JP5205440B2 - Amino acid transport protein and its gene - Google Patents

Description

  The present invention provides an amino acid transport protein or a part thereof, a DNA encoding the protein or a part thereof, an RNA encoding the protein or a part thereof, a DNA hybridizing to the DNA, an expression vector comprising the DNA, A transformed cell transformed with DNA or the vector, a cell introduced with the RNA, an antibody reactive with the protein or a part thereof, a part thereof, a cell producing the antibody, a part of the DNA A labeled DNA formed by radioactively labeling, a labeled RNA formed by radioactively labeling a part of the RNA, a labeled antibody formed by labeling the antibody or a part of the antibody, a kit comprising the labeled DNA, and the labeled RNA A kit comprising the labeled antibody, a pharmaceutical composition comprising a part of the DNA, a pharmaceutical composition comprising a part of the RNA , A pharmaceutical composition comprising the antibody or a part of the antibody, a method for measuring the presence or absence or expression level of the protein, a method for identifying a substance having the ability to inhibit the biological activity of the protein, the protein A method for identifying a substance having the ability to inhibit transcription of DNA encoding DNA into mRNA, a method for identifying a substance having the ability to inhibit the expression of the protein, a substance identified by the identification method, and the protein of the present invention The present invention relates to a transgenic mouse into which the encoding DNA has been introduced.

Amino acids not only serve as substrates for protein synthesis, but also serve as precursors in the formation of sugars and the biosynthesis of many biomolecules including porphyrins, purines and pyrimidines, and play an extremely important role.
Since such biosynthetic reactions are mainly carried out in cells, cells use various proteins collectively called amino acid transporter (amino acid transporter) for taking amino acids into the cell from outside the cell. It is provided in the cell membrane.

The amino acid transporter not only functions for the supply of amino acids to individual cells, but is also incorporated into tissues and epithelial transport of amino acids in the small intestine and renal tubules as well as the re-transmission of neurotransmitters in neural tissues. It is responsible for absorption and is located at an important position for the expression of specific functions in tissues.
The amino acid transport mechanism (amino acid transport system via amino acid transporter) has been identified and classified using cultured cells and cell membrane specimens since the 1960s, reflecting the diversity of amino acid molecules and different substrate specificities. An amino acid transport system via various amino acid transporters having sex has been identified (Physiol. Rev., Vol. 70, p. 43-77, 1990).
However, these transport systems do not function independently for each amino acid, and each molecule uses a plurality of amino acids as substrates and is responsible for intracellular transport of the plurality of amino acids.

Amino acids include basic amino acids having a positive charge (diamino / monocarboxylic acid), acidic amino acids having a negative charge (monoamino / dicarboxylic acid), and neutral amino acids (other than monoamino / monocarboxylic acid or basic amino acids or acidic amino acids) )are categorized. Because of this charge of amino acids, for example, neutral amino acids and negatively charged acidic amino acids are transported against cells with negative potential against a concentration gradient, so active transport coupled with some energy consumption There is a need to do.
From this point, the amino acid transporter, as with sugar transport system, and shows the sodium (Na ⁺⁾ dependent, and those showing a Na ⁺ -independent exist. The Na ⁺ -dependent transporter has a large concentration ability because it can transport amino acids against a concentration gradient by coupling amino acid transport and Na ⁺ transport, and thus has a large concentration through a cell membrane in vivo. It plays an important role in areas where it is required to form a difference (Annual Rev. Kidney, “Structure and function of kidney-specific organic solute transporters”, p.91-100, 1995, published by Chugai Medical) . Such Na ⁺ -dependent transporters can be further divided into two types of families, namely, the Na ⁺ / Cl ⁻ -dependent transporter family and the Na ⁺ / K ⁻ -dependent transporter family (Annual Rev Neurosci., Vol. 16, p. 73-93, 1993 and FASEB J., Vol. 7, p. 1450-1459, 1993).
In addition, the amino acid transporter, in combination with such charge properties, in terms of its substrate specificity, is a molecule having a basic amino acid (diamino / monocarboxylic acid) as a substrate, an acidic amino acid (monoamino / dicarboxylic acid). The molecule can be classified into a molecule that uses a substrate and a neutral amino acid (other than monoamino / monocarboxylic acid, basic amino acid, or acidic amino acid) as a substrate.

For example, basic amino acids having an amino group or an imidazole group in the side chain such as arginine, lysine and histidine (basic amino acids close to neutrality) are mainly transported by the Na ⁺ -independent amino acid transporter y ⁺ . (J. Membrane Biol., Vol. 66, p.213-225, 1982). It is known that acidic amino acids having a carboxyl group in the side chain, such as glutamic acid and aspartic acid, are transported by Na ⁺ -dependent amino acid transporters X ^- _{A and G} (Biochim. Biophys. Acta, Vol. 732). , p.24-31, 1983). In addition, in the transport of neutral amino acids to which many amino acids belong, Na ⁺ -independent amino acid transporter L (Ann. Rev. Physiol., Vol. 46, p.417-433, 1984), Na ⁺ -dependent amino acid transporter. Porter A and ASC (Ann. Rev. Physiol., Vol.46, p.417-433, 1984 and J. Membrane biol., Vol.52, p.83-92, 1980) play an important role. (Physiol. Rev., Vol.70, p.43-77, 1990 and the latest medicine, Vol.50, p.1997-2004, 1995).
As mentioned above, amino acids have an extremely important role as a raw material in the biosynthesis of various biological components carried out in cells. It can be inferred that they are involved.

  From previous studies, pathological conditions involving abnormal transport mechanisms of amino acids into cells include amino aciduria, which causes impaired reabsorption of amino acids from renal tubules, and impaired glutamate uptake and neuronal cell death. Amyotrophic lateral sclerosis is known (Annual Rev. Kidney, “Structure and function of kidney-specific organic solute transporter”, p.91-100, 1995, published by Chugai Medical; latest medicine, Vol. .50, p.1997-2004, 1995; and the latest medicine, Vol.51, p.64-70, 1996).

  Amino acid transporters play an essential and vital role in the uptake of amino acids necessary for the development, differentiation, proliferation and maintenance of all cells and are therefore involved in the development of more pathologies as well as those described above. It is considered a thing. In addition, considering the essentiality of amino acid transporters in the living body, it is unlikely that the uptake of various amino acids is mediated only by some of the transporters that have been identified so far. A porter is considered to exist.

The identification of unknown amino acid transporters that play an essential role in the survival and maintenance of such cells, tissues and organs, and in the living body, may be able to elucidate the cause of the onset of various diseases whose causes have not been elucidated. Have Furthermore, if the relationship between the amino acid transporter and various diseases is elucidated, it will be possible to effectively treat the disease by regulating the biological function or expression of the amino acid transporter. Therefore, there is an urgent need to identify a new amino acid transporter and elucidate the relationship between the transporter molecule and a disease state.
However, in spite of such medical and social needs, the current situation is that the identification of amino acid transporters and the elucidation of the amino acid transport mechanism have not progressed much.
That is, in order to identify an amino acid transporter molecule, it is necessary to purify the molecule, and in order to analyze the activity of the purified product, it is necessary to reconstitute the purified product into a cell membrane and reproduce the amino acid transport activity. There is. However, since amino acid transporter molecules have a relatively small expression level as a membrane protein and are not sufficiently reproducible in activity, there are technical difficulties in identifying new molecules.

  In addition, for example, an amino acid transporter that is specifically expressed in a non-normal cell (abnormal cell) directly related to a disease state such as a cancer cell (tumor cell) and is responsible for supplying amino acid to the abnormal cell is identified. It is extremely important in elucidating the mechanism of survival and proliferation of such pathologically related cells and in developing therapeutic methods for diseases such as cancer, but amino acid transporters are essential for normal cell survival. Therefore, it is not easy to identify such amino acid transporter molecules that are specifically expressed in abnormal cells.

  As the neutral amino acid transporter, ASCT1 and ASCT2 have been cloned as sodium-dependent transporters (Kanai, Curr. Opin. Cell Biol., 9, 565 (1997)). However, these have alanine, serine, cysteine, threonine, and glutamine as main substrates, and are different in substrate selectivity from the neutral amino acid transport system L. In addition, glycine transporter and proline transporter have been cloned, but the substrate selectivity is different from that of neutral amino acid transport system L. (Amara and Kuhar, Annu. Rev. Neurosci., 16, 73 (1993)).

  RBAT and 4F2hc cDNAs, which are type II membrane glycoproteins that have only one transmembrane structure, which is considered to be an activator of amino acid transporters, but not the transporter itself, have been cloned, When expressed in Xenopus oocytes, it is known to activate the uptake of basic amino acids as well as neutral amino acids (Palacin, J. Exp. Biol., 196, 123 (1994)).

Therefore, an unidentified amino acid transporter molecule that is specifically expressed in abnormal cells deeply related to such pathological conditions is identified, and the relationship between the molecule and the survival and proliferation of the abnormal cells is elucidated. This is an effective clue for providing a method for treating the disease state and the disease.
That is, the disease can be treated by controlling the biological activity of the amino acid transporter molecule or the expression of the molecule.

  In order to search for a novel amino acid transporter that is specifically expressed in such pathologically abnormal cells, particularly tumor cells, the present inventors have known cell membranes that are considered essential for tumor cell growth. By focusing on the surface molecule 4F2 (CD98), a novel amino acid transporter molecule named LAT1 (L-type amino acid transporter-1), which is particularly significantly expressed in tumor cells compared with that in normal cells, is identified. Succeeded.

  That is, in order for tumor cells to rapidly divide and continuously grow and proliferate, nutrients such as amino acids and sugars need to be supplied into the cells, and this supply is specific to the nutrients. It is thought to be borne by up-regulation of amino acid transporters (Physiol. Rev., Vol.70, p.43-77, 1990). In order to grow, proliferate and maintain tumor cells, protein biosynthesis needs to be carried out within the cell, so that the incorporation of essential amino acids into the cell (transportation from outside the cell into the cell) is particularly important.

From the studies so far, the growth of tumor cells is known as 4F2 (CD98), which is classified as a type II membrane glycoprotein that is considered to have a function of activating an amino acid transporter that has not yet been identified. It has been thought that cell membrane surface antigens may play an important role (J. Immunol., Vol. 126, p. 1409-1414, 1981; J. Immunol., Vol. 129, p. 623-628, 1982; Proc. Natl. Acad. Sci. USA., Vol.84, p.6526-6530, 1987; Cancer Res., Vol.46, p.1478-1484, 1986; J. Biol. Chem., Vol. 267, p.15285-15288, 1992; Proc. Natl. Acad. Sci. USA., Vol.89, p.5606-5610, 1992; Biochem. J., Vol.324, p.535-541, 1997; And J. Exp. Biol., Vol. 196, p. 123-137, 1994).
Accordingly, as a result of intensive studies on the identification of human tumor cell membrane surface molecules that are coupled or interact with 4F2 molecules, the present inventors have found a gene encoding a novel amino acid transporter LAT1 having the following characteristics: It came to complete.

The amino acid transporter LAT1 according to the present invention, in particular, the human-derived amino acid transporter LAT1 has the following characteristics.
(1) Northern blotting using human-derived tumor cells and human normal tissue-derived mRNA, stomach signet ring cell carcinoma (KATOIII), melanoma cell lines (malignant melanoma (G-361)) and lung small cell carcinoma (RERF-LC-MA) and a wide range of human-derived tumor cells are expressed as a band of about 4.8 kb. In normal human tissues, it is also expressed as a band of approximately 4.8 kb only in certain limited tissues (placenta, fetal liver, bone marrow, testis, brain, and peripheral blood leukocytes) that are vigorously born and proliferated. Is confirmed.
(2) The open reading frame (ORF, including the termination codon) has a base sequence consisting of 1,524 bases (base sequences 66 to 1589 of the base sequence described in SEQ ID NO: 1), The ORF encodes an amino acid sequence composed of 507 amino acids as a whole, and has a molecular weight of approximately 55 kDa (calculated value) (SEQ ID NO: 2).

(3) According to hydrophobicity plot analysis, human LAT1 has 12 transmembrane regions, and in the intracellular region, phosphorylation site by tyrosine protein kinase (119th Tyr in the amino acid sequence of SEQ ID NO: 2) and protein It is identified as a membrane surface molecule having a phosphorylation site by kinase C (the 189th Ser and the 346th Ser of the amino acid sequence of SEQ ID NO: 2).
(4) In cells in which human LAT1 and human 4F2hc (4F2 heavy chain) are coexpressed, neutral amino acids leucine (Leu), isoleucine (Ile), phenylalanine (Phe), methionine (Met), tyrosine (Tyr) , Tryptophan (Trp) and valine (Val), and histidine (His), a basic amino acid close to neutrality, are confirmed. In addition, significant uptake of threonine, cysteine, asparagine and glutamine, which are other neutral amino acids, is confirmed.

(5) In cells co-expressed with human LAT1 and human 4F2hc, not only the aforementioned amino acid incorporation, but also known drugs such as L-DOPA, which is a Parkinson therapeutic drug, and triiodothyronine (thyroid hormone) Uptake of physiologically active substances such as Furthermore, uptake of BCH (2-amino-2-norbornane-carboxylic acid) known as a neutral amino acid uptake inhibitor is also confirmed.
(6) In the Michaelis-Menten kinetic test, the Km value representing the affinity between human LAT1 and various substrates as described above is about 21 μM.
(7) The incorporation of various amino acids, drugs and physiologically active substances into cells via LAT1 described above does not depend on Na ⁺ ions or Cl ⁻ ions.

That is, the present invention is an amino acid transporter that is considered to be essential for the survival and proliferation of various tumor cells having a wide range of substrate specificities, as well as a specific expression in a wide range of tumor cells compared to normal cells. For the first time in the world.
Because of the above-described characteristics of the amino acid transporter molecule of the present invention, the molecule is extremely promising as a target in the development of, for example, an antitumor drug (anticancer drug). That is, a drug having the biological activity of the molecule or the activity of suppressing the expression of the molecule (antisense DNA drug, antisense RNA drug, antibody drug, antibody fragment drug, peptide antagonist drug, non-peptide antagonist such as low molecular weight compound, etc. Inhibiting the uptake of nutrients (various amino acids and physiologically active substances) through the molecule into tumor cells using drugs, etc., thereby making the tumor cells starved and inhibiting the survival and proliferation of tumor cells It is possible.

Accordingly, the protein of the present invention or a part thereof, DNA encoding the protein or part thereof, RNA encoding the protein or part thereof, DNA hybridizing to the DNA, expression vector containing the DNA, the DNA Or a transformed cell transformed with the vector, a cell into which the RNA has been introduced, an antibody or a part thereof reactive with the protein or a part thereof, a cell producing the antibody, a part of the DNA Radiolabeled DNA, radiolabeled RNA labeled part of the RNA, labeled antibody labeled with the antibody or part of the antibody, kit comprising the labeled DNA, consisting of the labeled RNA The kit and the kit comprising the labeled antibody are used as a drug having such an antitumor effect and / or for such drug development. It is extremely useful as a definitive reagent.
In addition, by using various substances such as the above-described DNA, RNA, or transformed cells of the present invention, various methods (or assay methods) for identifying such various drugs can be provided. Is possible.

Specifically, various inventions in the present application each have the following utility.
The DNA, RNA and transformed cells of the present invention are not only useful in producing the protein of the present invention as a recombinant protein using gene recombination techniques, but also control the biological activity of the protein of the present invention (activity) Agents, agents that inhibit transcription of the protein of the present invention into mRNA, agents that inhibit translation of the mRNA into the protein of the present invention, or inhibit the interaction of the protein with other molecules. It is extremely useful as a reagent (tool) for drug design, screening, and identification of drugs to be performed.

Specifically, the DNA of the present invention can be used not only in assays for identifying agents that control the biological activity of the proteins of the present invention, but also in assays for identifying agents that modulate the expression of the proteins of the present invention. Can also be used.
In the former assay, a cell such as a mammal is transformed with a DNA encoding the amino acid transporter molecule of the present invention to cause the cell to express the molecule, and the transformed cell is expressed as a test substance and a substrate of the molecule. The activity of the test substance for controlling the biological activity of the amino acid transporter of the present invention by comparing the amount of the substrate taken up into the cell with that in the control cell by culturing in the presence of (amino acid etc.) Is to evaluate.

  The latter assay includes so-called reporter gene assay commonly used in assay, screening and identification of such drugs, and so-called high-throughput screening in which screening is automatically performed using a machine (robot) based on the reporter gene assay. (High Throughput Screening) (Tissue Culture Engineering, Vol. 23, No. 13, p. 521-524; U.S. Pat. No. 5,670,113).

In the present invention, DNA encoding the amino acid transporter molecule of the present invention, DNA encoding the expression control region of the DNA, and DNA encoding a reporter protein molecule that emits fluorescence, such as luciferase, are included in the transporter molecule. Tested on transformed cells obtained by transforming cells commonly used in the production of recombinant proteins with an expression vector inserted so that the reporter protein molecule can be expressed depending on the expression. Contacting a compound and measuring the amount of transporter molecule expressed depending on the action of the compound indirectly by measuring the amount of fluorescence emitted by the reporter protein expressed simultaneously with the expression of the molecule Analysis of whether the compound affects transporter molecule expression Can Rukoto (U.S. Pat. No. 5,436,128; U.S. Pat. No. 5,401,629).
In addition, the RNA of the present invention can be used in an assay for identifying a drug that controls the biological activity of the amino acid transporter protein molecule of the present invention.

  In this assay, RNA encoding the amino acid transporter molecule of the present invention is injected into an Xenopus oocyte, for example, to express the transporter molecule in the cell, and the test substance and the substrate of the molecule (amino acid) Etc.), the amount of the substrate taken up into the cell is compared with the uptake in the control cell to evaluate the activity of the test substance against the biological activity of the amino acid transporter of the present invention. To do.

Part of the DNA and part of RNA of the present invention can be used as a probe for identifying DNA or RNA that hybridizes with them using a colony hybridization method or a plaque hybridization method. Furthermore, a part of the DNA of the present invention can be used as a primer for amplifying the gene encoding the DNA of the present invention or the transporter molecule of the present invention using PCR (Polymerase-chain reaction).
Furthermore, a part of the DNA of the present invention, a DNA complementary to the DNA, or a part of the RNA of the present invention is not only useful as the reagent described above, but also as a so-called antisense DNA drug or antisense RNA drug. Useful.

  That is, an antisense drug is a part of the base sequence of DNA, a part of the base sequence complementary to the base sequence of the DNA, or a part of the base sequence of RNA is complementary to the base sequence of each. It is a drug by a mechanism that inhibits transcription from DNA to mRNA or translation from mRNA to protein by utilizing the property of binding to DNA or RNA having a sequence. This antisense drug can modify properties such as increased half-life in blood, permeability into cells, or targeting efficiency to disease target sites by chemically modifying part of the antisense sequence. Is possible.

  As described above, the protein of the present invention is used to identify a drug that controls the biological activity of the protein of the present invention or the expression of the protein by utilizing the state in which the protein molecule is expressed on the cell surface. Can do. Moreover, based on the amino acid sequence of the protein, a peptide antagonist having the ability to inhibit the biological activity of the protein can be designed. The peptide antagonist designed in this way can competitively inhibit the binding of the amino acid transporter, which is the protein of the present invention, to various substrates, or the binding of the protein of the present invention to other molecules. It is useful as a medicine that prevents the biological function of the protein from being exerted.

The protein of the present invention and a part thereof, and cells such as transformed cells expressing the protein are useful as immunization antigens in the production of antibodies (antisera, monoclonal antibodies) against the protein of the present invention.
Antiserum (polyclonal antibody) and monoclonal antibody reactive to the amino acid transporter molecule which is the protein of the present invention are antibodies produced by inhibiting (neutralizing) the biological activity of the molecule by binding to the molecule. Useful as a medicine.
Further, the antibody is labeled with various substances capable of producing a detectable signal, thereby analyzing the expression state of the protein of the present invention in various biological samples (cells, tissues, organs, body fluids, etc.) ( It is useful as a reagent in immunohistochemical staining, Western blot, ELISA, etc.

