta, lymph node, pancreas, large intestine, blood, or kidney), commercially available mRNA (manufactured by Clontech) was used. As mRNA derived from the human clinical cancer tissues (lung cancer, gastric cancer, colorectal cancer, kidney cancer, liver cancer, uterine cancer, breast cancer, or esophageal cancer), commercially available RNA (manufactured by BioChain) was also used.
To 1 μg of poly A(+) RNA obtained in (2) or commercially available tissue-derived mRNA, 1 μL of 10 mmol/L dNTPs and 1 μL of a 0.5 μg/μL Oligo (dT)12-18 were added, and then DEPC water was added thereto to give a total volume of 7 μL. The reaction solution was heated to denaturation at 65° C. for 5 minutes, quenched on ice and allowed to stand for 1 minute or longer. To the mRNA solution, 10×RT buffer (2 μL), magnesium chloride (4 μL, 25 mmol/L), 0.1 mol/L DTT (2 μL) and RNase OUT Recombinant Ribonuclease Inhibitor (1 μL) were added and the temperature was kept at 42° C. for 2 minutes. SuperscriptII RT (1 μL) was further added thereto to carry out reverse transcription reaction at 42° C. for 50 minutes. The enzyme was inactivated by heating at 70° C. for 15 minutes. Then, 1 μL of E. coli RNase H was added to reaction solution and reacted at 37° C. for 20 minutes. DEPC water was added to the resulting solution to give a total volume of 1 mL.
Hereinafter, the reaction system of real-time PCR employed 10 μL of a five-fold dilution of the above-prepared solution.
(4) Quantitative Determination of Expression Levels of mRNA of the ASCT2 Gene in Cell Lines, Tumor Mass of Xenografts, and Normal Tissues by Real-Time PCR Method (Quantitative PCR Method, or Q-PCR Method)
To 10 μl, of each cDNA prepared in (3) (corresponding to 2 ng of poly A(+) RNA), a forward primer (Fw#1) for detecting a cDNA of the ASCT2 gene comprising the nucleotide sequence represented by SEQ ID NO:3 which was designed from SEQ ID NO:1, and a reverse primer (Rv#1) for detecting a cDNA of the ASCT2 gene comprising the nucleotide sequence represented by SEQ ID NO:4 (all manufactured by Proligo) were added to give a final concentration of 300 nmol/L for each of them. Furthermore, to the resulting solution, 10×R-PCR buffer (Mg2+-free, manufactured by Takara Bio, 2 μL), 250 mmol/L Mg2+ solution (0.2 μL), dNTPs (10 mmol/L, 0.6 μL), Ex Taq R-PCR (manufactured by Takara Bio, 0.2 μL) and SYBR Green I (manufactured by BMA; original solution product was diluted 2,500-fold, 1 μL) were added. DEPC water was added thereto to give a total volume of 20 μL.
PCR was carried out under the following reaction conditions: initially activation of Taq DNA polymerase and denaturation of a template DNA at 94° C. for 5 minutes, and then 45 cycles each consisting of three processes, denaturation at 94° C. for 30 seconds, annealing at 65° C. for 30 seconds, and DNA elongation at 72° C. for 30 seconds. The fluorescence intensity generated by SYBR Green I intercalated to the amplified product was measured by PRISM 7700 (manufactured by Applied Biosystems) and data were analyzed according to the software, Sequence Detector ver. 1.7a, attached to the instrument PRISM 7700. Whether the signal obtained by the above real-time PCR was the desired amplified fragment was judged by the size of the major amplified fragment obtained by subjecting the reaction solution after completion of the reaction to agarose gel electrophoresis. The above real-time PCR was carried out using a 96-well PCR plate. Besides the above cDNA-containing reaction solution, a negative control (sterile water) and a sample for the preparation of a calibration curve (1×10 to 1×106 copies/well) prepared using a plasmid HCHON2001712 (Homo sapiens cDNA FLJ43232 fis, The DNA Data Bank of Japan (DDBJ) Accession No. AK125222) encoding a partial fragment of ASCT2 purified by a Qiagen Plasmid Midi Kit (manufactured by Qiagen) were arranged as a sample in each well of the PCR plate and PCR was then carried out.
FIGS. 1(1) to (4) show the alignment of a full-length sequence of NM—005628 (SEQ ID NO:1, GenBank Accession No. NM—005628) which is a standard sequence of ASCT2 and the alignment of a full-length sequence of plasmid HCHON2001712 which is used as a template for the preparation of a calibration curve. There are different sites between full-length sequences of NM—005628 and HCHON2001712. Therefore, both of the forward primer (Fw#1) for detecting a cDNA comprising the nucleotide sequence represented by SEQ ID NO:3 and the reverse primer (Rv#1) for detecting a cDNA comprising the nucleotide sequence represented by SEQ ID NO:4 which were used for the measurement of an ASCT2 expression level were designed using the region in which sequences of NM—005628 and HCHON2001712 were completely identical therebetween.
Results of the expression level of mRNA of the ASCT2 gene in each of the thus obtained cell lines, xenograft tumor mass and normal tissues were shown in FIGS. 2(1) to (2). The mRNA expression level was shown as a relative ratio in terms of numbers of expressed ASCT2 molecules per 2 ng of poly A(+) RNA and the ASCT2 gene-expression level in the airway showing the highest expression in normal tissues was defined as 1. As compared with a normal airway, enhanced expression levels were recognized, that is, 10-fold or higher enhanced expression for blood cancer-derived cell lines KG-1 (AML-derived cell line), HSB-2 (ALL-derived cell line), K-562 (CML-derived cell line), and KMS-11 and ARH-77 (both are MM-derived cell lines), 5-fold or higher enhanced expression for Jurkat (ALL-derived cell line), IM-9, RPMI 8226 (both are MM-derived cell lines), HS-Sultan (BL-derived cell line), and ML-1 (HL-derived cell line), and 2-fold or higher enhanced expression for THP-1 (AML-derived cell line), CCRF-CEM (ALL-derived cell line), KU812 (CML-derived cell line), Raji (BL-derived cell line), U-937 (HS-derived cell line), gastric cancer tissues, and esophageal cancer tissues.
In accordance with the following procedure, a plasmid pCR4-SLC1A5-myc/His was obtained comprising the nucleotide sequence represented by SEQ ID NO:5 and the amino acid sequence represented by SEQ ID NO:6. Using this plasmid, a CHO cell line into which the human ASCT2-myc/His gene was introduced was obtained.
To the respective primers (10 μmol/L, each 1 μL) comprising each of the nucleotide sequences of SEQ ID NOs:7 to 10, 10×Ex Taq buffer (10 μL), dNTPs (2 mmol/L, 10 μL), and Ex Taq polymerase (1 μL, all manufactured by Takara Bio) were added, and sterile water was further added thereto to give a total volume of 100 μL. In a manner similar to the procedure described in Example 1(3), PCR was carried out under the following reaction conditions: reaction at 96° C. for 2 minutes, and then 35 cycles each consisting of three processes, reaction at 96° C. for 1 minute, reaction at 60° C. for 1 minute, and reaction at 72° C. for 1 minute. As a result, the nucleotide sequence (hereinafter, referred to as “N-SLC1A5”) was synthesized corresponding to the nucleotide sequence at positions 1 to 370 when the nucleotide at a translation initiation point of the human ASCT2 gene was defined as position 1. The reaction product was separated by agarose gel electrophoresis. The resulting about 0.4-kb amplified fragment was extracted using a QIAquick Gel Extraction Kit (manufactured by Qiagen), and then cloned into a pCR4TOPO vector using a TOPO TA cloning kit (manufactured by Invitrogen) (hereinafter, the resulting plasmid is referred to as “pCR4-N-SLC1A5”).
Next, to 10 ng of the plasmid HCHON2001712 (DDBJ Accession No. AK125222) comprising the human ASCT2 gene as a template, the respective primers (10 μmol/L, each 1 μL) comprising the nucleotide sequences represented by SEQ ID NOs:11 and 12, 10×Ex Taq buffer (10 μL), dNTPs (2 mmol/L, 10 μL), and Ex Taq polymerase (1 μL, all manufactured by Takara Bio) were added, and sterile water was further added thereto to give a total volume of 100 μL. Under the same reaction conditions as described above, PCR was carried out to synthesize a nucleotide sequence (hereinafter, referred to as “C-SLC1A5-myc/His”) encoding a fusion protein having an addition of myc/His to the C-terminal of a nucleotide sequence corresponding to the nucleotide sequence of the human ASCT2 gene at positions 365 to 1623. The reaction product was separated by agarose gel electrophoresis. The resulting about 1.2-kb amplified fragment was extracted using a QIAquick Gel Extraction Kit (manufactured by Qiagen). To the resulting extraction fragment as a template, the primers (10 μmol/L, each 1 μL) comprising the nucleotide sequences represented by SEQ ID NOs:11 and 13, 10×Ex Taq buffer (10 μL), dNTPs (2 mmol/L, 10 μL), and Ex Taq polymerase (1 μL, all manufactured by Takara Bio) were added, and sterile water was further added thereto to give a total volume of 100 μL. Under the same reaction conditions as described above, PCR was carried out to add NotI and SpeI restriction enzyme sites to the C-terminal of C-SLC1A5-myc/His. The resulting reaction product was separated by agarose gel electrophoresis. The resulting about 1.2-kb amplified fragment was extracted using a QIAquick Gel Extraction Kit (manufactured by Qiagen), and then cloned into a pCR4 TOPO vector using a TOPO TA cloning kit (manufactured by Invitrogen) (hereinafter, the resulting plasmid is referred to as “pCR4-C-SLC1A5-myc/His”). The resulting gene sequence exhibited no amino acid variation even though there was a substitution of T at position 1455 with C when a nucleotide at which a translation initiation point of the sequence represented by SEQ ID NO:1 was defined as position 1.
The resulting pCR4-N-SLC1A5 was digested with BssHII (manufactured by Takara Bio) and Spa (manufactured by Takara Bio) and separated by agarose gel electrophoresis. The resulting about 4.4-kb gene fragment was extracted using a QIAquick Gel Extraction Kit (manufactured by Qiagen). Similarly, the resulting pCR4-C-SLC1A5-myc/His was digested with BssHII (manufactured by Takara Bio) and SpeI (manufactured by Takara Bio), and an about 1.4-kb gene fragment was extracted. Each extraction fragments were ligated using a Ligation high (manufactured by Toyobo), and then Escherichia coli DH5α (manufactured by Toyobo) was transformed with the resulting vector, in accordance with the method of Cohen et al [Proc, Natl, Acad-Sci. USA. 69, 2110 (1972)]. A plasmid was extracted from the resulting transformant using an automated plasmid isolation system PI-50 (manufactured by Kurabo), and a plasmid pCR4-SLC1A5 myc/His comprising the nucleotide sequence represented by SEQ ID NO:5 and the amino acid sequence represented by SEQ ID NO:6 was obtained.
The resulting pCR4-SLC1A5 myc/His was digested with EcoRI (manufactured by Takara Bio) and KpnI (manufactured by Takara Bio), and in a manner similar to the above, a gene fragment was extracted to obtain a fragment comprising a nucleotide sequence (hereinafter, referred to as “SLC1A5-myc/His”) encoding a fusion protein in which myc/His was added to the C-terminal of the human ASCT2 gene.
