JP5167539B2 - Genetic polymorphism and its use - Google Patents

Description

  The present invention relates to a novel gene polymorphism that can serve as a prognosis marker for cancer, and to prognosis of cancer by detecting the polymorphism. The invention also relates to the treatment of cancer by inhibiting the activity or expression of the gene.

  Transforming growth factor β (TGF-β) is a potent growth inhibitor in most cell types, including epithelial cells. Secretion of TGF-β by tumor cells can contribute to tumor suppression by inhibiting autocrine growth, while promoting tumor progression by stimulating tumor invasion and angiogenesis and inhibiting immune responses It is possible to do. TGF-β is synthesized as a latent high molecular weight complex consisting of TGF-β, the amino terminal portion of the TGF-β precursor, and a latent TGF-β binding protein (LTBP). LTBP is a glycoprotein with a molecular weight greater than 190 kDa and has several repeats of a unique motif containing 16-18 EGF-like domains and 8 cysteine residues. In LTBP, four isoforms (LTBP-1-LTBP-4) have been found in mammalian species. LTBP-1 binds to TGF-β1 through one of the 8 cysteine motifs, and TGF-β1 assembly and secretion (see, for example, Non-Patent Document 1) and activation (see, for example, Non-Patent Document 2). Facilitate. From the results of immunoelectron microscopic observation, LTBP is one of the extracellular microfiber components, targeting TGF-β1 to the extracellular structure, and possibly concentrating latent TGF-β1 on the cell surface where activation occurs Is suggested to be involved in the activation of latent TGF-β1 (see, for example, Non-Patent Document 3).

  LTBP-1 is known to have two major isoforms called LTBP-1S (short) and LTBP-1L (long), respectively, between a separate promoter and LTBP-1S codons 145 and 146. Derived from alternative splicing (Non-patent Document 4). LTBP-1L has an N-terminal extension of 346 amino acids that is not found in LTBP-1S (Non-patent Document 5). This N-terminal extension contains an EGF-like domain that promotes matrix binding, and LTBP-1L has been confirmed to bind more efficiently to the extracellular matrix than LTBP-1S.

We have previously shown that LTBP-1 and TGF-β1 proteins are overexpressed in several ovarian cancer patients, primarily due to upregulation of LTBP-1L mRNA but not LTBP-1S mRNA (Non-patent document 6). This overexpression is thought to affect TGF-β1 function and its signaling, which may contribute to the development of ovarian cancer. However, little information is available on the transcriptional control of these genes.
Miyazono K. et al., “The Embo Journal (EMBO J.)”, UK, Vol. 10, pp. 1091-1101, 1991 Fraumenhaft R. et al., “The Journal of Cell Biol.”, USA, Vol. 120, pp. 995-1002, 1993 Taipale J. et al., “The Journal of Cell Biol.”, USA, Vol. 124, pp. 171-181, 1994 Koski C. et al., “The Journal of Biological Chemistry”, USA, Vol. 274, pp. 32619-32630, 1999 Olofsson A. et al., “The Journal of Biological Chemistry”, USA, Vol. 270, pp. 31294-31297, 1995 Higashi T. et al., “Japanese Journal of Cancer Res”, Japan, Vol. 92, pp. 506-515, 2001

  Therefore, an object of the present invention is to clarify the molecular mechanism of LTBP-1L overexpression in ovarian cancer and to elucidate the clinical significance of the overexpression.

As a result of intensive studies to achieve the above object, the present inventors have found that two novel SNPs (-202G / C and + 20A / C; numbers present in the 5'-regulatory region of the LTBP-1L gene; Shows the position based on the transcription start point +1), but it contributes to the overexpression of this gene, and that it affects the prognosis of cancer patients such as ovarian cancer, endometrial cancer, lung cancer, etc. I found it.
Furthermore, the present inventors have correlated these SNPs with susceptibility to cancers such as endometrial cancer, stomach cancer and lung cancer, and surprisingly alleles that cause poor prognosis in these cancers are rather protective (difficult) in terms of sensitivity. It was found that an allele that is a (susceptible) allele and has a relatively good prognosis is a disease (susceptible) allele.
As a result of further studies based on these findings, the present inventors have completed the present invention. That is, the present invention
[1] In a genomic DNA-containing sample collected from a cancer patient, a large number of nucleotides represented by base numbers 2014 and / or 2235 in the base sequence of the human LTBP-1L gene 5′-regulatory region represented by SEQ ID NO: 1 A test method for the prognosis of cancer, characterized by testing the type;
[2] The method according to [1] above, wherein the cancer is selected from the group consisting of ovarian cancer, cervical cancer, endometrial cancer, stomach cancer, colon cancer and lung cancer,
[3] The method according to [2] above, wherein the cancer is ovarian cancer, endometrial cancer or lung cancer,
[4] A partial base sequence of the human LTBP-1L gene, which comprises about 15 to about 500 consecutive bases including the base shown by base numbers 2014 and / or 2235 in the base sequence represented by SEQ ID NO: 1. A nucleic acid comprising a base sequence,
[5] One or more sets of nucleic acid probes capable of detecting each of the polymorphisms at the bases indicated by base numbers 2014 and 2235 in the base sequence of the human LTBP-1L gene 5′-regulatory region represented by SEQ ID NO: 1 and A test kit for prognosis of cancer, comprising a primer,
[6] The nucleic acid probe contains a base of the polymorphic site represented by base numbers 2014 and / or 2235 in the base sequence of the human LTBP-1L gene 5′-regulatory region represented by SEQ ID NO: 1; A nucleic acid comprising a continuous base sequence of about 500 bases, wherein the nucleic acid primer is a partial base sequence of the human LTBP-1L gene, the base number 2014 in the base sequence represented by SEQ ID NO: 1 and / or Or a kit according to the above [5], which is a pair of nucleic acids capable of amplifying a continuous base sequence of about 50 to about 1,000 bases including the base of the polymorphic site represented by 2235,
[7] The kit according to [5] or [6] above, wherein the cancer is selected from the group consisting of ovarian cancer, cervical cancer, endometrial cancer, stomach cancer, colon cancer and lung cancer,
[8] The kit according to [7] above, wherein the cancer is ovarian cancer, endometrial cancer or lung cancer,
[9] A cancer therapeutic agent comprising a nucleic acid comprising a base sequence complementary to or substantially complementary to the base sequence represented by SEQ ID NO: 2 or a part thereof,
[10] A nucleic acid comprising a base sequence complementary to or substantially complementary to the partial base sequence represented by base numbers 1 to 1038 in the base sequence represented by SEQ ID NO: 2 or a part thereof, The agent according to [9] above,
[11] A cancer therapeutic agent comprising a neutralizing antibody against a protein comprising the same or substantially the same amino acid sequence represented by SEQ ID NO: 3, or a partial peptide thereof,
[12] The agent according to [11] above, wherein the neutralizing antibody recognizes part of the amino acid sequence represented by amino acid numbers 1 to 346 in the amino acid sequence represented by SEQ ID NO: 3.
[13] A cancer therapeutic agent comprising the nucleic acid according to [4] above,
[14] The agent according to any one of [9] to [13] above, wherein the cancer is selected from the group consisting of ovarian cancer, cervical cancer, endometrial cancer, stomach cancer, colon cancer and lung cancer,
[15] The agent according to [14] above, wherein the cancer is ovarian cancer, endometrial cancer or lung cancer,
[16] In a genomic DNA-containing sample collected from a subject, a polymorphism in the base represented by base numbers 2014 and / or 2235 in the base sequence of the human LTBP-1L gene 5′-regulatory region represented by SEQ ID NO: 1 A test method for susceptibility to cancer selected from the group consisting of endometrial cancer, stomach cancer and lung cancer,
[17] One or more sets of nucleic acid probes capable of detecting each of the polymorphisms at the bases indicated by base numbers 2014 and 2235 in the base sequence of the human LTBP-1L gene 5′-regulatory region represented by SEQ ID NO: 1 and A kit for testing susceptibility to endometrial cancer, gastric cancer or lung cancer, comprising a primer, and [18] a nucleic acid probe represented by SEQ ID NO: 1, the base of the human LTBP-1L gene 5′-regulatory region A nucleic acid comprising a continuous base sequence of about 15 to about 500 bases, including the base of the polymorphic site represented by base number 2014 and / or 2235 in the sequence, wherein the nucleic acid primer is a human LTBP-1L gene A partial base sequence that can amplify a continuous base sequence of about 50 to about 1,000 bases including the base of the polymorphic site shown by base numbers 2014 and / or 2235 in the base sequence represented by SEQ ID NO: 1. [17] which is a pair of nucleic acids The kit according,
Etc.

Two SNPs in the LTBP-1L gene 5'-regulatory region affect the transcriptional activity of the gene and in cancer patients with homozygous genotypes of -202G and + 20A, patients with other genotypes Since the expression of LTBP-1 increases more and the life prognosis is poor, the prognosis of cancer becomes possible by examining the SNPs in cancer patients. In addition, by inhibiting the expression or activity of LTBP-1L protein, the therapeutic / progressive effect of cancer is exhibited.
On the other hand, these SNPs correlate with susceptibility to cancers such as endometrial cancer, gastric cancer and lung cancer, and the -202C and + 20C alleles are more effective against these cancers than More susceptible (susceptible). Therefore, the sensitivity to these cancers can be predicted by examining the SNPs in the subject.

FIG. 1A schematically shows two novel SNPs in the LTBP-1L promoter. In the figure, ATG represents the start codon. FIG. 1B shows the result of polymorphism analysis by PCR-RFLP method. The upper row shows the restriction enzyme cleavage site in each haplotype and the length of each generated fragment, and the lower row shows the band pattern of EcoRII and CspI digests in each genotype. In the figure, M represents a molecular weight marker, and D represents a heteroduplex produced by annealing with a heteroallelic mismatch. FIG. 2A schematically shows a luciferase reporter construct comprising an LTBP-1L promoter region with different haplotypes at the SNP site. FIG. 2B shows the relative luciferase activity of reporter plasmids with various haplotypes. Each column shows the average value of three independent experiments (each performed in triplicate), and the error bar shows the standard deviation. In the figure, * indicates P <0.01 (vs. C-C construct). C: It is a figure which shows the effect of Sp1 which acts on LTBP-1L promoter activity. Blank indicates an empty vector, Sp1 indicates a Sp1 expression vector, and DN-SP1 indicates a co-transfected vector expressing a dominant negative form of Sp3. Each column shows the average value of three independent experiments (each performed in triplicate), and the error bar shows the standard deviation. In the figure, * indicates P <0.0001 (vs. G-A blank), and # indicates P <0.0001 (vs. C-C blank). FIG. 3 shows EMSA results for Sp1 binding to the LTBP-1L promoter. In the figure, B represents a probe-Sp1 complex, S represents a probe-Sp1-anti-Sp1 antibody complex, and F represents a band corresponding to a free probe. FIG. 4 shows the survival curve for each genotype of the LTBP-1L promoter in ovarian cancer patients. In the figure, ○ indicates the occurrence of death cases. FIG. 5 shows survival curves for each genotype of LTBP-1L promoter in endometrial cancer patients. In the figure, ○ indicates the occurrence of death cases. FIG. 6 shows a survival curve for each LTBP-1L promoter genotype in lung cancer patients. In the figure, ○ indicates the occurrence of death cases.

  The cancer prognosis method of the present invention (hereinafter, sometimes simply referred to as “the diagnosis method of the present invention”. In this specification, the “test method for prognosis” refers to a medical decision stage by a doctor. It is synonymous with “prognostic method” except for the case where it is not included) and a method for predicting susceptibility to cancers such as endometrial cancer, stomach cancer, lung cancer and the like (hereinafter simply referred to as “prediction method of the present invention”). In the present specification, “sensitivity test method” is synonymous with “sensitivity prediction method” except that it does not include a medical decision stage by a doctor, and LTBP-1L gene 5′-regulation of a subject It is characterized by detecting a polymorphism in the region. As a polymorphism that can be used in the diagnosis and prediction method of the present invention, a nucleotide sequence represented by the novel polymorphism found in the present invention [“GenBank accession number AF171934 (AF171934.1 GI: 6424997)” is used. Among them, a single base substitution (SNP) polymorphism (SEQ ID NO: 1)] in which C shown by base number 2014 and C shown by 2235 are G and A, respectively, may be mentioned. Hereinafter, unless otherwise specified, the position of the nucleotide (base) of the polymorphic site is the estimated transcription start point (the base represented by base number 2216 in the base sequence represented by SEQ ID NO: 1). +1) as a reference (ie, the polymorphism at base number 2014 in the base sequence represented by SEQ ID NO: 1 is -202G / C, and the polymorphism at base number 2235 is + 20A / C) write).

Other polymorphisms that can be used in the diagnosis and prediction method of the present invention include polymorphisms in a linkage disequilibrium state with −202 G / C or +20 A / C and a linkage disequilibrium coefficient D ′ of 0.9 or more. It is done. Here, the “linkage disequilibrium coefficient D ′” refers to the four haplotypes (AB, where each allele of the first SNP is (A, a) and each allele of the second SNP is (B, b). , ab, aB, each frequency of _{ab) P aB, P ab,} P aB, when the P _ab, obtained by the following equation.
D ′ = (P _AB P _ab −P _Ab P _aB ) / Min [(P _AB + P _aB ) (P _aB + P _ab ), (P _AB + P _Ab ) (P _Ab + P _ab )]
[ _Wherein , Min [(P _AB + P _aB ) (P _aB + P _ab ), (P _AB + P _Ab ) (P _Ab + P _ab )] is (P _AB + P _aB ) (P _aB + P _ab ) and (P _AB + P _Ab ) (P _Ab + P _ab ) means that the smaller value is taken. ]
Preferably, a polymorphism in which D ′ is 0.95 or more, more preferably 0.99 or more, and most preferably 1 is mentioned. In addition, -202G / C and + 20A / C are completely linked (D '= 1) as long as examined in the Japanese population, that is, only GA or CC haplotypes exist.