Similar to such labeled antibodies, labeled DNA labeled with various substances capable of producing a detectable signal of the DNA of the present invention or a part thereof can be tested in the identification of the gene encoding the protein of the present invention ( For example, it is useful as a reagent in Southern blotting, FISH and the like.
Similarly, a radiolabeled RNA obtained by labeling the RNA of the invention or a part thereof with a radioisotope is a reagent for analyzing the expression state of mRNA encoding the protein of the present invention in cells, tissues or organs (such as Northern blotting). Useful as.
In addition, for the DNA of the present invention, for example, a transgenic animal as a model animal can be obtained by introducing a human-derived amino acid transporter DNA which is one aspect of the present invention into a mammal other than a human such as a mouse. Can be produced.

  Furthermore, using the gene encoding the human-derived amino acid transporter of the present invention as a probe, a gene encoding a homolog protein derived from rabbit or mouse is cloned, and based on the selected gene information, the gene of mouse or rabbit is cloned. A model animal (knockout animal) can be prepared by disrupting (inactivating) the endogenous gene encoding the homologous protein. By analyzing the physical, biological, pathological and genetic characteristics of these model animals, the function of the amino acid transporter according to the present invention can be elucidated in more detail.

  Further, a model animal having only a gene (DNA) encoding the human-derived amino acid transporter of the present invention is produced by mating the transgenic animal with the endogenous gene destroyed in this way. It is possible. By administering to this model animal an agent (antisense drug, peptide antagonist, low molecular non-peptide compound, antibody, etc.) that controls the biological activity of the amino acid transporter molecule of the present invention described above or the expression of the molecule, It is possible to evaluate the therapeutic effect of the drug.

That is, the present invention provides substances, medicaments, reagents and methods described in the following <1> to <55>, which have extremely high industrial utility as described above.
<1> A cell surface protein having the ability to mediate transport of amino acids into cells, which is leucine (Leu), isoleucine (Ile), phenylalanine (Phe), methionine (Met), tyrosine (Tyr), tryptophan ( Trp), a protein having the ability to mediate uptake of at least one amino acid selected from the group consisting of valine (Val) and histidine (His) into cells in a Na ⁺ -independent manner.
<2> The protein according to <1>, which is a protein having an ability to transport a neutral amino acid and a similar substance when coexisting with a 4F2 protein classified as a type II membrane glycoprotein or a part thereof.
<3> A 4F2 protein classified as a type II membrane glycoprotein is a protein having the amino acid sequence of SEQ ID NO: 6 or 8, or an amino acid sequence in which a part of the amino acids is deleted, substituted, or added. The protein according to <2>.
<4> The protein according to any one of <1> to <3>, which is a protein derived from human or rat.
<5> The protein according to any one of <1> to <4>, which has an amino acid sequence of any one of (1) and (2) below.
(1) the amino acid sequence described in SEQ ID NO: 2 or 4; or (2) the amino acid sequence described in SEQ ID NO: 2 or 4, wherein one or several amino acids are deleted, substituted or added.
<6> A polypeptide having a partial amino acid sequence in the amino acid sequence of SEQ ID NO: 2 or 4 and having antigenicity.
<7> DNA encoding the protein according to any one of <1> to <5>.
<8> The DNA according to <7>, which is human or rat-derived DNA.
<9> Hybridizes under stringent conditions to DNA having the nucleotide sequence of nucleotide numbers 66 to 1586 of the nucleotide sequence of SEQ ID NO: 1 or the nucleotide sequence of nucleotide numbers of 64 to 1599 of the nucleotide sequence of SEQ ID NO: 3. DNA encoding a cell surface protein having the ability to mediate uptake of at least one amino acid into a cell.
<10> The DNA according to <9>, which encodes a cell surface protein whose amino acid uptake into cells is mediated in the presence of a 4F2 protein classified as a type II membrane glycoprotein or a part thereof.
<11> A 4F2 protein classified as a type II membrane glycoprotein is a protein having the amino acid sequence of SEQ ID NO: 6 or 8, or an amino acid sequence in which a part of the amino acids is deleted, substituted, or added <10> The DNA according to <10>.
<12> RNA that can be derived from the DNA according to <7> to <11>.
<13> The RNA according to <12>, wherein the RNA has the base sequence described in SEQ ID NO: 26 or 27.
<14> An expression vector comprising the DNA according to any one of <7> to <11>.
<15> A transformed cell transformed with the expression vector according to <14>.
<16> The transformed cell is further represented by any one of the base sequence of base numbers 110 to 1696 of the base sequence described in SEQ ID NO: 5 and TAG, TGA, or TAA adjacent to the base of the base number 1696 The transformed cell according to <14> or <15>, wherein the transformed cell is transformed with a DNA comprising a base sequence consisting of a nonsense base sequence.
<17> The transformed cell according to any one of <14> to <16>, wherein the transformed cell is further transformed with a DNA encoding a reporter protein.
<18> A non-human cell, wherein the RNA according to <12> or <13> is introduced.
<19> The cell according to <18>, wherein the cell is a Xenopus oocyte.
<20> An antiserum or a polyclonal antibody having reactivity with the protein according to any one of <1> to <5> or the polypeptide according to <6>.
<21> A monoclonal antibody having a reactivity with the protein according to any one of <1> to <5> or the polypeptide according to <6> or a part of the monoclonal antibody.
<22> The above <21>, wherein the monoclonal antibody is a recombinant chimeric monoclonal antibody comprising a variable region of an immunoglobulin derived from a mammal other than a human and a constant region of an immunoglobulin derived from a human. The monoclonal antibody according to the description or a part of the monoclonal antibody.
<23> A part or all of complementarity determining regions of a hypervariable region of an immunoglobulin derived from a mammal other than a human, a framework region of a hypervariable region of a human-derived immunoglobulin, and a human-derived immunoglobulin. The monoclonal antibody according to <21> or a part of the monoclonal antibody, which is a recombinant human monoclonal antibody comprising a constant region of globulin.
<24> The monoclonal antibody according to any one of <21> to <23>, or a part of the monoclonal antibody, wherein the monoclonal antibody is a human monoclonal antibody.
<25> A cell that produces the monoclonal antibody according to any one of <21> to <24>.
<26> The above <25>, wherein the cell is a fusion cell obtained by fusing a B cell derived from a non-human mammal having the ability to produce the monoclonal antibody and a myeloma cell derived from a mammal. > Cells.
<27> A gene in which the cell is transformed by introducing either the DNA encoding the heavy chain of the monoclonal antibody or the DNA encoding the light chain thereof, or both DNAs into the cell The cell according to <25>, which is a recombinant cell.
<28> A pharmaceutical composition comprising the DNA according to any one of <7> to <11> and a pharmaceutically acceptable carrier.
<29> The pharmaceutical composition according to <28>, wherein the pharmaceutical composition is used for suppressing the growth of tumor cells.
<30> A pharmaceutical composition comprising the RNA according to <12> or <13> and a pharmaceutically acceptable carrier.
<31> The pharmaceutical composition according to <30>, wherein the pharmaceutical composition is used for suppressing the growth of tumor cells.
<32> A pharmaceutical composition comprising the antiserum or polyclonal antibody according to <20> above and a pharmaceutically acceptable carrier.
<33> The pharmaceutical composition according to <32>, wherein the pharmaceutical composition is used for suppressing the growth of tumor cells.
<34> A pharmaceutical composition comprising the monoclonal antibody according to any one of <21> to <24> or a part of the monoclonal antibody, and a pharmaceutically acceptable carrier.
<35> The pharmaceutical composition according to <34>, wherein the pharmaceutical composition is used for suppressing the growth of tumor cells.
<36> A labeled monoclonal antibody obtained by labeling the monoclonal antibody according to any one of <21> to <24>, alone or with a labeling substance capable of providing a detectable signal by reacting with another substance antibody.
<37> The labeled monoclonal antibody according to <36>, wherein the labeling substance is an enzyme, a fluorescent substance, a chemiluminescent substance, biotin, avidin, or a radioisotope.
<38> A kit for detecting a protein having the amino acid sequence set forth in SEQ ID NO: 2 or a fragment thereof, wherein the kit comprises the labeled monoclonal antibody according to <36> or <37>. .
<39> A method for examining the presence or absence or expression level of protein in a sample,
(1) contacting the sample with the labeled monoclonal antibody according to <36> or <37>; and (2) binding the amount of the labeled monoclonal antibody bound to the sample to the labeled monoclonal antibody. A step of measuring by detecting fluorescence, chemiluminescence or radioactivity according to the type of labeling substance,
A method comprising the steps of:
<40> The method according to <39>, wherein the sample is a tumor cell, a tumor tissue, an organ having a tumor, or a part thereof.
<41> A labeled DNA obtained by labeling the DNA according to any one of <7> to <11> or a fragment thereof with an enzyme, a fluorescent substance, a chemiluminescent substance, biotin, avidin, or a radioisotope.
<42> Radiolabeled RNA obtained by labeling the RNA according to <12> or <13> with a radioisotope.
<43> A kit for detecting a gene encoding the protein according to any one of <1> to <5>, wherein the labeled DNA according to <41> or the radioactivity according to <42> A kit comprising RNA. More specifically, a kit for detecting a gene encoding a protein having the amino acid sequence represented by SEQ ID NO: 2, comprising the radioactive DNA according to <41> or the radioactive RNA according to <42> The kit characterized by becoming.
<44> A method for detecting the action of a test substance as a substrate on the ability of the protein to transport neutral amino acids, using the protein according to any one of <1> to <5>.
<45> The method according to <44>, wherein the cell transformed with the DNA according to any one of <7> to <11> is used.
<46> The method according to <44>, wherein Xenopus oocytes are used.
<47> The method according to any one of <44> to <46>, wherein the test substance is a substance other than an amino acid.
<48> A method for screening a test substance having an action of inhibiting the ability to transport a neutral amino acid and a similar substance of the protein using the protein according to any one of <1> to <5>. . More specifically, the biological function of the protein having the amino acid sequence set forth in SEQ ID NO: 2, comprising leucine (Leu), isoleucine (Ile), phenylalanine (Phe), methionine (Met), tyrosine (Tyr), A method for identifying a substance having an ability to inhibit the ability to mediate intracellular uptake of any one amino acid selected from the group consisting of histidine (His), tryptophan (Trp) and valine (Val), The method comprises the following steps (1) and (2):
(1) Any one of the following cells (a) to (d) is added to the substance, leucine (Leu), isoleucine (Ile), phenylalanine (Phe), methionine (Met), tyrosine (Tyr), histidine (His) ), Tryptophan (Trp) and valine (Val) in the presence of any one of the amino acids selected from the group consisting of a radioisotope labeled with a radioisotope, or in the presence of the radiolabeled amino acid alone Step of culturing:
(A) a naturally occurring cell co-expressing the protein having the amino acid sequence set forth in SEQ ID NO: 2 and the protein having the amino acid sequence set forth in SEQ ID NO: 6;
(B) a base sequence comprising the base sequence of base numbers 66 to 1586 of the base sequence described in SEQ ID NO: 1 and any one nonsense base sequence represented by TAG, TGA or TAA adjacent to the base of the base number 1586 A base sequence of base numbers 110 to 1696 of the base sequence shown in SEQ ID NO: 5 and any one nonsense base sequence represented by TAG, TGA or TAA adjacent to the base of the base number 1696 A recombinant cell co-expressing the protein having the amino acid sequence set forth in SEQ ID NO: 2 and the protein having the amino acid sequence set forth in SEQ ID NO: 6 by co-transformation with DNA containing the base sequence;
(C) a base sequence consisting of the base sequence of base numbers 1 to 1521 of the base sequence shown in SEQ ID NO: 26 and any one nonsense base sequence represented by UAG, UGA or UAA adjacent to the base of base number 1521 And the nucleotide sequence of nucleotide numbers 1 to 1587 of the nucleotide sequence of SEQ ID NO: 27 and any one nonsense nucleotide sequence represented by UAG, UGA or UAA adjacent to the nucleotide of the nucleotide number 1587 A non-human-derived recombinant cell co-expressing the protein having the amino acid sequence set forth in SEQ ID NO: 2 and the protein having the amino acid sequence set forth in SEQ ID NO: 6 by introduction of RNA containing the base sequence together; or ( d) human-derived tumor cells; and (2) cells cultured in the presence of the substance and the radiolabeled amino acid. Step radioactivity and the radioactivity of cells cultured in the presence of only the radiolabeled amino acid has to compare the difference in each value included in the.
<49> The method according to <48>, wherein the cell transformed with the DNA according to any one of <7> to <11> is used.
<50> The method according to <48>, wherein Xenopus oocytes are used.
<51> Identification of a substance having the ability to inhibit transcription of the DNA according to any one of <7> to <11> to mRNA or the expression of the protein according to any one of <1> to <5> how to.
<52> A substance detected, screened or identified by the method according to any one of <44> to <51>.
<53> The substance according to <52>, wherein the substance is a substance having an ability to inhibit the growth of tumor cells.
<54> A transgenic mouse having a foreign gene, wherein the mouse is incorporated with a DNA encoding a protein having the amino acid sequence set forth in SEQ ID NO: 2 or 4 on the endogenous gene. A transgenic mouse comprising cells that express the expression in the body.
<55> The nonsense base sequence represented by the base sequence of base numbers 66 to 1586 of the base sequence shown in SEQ ID NO: 1 and TAG, TGA or TAA adjacent to the base of the base number 1586 of the DNA Or a nonsense base sequence represented by TAG, TGA or TAA adjacent to the base sequence of the base number 1599 and the base sequence of the base sequence of the base sequence shown in SEQ ID NO: 3 The transgenic mouse according to <54>, wherein the DNA is a DNA comprising the nucleotide sequence of

The amino acid transporter molecule of the present invention has an important biological function that mediates the uptake of various amino acids, which are nutrients essential for cell production and proliferation, and has a wider variety than the expression in normal cells. Therefore, the molecule is extremely promising as a target in the development of antitumor drugs (anticancer drugs), for example.
That is, a drug having the biological activity of the molecule or the activity of suppressing the expression of the molecule (antisense DNA drug, antisense RNA drug, antibody drug, antibody fragment drug, peptide antagonist drug, non-peptide antagonist such as low molecular weight compound, etc. Inhibiting the uptake of nutrients (various amino acids and physiologically active substances) through the molecule into tumor cells using drugs, etc., thereby making the tumor cells starved and inhibiting the survival and proliferation of tumor cells It is possible.
Accordingly, the protein of the present invention or a part thereof, DNA encoding the protein or part thereof, RNA encoding the protein or part thereof, DNA hybridizing to the DNA, expression vector containing the DNA, the DNA Or a transformed cell transformed with the vector, a cell into which the RNA has been introduced, an antibody or a part thereof reactive with the protein or a part thereof, a cell producing the antibody, a part of the DNA Radiolabeled DNA, radiolabeled RNA labeled part of the RNA, labeled antibody labeled with the antibody or part of the antibody, kit comprising the labeled DNA, consisting of the labeled RNA The kit and the kit comprising the labeled antibody are used as a drug having such an antitumor effect and / or for such drug development. It can be provided as a definitive reagent.
In addition, by using various substances such as the DNA, RNA or transformed cells of the present invention, agents that control (activate, suppress, inhibit) the biological activity of the protein of the present invention, transcription of the protein of the present invention to mRNA Drug design, screening (for example, reporter gene assay, etc.), such as an agent that inhibits the translation of the mRNA into the protein of the present invention, or an agent that inhibits the interaction of the protein with other molecules, and Identification becomes possible.
Furthermore, a part of the DNA and a part of the RNA of the present invention can be provided as a probe for identifying DNA or RNA that hybridizes with them using a colony hybridization method or a plaque hybridization method. Furthermore, a part of the DNA of the present invention can be provided as a primer for amplifying the gene encoding the DNA of the present invention or the transporter molecule of the present invention using PCR.
Furthermore, a part of the DNA of the present invention, a DNA complementary to the DNA, or a part of the RNA of the present invention is not only provided as the aforementioned reagent, but also as a so-called antisense DNA drug or antisense RNA drug. Can be provided.
As described above, the protein of the present invention is used to identify a drug that controls the biological activity of the protein of the present invention or the expression of the protein by utilizing the state in which the protein molecule is expressed on the cell surface. Can do. Moreover, based on the amino acid sequence of the protein, a peptide antagonist having the ability to inhibit the biological activity of the protein can be designed. The peptide antagonist designed in this way can competitively inhibit the binding of the amino acid transporter, which is the protein of the present invention, to various substrates, or the binding of the protein of the present invention to other molecules. It can be provided as a pharmaceutical that prevents the biological function of the protein from being exerted.
The protein of the present invention and a part thereof, and cells such as transformed cells expressing the protein can be provided as an immunization antigen in the production of an antibody (antiserum, monoclonal antibody) against the protein of the present invention.
Antiserum (polyclonal antibody) and monoclonal antibody reactive to the amino acid transporter molecule which is the protein of the present invention are antibodies produced by inhibiting (neutralizing) the biological activity of the molecule by binding to the molecule. It can be provided as a medicine.
Furthermore, the antibody can be labeled with various substances capable of giving a detectable signal, so that the expression state of the protein of the present invention in various biological samples (cells, tissues, organs, body fluids, etc.) can be obtained. It can be provided as a reagent in analysis (immunohistological staining, Western blot, ELISA, etc.).
Similar to such labeled antibodies, labeled DNA labeled with various substances capable of producing a detectable signal of the DNA of the present invention or a part thereof can be tested in the identification of the gene encoding the protein of the present invention ( For example, it can be provided as a reagent in Southern blotting, FISH and the like.
Similarly, a radiolabeled RNA obtained by labeling the RNA of the invention or a part thereof with a radioisotope is a reagent for analyzing the expression state of mRNA encoding the protein of the present invention in cells, tissues or organs (such as Northern blotting). Can be provided as

It is a figure which shows the homology of each amino acid sequence of human amino acid transporter LAT1 and rat amino acid transporter LAT1. It is a figure which shows the hydrophilic property and hydrophobic region of the human amino acid transporter LAT1 by hydrophobicity plot analysis. It is a figure which shows the expression state of mRNA of the human amino acid transporter LAT1 in various human tissues by Northern blotting. It is a figure which shows the uptake | capture activity of the leucine in the cell in the Xenopus oocyte which coexpressed the human amino acid transporter LAT1 and the human cell membrane surface molecule 4F2hc. It is a figure which shows the uptake | capture amount of the leucine in the cell at the time of culture | cultivating the Xenopus oocyte which coexpressed the human amino acid transporter LAT1 and the human cell membrane surface molecule 4F2hc in presence of various salts. It is a figure which shows the affinity with respect to the substrate of the human amino acid transporter LAT1 by a Michaelis-Menten dynamic test. The figure which shows the uptake | capture amount of the radioactive label leucine as a substrate when the Xenopus oocyte which co-expressed human amino acid transporter LAT1 and the human cell membrane surface molecule 4F2hc was cultured in presence of various amino acids into a cell. It is. The figure which shows the uptake | capture amount of the radiolabeled phenylalanine as a substrate when the Xenopus oocyte which co-expressed human amino acid transporter LAT1 and the human cell membrane surface molecule 4F2hc was cultured in the presence of an amino acid or a drug. It is. When Xenopus oocytes coexpressed with human amino acid transporter LAT1 and human cell membrane surface molecule 4F2hc were cultured in the presence of amino acids or physiologically active substances, the amount of radioactively labeled leucine as a substrate incorporated into the cells was measured. FIG. It is a figure which shows the uptake | capture amount into the cell of various radiolabeled amino acids as a substrate to the Xenopus oocyte which co-expressed human amino acid transporter LAT1 and the human cell membrane surface molecule 4F2hc. It is a figure which shows the result of a leucine uptake | capture experiment by the oocyte which injected rat C6 glioma origin mRNA and / or rat 4F2hc gene cRNA. It is a figure which shows the hydrophobicity plot of rat neutral amino acid transporter LAT1. It is the photograph replaced with drawing which showed the result of having analyzed the expression of LAT1 gene mRNA in each organ tissue of a rat by Northern blotting. It is a photograph replacing a drawing showing the results of comparing the expression of LAT1 gene mRNA in each cultured cell line of rat and the expression of LAT1 gene mRNA in rat liver by Northern blotting. It is the photograph replaced with drawing which showed the result of having analyzed the expression of LAT1 gene mRNA in each human cultured cell line by Northern blotting. It is a figure which shows the result of having performed the leucine uptake | capture experiment by the oocyte which injected cRNA of the rat LAT1 gene and / or rat 4F2hc gene on the 2nd or 5th day after cRNA injection. It is a figure which shows the result of having investigated the influence of the salt added in the leucine uptake | capture experiment by the oocyte which injected cRNA of rat LAT1 gene and cRNA of rat 4F2hc gene. It is a figure which shows the result of having investigated the influence of the density | concentration of a substrate leucine in the leucine uptake | capture experiment by the oocyte which injected cRNA of the rat LAT1 gene and cRNA of the rat 4F2hc gene. In the leucine uptake experiment by the oocyte which injected the cRNA of rat LAT1 gene and rat 4F2hc gene, it is a figure which shows the result of having investigated the influence of various amino acids or its analog addition to a system | strain. In a cultured rat hepatocyte cell line, it is a figure which shows the result of having investigated the influence on the cell growth of D-leucine or BCH addition to a culture medium. It is a photograph replacing a drawing showing the results of analyzing the expression of LAT1 gene mRNA and 4F2hc gene mRNA in a human cultured tumor cell line by Northern blotting. It is the photograph replaced with drawing which showed the result of having analyzed the expression of LAT1 gene mRNA and 4F2hc gene mRNA in a human cultured tumor cell line (leukemia cell) by Northern blotting. It is a figure which shows the result of having investigated the Na ^<+> dependence of the leucine uptake | capture of T24 cell. Upper: The figure which showed the concentration dependence (Michaelis-Menten kinetic test) of the leucine uptake of T24 cell. Bottom: It is the figure which showed the result of the analysis by Eadie-Hoffstee plot of the concentration dependence of the leucine uptake | capture of T24 cell. It is a figure which shows the result of having investigated the influence of various amino acids or its analog addition to a type | system | group in the leucine uptake | capture experiment by T24 cell. It is a figure which shows the result of having analyzed the effect of BCH using the double reciprocal plot in the leucine uptake | capture experiment by T24 cell. It is the figure which showed the effect of BCH with respect to the proliferation of T24 cell (A) and Daudi cell (B). It is a figure which shows the result of having transplanted mouse | mouth sarcoma180 cell intraperitoneally to an ICR mouse | mouth, and examining the life prolonging effect of BCH, D-Leu, and D-Ala.