The resulting fragment containing SLC1A5-myc/His was ligated into a pKANTEX93 vector (WO97/10354) which had been previously digested with EcoRI (manufactured by Takara Bio) and KpnI (manufactured by Takara Bio). In a manner similar to the above, Escherichia coli DH5α (manufactured by Toyobo) was transformed with the ligation product. After obtaining a transformant, an expression plasmid pKANTEX-SLC1A5-myc/His was obtained using a plasmid isolation kit (manufactured by Qiagen).
In accordance with electroporation [Cytotechnology, 3, 133 (1990)], introduction of pKANTEX-SLC1A5-myc/His into a CHO/DG44 cell [Somatic Cell and Molecular Genetics. 12, 555 (1986)] was carried out in the following manner. The cells used herein were those subcultured in a medium where 1×HT supplement (manufactured by Invitrogen) was added to IMDM (manufactured by Invitrogen) containing 10% FBS (manufactured by Life Technologies) and gentamicin (50 μg/mL, manufactured by Nacalai Tesque) (hereinafter, referred to as “A3 medium”). CHO/DG44 cells were suspended in buffer containing potassium chloride (137 nmol/L), sodium chloride (2.7 nmol/L), disodium hydrogen phosphate (8.1 mmol/L), sodium dihydrogen phosphate (1.5 nmol/L) and magnesium chloride (4 mmol/L) (hereinafter, referred to as “K-PBS”) to give a cell density of 8×106 cells/mL, and the resulting cell suspension (200 μL, 1.6×106 cells in terms of cell count) was mixed with an expression plasmid pKANTEX-SLC1A5-myc/His (10 μg). The mixture was transferred into a cuvette (interelectrode distance: 2 mm), and gene introduction was carried out using a GenePulser II (manufactured by Bio-Rad) at a pulse voltage of 0.35 kV and an electric capacity of 250 μF. The cuvette was allowed to stand on ice, and a cell suspension in the cuvette was suspended in a cell culture vessel containing an A3 medium and cultured in a 5% CO2 incubator at 37° C. Four days after the culturing, the culture medium was exchanged with an A3 medium containing G418 (manufactured by Nacalai Tesque; 0.5 mg/mL), followed by cell culture. With medium exchange and subculturing during the cell culture, a transformed cell resistant to G418 was obtained about two weeks after the gene introduction.
The resulting G418-resistant transformed cells were diluted to give a cell density of 5 cells/10 mL in an A3 medium containing 0.5 mg/L of G418 (manufactured by Nacalai Tesque), and 100 μL/well of the diluted cell suspension was dispensed into a 96-well plate, followed by growing in stepwise increasing concentrations of methotrexate. In this manner, a clone showing a high expression level of ASCT2-myc/His was selected, from which a CHO cell line with the introduction of human ASCT2-myc/His gene was then obtained.
The resulting human ASCT2-myc/His gene-introduced CHO cells (1×105 to 5×105 cells) were suspended in 70% ethanol-PBS (1 mL) and fixed at ice temperature for 30 minutes. After dispensing at 1×106 to 5×106 cells/well into a 96-well U-bottom plate and subsequent centrifugation at 1,500 rpm for 5 minutes, the supernatant was discarded, followed by blocking with 1% BSA-PBS at ice temperature for 30 minutes. After removing the supernatant by centrifugation, an anti-myc antibody PL14 (manufactured by Medical & Biological Laboratories), an anti-His antibody (manufactured by Qiagen) and a mouse IgG1 isotype control (manufactured by Dako), as the primary antibodies, were diluted with 1% BSA-PBS to give final concentrations of 1.0, 0.1 and 0.1 μg/mL, respectively, and dispensed at 100 μL/well to react at ice temperature for 60 minutes. After the plate was washed once with 1% BSA-PBS, 100 μL/well of an FITC-labeled anti-mouse immunoglobulin G (H+L) (manufactured by DAKO) diluted 50-fold with 1% BSA-PBS, as the secondary antibody, was added and react at ice temperature for 30 minutes under shading. After washing once again with 1% BSA-PBS, the cells were suspended in PBS and the fluorescence intensity was measured by a flow cytometer (Cytomics FC500MPL, manufactured by Beckman Coulter). The results were shown in
(1) Preparation of Immunogen
For binding to a carrier protein, an N-terminal partial peptide of the human ASCT2 gene represented by SEQ ID NO:14 (amino acid residues at positions 2 to 16 from the N-terminal) in which Cys was added to the C-terminal was synthesized using an automated synthesizer (PSSM-8, manufactured by Shimadzu).
In order to enhance the immunogenity, a conjugate with KLH (manufactured by Wako Pure Chemical) was prepared as follows and used as an immunogen. That is, KLH was dissolved in PBS to give a concentration of 10 mg/mL and a 1/10 volume of N-(m-maleimide benzoyloxy)succinimide (MBS, manufactured by Nacalai Tesque, 25 mg/mL) was then added thereto dropwise, followed by reaction under stirring for 30 minutes. The reaction solution was passed through a gel filtration column (Sephadex G-25 column, manufactured by GE Healthcare) which had been previously equilibrated with PBS, and non-reacted MBS was removed to obtain KLH-MBS. The N-terminal partial peptide of human ASCT2 to which Cys was added (1 mg) was dissolved in sodium phosphate buffer (0.1 mol/L, pH 7.0) and 2.5 mg of KLH-MBS was added thereto, followed by reaction under stirring at room temperature for 3 hours. After the reaction was complete, the reaction solution was dialyzed against PBS, thereby obtaining a human ASCT2 N-terminal peptide-KLH conjugate as an immunogen.
(2) Immunization of Animals and Preparation of Antibody-Producing Cell
The human ASCT2 N-terminal peptide-KLH conjugate (100 μg) obtained in the above (1) together with 2 mg of aluminum gel and 1×109 cells of a pertussis vaccine (manufactured by Chiba Serum Institute) was administered to 4-week old female SD rats (manufactured by Japan SLC). Two weeks after the first administration, the conjugate (100 μg) was additionally administered to the rats once a week, four times in total. Blood was collected from caudal veins of the rats, and the reactivity thereof with the human ASCT2 partial peptide was investigated by the following enzyme immunoassay. Three days after the final immunization, the spleen was excised from the rat which showed sufficient antibody titer.
The spleen was minced into small pieces in MEM (manufactured by Nissui Pharmaceutical), loosened with forceps, and centrifuged at 1,200 rpm for 5 minutes (CR5B, manufactured by Hitachi). To the obtained precipitated fraction, Tris-ammonium chloride buffer (pH 7.65) was added and reacted for 1 to 2 minutes, whereby red blood cells were removed. The cell fraction obtained as a precipitation fraction was washed three times with MEM, thereby preparing antibody-producing cells.
(3) Enzyme Immunoassay
As an assay antigen, the Cys-added N-terminal partial peptide of human ASCT2 presented by SEQ ID NO:14 was prepared in the form of a conjugate with thyroglobulin (hereinafter, referred to as “THY”), according to the following procedure. The construction method of the conjugate was the same as in (1), but succinimidyl 4-[N-maleimide methyl]-cyclohexane-1-carboxylate (SMCC, manufactured by Sigma) was used instead of MBS.
The resulting human ASCT2 N-terminal peptide-THY conjugate (10 μg/mL, at 50 μL/well) was dispensed into a 96-well EIA plate (manufactured by Greiner), and allowed to stand at 4° C. overnight for adsorption. After the non-adsorbed conjugate was washed, 1% BSA-PBS (100 μL/well) was added to the plate, followed by reaction at room temperature for 1 hour, and the remaining active groups were blocked. After the non-reacted BSA-PBS was washed, 50 μL/well of the test material such as antiserum or culture supernatant, as the primary antibody, was dispensed to the plate, followed by reaction for 2 hours. The plate was washed with 0.05% Tween-PBS and 50 μL/well of a diluted peroxidase-labeled anti-rat immunoglobulin (manufactured by Dako) as the secondary antibody was added to the plate, followed by reaction at room temperature for 1 hour. After the plate was washed with 0.05% Tween-PBS, a 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)ammonium (ABTS) substrate solution [ABTS (manufactured by Wako Pure Chemical, 1 mmol/L), citrate buffer (0.1 mol/L, pH 4.2), and H2O2 (0.1%)] was added to the well and then color-developed. An absorbance (OD415-OD490) at a sample wavelength of 415 nm and then a reference wavelength of 490 nm was measured using a plate reader (Emax microplate reader, Molecular Devices).
(4) Preparation of Mouse Myeloma Cell
The 8-azaguanine-resistant mouse myeloma cell line P3-U1 [P3X63Ag8U.1, ATCC Accession No. CRL-1597, European Journal of Immunology, 6, 511 (1976)] was cultured in a medium (hereinafter referred to as “normal medium”) where glutamine (1.5 mmol/L), 2-mercaptoethanol (5×10−5 mol/L), gentamicin (10 μg/mL) and FBS (10%) were added to an RPMI 1640 medium (manufactured by Invitrogen) to ensure the cell count of 2×107 or more necessary for cell fusion, and was provided as parent cells in the cell fusion.
(5) Preparation of Hybridoma
The antibody-producing cells obtained in (2) and the myeloma cells obtained in (4) were mixed at a ratio of 10:1 and the obtained cells were centrifuged at 1,200 rpm for 5 minutes (CR5B, manufactured by Hitachi). The supernatant was discarded and the precipitated cells were well loosened. A mixture of PEG 1000 (1 g), MEM (manufactured by Invitrogen, 1 mL) and dimethyl sulfoxide (0.35 mL) was added thereto at 0.5 mL/1×108 antibody-producing cells and at 37° C. under stirring, and MEM (1 mL) was added several times to the suspension every 1 to 2 minutes and then MEM was added to give a total volume of 50 mL. After centrifugation at 900 rpm for 5 minutes (CR5B, manufactured by Hitachi), the supernatant was discarded and the precipitated cells were slowly loosened. Then, the cells were suspended in 100 mL of a normal medium to which HAT Media Supplement (manufactured by Invitrogen) was added (hereinafter, referred to as “HAT medium”) by gently pipetting up and down.
Into a 96-well culture plate, 200 μL/well of the obtained suspension was dispensed and cultured in a 5% CO2 incubator for 10 to 14 days at 37° C.
After culturing, the culture supernatant was examined by the enzyme immunoassay described in (3), the wells which specifically reacted to the N-terminal partial peptide of human ASCT2 were selected. The cells contained in the selected wells were subjected to cloning by a limiting dilution method twice to give a hybridoma KM3842 which produces a monoclonal antibody against the N-terminal partial peptide of ASCT2. The antibody subclass of KM3842 was determined to be rat IgG2a by using a subclass typing kit (manufactured by ABT Sarotech) (
(6) Obtaining of Purified Monoclonal Antibody
The hybridoma (5×106 to 20×106 cells/animal) obtained in (5) was intraperitoneally injected into pristane-treated 8-week-old female nude mice (Balb/c, manufactured by Japan SLC). After 10 to 21 days, the hybridoma became to ascites carcinoma. The ascitic fluid (1 to 8 mL/animal) was collected from the mice which had produced ascites. Then, the ascites was centrifuged at 3,000 rpm for 5 minutes (CR5B, manufactured by Hitachi) to remove solids. The resulting solution was purified by the caprylic acid precipitation method [Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988)] to obtain a purified KM3842 antibody.