The prognostic marker polymorphisms -202G / C and + 20A / C used in the diagnosis and prediction method of the present invention are related to the transcriptional activity of the LTBP-1L gene and the binding activity to the transcription factor Sp1, as shown in the Examples below It produces differences between alleles (-202G / C overlaps with the Sp1 binding motif GC box, + 20A / C is adjacent to the GC box). That is, in the -202G and + 20A alleles, both the above activities are remarkably increased as compared to the -202C and + 20C alleles (the effect on both activities is greater in -202G / C). In addition, the GA allele is a major allele in certain cancer (eg, ovarian cancer, colon cancer) patient populations and healthy populations, and it has been shown that there is no significant difference in the allele frequency in these populations. However, cancer patients with GA / GA homozygous genotypes tend to have a lower cumulative survival rate than patients with CC / GA heterozygous and CC / CC homozygous genotypes Indicates. These facts indicate that LTBP-1L expression plays an important role in cancer progression, and GA / GA genotype holders have increased Sp1 binding activity to LTBP-1L promoter and LTBP-1L transcriptional activity. Therefore, it is strongly suggested that cancer is likely to progress (poor prognosis). Therefore, the -202G / C and + 20A / C polymorphisms are polymorphisms that define the quality of life prognosis in cancer patients.
On the other hand, the CC allele frequency is significantly higher (p <0.1) in another specific cancer (for example, endometrial cancer, gastric cancer, lung cancer) patient population compared to the healthy population. In particular, according to the examples below, for CC allele holders (CC / GA heterozygous and CC / CC homozygous) in endometrial cancer and gastric cancer to non-carriers (GA / GA homozygous) Since the odds ratio is 2 to 3 or more (p <0.05), the CC allele is one of the genetic risk factors for these cancers. Conversely, GA / GA genotypes have a significantly lower risk of developing endometrial and gastric cancer, despite having a poor prognosis for advanced stage cancers. This may suggest that high expression of LTBP-1L rather contributes to the suppression of cancer cell proliferation before or early onset of cancer. In any case, the -202G / C and + 20A / C polymorphisms are polymorphisms that can predict the risk of developing cancers such as endometrial cancer, gastric cancer, and lung cancer in humans.

LTBP-1L forms a complex with latent TGF-β1 and plays an important role in the activation of TGF-β1. TGF-β can act as both a tumor suppressor and a stimulator of cancer progression. At the early stage of carcinogenesis, since the growth inhibitory response of cells to TGF-β is maintained, TGF-β directly suppresses tumor growth, but as the stage progresses, cancer cells inhibit growth by TGF-β. It becomes resistant and TGF-β is rather thought to contribute to tumor invasion and metastasis. Accordingly, examples of cancer that can be diagnosed prognostically by the diagnostic method of the present invention include, but are not limited to, advanced stage cancers. Preferably, the diagnostic method of the present invention comprises ovarian cancer, cervical cancer, endometrial cancer, stomach cancer, colon cancer, lung cancer, and other solid cancers (eg, liver cancer, pancreatic cancer, gallbladder cancer, kidney cancer, etc.) and more. Preferably, it is useful for prognosis of ovarian cancer, cervical cancer, endometrial cancer, stomach cancer, colon cancer and lung cancer, particularly preferably ovarian cancer, endometrial cancer and lung cancer.
On the other hand, examples of cancers whose sensitivity can be predicted by the prediction method of the present invention include endometrial cancer, stomach cancer and lung cancer.

The polymorphism detected in the diagnosis and prediction method of the present invention may be either one or both of the above-described polymorphisms.
In the diagnosis and prediction method of the present invention, any of known SNP detection methods can be used for polymorphism detection. As a classic detection method, for example, genomic DNA extracted from a subject's cells or the like is used as a sample, and the base number 2014 and / or in the base sequence of the human LTBP-1L gene 5′-regulatory region represented by SEQ ID NO: 1 is used. Alternatively, a nucleic acid containing a continuous base sequence of about 15 to about 500 bases including the base of the polymorphic site represented by 2235 is used as a probe, for example, Wallace et al. (Proc. Natl. Acad. Sci. USA, 80 , 278-282 (1983)), by performing hybridization while accurately controlling stringency, and detecting only a sequence completely complementary to the probe, or the base of the polymorphic site in the nucleic acid and the nucleic acid. Gradually from the denaturation temperature using a mixed probe in which either one of the nucleic acids in which (-202G and / or + 20A) is substituted with another base (-202C and / or + 20C) is labeled and the other is unlabeled To lower reaction temperature The method may include a method in which hybridization is performed while lowering, and a sequence completely complementary to one probe is first hybridized to prevent cross-reaction with a probe having a mismatch. Here, as the labeling agent, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance, or the like is used. As the radioisotope, for example, [ ¹²⁵ I], [ ¹³¹ I], [ ³ H], [ ¹⁴ C] and the like are used. As the enzyme, a stable enzyme having a large specific activity is preferable. For example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like are used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate and the like are used. As the luminescent substance, for example, luminol, luminol derivatives, luciferin, lucigenin and the like are used.

  Preferably, the detection of the polymorphism is performed by various methods described in, for example, WO 03/023063, such as RFLP method, PCR-SSCP method, ASO hybridization, direct sequencing method, ARMS method, denaturing agent concentration gradient gel electrophoresis. Electrophoresis, RNaseA cleavage method, chemical cleavage method, DOL method, TaqMan PCR method, invader method, MALDI-TOF / MS method, TDI method, molecular beacon method, dynamic allele specific hybridization method, padlock probe method, It can be carried out by UCAN method, nucleic acid hybridization method using DNA chip or DNA microarray, ECA method and the like (see WO 03/023063, page 17, line 5 to page 28, line 20). Hereinafter, the TaqMan PCR method and the invader method will be described in more detail as representative methods.

(1) TaqMan PCR method
The TaqMan PCR method uses a fluorescently labeled allele-specific oligonucleotide (TaqMan probe) and PCR using Taq DNA polymerase. As the TaqMan probe, an oligonucleotide having a partial base sequence of the human LTBP-1L gene and comprising a continuous base sequence of about 15 to about 30 bases including the bases of any of the polymorphic sites described above is used. The probe is labeled with a fluorescent dye such as FAM or VIC at the 5 'end and labeled with a quencher (quenching substance) such as TAMRA at the 3' end, and the quencher absorbs the fluorescence energy as it is. Therefore, fluorescence is not detected. Probes are preferably prepared for both alleles and labeled with fluorescent dyes having different fluorescence wavelengths (for example, one allele is FAM and the other is VIC) for batch detection. In addition, the 3 ′ end is phosphorylated so that a PCR extension reaction from the TaqMan probe does not occur. When PCR is performed with a primer designed to amplify a partial sequence of genomic DNA containing a region that hybridizes with the TaqMan probe and Taq DNA polymerase, the TaqMan probe hybridizes with the template DNA and simultaneously extends from the PCR primer. However, when the extension reaction proceeds, the TaqMan probe hybridized by the 5 ′ nuclease activity of Taq DNA polymerase is cleaved, the fluorescent dye is released, and it is not affected by the quencher, and fluorescence is detected. The fluorescence intensity increases exponentially by amplification of the template.
For example, in the detection of -202G / C polymorphism, an allele-specific oligonucleotide containing the base (about 15 to about 30 bases in length; G allele is FAM, C allele is 5 'end-labeled with VIC, 3' When both ends are labeled with TAMRA) as a TaqMan probe, if the subject's genotype is GG or CC, strong fluorescence intensity of FAM or VIC is observed, and the other fluorescence is hardly observed. On the other hand, if the subject's genotype is GC, both FAM and VIC fluorescence are detected.

(2) Invader method Unlike the TaqMan PCR method, the invader method does not label the allele-specific oligonucleotide (allele probe) itself, and a sequence (flap) that is not complementary to the template DNA on the 5 'side of the base of the polymorphic site. And a complementary sequence specific to the template on the 3 ′ side. In the invader method, an oligonucleotide having a specific complementary sequence on the 3 ′ side of the polymorphic site of the template (invader probe; the base corresponding to the polymorphic site at the 5 ′ end of the probe is arbitrary), The 5 'side has a sequence that can take a hairpin structure, and when the hairpin structure is formed, the sequence that is continuous 3' from the base paired with the 5 'end base is a sequence complementary to the allele probe flap. FRET (fluorescence resonance energy transfer) probes characterized by this are used. The 5 ′ end of the FRET probe is fluorescently labeled (for example, FAM, VIC, etc.), and a quencher (for example, TAMRA, etc.) is bound in the vicinity thereof, and fluorescence is not detected as it is (hairpin structure).
When the allele probe and the invader probe are reacted with the genomic DNA as a template, the 3 ′ end of the invader probe enters the polymorphic site when the three members complementarily bind. Using the enzyme that recognizes the structure of this polymorphic site (cleavase), the single-stranded part of the allele probe (ie, the 5'-side flap from the base of the polymorphic site) is excised, and the flap is complementary to the FRET probe. And the polymorphic site of the flap enters the hairpin structure of the FRET probe. When this structure is recognized and cleaved by cleavase, the end-labeled fluorescent dye of the FRET probe is released, and the fluorescence is detected without being affected by the quencher. An allele probe whose base at the polymorphic site does not match the template is not cleaved by cleavase, but an allele probe that is not cleaved can also hybridize with the FRET probe, and thus fluorescence is detected in the same manner. However, since the reaction efficiencies are different, the allele probe that matches the base of the polymorphic site has significantly higher fluorescence intensity than the allele probe that does not match.
Usually, before reacting with the three types of probes and cleavase, the template DNA is preferably amplified by PCR using a primer that can amplify a region containing a portion to which the allele probe and invader probe hybridize.

(3) RFLP method
Since the LTBP-1L gene has a difference in restriction enzyme fragment length due to the polymorphisms of -202G / C and + 20A / C, the polymorphism can be easily detected using this. That is, the -202G allele is cleaved with EcoRII, but the -202C allele is not cleaved. On the other hand, the + 20A allele is not cleaved by CspI, whereas the + 20C allele is cleaved by CspI. Therefore, as shown in the examples below, the LTBP-1L gene fragment containing -202 to +20 is amplified by PCR and double-digested with EcoRII and CspI to compare the restriction enzyme fragment lengths. Thus, the polymorphism can be detected.

As a result of examining the polymorphism as described above, when it is determined that the -202G and / or + 20A allele is possessed, particularly when it is determined that the allele is a homozygote, the subject is It can be diagnosed that the prognosis of cancer is likely to be poor. In addition, when it is determined that the subject possesses the -202C and / or + 20C allele, it can be predicted that the subject is susceptible to cancer such as endometrial cancer, gastric cancer, and lung cancer.
When two polymorphisms are examined, if one result is in conflict with the other results (ie, when a GC allele or CA allele is detected), the -202G / C polymorphism has priority. (However, as far as examined in the examples below, the GC allele and CA allele are not detected).

As described above, the -202G / C and + 20A / C polymorphisms are novel polymorphisms found for the first time in the present invention, and can be used for prognosis of cancer and prediction of sensitivity of specific cancers. It is. More specifically, in the base sequence represented by GenBank accession number AF171934, the base represented by base number 2014 and the base represented by base number 2235 are both C. In G, new SNPs were identified in which the latter was replaced from C to A, respectively.
Therefore, the present invention also includes a partial base sequence of the human LTBP-1L gene, comprising about 15 to about 500 comprising the base represented by base numbers 2014 and / or 2235 in the base sequence represented by SEQ ID NO: 1. Provided is a nucleic acid comprising a continuous base sequence of bases (preferably about 15 to about 200 bases, more preferably about 15 to about 50 bases). Such a novel nucleic acid containing an SNP site can be preferably used for detecting a polymorphism in the base in the above-described diagnosis and prediction method of the present invention.

  The present invention also provides a kit for use in the diagnosis and prediction method of the present invention. That is, the diagnostic and prediction kit of the present invention detects each of the polymorphisms in the bases indicated by base numbers 2014 and 2235 in the base sequence of the human LTBP-1L gene 5′-regulatory region represented by SEQ ID NO: 1. One or more sets of nucleic acid probes and / or primers that can be used.

  Specifically, the nucleic acid probe used in the diagnostic and prediction kit of the present invention has the base number 2014 and / or 2235 in the base sequence of the human LTBP-1L gene 5′-regulatory region represented by SEQ ID NO: 1. Containing a continuous base sequence of about 15 or more, preferably about 15 to about 500 bases, more preferably about 15 to about 200 bases, more preferably about 15 to about 50 bases, including the bases of the indicated polymorphic site It is a nucleic acid. Here, the base represented by the base number 2014 (namely, -202 position) is G or C, and the base represented by the base number 2235 (namely, +20 position) is A or C, depending on the polymorphism detection method used. In addition, a nucleic acid having any one base for each polymorphic site can be used, or two kinds of nucleic acids having a base corresponding to each allele can be used. In the invader probe used in the above invader method, the base of the polymorphic site (that is, the base at the 3 'end) may be any base.

The probe may contain additional sequences suitable for detection of polymorphisms (sequences that are not complementary to genomic DNA). For example, the allele probe used in the invader method has an additional sequence called a flap at the 5 ′ end of the base at the polymorphic site.
The probe may be an appropriate labeling agent such as a radioisotope (eg, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, etc.), an enzyme (eg, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase). , Malate dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluorescein isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.). Alternatively, a quencher (quenching substance) that absorbs fluorescence energy emitted by the fluorescent substance may be further bound in the vicinity of the fluorescent substance (eg, FAM, VIC, etc.). In such an embodiment, the fluorescence is detected by separating the fluorescent substance and the quencher during the detection reaction.

  The nucleic acid primer used in the diagnostic and prediction kit of the present invention was designed to specifically amplify a region of genomic DNA containing the base of the polymorphic site to be detected in the diagnostic and prediction method of the present invention. Any thing can be used. For example, the nucleic acid primer is a partial base sequence of the human LTBP-1L gene and is a polymorphic site to be detected, that is, a polymorphic site represented by base number 2014 or 2235 in the base sequence represented by SEQ ID NO: 1. A nucleic acid containing a base sequence of about 15 to about 50 bases, preferably about 15 to about 30 bases, which hybridizes to a part of the complementary strand sequence on the 5 'side, and 3' to the base of the polymorphic site A combination with a nucleic acid comprising a base sequence of about 15 to about 50 bases, preferably about 15 to about 30 bases, which hybridizes to a part of the sequence, and the fragment length of the nucleic acid amplified by them is about 50 to about A pair of nucleic acids having 1,000 bases, preferably about 50 to about 500 bases, more preferably about 50 to about 200 bases, can be mentioned.