  Hereinafter, the meaning of the terms used in the present invention, and general production of the DNA, protein, antibody, antibody-producing cell, transformant, labeled DNA, labeled RNA, labeled antibody, pharmaceutical composition, and transgenic mouse of the present invention By clarifying the method, the present invention will be described in detail.

As used herein, the term “mammal” refers to any mammal, such as humans, cows, horses, pigs, goats, sheep, dogs, cats, chickens, rabbits, rats, hamsters, guinea pigs, and mice, Preferred are humans, cows, horses, pigs, goats, sheep, dogs, cats, chickens, rabbits, rats, hamsters, guinea pigs, and mice, and particularly preferred are humans, rats, hamsters, guinea pigs, and mice. .
The terms “mammal other than human” and “non-human mammal” used in the present invention are synonymous with each other and mean any mammal other than a human in the mammals defined above.

The term “amino acid” used in the present invention means any amino acid that exists in nature, and is preferably represented as follows according to the three-letter code or single-letter code of the alphabet used to represent the amino acid. Means an amino acid.
Glycine (Gly / G), Alanine (Ala / A), Valine (Val / V), Leucine (Leu / L), Isoleucine (Ile / I), Serine (Ser / S), Threonine (Thr / T), Asparagine Acid (Asp / D), glutamic acid (Glu / E), asparagine (Asn / N), glutamine (Glu / Q), lysine (Lys / K), arginine (Arg / R), cysteine (Cys / C), methionine (Met / M), phenylalanine (Phe / F), tyrosine (Tyr / Y), tryptophan (Trp / W), histidine (His / H), proline (Pro / P).

Amino acids are classified into acidic amino acids, basic amino acids and neutral amino acids according to the charge and polarity state they have. If the above amino acids are classified according to the said classification, they are classified as follows. However, when further subdividing the degree of charge and polarity, or when considering other parameters, the classification is not necessarily as follows. Some amino acids are not suitable for this classification.
(Acidic amino acids)
Aspartic acid (Asp / D), glutamic acid (Glu / E)
(Basic amino acids)
Lysine (Lys / K), Arginine (Arg / R), Histidine (His / H)
(Neutral amino acid)
Leucine (Leu / L), isoleucine (Ile / I), phenylalanine (Phe / F), methionine (Met / M), tyrosine (Tyr / Y), tryptophan (Trp / W), valine (Val / V), histidine (His / H), Threonine (The / T), Cysteine (Cys / C), Asparagine (Asn / N), Glutamine (Gln / Q), Glycine (Gly / G), Alanine (Ala / A), Serine ( Ser / S), proline (Pro / P)

The term “protein” used in the present invention means a molecule derived from the above-mentioned mammal and having a unique amino acid sequence consisting of the above-mentioned amino acids.
The “protein” of the present invention is a protein as described in any one of <1> to <5>. More specifically,
“A cell surface protein having the ability to mediate transport of amino acids into cells, wherein the protein is expressed in cells expressing leucine (Leu), isoleucine (1) or (2) below. Ile), phenylalanine (Phe), methionine (Met), tyrosine (Tyr), histidine (His), tryptophan (Trp), and any one amino acid selected from the group consisting of valine (Val) A protein characterized by having the ability to mediate:
(1) a protein having the amino acid sequence set forth in SEQ ID NO: 6 or 8, or (2) a homologous protein of the protein having the amino acid sequence set forth in SEQ ID NO: 6 or 8, wherein the base is set forth in SEQ ID NO: 5 or 7 A protein encoded by DNA that hybridizes under stringent conditions to DNA containing the sequence. "
The above-described protein of the present invention, that is, the protein of the present invention together on the cell membrane on which the human-derived cell membrane surface molecule 4F2hc having the amino acid sequence shown in SEQ ID NO: 6 or its homologous protein derived from a non-human animal is expressed. It means a protein that, when expressed, can give the cell such a property as to induce the uptake of the at least one amino acid into the cell.
Here, the “homologous protein” has sequence homology to the amino acid sequence (SEQ ID NO: 6) of the human-derived cell membrane surface molecule 4F2hc and is considered to be derived from an evolutionary common ancestor protein. Means a protein derived from a non-human animal species having the same physiological function.

The protein of the present invention is preferably one of the following proteins (1) or (2).
(1) a protein having the amino acid sequence set forth in SEQ ID NO: 2; or (2) a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence set forth in SEQ ID NO: 2. And, in a cell expressing a protein having the amino acid sequence shown in SEQ ID NO: 6, leucine (Leu), isoleucine (Ile), phenylalanine (Phe), methionine (Met), tyrosine (Tyr), A protein having the ability to mediate intracellular uptake of any one amino acid selected from the group consisting of histidine (His), tryptophan (Trp), and valine (Val).

Here, “several amino acids” mean a plurality of amino acids, specifically 1 to 40 amino acids, preferably 1 to 30 amino acids, more preferably 1 to 20 amino acids. Particularly preferred is 1 to 10 amino acids.
The partial modification (deletion, substitution, insertion, addition) of the amino acid in the amino acid sequence of the protein of the present invention as described above can be introduced by partially modifying the base sequence encoding the protein. . This partial modification of the nucleotide sequence can be introduced by a conventional method using a known site-specific mutagenesis method (Proc. Natl. Acad. Sci., USA, Vol. 81, p. .5662-5666, 1984).

The “partial amino acid sequence” in the present invention is one embodiment of the protein of the present invention described above, and means any partial amino acid sequence (protein fragment) in the amino acid sequence. Preferably, a partial sequence containing a site necessary for the protein of the present invention to exert its biological function, or a site where the protein of the present invention binds or interacts with other protein molecules (receptors and ligands) is preferred. Can be mentioned.
Further, the “polypeptide having a partial amino acid sequence and having antigenicity” in the present invention is a polypeptide including the partial amino acid sequence, and the polypeptide is administered into the body of the aforementioned mammal. In some cases, it means a polypeptide that is recognized as non-self or foreign by the immune response mechanism of the mammal and can produce antibodies against the polypeptide in the mammal body.

The protein of the present invention or a polypeptide containing a partial amino acid in the amino acid sequence of the protein of the present invention may be used in the technical field such as a chemical synthesis method, a cell culture method, etc. in addition to the gene recombination technology described below. It can be expressed by appropriately using a known method or its modification method.
In addition, the protein of the present invention is injected into various cells such as, for example, Xenopus oocytes, without causing transcription from DNA to mRNA. It can also be expressed by directly translating the injected RNA into protein (Experimental Medicine Extra Number, “Biosignal Experimental Method”, Vol.11, No.3, p.30-38, 1993).

  The “DNA” of the present invention is the DNA as described in any one of <7> to <11>. As a preferred embodiment, the DNA encoding the protein or polypeptide of the present invention described above and any base such as cDNA, DNA complementary to the cDNA, and genomic DNA can be used as long as the DNA can encode the protein of the present invention. DNA having a sequence is also included. Furthermore, the DNA of the present invention includes DNA composed of any codon as long as it is a codon encoding the same amino acid. One of the preferred embodiments of the DNA of the present invention is DNA encoding the human-derived protein of the present invention.

More specifically, the DNA of the present invention is the DNA described in any of (1) to (3) below.
(1) A DNA encoding the protein according to any one of <1> to <5>.
Here, the DNA includes any nucleotide sequence such as cDNA, DNA complementary to the cDNA, and genomic DNA as long as the protein encodes the protein, for example, a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or 4. Also included are DNAs having

(2) The base sequence of base numbers 66 to 1586 of the base sequence described in SEQ ID NO: 1 and any one nonsense base sequence represented by TAG, TGA or TAA adjacent to the base of the base number 1586, or SEQ ID NO: A base sequence consisting of a base sequence consisting of the base sequence of base numbers 64 to 1599 of the base sequence described in 3, and any one nonsense base sequence represented by TAG, TGA or TAA adjacent to the base of the base number 1599 Contains DNA.
Here, the “nonsense base sequence” means any base sequence of TAG, TGA or TAA, which is also called a termination codon, termination codon, nonsense codon, termination codon or termination signal, and encodes the termination point of protein synthesis. It is a base sequence.

(3) The base sequence of base numbers 66 to 1586 of the base sequence described in SEQ ID NO: 1, or the base sequence of base numbers 64 to 1599 of the base sequence of SEQ ID NO: 3 and the TAG adjacent to the base of the base number 1599 The ability to hybridize under stringent conditions to mediate uptake of at least one amino acid into a cell, to DNA having a base sequence consisting of any one nonsense base sequence represented by TGA or TAA A DNA encoding a cell surface protein having the amino acid, wherein the incorporation of the amino acid into a cell is mediated without depending on the coexistence of any of the following proteins (A) or (B): :
(A) a protein having the amino acid sequence of SEQ ID NO: 6 or 8;
(B) A protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of SEQ ID NO: 6 or 8.

Here, “several amino acids” have the meaning as defined above.
In addition, “under stringent conditions” here means conditions for causing hybridization, and specifically means temperature and salt concentration. The temperature is generally about 36 to about 42 ° C., but can also be set as follows according to the length of the probe used and the degree of complementation.
For example, when hybridization is performed under 0.9% NaCl using a probe of 50 bases or more, a standard for the temperature (Tm) at which 50% dissociation occurs is obtained from the following formula, and the hybridization temperature is determined as follows: It can be set like a calculation formula.

Tm = 82.3 ℃ + 0.41 × (G + C)% − 500 / n−0.61 × (formamide)%
(N indicates the number of bases of the probe.)
Temperature = Tm-25 ° C
Moreover, when using a probe of 100 bases or more (in the case of G + C = 40 to 50%), it is a guide that Tm changes as shown in (1) and (2) below.
(1) Tm decreases by about 1 ° C. for every 1% mismatch.
(2) Tm decreases by 0.6 to 0.7 ° C. for every 1% formamide.
Therefore, the temperature conditions in the case of a combination of completely complementary strands can be as follows.
(A) 65-75 ° C. (no formamide added)
(B) 35-45 ° C. (in the presence of 50% formamide)
Moreover, the temperature conditions in the case of a combination of incomplete complementary strands can be as follows.
(A) 45-55 ° C. (no formamide added)
(B) 35-42 ° C. (in the presence of 30% formamide)
Further, the temperature condition when using a probe of 23 bases or less can be 37 ° C., and the following calculation formula can be used as a guide.
Temperature = 2 ° C. × (number of A + T) + 4 ° C. × (number of C + G) −5 ° C.
The salt concentration is usually set to 5 × SSC or a salt concentration equivalent thereto.
Therefore, the temperature in the hybridization in the present invention can be set to about 37 ° C., for example. The salt concentration can be set to 5 × SSC or a salt concentration equivalent thereto.
The DNA of the present invention described above may be obtained by any method. For example, complementary DNA (cDNA) prepared from mRNA, DNA prepared from genomic DNA, DNA obtained by chemical synthesis, DNA obtained by amplifying by PCR using RNA or DNA as a template, and appropriate combinations of these methods Any DNA constructed in this manner is encompassed by the DNA of the present invention.

The DNA encoding the protein of the present invention can be obtained by a method of cloning cDNA from the mRNA of the protein of the present invention according to a conventional method, a method of splicing genomic DNA, a method of chemical synthesis, or the like.
(1) For example, the following method is exemplified as a method for cloning cDNA from mRNA of the protein of the present invention.
First, mRNA encoding the protein of the present invention is prepared from the aforementioned tissue or cell that expresses and produces the protein of the present invention. The mRNA is prepared using a known method such as the guanidine thiocyanate method (Chirgwin et al., Biochemistry, Vol. 18, page 5294, 1979), the hot phenol method or the AGPC method. The obtained total RNA can be subjected to affinity chromatography using oligo (dT) cellulose, poly U-sepharose or the like.

  Then, using the obtained mRNA as a template, a known method such as using reverse transcriptase, for example, the method of Okayama et al. (Mol. Cell. Biol., Volume 2, page 161, 1982) And the same volume, Vol. 3, 280, 1983) and the method of Hoffman et al. (Gene, Vol. 25, 263, 1983), etc. Conversion to double-stranded cDNA is performed. This cDNA is incorporated into a plasmid vector or a phage vector, and E. coli is transformed, or after in vitro packaging, E. coli is transfected (transfected) to prepare a cDNA library.

The plasmid vector used here is not particularly limited as long as it can be replicated and retained in the host, and any phage vector that can be propagated in the host may be used. Examples of commonly used cloning vectors include λZipLox, pUC19, λgt10, and λgt11. However, when it is subjected to immunological screening described later, it is preferably a vector having a promoter capable of expressing a gene encoding the protein of the present invention in a host.
As a method for incorporating cDNA into a plasmid, for example, the method of Maniatis et al. (Molecular Cloning, A Laboratory Manual, Second Edition), Cold Spring Harbor Laboratory (Cold Spring Harbor Laboratory), 1.53, 1989). Examples of a method for incorporating cDNA into a phage vector include the method of Hyunh et al. (DNA cloning, a practical approach, Vol. 1, page 49, 1985). . For convenience, a commercially available cloning kit (for example, manufactured by GIBCO or Takara Shuzo) can also be used. The recombinant plasmid and phage vector thus obtained are introduced into a suitable host such as a prokaryotic cell (for example, E. coli: HB101, DH5α, MC1061 / P3, etc.).

  As a method for introducing a plasmid into a host, (Molecular Cloning, A Laboratory Manual (second edition), Cold Spring Harbor Laboratory, page 1.74, 1989) Examples include the calcium chloride method, calcium chloride / rubidium chloride method, electroporation method and the like. Examples of a method for introducing a phage vector into a host include a method for introducing phage DNA into an expanded host after in vitro packaging. In vitro packaging can be easily performed by using a commercially available in vitro packaging kit (for example, manufactured by Stratagene, Amersham, etc.).

The method for isolating cDNA encoding the protein of the present invention from the cDNA library prepared by the above method can be performed by combining general cDNA screening methods.
For example, after separately preparing DNA containing a part or all of the base sequence encoding the amino acid sequence of the protein of the present invention, or DNA having homology with the base sequence, this is prepared as ³² P or [α- ³² P ] Labeled with dCTP to form a probe, known colony hybridization method (Crunstein et al., Proc. Natl. Acid. Sci. USA), 72, 3961 1975) or a plaque hybridization method (Molecular Cloning, A Laboratory Manual, second edition, Cold Spring Harbor Laboratory, page 2.108, 1989), or a method for screening a clone containing a target cDNA, Producing and amplifying a specific region of the protein of the present invention by PCR, And a method of selecting a clone having a DNA fragment coding for.

In addition, when a cDNA library prepared using a vector capable of expressing cDNA (eg, λZipLox, λgt11 phage vector) is used, an antigen-antibody reaction using an antibody reactive with the protein of the present invention is used. The target clone can be selected. When a large amount of clones are processed, it is preferable to use a screening method using the PCR method.
The base sequence of the DNA thus obtained is the Maxam-Gilbert method (Maxam et al., Proc. Natl. Acad. Sci. USA., 74, 560, 1977), using phage M13. Dideoxynucleotide synthesis chain termination method (Sanger et al., Proc. Natl. Acad. Sci. USA., 74, 5463-5467, 1977) or dye terminator cycle sequencing method (Applied Biosysytems). The gene encoding the protein of the present invention can be obtained by cutting out all or part of the gene from the clone obtained as described above with a restriction enzyme or the like.

  (2) Moreover, as a preparation method by isolating DNA encoding the protein of the present invention from the genomic DNA derived from the cell expressing the protein of the present invention as described above, for example, the following method is exemplified. . The cells are preferably lysed using SDS or proteinase K or the like, and extraction with phenol is repeated to deproteinize DNA. RNA is preferably digested with ribonuclease. The obtained DNA is partially digested with an appropriate restriction enzyme, and the obtained DNA fragment is amplified with an appropriate phage or cosmid to prepare a library. A clone having the target sequence is detected by, for example, a method using a radiolabeled DNA probe, and all or a part of the gene encoding the protein of the present invention is cut out from the clone with a restriction enzyme or the like.

  (3) In addition, a cDNA encoding a protein having the amino acid sequence set forth in SEQ ID NO: 2 which is an embodiment of the DNA of the present invention can be produced by a conventional method based on the base sequence set forth in SEQ ID NO: 1. Can be done according to.

For example, rat C6 glioma cells are used as a gene source, and mRNA (poly (A) ⁺ RNA) is prepared therefrom. This is fractionated and each fraction is introduced into Xenopus oocytes along with 4F2hc cRNA.
The cDNA for the 4F2hc gene has already been reported [Broer et al., Biochem. J. et al. 312, 863, 1995], it is possible to easily obtain a 4F2hc gene from this sequence information by using a PCR method or the like. From the obtained 4F2hc cDNA, RNA (cRNA) complementary to this can be synthesized using T3 or T7 RNA polymerase or the like.

  For oocytes introduced with mRNA and cRNA of 4F2hc, for example, using leucine or the like as a substrate, measuring the transport (uptake) of the substrate into the cell, and selecting the mRNA fraction showing high uptake activity, LAT1 mRNA can be concentrated. A cDNA library is prepared based on the concentrated mRNA. About 500 clones were prepared from the library cDNA as a group, and cRNAs (capped ones) were prepared. Each group was introduced into an oocyte together with 4F2hc cRNA, and the substrate uptake activity was used as an index. Select a positive group. If a positive group is found, it is further divided into subgroups, and the same operation is repeated to obtain a clone containing the LAT1 gene cDNA.

Regarding the obtained cDNA, the base sequence can be determined by a conventional method, the translation region can be analyzed, and the amino acid sequence of the protein encoded thereby, that is, LAT1 can be determined.
It can be verified, for example, as follows, that the obtained cDNA is a neutral amino acid transporter gene cDNA, that is, the gene product encoded by the cDNA is a neutral amino acid transporter. That is, cRNA prepared from cDNA of the obtained LAT1 gene is introduced into oocytes together with 4F2hc cRNA for expression, and the ability to transport (incorporate) neutral amino acids into the cell is the same as described above. This can be confirmed by measuring the uptake of the substrate into cells by a normal uptake test using a neutral amino acid as a substrate (Kanai and Hediger, Nature, 360, 467-471 (1992)).