The reactivity of a monoclonal antibody KM3842 with the N-terminal partial peptide of ASCT2 was examined in accordance with the enzyme immunoassay described in Example 3(3). The purified antibody KM3842 obtained in Example 3(6) was diluted with 1% BSA-PBS to give concentrations of 10, 1, 0.1, 0.01, 0.001, and 0.0001 μg/mL, respectively, and used as a primary antibody. As a result of the measurement, as shown in
(1) Preparation of Immunogen
The human ASCT2-myc/His gene-introduced CHO cell line obtained in Example 2 was cultured in IMDM (manufactured by Invitrogen) containing 10% FBS for 2 to 3 days and was peeled off using a 0.02% ethylenediaminetetraacetic acid (EDTA) solution (manufactured by Nacalai Tesque). The cells were suspended in PBS to give a cell count of 6×106 to 1×107 cells per one immunized animal.
(2) Immunization of Animal and Preparation of Antibody-Producing Cell
The cell suspension obtained in (1) was administered to 6-week old male BXSB mice (n=3/group, manufactured by Japan SLC) or 4-week old female SD rats (n=3/group, manufactured by Japan SLC) together with 1×109 cells of a pertussis vaccine (manufactured by Chiba Serum Institute). One week after the administration, the cell suspension was administered to the animals once a week, four times in total. Thereafter, blood was collected from the fundus oculi of mice or caudal veins of rats. The antibody titer thereof in the blood was measured by a fluorescent cell staining method using the following cell-based assay system (ABI 8200 Cellular Detection System, manufactured by Applied Biosystems) or flow cytometer (Cytomics FC500 MPL, manufactured by Beckman Coulter). Three days after the final immunization, the spleen was excised from a mouse or rat which showed sufficient antibody titer.
In the same manner as Example 3(2), the antibody-producing cells were prepared from the obtained spleen.
(3) Fluorescent Cell Staining Method—1 (Cell-Based Assay System)
The ASCT2-myc/His gene-introduced CHO cell line obtained in Example 2 and the vector-introduced CHO cells were used as assay cells. Each cell were cultured in IMDM (manufactured by Invitrogen) containing 10% FBS for 2 to 3 days and peeled off with a trypsin-EDTA solution (manufactured by Invitrogen) was suspended in the same medium, seeded into an ABI8200 black 96-well plate at a cell density of 1×104 cells/100 μL medium/well, and cultured overnight. The test substance such as antiserum or culture supernatant (10 μL/well) was dispensed to the plate as the primary antibody, 100 μL/well of ALEXA647-labeled anti-mouse immunoglobulin G (H+L) or ALEXA647-labeled anti-rat immunoglobulin G (H+L) (all manufactured by Invitrogen) was then added as the secondary antibody, followed by allowing to stand for 4 hours under shading. Fluorescence of 650 to 685 nm excited with a laser (633 nm He/Ne) was measured by the ABI 8200 Cellular Detection System (manufactured by Applied Biosystems).
(4) Fluorescent Cell Staining Method—2 (Flow Cytometer)
The human ASCT2-myc/His gene-introduced CHO cell line obtained in Example 2 and vector-introduced CHO cells were used as assay cells. Each cells which were cultured in IMDM (manufactured by Invitrogen) containing 10% FBS for 2 to 3 days and peeled off with a 0.02% EDTA solution (manufactured by Nacalai Tesque) were washed with PBS and were blocked for 20 minutes at ice temperature using 1% BSA-PBS in order to avoid the non-specific adsorption of antibodies. The resulting cells were seeded into a 96-well U-bottom plate at cell density of 5×105 cells/50 μL/well, followed by centrifugation at 1,800 rpm for 2 minutes (05PR-22, manufactured by Hitachi Koki), and then the supernatant was removed. The test substance (50 μL/well) such as antiserum or culture supernatant was dispensed as the primary antibody, followed by reaction at ice temperature for 30 minutes. Washing was carried out 3 times by a centrifugation method using PBS and 50 μL/well of ALEXA 488-labeled anti-mouse immunoglobulin G (G+L) or ALEXA488-labeled anti-rat immunoglobulin G (G+L) (all manufactured by Invitrogen) was added as the secondary antibody, followed by reaction at ice temperature for 30 minutes under shading. After the cells were washed again using PBS three times by centrifugation, the cells were suspended in PBS, and the fluorescence of 510 to 530 nm excited with a 488 nm Ar laser was measured by a flow cytometer (Cytomics FC500 MPL, manufactured by Beckman Coulter).
(5) Construction of Hybridoma
In the same manner as Example 3(5), the cell fusion was carried out between the antibody-producing cell obtained in (2) and the myeloma cell obtained in Example 3(4).
Next, the cells obtained from the cell fusion were suspended in an HAT medium. Into a 96-well culture plate, 200 μL/well of the suspension of the resulting cell was dispensed and the cells were cultured in a 5% CO2 incubator for 8 to 10 days at 37° C.
The reactivity of the post-culture supernatant was confirmed by the fluorescent cell staining method described in the above (3) and (4) and the well which reacts with the human ASCT2-myc/His gene-introduced CHO cell line and does not react with the vector-introduced CHO cells was selected. Then, the cells contained in the selected wells were subjected to cloning by a limiting dilution method twice to give hybridomas KM3998, KM4000, KM4001, KM4008, KM4012, and KM4018 which produce monoclonal antibodies against the extracellular region of ASCT2.
The determination of the subclass of a mouse monoclonal antibody among the obtained hybridomas was carried out in accordance with the following procedure.
Anti-mouse immunoglobulin rabbit polyclonal antibodies (manufactured by Dako, 10 μg/mL, 50 μL/well) was dispensed into a 96-well EIA plate (manufactured by Greiner) and allowed to stand at 4° C. overnight for adsorption. After the non-absorbed conjugate was washed, 100 μL/well of 1% BSA-PBS was added to the plate, followed by reaction at room temperature for 1 hour in order to block the remaining active groups. After the non-reacted BSA-PBS was washed, 50 μL/well of the test material was dispensed to the plate, followed by reaction for 2 hours. The plate was washed with 0.05% Tween-PBS and a diluted subclass-specific peroxidase-labeled anti-mouse immunoglobulin (manufactured by Invitrogen; 50 μL/well) was added to the plate as the secondary antibody, followed by reaction at room temperature for 1 hour. The plate was washed with 0.05% Tween-PBS and color-developed by adding the ABTS substrate solution used in Example 3(3). Then, an absorbance (OD415-OD490) at a sample wavelength of 415 nm and a reference wavelength of 490 nm was measured using a plate reader (Emax microplate reader, Molecular Devices). The subclass of the mouse monoclonal antibody whose subclass could not be determined by the above-mentioned method was determined using a mouse rat monoclonal isotyping kit (manufactured by Dainippon Sumitomo Pharma). Further, the subclass of the rat monoclonal antibody was determined using a rat monoclonal isotyping kit (manufactured by Dainippon Sumitomo Pharma).
(6) Obtaining of Purified Monoclonal Antibodies—1
Purified antibodies of KM3998 were obtained in accordance with the following procedure.
The hybridoma (5×106 to 20×106 cells/animal) obtained in (5) was intraperitoneally injected into pristane-treated 8-week-old female nude mice (Balb/c, manufactured by Japan SLC). The ascitic fluid (1 to 8 mL/animal) was collected from the mice when the hybridoma developed ascites tumor in 10 to 21 days. Then, the ascites was subjected to filtration [5 μm, polyethersulfone (PES) membrane, manufactured by Pall] to remove solids, followed by purification with the caprylic acid precipitation method [Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory (1988)].
(7) Obtaining of Purified Monoclonal Antibodies—2
Each of the purified antibodies of KM4000, KM4001, KM4008, KM4012 and KM4018 were obtained in accordance with the following procedure.
The individual hybridomas obtained in the above (5) were cultured in RPMI 1640 containing 10% FBS in a 5% CO2 incubator at 37° C. At the point of the cell count of 5×107 cells, the supernatant was discarded by centrifugation at 1,200 rpm for 5 minutes (CR5B, manufactured by Hitachi). The resulting cells were suspended at a cell density of 1×105 cells/mL in a Hybridoma-SFM (manufactured by Gibco) comprising 5% Daigo's GF21 (manufactured by Wako Pure Chemical) to give a volume of 500 mL and then cultured in a 5% CO2 incubator for three days at 37° C. The resulting cell suspension was centrifuged at 1,500 rpm for 5 minutes and the resulting supernatant was filtered through a bottle top filter (0.2 μm, PES membrane, manufactured by Corning).
Purification was carried out using a Protein A-conjugated resin (manufactured by Millipore) for KM4000, KM4001, KM4008 and KM4012 and using a Protein G-conjugated resin (manufactured by Millipore) for KM4018. Each resin was packed into a 1 mL mini-column and 10 mL of equilibration buffer was passed through it to achieve equilibration. The equilibration buffer to be used was glycine (1 mol/L)-sodium chloride (0.15 mol/L) (pH 8.6) for the Protein A-conjugated resin and glycine (0.5 mol/L)-PBS (pH 7.4) for the Protein G-conjugated resin. The culture supernatant which was obtained by the filtration treatment was passed through the column at a flow rate of about 100 mL/h and then the column was washed with 10 mL of the equilibration buffer. Thereafter, the antibodies adsorbed to the column were eluted with citrate buffer (0.1 mol/L, pH 3.0). Into tubes in which 80 μL of Tris (2 mol/L, pH 8.0) had been previously filled, and then 500 μL/tube of the eluted antibodies were aliquoted. Then, an absorbance (OD280 nm) of each tube was measured using a UV-V spectrophotometer (UV-1600, manufactured by Shimadzu) and the fraction in which the protein was detected was recovered. After overnight dialysis in PBS at 4° C., the fraction was filtrated (0.2 μm, PES membrane, manufactured by Pall) and the antibody concentration was calculated from the absorbance at 280 nm (OD280 nm) [OD280 nm measurement value (A)/1.4=protein concentration (mg/mL)].
(1) Fluorescent Cell Staining Method (Flow Cytometer)
The human ASCT2-myc/His gene-introduced CHO cell obtained in Example 2, the vector-introduced CHO cell and the multiple myeloma cell line KMS-11 (HSRRB No. JCRB1179) were respectively cultured in a 5% CO2 incubator for 3 to 4 days at 37° C. The human ASCT2-myc/His gene-introduced CHO cells and the vector-introduced CHO cells were peeled off with a 0.02% EDTA solution (manufactured by Nacalai Tesque) and washed with a mixed solution of PBS, 0.02% EDTA and 0.05% sodium azide. Additionally, in order to avoid the non-specific adsorption of antibodies, the cells were blocked for 30 minutes at ice temperature using 1% BSA-PBS for the human ASCT2-myc/His gene-introduced CHO cells and the vector-introduced CHO cells, and using 100 μg/mL of human IgG (manufactured by Sigma) for KMS-11. The cells were seeded into a 96-well U-bottom plate so as to give a density of 1×105 to 5×105 cells/100 μL/well and centrifuged at 1,500 rpm for 5 minutes (05PR-22, manufactured by Hitachi Koki) and then the supernatant was removed. The test substances as the primary antibody, i.e., the purified antibodies and rat IgG2a-UNLB (negative control, manufactured by Beckman Coulter) or KM511 {negative control, anti-G-CSF derivative antibody, [Agric. Biol. Chem., 53, 1095 (1989)], mouse IgG1} were diluted to give a final concentration of 10 μg/mL in a mixed solution (hereinafter, referred to as “Dilution Solution A”) of 1% BSA-PBS, 0.02% EDTA and 0.05% sodium azide and the resulting solution was added at the volume of 100 μL/well, followed by reaction for 60 minutes at ice temperature. After washing twice with the Dilution Solution A, 100 μL/well of ALEXA488-labeled anti-mouse immunoglobulin G (G-1-L) (manufactured by Invitrogen) or 100 μL/well of ALEXA488-labeled anti-rat immunoglobulin G (G+L) (manufactured by Invitrogen) or 100 μL/well of an FITC-labeled anti-mouse kappa-chain antibody (manufactured by Southern Biotech) or an FITC-labeled anti-rat kappa-chain antibody (manufactured by Southern Biotech), diluted with Dilution Solution A, was added as the secondary antibody, followed by reaction for 30 minutes at ice temperature under shading. Washing with Dilution Solution A was carried out three times again, the cells were suspended in PBS and the fluorescence intensity was measured by a flow cytometer (Cytomics FC500 MPL, manufactured by Beckman Coulter).