The primer may contain additional sequences suitable for detection of polymorphisms (sequences that are not complementary to genomic DNA), such as linker sequences.
The primer may be an appropriate labeling agent such as a radioisotope (eg, ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C, etc.), an enzyme (eg, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase). , Malate dehydrogenase, etc.), fluorescent materials (eg, fluorescamine, fluorescein isothiocyanate, etc.), luminescent materials (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.).

The nucleic acid probe or primer used in the diagnostic and prediction kit of the present invention may be DNA or RNA, and may be single-stranded or double-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA, or a DNA / RNA hybrid. Therefore, when describing a nucleic acid having a certain base sequence in the present specification, unless otherwise specified, a single-stranded nucleic acid having the base sequence, a single-stranded nucleic acid having a sequence complementary to the base sequence, and a hybrid thereof It should be understood that the term is used to encompass all double-stranded nucleic acids.
The nucleic acid probe or primer can be synthesized according to a conventional method using a DNA / RNA automatic synthesizer based on the information on the nucleotide sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2, for example.

The above nucleic acid probe and / or primer are each separately (or mixed if possible) in water or in an appropriate buffer (eg, TE buffer) at an appropriate concentration (eg, 2 × to 20 × concentration). 1-50 μM, etc.) and can be stored at about -20 ° C.
The kit for diagnosis and prediction of the present invention may further contain other components necessary for carrying out the method as a component depending on the polymorphism detection method. For example, when the kit is used for polymorphism detection by TaqMan PCR method, the kit includes 10 × PCR reaction buffer, 10 × MgCl ₂ aqueous solution, 10 × dNTPs aqueous solution, Taq DNA polymerase (5 U / μL), etc. Can further be included. When the kit is for polymorphism detection by the RFLP method, the kit can further contain restriction enzymes EcoRII and CspI, the restriction enzyme reaction buffer, and the like.

The present invention also provides a cancer, preferably a stage advanced cancer, such as ovarian cancer, cervical cancer, endometrial cancer, gastric cancer, colon cancer, lung cancer, by inhibiting the expression and / or activity of LTBP-1L. Other solid cancers (eg, liver cancer, pancreatic cancer, gallbladder cancer, kidney cancer, etc.), more preferably ovarian cancer, cervical cancer, uterine cancer, stomach cancer, colon cancer and lung cancer, particularly preferably ovarian cancer, uterine body It relates to the treatment of cancer and lung cancer.
The human LTBP-1L protein that is a therapeutic target in the present invention is a protein having the same or substantially the same amino acid sequence as the amino acid sequence represented by SEQ ID NO: 3. The protein is a human cell [for example, hepatocyte, spleen cell, nerve cell, glial cell, pancreatic β cell, bone marrow cell, mesangial cell, Langerhans cell, epidermal cell, epithelial cell, goblet cell, endothelial cell, smooth muscle cell. , Fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), Megakaryocytes, synovial cells, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells or stromal cells, or precursor cells, stem cells or cancer cells of these cells] or any tissue in which these cells are present Or organs [eg, brain, brain parts (eg, olfactory bulb, amygdala, basal sphere, hippocampus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord, pituitary, stomach, pancreas, kidney, liver Gonad, Thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood, prostate, testis, ovary, placenta, uterus, Natural protein isolated from bone, joint, intervertebral disc, adipose tissue (eg brown adipose tissue, white adipose tissue, etc.)] and can be produced chemically or using a cell-free protein synthesis system. It may be a chemically synthesized protein. Alternatively, it may be a recombinant protein produced from a transformant introduced with a nucleic acid having a base sequence encoding the amino acid sequence.

The amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 3 is about 80% or more, preferably about 90% or more, more preferably about 95%, with the amino acid sequence represented by SEQ ID NO: 3. As described above, most preferably an amino acid sequence having a homology of about 98% or more, wherein the protein having the amino acid sequence has substantially the same activity as the protein containing the amino acid sequence represented by SEQ ID NO: 3. Such an array. As used herein, “homology” refers to an optimal alignment when two amino acid sequences are aligned using a mathematical algorithm known in the art (preferably the algorithm uses a sequence of sequences for optimal alignment). The percentage of identical and similar amino acid residues relative to all overlapping amino acid residues in which one or both of the gaps can be considered). "Similar amino acids" means amino acids that are similar in physicochemical properties, such as aromatic amino acids (Phe, Trp, Tyr), aliphatic amino acids (Ala, Leu, Ile, Val), polar amino acids (Gln, Asn) ), Basic amino acids (Lys, Arg, His), acidic amino acids (Glu, Asp), amino acids with hydroxyl groups (Ser, Thr), amino acids with small side chains (Gly, Ala, Ser, Thr, Met), etc. Examples include amino acids classified into groups. It is expected that substitution with such similar amino acids will not change the phenotype of the protein (ie, is a conservative amino acid substitution). Specific examples of conservative amino acid substitutions are well known in the art and are described in various literature (see, for example, Bowie et al., Science, 247: 1306-1310 (1990)).
The homology of amino acid sequences in the present specification is determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gap; matrix = BLOSUM62; filtering) = OFF). Other algorithms for determining amino acid sequence homology include, for example, the algorithm described in Karlin et al., Proc. Natl. Acad. Sci. USA, 90: 5873-5877 (1993) [the algorithms are NBLAST and XBLAST] Embedded in the program (version 2.0) (Altschul et al., Nucleic Acids Res., 25: 3389-3402 (1997))], Needleman et al., J. Mol. Biol., 48: 444-453 (1970) [The algorithm is incorporated into the GAP program in the GCG software package], the algorithm described in Myers and Miller, CABIOS, 4: 11-17 (1988) [the algorithm is part of the CGC sequence alignment software package Embedded in the ALIGN program (version 2.0), Pearson et al., Proc. Natl. Acad. Sci. USA, 85: 2444-2448 (1988) [the algorithm is FASTA in the GCG software package. Program Built-in 'or the like include, but are not limited to.

Examples of substantially the same activity include binding activity to latent TGF-β1, activation promoting activity of TGF-β1, and the like. “Substantially homogeneous” means that their properties are qualitatively (eg, physiologically or pharmacologically) equivalent. Accordingly, it is preferable that the quantitative factors such as the above-mentioned degree of activity are equivalent, but may be different (for example, about 0.01 to about 100 times, preferably about 0.1 to about 10 times, more preferably about 0.5 times). ~ About 2 times).
The activity of LTBP-1L can be measured according to a method known per se. Examples include, but are not limited to, binding tests with TGF-β1.

In addition, as LTBP-1L used in the present invention, for example, (1) 1 or 2 or more (preferably about 1 to 30, preferably 1 to 10) in the amino acid sequence represented by SEQ ID NO: 3 (2) one or more amino acid sequences represented by SEQ ID NO: 3 (preferably about 1 to 30 amino acids, preferably about 1 to 5 amino acids) Is an amino acid sequence added with about 1 to 10 amino acids, more preferably 1 to 5 amino acids, and (3) one or more amino acid sequences represented by SEQ ID NO: 3 (preferably 1 to 30 amino acids) (4) 1 or 2 or more (preferably in the amino acid sequence represented by SEQ ID NO: 3) (preferably about 1-10, more preferably about 1-5) 1-30, preferably 1-10, and more preferably 1-5 amino) A protein comprising an amino acid sequence in which an acid is substituted with another amino acid, or (5) an amino acid sequence obtained by combining them, wherein the protein is substantially the same as the protein comprising the amino acid sequence represented by SEQ ID NO: 3. Proteins having activity are also included.
When the amino acid sequence is inserted, deleted or substituted as described above, the position of the insertion, deletion or substitution is not particularly limited as long as the activity of the protein is not impaired.

In the present specification, the protein specified by the amino acid sequence is the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end according to the convention of peptide designation. The LTBP-1L used in the present invention including the protein containing the amino acid sequence represented by SEQ ID NO: 3 has a C-terminal carboxyl group (—COOH), carboxylate (—COO ⁻ ), amide (— Either CONH ₂ ) or ester (—COOR) may be used.
Here, as R in the ester, for example, a C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, for example, a C _3-8 cycloalkyl group such as cyclopentyl, cyclohexyl, etc., for example, phenyl C _6-12 aryl groups such as α-naphthyl, for example, C _7- such as phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl ₁₄ aralkyl group, pivaloyloxymethyl group is used.
When the protein used in the present invention has a carboxyl group (or carboxylate) other than the C-terminus, a protein in which the carboxyl group is amidated or esterified is also included in the protein used in the present invention. As the ester in this case, for example, the above-mentioned C-terminal ester or the like is used.
Furthermore, the protein used in the present invention, amino acid residues at the N-terminus: the amino group protecting group (e.g. methionine residue) (e.g., formyl group, such as C _1-6 alkanoyl such as acetyl group C _{1- 6} protected with an acyl group, N-terminal glutamine residue produced by cleavage in vivo is pyroglutamine oxidized, and a substituent on the side chain of an amino acid in the molecule (eg, —OH, — SH, amino group, imidazole group, indole group, guanidino group, etc.) is protected with a suitable protecting group (e.g., formyl group, C _1-6 acyl group such as C _1-6 alkanoyl group such as acetyl group) Or a complex protein such as a so-called glycoprotein to which a sugar chain is bound.
Specific examples of the protein used in the present invention include human LTBP-1L (GenBank accession registration number: NP_996826.1) consisting of the amino acid sequence represented by SEQ ID NO: 3.

The LTBP-1L partial peptide used in the present invention has the same or substantially the same amino acid sequence as the partial amino acid sequence of the amino acid sequence represented by SEQ ID NO: 3, and has a specific affinity for LTBP-1L. Any one can be used as long as it can be an antigenic peptide for producing an antibody having the property.
Specifically, as the partial peptide, a peptide containing at least 3 and preferably 6 or more consecutive amino acid sequences of the constituent amino acid sequences of LTBP-1L used in the present invention is used.

  LTBP-1 has two isoforms produced from separate promoters, and LTBP-1L is characterized by containing an extension of 346 amino acids on the N-terminal side compared to LTBP-1S. Therefore, when LTBP-1L is used as a cancer therapeutic target and its expression and / or activity is inhibited, it is preferable to target the N-terminal 346 amino acids. Therefore, the partial peptide of LTBP-1L used in the present invention preferably contains either all or a part of the N-terminal 346 amino acids.

In the partial peptide of LTBP-1L used in the present invention, the C-terminus may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ), or an ester (—COOR). . Here, examples of R in the ester include the same as those described above for LTBP-1L. In addition, when the partial peptide has a carboxyl group (or carboxylate) other than the C-terminal, a peptide in which the carboxyl group is amidated or esterified is also an LTBP-1L partial peptide used in the present invention. included. Examples of the ester in this case are the same as the C-terminal ester. Furthermore, as in the case of LTBP-1L, in this partial peptide, the amino group of the N-terminal amino acid residue (eg, methionine residue) is protected with a protecting group, and the N-terminal side is cleaved in vivo. The resulting glutamine residue is pyroglutamine oxidized, the substituent on the side chain of the amino acid in the molecule is protected with an appropriate protecting group, or a complex peptide such as a so-called glycopeptide with a sugar chain attached Is also included.

  LTBP-1L or a partial peptide thereof used in the present invention may be in the form of a salt. For example, a salt with a physiologically acceptable acid (eg, inorganic acid, organic acid) or a base (eg, alkali metal salt) is used, and a physiologically acceptable acid addition salt is particularly preferable. Such salts include, for example, salts with inorganic acids (eg hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid) or organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid) and the like.

  LTBP-1L or a salt thereof used in the present invention can be prepared from the aforementioned human cells or tissues by a known protein purification method. Specifically, after homogenizing the animal tissue or cells, extraction with an acid or the like is performed, and the extract is purified and isolated by combining chromatography such as reverse phase chromatography or ion exchange chromatography. it can.

LTBP-1L or a partial peptide or a salt thereof used in the present invention can also be produced according to a known peptide synthesis method.
The peptide synthesis method may be, for example, either a solid phase synthesis method or a liquid phase synthesis method. When the partial peptide or amino acid capable of constituting the protein of the present invention is condensed with the remaining part, and the product has a protecting group, the target protein can be produced by removing the protecting group. Here, the condensation and the removal of the protecting group are performed according to a method known per se, for example, the methods described in the following (1) to (5).
(1) M. Bodanszky and MA Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(2) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(3) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(4) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
(5) Supervised by Yajima Haruaki, Development of follow-up drugs, Volume 14, Peptide synthesis, Hirokawa Shoten

  The LTBP-1L partial peptide or salt thereof used in the present invention can also be produced by cleaving LTBP-1L or a salt thereof obtained by any of the methods described above or below with an appropriate peptidase.

  Thus obtained LTBP-1L or a partial peptide thereof can be purified and isolated by a known purification method. Here, examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, recrystallization, and combinations thereof. When the protein or partial peptide obtained by the above method is a free form, the free form can be converted to an appropriate salt by a known method or a method analogous thereto, and conversely, the protein was obtained as a salt. In some cases, the salt can be converted to a free form or other salt by a known method or a method analogous thereto.