In addition, regarding the expressed cells, the same uptake experiment can be applied to examine the characteristics of LAT1, for example, the characteristic that LAT1 exchanges amino acids and the substrate specificity of LAT1.
Isolation of homologous genes and chromosomal genes from different tissues and organisms by screening appropriate cDNA libraries or genomic DNA libraries prepared with different gene sources using the obtained cDNA of LAT1 gene can do.
In addition, a normal PCR (Polymerase Chain Reaction) method using a synthetic primer designed based on the information of the disclosed base sequence of the gene of the present invention (base sequence shown in SEQ ID NO: 1 or a part thereof) A gene can be isolated from a cDNA library or a genomic DNA library.

  SEQ ID NO: 3 in the sequence listing below shows the full-length cDNA base sequence (about 3.5 kbp) of the gene of the neutral amino acid transporter (rat LAT1) derived from the rat C6 glioma cell line, and the protein encoded in the translation region thereof. Represents the amino acid sequence (512 amino acids). SEQ ID NO: 4 in the sequence listing represents the amino acid sequence (512 amino acids) of the neutral amino acid transporter (rat LAT1) derived from the rat C6 glioma cell line.

  DNA libraries such as cDNA libraries or genomic DNA libraries are disclosed in, for example, “Molecular cloning” (Sambrook, J., Fritsh, EF and Mantis, T., published by Cold Spring Harbor Press in 1989). It can be prepared by the method described. Alternatively, if there is a commercially available library, it may be used.

  In order to obtain cDNA encoding human-derived protein, a cosmid library ("Lab Manual Human Genome Mapping", edited by Masaaki Hori and Yusuke Nakamura, Maruzen Publishing) with further introduction of human genomic DNA (chromosomal DNA, genomic DNA) By preparing and screening the cosmid library, a positive clone containing DNA of the coding region of the target protein is obtained, and the aforementioned cDNA library is screened using the coding DNA cut out from the positive clone as a probe. Can also be prepared.

SEQ ID NO: 1 in the sequence listing below shows the full-length cDNA base sequence (about 4.5 kbp) of the gene of human-derived neutral amino acid transporter (human LAT1), and the amino acid sequence of the protein encoded by the translation region (507) Amino acid). Sequence number 2 of a sequence table represents the amino acid sequence (507 amino acids) of a human-derived neutral amino acid transporter (human LAT1).
SEQ ID NO: 5 in the sequence listing represents the full-length cDNA base sequence (about 1.8 kbp) of the gene of the human-derived 4F2hc protein and the amino acid sequence (529 amino acids) of the protein encoded in the translation region. SEQ ID NO: 6 represents the amino acid sequence of the human-derived 4F2hc protein (529 amino acids). SEQ ID NO: 7 in the sequence listing represents the full-length cDNA base sequence (about 1.8 kbp) of the rat-derived 4F2hc protein gene and the amino acid sequence (527 amino acids) of the protein encoded by the translation region. No. 8 represents the amino acid sequence (527 amino acids) of rat-derived 4F2hc protein.

  The “expression vector” of the present invention means a recombinant vector containing the above-described DNA of the present invention. The recombinant vector of the present invention is not particularly limited as long as it can be replicated and maintained in various prokaryotic and / or eukaryotic hosts, and includes plasmid vectors and phage vectors.

  The recombinant vector can be conveniently prepared by ligating the DNA of the present invention to a recombination vector (plasmid DNA and bacterial phage DNA) available in the art by a conventional method. Specific examples of the recombination vector used include pBR322, pBR325, pUC12, pUC13, and pUC19 as plasmids derived from E. coli, pSH19, pSH15, and the like as yeast-derived plasmids, and pUB110, pTP5, and pC194 as Bacillus subtilis-derived plasmids. Etc. are exemplified. Examples of the phage include bacteriophages such as λ phage, and animal and insect viruses (pVL1393, manufactured by Invitrogen) such as retrovirus, vaccinia virus, and nuclear polyhedrosis virus. Furthermore, pZL1 can be mentioned.

  For the purpose of expressing the protein of the present invention, an expression vector is useful. The expression vector is not particularly limited as long as it has an ability to express the protein of the present invention in various prokaryotic and / or eukaryotic host cells. For example, pMAL C2, pEF-BOS (Nucleic Acid Research, Vol. 18, p. 5322, 1990, etc.) or pME18S (Experimental Medicine separate volume “Gene Engineering Handbook”, 1992, etc.) be able to.

When bacteria, particularly Escherichia coli, is used as a host cell, an expression vector generally comprises at least a promoter-operator region, a start codon, DNA encoding the protein of the present invention, a stop codon, a terminator region, and a replicable unit.
When yeast, animal cells, or insect cells are used as the host, the expression vector preferably contains at least a promoter, a start codon, DNA encoding the protein of the present invention, and a stop codon. In addition, DNA encoding signal peptide, enhancer sequence, 5 ′ and 3 ′ untranslated regions of gene encoding protein of the present invention, splicing junction, polyadenylation site, selectable marker region or replicable unit, etc. May be included. Moreover, the gene amplification gene (marker) normally used according to the objective may be included.

The promoter-operator region for expressing the protein of the present invention in bacteria contains a promoter, an operator, and a Shine-Dalgarno (SD) sequence (for example, AAGG). For example, when the host is an Escherichia genus, preferred examples include those containing Trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, tac promoter and the like. Examples of promoters for expressing the protein of the present invention in yeast include PH05 promoter, PGK promoter, GAP promoter, and ADH promoter. When the host is Bacillus, SL01 promoter, SP02 promoter, penP promoter, etc. Can be mentioned. When the host is a eukaryotic cell such as a mammalian cell, examples include SV40-derived promoters, retrovirus promoters, heat shock promoters, and the like. SV-40 and retrovirus are preferred. However, it is not particularly limited to these. In addition, the use of an enhancer is an effective method for expression.
A suitable initiation codon is exemplified by methionine codon (ATG).
Examples of stop codons include commonly used stop codons (eg, TAG, TGA, TAA).

As the terminator region, a commonly used natural or synthetic terminator can be used.
A replicable unit refers to a DNA that has the ability to replicate its entire DNA sequence in a host cell, a natural plasmid, an artificially modified plasmid (a DNA fragment prepared from a natural plasmid) and Synthetic plasmids and the like are included. Suitable plasmids include E. coli. In Coli, plasmid pBR322, or an artificial modification thereof (DNA fragment obtained by treating pBR322 with an appropriate restriction enzyme), yeast 2μ plasmid or yeast chromosomal DNA in yeast, and plasmid pRSVneo (ATCC) in mammalian cells. 37198), plasmid pSV2dhfr (ATCC 37145), plasmid pdBPV-MMTneo (ATCC 37224), plasmid pSV2neo (ATCC 37149) and the like.
As the enhancer sequence, polyadenylation site and splicing junction site, those commonly used by those skilled in the art, such as those derived from SV40, respectively, can be used.

As the selection marker, a commonly used marker can be used by a conventional method. For example, antibiotic resistance genes such as tetracycline, ampicillin, neomycin or kanamycin are exemplified.
The gene amplification gene includes dihydrofolate reductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamate synthase gene, adenosine deaminase gene, ornithine decarboxylase gene, hygromycin-B-phosphotransferase gene, aspartate transcarbamylase gene Etc. can be illustrated.

The expression vector of the present invention can be prepared by linking at least the above promoter, start codon, DNA encoding the protein of the present invention, stop codon and terminator region continuously and circularly to suitable replicable units. it can. At this time, an appropriate DNA fragment (for example, a linker, other restriction sites, etc.) can be used by a conventional method such as digestion with a restriction enzyme or ligation using T4 DNA ligase, if desired.
The “transformed cell” of the present invention is a cell transformed with the above-described DNA or expression vector of the present invention, and is prepared by introducing the DNA or expression vector into a prokaryotic cell or eukaryotic cell as a host cell. can do.

The host cell used in the present invention is not particularly limited as long as it is compatible with the above-described expression vector and can be transformed, and is a natural cell or artificially established usually used in the technical field of the present invention. Examples include various cells such as recombinant cells (eg, bacteria (genus Escherichia, Bacillus), yeast (genus Saccharomyces, Pichia, etc.), animal cells or insect cells.
Preferred are E. coli or animal cells, specifically E. coli (DH5α, TB1, HB101, etc.), mouse-derived cells (COP, L, C127, Sp2 / 0, NS-1 or NIH3T3, etc.), rat-derived cells (PC12). , PC12h, etc.), hamster-derived cells (BHK, CHO, etc.), monkey-derived cells (COS1, COS3, COS7, CV1, Velo, etc.) and human-derived cells (Hela, diploid fibroblast-derived cells, HEK293 cells , Myeloma cells and Namalwa, etc.).

Introduction (transformation (transfection)) of an expression vector into a host cell can be carried out using a conventionally known method.
For example, in the case of bacteria (E. coli, Bacillus subtilis, etc.), for example, the method of Cohen et al. (Proc. Natl. Acad. Sci. USA., Vol. 69, p. 2110, 1972), the protoplast method (Mol. Gen Genet., Vol.168, p.111, 1979) and competent methods (J. Mol. Biol., Vol.56, p.209, 1971), for example in the case of Saccharomyces cerevisiae, (Proc. Natl. Acad. Sci. USA., Vol.75, p.1927, 1978) or lithium method (J. Bacteriol., Vol.153, p.163, 1983) For example, Graham's method (Virology, Vol. 52, p. 456, 1973). In the case of insect cells, for example, Summers et al.'S method (Mol. Cell. Biol., Vol. 3, p. 2156-2165, 1983), respectively.

The “protein” of the present invention can be expressed by culturing a transformed cell containing the expression vector prepared as described above (hereinafter used to include a transfectant) in a nutrient medium.
The nutrient medium preferably contains a carbon source, an inorganic nitrogen source or an organic nitrogen source necessary for the growth of the host cell (transformant). Examples of the carbon source include glucose, dextran, soluble starch, and sucrose. Examples of the inorganic or organic nitrogen source include ammonium salts, nitrates, amino acids, corn steep liquor, peptone, casein, meat extract, large extract, and the like. Examples include soybean cake, potato extract and the like. In addition, other nutrients (for example, inorganic salts (for example, calcium chloride, sodium dihydrogen phosphate, magnesium chloride), vitamins, antibiotics (for example, tetracycline, neomycin, ampicillin, kanamycin, etc.)) may be included as desired. .

Culturing is performed by methods known in the art. The culture conditions such as temperature, medium pH and culture time are appropriately selected so that the protein of the present invention is expressed in large quantities.
In addition, although the specific culture medium and culture | cultivation conditions used according to a host cell below are illustrated, it is not limited to these at all.
When the host is a bacterium, actinomycetes, yeast or filamentous fungus, for example, a liquid medium containing the above nutrient source is suitable. Preferably, the medium has a pH of 5-8.
When the host is E. coli, preferred media include LB medium, M9 medium (Miller et al., Exp. Mol. Genet, Cold Spring Harbor Laboratory, p.431, 1972) and the like. In such a case, the culture can be performed usually at 14 to 43 ° C. for about 3 to 24 hours with aeration and agitation as necessary.

When the host is Bacillus genus, the treatment can be performed usually at 30 to 40 ° C. for about 16 to 96 hours with aeration and stirring as necessary.
When the host is yeast, examples of the medium include Burkholder's minimum medium (Bostian, Proc. Natl. Acad. Sci. USA, Vol. 77, p. 4505, 1980), and the pH is 5-8. It is desirable to be. The culture is usually performed at about 20 to 35 ° C. for about 14 to 144 hours, and if necessary, aeration and agitation can be performed.
When the host is an animal cell, for example, a MEM medium (Science, Vol. 122, p. 501, 1952) containing about 5 to 20% fetal bovine serum, a DMEM medium (Virology, Vol. 8, p. 396, 1959), RPMI 1640 medium (J. Am. Med. Assoc., Vol. 199, p. 519, 1967), 199 medium (proc. Soc. Exp. Biol. Med., Vol. 73, p. 1, 1950) Etc. can be used. The pH of the medium is preferably about 6 to 8, and the culture is usually performed at about 30 to 40 ° C. for about 15 to 72 hours, and if necessary, aeration and agitation can be performed.
When the host is an insect cell, for example, Grace's medium containing fetal bovine serum (Proc. Natl. Acad. Sci. USA, Vol. 82, p. 8404, 1985) can be mentioned, and the pH is about 5-8. Is preferred. The culture is usually performed at about 20 to 40 ° C. for 15 to 100 hours, and aeration and agitation can be performed as necessary.

The protein of the present invention can be expressed by culturing the transformant prepared as described above using the above-described DNA or expression vector of the present invention under the culture conditions as described above.
When the protein of the present invention is prepared as a soluble protein, after the cell culture, the cells are collected and suspended in an appropriate buffer, and the cell walls of the cells and / or the like and / or the like by a method such as ultrasound, lysozyme, and freeze-thawing. After disrupting the cell membrane, a membrane fraction containing the protein of the present invention is obtained by a method such as centrifugation or filtration. The membrane fraction is solubilized using a surfactant such as Triton-X100 to obtain a crude solution. By subjecting the crude solution to a purification method generally used for purifying and isolating protein, the protein of the present invention can be isolated as a soluble protein.

  As an isolation and purification method, for example, a method using solubility such as salting out, solvent precipitation, dialysis, ultrafiltration, gel filtration, a method using molecular weight difference such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Methods using charges such as ion exchange chromatography and hydroxylapatite chromatography, methods using specific affinity such as affinity chromatography, methods using hydrophobic differences such as reversed-phase high-performance liquid chromatography, isoelectricity Examples thereof include a method using a difference in isoelectric point such as point electrophoresis.

The “RNA” of the present invention is the RNA as described in <5> above.
“Including the base sequence consisting of the base sequence of base numbers 1 to 1521 of the base sequence set forth in SEQ ID NO: 26 and any one nonsense base sequence represented by UAG, UGA or UAA adjacent to the base of the base number 1521 RNA. "
Here, the “nonsense base sequence” means any base sequence of UAG, UGA or UAA, which is also called termination codon, termination codon, nonsense codon, termination codon or termination signal, and is the termination point of translation into protein. Is a base sequence encoding.
The RNA of the present invention comprises the DNA according to the above <1>, that is, “a base sequence of base numbers 66 to 1586 of the base sequence of SEQ ID NO: 1 and a TAG, TGA or TAA adjacent to the base of the base number 1586”. A DNA comprising a nucleotide sequence consisting of any one nonsense nucleotide sequence represented by the above, and using a commercially available RNA polymerase (for example, T7 RNA polymerase) as a template, and preparing it by a conventional method. Can do.
The RNA of the present invention can be used for expressing the protein of the present invention in various cells. That is, by injecting the RNA of the present invention into, for example, Xenopus oocytes, the cells of the present invention can be expressed directly from the injected RNA without undergoing transcription from DNA to mRNA. Yes (Experimental Medicine Extra Number, “Biosignal Experiment”, Vol.11, No.3, p.30-38, 1993).

Another one of the present invention is the following DNA described in <4> above.
“DNA containing a partial base sequence in the base sequence shown in SEQ ID NO: 1 or DNA in which a part of the DNA is chemically modified, DNA containing a base sequence complementary to the partial base sequence, or one of the DNAs DNA whose part is chemically modified and having the following characteristics (1) and (2):
(1) The partial base sequence is a base sequence that does not completely match the partial base sequence in the base sequence set forth in SEQ ID NO: 3; and (2) the DNA or the chemically modified DNA is a sequence It hybridizes to a gene encoding a protein having the amino acid sequence described in No. 2. "

Here, the “partial base sequence in the base sequence described in SEQ ID NO: 1” means a partial base sequence composed of an arbitrary number of bases at an arbitrary site included in the base sequence described in SEQ ID NO: 1. .
The DNA is useful as a probe in DNA hybridization or RNA hybridization procedures. For the purpose of using the DNA as a probe, the partial base sequence includes a continuous partial base sequence of 20 bases or more, preferably a continuous partial base sequence of 50 bases or more, more preferably continuous 100 bases. The partial base sequence described above, more preferably a continuous partial base sequence of 200 bases or more, particularly preferably a continuous partial base sequence of 300 bases or more.

The above DNA is also useful as a primer in PCR. For the purpose of using the DNA as a PCR primer, the partial base sequence includes a continuous partial base sequence of 5 to 100 bases, preferably a continuous partial base sequence of 5 to 70 bases, more preferably continuous. 5 to 50 base partial base sequences, more preferably continuous 5 to 30 base partial base sequences.
Furthermore, the DNA is useful as an antisense drug. That is, the DNA can also inhibit transcription of the DNA into mRNA or translation of the mRNA into protein by hybridizing to DNA or RNA encoding a protein having the amino acid sequence of SEQ ID NO: 2. .

For the purpose of using the DNA as an antisense drug, the partial base sequence includes a continuous partial base sequence of 5 to 100 bases, preferably a continuous partial base sequence of 5 to 70 bases, more preferably Examples include a continuous partial base sequence of 5 to 50 bases, more preferably a continuous partial base sequence of 5 to 30 bases.
In addition, when this DNA is used as an antisense drug, it increases blood half-life (stability) when the DNA is administered into a patient's body, increases the permeability of an intracellular membrane, or is administered orally. For the purpose of increasing degradation resistance in the digestive organs or increasing absorption in some cases, it is possible to chemically modify a part of the base sequence of the DNA. Examples of the chemical modification include chemical modifications such as phosphate bond, ribose, nucleobase, sugar moiety, 3 ′ and / or 5 ′ end in the structure of the oligonucleotide.
Phosphate bond modifications include one or more of these bonds, phosphodiester bond (D-oligo), phosphorothioate bond, phosphorodithioate bond (S-oligo), methylphosphonate bond (MP-oligo), phosphoramido. Mention may be made of any of the date bonds, non-phosphate bonds and methylphosphonothioate bonds or combinations thereof. Examples of the modification of ribose include a change to 2′-fluororibose or 2′-O-methylribose. Nucleobase modifications include changes to 5-propynyluracil or 2-aminoadenine.

Another one of the present invention is the RNA as described in <6> above.
“RNA containing a partial base sequence in the base sequence of RNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 26, or RNA in which a part of the RNA is chemically modified, the RNA or the RNA The RNA that is chemically modified hybridizes to RNA that encodes a protein having the amino acid sequence set forth in SEQ ID NO: 2. "

Here, the “partial base sequence” means a partial base sequence composed of an arbitrary number of bases at an arbitrary site.
The above DNA is useful as an antisense drug. That is, the RNA can inhibit transcription of the DNA into mRNA or translation of the mRNA into protein by hybridizing to DNA or RNA encoding the protein having the amino acid sequence shown in SEQ ID NO: 2. it can.
For the purpose of using the RNA as an antisense drug, the partial base sequence includes a continuous partial base sequence of 5 to 100 bases, preferably a continuous partial base sequence of 5 to 70 bases, more preferably Examples include a continuous partial base sequence of 5 to 50 bases, more preferably a continuous partial base sequence of 5 to 30 bases.

  In addition, when this RNA is used as an antisense drug, the blood half-life is increased when the RNA is administered into the body of a patient, the permeability of the intracellular membrane is increased, or the digestive organ when administered orally. It is possible to chemically modify a part of the base sequence of the RNA for the purpose of increasing degradation resistance or increasing absorption. Examples of the chemical modification include chemical modification applied to the above-described antisense DNA.

The “antibody” of the present invention is a polyclonal antibody (antiserum) or a monoclonal antibody, preferably a monoclonal antibody.
Specifically, it is an antibody having reactivity with the protein of the present invention or a part thereof.
The “antibody” of the present invention is a protein of the present invention or a part thereof (including a natural body, a recombinant body, a chemical composition), or a cell (natural cell, transformed cell, normal cell or Obtained by immunizing non-human mammals such as mice, rats, hamsters, guinea pigs, chickens, rabbits, goats or sheep, using an immunogen (antigen) of any kind as an immunogen (antigen). Natural antibodies, recombinant chimeric monoclonal antibodies and recombinant human monoclonal antibodies (CDR-grafted antibodies) that can be produced using gene recombination technology, and human antibodies that can be produced using human antibody-producing transgenic animals, etc. Include.
In the case of a monoclonal antibody, a monoclonal antibody having any isotype such as IgG, IgM, IgA, IgD, or IgE is also included. Preferably, it is IgG or IgM.