The measurement results are shown in
As shown in
Further, as shown in
(2) Western Blotting
To each of the human ASCT2-myc/His gene-introduced CHO cell obtained in Example 2, the vector-introduced CHO cell, the multiple myeloma cell line KMS-11 (HSRRB No. JCRB1179) and the colorectal cancer cell line WiDr (ATCC Accession No. CCL-218), a mixed solution (hereinafter, referred to as “cell lysis buffer A”) of Tris-HCl (50 mmol/L, pH 7.2), 1% Triton X-100, sodium chloride (150 mmol/L), magnesium chloride (2 mmol/L), calcium chloride (2 mmol/L), 0.1% sodium azide, phenylmethanesulfonyl fluoride (PMSF, 5 μmol/L), N-ethyl maleimide (50 mmol/L), leupeptin (1 mg/mL) and dithiothreitol (0.1 mmol/L) was added at a volume of 1 mL per 5×107 cells, and the resulting solution was allowed to stand at 4° C. for 2 hours. The solution was centrifuged to obtain the supernatant which was used as a cell lysate. Each of cell lysates of 5×104 cells/lane was fractionated using SDS-polyacrylamide electrophoresis (PAGEL, manufactured by Atto) and transferred to a PVDF membrane (manufactured by Millipore). After the transferred PVDF membrane was blocked with 10% BSA-PBS, each of the test substances as the primary antibody, i.e., the purified antibodies, KM3842 (positive control) obtained in Example 3, rat IgG2a-UNLB (negative control, manufactured by Beckman Coulter) and KM511 (negative control) was diluted to give a concentration of 10 μg/mL in 1% BSA-PBS, followed by reaction at 4° C. overnight. The resulting PVDF membrane was thoroughly washed with 0.1% Tween-PBS (hereinafter, referred to as “PBST”), followed by reaction with the secondary antibody, i.e., peroxidase-labeled anti-mouse immunoglobulin G (H+L) (manufactured by Zymed) or peroxidase-labeled anti-rat immunoglobulin G (H+L) (manufactured by Dako) for 1 hour at room temperature. The PVDF membrane was thoroughly washed again with PBST and the antibody-bound band was detected using ECL Western Blotting Detection Reagents (manufactured by Amersham Pharmacia).
KM3842 could detect a band around a molecular weight of 75 kDa corresponding to a molecular weight of ASCT2 in the human ASCT2-myc/His gene-introduced CHO cells, ASCT2 mRNA-expressing KMS-11 cells and WiDr cells, but KM3998, KM4000, KM4001, KM4008, KM4012 and KM4018 could not detect ASCT2. Further, the reactivity of any antibody was not recognized against the vector-introduced CHO cells.
From the results of the above flow cytometry and Western blotting, it is considered that the each of binding activity of KM3998, KM4000, KM4001, KM4008, KM4012 and KM4018 to ASCT2 was lost by SDS denaturation of ASCT2 and it was demonstrated that KM3998, KM4000, KM4001, KM4008, KM4012 and KM4018 were the antibodies which recognize and bind to the native three-dimensional structure of ASCT2.
(3) Immunoprecipitation Method
To each of the human ASCT2-myc/His gene-introduced CHO cell obtained in Example 2 and the vector-introduced CHO cells, a mixed solution (hereinafter, referred to as “cell lysis buffer B”) of Tris-HCl (50 mmol/L, pH 7.5), 1% Triton X-100, sodium chloride (150 mmol/L), EDTA (5 mmol/L), 0.1% SDS, 0.5% sodium deoxycholate, Protease inhibitor cocktail (manufactured by Roche Diagnostics) and Phosphatase inhibitor cocktail (manufactured by Roche Diagnostics) was added at a volume of 1 mL per 2×107 cells and the resulting solution was stirred at 4° C. for 30 minutes, followed by centrifugation (CF15D, manufactured by Hitachi Koki). A protein concentration of the resulting supernatant was measured using a protein assay reagent (manufactured by Bio-Rad) and adjusted to 5 mg/mL by cell lysis buffer B to be used as a cell lysate.
Then, 2 μg of each test substance, i.e., the purified antibody, KM3842 (positive control) obtained in Example 3, rat IgG2a-UNLB (negative control, manufactured by Beckman Coulter) and KM511 (negative control), was mixed with 1 mg of the resulting cell lysate at 4° C. for 1 hour.
With 0.1% BSA-PBST (300 μL), Protein G-Sepharose beads (manufactured by Amersham; 30 μL) or Protein A-Sepharose beads (manufactured by Amersham; 30 μL) was pre-treated for 30 minutes or more, followed by centrifugation to remove the supernatant and the beads were suspended in a mixed solution (hereinafter, referred to as “bead wash buffer”) (90 μL) of Tris-HCl (50 mmol/L, pH 7.5), 1% Triton X-100, sodium chloride (150 mmol/L), EDTA (5 mmol/L) and Protease inhibitor cocktail (manufactured by Roche Diagnostics).
To the bead suspension, a mixed solution (100 μL) of antibodies and cell lysate was added and mixed at 4° C. for 2 hours and then the beads were recovered by centrifugation. The recovered beads were washed three to five times with the bead wash buffer and dissolved in a mixed solution (hereinafter, referred to as “SDS-PAGE sample buffer”) of 2% SDS, Tris-HCl (62 mmol/L, pH 6.8) and 10% glycerol. The resulting solution was analyzed by the following immunoblotting.
The obtained solution was fractionated by SDS-polyacrylamide electrophoresis and transferred to a PVDF membrane (manufactured by Millipore). The resulting PVDF membrane was blocked with 5% skim milk-PBST and allowed to react with 3.5 μg/mL of KM3842 (positive control) obtained in Example 3 as a primary antibody for 2 hours at room temperature. The resulting PVDF membrane was thoroughly washed with PBST and allowed to react with peroxidase-labeled anti-rat immunoglobulin G (H+L) (manufactured by Dako) at room temperature for 1 hour. The PVDF membrane was thoroughly washed again with PBST and the antibody-bound band was detected using ECL Western Blotting Detection Reagents (manufactured by Amersham Pharmacia).
KM4000, KM4001, KM4008, KM4012, KM4018 and positive control antibody KM3842 could detect a band around a molecular weight of 75 kDa. As a result, it was demonstrated that KM4000, KM4001, KM4008, KM4012 and KM4018 are antibodies which can detect ASCT2 by the immunoprecipitation reaction and recognize the three-dimensional structure of ASCT2.
(4) Inhibitory Activity on Intracellular Uptake of Amino Acids by ASCT2
The human colorectal cancer cell line WiDr (ATCC Accession No. CCL-218) was adjusted to give a cell density of 2×104 cells/mL in a Dulbecco's modified Eagle's medium (DMEM, manufactured by Invitrogen) containing 10% inactivated dialyzed fetal bovine serum (hereinafter, referred to as dFBS, manufactured by Invitrogen) (hereinafter, referred to as “glutamine-free medium”) and 100 μL/well of the resulting suspension was seeded into a 96-well plate, followed by culturing in a 5% CO2 incubator at 37° C. for 24 hours.
Each test substance, i.e., purified antibody, rat IgG2a-UNLB (negative control, manufactured by Beckman Coulter), KM511 (negative control) or a glutamine-competitive amino acid mixture [AST mixture, a mixture of alanine, serine and threonine (all manufactured by Sigma), 3.3 mmol/L for each)], was diluted with PBS to give a final concentration of 31.6 to 0.03 μg/mL. After, 20 μL/well of the resulting solution was added to the well, 20 μL/well of a glutamine solution (manufactured by Invitrogen) which was prepared to have a final concentration of 0.2 mmol/L in a glutamine-free medium was added. To the wells of the control and the wells of the blank plate, PBS (20 μL/well) and the above glutamine solution (20 μL/well) were added. After addition of antibodies, the plates except for the blank plate were incubated in a 5% CO2 incubator at 37° C. for 72 hours.
Furthermore, 20 μL/well of {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate} diluted to 50% in a glutamine-free medium (hereinafter, referred to as “WST-1 reagent”, manufactured by Roche Diagnostics) was added thereto, followed by further incubation at 37° C. for 2 hours.
An absorbance at 450 nm (control wavelength 650 nm) was measured using a microplate spectrophotometer (Emax microplate reader, manufactured by Molecular Devices). A relative proliferation rate (%) of the antibody-added well was calculated by regading an absorbance of the non-antibody added control well as 100% and an absorbance of the blank plate well as 0%.
The measurement results were shown in
As shown in
Further, as shown in
(1) Preparation of mRNA from Anti-ASCT2 Monoclonal Antibody-Producing Hybridoma Cell
From 5×107 cells of the respective hybridomas KW4008, KM4012 and KM4018 obtained in Example 5(5), about 6 μg of mRNA was prepared using an RNeasy Maxi Kit (manufactured by Qiagen) and an Oligotex-dT30<Super>mRNA Purification Kit (manufactured by Takara Bio) in accordance with the instructions attached thereto.
(2) Gene Cloning of H Chain and L Chain Variable Regions of Anti-ASCT2 Monoclonal Antibody
Using a BD SMART RACE cDNA Amplification Kit (manufactured by BD Biosciences) in accordance with the instructions attached thereto, cDNAs each comprising the nucleotide sequence of BD SMART II A Oligonucleotide attached to the kit at the 5′-terminal were obtained from 0.6 μg of mRNA of KM4008, KM4012 and KM4018 obtained in (1).
The resulting cDNA was used as a template and PCR was carried out using the universal primer A mix attached to the kit and the mouse Ig(γ)-specific primer (mG3a2 or mG2ba1) represented by SEQ ID NOs:15 and 16 or the rat Ig(γ)-specific primer (rG2a) represented by SEQ ID NO:43 so that the cDNA fragment of VH was amplified. Another PCR was carried out using the mouse Ig(κ)-specific primer (mKa1) represented by SEQ ID NO:17 or the rat Ig(κ)-specific primer (rKa2) represented by SEQ ID NO:44 in place of the Ig(γ)-specific primer to amplify the cDNA fragment of VL.