Human LTBP-1L (or a partial peptide thereof; hereinafter may be simply referred to as LTBP-1L) is produced by culturing a transformant introduced with an expression vector containing a nucleic acid encoding them to produce LTBP-1L, It can also be produced by separating and purifying LTBP-1L from the obtained culture.
The nucleic acid encoding human LTBP-1L or a partial peptide thereof may be any nucleic acid as long as it contains the aforementioned LTBP-1L amino acid sequence or a base sequence encoding the partial amino acid sequence. The nucleic acid may be DNA, RNA, or a DNA / RNA chimera, preferably DNA. The nucleic acid may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA, or a DNA: RNA hybrid.
DNA encoding LTBP-1L or a partial peptide thereof is genomic DNA, human cells [eg, hepatocytes, spleen cells, neurons, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, Epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibroblasts, muscle cells, fat cells, immune cells (eg macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, Basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells or stromal cells, or progenitors of these cells, stem cells or cancer Cells, etc.] or any tissue or organ in which those cells are present [eg, brain, brain regions (eg, olfactory bulb, amygdala, basal sphere, hippocampus, hypothalamus, cerebral cortex, medulla, cerebellum), Spine , Pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, muscle, lung, gastrointestinal tract (eg large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, Peripheral blood, prostate, testicle, ovary, placenta, uterus, bone, joint, intervertebral disc, adipose tissue (eg, brown adipose tissue, white adipose tissue)], cDNA, synthetic DNA, and the like. Genomic DNA and cDNA encoding LTBP-1L or its partial peptide were prepared using Polymerase Chain Reaction (hereinafter referred to as “PCR method”) using the genomic DNA fraction and total RNA or mRNA fraction prepared from the cells and tissues described above as templates, respectively. And a reverse transcriptase-PCR (hereinafter abbreviated as “RT-PCR method”). Alternatively, genomic DNA and cDNA encoding LTBP-1L or its partial peptides can be prepared by inserting genomic DNA prepared from the cells and tissues described above and total RNA or mRNA fragments into an appropriate vector. It can also be cloned from a library and a cDNA library by colony or plaque hybridization method or PCR method, respectively. The vector used for the library may be any of bacteriophage, plasmid, cosmid, phagemid and the like.

As DNA encoding human LTBP-1L, for example, DNA containing the base sequence shown as the coding region in the base sequence represented by SEQ ID NO: 2, or hybridizes with the base sequence under stringent conditions Contains a nucleotide sequence and has substantially the same activity as a protein containing the amino acid sequence represented by SEQ ID NO: 3 (for example, TGF-β1 binding activity, TGF-β1 activation promoting activity, etc.) Examples thereof include DNA encoding a protein possessed.
The DNA that can hybridize under stringent conditions with the base sequence shown as the coding region in the base sequence represented by SEQ ID NO: 2 is, for example, shown as the coding region in the base sequence represented by SEQ ID NO: 2. A DNA containing a base sequence having a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more with the base sequence is used.
The homology of the nucleotide sequences in the present specification uses the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) and the following conditions (expected value = 10; allow gap; filtering = ON; match) It can be calculated by score = 1; mismatch score = -3). As another algorithm for determining the homology of a base sequence, the above-mentioned algorithm for calculating homology of amino acid sequences is also preferably exemplified.

Hybridization is carried out according to a method known per se or a method analogous thereto, for example, the method described in Molecular Cloning 2nd edition (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). Can do. When a commercially available library is used, hybridization can be performed according to the method described in the attached instruction manual. Hybridization can be preferably performed according to stringent conditions.
Examples of stringent conditions include a hybridization reaction at 45 ° C. in 6 × SSC (sodium chloride / sodium citrate), followed by one or more washings at 65 ° C. in 0.2 × SSC / 0.1% SDS. It is done. A person skilled in the art may appropriately change the salt concentration of the hybridization solution, the temperature of the hybridization reaction, the probe concentration, the length of the probe, the number of mismatches, the time of the hybridization reaction, the salt concentration of the washing solution, the washing temperature, etc. Thus, the desired stringency can be easily adjusted.

  The DNA encoding LTBP-1L is preferably human LTBP-1L cDNA (GenBank accession number: NM_206943.1) or an allele thereof containing the base sequence shown as the coding region in the base sequence represented by SEQ ID NO: 2. Mutant etc. are mentioned.

The DNA encoding the partial peptide of LTBP-1L is any DNA as long as it contains a base sequence encoding the same or substantially the same amino acid sequence as part of the amino acid sequence represented by SEQ ID NO: 3. May be. Further, any of genomic DNA, cDNA derived from the cells and tissues described above, and synthetic DNA may be used.
Specifically, as the DNA encoding the partial peptide, for example,
(1) a partial base sequence of DNA having a base sequence shown as a coding region in the base sequence represented by SEQ ID NO: 2, or (2) a base shown as a coding region in the base sequence represented by SEQ ID NO: 2. A DNA encoding a peptide having a base sequence that hybridizes with a DNA having a sequence under stringent conditions is used.

  The DNA encoding LTBP-1L or a partial peptide thereof is amplified by PCR using a synthetic DNA primer having a part of the base sequence encoding the protein or peptide, or is incorporated into a suitable expression vector, It can be cloned by hybridization with a labeled DNA fragment or synthetic DNA encoding a part or the entire region of LTBP-1L. Hybridization can be performed, for example, according to the method described in Molecular Cloning Second Edition (described above). When a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.

The DNA base sequence can be determined using known kits such as Mutan ^™ -super Express Km (Takara Shuzo), Mutan ^™ -K (Takara Shuzo), etc., using the ODA-LA PCR method, the Gapped duplex method, Conversion can be performed according to a method known per se such as the Kunkel method or a method analogous thereto.

  The cloned DNA can be used as it is depending on the purpose, or after digestion with a restriction enzyme or addition of a linker, if desired. The DNA may have ATG as a translation initiation codon on the 5 'end side and TAA, TGA or TAG as a translation termination codon on the 3' end side. These translation initiation codon and translation termination codon can be added using an appropriate synthetic DNA adapter.

The protein or peptide can be produced by transforming a host with an expression vector containing DNA encoding the above LTBP-1L or a partial peptide thereof, and culturing the resulting transformant.
An expression vector containing a DNA encoding LTBP-1L or a partial peptide thereof is, for example, excising a target DNA fragment from DNA encoding LTBP-1L and ligating the DNA fragment downstream of a promoter in an appropriate expression vector Can be manufactured.
The promoter may be any promoter as long as it is appropriate for the host used for gene expression.
For example, when the host is an animal cell, a cytomegalovirus (CMV) -derived promoter (eg, CMV immediate early promoter), a human immunodeficiency virus (HIV) -derived promoter (eg, HIV LTR), or rous sarcoma virus (RSV) Promoter (eg RSV LTR), mouse mammary tumor virus (MMTV) derived promoter (eg MMTV LTR), Moloney murine leukemia virus (MoMLV) derived promoter (eg MMTV LTR), herpes simplex virus (HSV) derived promoter (eg: HSV thymidine kinase (TK) promoter), SV40 promoter (eg, SV40 early promoter), Epstein Barr virus (EBV) promoter, adeno-associated virus (AAV) promoter (eg, AAV p5 promoter), adenovirus (AdV) Origin promoter (Ad2 or Ad5 major late promoter) I can.
When the host is a bacterium of the genus Escherichia, trp promoter, lac promoter, recA promoter, .lambda.P _L promoter, lpp promoter, T7 promoter and the like are preferable.
When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable.
When the host is yeast, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, etc. are preferable.
When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable.

In addition to the above, an expression vector containing an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 origin of replication, and the like can be used as desired. Examples of the selection marker include dihydrofolate reductase (dhfr) gene [methotrexate (MTX) resistance], ampicillin resistance (Amp ^r ) gene, neomycin resistance (Neo ^r ) gene (G418 resistance), and the like. In particular, when dhfr gene-deficient Chinese hamster (CHO-dhfr ⁻ ) cells are used and the dhfr gene is used as a selection marker, the target gene can be selected using a medium not containing thymidine.
If necessary, a base sequence (signal codon) encoding a signal sequence suitable for the host may be added to the 5 ′ end of DNA encoding LTBP-1L or a partial peptide thereof. When the host is Escherichia, PhoA signal sequence, OmpA signal sequence, etc., when the host is Bacillus, α-amylase signal sequence, subtilisin signal sequence, etc., when the host is yeast, MFα When the signal sequence, SUC2 signal sequence or the like is an animal cell, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence or the like is used.

As the host, for example, Escherichia, Bacillus, yeast, insect cells, insects, animal cells and the like are used.
Examples of the genus Escherichia include Escherichia coli K12, DH1, JM103, JA221, HB101, C600, and the like.
Examples of the Bacillus genus include Bacillus subtilis MI114, 207-21, and the like.
Examples of the yeast include Saccharomyces cerevisiae AH22, AH22R ⁻ , NA87-11A, DKD-5D, 20B-12, Schizosaccharomyces pombe NCYC1913, NCYC2036, Pichia pastoris (Pichia pastoris (Pichia pastoris) KM71 etc. are used.

Insect cells include, for example, when the virus is AcNPV, larvae-derived cell lines (Spodoptera frugiperda cells; Sf cells), MG1 cells derived from the midgut of Trichoplusia ni, High Five ^TM cells derived from eggs of Trichoplusia ni , Cells derived from Mamestra brassicae, cells derived from Estigmena acrea, and the like are used. When the virus is BmNPV, insect-derived cell lines (Bombyx mori N cells; BmN cells) and the like are used as insect cells. Examples of the Sf cells include Sf9 cells (ATCC CRL 1711), Sf21 cells (Vaughn, JL et al., In Vivo, 13, 213-217 (1977)).
Examples of insects include silkworm larvae.

Examples of animal cells include monkey-derived cells (eg, COS-1, COS-7, CV-1, Vero), hamster-derived cells (eg, BHK, CHO, CHO-K1, CHO-dhfr ⁻ ), mouse-derived cells. Cells (eg: NIH3T3, L, L929, CTLL-2, AtT-20), rat-derived cells (eg: H4IIE, PC-12, 3Y1, NBT-II), human-derived cells (eg: HEK293, A549, HeLa, HepG2, HL-60, Jurkat, U937) are used.

Transformation can be performed according to a known method depending on the type of host.
Escherichia can be transformed, for example, according to the method described in Proc. Natl. Acad. Sci. USA, 69, 2110 (1972), Gene, 17, 107 (1982), and the like.
Bacillus can be transformed, for example, according to the method described in Molecular and General Genetics, 168, 111 (1979).
Yeast can be transformed according to the method described in, for example, Methods in Enzymology, 194, 182-187 (1991), Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).
Insect cells and insects can be transformed according to the method described in, for example, Bio / Technology, 6, 47-55 (1988).
Animal cells can be transformed, for example, according to the method described in Cell Engineering Supplement 8 New Cell Engineering Experimental Protocol, 263-267 (1995) (published by Shujunsha), Virology, 52, 456 (1973).

The culture of the transformant can be performed according to a known method depending on the type of the host.
For example, when culturing a transformant whose host is an Escherichia or Bacillus genus, a liquid medium is preferable as a medium used for the culture. Moreover, it is preferable that a culture medium contains a carbon source, a nitrogen source, an inorganic substance, etc. which are required for growth of a transformant. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose; examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein, meat extract, soybean meal, Inorganic or organic substances such as potato extract; examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, and the like. In addition, yeast extract, vitamins, growth promoting factors and the like may be added to the medium. The pH of the medium is preferably about 5-8.
As a medium for culturing a transformant whose host is an Escherichia bacterium, for example, an M9 medium containing glucose and casamino acids is preferable. If necessary, an agent such as 3β-indolylacrylic acid may be added to the medium in order to make the promoter work efficiently. The culture is usually performed at about 15 to 43 ° C. for about 3 to 24 hours. If necessary, aeration or agitation may be performed.
Culture of transformants whose host is Bacillus is usually performed at about 30 to 40 ° C. for about 6 to 24 hours. If necessary, aeration or agitation may be performed.
Examples of the medium for culturing a transformant whose host is yeast include a Burkholder minimum medium and an SD medium containing 0.5% casamino acid. The pH of the medium is preferably about 5-8. The culture is usually performed at about 20 ° C. to 35 ° C. for about 24 to 72 hours. Aeration and agitation may be performed as necessary.
As a medium for culturing a transformant whose host is an insect cell or an insect, for example, a medium obtained by appropriately adding an additive such as 10% bovine serum inactivated to Grace's Insect Medium is used. The pH of the medium is preferably about 6.2 to 6.4. The culture is usually performed at about 27 ° C. for about 3 to 5 days. You may perform ventilation | gas_flowing and stirring as needed.
As a medium for culturing a transformant whose host is an animal cell, for example, a minimum essential medium (MEM) containing about 5 to 20% fetal bovine serum, Dulbecco's modified Eagle medium (DMEM), RPMI1640 medium, 199 A medium or the like is used. The pH of the medium is preferably about 6-8. The culture is usually performed at about 30 ° C. to 40 ° C. for about 15 to 60 hours. You may perform ventilation | gas_flowing and stirring as needed.
As described above, LTBP-1L can be produced inside or outside the transformant.

LTBP-1L can be separated and purified from a culture obtained by culturing the transformant according to a method known per se.
For example, when LTBP-1L is extracted from cultured cells or cells, the cells or cells collected from the culture by a known method are suspended in an appropriate buffer, and the bacteria are obtained by ultrasound, lysozyme and / or freeze-thawing. After the body or cells are destroyed, a method for obtaining a crude extract of soluble protein by centrifugation or filtration is appropriately used. The buffer solution may contain a protein denaturant such as urea or guanidine hydrochloride and a surfactant such as Triton X-100 ^™ . On the other hand, when LTBP-1L is secreted extracellularly, the culture supernatant is recovered from the culture by centrifugation or filtration.
Isolation and purification of LTBP-1L contained in the thus obtained soluble fraction or culture supernatant can be performed according to a method known per se. Such methods include methods that utilize solubility such as salting out and solvent precipitation; mainly using differences in molecular weight such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis. A method utilizing a difference in charge such as ion exchange chromatography; a method utilizing a specific affinity such as affinity chromatography; a method utilizing a difference in hydrophobicity such as reverse phase high performance liquid chromatography; A method using a difference in isoelectric point, such as point electrophoresis, is used. These methods can be combined as appropriate.

When LTBP-1L or a partial peptide thereof thus obtained is a free form, the free form can be converted into a salt by a method known per se or a method analogous thereto, and the protein or peptide is obtained as a salt. In some cases, the salt can be converted into a free form or other salt by a method known per se or a method analogous thereto.
The LTBP-1L produced by the transformant can be arbitrarily modified or the polypeptide can be partially removed by allowing an appropriate protein modifying enzyme to act before or after purification. Examples of the protein modifying enzyme include trypsin, chymotrypsin, arginyl endopeptidase, protein kinase, glycosidase and the like.
The presence of LTBP-1L thus obtained can be confirmed by enzyme immunoassay or Western blotting using a specific antibody.