The polyclonal antibody (antiserum) or monoclonal antibody referred to in the present invention can be produced by an existing general production method.
That is, for example, an immunogen (antigen) as described above, together with Freund's Adjuvant as necessary, a mammal, preferably a mouse, rat, hamster, guinea pig, rabbit, chicken, cat, dog, pig , Goats, horses or cows, more preferably mice, rats, hamsters, guinea pigs or rabbits.
A polyclonal antibody (antiserum) can be obtained from the serum obtained from the immunized animal.
Monoclonal antibodies include antibody-producing cells (spleen, lymph nodes, bone marrow, tonsils, etc., preferably splenic B cells) obtained from the immunized animal and myeloma cells (myeloma cells) having no autoantibody-producing ability. By preparing a hybridoma from the mouse, cloning the hybridoma, and selecting a clone producing a monoclonal antibody exhibiting specific affinity for the antigen used for immunization of the mammal by an immunoassay (such as ELISA) Manufactured.

  Specifically, the monoclonal antibody can be produced as follows. That is, the protein of the present invention or a part thereof (including a natural body, a recombinant body, a chemical composition), or a cell expressing the protein (a natural cell, a transformed cell, a normal cell, a tumor cell, etc.) Any immunogen) and, if necessary, Freund's Adjuvant, mouse, rat, hamster, guinea pig, chicken or rabbit, preferably mouse, rat or hamster (human antibody-producing transgenic) (Including transgenic animals engineered to produce antibodies from other animals such as mice) injected or implanted subcutaneously, intramuscularly, intravenously, in the Hoodpad or intraperitoneally one or several times To immunize. Usually, immunization is performed 1 to 4 times about every 1 to 14 days from the initial immunization, and antibody-producing cells can be obtained from the immunized mammal about 1 to 5 days after the final immunization.

Hybridomas that secrete monoclonal antibodies can be prepared according to the method of Köhler and Milstein et al. (Nature, Vol. 256, 495-497, 1975) and modifications according thereto.
That is, antibody-producing cells contained in the spleen, lymph node, bone marrow, tonsil, etc., preferably from the spleen obtained from the immunized mammal as described above, and preferably mouse, rat, guinea pig, hamster, rabbit or human It is prepared by cell fusion with a myeloma cell having no autoantibody producing ability derived from a mammal such as mouse, rat or human.

  Examples of myeloma cells used for cell fusion include mouse-derived myeloma P3 / X63-AG8.653 (653; ATCC No. CRL1580), P3 / NSI / 1-Ag4-1 (NS-1), P3 / X63-Ag8. . U1 (P3U1), SP2 / 0-Ag14 (Sp2 / 0, Sp2), PAI, F0 or BW5147, rat-derived myeloma 210RCY3-Ag. 2.3. Human-derived myeloma U-266AR1, GM1500-6TG-A1-2, UC729-6, CEM-AGR, D1R11 or CEM-T15 can be used.

The screening of hybridoma clones producing monoclonal antibodies is carried out by cultivating the hybridomas in, for example, a microtiter plate, and determining the reactivity of the culture supernatant of the wells in which proliferation has been observed with the immunizing antigen used in the aforementioned mouse immunization. For example, it can be carried out by measuring by an enzyme immunoassay such as RIA or ELISA.
Production of monoclonal antibodies from hybridomas is carried out in vitro or in vivo in mice, rats, guinea pigs, hamsters or rabbits, preferably mice or rats, more preferably mice ascites, and the resulting culture supernatant. Or by isolation from ascites of mammals.
When cultivating in vitro, in order to grow, maintain and preserve the hybridoma according to various conditions such as the characteristics of the cell type to be cultured, the purpose of the research and the culture method, etc., to produce a monoclonal antibody in the culture supernatant It is possible to carry out using any nutrient medium that is derived from a known nutrient medium or a known basic medium as used in the above.

Examples of the basic medium include a low calcium medium such as Ham'F12 medium, MCDB153 medium or low calcium MEM medium, and a high calcium medium such as MCDB104 medium, MEM medium, D-MEM medium, RPMI1640 medium, ASF104 medium or RD medium. The basic medium can contain, for example, serum, hormones, cytokines and / or various inorganic or organic substances depending on the purpose.
Isolation and purification of the monoclonal antibody can be carried out by using the above-mentioned culture supernatant or ascites, saturated ammonium sulfate, euglobulin precipitation method, caproic acid method, caprylic acid method, ion exchange chromatography (DEAE or DE52, etc.), anti-immunoglobulin column or It can be performed by subjecting to affinity column chromatography such as a protein A column.
In addition, a gene encoding a monoclonal antibody is cloned from the hybridoma, and a transgenic animal, goat, sheep or pig in which the antibody-coding gene is incorporated into an endogenous gene using a transgenic animal production technique is produced. It is also possible to obtain a large amount of a monoclonal antibody derived from the antibody gene from milk of a transgenic animal (Nikkei Science, April 1997, pages 78 to 84).

The “recombinant chimeric monoclonal antibody” of the present invention is a monoclonal antibody produced by genetic engineering. Specifically, for example, the variable region is a variable region derived from mouse immunoglobulin, and the constant region. Means a chimeric monoclonal antibody such as a mouse / human chimeric monoclonal antibody characterized by being a constant region derived from human immunoglobulin.
The constant regions derived from human immunoglobulins each have a unique amino acid sequence depending on isotypes such as IgG, IgM, IgA, IgD and IgE. It may be a constant region of groggrin. Preferably, it is a constant region of human IgG.

The chimeric monoclonal antibody in the present invention can be produced, for example, as follows. However, it goes without saying that it is not limited to such a manufacturing method.
For example, a mouse / human chimeric monoclonal antibody can be prepared with reference to experimental medicine (special issue), Vol. 1.6, No. 10, 1988, and Japanese Patent Publication No. 3-73280.

That is, downstream of the active V _H gene (rearranged VDJ gene encoding the heavy chain variable region) obtained from the DNA encoding the mouse monoclonal antibody isolated from the hybridoma producing the mouse monoclonal antibody, C _H gene (H-chain constant region C gene encoding), also active V _L genes obtained from DNA encoding the mouse monoclonal antibody isolated from the hybridoma obtained from the DNA encoding the Guromurin (L chain variable the C gene) encoding the C _L gene (L-chain constant region obtained from DNA encoding human immunoglobulin Guromu phosphorus downstream of rearranged VJ gene) encoding region, and arranged so as to each expressible 1 Inserting into one or separate expression vectors, transforming host cells with the expression vectors, It can be produced by culturing the transformed cells.

  Specifically, first, DNA is extracted from a mouse monoclonal antibody-producing hybridoma by a conventional method, and then the DNA is digested with an appropriate restriction enzyme (for example, EcoRI, HindIII, etc.) and subjected to electrophoresis (for example, 0.7 Perform Southern blotting using a% agarose gel. The electrophoresed gel is stained with, for example, ethidium bromide, and after taking a photograph, the marker is marked, the gel is washed twice, and immersed in a 0.25M HCl solution for 15 minutes. Then, immerse in 0.4N NaOH solution for 10 minutes while gently shaking. Transfer to a filter by a conventional method, collect the filter after 4 hours, and wash twice with 2 × SSC. After the filter is sufficiently dried, baking (75 ° C., 3 hours) is performed. After baking, the filter is placed in a 0.1 × SSC / 0.1% SDS solution and treated at 65 ° C. for 30 minutes. Then, soak in 3 × SSC / 0.1% SDS solution. The obtained filter is put into a plastic bag together with the prehybridization solution and treated at 65 ° C. for 3 to 4 hours.

Next, ³² P-labeled probe DNA and a hybridization solution are put into this and reacted at 65 ° C. for about 12 hours. After completion of hybridization, the filter is washed under an appropriate salt concentration, reaction temperature and time (for example, 2 × SSC-0.1% SDS solution, room temperature, 10 minutes). Place the filter in a plastic bag, add a small amount of 2 × SSC, seal, and perform autoradiography.
By the Southern blotting method, rearranged VDJ gene and VJ gene encoding the H chain and L chain of mouse monoclonal antibody, respectively, are identified. The region containing the identified DNA fragment is fractionated by sucrose density gradient centrifugation and incorporated into a phage vector (for example, Charon 4A, Charon 28, λEMBL3, λEMBL4, etc.), and E. coli (for example, LE392, NM539, etc.) ) To produce a genomic library. For example, the Benton Davis method (Science, Vol. 196, pp. 180-182, 1977) can be obtained by using an appropriate probe (H chain J gene, L chain (κ) J gene, etc.). ), Plaque hybridization is performed to obtain positive clones each containing the rearranged VDJ gene or VJ gene. A restriction enzyme map of the obtained clone is prepared, the nucleotide sequence is determined, and it is confirmed that the gene containing the target rearranged V _H (VDJ) gene or _VL (VJ) gene is obtained. .

On the other hand, isolated Separately, human C _H gene and human C _L gene used for chimerization. For example, when a chimeric antibody with human IgG1 is produced, isolating the Cκ gene is C gamma ₁ gene and the C _L gene is C _H gene. These genes using mouse C gamma ₁ gene and mouse Cκ gene, corresponding to human C gamma ₁ gene and human Cκ gene by utilizing high homology in nucleotide sequence of the mouse immunoglobulin gene and the human immunoglobulin genes as a probe, the human genome live It can be obtained by isolation from a rally.

Specifically, for example, a 3 kb HindIII-BamHI fragment from clone Ig146 (Procedural National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 75, 4709-4713, 1978). And a 6.8 kb EcoRI fragment from clone MEP10 (Proc. Natl. Acad. Sci. USA, Vol. 78, pp. 474-478, 1981) as a probe. A DNA fragment containing a human Cκ gene and containing an enhancer region from the HaeIII-AluI genomic library of lambda Charon 4A (Cell, Vol. 15, pp. 1157 to 1174, 1978) Isolate. Moreover, the human C gamma ₁ gene, for example, a human fetal hepatocyte DNA is digested with HindIII, after fractionated by agarose gel electrophoresis, and inserted into a band of 5.9kb in Ramuda788, isolated with the probes .

Thus the mouse V _H gene was to isolated and mouse V _L gene, and human C _H gene and using human C _L gene, human downstream of the mouse V _H gene while considering the promoter region and enhancer region a C _H gene, also a human C _L gene downstream of a mouse V _L gene, with appropriate restriction enzymes and DNA ligase, incorporated in a conventional manner, for example, in the expression vector such as pSV2gpt or pSV2neo. In this case, chimeric genes of mouse V _H gene / human C _H gene and mouse V _L gene / human C _L gene can be arranged simultaneously on one expression vector, may each be placed in separate expression vectors it can.
The chimeric gene insertion expression vector thus prepared was used as a protoplast fusion method, DEAE-dextran method, calcium phosphate method or electrolysis method for myeloma cells that did not produce antibodies themselves, such as P3X63 / Ag8 / 653 cells or SP210 cells. Introduced by drilling method. The transformed cells are selected by culturing in a drug-containing medium corresponding to the drug resistance gene introduced into the expression vector to obtain the desired chimeric monoclonal antibody-producing cells.
The target chimeric monoclonal antibody is obtained from the culture supernatant of the antibody-producing cells thus selected.

  The “human antibody (CDR-grafted antibody)” of the present invention is a monoclonal antibody produced by genetic engineering, specifically, for example, a part or all of the complementarity determining region of the hypervariable region. Is the complementarity determining region of the hypervariable region derived from a mouse monoclonal antibody, the framework region of the variable region is the framework region of the variable region derived from human immunoglobulin, and the constant region is a constant region derived from human immunoglobulin It means a human monoclonal antibody characterized by

  Here, the complementarity-determining regions of the hypervariable regions are the three regions (Complementarity-determining residues; CDR1, CDR2) that exist in the hypervariable regions in the variable region of the antibody and directly bind to the antigen in a complementary manner. , CDR3), and the framework region of the variable region refers to four relatively conserved regions (Framework: FR1, FR2, FR3, FR4) interposed before and after the three complementarity determining regions.

In other words, for example, a monoclonal antibody in which all regions other than part or all of the complementarity determining region of the hypervariable region of a mouse monoclonal antibody are replaced with the corresponding region of human immunoglobulin.
The constant region derived from human immunoglobulin has a unique amino acid sequence depending on the isotype such as IgG, IgM, IgA, IgD, and IgE. It may be a constant region of glycine. Preferably, it is a constant region of human IgG. Further, the framework region of the variable region derived from human immunoglobulin is not limited.

The human monoclonal antibody in the present invention can be produced, for example, as follows. However, it goes without saying that it is not limited to such a manufacturing method.
For example, a recombinant human monoclonal antibody derived from a mouse monoclonal antibody can be prepared by genetic engineering with reference to JP-T-4-506458 and JP-A-62-2296890.
That is, from a hybridoma producing a mouse monoclonal antibody, at least one mouse H chain CDR gene and at least one mouse L chain CDR gene corresponding to the mouse H chain CDR gene are isolated, and the mouse H chain CDR gene is isolated from a human immunoglobulin gene. A human H chain gene encoding the entire region other than the human H chain CDR corresponding to the chain CDR and a human L chain gene encoding the entire region other than the human L chain CDR corresponding to the pre-mouse L chain CDR are isolated.

  The isolated mouse H chain CDR gene and the human H chain gene are introduced into an appropriate expression vector so that they can be expressed, and similarly, the mouse L chain CDR gene and the human L chain gene are also expressed appropriately. Into another expression vector. Alternatively, the mouse H chain CDR gene / human H chain gene and the mouse L chain CDR gene / human L chain gene can also be introduced so that they can be expressed in the same expression vector. By transforming host cells with the expression vector thus prepared, human-type monoclonal antibody-producing transformed cells are obtained, and by culturing the transformed cells, the desired human-type monoclonal antibody is obtained from the culture supernatant. obtain.

The “human antibody” of the present invention means that all regions including the variable region of the heavy chain and the constant region of the heavy chain, and the variable region of the light chain and the constant region of the light chain encode human immunoglobulin. It is an immunoglobulin derived from a gene.
The human antibody can be obtained by immunizing a transgenic animal prepared by incorporating at least a human immunoglobulin gene into a locus of a mammal other than human, such as a mouse, with an antigen according to a conventional method. It can be produced in the same manner as the polyclonal antibody or monoclonal antibody production method.

For example, transgenic mice that produce human antibodies include Nature Genetics, Vol. 15, p.146-156, 1997); Nature Genetics, Vol. 7, p.13-21, 1994; International Publication WO94 / 25585; Nikkei Science, June, 40-50, 1995; Nature, Vol. 368, p.856-859, 1994; and JP 6-500233 It can be produced according to the method described in the publication.
The “part of the antibody” in the present invention means a region of a part of the above-mentioned antibody, preferably a monoclonal antibody in the present invention. Specifically, F (ab ′) ₂ , Fab ′, Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv) or dAb (single domain antibody) (Exp. Opin. Ther. Patents, Vol. 6, No. 5, p. 441-456, 1996).

Here, “F (ab ′) ₂ ” and “Fab ′” are produced by treating immunoglobulin (monoclonal antibody) with a protease such as pepsin or papain, and the two in the hinge region. An antibody fragment produced by digestion before and after the disulfide bond existing between the heavy chains. For example, when IgG is treated with papain, it is cleaved upstream of a disulfide bond existing between two H chains in the hinge region, and _L consisting of V _L (L chain variable region) and C _L (L chain constant region). And two homologous antibody fragments in which the H chain fragment consisting of the chain and V _H (H chain variable region) and CHγ ₁ (γ1 region in the H chain constant region) are linked by a disulfide bond in the C-terminal region. Can do. Each of these two homologous antibody fragments is called Fab ′.

In addition, when IgG is treated with pepsin, an antibody fragment that is cleaved downstream of the disulfide bond existing between the two heavy chains in the hinge region to produce a slightly larger antibody fragment than the two Fab's connected in the hinge region is produced. Can do. This antibody fragment is called F (ab ') ₂ .

The “cell producing the monoclonal antibody” of the present invention means any cell that produces the above-described monoclonal antibody of the present invention. Specifically, for example, the cells described in any of (1) to (3) below can be mentioned.
(1) As described above, the protein of the present invention, a part thereof, or a monoclonal antibody having reactivity to the protein of the present invention or a part thereof obtained by immunizing a non-human mammal with a cell or the like expressing the protein A monoclonal antibody-producing B cell derived from the non-human mammal that produces
(2) The aforementioned hybridoma (fused cell) obtained by cell fusion of the antibody-producing B cell thus obtained with a myeloma cell derived from a mammal.
(3) By a gene encoding the monoclonal antibody isolated from the monoclonal antibody-producing B cell or the monoclonal antibody-producing hybridoma (either a gene encoding a heavy chain or a gene encoding a light chain, or both genes) Monoclonal antibody-producing transformed cells (genetically modified cells) obtained by transforming cells other than the B cells and hybridomas.

  Here, the monoclonal antibody-producing transformed cell (gene recombinant cell) described in the above (3) is a monoclonal antibody gene recombinant produced by the B cell of (1) or the hybridoma of (2). It means a transgenic cell to be produced. This recombinant monoclonal antibody-producing cell can be produced in the same manner as that used in the production of the chimeric monoclonal antibody and human antibody described above.

The “pharmaceutical composition” of the present invention is a pharmaceutical composition comprising, for example, any of the following (a) to (c) and a pharmaceutically acceptable carrier.
(A) An antibody as defined above (preferably a monoclonal antibody, not limited to a naturally occurring antibody or a recombinant antibody) or a part of the antibody.
(B) DNA fragment useful as an antisense drug: For example, the following DNA:
“A partial base sequence in the base sequence shown in SEQ ID NO: 1 or 3, preferably a DNA containing a partial base sequence of 14 bases or more, or a DNA in which a part of the DNA is chemically modified, or complementary to the partial base sequence DNA containing a typical base sequence or DNA in which a part of the DNA is chemically modified "
(C) RNA fragment useful as an antisense drug: for example, the following RNA:
“RNA containing a partial base sequence in the base sequence of RNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 26 or 27, or RNA in which a part of the RNA is chemically modified, Alternatively, the chemically modified RNA hybridizes to RNA encoding a protein having the amino acid sequence set forth in SEQ ID NO: 2. "

As used herein, “pharmaceutically acceptable carrier” refers to excipients, diluents, extenders, disintegrants, stabilizers, preservatives, buffers, emulsifiers, fragrances, colorants, sweeteners, thickeners. , Flavoring agents, solubilizers or other additives. By using one or more of such carriers, pharmaceuticals in the form of tablets, pills, powders, granules, injections, solutions, capsules, trowels, elixirs, suspensions, emulsions or syrups, etc. A composition can be prepared.
These pharmaceutical compositions can be administered orally or parenterally. Other forms for parenteral administration include topical solutions containing one or more active substances and prescribed by conventional methods, suppositories for enteric administration, pessaries, and the like.

The dose varies depending on the patient's age, sex, weight and symptoms, therapeutic effect, administration method, treatment time, type of active ingredient (such as the above-mentioned protein or antibody) contained in the pharmaceutical composition, etc. The dose can be administered in a range of 10 μg to 1000 mg (or 10 μg to 500 mg) per person. However, since the dose varies depending on various conditions, a dose smaller than the above dose may be sufficient, or a dose exceeding the above range may be required.
In particular, in the case of injections, the concentration is 0.1 μg antibody / ml carrier to 10 mg antibody / ml carrier in a non-toxic pharmaceutically acceptable carrier such as physiological saline or commercially available distilled water for injection. It can be produced by dissolving or suspending in the solution.