PCR for the rat Ig(κ)-specific primer (rKa2) was carried out by heating at 94° C. for 5 minutes; 40 cycles each consisting of reaction at 94° C. for 15 seconds, reaction at 68° C. for 30 seconds and reaction at 72° C. for 3 minutes; and then reaction at 72° C. for 10 minutes. Other PCR was carried out by heating at 94° C. for 5 minutes; 5 cycles each consisting of reaction at 94° C. for 15 seconds and reaction at 72° C. for 3 minutes; 5 cycles each consisting of reaction at 94° C. for 15 seconds and reaction at 72° C. for 3 minutes and 30 seconds; and 30 cycles each consisting of reaction at 94° C. for 15 seconds, reaction at 68° C. for 30 seconds and reaction at 72° C. for 3 minutes, followed by reaction at 72° C. for 10 minutes. The PCR was carried out using a PTC-200 DNA Engine (manufactured by Bio-Rad). The resulting PCR product of H chain and L chain had a size of about 700 bp and about 800 bp in the H chain and the L chain, respectively.
In order to determine the nucleotide sequence of the resulting PCR product, the PCR product was separated by agarose gel electrophoresis and extracted using a Gel Extraction Kit (manufactured by Qiagen). The resulting extraction fragment was ligated into a pCR4TOPO vector using a TOPO TA cloning kit (manufactured by Invitrogen), and Escherichia coli DH5α (manufactured by Toyobo) was transformed with the resulting vector, using a method of Cohen et al [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)]. A plasmid was extracted from the resulting transformant using an automated plasmid isolation system PI-50 (manufactured by Kurabo) to obtain a plasmid, followed by reaction using a Big Dye Terminator Cycle Sequencing FS Ready Reaction Kit (manufactured by PE Biosystems) in accordance with the instructions attached thereto and then the nucleotide sequence was analyzed using a DNA sequencer ABI PRISM 3700 (manufactured by PE Biosystems).
As a result, a plasmid 08H2b10 comprising the full-length H chain cDNA of KM4008, a plasmid 08La4 comprising the full-length L chain cDNA of KM4008, a plasmid 12Ha5 comprising the full-length H chain cDNA of KM4012, a plasmid 12La4 comprising the full-length L chain cDNA of KM4012, a plasmid 18rHa1 comprising the full-length H chain cDNA of KM4018 and a plasmid 18Lb3 comprising the full-length L chain cDNA of KM4018 in which an MG sequence presumed to be an initiation codon was present at the 5′ terminal of cDNA were prepared.
A full-length nucleotide sequence of VH contained in the plasmid 08H2b10 comprising the H chain cDNA of KM4008 was represented by SEQ ID NO:18, a full-length amino acid sequence of secretory VH comprising a signal sequence deduced from the full-length nucleotide sequence of VH contained in the plasmid 08H2b10 was represented by SEQ ID NO:19, a full-length nucleotide sequence of VL contained in the plasmid 08La4 comprising the L chain cDNA of KM4008 was represented by SEQ ID NO:20, a full-length amino acid sequence of secretory VL comprising a signal sequence deduced from the full-length nucleotide sequence of VL contained in the plasmid 08La4 was represented by SEQ ID NO:21, a full-length nucleotide sequence of VH contained in the plasmid 12Ha5 comprising the H chain cDNA of KM4012 was represented by SEQ ID NO:22, a full-length amino acid sequence of secretory VH comprising a signal sequence deduced from the full-length nucleotide sequence of VH contained in the plasmid 12Ha5 was represented by SEQ ID NO:23, a full-length nucleotide sequence of VL contained in the plasmid 12La4 comprising the L chain cDNA of KM4012 was represented by SEQ ID NO:24, a full-length amino acid sequence of secretory VL comprising a signal sequence deduced from the full-length nucleotide sequence of VL contained in the plasmid 12La4 was represented by SEQ ID NO:25, a full-length nucleotide sequence of VH contained in the plasmid 18rHa1 comprising the H chain cDNA of KM4018 was represented by SEQ ID NO:45, a full-length amino acid sequence of secretory VH comprising a signal sequence deduced from the full-length nucleotide sequence of VH contained in the plasmid 18rHa1 was represented by SEQ ID NO:46, a full-length nucleotide sequence of VL contained in the plasmid 18Lb3 comprising the L chain cDNA of KM4018 was represented by SEQ ID NO:47, and a full-length amino acid sequence of secretory VL comprising a signal sequence deduced from the full-length nucleotide sequence of VL contained in the plasmid 18Lb3 was represented by SEQ ID NO:48, respectively.
(3) Analysis of Amino Acid Sequence of V Region of Anti-ASCT2 Monoclonal Antibody
The N-terminal amino acid sequences of the H chain and L chain in the purified monoclonal antibodies of KM4008, KM4012 and KM4018 obtained in Example 5(7) were analyzed using a protein sequencer (PPSQ-10, manufactured by Shimadzu) and about 20 residues were determined. From the comparison of the obtained analysis results with the amino acid sequences deduced from the nucleotide sequences of individual antibodies obtained in (2), it was found that the individual sequences were identical to the corresponding sequence and thus confirmed that the nucleotide sequences obtained in the (2) were nucleotide sequences of the desired antibodies. Further, from the comparison with amino acid sequence data of known mouse antibodies or rat antibodies [SEQUENCES of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)], it became clear that each of the isolated cDNAs was a full-length cDNA encoding the anti-ASCT2 monoclonal antibody KM4008, KM4012 or KM4018 comprising a secretory signal sequence; the secretory signal sequence of the H chain and L chain of KM4008 were the amino acid sequence from positions 1 to 19 in the amino acid sequence represented by SEQ ID NO:19 and the amino acid sequence from positions 1 to 20 in the amino acid sequence represented by SEQ ID NO:21, respectively; the secretory signal sequence of the H chain and L chain of KM4012 were the amino acid sequence from positions 1 to 19 in the amino acid sequence represented by SEQ ID NO:23 and the amino acid sequence from positions 1 to 20 in the amino acid sequence represented by SEQ ID NO:25, respectively; the secretory signal sequence of the H chain and L chain of KM4018 were the amino acid sequence from positions 1 to 19 in the amino acid sequence represented by SEQ ID NO:46 and the amino acid sequence from positions 1 to 20 in the amino acid sequence represented by SEQ ID NO:48, respectively.
Then, using the amino acid sequences of VH and VL of the anti-ASCT2 monoclonal antibodies KM4008, KM4012 and KM4018, amino acid sequence database of known proteins were searched by the BLASTP method [Nucleic Acid Res. 25, 3389 (1997)]. As a result, no completely identical amino acid sequence was found for both VH and VL and it was confirmed that VH and VL of the anti-ASCT2 monoclonal antibodies KM4008, KM4012, and KM4018 had novel amino acid sequences.
Further, the CDR sequences of VH and VL of the anti-ASCT2 monoclonal antibodies KM4008, KM4012, and KM4018 were identified by comparing them with the amino acid sequences of known antibodies. Amino acid sequences of CDR1, CDR2 and CDR3 of VH of the anti-ASCT2 monoclonal antibody KM4008 were represented by SEQ ID NOs:26, 27 and 28, respectively and amino acid sequences of CDR1, CDR2 and CDR3 of VL thereof were represented by SEQ ID NOs:29, 30 and 31, respectively; amino acid sequences of CDR1, CDR2 and CDR3 of VH of the anti-ASCT2 monoclonal antibody KM4012 were represented by SEQ ID NOs:32, 33 and 34, respectively, and amino acid sequences of CDR1, CDR2 and CDR3 of VL thereof were represented by SEQ ID NOs:35, 36 and 37, respectively; and amino acid sequences of CDR1, CDR2 and CDR3 of VH of the anti-ASCT2 monoclonal antibody KM4018 were represented by SEQ ID NOs:49, 50 and 51, respectively, and amino acid sequences of CDR1, CDR2 and CDR3 of VL thereof were represented by SEQ ID NOs:52, 53 and 54, respectively.
(1) Construction of Anti-ASCT2 Human Chimeric Antibody-Expressing Vector cKM4008—93
Using a vector pKANTEX93 (WO97/10354) for the expression of a humanized antibody and a plasmid 08H2b10 comprising an H chain cDNA of KM4008 and a plasmid 08La4 comprising an L chain cDNA of KM4008 each of which obtained in Example 7(2), an anti-ASCT2 human chimeric antibody-expressing vector cKM4008—93 was constructed as follows.
The PCR was carried out in the same manner as in Example 2. To the plasmid 08H2b10 (100 ng) as a template, primers (10 μmol/L, each of 1 μL) having the nucleotide sequences represented by SEQ ID NOs:38 and 39, 10×Ex Taq buffer (5 μL), dNTPs (2.5 mmol/L, 4 μL), and Ex Taq polymerase (1 μL, all manufactured by Takara Bio) were added, followed by adding sterile water to give a total volume of 50 μL. The PCR was carried out by denaturation at 96° C. for 2 minutes; 30 cycles each consisting of reaction at 94° C. for 1 minute, reaction at 55° C. for 1 minute, and reaction at 72° C. for 1 minute; and then reaction at 72° C. for 5 minutes. As a result, a gene fragment encoding VH of KM4008 was amplified in which a restriction enzyme recognizing sequence for insertion into pKANTEX93 was added.
In addition, using the plasmid 08La4 (100 ng) as a template, primers (10 μmol/L, each of 1 μL) having the nucleotide sequences represented by SEQ ID NOs:40 and 41, 10×Ex Taq buffer (5 μL), dNTPs (2.5 mmol/L, 4 μL) and Ex Taq polymerase (1 μL, all manufactured by Takara Bio), the PCR was carried out in the same manner as above to amplify a gene fragment encoding VL of KM4008 in which a restriction enzyme recognizing sequence for insertion into pKANTEX93 was added.
Each of the resulting reaction products was separated by agarose gel electrophoresis and the about 0.5-kbp amplified fragment was extracted using a Gel Extraction Kit (manufactured by Qiagen). The obtained gene fragment was ligated into a pCR4TOPO vector using a TOPO TA cloning kit (manufactured by Invitrogen), and Escherichia coli DH5α (manufactured by Toyobo) was transformed with the ligation product, as a same manner in Example 7(2), thereby obtaining a plasmid 08VH3 comprising a nucleotide sequence encoding VH of KM4008 and a plasmid 08VL6 comprising a nucleotide sequence encoding VL of KM4008.
The resulting plasmid 08VH3 was digested with restriction enzymes ApaI (manufactured by Takara Bio) and NotI (manufactured by Takara Bio), and the plasmid 08VL6 was digested with restriction enzymes EcoRI (manufactured by Takara Bio) and BsiWI (manufactured by Takara Bio) and separated by agarose gel electrophoresis separation of the plasmids. The resulting about 0.5-kbp gene fragment was extracted using a Gel Extraction Kit (manufactured by Qiagen).
The resulting digested fragment of the plasmid 08VL6 was ligated into the pKANTEX93 vector digested with the same restriction enzymes EcoRI (manufactured by Takara Bio) and BsiWI (manufactured by Takara Bio). Escherichia coli DH5α (manufactured by Toyobo) was transformed with the ligation product, in the same manner as Example 7(2), and the pKANTEX93 vector in which VL of KM4008 were inserted was obtained using a plasmid isolation kit (manufactured by Qiagen).
The pKANTEX93 vector with insertion of VL of KM4008 was digested with ApaI (manufactured by Takara Bio) and NotI (manufactured by Takara Bio) and then ligated into the above plasmid 08VH3-digested fragment. Then, Escherichia coli DH5α (manufactured by Toyobo) was transformed with the ligation product, as a same manner in Example 7(2), and an anti-ASCT2 human chimeric antibody expression vector cKM4008—93 in which VH and VL of KM4008 were inserted was obtained using a plasmid isolation kit (manufactured by Qiagen).