Furthermore, LTBP-1L or its partial peptide can be translated in vitro using a cell-free protein translation system consisting of rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate, etc. using RNA corresponding to the DNA encoding it as a template. Can also be synthesized. Alternatively, a cell-free transcription / translation system further containing RNA polymerase can be used to synthesize DNA encoding LTBP-1L or a partial peptide thereof as a template. Cell-free protein (transcription / translation) translation systems can be commercially available, and methods known per se, such as Escherichia coli extract, edited by Pratt JM et al., “Transcription and Tranlation”, Hames BD and Higgins SJ , IRL Press, Oxford 179-209 (1984). Examples of commercially available cell lysates include E. coli S30 extract system (Promega) and RTS 500 Rapid Tranlation System (Roche), which are derived from E. coli. Rabbit Reticulocyte Lysate System is derived from rabbit reticulocytes. (Promega), and wheat germ-derived ones include PROTEIOS ^™ (TOYOBO). Of these, those using wheat germ lysate are preferred. As a method for producing wheat germ lysate, for example, the method described in Johnston FB et al., Nature, 179, 160-161 (1957) or Erickson AH et al., Meth. Enzymol., 96, 38-50 (1996), etc. should be used. Can do.
As a system or apparatus for protein synthesis, there are a batch method (Pratt, JM et al. (1984) mentioned above) and a continuous cell-free protein synthesis system (Spirin AS et al. , Science, 242, 1162-1164 (1988)), dialysis (Kikawa et al., 21st Japan Molecular Biology Society, WID6) or multi-layer method (PROTEIOS ^TM Wheat germ cell-free protein synthesis core kit instruction manual: TOYOBO Etc.). Furthermore, a method of supplying template RNA, amino acid, energy source and the like to the synthesis reaction system when necessary, and discharging a synthesized product or a decomposed product when necessary (Japanese Patent Laid-Open No. 2000-333673) can be used.

  An antibody to the LTBP-1L protein or a partial peptide thereof used in the present invention (hereinafter sometimes abbreviated as the antibody of the present invention) is polyclonal as long as it is an antibody that can recognize the LTBP-1L protein or a partial peptide thereof. Either an antibody or a monoclonal antibody may be used. The isotype of the antibody is not particularly limited, but preferably IgG, IgM or IgA, particularly preferably IgG.

The antibody of the present invention is not particularly limited as long as it has at least a complementarity determining region (CDR) for specifically recognizing and binding to a target antigen. In addition to a complete antibody molecule, for example, Fab, Fab ′ , F (ab ') ₂ fragments, scFv, scFv-Fc, minibodies, diabodies and other conjugation molecules prepared by genetic engineering, or molecules having protein stabilizing action such as polyethylene glycol (PEG) The derivative | guide_body modified by these etc. may be sufficient.

An antibody against the LTBP-1L protein or a partial peptide thereof (hereinafter, these may be simply abbreviated as LTBP-1L protein in the description of the antibody) can be produced according to a known method for producing an antibody or antiserum. it can.
Hereinafter, a method for preparing an immunogen of the antibody of the present invention and a method for producing the antibody will be described.

(1) Preparation of antigen The antigen used for preparing the antibody of the present invention has one or more of the above-described LTBP-1L protein or a partial peptide thereof, or the same antigenic determinant (synthesis) ) Any peptide or the like can be used (hereinafter, these may be simply referred to as the antigen of the present invention).
As described above, the human LTBP-1L protein or a partial peptide thereof is prepared by, for example, (a) using a known method from human tissues or cells or a method equivalent thereto, and (b) a known method using a peptide synthesizer or the like. Chemical synthesis by peptide synthesis method, (c) culturing a transformant containing DNA encoding human LTBP-1L protein or its partial peptide, or (d) encoding human LTBP-1L protein or its partial peptide It is produced by biochemical synthesis using a nucleic acid as a template and a cell-free transcription / translation system.
(A) When preparing LTBP-1L protein from human tissues or cells, the homogenized tissues or cells can be used as crude antigens (eg, membrane fraction, soluble fraction) as antigens. . Alternatively, extraction with an acid, surfactant, alcohol, or the like is performed, and the extract is purified by a combination of chromatography such as salting out, dialysis, gel filtration, reverse phase chromatography, ion exchange chromatography, and affinity chromatography. It can also be separated. The obtained LTBP-1L protein can be used as an immunogen as it is, or a partial peptide can be prepared by limited degradation using a peptidase or the like and used as an immunogen.
(B) When the antigen of the present invention is chemically prepared, the synthetic peptide includes, for example, a peptide having the same structure as the LTBP-1L protein purified from a natural material using the method (a) described above. Specifically, a peptide containing one or two or more amino acid sequences identical to the amino acid sequence at any position consisting of at least 3 amino acids, preferably 6 amino acids in the amino acid sequence of the protein is used.
(C) When producing the antigen of the present invention using a transformant containing DNA, the DNA can be obtained by a known cloning method [for example, Molecular Cloning 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab The method described in Press, 1989)]. The cloning method includes (1) isolating DNA encoding the antigen from a human cDNA library by a hybridization method using a DNA probe designed based on a gene sequence encoding human LTBP-1L protein, 2) Using a DNA primer designed based on the gene sequence encoding human LTBP-1L protein, preparing DNA encoding the antigen by PCR using human cDNA as a template, and inserting the DNA into an expression vector suitable for the host The method of doing is mentioned. A desired antigen can be obtained by culturing a transformant obtained by transforming a host with the expression vector in an appropriate medium.
(D) When a cell-free transcription / translation system is used, an expression vector inserted with a DNA encoding an antigen prepared by the same method as in (c) above (for example, the DNA is under the control of a T7, SP6 promoter, etc. After synthesizing mRNA using a transcription reaction solution containing RNA polymerase and substrates (NTPs) compatible with the promoter using the expressed expression vector as a template, a known cell-free translation system (eg, using the mRNA as a template) : Extraction method of Escherichia coli, rabbit reticulocyte, wheat germ, etc.) and the like. By appropriately adjusting the salt concentration and the like, the transcription reaction and the translation reaction can also be carried out collectively in the same reaction solution.

  As an immunogen, a complete human LTBP-1L protein molecule or a peptide having a partial amino acid sequence thereof can be used. Examples of the partial amino acid sequence include those consisting of 3 or more consecutive amino acid residues, preferably 4 or more, more preferably 5 or more, and even more preferably 6 or more consecutive amino acid residues. It is done. Alternatively, the amino acid sequence includes, for example, 20 or less consecutive amino acid residues, preferably 18 or less, more preferably 15 or less, and even more preferably 12 or less consecutive amino acid residues. Is mentioned. Some of these amino acid residues (eg, 1 to several) may be substituted with a substitutable group (eg, Cys, hydroxyl group, etc.). A peptide used as an immunogen has an amino acid sequence containing 1 to several such partial amino acid sequences.

  Alternatively, human cells that express the LTBP-1L protein itself can also be used directly as the antigen of the present invention. As human cells, natural cells as described in the above section (a), cells transformed by the method as described in the above section (c), and the like can be used. The host used for transformation may be any cell collected from humans, monkeys, rats, mice, hamsters, chickens, etc., HEK293, COS7, CHO-K1, NIH3T3, Balb3T3, FM3A, L929, SP2 / 0, P3U1, B16, or P388 is preferably used. Natural human cells or transformed warm-blooded animal cells expressing human LTBP-1L protein were suspended in a medium (eg, RPMI1640) or buffer (eg, Hanks' Balanced Salt Solution) used for tissue culture The immunized animal can be injected in a state. As an immunization method, any method can be used as long as it can promote antibody production, and intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, and the like are preferably used.

  The antigen of the present invention can be directly immunized as long as it has immunogenicity, but it has a low molecular weight (for example, a molecular weight of about 3,000 or less) having only one or several antigenic determinants in the molecule. ) Antigens (ie, LTBP-1L partial peptides), since these antigens are usually low immunogenic hapten molecules, they are immunized as a complex bound or adsorbed to an appropriate carrier. be able to. A natural or synthetic polymer can be used as the carrier. Examples of natural polymers include serum albumin of mammals such as cows, rabbits, and humans, and thyroglobulin of mammals such as cows and rabbits, such as chicken ovalbumin, such as mammals such as cows, rabbits, humans, and sheep. Hemoglobin, keyhole limpet hemocyanin (KLH), etc. are used. Examples of the synthetic polymer include various latexes such as polymers or copolymers such as polyamino acids, polystyrenes, polyacryls, polyvinyls, and polypropylenes.

  The mixing ratio of the carrier and the hapten is such that any antibody can be bound or adsorbed at any ratio as long as an antibody against the antigen bound or adsorbed to the carrier is efficiently produced. The above-mentioned natural or synthetic polymer carrier, which is commonly used in the production of the above, can be used which is bound or adsorbed at a weight ratio of 0.1 to 100 with respect to hapten 1.

  Various coupling agents can be used for coupling the hapten and the carrier. For example, diazonium compounds such as bisdiazotized benzidine that crosslinks tyrosine, histidine, and tryptophan, dialdehyde compounds such as glutaraldehyde that crosslink amino groups, diisocyanate compounds such as toluene-2,4-diisocyanate, and thiol groups A dimaleimide compound such as N, N′-o-phenylene dimaleimide, a maleimide active ester compound that crosslinks an amino group and a thiol group, a carbodiimide compound that crosslinks an amino group and a carboxyl group, and the like are advantageously used. In addition, when cross-linking amino groups, an active ester reagent having a dithiopyridyl group on one of the amino groups (for example, N-succinimidyl (SPDP) 3- (2-pyridyldithio) propionate) was reacted. It is also possible to introduce a thiol group by post-reduction, introduce a maleimide group into the other amino group with a maleimide active ester reagent, and then react both.

(2) Production of monoclonal antibody (a) Production of monoclonal antibody-producing cell The antigen of the present invention is administered to a warm-blooded animal by, for example, intraperitoneal injection, intravenous injection, subcutaneous injection, intradermal injection, or the like. It is administered to a site where production is possible by itself or together with a carrier or diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every 1 to 6 weeks, 2 to 10 times in total. Examples of warm-blooded animals used include monkeys, rabbits, dogs, guinea pigs, mice, rats, hamsters, sheep, goats, donkeys, and chickens. In order to avoid the problem of anti-Ig antibody production, it is preferable to use a mammal of the same species as the administration subject, but mice and rats are generally preferably used for the production of monoclonal antibodies.

  Since human immunization to humans is ethically difficult, when the antibody of the present invention is to be administered to humans, (i) a human antibody-producing animal prepared according to the method described later (eg, mouse) ) To obtain a human antibody, (ii) preparing a chimeric antibody, humanized antibody or fully human antibody according to the method described later, or (iii) in vitro immunization and cell immortalization by virus, human-human (or It is preferable to obtain a human antibody by combining a mouse) hybridoma production technique, a phage display method and the like. In addition to the possibility of obtaining an antigen against an antigen whose antibody production is suppressed by normal immunization, the in vitro immunization method can obtain an antibody with an antigen amount on the order of ng to μg, Can be preferably used also in the case of preparing an antibody derived from a non-human animal as a method for obtaining an antibody against an unstable antigen that is difficult to prepare in large quantities.

Examples of animal cells used for in vitro immunization include lymphocytes isolated from the peripheral blood, spleen, lymph nodes, etc. of humans and warm-blooded animals (preferably mice and rats) described above, preferably B lymphocytes and the like. . For example, in the case of mouse or rat cells, the spleen is removed from an animal of about 4 to 12 weeks of age, and the spleen cells are isolated, and an appropriate medium (eg Dulbecco's modified Eagle medium (DMEM), RPMI1640 medium, Ham F12 medium, etc.) After washing with ( _{2), the} cells are suspended in a medium supplemented with fetal calf serum (FCS; about 5 to 20%) containing the antigen and cultured for about 4 to 10 days using a CO ₂ incubator or the like. Examples of the antigen concentration include, but are not limited to, 0.05 to 5 μg. It is preferable to prepare a thymocyte culture supernatant of an animal of the same strain (preferably about 1 to 2 weeks of age) according to a conventional method and add it to the medium.

  In vitro immunization of human cells, it is difficult to obtain a thymocyte culture supernatant, so several cytokines such as IL-2, IL-4, IL-5, and IL-6, and adjuvant substances (eg, mura) It is preferable to perform immunization by adding a mildipeptide or the like) together with the antigen to the medium.

  When producing a monoclonal antibody, select an individual or a cell population in which an antibody titer has been increased from a warm-blooded animal (eg, mouse, rat) or animal cell (eg: human, mouse, rat) immunized with the antigen. Collect spleen or lymph nodes 2 to 5 days after the final immunization or culture for 4 to 10 days after in vitro immunization, collect cells, isolate antibody-producing cells, and fuse this with myeloma cells Production hybridomas can be prepared. The antibody titer in serum can be measured, for example, by reacting a labeled antigen with antiserum and then measuring the activity of the labeling agent bound to the antibody.

  The myeloma cell is not particularly limited as long as it can produce a hybridoma that secretes a large amount of antibody, but it preferably does not produce or secrete an antibody itself, and more preferably has high cell fusion efficiency. In order to facilitate selection of hybridomas, it is preferable to use a HAT (hypoxanthine, aminopterin, thymidine) sensitive strain. For example, mouse myeloma cells include NS-1, P3U1, SP2 / 0, AP-1, etc., rat myeloma cells include R210.RCY3, Y3-Ag 1.2.3, and human myeloma cells include SKO- 007, GM 1500-6TG-2, LICR-LON-HMy2, UC729-6, and the like.