The thus-prepared injection is administered to a human patient in need of treatment at a rate of 1 μg to 100 mg per kg body weight in a single administration, preferably at a rate of 50 μg to 50 mg per day. Can be administered several times to several times. Examples of administration forms include medically suitable administration forms such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, or intraperitoneal injection. Intravenous injection is preferred.
In addition, injections can be prepared as non-aqueous diluents (for example, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, etc.), suspensions or emulsions. .
Such sterilization of the injection can be performed by filtration sterilization through a bacteria-retaining filter, blending of a bactericide, or irradiation. The injection can be produced as a form prepared at the time of use. That is, it can be used as a sterile solid composition by lyophilization, etc., and dissolved in sterile water for injection or other solvents before use.
The pharmaceutical composition of the present invention inhibits, for example, the biological activity of the amino acid transporter molecule of the present invention or the expression of the molecule, and the uptake of amino acids as nutrients essential for the survival or growth of tumor cells into the cells. For example, it can be used for the treatment of cancer.

In the “transgenic mouse” of the present invention, a DNA (cDNA or genomic DNA) encoding a human-derived protein included in the protein of the present invention is integrated on the endogenous locus of the mouse, It is a transgenic mouse that expresses the protein of the present invention in the body.
Specifically, it is a transgenic mouse as described in the following <1> or <2> described in <52> or <53>.
(1) A transgenic mouse having a foreign gene, wherein the mouse expresses the protein by incorporating DNA encoding the protein having the amino acid sequence of SEQ ID NO: 2 on the endogenous gene. A transgenic mouse characterized by having a cell to do in the body.
(2) The nonsense base sequence represented by TAG, TGA or TAA adjacent to the base sequence of base numbers 66 to 1586 and the base of base number 1586 of the base sequence described in SEQ ID NO: 1 The transgenic mouse according to (1) above, which is a DNA comprising a base sequence consisting of

The transgenic mouse is prepared by a conventional method as commonly used in the production of transgenic animals (for example, the latest animal cell experiment manual, published by L.I.C., Chapter 7, pages 361-408, 1990). Reference).
Specifically, an embryonic stem cell (Embryonic Stem Cell, ES Cell) obtained from a normal mouse blastcyst is a gene and marker encoding a protein having the amino acid sequence of SEQ ID NO: 2 of the present invention. A gene (for example, neomycin resistance gene) is transformed with an expression vector inserted so that it can be expressed. ES cells in which the gene encoding the protein is integrated on the endogenous gene are selected by a conventional method based on the presence or absence of the expression of the marker gene. Next, the selected ES cells are microinjected into fertilized eggs (alveolar vesicles) obtained from another normal mouse (Proc. Natl. Acad. Sci. USA, Vol. 77, No. 12, pp. 7380-7384). 1980; US Pat. No. 4,873,191). The blastocyst is transplanted into the uterus of another normal mouse as a foster parent. Thus, a founder mouse (child mouse) is born from the temporary parent mouse. A heterotransgenic mouse is obtained by crossing the founder mouse with a normal mouse. By crossing the heterogeneic transgenic mice, homogeneic transgenic mice can be obtained according to Mendel's law.

Further, DNA encoding a mouse-derived protein included in the present invention (particularly genomic DNA), that is, DNA encoding a mouse homologue of a human-derived amino acid transporter having the amino acid sequence set forth in SEQ ID NO: 2 (particularly, A so-called “knockout mouse” can be prepared based on the base sequence of genomic DNA.
The knockout mouse is a mouse in which an endogenous gene encoding the mouse homolog protein is knocked out (inactivated). For example, a positive negative selection method using homologous recombination (US Pat. No. 5,627,059, Proc. Natl. Acad. Sci. USA, Vol. 86, 8932-8935, 1989, Nature, Vol. 342, 435-438, 1989, etc.). Such a knockout mouse is also an embodiment of the present invention.

The “labeled DNA” of the present invention is an enzyme, fluorescent substance, chemiluminescent substance, biotin, avidin, or radioisotope ( ³ H, ¹⁴ C, ¹²⁵ I or ¹³¹ I etc.).
For example, radiolabeled DNA labeled with a radioisotope is used for various test methods such as Southern blotting (experimental medicine / separate volume, “Gene Engineering Handbook”, published by Yodosha). , P.133-140, 1992).
In addition, labeled DNA labeled with a radioactive substance or a non-radioactive substance such as biotin is used for in situ hybridization (for example, FISH (Fluorescence in situ) for analyzing the position of genomic DNA encoding the protein of the present invention on the chromosome. hybridization), experimental medicine / separate volume, “Genetic Engineering Handbook”, published by Yodosha, 1992, pp. 271 to 277).

The “radiolabeled RNA” of the present invention is DNA obtained by labeling the RNA of the present invention with a radioisotope such as ³ H, ¹⁴ C, ¹²⁵ I or ¹³¹ I.
The radiolabeled RNA is analyzed for the expression state of mRNA encoding the protein of the present invention in cells, tissues, or organs, for example, Northern blotting (Experimental Medicine / separate volume, “Genetic Engineering Handbook”, published by Yodosha, p.133). -140, 1992).

The “labeled substance capable of producing a detectable signal by reacting alone or with another substance” for labeling the “labeled monoclonal antibody” of the present invention refers to a monoclonal antibody antibody as defined above It means a substance that is used to make the presence of the monoclonal antibody detectable by binding to it by physicochemical binding or the like.
Specifically, an enzyme, a fluorescent substance, a chemiluminescent substance, biotin, avidin, a radioactive isotope, or the like.

More specifically, peroxidase (for example, horseradish peroxidase), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, Enzymes such as apoglucose oxidase, urease, luciferase or acetylcholinesterase, fluorescent substances such as fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelates, dansyl chloride or tetramethylrhodamine isothiocyanate, ³ H, ¹⁴ C, ¹²⁵ I or A radioactive isotope such as ¹³¹ I, biotin, avidin, or a chemiluminescent substance.

  Here, the radioisotope and the fluorescent substance can provide a detectable signal alone. On the other hand, enzymes, chemiluminescent substances, biotin, and avidin alone cannot produce a detectable signal, and thus, react with one or more other substances to produce a detectable signal. For example, in the case of an enzyme, at least a substrate is required, and various substrates are used depending on a method for measuring enzyme activity (colorimetric method, fluorescence method, bioluminescence method, chemiluminescence method, etc.). For example, in the case of peroxidase, hydrogen peroxide is used as a substrate. In the case of biotin, the reaction is generally carried out using at least avidin or enzyme-modified avidin (for example, streptavidin-β-galactosidase) as a substrate, but this is not a limitation. Various color-developing substances depending on the substrate are used as necessary. For example, when streptavidin-β-galactosidase is used as the substrate for biotin, 4-methyl-umbelliferyl-β-D-galactoside should be used as the chromogenic substance. Can do.

The “labeled monoclonal antibody” and the aforementioned “labeled DNA” of the present invention mean monoclonal antibodies and DNAs labeled with various labeling substances as described above.
The labeled monoclonal antibody can be used for detecting or quantifying the aforementioned protein of the present invention. Specifically, various biological samples such as cells (regardless of normal cells, abnormal cells such as tumor cells derived from diseases, natural cells, or genetically modified cells), tissues Presence or absence of expression of the protein of the present invention (regardless of the origin of a healthy organism or a living organism suffering from a disease) or an organ (regardless of the origin of a healthy organism or a living organism suffering from a disease) Or it can use for the measurement of an expression level. The measurement can be performed according to a conventional method using a conventional immunohistological technique (Experimental Medicine separate volume, “Cell Engineering Handbook”, Yodosha, p.207-213, 1992).
In addition, the labeled monoclonal antibody of the present invention is prepared not only by the immunohistological test described above, but also by preparing a soluble membrane protein from the cells, tissues or organs or a part thereof as a test sample according to a conventional method. It can also be used in Western blotting, which comprises confirming the presence or absence of the protein of the present invention in the soluble membrane protein by reacting the labeled monoclonal antibody with the membrane protein (Experimental Medicine separate volume, “Cell Engineering Handbook” "Yodosha, p.201-206, 1992).
In the immunological measurement method as described above, a labeled monoclonal antibody labeled with any of the above-mentioned labeling substances can be used, but considering the high detection sensitivity or quantitative sensitivity and the convenience of operation. It is preferable to use a monoclonal antibody labeled with an enzyme such as peroxidase or biotin.

The present invention also relates to a method for detecting or quantifying the protein of the present invention by an immunohistological technique using the labeled monoclonal antibody described above. Specifically, it is a method as described above, for example, a method including the following steps (1) and (2).
(1) contacting the sample with the labeled monoclonal antibody of the present invention; and (2) the amount of the labeled monoclonal antibody bound to the sample depending on the type of the labeling substance bound to the labeled monoclonal antibody, A step of measuring by detecting fluorescence, chemiluminescence or radioactivity.

Here, “cell” refers to a primary culture cell obtained from a human body, a cell line established so as to be passaged, or a genetically modified cell (transformed cell). And is preferably a primary cultured cell. In addition, the cells include normal cells and abnormal cells obtained from the living body of a patient suffering from a disease. Examples of the abnormal cells include various tumor cells. The “tissue” means any tissue derived from a living body of a healthy animal or a patient suffering from a disease, and examples of the tissue derived from the living body of the patient include tumor tissue. The term “organ or part thereof” means any organ or part thereof derived from the living body of a healthy animal or a patient suffering from a disease. Examples of the organ derived from the patient include an organ having a tumor.
More specifically, the method of the present invention can include, for example, the following steps, but is not limited thereto.

(Step 1) For example, normal cells, normal tissues or normal organs or parts thereof derived from healthy individuals that are excised and eliminated during surgery, or tumor cells, tumor tissues, or organs having tumors derived from cancer patients Or a part thereof (an organ or a part thereof can be sliced into slices if necessary) is immobilized with paraformaldehyde or the like to prepare an immobilized sample;
(Step 2) A step of adding the labeled monoclonal antibody of the present invention labeled with an enzyme such as biotin or peroxidase to the immobilized sample to cause an antigen-antibody reaction;
(Step 3) Next, after washing the immobilized sample as necessary, depending on the type of enzyme used, various substrates or enzyme-modified avidin such as avidin or streptavidin-β-galactosidase was added, A step of reacting avidin or enzyme-modified avidin with a labeling substance on the labeled antibody (for example, in the case of using a labeled antibody labeled with an enzyme such as peroxidase in step 2, diaminobenzidine, 4-chloro-1 Hydrogen peroxide can be added with either -naphthol or aminoethylcarbazole, and avidin or enzyme-modified avidin is used when using a labeled antibody labeled with biotin in step 2);
(Step 4) When enzyme-modified avidin is added in Step 3, various substrates (for example, 4-methyl-umbelliferyl-β-D-galactoside, etc.) depend on the type of enzyme used for the modification. And reacting the substrate bound with the enzyme bound to avidin;
(Step 5) A step of washing the immobilized sample as necessary to stop the enzyme reaction and the color reaction; and (Step 6) observing the immobilized sample under a microscope to obtain a colorimetric intensity, a fluorescence intensity, or A step of measuring luminescence intensity.
Since the amino acid transporter protein of the present invention is observed specifically for a wide variety of tumor cells compared to the expression in normal cells, it can be used in various ways derived from living organisms using the immunohistological method as described above. By detecting the presence or absence of expression of the protein in a cell or tissue, it may be possible to determine whether the test cell or test tissue is a normal cell or an abnormal cell such as a tumor cell.

Another one of the present invention is a method for identifying a substance having the ability to inhibit the biological activity of the protein of the present invention. Specifically, for example, the method described above.
“The biological function of the protein having the amino acid sequence shown in SEQ ID NO: 2 or 4, which is leucine (Leu), isoleucine (Ile), phenylalanine (Phe), methionine (Met), tyrosine (Tyr), histidine ( His), tryptophan (Trp), and valine (Val), a method for identifying a substance having the ability to inhibit the ability to mediate intracellular uptake of any one amino acid selected from the group consisting of: The method comprises the following steps (1) and (2):
(1) Any one of the following cells (a) to (d) is added to the substance, leucine (Leu), isoleucine (Ile), phenylalanine (Phe), methionine (Met), tyrosine (Tyr), histidine (His) ), Tryptophan (Trp) and valine (Val) in the presence of any one of the amino acids selected from the group consisting of a radioisotope labeled with a radioisotope, or in the presence of the radiolabeled amino acid alone Step of culturing:
(A) a naturally occurring cell co-expressing the protein having the amino acid sequence set forth in SEQ ID NO: 2 or 4 and the protein having the amino acid sequence set forth in SEQ ID NO: 6;
(B) A protein having the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 6 or 8 are obtained by co-transformation with DNA containing the base sequence of the translation region of the base sequence of SEQ ID NO: 1 or 3. A recombinant cell co-expressing a protein having the amino acid sequence described;
(C) a base sequence consisting of the base sequence of base numbers 1 to 1521 of the base sequence shown in SEQ ID NO: 26 and any one nonsense base sequence represented by UAG, UGA or UAA adjacent to the base of base number 1521 And the nucleotide sequence of nucleotide numbers 1 to 1587 of the nucleotide sequence of SEQ ID NO: 27 and any one nonsense nucleotide sequence represented by UAG, UGA or UAA adjacent to the nucleotide of the nucleotide number 1587 A non-human-derived recombinant cell co-expressing the protein having the amino acid sequence set forth in SEQ ID NO: 2 and the protein having the amino acid sequence set forth in SEQ ID NO: 6 by introduction of RNA containing the base sequence together; or ( d) human-derived tumor cells; and (2) cells cultured in the presence of the substance and the radiolabeled amino acid. Step radioactivity and the radioactivity of cells cultured in the presence of only the radiolabeled amino acid has to compare the difference in each value included in the. "
That is, this method co-expresses the amino acid transporter protein of the present invention (having the amino acid sequence described in SEQ ID NO: 2) and the human-derived cell membrane surface molecule 4F2hc (having the amino acid sequence described in SEQ ID NO: 6). Cells have at least one amino acid of leucine (Leu), isoleucine (Ile), phenylalanine (Phe), methionine (Met), tyrosine (Tyr), histidine (His), tryptophan (Trp) or valine (Val) It is a method characterized by utilizing the property of having the ability to capture.

That is, the inhibitory activity of the test substance is determined by culturing the cell in the presence of any of the above amino acids labeled with a radioisotope ( ³ H, ¹⁴ C, ¹²⁵ I, ¹³¹ I, etc.) and the test substance. The amount of the labeled amino acid taken up by the cells can be measured by comparing the amount of the labeled amino acid taken up by cells cultured in the presence of only the labeled amino acid without containing the test substance.
As the cell, any cell can be used as long as it is a cell co-expressing the two protein molecules. For example, a natural cell as described in (a) above, a transformed cell (genetical recombination cell) transformed with two DNAs each encoding the both protein molecules as described in (b) Either a cell into which RNA encoding each of the protein molecules as described in (c) is introduced or a human-derived tumor cell as described in (d) can be used.
As the host cell used for the preparation of the transformed cell, various cells described in the detailed description of the method for expressing the protein of the present invention using the DNA of the present invention described above can be used.

For example, various cells (for example, bacteria (Escherichia, Bacillus), yeast (Saccharomyces, Pichia, etc.) such as natural cells or artificially established recombinant cells that are usually used in the technical field of the present invention. )), Animal cells or insect cells.
Preferred are E. coli or animal cells, specifically E. coli (DH5α, TB1, HB101, etc.), mouse-derived cells (COP, L, C127, Sp2 / 0, NS-1 or NIH3T3, etc.), rat-derived cells (PC12). , PC12h, etc.), hamster-derived cells (BHK, CHO, etc.), monkey-derived cells (COS1, COS3, COS7, CV1, Velo, etc.) and human-derived cells (Hela, cells derived from diploid fibroblasts, HEK293 cells , Myeloma cells, Namalwa and the like).
Examples of cells into which the RNA is injected include Xenopus oocytes (Experimental Medicine Extra Number, “Biosignal Experimental Method”, Vol. 11, No. 3, p. 30-38, 1993) .
The human-derived tumor cell may be any tumor cell, and both the protein having the amino acid sequence shown in SEQ ID NO: 2 and the protein having the amino acid sequence shown in SEQ ID NO: 6 are expressed. It is preferable to use tumor cells that have been confirmed.

Here, the “substance” means a natural substance existing in nature or an arbitrary substance prepared artificially. The substance can be roughly classified into “peptidic substance” and “non-peptidic substance”.
The “peptidic substance” includes the above-described antibody of the present invention (preferably a monoclonal antibody, particularly preferably a recombinant human monoclonal antibody or a human monoclonal antibody), an oligopeptide, or a chemical modification thereof. Can be mentioned. Examples of oligopeptides include peptides consisting of 5 to 30 amino acids, preferably 5 to 20 amino acids. The chemical modification can be designed according to various purposes such as an increase in blood half-life when administered to a living body or an increase in resistance to or absorption in the digestive tract upon oral administration. .

The “non-peptide substance” includes “DNA containing a partial base sequence or chemically modified DNA obtained by chemically modifying them” useful as an antisense drug described in detail in the definition of the invention <4> above, 6> “RNA containing a partial base sequence or chemically modified RNA obtained by chemically modifying them” or any chemically synthesized “compound” useful as an antisense drug described in detail in the definition of the invention of 6> . Here, the “compound” is a compound excluding DNA, RNA, and the above peptidic substances, and is a compound having a molecular weight of about 100 to about 1000 or less, preferably a compound having a molecular weight of about 100 to about 800, more preferably. May include compounds having a molecular weight of about 100 to about 600.
The substance identified by the identification method of the present invention is preferably a substance having the ability to inhibit the growth of any tumor cell that occurs in any tissue of the human body. Examples of the tissue include brain, cervix, liver, pancreas, kidney, large intestine, small intestine, duodenum, prostate, lung, stomach, heart, skin, bone marrow, uterus, ovary, testis, oral cavity, tongue, bone or chest. Can be mentioned.

Yet another one of the present invention is a method for identifying a substance having the ability to inhibit transcription of a gene encoding the protein of the present invention into mRNA or expression of the protein of the present invention. Specifically, for example, the method is as described above.
“Method for identifying a substance having the ability to inhibit transcription of a gene encoding a protein having the amino acid sequence described in SEQ ID NO: 2 or 4 into mRNA or expression of the protein having the amino acid sequence described in SEQ ID NO: 2 or 4 A method comprising the following steps:
(1) A cell cotransformed with the following DNAs (a), (b) and (c), wherein the cell has the amino acid sequence of SEQ ID NO: 2 encoded by the DNA of (a) Depending on the expression of the protein possessed, a cell transformed to express the reporter protein encoded by the DNA of (c) at the same time in the presence or absence of the substance. Step of culturing:
(A) DNA containing the base sequence of the translation region of the base sequence set forth in SEQ ID NO: 1 or 3;
(B) DNA containing the base sequence of the translation region of the base sequence set forth in SEQ ID NO: 5 or 7;
(C) DNA encoding a reporter protein; and (2) a step of measuring and comparing the expression level of the reporter protein in each of cells cultured in the presence of the substance and cells cultured in the absence of the substance. . "

  That is, this method is a method collectively called a so-called reporter gene assay. Specifically, DNA encoding the amino acid transporter molecule of the present invention, DNA encoding the expression control region of the DNA And a DNA encoding a fluorescent reporter protein (such as luciferase derived from firefly or Renilla, or GFP (Green Fluorescence Protein) derived from jellyfish), depending on the expression of the transporter molecule, A test compound is brought into contact with a transformed cell obtained by transforming a cell generally used in the production of a recombinant protein with an expression vector inserted so that the compound can be expressed. The amount of transporter molecule expressed in dependence on the expression of the molecule A method of analyzing whether the compound affects the expression of a transporter molecule by indirectly measuring the amount of fluorescence emitted by the reporter protein to be expressed (for example, the United States See US Pat. No. 5,436,128 and US Pat. No. 5,401,629).

In addition, the identification of the compound using this assay can be performed manually, but so-called high throughput screening (tissue culture engineering, Vol. 23, No. .13, p.521-524; US Pat. No. 5,670,113) can be performed more quickly and easily.
The terms “cell” and “substance” used in the above method have the meanings as defined above.

Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited only to the embodiments described in the examples.
In the following examples, each operation is described in “Molecular cloning” (Sambrook, J., Fritsh, EF and Mantis, T., published by Cold Spring Harbor Press in 1989) unless otherwise specified. In the case of using a commercially available reagent or kit, it was used according to the instructions for the commercially available product.