(2) Construction of Anti-ASCT2 Human Chimeric Antibody-Expressing Vector cKM4012—93
In the same manner as (1), an anti-ASCT2 human chimeric antibody-expressing vector cKM4012—93 was constructed from the vector pKANTEX93 for the expression of a humanized antibody (WO97/10354), and the plasmid 12Ha5 comprising the H chain cDNA of KM4012 and the plasmid 12La4 comprising the L chain cDNA of KM4012 obtained in Example 7(2).
Using plasmids 12Ha5 and 12La4 as templates, the primers for the amplification of VH having the nucleotide sequences represented by SEQ ID NOs:38 and 42 and the primers for amplification of VL having the nucleotide sequences represented by SEQ ID NOs:40 and 41, the PCR was carried out to amplify a gene fragment. Each of the resulting reaction products was separated, extracted and ligated into a pCR4TOPO vector. Escherichia coli DH5α (manufactured by Toyobo) was transformed with the ligation product, thereby obtaining a plasmid 12VH1 comprising the nucleotide sequence encoding VH of KM4012, and a plasmid 12VL11 comprising the nucleotide sequence encoding VL of KM4012.
The resulting plasmids 12VH1 and 12VL11 were digested with restriction enzymes, respectively, separated and extracted to obtain fragments of the plasmid 12VH1 and the plasmid 12VL11. Each of the resulting fragments was ligated into the restriction enzyme-digested pKANTEX93 vector. Then, Escherichia coli DH5α (manufactured by Toyobo) was transformed with the ligation product and an anti-ASCT2 human chimeric antibody expression vector cKM4012—93 in which VH and VL of KM4012 were inserted was obtained.
(3) Construction of Anti-ASCT2 Human Chimeric Antibody-Expressing Vector cKM4018—93
In a same manner as the (1), an anti-ASCT2 human chimeric antibody expression vector cKM14018—93 was constructed from the vector pKANTEX93 for the expression of a humanized antibody (WO97/10354), and the plasmid 18rHa1 comprising the H chain cDNA of KM4018 and the plasmid 18Lb3 comprising the L chain cDNA of KM4018 obtained in Example 7(2).
Using the plasmids 18rHa1 and 18Lb3 as templates, the primers for the amplification of VH having the nucleotide sequences represented by SEQ ID NOs:55 and 56 and primers for the amplification of VL having the nucleotide sequences represented by SEQ ID NOs:57 and 58, PCR was carried out to amplify the gene fragment. Each of the resulting reaction products was separated, extracted, and ligated into a pCR4 TOPO vector, and Escherichia coli DH5α (manufactured by Toyobo) was transformed with the ligation product, thereby obtaining a plasmid 18VH5 comprising a nucleotide sequence encoding VH of KM4018, and a plasmid 18V2L1 comprising a nucleotide sequence encoding VL of KM4018.
The resulting plasmids 18VH5 and 18V2L1 were digested with restriction enzymes, respectively, separated, and extracted to obtain fragments of the plasmid 18VH5 and the plasmid 18V2L1. Each of the resulting fragments was ligated into the restriction enzyme-digested pKANTEX93 vector. Then, Escherichia coliDH5α (manufactured by Toyobo) was transformed with the ligation product and an anti-ASCT2 human chimeric antibody expression vector cKM4018—93 was obtained in which VH and VL of KM4018 were instructed.
(4) Expression of Anti-ASCT2 Human Chimeric Antibody in Animal Cell
Using the anti-ASCT2 human chimeric antibody-expressing vector cKM4008—93 obtained in the above (1), expression of the anti-ASCT2 human chimeric antibody in an animal cell was carried out by a conventional method [Antibody Engineering, A Practical Guide, W.H. Freeman and Company (1992)]. Similarly, using the anti-ASCT2 human chimeric antibody-expressing vector cKM4012—93, or the anti-ASCT2 human chimeric antibody-expressing vector cKM4018—93, expression of individual anti-ASCT2 human chimeric antibodies in an animal cell was carried out. In this manner, transformants were obtained which produce an anti-ASCT2 human chimeric antibodies.
(5) Obtaining of Purified Antibody
After each of transformants obtained in the above (4) was cultured by a conventional culturing method, the cell suspensions were collected and centrifuged at 3,000 rpm and 4° C. for 15 minutes to recover the supernatants. The culture supernatants were subjected to sterile filtration using a 0.22-μm Millex GV filter (manufactured by Millipore). The anti-ASCT2 human chimeric antibodies cKM4008, cKM4012 and cKM4018 (hereinafter referred to as cKM4008, cKM4012, and cKM4018, respectively) were purified from the resulting culture supernatants, using a Protein A High-capacity resin (manufactured by Millipore) column in accordance with the instructions attached thereto.
The degree of purification and expressed molecular size of purified preparations of the resulting cKM4008, cKM4012, cKM4018 were confirmed by SDS-PAGE using a gradient gel (Catalog Number: E-T520L, manufactured by Atto) in accordance with the instructions attached thereto. With regard to phoretic patterns of the purified anti-ASCT2 human chimeric antibodies, one band was found around a molecular weight of 150 to 200 kilodaltons (hereinafter, referred to as “kDa”) under non-reducing conditions, and two bands of about 50 kDa and about 25 kDa were found under reducing conditions [Antibodies—A Laboratory Manual, Cold Spring Harbor Laboratory, Chapter 14 (1988), Monoclonal Antibodies—Principles and Practice, Academic Press Limited (1996)]. Those phoretic patterns were consistent with the results for antibodies of the IgG class, obtained by SDS-PAGE under the same conditions. Thus, it was confirmed that the anti-ASCT2 human chimeric antibodies cKM4008, cKM4012 and cKM4018 were expressed as antibody molecules having a correct structure.
(1) Fluorescent Cell Staining Method (Flow Cytometer)
Each of the human ASCT2-myc/His gene-introduced CHO cell obtained in Example 2, and the multiple myeloma cell line OPM-2 (DSMZ Accession No. ACC50) was cultured in a 5% CO2 incubator for 3 to 4 days at 37° C. The human ASCT2-myc/His gene-introduced CHO cells were peeled off with a 0.02% EDTA solution (manufactured by Nacalai Tesque), and in order to avoid the non-specific adsorption of antibodies, the cells were blocked for 30 minutes at ice temperature using 1% BSA-PBS. The cells were seeded into a 96-well U-bottom plate so as to give a density of 1×105 to 5×105 cells/100 μL/well. The plates were centrifugation at 1,500 rpm for 5 minutes (05PR-22, manufactured by Hitachi Koki), and then the supernatant was removed. As the primary antibody, the purified antibody which was a test substance was diluted to give a final concentration of 0.01 μg/mL to 10 μg/mL in 1% BSA-PBS, and dispensed to the plate at 100 μL/well, followed by reaction at ice temperature for 60 minutes. After washing the cells with 1% BSA-PBS, FITC-labeled anti-human immunoglobulin G (H+L) (manufactured by Jackson Laboratories) diluted with 1% BSA-PBS was added as the secondary antibody, followed by reaction at ice temperature for 30 minutes under shading. The cells were washed with 1% BSA-PBS and then suspended in PBS. The fluorescence intensity of the cells was measured by a flow cytometer (Cytomics FC500 MPL, manufactured by Beckman Coulter).
The measurement results are given in
(2) ADCC Activity
The multiple myeloma cell line KMS-11 (HSRRB No. JCRB 1179) was used as a target cell. The cell was adjusted to give a cell density of 2×105 cells/mL using RPMI 1640 medium (manufactured by Invitrogen) containing 5% FBS (manufactured by Invitrogen) and containing no Phenol Red (hereinafter, referred to as “ADCC activity assay medium”) and was used as a target cell solution. Lymphoprep (manufactured by Nycomed) was used for the preparation of an effector cell solution, and, a peripheral blood mononuclear cell (PBMC) fraction was separated from healthy human peripheral blood in accordance with the instructions attached thereto. The separated PBMC fraction was washed in an ADCC activity assay medium by centrifugation twice, was adjusted to give a cell density of 2×106 cells/mL and then used as an effector cell solution.
Into a 96-well U-bottom plate (manufactured by Falcon), 50 μL (1×104 cells/well) of the target cell solution was dispensed and then 50 μL of the effector cell solution (ratio of effector cells:target cells=10:1) was added. In addition, the anti-ASCT2 human chimeric antibody cKM4008, cKM4012 or cKM4018 was diluted in an ADCC activity assay medium. The diluted antibody was added to the plate to give a total volume of 150 μL at a final concentration of 0.01 ng/mL to 1000 ng/mL, followed by reaction at 37° C. for 4 hours. After the reaction was complete, the plate was centrifuged and a lactic acid dehydrogenase (LDH) activity in the supernatant was detected by measuring the absorbance using an LDH-Cytotoxic Test (manufactured by Wako Pure Chemical) in accordance with the instructions attached thereto. The absorbance of spontaneous release of the target cells was obtained using an ADCC activity assay medium in place of the effector cells solution and the antibody solution, and the absorbance data of spontaneous release of the effector cells was obtained using an ADCC activity assay medium in place of the target cell solution and the antibody solution, followed by carrying out the same operation as above. The absorbance of total release of the target cells was obtained using an ADCC activity assay medium in place of the antibody solution and the effector cell solution, by adding 20 μL of a 9% Triton X-100 solution at 45 minutes before completion of the reaction and carrying out the same operation as above. The ADCC activity was calculated by the following formula.
ADCC activity(%)=[(absorbance of sample)−(absorbance of spontaneous release of effector cells/target cells)]/(absorbance of total release of target cells)−(absorbance of spontaneous release of target cells)]×100 (Formula)
The measurement results are shown in
(3) CDC Activity
As target cells, the human ASCT2-myc/His gene-introduced CHO cell obtained in Example 2 and the colorectal cancer cell line Colo205 (ATCC Accession No. CCL-222) were peeled off using a 0.02%-EDTA Solution (manufactured by Nacalai Tesque), washed in RPMI 1640 medium (manufactured by Invitrogen) containing 1.4% BSA (manufactured by Invitrogen) and 50 μg/mL gentamicin (manufactured by Nacalai Tesque), which was prepared as a CDC assay medium and then suspended in the same medium at a cell density of 2×105 cells/mL. The resulting suspension was used as a target cell solution.
The human complement serum (S2257-5ML, manufactured by Sigma) was dissolved in 5 mL of deionized water and was two-fold diluted by adding an equal volume of a CDC assay medium. The resulting dilution was used as a human complement solution.
Into a 96-well flat-bottom plate (manufactured by Sumitomo Bakelite), 50 μL/well of the complement solution was dispensed. Then, to the well, 50 μL of the target cell solution was added. Then, 50 μL of each of antibody solutions diluted with a CDC assay medium was additionally added to give a total volume of 150 μL and reacted at 37° C. for 2 hours in the presence of 5% CO2. To the well, 15 μL/well of a WST-1 reagent (manufactured by Roche Diagnostics) was added. The resulting solution was stirred with a plate mixer, followed by at 37° C. for 2 hours in the presence of 5% CO2. The absorbance at 450 nm (control wavelength 650 nm) was measured using a microplate spectrophotometer (Emax microplate reader, manufactured by Molecular Devices). The absorbance of a well to which 50 μL of the complement solution and 100 μL of the CDC medium were added, was measured as blank. The absorbance of a well to which 50 μL of the target cells, 50 μL of the complement solution and 50 μL of the CDC assay medium were added (antibody not added) were measured. The CDC activity was calculated by the following formula.