The fusion operation can be performed according to a known method, for example, the method of Kohler and Milstein [Nature, 256, 495 (1975)]. Examples of the fusion promoter include polyethylene glycol (PEG) and Sendai virus. Preferably, PEG is used. The molecular weight of PEG is not particularly limited, but PEG1000 to PEG6000 having low toxicity and relatively low viscosity are preferable. Examples of the PEG concentration include about 10 to 80%, preferably about 30 to 50%. Various solutions such as serum-free medium (eg, RPMI1640), complete medium containing about 5 to 20% serum, phosphate buffered saline (PBS), Tris buffer, etc. can be used as the PEG dilution solution. it can. If desired, DMSO (eg, about 10 to 20%) can be added. The pH of the fusion solution is, for example, about 4 to 10, preferably about 6 to 8.
The preferred ratio between the number of antibody-producing cells (spleen cells) and the number of bone marrow cells is usually about 1: 1 to 20: 1, and is usually incubated at 20 to 40 ° C., preferably 30 to 37 ° C. for usually 1 to 10 minutes. Cell fusion can be carried out efficiently.

  Antibody-producing cell lines can also be obtained by infecting antibody-producing cells with a virus capable of transforming lymphocytes to immortalize the cells. Examples of such viruses include Epstein-Barr (EB) virus. The vast majority of people have immunity because they have been infected with this virus as an asymptomatic infection of infectious mononucleosis, but virus particles are also produced when using normal EB virus. Appropriate purification should be performed. Recombinant EB virus that retains the ability to immortalize B lymphocytes but lacks the ability to replicate viral particles (for example, switching from a latent infection state to a lytic infection state) as an EB system without the possibility of viral contamination It is also preferable to use a deficiency in the gene.

Since B95-8 cells derived from marmoset secrete EB virus, B lymphocytes can be easily transformed using the culture supernatant. After culturing the cells in, for example, serum and penicillin / streptomycin (P / S) -added medium (eg RPMI1640) or serum-free medium added with cell growth factor, the culture supernatant is separated by filtration or centrifugation, etc. Antibody production by suspending antibody-producing B lymphocytes at an appropriate concentration (eg, about 10 ⁷ cells / mL) and incubating usually at 20 to 40 ° C., preferably at 30 to 37 ° C. for usually about 0.5 to 2 hours. B cell lines can be obtained. When human antibody-producing cells are provided as mixed lymphocytes, most people have T lymphocytes that are toxic to EB virus-infected cells, so in order to increase the frequency of transformation, For example, it is preferable to remove T lymphocytes in advance by forming E rosette with sheep erythrocytes or the like. Alternatively, sheep erythrocytes bound with a soluble antigen can be mixed with antibody-producing B lymphocytes, and a lymphocyte specific for the target antigen can be selected by separating rosettes using a density gradient such as Percoll. Furthermore, by adding a large excess of antigen, antigen-specific B lymphocytes are capped and no longer present IgG on the surface, so when mixed with sheep erythrocytes bound with anti-IgG antibodies, antigen-nonspecific B lymphocytes Only form a rosette. Therefore, antigen-specific B lymphocytes can be selected by collecting a non-rosette-forming layer using a density gradient such as Percoll from this mixture.

  Human antibody-secreting cells that have acquired infinite growth ability by transformation can be back-fused with mouse or human myeloma cells in order to stably maintain the antibody-secreting ability. As the myeloma cells, the same ones as described above can be used.

Hybridoma screening and breeding is usually performed in animal cell culture medium (eg RPMI1640) containing 5-20% FCS or serum-free medium supplemented with cell growth factor, with the addition of HAT (hypoxanthine, aminopterin, thymidine). Is called. Examples of the concentration of hypoxanthine, aminopterin, and thymidine include about 0.1 mM, about 0.4 μM, and about 0.016 mM, respectively. For selection of human-mouse hybridomas, ouabain resistance can be used. Since human cell lines are more sensitive to ouabain than mouse cell lines, unfused human cells can be eliminated by adding them to the medium at about 10 ⁻⁷ to 10 ⁻³ M.

For selection of hybridomas, it is preferable to use feeder cells or certain cell culture supernatants. Feeder cells can be irradiated with different types of cells that have a limited life span so that they can die by helping the emergence of hybridomas, or cells that can produce large amounts of growth factors useful for the appearance of hybridomas. Those having reduced proliferation ability are used. For example, mouse feeder cells include spleen cells, macrophages, blood, thymocytes, etc., and human feeder cells include peripheral blood mononuclear cells. Examples of the cell culture supernatant include primary culture supernatants of the above-mentioned various cells and culture supernatants of various cell lines.
Hybridomas can also be selected by separating the cells that bind to the antigen using a fluorescence activated cell sorter (FACS) after the antigen is fluorescently labeled and reacted with the fused cells. In this case, since a hybridoma that produces an antibody against the target antigen can be directly selected, the cloning effort can be greatly reduced.

Various methods can be used for cloning of hybridomas producing monoclonal antibodies against the target antigen.
Since aminopterin inhibits many cell functions, it is preferably removed from the medium as soon as possible. In the case of mice and rats, most myeloma cells die within 10-14 days, so aminopterin can be removed after 2 weeks of fusion. However, human hybridomas are usually maintained in an aminopterin-added medium for about 4 to 6 weeks after fusion. It is desirable to remove hypoxanthine and thymidine at least 1 week after removal of aminopterin. That is, in the case of mouse cells, for example, a complete medium supplemented with hypoxanthine and thymidine (HT) (eg, RPMI1640 supplemented with 10% FCS) is added or exchanged 7 to 10 days after the fusion. Visible clones appear about 8-14 days after fusion. If the clone diameter is about 1 mm, the amount of antibody in the culture supernatant can be measured.

The amount of antibody can be measured, for example, by hybridoma culture supernatant on a solid phase (eg, microplate) on which a target antigen or derivative thereof or a partial peptide thereof (including a partial amino acid sequence used as an antigenic determinant) is adsorbed directly or with a carrier , Then radioactive material (eg ¹²⁵ I, ¹³¹ I, ³ H, ¹⁴ C), enzyme (eg β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase), fluorescence Anti-immunoglobulin (IgG) antibodies labeled with substances (eg fluorescamine, fluorescein isothiocyanate), luminescent substances (eg luminol, luminol derivatives, luciferin, lucigenin), etc. (Antibodies to IgG from the same species of animal are used) or protein A is added and bound to the solid phase. A method for detecting an antibody against a combined target antigen (antigenic determinant), a hybridoma culture supernatant added to a solid phase adsorbed with anti-IgG antibody or protein A, and a target antigen labeled with the same labeling agent as described above or its target antigen A derivative or a partial peptide thereof is added, and a method for detecting an antibody against a target antigen (antigenic determinant) bound to a solid phase can be performed.

The limiting dilution method is usually used as a cloning method, but cloning using soft agar or cloning using FACS (described above) is also possible. Cloning by the limiting dilution method can be performed, for example, by the following procedure, but is not limited thereto.
The positive well is selected by measuring the amount of antibody as described above. Appropriate feeder cells are selected and added to a 96-well plate. Cells are aspirated from antibody-positive wells, suspended in complete medium (eg RMPI1640 supplemented with 10% FCS and P / S) to a density of 30 cells / mL, and 0.1 mL (3 Cells / well), dilute the remaining cell suspension to 10 cells / mL and sprinkle into another well (1 cell / well), and dilute the remaining cell suspension to 3 cells / mL. Seed into another well (0.3 cells / well). Incubate for 2-3 weeks until a visible clone appears, measure the amount of antibody, select positive wells, and clone again. Because human cells are relatively difficult to clone, prepare a 10-cell / well plate. Usually, monoclonal antibody-producing hybridomas can be obtained by subcloning twice, but it is desirable to re-clone regularly for several months to confirm their stability.

Hybridomas can be cultured in vitro or in vivo.
As an in vitro culture method, the monoclonal antibody-producing hybridoma obtained as described above is used in a well plate while maintaining the cell density at, for example, about 10 ⁵ to 10 ⁶ cells / mL and gradually reducing the FCS concentration. There is a method of gradually increasing the scale.
As an in vivo culture method, for example, a mouse in which plasma oil (MOPC) was induced by injecting mineral oil into the abdominal cavity (mouse compatible with the hybridoma parent strain) was used after 10 ⁶ to 10 ⁶ Examples include a method in which a hybridoma of about 10 ⁷ cells is injected intraperitoneally, and ascites is collected under anesthesia 2 to 5 weeks later.

(B) Purification of monoclonal antibody Separation and purification of monoclonal antibody can be performed by a method known per se, for example, separation and purification of immunoglobulin [Example: salting-out method, alcohol precipitation method, isoelectric precipitation method, electrophoresis method, ion exchange Absorbing and desorbing by body (eg DEAE, QEAE), ultracentrifugation, gel filtration, antigen-binding solid phase or active adsorbent such as protein A or protein G, and collecting antibody alone to dissociate the antibody Can be carried out according to the specific purification method obtained.

  As described above, a monoclonal antibody can be produced by culturing a hybridoma in vivo or ex vivo of a warm-blooded animal and collecting the antibody from the body fluid or culture.

  When the antibody of the present invention is used for cancer prevention / treatment, since the antibody must have antitumor activity, it is necessary to examine the degree of antitumor activity of the obtained monoclonal antibody. Antitumor activity can be measured by comparing the proliferation of cancer cells, the induction of apoptosis, etc. in the presence and absence of antibodies.

  In a preferred embodiment, since the antibody of the present invention is used as a pharmaceutical for human administration, the antibody of the present invention (preferably a monoclonal antibody) has a reduced risk of showing antigenicity when administered to a human. Antibodies, specifically, fully human antibodies, humanized antibodies, mouse-human chimeric antibodies, etc., particularly preferably fully human antibodies. Humanized antibodies and chimeric antibodies can be produced by genetic engineering according to the methods described below. Although a fully human antibody can be produced from the above-described human-human (or mouse) hybridoma, in order to provide a large amount of antibody stably and at low cost, a human antibody-producing animal (described later) Example: mouse) or phage display method is preferred.

"Chimeric antibody" produced herein (i) a chimeric antibody derived from a mammalian species where there is a sequence of the variable region of H chain and L chain (V _H and V _L), the constant region (C _H and An antibody whose sequence of C _L ) is derived from another mammalian species. The sequence of the variable region is preferably derived from an animal species that can easily produce a hybridoma such as a mouse, and the sequence of the constant region is preferably derived from the mammalian species to be administered.

Examples of the method for producing a chimeric antibody include the method described in US Pat. No. 6,331,415 or a method obtained by partially modifying it. Specifically, first, mRNA or total RNA is prepared from a monoclonal antibody-producing hybridoma (for example, mouse-mouse hybridoma) obtained as described above according to a conventional method, and cDNA is synthesized. Using the cDNA as a template, an appropriate primer (for example, an oligo DNA containing a nucleotide sequence encoding each N-terminal sequence of V _H and V _L as a sense primer, and an N-terminal sequence of C _H and C _L as an antisense primer) DNA encoding _VH and _VL is amplified and purified by PCR according to a conventional method using an oligo DNA that hybridizes with the base sequence to be synthesized (see, for example, Bio / Technology, 9: 88-89, 1991). By the same method, other mammalian (e.g. human) DNA to amplify and purify encoding a C _H and C _L by RT-PCR from RNA was prepared from lymphocytes or the like. V _H and C _H , _VL and C _L were ligated using a conventional method, and the resulting chimeric H chain DNA and chimeric L chain DNA were respectively converted into appropriate expression vectors (for example, CHO cells, COS cells, mice). It is inserted into a promoter (eg, a vector containing a CMV promoter, SV40 promoter, etc.) having transcription activity in myeloma cells or the like. DNAs encoding both strands may be inserted into separate vectors or tandemly inserted into one vector. Host cells are transformed with the obtained chimeric H chain and chimeric L chain expression vectors. Examples of host cells include animal cells such as the above-described mouse myeloma cells, Chinese hamster ovary (CHO) cells, monkey-derived COS-7 cells, Vero cells, and rat-derived GHS cells. For transformation, any method applicable to animal cells may be used, preferably electroporation. After culturing in a medium suitable for host cells for a certain period, the culture supernatant is recovered and purified by the same method as described above, whereby a chimeric monoclonal antibody can be isolated. Alternatively, transgenic animals such as cattle, goats and chickens have been established as host cells, and germline cells of animals that have accumulated extensive breeding know-how as livestock (poultry) are used to produce transgenic animals by conventional methods. By doing so, a chimeric monoclonal antibody can also be obtained easily and in large quantities from the milk or egg of the animal obtained. In addition, transgenic cells such as corn, rice, wheat, soybean, and tobacco have been established, and plant cells grown in large quantities as main crops are used as host cells for microinjection, electroporation, and intact cells into protoplasts. It is also possible to produce a transgenic plant using the particle gun method, Ti vector method, etc., and obtain a chimeric monoclonal antibody in large quantities from the seeds and leaves obtained.
Fab can be obtained by digesting the obtained chimeric monoclonal antibody with papain, and F (ab ') ₂ can be obtained by digesting with pepsin.

In addition, DNA encoding mouse _VH and _VL is converted into a suitable linker, for example, a peptide comprising 1 to 40 amino acids, preferably 3 to 30 amino acids, more preferably 5 to 20 amino acids (eg, [Ser- (Gly) m scFv by ligating via DNA encoding] n or [(Gly) m-Ser] n (m is an integer of 0 to 10, n is an integer of 1 to 5), etc. or a minidody monomer by ligating the DNA encoding the C _H3 via a suitable linker may also be a scFv-Fc by ligating a DNA encoding a C _H full length through a suitable linker. DNA encoding such genetically modified (conjugated) antibody molecules can be expressed in microorganisms such as Escherichia coli and yeast by placing them under the control of an appropriate promoter, producing large amounts of antibody molecules can do.

When DNA encoding mouse _VH and _VL is inserted in tandem downstream of one promoter and introduced into E. coli, a dimer called Fv is formed by monocistronic gene expression. Moreover, when an appropriate amino acid in FR of _VH and _VL is substituted with Cys using molecular modeling, a dimer called dsFv is formed by an intermolecular disulfide bond between both chains.