Example 1 Isolation of cDNA of Human Cell Membrane Surface Molecule 4F2hc and Preparation of cRNA (1) Preparation of cDNA Fragment Encoding Rat 4F2hc by RT-PCR According to a conventional method, poly (A) ⁺ RNA purified from rat liver was prepared did. Further, a 5 ′ primer (SEQ ID NO: 9) and a 3 ′ primer (SEQ ID NO: 10) were synthesized based on a cDNA sequence encoding rat 4F2hc (Biochem. J., Vol. 312, p. 863, 1995).
Using the poly (A) ⁺ RNA as a template, RT-PCR (Reverse transcription-polymerase chain reaction; Experimental Medicine / Special Issue, “PCR and its application”, No. 8 using the two primers and Taq polymerase (manufactured by TAKARA) Vol. 9, No., 1990; and “Gene Amplification PCR Method: Fundamentals and New Development”, published by Kyoritsu Shuppan Co., Ltd., 1992). The reaction was performed using a DNA thermal cycler (Perkin Elmer Cetus) according to the protocol attached to the polymerase.
The amplified cDNA was subjected to agarose gel electrophoresis and purified using a DNA extraction kit (manufactured by Qiagen) to obtain a rat 4F2hc gene fragment (positions 34 to 479 of the base sequence described in SEQ ID NO: 7).
The cDNA sequence encoding rat 4F2hc and the corresponding amino acid sequence are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

(2) Acquisition of cDNA encoding human 4F2hc and preparation of cRNA Using a kit for cDNA synthesis (trade name: Superscript Choice System, manufactured by Gibco), according to the experimental operation method attached to the kit, A) A human cDNA was prepared from ⁺ RNA (manufactured by Clontech), and the cDNA was incorporated into a restriction enzyme EcoRI cleavage site of a phage vector λZipLox (manufactured by Gibco) using a DNA ligase (manufactured by Gibco) to prepare a human cDNA library. .
The rat 4F2hc gene fragment obtained in (1) above was labeled with ³² P-dCTP and used as a probe for plaque hybridization.
Using the probe, the human cDNA library prepared above was screened as follows.
The cDNA library was spread on an agar plate, and a replica was prepared using a commercially available filter membrane. Using this replica and the radioactive probe, hybridization was performed overnight at 37 ° C. in a hybridization solution. As a solution for hybridization, 5 × SSC, 3 × Denhard's solution (Denhard's solution) 0.2% SDS, 10% dextran sulfate, 50% formamide, 0.01% Abform B (antifoaming agent, manufactured by Sigma), 0.2 mg A pH 6.5 buffer solution containing / ml salmon sperm denatured DNA, 2.5 mM sodium pyrophosphate, and 25 mM MES was used. The filter membrane was washed with 0.1 × SSC / 0.1% SDS at 37 ° C.
A positive clone selected by the hybridization was isolated with a single plaque, then subjected to in vivo excision, recombined with plasmid pZL1 (Gibco) and recovered as plasmid DNA. The plasmid DNA was further subcloned into pBlueScriptII SK (-) (Stratagene).
In order to determine the base sequence of human 4F2hc cDNA contained in the obtained clone, seven types of primers were synthesized (SEQ ID NO: 11 to SEQ ID NO: 17). The cDNA base sequence was determined by the dye terminator cycle sequencing method (Applied Biosystems) using the seven synthetic primers and the commercially available universal primers T7 primer and SP6 primer (Stratagene). As a result, it was confirmed that the cloned cDNA was that of the human 4F2hc gene.
The cDNA sequence encoding human 4F2hc and the corresponding amino acid sequence are shown in SEQ ID NO: 5 and SEQ ID NO: 6, respectively.
From the plasmid containing human 4F2hc cDNA obtained above, cRNA (RNA complementary to cDNA, SEQ ID NO: 27) was prepared according to a conventional method using T7 RNA polymerase (Stratagene) (Experimental Medical Supplement, “ Biosignal experiment method ", Vol.11, No.3, p.33-34, 1993).

Example 2 Isolation of cDNA of Human Amino Acid Transporter LAT1 and Preparation of cRNA Using a kit for cDNA synthesis (trade name: Superscript Choice System, manufactured by Gibco Co., Ltd.), according to the experimental operation method attached to the kit, Human cDNA is prepared from normal cell line PA-1-derived poly (A) ⁺ RNA (purchased from Clontech), and the DNA is ligated (manufactured by Gibco) and the cDNA is used as a restriction enzyme EcoRI of phage vector λZipLox (manufactured by Gibco). An integrated human cDNA library was prepared at the cleavage site.
CDNA encoding rat amino acid transporter LAT1 (DDBJ / EMBL / GenBank accession number: AB015432, segment corresponding to nucleotide numbers 1135 to 1529 in SEQ ID NO: 3) was excised with the restriction enzyme BamHI. The amino acid sequence of rat amino acid transporter LAT1 is shown in SEQ ID NO: 4.
This DNA segment was labeled with ³² P-dCTP and used as a probe for plaque hybridization.
Using the probe, the human cDNA library prepared above was screened as follows.
The cDNA library was spread on an agar plate, and a replica was prepared using a commercially available filter membrane. Using this replica and the radioactive probe, hybridization was performed overnight at 37 ° C. in a hybridization solution. As a solution for hybridization, 5 × SSC, 3 × Denhard's solution (Denhard's solution) 0.2% SDS, 10% dextran sulfate, 50% formamide, 0.01% Abform B (antifoaming agent, manufactured by Sigma), 0.2 mg A pH 6.5 buffer solution containing / ml salmon sperm denatured DNA, 2.5 mM sodium pyrophosphate, and 25 mM MES was used. The filter membrane was washed with 0.1 × SSC / 0.1% SDS at 37 ° C.
A positive clone selected by the hybridization was isolated with a single plaque, subjected to in vivo excision, recombined with plasmid pZL1 (Stratagene), and recovered as plasmid DNA. The plasmid DNA was cleaved with the restriction enzyme PstI to obtain three cDNA fragments having sizes of 1.8 kb, 2.5 kb and 4.3 kb. The 1.8 kb and 2.5 kb fragments were each subcloned into pBlueScriptII SK (-) (Stratagene). In addition, the 4.3 kb cDNA fragment was self-ligated.
In order to determine the nucleotide sequence of the cDNA of the human amino acid transporter LAT1 contained in each plasmid having the obtained three cDNA fragments, eight kinds of primers were synthesized (SEQ ID NO: 18 to SEQ ID NO: 25). The cDNA base sequence was determined by the dye terminator cycle sequencing method (Applied Biosystems) using the 8 synthetic primers and the commercially available universal primers M13 forward primer and M13R reverse primer (Stratagene).
The sequence of the full-length cDNA encoding the obtained human amino acid transporter LAT1 and the corresponding amino acid sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
Furthermore, cRNA (SEQ ID NO: 26, RNA complementary to cDNA) was prepared from the plasmid containing cDNA encoding the human amino acid transporter LAT1 using T3 RNA polymerase (Stratagene).
Analysis of the homology of the respective amino acid sequences of rat amino acid transporter LAT1 and human amino acid transporter LAT1 revealed that human LAT1 had about 91% amino acid homology with rat LAT1. The results are shown in FIG.
As a result of analyzing the amino acid sequence of the human amino acid transporter LAT1 by hydrophobicity plot analysis (Kyte-Doolittle hydropathy analysis), it is confirmed that human LAT1 is a cell membrane surface molecule having 12 membrane-spanning domains. Estimated. The results are shown in FIG.

Example 3 Analysis of mRNA expression of human amino acid transporter LAT1 in various human tissues cDNA encoding human amino acid transporter LAT1 (cDNA fragment corresponding to bases 649 to 1128 of SEQ ID NO: 1) was restricted. It was cut out with SmaI and labeled with ³² P-dCTP to obtain a hybridization probe. Using this probe, Northern blotting on various human tissues was performed as follows.
Nylon membrane (trade name: MTN Blot: manufactured by Clontech) blotted with human poly (A) ⁺ RNA was hybridized and washed using the ³² P-dCTP-labeled human LAT1 probe according to the protocol attached to the kit. It was. The results are shown in FIG. 3 (fractions (a) to (c)).
As a result, expression of human LAT1 mRNA having a size of about 4.8 kb was observed in the placenta, brain, testis, bone marrow, and fetal liver. Weak mRNA expression was also observed in peripheral blood leukocytes.

Example 4 Analysis of biological activity of human amino acid transporter LAT1 (1) Analysis of ability to mediate transport of amino acids into cells Based on previous studies on tumor cell growth, heavy proteins classified as type II membrane glycoproteins Heavy chain (4F2hc), a known cell membrane surface antigen named 4F2 (CD98), a heterodimer of chain and light chain, plays an important role in activating an unidentified amino acid transporter (J. Immunol., Vol. 126, p. 1409-1414, 1981; J. Immunol., Vol. 129, p. 623-628, 1982; Proc. Natl. Acad. Sci. USA., Vol.84, p.6526-6530, 1987; Cancer Res., Vol.46, p.1478-1484, 1986; J. Biol. Chem., Vol.267, p.15285-15288 1992; Proc. Natl. Acad. Sci. USA., Vol. 89, p. 5606-5610, 1992; Biochem. J., Vol. 324, p. 535-541, 1997; and J. Exp. Biol. , Vol.196, p.123-137, 1994).
Therefore, whether or not the amino acid transporter LAT1 of the present invention is responsible for transporting amino acids into cells is determined using both cells expressing only human LAT1 and cells expressing both human LAT1 and human 4F2hc. The amount of leucine (neutral amino acid) taken up in the cells was measured and analyzed.
This test is based on the method using Xenopus oocytes, which is widely used in the uptake test of various substances into cells (Experimental Medicine Extra Number, “Biosignal Experimental Method”, Vol.11, No. 3, p.30-38, 1993).
CRNA (25 ng) encoding human LAT1 prepared in the above example alone, cRNA (25 ng) encoding human 4F2hc alone prepared in the above example, or cRNA (17.5 ng) encoding human LAT1 and human 4F2hc An encoded cRNA (7.5 ng) is injected into Xenopus oocytes and cultured for 2 days or 5 days, whereby an oocyte expressing only human LAT1 or an oocyte expressing only human 4F2hc, And oocytes co-expressing human LAT1 and human 4F2hc were prepared.
Using ^14C radiolabeled radiolabeled leucine as a substrate, the incorporation of the labeled leucine in each oocyte was carried out by the method of Kanai et al. (Kanai and Hediger, Nature, Vol. Accordingly, the procedure was performed as follows.
Specifically, each oocyte consists of a choline chloride uptake solution (100 mM choline chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride and 5 mM HEPES) containing ¹⁴ C-labeled leucine (50 μM). The amount of ¹⁴ C-labeled leucine incorporated into the cells by culturing for 30 minutes in .4) was determined by measuring the radioactivity of the cells with a scintillation counter. As a control, an experiment was similarly performed using an oocyte in which none of the above RNAs were injected and water alone was injected. The results are shown in FIG.
As a result, the oocytes expressing only human LAT1 showed almost no leucine uptake as in the oocytes injected with water as a control, but the oocytes expressed both human LAT1 and human 4F2hc. Large uptake of leucine was confirmed in the cells. From this result, it was considered that human 4F2hc is necessary for the human amino acid transporter LAT1 to exert a function of mediating amino acid uptake.

(2) Analysis of salt dependency of amino acid transport into cells The presence or absence of salt dependency of the ability of human amino acid transporter LAT1 to mediate transport of amino acids into cells was analyzed as follows. Specifically, in Example 4 (1), analysis was performed by observing changes in the amount of leucine taken up into the cells caused by changing the type of uptake solution for culturing oocytes.
Xenopus oocytes co-expressing human LAT1 and human 4F2hc prepared in Example 4 (1), including choline chloride uptake solution containing the aforementioned ¹⁴ C-labeled leucine (50 [mu] ^{M), 14} C-labeled leucine (50 [mu] M) Sodium uptake solution (100 mM choline chloride in the above choline uptake solution changed to 100 mM sodium chloride), or gluconic acid uptake solution containing ¹⁴ C-labeled leucine (50 μM) (100 mM sodium chloride in the sodium uptake solution is changed to 100 mM gluconic acid) Incubate for 30 minutes.
The amount of ¹⁴ C-labeled leucine incorporated into the cells was determined by measuring the radioactivity of the cells with a scintillation counter. The results are shown in FIG.
As a result, even when extracellular choline was changed to sodium and extracellular chloride ion was changed to gluconate ion, leucine uptake into the cell was not affected at all. From this, it was shown that the human amino acid transporter LAT1 is a transporter molecule that works independently of sodium ions and chloride ions.

(3) Affinity of the human amino acid transporter LAT1 to the substrate In order to analyze the affinity of the human amino acid transporter LAT1 to the substrate, the Michaelis-Menten kinetic test (Biochemical Dictionary, 2nd edition, 1307-1308, 4th printing, 1992).
This kinetic test was conducted by examining the change in the leucine uptake rate depending on the concentration of leucine as a substrate.
The leucine uptake experiment was performed according to the method described in Example 4 (1) above using Xenopus oocytes co-expressing human LAT1 and human 4F2hc. The results are shown in FIG.
As a result, the Michaelis constant (Km) was about 21 μM.

(4) Analysis of substrate specificity of human amino acid transporter LAT1 (Part 1)
The substrate specificity of the human amino acid transporter LAT1 (the type of substrate incorporated into cells via LAT1) was analyzed by a competitive antagonism test.
Specifically, Xenopus oocytes co-expressing human LAT1 and human 4F2hc are cultured in the presence of a test substance (various amino acids, drugs, physiologically active substances, or other low-molecular synthetic compounds). Analysis was performed by measuring changes in the amount of ¹⁴ C-labeled leucine incorporated into the cells as a substrate. When the amount of ¹⁴ C-labeled leucine taken up is reduced compared to the control without the addition of the test substance, it is indicated that the test substance is taken into the cell via the human amino acid transporter LAT1.
Xenopus oocytes co-expressing human LAT1 and human 4F2hc prepared in Example 4 (1) were mixed in a choline chloride uptake solution containing ¹⁴ C-labeled leucine (20 μM) and one of the following test substances (2 mM). For 30 minutes.
In addition, the culture | cultivation in the ¹⁴ C labeling leucine containing choline uptake solution which does not contain any test substance as a control was performed similarly.
[Test substance]
Glycine, alanine, serine, threonine, cysteine, leucine, isoleucine, phenylalanine, methionine, tyrosine, histidine, tryptophan, valine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, proline, and BCH (2-amino-2- norbornane-carboxylic acid). The amount of ¹⁴ C-labeled leucine incorporated into the cells was determined by measuring the radioactivity of the cells with a scintillation counter. The results are shown in FIG.
Furthermore, instead of the ¹⁴ C-labeled leucine, ¹⁴ C-labeled phenylalanine was used to examine the following test substance uptake into cells according to the method described above. As a control, culture in a ¹⁴ C-labeled phenylalanine-containing choline uptake solution not containing any test substance was performed in the same manner.
[Test substance]
L-DOPA (parkinson's disease drug) and triiodothyronine (thyroid hormone).
The results are shown in FIGS.
As a result, a cis-inhibiting effect on the incorporation of ¹⁴ C-labeled leucine or ¹⁴ C-labeled phenylalanine into cells was observed with various amino acids, drugs and physiologically active substances. In particular, leucine, isoleucine, phenylalanine, methionine, tyrosine, histidine, tryptophan, and valine strongly inhibited the uptake of ¹⁴ C-labeled leucine via human LAT1, so that any of these amino acids enter the cell via human LAT1. It was strongly suggested to be transported. In addition, 2-amino-2-norbornane-carboxylic acid (BCH), known as a neutral amino acid uptake inhibitor, also inhibited the uptake of ¹⁴ C-labeled leucine. Incorporation of ¹⁴ C-labeled phenylalanine into cells was strongly inhibited by drugs such as L-DOPA (parkinson's disease therapeutic drug) and physiologically active substances such as triiodothyronine (thyroid hormone). On the other hand, when acidic amino acids (glutamic acid, aspartic acid) or basic amino acids (lysine, arginine) were used as test substances, there was no effect on the uptake of ¹⁴ C-labeled leucine via human LAT1.
As a result, the human amino acid transporter LAT1 has been introduced into cells such as various amino acids (particularly neutral or nearly neutral amino acids), various drugs, various physiologically active substances, and other low-molecular synthetic compounds. It strongly suggests that it mediates transport.

(5) Analysis of substrate specificity of human amino acid transporter LAT1 (Part 2)
Based on the knowledge in Example 4 (4), the presence or absence of uptake of leucine, isoleucine, phenylalanine, methionine, tyrosine, histidine, tryptophan and valine into cells via human LAT1 was analyzed.
This test was carried out in the same manner as in Example 2 (1), using the following ¹⁴ C-labeled amino acids, each of which was labeled with ¹⁴ C, as a substrate instead of ¹⁴ C-labeled leucine.
[ ¹⁴ C-labeled amino acid]
¹⁴ C-labeled leucine, ¹⁴ C-labeled isorosine, ¹⁴ C-labeled phenylalanine, ¹⁴ C-labeled methionine, ¹⁴ C-labeled tyrosine, ¹⁴ C-labeled histidine, ¹⁴ C-labeled tryptophan, or ¹⁴ C-labeled valine.
For comparison, ¹⁴ C-labeled glycine, ¹⁴ C-labeled serine, ¹⁴ C-labeled D-leucine, and ¹⁴ C-labeled D-phenylalanine were also tested in the same manner. The results are shown in FIG.
As a result, it was confirmed that leucine, isoleucine, phenylalanine, methionine, tyrosine, histidine, tryptophan and valine were significantly incorporated into oocytes.

Example 5 Analysis of mRNA expression of human amino acid transporter LAT1 in various human-derived tumor cells Using ISOGEN (trade name: manufactured by Nippon Gene) from various human-derived tumor cells, total RNA was obtained by a conventional method, The RNA is subjected to agarose electrophoresis according to a conventional method and blotted on a nitrocellulose membrane.
Northern blotting is performed using a hybridization probe prepared by labeling the cDNA fragment encoding the human amino acid transporter LAT1 prepared in Example 3 with ³² P-dCTP. The Northern blotting is performed according to a protocol attached to a commercially available nylon membrane for Northern blotting (for example, trade name: MTN Blot: manufactured by Clontech) in which various poly (A) ⁺ RNAs are blotted.
By this northern blotting, the expression of mRNA encoding human LAT1 in various human-derived tumor cells can be confirmed.

Example 6 (Cloning of rat neutral amino acid transporter)
(1) Isolation of rat 4F2hc cDNA and preparation of cRNA The cDNA library was prepared from a poly (A) ⁺ RNA purified from rat liver using a cDNA synthesis kit (trade name: Superscript Choice System, manufactured by Gibco). And was incorporated into the restriction enzyme EcoRI cleavage site of the phage vector λZipLox (Gibco). By PCR, a segment corresponding to nucleotides 135 to 580 of the rat 4F2hc gene (Broer et al., Biochem. J., Vol. 312, 863, 1995) was amplified, and this was amplified with ³² P-dCTP. A rat liver cDNA library was screened using the labeled and probe. Hybridization was performed overnight in a hybridization solution at 37 ° C., and the filter membrane was washed with 0.1 × SSC / 0.1% SDS at 37 ° C. As a solution for hybridization, 5 × SSC, 3 × denhard's solution (Denhard's solution) 0.2% SDS, 10% dextran sulfate, 50% formamide, 0.01% Abform B (trade name, Sigma) (antifoaming agent) ), A buffer solution of pH 6.5 containing 0.2 mg / ml salmon sperm denatured DNA, 2.5 mM sodium pyrophosphate, 25 mM MES was used. The cDNA portion of λZipLox phage incorporating the cDNA was incorporated into the plasmid pZL1 and further subcloned into the plasmid pBluescriptIISK- (Stratagene).

About the obtained clone, ie, clone containing rat 4F2hc cDNA, dideoxy method using synthetic primer for base sequence determination, base sequence determination kit (trade name: Sequenase ver. 2.0, manufactured by Amersham) Thus, the nucleotide sequence of cDNA was determined. This confirmed that the cloned cDNA was that of the rat 4F2hc gene. The obtained base sequence of 4F2hc is shown in SEQ ID NO: 2 in the Sequence Listing described later.
From the plasmid containing rat 4F2hc cDNA obtained above, using T7 RNA polymerase. cRNA (RNA complementary to cDNA) was prepared.