CDC activity(%)={1−[(absorbance of antibody-added sample)−(absorbance of blank)]/[(absorbance of non-antibody added sample)−(absorbance of blank)]×100} (Formula)
The measurement results are shown in
(4) Inhibitory Activity on Intracellular Uptake of Amino Acids by ASCT2
Into a 96-well plate, 100 μL/well of the human colorectal cancer cell line WiDr (ATCC Accession No. CCL-218), which was adjusted to give a cell density of 1×104 cells/mL in a DMEM (manufactured by Invitrogen) containing 10% dFBS (manufactured by Invitrogen) (hereinafter, referred to as “glutamine-free medium”), was seeded and cultured in a 5% CO2 incubator at 37° C. for 24 hours.
To the well, 20 μL/well of each test substance, i.e., the anti-ASCT2 human chimeric antibody, KM511 (negative control) or a glutamine-competitive amino acid mixture [AST mixture, a mixture of alanine, serine and threonine (all manufactured by Sigma), 3.3 mmol/L for each)], which was diluted with PBS to give a final concentration of 10 to 0.01 μg/mL, was added, followed by further addition of 20 μL/well of a glutamine solution (manufactured by Invitrogen) which was prepared to give a final concentration of 0.2 mmol/L in a glutamine-free medium. To the wells of the control and the wells of the blank plate, 20 μL/well of PBS and 20 μL/well of the above glutamine solution were added. After addition of antibodies, the plates except for the blank plate were incubated in a 5% CO2 incubator at 37° C. for 72 hours.
Then, 20 μL/well of a WST-1 reagent (manufactured by Roche Diagnostics) diluted to 50% in the glutamine-free medium was added thereto and further incubated at 37° C. for 2 hours.
The absorbance at 450 nm (control wavelength 650 nm) was measured using a microplate spectrophotometer (Emax microplate reader, manufactured by Molecular Devices). A relative proliferation rate (%) of the well with adding the antibody was calculated by regarding the absorbance of the well of the control without adding the antibody as 100% and regarding an absorbance of the well of the blank plate as 0%.
The measurement results are shown in
In accordance with the following procedure, the plasmid pBluescript II SK (−)-Mouse_ASCT2-myc/His comprising the nucleotide sequence represented by SEQ ID NO:59 and the amino acid sequence represented by SEQ ID NO:60 was obtained, and a mouse ASCT2-myc/His gene-introduced CHO cell line was obtained using this plasmid.
Into 50 mL of an ampicillin-containing LB medium, 10 μl, of the solution of an E. coli comprising a mouse ASCT2 gene clone (Clone ID: 4192790, manufactured by Open Biosystems) was seeded, cultured under stirring overnight and then centrifuged (CR2DGII, manufactured by Hitachi Koki, 6,000 rpm, 10 minutes) to recover bacteria. A plasmid comprising mouse ASCT2 gene was prepared from the obtained bacteria using a plasmid isolation kit (manufactured by Qiagen). Total 1004 of a solution containing 100 ng of the obtained plasmid as a template, 10×KOD buffer I (10 μL), dNTPs (2 mmol/L, 5 μL), MgCl2 (25 mmol/L, 4 μL), primers (10 μmol/L, each of 1 μL) having the nucleotide sequences represented by SEQ ID NOs:61 and 62, KOD polymerase (1 μL, manufactured by Toyobo), and dimethyl sulfoxide (5 μL) was prepared, and the PCR was carried out by reaction at 96° C. for 3 minutes; 35 cycles each consisting of reaction at 95° C. for 1 minute, reaction at 50° C. for 1 minute, and reaction at 72° C. for 1.5 minutes; and then reaction at 72° C. for 7 minutes. The reaction product was separated by agarose gel electrophoresis, and the resulting about 1.7-kb amplified fragment was extracted using a QIAquick Gel Extraction Kit (manufactured by Qiagen). The resulting extraction fragment was digested with EcoRI (manufactured by Takara Bio) and KpnI (manufactured by Takara Bio) and then re-extracted using a QIAquick Gel Extraction Kit. The extraction fragment was ligated into a pBluescript II SK (−) vector digested with EcoRI (manufactured by Takara Bio) and KpnI (manufactured by Takara Bio) using a Ligation high (manufactured by Toyobo), and then Escherichia coli DH5α (manufactured by Toyobo) was transformed with the ligation product in accordance with the method of Cohen et al [Proc, Natl, Acad-Sci. USA. 69, 2110 (1972)]. A plasmid was extracted from the resulting transformant using an automated plasmid isolation system PI-50 (manufactured by Kurabo), and a plasmid pBluescript II SK (−)-Mouse_ASCT2-myc/His comprising the nucleotide sequence represented by SEQ ID NO:59 and the amino acid sequence represented by SEQ ID NO:60 was obtained.
The resulting pBluescript II SK (−)-Mouse_ASCT2-myc/His was digested with EcoRI (manufactured by Takara Bio) and KpnI (manufactured by Takara Bio), and a gene fragment was extracted in the same manner as in the above and ligated into a pKANTEX93 vector (WO97/10354) which had been previously digested with EcoRI and KpnI. Then, Escherichia coli DH5α (manufactured by Toyobo) was transformed with the ligation product in the same manner as in the above, thereby obtaining a transformant. The plasmid pKANTEx-Mouse_ASCT2-myc/His was obtained from the transformant using a plasmid isolation kit (manufactured by Qiagen).
In accordance with the following procedure, the pKANTEX-Mouse_ASCT2-myc/His was introduced into CHO/DG44 cells [Somatic cell and Molecular Genetics, 12, 555 (1986)] by an electroporation method [Cytotechnology, 3, 133 (1990)]. The cells used herein are those subcultured in a medium where 1×FIT supplement (manufactured by Invitrogen) was added to IMDM (manufactured by Invitrogen) containing 10% dFBS (manufactured by Invitrogen) and gentamicin (manufactured by Nacalai Tesque, 50 μg/mL) (hereinafter, referred to as “A4 medium”). The CHO/DG44 cells were suspended in buffer containing potassium chloride (137 nmol/L), sodium chloride (2.7 nmol/L), disodium hydrogen phosphate (8.1 mmol/L), sodium dihydrogen phosphate (1.5 nmol/L) and magnesium chloride (4 mmol/L) (hereinafter, referred to as “K-PBS”) to give a density of 8×106 cells/mL, and the resulting cell suspension (200 μL, 1.6×106 cells in terms of cell count) was mixed with an expression plasmid pKANTEX-Mouse_ASCT2-myc/His (10 μg). The mixture was transferred into a cuvette (interelectrode distance: 2 mm), and the gene introduction was carried out using a GenePulser II (manufactured by Bio-Rad) at a pulse voltage of 0.35 kV and an electric capacity of 250 μF. The cuvette was allowed to stand on ice, and a cell suspension in the cuvette was suspended in a cell culture vessel containing an A4 medium and cultured in a 5% CO2 incubator at 37° C. After four day culturing, the culture medium was exchanged with an A4 medium containing 0.5 mg/mL of G418 (manufactured by Nacalai Tesque) and continued to culture the cells. With medium exchange and subculturing during the cell culture, a transformed cell line resistant to G418 was obtained about two weeks after the gene introduction.
The resulting G418-resistant transformant cells were diluted to give a cell density of 5 cells/mL in an A4 medium containing 0.5 mg/mL of G418 (manufactured by Nacalai Tesque), and 100 μL/well of the diluted cell suspension was dispensed into a 96-well plate, and treated with a stepwise increasing concentration of methotrexate. In this manner, a clone which highly expresses mouse ASCT2-myc/His was selected, thereby obtaining mouse ASCT2/CHO.
(1) Construction of Variant Human ASCT2 Gene
PfuTubo DNA polymerase (1 μl) was added to total 50 μL of a solution containing pCR4-SLC1A5-myc/His constructed in Example 2 as a template, the primers (0.1 μg/mL, 2.5 μL) comprising the nucleotide sequence represented by SEQ ID NOs:63 and 64, 10× reaction buffer (5 μL) attached to QuikChange Site-Directed Mutagenesis Kit and dNTPs (1 μL), and PCR was carried out by reaction at 95° C. for 30 seconds; 18 cycles each consisting of three processes, reaction at 95° C. for 30 seconds, reaction at 55° C. for 1 minute, and reaction at 68° C. for 5 minute; and then reaction at 37° C. for 1 hour with addition of 1 μL of DnpI after completion of the reaction. Escherichia coli XL1-Blue (manufactured by Stratagene) attached to a QuickChange Site-Directed Mutagenesis Kit was transformed with 2 μL of the reaction product. A plasmid was extracted from the resulting transformant using an automated plasmid isolation system PI-50 (manufactured by Kurabo) and a plasmid pCR4-hNco1KO_ASCT2-myc/His was obtained. Using this plasmid as a template, and the primers (0.1 μg/mL, 2.5 μL) comprising the nucleotide sequences represented by SEQ ID NOs:65 and 66, respectively, a plasmid pCR4-hBam3KI_ASCT2-myc/His was obtained in the same manner as in the above. The obtained plasmid was digested with EcoRI (manufactured by Takara Bio) and KpnI (manufactured by Takara Bio), extracted using a QIAquick Gel Extraction Kit, and was ligated into a pBluescript II SK (−) vector (manufactured by Stratagene) which had been previously digested with EcoRI and KpnI, using a Ligation high (manufactured by Toyobo). Then, Escherichia coli DH5α (manufactured by Toyobo) was transformed with the ligation product, in accordance with the method of Cohen et al [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], and a plasmid pBluescript II SK (−) hBam3KI_ASCT2-myc/His was obtained in the same manner as above.
(2) Construction of Variant Mouse ASCT2 Gene
Using pBluescript II SK (−)-Mouse-ASCT2-myc/His constructed in Example 10 as a template, and primers comprising the nucleotide sequences represented by SEQ ID NOs:67 and 68, respectively, a plasmid pBluescript II SK (−)-mNcoKO_ASCT2-myc/His was obtained in the same manner as in Example 11(1).
(3) Construction of Mouse/Human Chimeric ASCT2/CHO
A plasmid, which was obtained by re-transforming dam-Escherichia coli (Catalog Number: C2925, manufactured by New England Biolabs) with the plasmids obtained in (1) and (2), was digested with EcoRI (manufactured by Takara Bio) and BclI (equivalent to FbaI manufactured by Takara Bio), and ligated into a plasmid comprising a human ASCT2 gene as a vector, using a mouse ASCT2 gene as an insert. Then, Escherichia coli DH5α (manufactured by Toyobo) was transformed with the ligation product to obtain a plasmid pBluescript II SK (−)-mEL1_ASCT2-myc/His in which EL1 was replaced with a mouse type of EL1. Similarly, a plasmid pBluescript II SK (−)-mEL2_ASCT2-myc/His and a plasmid pBluescript II SK (−)-mEL3_ASCT2-myc/His were obtained by replacing EL2 with a mouse type of EL2 at Bell and NcoI sites and replacing EL3 with a mouse type of EL3 at NcoI and BamIII sites, respectively.