(Ii) Humanized antibody As used herein, the term “humanized antibody” refers to all regions other than the complementarity determining region (CDR) present in the variable region (that is, the framework region (FR) in the constant region and the variable region. )) Sequences are derived from humans, and only CDR sequences are derived from other mammalian species. As other mammalian species, for example, an animal species that can easily produce a hybridoma such as a mouse is preferable.
Examples of the method for producing a humanized antibody include the methods described in US Pat. Nos. 5,225,539, 5,585,089, 5,693,761 and 5,693,762, or methods obtained by partially modifying them. Specifically, in the same manner as in the case of the chimeric antibody, DNAs encoding V _H and V _L derived from mammalian species other than humans (eg, mice) are isolated, and then an automatic DNA sequencer ( Example: Applied Biosystems, etc.) and the obtained nucleotide sequence or the amino acid sequence deduced therefrom is used as a known antibody sequence database [for example, Kabat database (Kabat et al., “Sequences of Proteins of Immunological Interest”] , US Department of Health and Human Services, Public Health Service, NIH, 5th edition, 1991) etc.] to determine the CDR and FR of both strands. Nucleotide sequences encoding L and H chains of human antibodies having an FR sequence similar to the determined FR sequence [eg, human κ-type L chain subgroup I and human H chain subgroup II or III (Kabat et al., 1991 (See above))] is designed by replacing the CDR coding region with the determined base sequence encoding the heterologous CDR, and the base sequence is divided into fragments of about 20 to 40 bases. A sequence complementary to the sequence is divided into fragments of about 20 to 40 bases so as to alternately overlap with the fragments. By synthesizing each fragment using a DNA synthesizer and hybridizing and ligating them according to a conventional method, DNA encoding V _H and V _L having FRs derived from human and CDRs derived from other mammalian species is constructed. can do. To transplant CDRs from other mammalian species into human-derived _VH and _VL more rapidly and efficiently, it is preferable to use site-directed mutagenesis by PCR. Examples of such a method include a sequential CDR transplantation method described in JP-A-5-227970. The DNA encoding V _H and V _L thus obtained is introduced into an appropriate host cell by ligating with DNA encoding human _H C and C _L in the same manner as in the case of the chimeric antibody. By doing so, cells or transgenic animals and plants that produce humanized antibodies can be obtained.

Humanized antibodies can also be modified into scFv, scFv-Fc, minibody, dsFv, Fv, etc. using genetic engineering techniques as well as chimeric antibodies, and can be produced in microorganisms such as Escherichia coli and yeast using appropriate promoters. Can be made.
The humanized antibody production technique can also be applied to, for example, production of a monoclonal antibody that can be preferably administered to other animal species for which hybridoma production techniques have not been established. For example, animals that are widely bred as domestic animals (poultry) such as cattle, pigs, sheep, goats, chickens, and pet animals such as dogs and cats can be mentioned.

(Iii) Production of fully human antibodies using human antibody-producing animals A functional human Ig gene is introduced into a non-human warm-blooded animal knocked out (KO) of an endogenous immunoglobulin (Ig) gene, and this is immunized with an antigen. Thus, human antibodies are produced instead of the animal-derived antibodies. Therefore, using an animal with established hybridoma production technology, such as a mouse, it is possible to obtain a fully human monoclonal antibody by the same method as the production of a conventional mouse monoclonal antibody. First, a mouse in which human Ig H-chain and L-chain minigenes were introduced using normal transgenic (Tg) technology, and a mouse in which the endogenous mouse Ig gene was inactivated using normal KO technology. Some of the human monoclonal antibodies produced using human antibody-producing mice obtained by mating (see Immunol. Today, 17: 391-397, 1996) are already in the clinical stage, and so far anti-human Production of Ig human antibody (HAHA) has not been reported.

  Thereafter, Abgenix [trade name: XenoMouose (Nat. Genet., 15: 146-156, 1997; see US Pat. No. 5,939,598, etc.)] and Medarex [trade name: Hu-Mab Mouse (Nat. Biotechnol., 14: 845-851, 1996; see US Pat. No. 5,545,806, etc.)] using a yeast artificial chromosome (YAC) vector to create a Tg mouse into which a larger human Ig gene was introduced, and a human antibody with a richer repertoire. Can be produced. However, for example, in the case of the H chain, the human Ig gene has its VDJ exon in which various combinations of about 80 V fragments, about 30 D fragments, and 6 J fragments encode antigen binding sites. Because of the diversity, the total length of H chain is about 1.5 Mb (Chromosome 14), κ L chain is about 2 Mb (Chromosome 2), and λ L chain is about 1 Mb (Chromosome 22). In order to reproduce various antibody repertoires similar to those in humans with other animal species, it is desirable to introduce the full length of each Ig gene, but conventional gene transfer vectors (plasmids, cosmids, BACs, YACs, etc.) The DNA that can be inserted into is usually several kb to several hundred kb, and it has been difficult to introduce the full length by the conventional transgenic animal production technique in which the cloned DNA is injected into a fertilized egg.

Tomizuka et al. (Nat. Genet., 16: 133-143, 1997) introduced a human chromosome natural fragment (hCF) carrying an Ig gene into a mouse (chromosome transfer (TC) mouse) and introduced a full-length human Ig. Mice carrying the gene were produced. That is, first, a human-mouse hybrid cell having a human chromosome obtained by labeling the chromosome 14 containing the H chain gene and the chromosome 2 containing the κ L chain gene with, for example, a drug resistance marker or the like is used as a spindle thread formation inhibitor (eg : Colcemid) to prepare microcells in which one to several chromosomes or fragments thereof are encapsulated in the nuclear membrane, and introduce the chromosomes into mouse ES cells by the micronucleus fusion method. Hybrid ES cells retaining a chromosome having a human Ig gene or a fragment thereof are selected using a medium containing a drug, and microinjected into a mouse embryo in the same manner as in the case of normal KO mouse production. TC mouse strains that transmit the human chromosome 14 fragment (TC (hCF14)) and TC mouse strains that transmit the human chromosome 2 fragment by selecting germline chimeras from the resulting chimeric mouse using the coat color as an index, etc. (TC (hCF2)) is established. A mouse (KO (IgH) and KO (Igκ)) in which the endogenous heavy chain gene and κ light chain gene are KOed by a conventional method is prepared, and all four kinds of genetic modifications are carried out by repeating crosses of these four strains. A mouse strain (double TC / KO) can be established.
An antigen-specific human monoclonal antibody-producing hybridoma can be produced by applying the same method as that for producing a normal mouse monoclonal antibody to the double TC / KO mouse produced as described above. However, since hCF2 containing the κ light chain gene is unstable in mouse cells, there is a drawback that the efficiency of obtaining hybridomas is lower than that of normal mice.

  On the other hand, the Hu-Mab Mouse contains about 50% of the κ light chain gene, but has a structure in which the variable region cluster is doubled, and thus exhibits the same diversity of κ chain as when it contains the full length (on the other hand, the H chain gene). Contains only about 10%, so the diversity of the heavy chain is low, and the responsiveness to the antigen is insufficient), and it is inserted into the mouse chromosome by the YAC vector (Igκ-YAC). It is kept stable. Taking advantage of this advantage, TC (hCF14) mouse and Hu-Mab Mouse are crossed to create a mouse (trade name: KM mouse) that stably retains hCF14 and Igκ-YAC. The efficiency of obtaining the hybridoma and the antigen affinity of the antibody can be obtained.

  Furthermore, in order to more fully reproduce various antibody repertoires in humans, human antibody-producing animals into which a λL chain gene has been further introduced can be produced. Such an animal produced a TC mouse (TC (hCF22)) into which human chromosome 22 carrying a λL chain gene or a fragment thereof was introduced in the same manner as described above, and the double TC / KO mouse or KM mouse. Or can be obtained by constructing a human artificial chromosome (HAC) containing, for example, an H chain locus and a λ L chain locus and introducing it into mouse cells (Nat. Biotechnol., 18: 1086-1090, 2000).

  When the antibody of the present invention is used as a pharmaceutical, it is preferably a monoclonal antibody, but may be a polyclonal antibody. When the antibody of the present invention is a polyclonal antibody, the use of a hybridoma is not required. Therefore, a hybridoma production technique is not established, but an animal species for which a transgenic technique is established, preferably an ungulate such as a cow is selected. If a human antibody-producing animal is produced using the same method as described above, a larger amount of human antibody can be produced at a low cost (see, for example, Nat. Biotechnol., 20: 889-894, 2002). . The resulting human polyclonal antibody can be purified by collecting blood, ascites, milk, eggs, etc., preferably milk and eggs of human antibody-producing animals, and combining the same purification techniques as described above.

(Iv) Production of fully human antibody using phage display human antibody library Another approach for producing fully human antibody is a method using phage display. In this method, PCR mutations may be introduced in addition to CDRs, so there are few reports of HAHA production in the clinical stage, but there is no risk of infection with heterologous viruses derived from the host animal. And the specificity of the antibody is infinite (antibodies against prohibited clones, sugar chains, etc. can be easily produced).

Examples of the method for preparing a phage display human antibody library include, but are not limited to, the following.
The phage to be used is not particularly limited, but usually filamentous phage (Ff bacteriophage) is preferably used. Examples of a method for displaying a foreign protein on the phage surface include a method of expressing and displaying on a coat protein as a fusion protein with any of the coat proteins g3p and g6p to g9p, but g3p or g8p is often used. This is a method of fusing to the N-terminal side. The phage display vector includes 1) a foreign protein fused to the coat protein gene of the phage genome, and all the coat proteins displayed on the phage surface are presented as fusion proteins with the foreign protein. ) A gene encoding the fusion protein is inserted separately from the wild type coat protein gene to express the fusion protein and the wild type coat protein simultaneously, and 3) E. coli having a phagemid vector having the gene encoding the fusion protein Infecting a helper phage having a wild-type coat protein gene to produce phage particles that simultaneously express the fusion protein and the wild-type coat protein. Because of the loss of infectivity, 2) or Type 3) is used.

Specific examples of the vector include those described in Holt et al. (Curr. Opin. Biotechnol., 11: 445-449, 2000). For example, pCES1 (see J. Biol. Chem., 274: 18218-18230, 1999) is a DNA encoding a κ light chain constant region downstream of a g3p signal peptide under the control of one lactose promoter and a g3p signal peptide. This is a Fab expression type phagemid vector in which DNA encoding CH3, His-tag, c-myc tag, and g3p coding sequence are arranged via an amber stop codon (TAG) downstream. When introduced into Escherichia coli having an amber mutation, Fab is displayed on the g3p coat protein, but when expressed in an HB2151 strain without an amber mutation, a soluble Fab antibody is produced. Moreover, as the scFv expression type phagemid vector, for example, pHEN1 (J. Mol. Biol., 222: 581-597, 1991) is used.
On the other hand, examples of the helper phage include M13-KO7 and VCSM13.
As another phage display vector, a sequence containing a codon encoding cysteine is linked to the 3 ′ end of the antibody gene and the 5 ′ end of the coat protein gene, respectively, and both genes are separated separately (not as a fusion protein). Examples include those designed to express antibodies and present antibodies on the coat protein on the phage surface via SS bonds between the introduced cysteine residues (CysDisplay ^™ technology from Morphosys).

Examples of human antibody libraries include naive / non-immunized libraries, synthetic libraries, immune libraries, and the like.
A naive / non-immunized library is a library obtained by RT-PCR of _VH and _VL genes possessed by normal humans and randomly cloning them into the above phage display vectors. It is. Normally, mRNA derived from lymphocytes such as normal human peripheral blood, bone marrow, and tonsils is used as a template. In order to eliminate V gene bias such as disease history, an amplification of only IgM-derived mRNA that has not undergone class switching due to antigen sensitization is called a naive library. Representative examples include CAT libraries (see J. Mol. Biol., 222: 581-597, 1991; Nat. Biotechnol., 14: 309-314, 1996), MRC libraries (Annu Rev. Immunol., 12: 433-455, 1994), Dyax library (J. Biol. Chem., 1999 (supra); Proc. Natl. Acad. Sci. USA, 14: 7969-7974, 2000).
Synthetic library selects functional specific antibody genes in human B cells and replaces part of antigen gene binding region such as CDR3 of V gene fragment with DNA encoding random amino acid sequence of appropriate length It is a library. Since a library can be constructed from a combination of _VH and _VL genes that produce functional scFv and Fab from the beginning, it is said to be excellent in antibody expression efficiency and stability. Representative examples include Morphosys' HuCAL library (see J. Mol. Biol., 296: 57-86, 2000), BioInvent's library (see Nat. Biotechnol., 18: 852, 2000), And a library of Crucell (see Proc. Natl. Acad. Sci. USA, 92: 3938, 1995; J. Immunol. Methods, 272: 219-233, 2003).
An immunized library is a lymphocyte collected from humans with increased blood antibody titers against the target antigen, such as patients with cancer, autoimmune diseases, infectious diseases, etc. Prepare mRNA from human lymphocytes artificially immunized with the target antigen in the same manner as in the above naive / non-immunized library, and amplify the _VH and _VL genes by RT-PCR method to make a library It is a thing. Since the target antibody gene is contained in the library from the beginning, the target antibody can be obtained even from a relatively small size library.

The greater the diversity of the library, the better. However, in reality, the number of phages that can be handled by the following panning operations (10 ¹¹ to 10 ¹³ phages) and the number of phages necessary for isolation and amplification of clones by normal panning (100 In consideration of (˜1,000 phage / clone), about 10 ⁸ to 10 ¹¹ clones are appropriate, and an antibody having a Kd value of usually about 10 ⁻⁹ order can be screened with a library of about 10 ⁸ clones.

The process of selecting antibodies against the target antigen by the phage display method is called panning. Specifically, for example, a carrier on which an antigen is immobilized is contacted with a phage library, and after washing away unbound phage, the bound phage is eluted from the carrier, and the phage is propagated by infecting E. coli. The phages displaying antigen-specific antibodies are concentrated by repeating a series of operations 3 to 5 times. As a carrier for immobilizing an antigen, various carriers used in normal antigen-antibody reaction and affinity chromatography, for example, insoluble polysaccharides such as agarose, dextran, and cellulose, synthetic resins such as polystyrene, polyacrylamide, and silicon, or glass, Examples include microplates, tubes, membranes, columns, beads, and the like made of metal, and surface plasmon resonance (SPR) sensor chips. For the immobilization of the antigen, physical adsorption may be used, or a method using a chemical bond usually used to insolubilize and immobilize a protein or an enzyme may be used. For example, a biotin- (strept) avidin system is preferably used. When the endogenous ligand that is the target antigen is a small molecule such as a peptide, special care must be taken so that the moiety used as the antigenic determinant is not covered by binding to the carrier. For washing of unbound phage, blocking solution such as BSA solution (1-2 times), PBS containing a surfactant such as Tween (3-5 times) and the like can be used sequentially. There are also reports that the use of citrate buffer (pH 5) and the like is preferable. For elution of specific phage, acid (eg 0.1M hydrochloric acid) is usually used, but cleavage by specific protease (for example, gene sequence encoding a trypsin cleavage site at the junction between antibody gene and coat protein gene) In this case, since the wild-type coat protein is displayed on the surface of the phage to be eluted, even if all of the coat protein is expressed as a fusion protein, infection and proliferation of E. coli are possible) Competitive elution with soluble antigen, or reduction of SS binding (for example, with CysDisplay ^™ , after panning, antigen-specific phages are recovered by dissociating the antibody and coat protein using an appropriate reducing agent. Elution) is also possible. In the case of elution with acid, after neutralization with Tris or the like, the eluted phage is infected with E. coli, and after culturing, the phage is recovered by a conventional method.