(2) Cloning of rat neutral amino acid transporter LAT1 According to the method of Kanai et al. (Kanai and Hediger, Nature, Vol. 360, 467-471 Mitsugu, 1992), the expression cloning method was used as follows. It was.
400 μg of rat C6 glioma cell poly (A) ⁺ RNA was fractionated by gel electrophoresis.
Each fraction obtained by fractionation was injected into the oocyte together with the rat 4F2hc cRNA obtained in (1) above and cultured for 2 days.
For the oocytes injected with RNA, leucine was used as a substrate, and the substrate uptake experiment was performed according to the method of Kanai et al. (Kanai and Hediger, Nature, Vol. 360, 467-471 Mitsugu, 1992) as follows. Went to. 30 in choline chloride uptake solution (100 mM choline chloride, 2 mM potassium chloride, 1.8 mM calcium chloride, 1 mM magnesium chloride, 5 mM HEPES, pH 7.4) containing ¹⁴ C-leucine (50 μM) as substrate. The oocytes were cultured, and the substrate uptake rate was measured by counting the radioactivity incorporated into the cells. In this system, the oocytes injected with both rat C6 glioma cell poly (A) ⁺ RNA (mRNA) and rat 4F2hc cRNA are synergistically uptake compared to the oocytes injected alone. It was confirmed that enhancement was observed (FIG. 11).

Among the RNA fractions obtained by fractionation, the fraction in which the oocyte injected with RNA showed the highest leucine uptake rate was selected. Using this fraction poly (A) ⁺ RNA (2.8-4.0 kb), a cDNA library was prepared using a cDNA synthesis and plasmid cloning kit (trade name: Superscript Plasmid System, manufactured by Gibco) did. These DNAs are incorporated into restriction enzyme Sal1 and Not1 recognition sites of plasmid pSPORT1 (manufactured by Gibco), and the resulting recombinant plasmid DNA is transferred to a competent cell of E. coli DH10B strain (trade name: Electro Max DH10B Competent cell, manufactured by Gibco). Introduced. The obtained transformant was cultured on a nitrocellulose membrane, and about 500 colonies were obtained per plate. Plasmid DNA was prepared from these colonies, and cut with the restriction enzyme NotI. Using the obtained DNA, capped cRNA was synthesized by in vitro transcription.

The obtained cRNA (about 45 ng) was injected into the oocyte together with the rat 4F2hc cRNA obtained in (1) above (5 ng). These oocytes were screened for positive clones by conducting a leucine uptake experiment in the same manner as described above. In screening, a group in which DNA extracted from a plurality of clones was pooled was examined. When leucine uptake was confirmed in a certain group, it was further divided into a plurality of groups and further screening was performed.
About the obtained clone, that is, a clone containing the rat neutral amino acid transporter LAT1 cDNA, a synthetic primer for base sequence determination, a base sequence determination kit (trade name: Sequenase ver. 2.0, manufactured by Amersham) Was used to determine the base sequence of cDNA by the dideoxy method.
Thereby, the base sequence of the rat neutral amino acid transporter LAT1 gene was obtained. Moreover, the base sequence of cDNA was analyzed by a conventional method, and the translation region of cDNA and the amino acid sequence of LAT1 encoded there were determined. The translation region is bases 64-1599.

These sequences are shown in SEQ ID NOs: 4 (amino acid sequence) and 3 (base sequence) in the sequence listing below.
As a result of analyzing the amino acid sequence of LAT1 by Kyte-Doolittle hydropathy analysis (hydrophobicity plot), as shown in FIG. 12, 12 membrane-spanning domains were predicted. Further, there were two sites considered to be tyrosine phosphorylation sites in the second hydrophilic loop and protein kinase C-dependent phosphorylation sites in the fourth and eighth hydrophilic loops.

(3) Expression of LAT1 gene in various rat tissues and rat cell lines (analysis by Northern blotting)
A cDNA fragment corresponding to nucleotides 202 to 1534 of the rat LAT1 gene was labeled with ³² P-dCTP and used as a probe to extract RNA from various tissues of rats and cultured tumor cell lines derived from rats. On the other hand, Northern blotting was performed as follows. 3 μg of poly (A) ⁺ RNA was electrophoresed on a 1% agarose / formaldehyde gel and then transferred to a nitrocellulose filter. This filter was hybridized overnight at 42 ° C. with a hybridization solution containing the LAT1 cDNA fragment labeled with ³² P-dCTP. The filter was washed with 0.1 × SSC containing 0.1% SDS at 65 ° C.
As a result of Northern blotting (FIG. 13), a band was detected in the vicinity of 3.8 kb in the C6 glioma cells, placenta, brain, spleen, large intestine, and testis, and in the placenta, a band was detected in the vicinity of 2.6 kb, and expression was observed. Although expression was very weak in normal liver, a strong band was detected around 3.8 kb in the transformed rat hepatocyte cell line and hepatocellular carcinoma cell line, and expression was observed (FIG. 14).
Furthermore, it was exposed to light for a long time, and a faint band was detected near 3.8 kb in other tissues.

(4) Expression of LAT1 gene in human tumor cell lines (analysis by Northern blotting)
A cDNA fragment corresponding to nucleotides 202 to 1534 of the rat LAT1 gene was labeled with ³² P-dCTP, and using this as a probe, Northern blotting was performed on RNA extracted from a human-derived cultured tumor cell line as follows. I went as follows. 3 μg of poly (A) ⁺ RNA was electrophoresed on a 1% agarose / formaldehyde gel and then transferred to a nitrocellulose filter. This filter was hybridized overnight at 37 ° C. with a hybridization solution containing a rat LAT1 cDNA fragment labeled with ³² P-dCTP. The filter was washed with 0.1 × SSC containing 0.1% SDS at 37 ° C.
As a result of Northern blotting (FIG. 15), a strong band of 4.0 kb was observed in the gastric signet ring cell carcinoma cell line, small lung cell carcinoma cell line, and melanoma cell line, and a weak band of 4.0 kb was present in the neuroblastoma cell line. Detected and expression was observed.

Example 7 (Characterization of neutral amino acid transporter LAT1)
(1) Role of 4F2hc in LAT1 transport activity Leucine uptake activity when rat LAT1 gene cRNA is expressed alone in oocytes and when both rat LAT1 gene cRNA and 4F2hc gene cRNA are expressed in oocytes Compared.
Rat LAT1 gene cRNA (25 ng), rat 4F2hc gene cRNA (25 ng), or rat LAT1 gene cRNA (12.5 ng) / rat 4F2hc gene cRNA (12.5 ng) were expressed by injection into oocytes and cultured for 2 or 5 days.
The leucine uptake experiment was performed as follows according to the method described in Example 6 (2). That is, an oocyte injected with rat LAT1 gene cRNA, rat 4F2hc gene cRNA, or rat LAT1 gene cRNA and rat 4F2hc gene cRNA was cultured in an uptake solution containing ¹⁴ C-leucine (50 μM) for 30 minutes. The uptake of radioactivity into the cells was measured.
As a result (FIG. 16), leucine uptake was the same level in the oocytes that expressed only LAT1 as in the oocytes injected with water as a control, but the oocytes that expressed both LAT1 and 4F2hc. The cells showed a large uptake of leucine, and it was considered that 4F2hc is necessary for LAT1 to exert its function.

(2) Salt Dependence of LAT1 Transport Activity Both rat LAT1 gene cRNA and 4F2hc gene cRNA were examined for the effect of salts added to the medium in leucine uptake experiments by oocytes.
The leucine uptake experiment was performed according to the method described in Example 6 (2) above using oocytes injected with both rat LAT1 gene cRNA and rat 4F2hc gene cRNA. However, as the uptake solution, when the influence of sodium ions was observed, a sodium uptake solution (100 mM choline chloride was changed to 100 mM sodium chloride) was used instead of the choline chloride uptake solution. When examining the influence of chloride ions, a solution for gluconic acid uptake (100 mM sodium chloride was changed to 100 mM sodium gluconate) was used instead of the sodium uptake solution.
As a result (FIG. 17), changing extracellular choline to sodium or changing extracellular chloride ion to gluconate ion had no effect on leucine uptake. This indicates that LAT1 is a transporter that works independently of sodium ions and chloride ions.

(3) Michaelis-Menten kinetic test of LAT1 Michaelis-Menten kinetic test of neutral amino acid transporter was performed. The Michaelis-Menten kinetic test of the neutral amino acid transporter was performed by examining the change in the leucine uptake rate due to the difference in the concentration of the substrate leucine.
The leucine uptake experiment was performed according to the method described in Example 6 (2) above using oocytes injected with both rat LAT1 gene cRNA and rat 4F2hc gene cRNA. As a result (FIG. 18), the Km value was about 24 μM.

(4) Substrate selectivity of LAT1 (inhibition experiment by adding amino acids and similar substances)
In an uptake experiment of leucine by an oocyte injected with both rat LAT1 gene cRNA and rat 4F2hc gene cRNA, the influence of various amino acids and similar substances added to the system was examined.
The leucine uptake experiment was performed according to the method described in Example 6 (2) above using oocytes injected with both rat LAT1 gene cRNA and rat 4F2hc gene cRNA. However, uptake of ¹⁴ C-leucine (20 μM) was measured using a choline uptake solution in the presence and absence of 2 mM of various compounds (unlabeled).
As a result (FIG. 19), cis-inhibitory effects were observed with various neutral amino acids. In particular, leucine, isoleucine, phenylalanine, methionine, tyrosine, histidine, tryptophan, and valine strongly inhibited ^14C- leucine uptake via LAT1. In addition to substances other than the standard amino acids, drugs such as L-DOPA (parkinson's disease drug), melphalan (antitumor drug), triiodothyronine (thyroid hormone), thyroxine (thyroid hormone), and physiological activities The substance also inhibited ^14C- leucine uptake via LAT1. Furthermore, 2-amino-2-norbornane-carboxylic acid (BCH), which was known as a neutral amino acid uptake inhibitor, also inhibited ¹⁴ C-leucine uptake via LAT1. did. Acidic amino acids and basic amino acids did not affect the uptake of ¹⁴ C-leucine via LAT1.

(5) Substrate selectivity of LAT1 (uptake test using various amino acids and similar substances as substrates)
Uptake by LAT1 was examined using various amino acids and similar substances as substrates.
The uptake experiment of various amino acids and similar substances was carried out according to the method described in Example 6 (2) above using oocytes injected with both rat LAT1 gene cRNA and rat 4F2hc gene cRNA. However, as the substrate, various radioactively labeled compounds were used instead of ¹⁴ C-leucine.
As a result, leucine ( ^14C compound), isorosine ( ^14C compound), phenylalanine ( ^14C compound), methionine ( ^14C compound), tyrosine ( ^14C compound), histidine ( ^14C compound), tryptophan ( ^14C compound) ) And uptake into oocytes was observed when valine ( ^14C compound) was used as a substrate.

Example 8 Control of Cell Growth by Inhibition of Neutral Amino Acid Transporter LAT1 (1) Cell Growth Inhibitory Effect of LAT1 Inhibition The inhibitory effect on LAT1 inhibition by LAT1 inhibitor on cell proliferation was examined.
A rat liver cell line that highly expresses LAT1 is cultured in William's medium, D-leucine or BCH that inhibits uptake through LAT1 is added to 20 mM medium, cultured for 48 hours, and the number of cells is counted as Cell Counting Kit-8. (Dojindo Laboratories) was used. Cell number was measured as absorbance at 450 nm (OD 450).
As a result (FIG. 20), the group to which D-leucine or BCH was added showed a decrease in the number of cells compared to the control group to which D-leucine or BCH was not added, and the neutral amino acid uptake inhibition by LAT1 suppression It was thought that cell proliferation was suppressed.

Example 9 Expression of LAT1 gene and 4F2hc gene in various human tumor cell lines (analysis by Northern blotting)
A cDNA fragment corresponding to the 649th to 1128th bases of the hLAT1 gene was excised with the restriction enzyme SmaI, labeled with ³² P-dCTP as a probe, and Northern blotting was carried out on human tumor cell lines as follows. Poly (A) ⁺ RNA was extracted from various human tumor cell lines, and hybridization and washing were performed with a ³² P-dCTP-labeled LAT1 probe.
A cDNA fragment corresponding to the 106th to 645th bases of the h4F2hc gene was excised with the restriction enzyme PstI, labeled with ³² P-dCTP, and used as a probe for Northern blotting on human tumor cell lines in the same manner.
As a result of Northern blotting, LAT1 expression was observed in the vicinity of 4.8 kb in all of the examined tumor cell lines shown in FIGS. With regard to 4F2hc, expression was observed in the vicinity of 2.2 kb in most tumor cell lines, but there were strong and weak depending on the expression, especially in leukemia cell lines Daudi, CCRF-SB, P30 / OHK, the signal by Northern blotting was It was not detected (FIG. 22).

Example 10 Significance of human bladder cancer-derived T24 cells as an LAT1 inhibitor evaluation system Human bladder cancer-derived T24 cells were cultured and maintained in Eagle's minimal essential medium containing 10% fetal bovine serum. The amino acid incorporation test into T24 cells was performed in a state where T24 cells were cultured in 24-well plates and reached confluent. The amino acid uptake test was started by removing the culture solution and adding Dulbecco's PBS containing ¹⁴ C-amino acid (Gibco), removing it and washing with ice-cooling and Dulbecco's PBS. After washing, the product was dissolved with 0.1N NaOH, and the radioactivity was measured with a liquid scintillation counter.

(1) Na ⁺ dependence of leucine uptake of T24 cells In the leucine uptake experiment by T24 cells, the influence of sodium ions in the medium was examined.
When the effect of sodium ion in the medium on the uptake of leucine was observed, the uptake solution (in which sodium chloride was replaced with choline chloride) was used instead of Dulbecco's PBS.
As a result (FIG. 23), changing extracellular choline to sodium did not affect leucine uptake. From this, it was shown that the uptake of leucine in T24 cells is carried by a transport system independent of sodium ions.

(2) Michaelis-Menten kinetic test of leucine uptake of T24 cells Michaelis-Menten kinetic test of leucine uptake of T24 cells was performed. The Michaelis-Menten kinetic test was performed by examining the change in the leucine uptake rate due to the difference in the concentration of the substrate leucine.
As a result (FIG. 24), the Km value was 100.3 mM, and the Vmax value was 23,870 pmol / mg protein / min.

(3) Inhibition experiment of leucine uptake by T24 cells by addition of amino acid and its similar substances In the leucine uptake experiment by T24 cells, the influence of various amino acids and its similar substances addition to the system was examined.
In the leucine uptake experiment, ¹⁴ C-leucine (20 mM) uptake was measured in the presence and absence of 2 mM of various compounds (unlabeled).
As a result (FIG. 25), a strong cis-inhibitory effect was observed with methinein, leucine, isoleucine, valine, phenylalanine, tyrosine, tryptophan, histidine and cysteine. Amino acid transport system L-specific inhibitor BCH strongly inhibited leucine uptake. The results of this inhibition experiment are consistent with the results when LAT1 is expressed in Xenopus oocytes.

(4) Inhibition mode of T24 cell leucine incorporation uptake inhibitor BCH The concentration dependence of ^14C- leucine uptake of T24 cells was measured in the absence of BCH, in the presence of 50 mM of BCH, and in the presence of 100 mM of BCH. The mode of inhibition was examined using a multiple reciprocal plot.
As a result (FIG. 26), it became clear that inhibition by BCH was competitive inhibition, and its Ki value was 156 mM.

Example 11 Effect of amino acid incorporation inhibitor BCH on growth of human tumor cell line Human bladder cancer-derived T24 cells were maintained in culture in Eagle's minimal essential medium containing 10% fetal bovine serum. Human Daudi cells were maintained in culture in RPMI medium containing 20% fetal bovine serum. T24 cells or Daudi cells were cultured in a 24-well plate (800 / well) for 5 days in a medium with or without 20 mM BCH, and the number of cells was counted.
As a result (FIG. 27), T24 cells have a rapid cell growth compared to Daudi cells, and BCH highly suppresses T24 proliferation, but shows little growth inhibitory effect on Daudi cells. It became clear.
T24 cells are strongly expressed in both LAT1 and 4F2hc (FIG. 21). As shown in Example 10, LAT1 shows strong functional activity in T24 cells. In contrast, in Daudi cells, LAT1 is strongly expressed, but the expression of 4F2hc necessary for the functional expression of LAT1 is not detected (FIG. 22). Therefore, it is considered that LAT1 does not function in Daudi cells. The growth of T24 cells with strong LAT1 functional activity is fast, BCH shows a high degree of cell growth inhibition, the growth of Daudi cells considered to be non-functioning LAT1 is slow, and BCH does not show the growth inhibitory effect. It supports the hypothesis that essential amino acid uptake via LAT1 forms one rate-limiting step of cell growth, and that inhibition indicates cell growth inhibition.
In CCRF-SB cells and P30 / OHK cells (FIG. 22) in which the expression of 4F2hc is not detected as in Daudi cells, proliferation is slow as in Daudi cells, and the effect of BCH is weak, and LAT1 and 4F2hc are similar to those in T24 cells. It was confirmed that the cells that both expressed strongly grew rapidly and BCH showed a strong inhibitory effect.

Example 13 Life Prolonging Effect of Amino Acid Transporter Inhibitor BCH in Tumor-Inoculated Mice Mouse Sarcoma 180 cells were intraperitoneally transplanted (1 × 10 ⁶ ) into male ICR mice, and amino acid transporter inhibitor BCH, amino acid transporter inhibitory effect from the day after transplantation D-Leu and D-Ala having no inhibitory effect were administered at a dose of 300 mg / kg for 10 days. Every day after transplantation, the mice were confirmed to be alive or dead.
As a result of observation for 19 days, all the untreated controls survived, but the survival period was significantly shortened in the sarcoma 180 cell-inoculated group, the sarcoma 180-cell inoculated group, and the vehicle-administered group after inoculation with sarvosa 180 cells compared to the control ( FIG. 28). In contrast, in the group administered with BCH and the group administered with D-Leu after inoculating sasarcoma 180 cells, a significant life-prolonging effect due to the drug treatment was observed (FIG. 28). D-Ala did not show a life-prolonging effect (FIG. 28).

Claims (3)

A cytostatic pharmaceutical composition for tumor cells expressing LAT1 and 4F2 protein cell surface,
At least one amino acid selected from the group consisting of leucine (Leu), isoleucine (Ile), phenylalanine (Phe), methionine (Met), tyrosine (Tyr), tryptophan (Trp), valine (Val) and histidine (His) A pharmaceutical composition comprising a D-amino acid or BCH corresponding to 1 as an active ingredient and a pharmaceutically acceptable carrier. A cytostatic pharmaceutical composition for tumor cells expressing LAT1 and 4F2 protein cell surface,
An antigenic polypeptide having reactivity to the neutral amino acid transporter LAT1 protein comprising the amino acid sequence of either (1) or (2) below and comprising a partial sequence of the LAT1, Tyr which corresponds to the 119-th number 2, 189th or comprising at least one antigenic polypeptide intracellular region comprising the amino acid of the corresponding Ser to 346th (including the "KPELERPIKVNL" and "WWKNKPKWILQ" when comprising antisera or polyclonal antibodies that have a reactivity, and a pharmaceutically acceptable carrier relative to the exception), the pharmaceutical compositions;
(1) an amino acid sequence described in SEQ ID NO: 2 or 4; or (2) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 2 or 4. A cytostatic pharmaceutical composition for tumor cells expressing LAT1 and 4F2 protein cell surface,
An antigenic polypeptide having reactivity to the neutral amino acid transporter LAT1 protein comprising the amino acid sequence of either (1) or (2) below and comprising a partial sequence of the LAT1, Tyr which corresponds to the 119-th number 2, 189th or comprising at least one antigenic polypeptide intracellular region comprising the amino acid of the corresponding Ser to 346th (including the "KPELERPIKVNL" and "WWKNKPKWILQ" comprising an antigen-binding fragment of a monoclonal antibody or the monoclonal antibody to have a reactive and a pharmaceutically acceptable carrier relative unless) the pharmaceutical composition;
(1) an amino acid sequence described in SEQ ID NO: 2 or 4; or (2) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence described in SEQ ID NO: 2 or 4.