Using the pBluescript II SK (−) hBam3KI_ASCT2-myc/His obtained in (1) as a template, and the primers having the nucleotide sequences represented by SEQ ID NOs:69 and 70, the plasmid pBluescript II SK (−)-mEL4_ASCT2-myc/His in which EL4 was replaced with a mouse type of EL4 was obtained in the same manner as in Example 11(1).
The resulting pBluescript II SK (−)-mEL1_ASCT2-myc/His, pBluescript II SK (−)-mEL2_ASCT2-myc/His, pBluescript II SK (−)-mEL3_ASCT2-myc/His and pBluescript II SK (−)-mEL4_ASCT2-myc/His were digested with EcoRI (manufactured by Takara Bio) and KpnI (manufactured by Takara Bio) and each of gene fragments was extracted in the same manner as above, and ligated into a pKANTEX93 vector (WO97/10354) which had been previously digested with EcoRI and KpnI. Then, Escherichia coli DH5α (manufactured by Toyobo) was transformed with the ligation product to obtain transformants. Then, the expression plasmids pKANTEX-mEL1_ASCT2-myc/His, pKANTEX-mEL2_ASCT2-myc/His, pKANTEX-mEL3_ASCT2-myc/His and pKANTEX-mEL4_ASCT2-myc/His were obtained from the respective transformants using a plasmid isolation kit (manufactured by Qiagen).
In accordance with the electroporation method, pKANTEX-mEL1-ASCT2-myc/His, pKANTEX-mEL2_ASCT2-myc/His, pKANTEX-mEL3_ASCT2_myc/His and pKANTEX-mEL4_ASCT2-myc/His were introduced into CHO/DG44 cells respectively in the same manner as in Example 10, and thereby obtained a mouse EL1 type ASCT2-myc/His gene-introduced CHO cell line (hereinafter, referred to as “mEL1/CHO”), a mouse EL2 type ASCT2-myc/His gene-introduced CHO cell line (hereinafter, referred to as “mEL2/CHO”), a mouse EL3 type ASCT2-myc/His gene-introduced CHO cell line (hereinafter, referred to as “mEL3/CHO”) and a mouse EL4 type ASCT2-myc/His gene-introduced CHO cell line (hereinafter, referred to as “mEL4/CHO”), respectively.
Using the mouse ASCT2/CHO and mouse/human chimeric ASCT2/CHO cells constructed in Examples 10 and 11 and FITC-labeled anti-mouse immunoglobulin G (H+L) (manufactured by Dako) or ALEXA488-labeled anti-rat immunoglobulin G (G+L) (manufactured by Invitrogen) as the secondary antibody, the reactivity of anti-ASCT2 monoclonal antibodies KM4008, KM4012 and KM4018 was measured in the same manner as in Example 9(1).
The measurement results are shown in
From these results, it was demonstrated that the anti-ASCT2 monoclonal antibodies KM4008 and KM4012 recognize and bind to EL2, and the anti-ASCT2 monoclonal antibody KM4018 recognizes and binds to a three-dimensional structure comprising EL1, EL2 and EL3.
(1) Design of Amino Acid Sequences of VH and VL of Anti-ASCT2 Humanized Antibody
An amino acid sequence of VH of an anti-ASCT2 humanized antibody was designed as follows.
Firstly, the amino acid sequence of FR of VH of a human antibody was selected in order to graft amino acid sequences of CDR1 to CDR3 of KM4008VH represented by SEQ ID NOs:26 to 28, respectively, obtained in Example 7(3). Using a GCG Package (manufactured by Genetics Computer Group) as a sequence analysis system, based on the amino acid sequence database of conventional proteins by the BLASTP method [Nucleic Acids Res., 25, 3389 (1997)], a human antibody having a high homology with KM4008 was searched. When the homology score was compared with the homology of an actual amino acid sequence, SWISSPROT database accession no. BAC01342, the immunoglobulin heavy chain VHDJ region (hereinafter, referred to as “BAC01342”) exhibited a homology of 77.0%, and it was a human antibody which had the highest homology, so the amino acid sequence of FR of this antibody was selected.
Amino acid sequences of CDRs of VH of KM4008 represented by SEQ ID NOs:26 to 28 were grafted into an appropriate position of the thus determined amino acid sequence of FR of the human antibody. In this manner, an amino acid sequence HV0 of VH of the anti-ASCT2 humanized antibody represented by SEQ ID NO:71 was designed.
Next, an amino acid sequence of VL of the anti-ASCT2 humanized antibody was designed as follows.
An amino acid sequence of FR of VL of a human antibody was selected in order to graft amino acid sequences of CDR1 to CDR3 of KM4008VL represented by SEQ ID NOs:29 to 31, respectively, obtained in Example 7(3). Kabat et at have classified VL of conventionally known various human antibodies into four subgroups (HSG I to IV) based on the homology of their amino acid sequences and reported on the consensus sequences for each of the subgroups [Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services (1991)]. Accordingly, the homology was examined between the amino acid sequences of FR of consensus sequences of subgroups I to IV of VL of the human antibody and the amino acid sequence of FR of VL of KM4008.
As a result of the homology analysis, the homology of HSG I, HSG II, HSG III, and HSG IV was 77.5%, 60.0%, 63.8%, and 68.8%, respectively. Therefore, the amino acid sequence of FR of KM4008VL had the highest homology with the subgroup I.
Based on these results, the amino acid sequence of CDR of VL of KM4008 was grafted into an appropriate position of an amino acid sequence of FR of the consensus sequence of subgroup I of VL of the human antibody. However, since Leu at position 124 in the amino acid sequence of VL of KM4008 represented by SEQ ID NO:21 was not the amino acid residue having the highest use frequency in the region which corresponded to the amino acid sequence of the human antibody FR cited by Kabat, but is an amino acid residue which was used at a relatively high frequency, the above amino acid residue recognized in the amino acid sequence of KM4008 was used. In this manner, the amino acid sequence LV0 of VL of an anti-ASCT2 humanized antibody represented by SEQ ID NO:72 was designed.
The amino acid sequence HV0 of VH and amino acid sequence LV0 of VL of the anti-ASCT2 humanized antibody designed in the above are sequences in which the amino acid sequence of CDR of the anti-ASCT2 mouse monoclonal antibody KM4008 alone was grafted into the selected amino acid sequence of FR of the human antibody. However, it is known that when a humanized antibody is produced by simply grafting only CDRs of a mouse antibody into FRs of a human antibody, its antigen-binding activity is lower than that of the original mouse antibody. For these reasons, in order to avoid lowering of the binding activity, the amino acid residues which are considered to have an influence on the binding activity, among the amino acid residues of FRs which are different between human antibodies and mouse antibodies, are usually modified together with the grafting of the amino acid sequence of CDRs. Thus, in this Example, the amino acid residues of FRs which is considered to influence on the binding activity were identified as follows.
Firstly, a three-dimensional structure of V region of the antibody (hereinafter, referred to as “HV0LV0”) comprising the amino acid sequence of HV0 of VH and amino acid sequence of LV0 of VL of anti-ASCT2 humanized antibody designed in the above was constructed using a computer modeling technique. The three dimensional structure was prepared and displayed using Discovery Studio (manufactured by Accelrys) in accordance with instructions attached thereto. In addition, a computer model of the three-dimensional structure of the V region of the anti-ASCT2 mouse monoclonal antibody KM4008 was also constructed in the same manner. Further, in the amino acid sequence of FRs of VH and VL of HV0LV0, amino acid residues which were different from those of KM4008 were selected and replaced with the corresponding amino acid residues of KM4008, and the three-dimensional structure model was constructed based on the resulting amino acid sequence in the same manner. The amino acid residues predicted to influence on the binding activity of the antibody were identified by comparing three-dimensional structures of the V regions of the constructed KM4008, HV0LV0 and modified product.
As a result, as the amino acid residues among amino acid residues of FR of HV0LV0, which are considered to change a three-dimensional structure of the antigen binding region and therefore influence on the binding activity of the antibody, Val at position 2, Ser at position 9, Val at position 20, Ser at position 30, Arg at position 38, Glu at position 46, Leu at position 86, Val at position 93, Tyr at position 95 and Val at position 116 in the HV0 sequence, and Pro at position 8, Val at position 15, Gln at position 38, Ala at position 43, Pro at position 44, Phe at position 71, and Tyr at position 87 in the LV0 sequence were respectively selected.
By modifying at least one or more amino acid sequences of these selected amino acid residues into the amino acid residues which are present at the same positions of KM4008, VH and VL of a humanized antibody having various modifications were designed.
Specifically, regarding VH, at least one modification among the amino acid modifications for substituting Val at position 2 with Ile, Ser at position 9 with Pro, Val at position 20 with Ile, Ser at position 30 with Thr, Arg at position 38 with Lys, Glu at position 46 with Lys, Leu at position 86 with Val, Val at position 93 with Thr, Tyr at position 95 with Phe, and Val at position 116 with Leu was introduced in the amino acid sequence represented by SEQ ID NO:71. Further, regarding VL, at least one modification among the amino acid modifications for substituting Pro at position 8 with Thr, Val at position 15 with Leu, Gln at position 38 with Arg, Ala at position 43 with Thr, Pro at position 44 with Val, Phe at position 71 with Tyr, and Tyr at position 87 with Phe was introduced in the amino acid sequence represented by SEQ ID NO:72.
As a result, HV0LV3, HV2LV0, HV2LV3, HV4LV0, HV4LV3, HV0LV0, HV7LV3, HV10LV0, and HV10LV3 were respectively designed as antibody V regions of the anti-ASCT2 humanized antibody with modifications of at least one of amino acid residues present in FR of HV0LV0. The amino acid sequences of H chain variable regions HV2, HV4, HV7 and HV10, and L chain variable region LV3 were represented by SEQ ID NOs:76, 78, 80, 82 and 84, respectively.
(2) Preparation of Anti-ASCT2 Humanized Antibody
A DNA encoding the amino acid sequence of the variable region of the anti-ASCT2 humanized antibody was prepared in mammalian cells using a codon which was used at a high frequency, when amino acid modification(s) were carried out using a codon which is used as a DNA encoding the amino acid sequence of VH or VL of KM4008. The nucleotide sequences encoding the amino acid sequence of HV0 and LV0 of the anti-ASCT2 humanized antibody were represented by SEQ ID NOs:73 and 74, respectively, whereas the nucleotide sequences encoding the amino acid sequences of variable regions HV2, HV4, HV7, HV10 and LV3 on which amino acid modification(s) were made were represented by SEQ ID NOs:75, 77, 79, 81 and 83, respectively.
Using these nucleotide sequences, humanized antibody-expressing vectors were constructed and humanized antibodies were respectively expressed in the same manner as in Example 8.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skill in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.
This application is based on Japanese application No. 2008-185790, filed on Jul. 17, 2008, and U.S. provisional application No. 61/081,820, filed on Jul. 18, 2008, the entire contents of which are incorporated hereinto by reference. All references cited herein are incorporated in their entirety.
| Number | Date | Country | Kind |
|---|---|---|---|
| P.2008-185790 | Jul 2008 | JP | national |
| Number | Date | Country |
|---|---|---|
| 2004-032966 | Apr 2004 | WO |
| WO 2004-101772 | Nov 2004 | WO |
| 2005020888 | Mar 2005 | WO |
| 2007109376 | Sep 2007 | WO |
| WO 2010-008075 | Jan 2010 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 20100196392 A1 | Aug 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61081820 | Jul 2008 | US |