  When the phages displaying antigen-specific antibodies are concentrated by panning, they are infected with E. coli and then seeded on a plate for cloning. The phages are collected again, and the antigen-binding activity is confirmed by the above-described antibody titer measurement method (eg, ELISA, RIA, FIA, etc.) or measurement using FACS or SPR.

Isolation and purification of antibodies from phage clones displaying selected antigen-specific antibodies can be achieved, for example, when using a vector in which an amber stop codon is introduced at the junction between the antibody gene and the coat protein gene as a phage display vector. When the phage is infected with E. coli without an amber mutation (eg, strain HB2151), soluble antibody molecules are produced and secreted into the periplasm or medium. Can be recovered and purified using the same purification technique as described above. If His-tag or c-myc tag is introduced, it can be easily purified using IMAC or anti-c-myc antibody column. Also,
In the case of utilizing cleavage by a specific protease during panning, the antibody is separated from the phage surface when the protease is allowed to act. Therefore, the target antibody can be purified by carrying out the same purification operation. it can.

  Fully human antibody production techniques using human antibody-producing animals and phage display human antibody libraries can also be applied to produce monoclonal antibodies of other animal species. For example, animals that are widely bred as domestic animals (poultry) such as cattle, pigs, sheep, goats, chickens, and pet animals such as dogs and cats can be mentioned. In non-human animals, the use of an immune library is more effective because there are few ethical problems with respect to artificial immunity of the target antigen.

(3) Production of polyclonal antibody The polyclonal antibody of the present invention can be produced according to a method known per se or a method analogous thereto. For example, an immune antigen (protein or peptide antigen) itself or a complex of it and a carrier protein is prepared, and a warm-blooded animal is immunized in the same manner as the above-described monoclonal antibody production method. From the immunized animal to the LTBP-1L protein It can be produced by collecting the antibody-containing material and separating and purifying the antibody.

  Regarding the complex of immunizing antigen and carrier protein used to immunize warm-blooded animals, the type of carrier protein and the mixing ratio of carrier protein and hapten are such that antibodies against hapten immunized by cross-linking to carrier protein Any one can be cross-linked at any ratio as long as it is efficient.For example, bovine serum albumin, bovine thyroglobulin, hemocyanin and the like are about 0.1 to about 20, preferably about A method of coupling at a rate of 1 to about 5 is used.

Various coupling agents can be used for coupling of the hapten and the carrier protein, but an active ester reagent containing glutaraldehyde, carbodiimide, maleimide active ester, thiol group, dithiobilidyl group or the like is used.
The condensation product is administered to warm-blooded animals at the site where antibody production is possible, or with a carrier and a diluent. Complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance antibody production ability upon administration. The administration is usually performed once every about 1 to about 6 weeks, about 2 to about 10 times in total.

Polyclonal antibodies can be collected from blood, ascites, etc., preferably from blood of warm-blooded animals immunized by the above method.
The polyclonal antibody titer in the antiserum can be measured in the same manner as the antibody titer in the antiserum described above. Separation and purification of the polyclonal antibody can be performed according to the same immunoglobulin separation and purification method as the above-described monoclonal antibody separation and purification.

A nucleic acid containing a base sequence complementary to the target region of the target nucleic acid, that is, a nucleic acid that can hybridize with the target nucleic acid can be said to be “antisense” to the target nucleic acid.
In the nucleotide sequence of a nucleic acid encoding human LTBP-1L protein (eg, DNA (hereinafter, in the description of antisense nucleic acid, DNA encoding human LTBP-1L may be abbreviated as DNA of the present invention)) As an antisense nucleic acid having a complementary or substantially complementary base sequence or a part thereof, a base complementary or substantially complementary to the base sequence of a nucleic acid (eg, DNA) encoding human LTBP-1L Any antisense nucleic acid may be used as long as it contains a sequence or a part thereof and has an action capable of suppressing the expression of the nucleic acid.

  The base sequence substantially complementary to the DNA of the present invention refers to, for example, a region overlapping with the base sequence of the base sequence complementary to the DNA of the present invention (that is, the complementary strand of the DNA of the present invention). It is a base sequence having a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more. Here, “homology” is synonymous with the DNA of the present invention described above. In particular, among the entire base sequences of the complementary strand of the DNA of the present invention, in the case of (a) an antisense nucleic acid directed to translation inhibition, the base sequence of the portion encoding the N-terminal site of the LTBP-1L protein (for example, the start) An antisense nucleic acid having a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more with a complementary strand of a base sequence in the vicinity of a codon, etc. (b) directs RNA degradation by RNaseH In the case of an antisense nucleic acid, an antisense nucleic acid having a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more with the complementary strand of the entire base sequence of the DNA of the present invention including introns. Each is suitable.

  Specifically, an antisense nucleic acid comprising a base sequence complementary or substantially complementary to the base sequence represented by SEQ ID NO: 2 or a part thereof, preferably, for example, a base represented by SEQ ID NO: 2. Examples include antisense nucleic acids containing a base sequence complementary to the sequence or a part thereof.

  An antisense nucleic acid having a base sequence complementary to or substantially complementary to the base sequence of the DNA of the present invention or a part thereof (hereinafter also referred to as the antisense nucleic acid of the present invention) has been cloned or determined. It can be designed and synthesized based on the nucleotide sequence information of DNA encoding LTBP-1L. Such antisense nucleic acids can inhibit the replication or expression of the LTBP-1L gene. That is, the antisense nucleic acid of the present invention can hybridize with RNA (mRNA or initial transcription product) transcribed from the LTBP-1L gene and inhibits mRNA synthesis (processing) or function (translation into protein). can do.

The length of the target region of the antisense nucleic acid of the present invention is not particularly limited as long as the antisense nucleic acid hybridizes, and as a result, the translation into the LTBP-1L protein is inhibited. The entire sequence or partial sequence of the encoded mRNA may be used, and may be a short sequence of about 10 bases and a long sequence of mRNA or the initial transcript. In view of ease of synthesis and antigenicity, an oligonucleotide consisting of about 10 to about 40 bases, particularly about 15 to about 30 bases is preferable, but not limited thereto. Specifically, the LTBP-1L gene 5 'end hairpin loop, 5' end 6-base pair repeat, 5 'end untranslated region, translation initiation codon, protein coding region, ORF translation stop codon, 3' end non A translation region, 3 ′ end palindromic region, 3 ′ end hairpin loop, or the like can be selected as a preferred target region of an antisense nucleic acid, but any region within the LTBP-1L gene can be selected as a target. For example, the intron portion of the gene can be used as the target region.
Furthermore, the antisense nucleic acid of the present invention not only hybridizes with LTBP-1L mRNA or initial transcription product to inhibit translation into protein, but also binds to the LTBP-1L gene, which is a double-stranded DNA, to form a triplex. It may form a chain (triplex) and inhibit RNA transcription. Alternatively, a DNA: RNA hybrid may be formed to induce degradation by RNaseH.

  In order to prevent degradation by a hydrolase such as nuclease, phosphate residues (phosphates) of each nucleotide constituting an antisense nucleic acid are converted to chemically modified phosphate residues such as phosphorothioate, methylphosphonate, and phosphorodithionate. May be substituted. In addition, the sugar (deoxyribose) of each nucleotide may be substituted with a chemically modified sugar structure such as 2′-O-methylation, and the base portion (pyrimidine, purine) is also chemically modified. Any one may be used as long as it hybridizes with DNA having the base sequence represented by SEQ ID NO: 2.

  Antisense nucleic acids include polynucleotides containing 2-deoxy-D-ribose, polynucleotides containing D-ribose, other types of polynucleotides that are N-glycosides of purine or pyrimidine bases, non-nucleotides Other polymers with backbones (eg, commercially available protein nucleic acids and synthetic sequence specific nucleic acid polymers) or other polymers containing special linkages, provided that the polymer is a pair of bases as found in DNA or RNA And a nucleotide having a configuration that allows attachment of a ring or a base). They may be double-stranded DNA, single-stranded DNA, double-stranded RNA, single-stranded RNA, DNA: RNA hybrids, unmodified polynucleotides (or unmodified oligonucleotides), known modifications Additions, such as those with labels known in the art, capped, methylated, one or more natural nucleotides replaced with analogs, intramolecular nucleotide modifications Such as those having uncharged bonds (eg methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged bonds or sulfur-containing bonds (eg phosphorothioates, phosphorodithioates, etc.) Things, eg proteins (eg, nucleases, nuclease inhibitors, toxins, antibodies, signals Peptides, poly-L-lysine, etc.) and sugars (eg, monosaccharides, etc.) having side chain groups, intercurrent compounds (eg, acridine, psoralen, etc.), chelate compounds (eg, Metals, radioactive metals, boron, oxidizing metals, etc.), alkylating agents, and modified bonds (eg, α-anomeric nucleic acids) Good. Here, the “nucleoside”, “nucleotide” and “nucleic acid” may include not only purine and pyrimidine bases but also those having other modified heterocyclic bases. Such modifications may include methylated purines and pyrimidines, acylated purines and pyrimidines, or other heterocycles. Modified nucleosides and modified nucleotides may also be modified at the sugar moiety, for example, one or more hydroxyl groups are replaced by halogens, aliphatic groups, etc., or functional groups such as ethers, amines, etc. It may have been converted.

  The antisense nucleic acid of the present invention is RNA, DNA or a modified nucleic acid (RNA, DNA). Specific examples of the modified nucleic acids include nucleic acid sulfur derivatives, thiophosphate derivatives, polynucleoside amides and oligonucleoside amides that are resistant to degradation. The antisense nucleic acid of the present invention can be designed, for example, as follows. That is, make the antisense nucleic acid more stable in the cell, increase the cell permeability of the antisense nucleic acid, increase the affinity for the target sense strand, and if it is toxic In this case, the toxicity of the antisense nucleic acid is made smaller. Many such modifications have been reported in, for example, Pharm Tech Japan, 8, 247 or 395, 1992, Antisense Research and Applications, CRC Press, 1993, and the like.

  The antisense nucleic acid of the present invention may be altered or contain modified sugars, bases and bonds, provided in special forms such as liposomes, microspheres, applied by gene therapy, It can be given in an added form. In this way, the additional form includes polycationic substances such as polylysine that acts to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( Examples include hydrophobic ones such as phospholipid and cholesterol. Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.). These can be attached to the 3 'or 5' end of nucleic acids and can be attached via bases, sugars, intramolecular nucleoside linkages. Examples of the other group include a cap group specifically arranged at the 3 'end or 5' end of a nucleic acid, which prevents degradation by nucleases such as exonuclease and RNase. Such capping groups include, but are not limited to, hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol.

  A ribozyme capable of specifically cleaving the mRNA or initial transcript encoding LTBP-1L within the coding region (including the intron portion in the case of the initial transcript) can also be included in the antisense nucleic acid of the present invention. . “Ribozyme” refers to RNA having an enzyme activity that cleaves nucleic acid. Recently, it has been clarified that oligo DNA having the base sequence of the enzyme active site also has a nucleic acid cleavage activity. As long as it has sequence-specific nucleic acid cleavage activity, it is used as a concept including DNA. The most versatile ribozyme is self-splicing RNA found in infectious RNA such as viroid and virusoid, and the hammerhead type and hairpin type are known. The hammerhead type exhibits enzyme activity at about 40 bases, and several bases at both ends adjacent to the part that takes the hammerhead structure (about 10 bases in total) are made complementary to the desired cleavage site of mRNA. By doing so, it is possible to specifically cleave only the target mRNA. This type of ribozyme has the additional advantage of not attacking genomic DNA since it only uses RNA as a substrate. When LTBP-1L mRNA itself has a double-stranded structure, the target sequence is made single-stranded by using a hybrid ribozyme linked to an RNA motif derived from a viral nucleic acid that can specifically bind to an RNA helicase. [Proc. Natl. Acad. Sci. USA, 98 (10): 5572-5577 (2001)]. Furthermore, when the ribozyme is used in the form of an expression vector containing the DNA encoding the ribozyme, in order to promote the transfer of the transcription product to the cytoplasm, the ribozyme should be a hybrid ribozyme further linked with a tRNA-modified sequence. [Nucleic Acids Res., 29 (13): 2780-2788 (2001)].

  In the present specification, a double strand comprising an oligo RNA complementary to LTBP-1L mRNA or a partial sequence in the coding region of the initial transcript (including an intron in the case of the initial transcript) and its complementary strand RNA, so-called siRNA, is also defined as encompassed by the antisense nucleic acid of the invention. When a short double-stranded RNA is introduced into a cell, a phenomenon called RNA interference (RNAi), in which mRNA complementary to the RNA is degraded, has been known in nematodes, insects, plants, etc. Since this phenomenon was confirmed to occur widely in animal cells [Nature, 411 (6836): 494-498 (2001)], it has been widely used as an alternative to ribozyme. siRNA can be appropriately designed using commercially available software (eg, RNAi Designer; Invitrogen) based on the base sequence information of the target mRNA.

  The antisense oligonucleotide and ribozyme of the present invention determine the target sequence of mRNA or initial transcript based on the cDNA sequence or genomic DNA sequence of LTBP-1L, and are commercially available DNA / RNA automatic synthesizers (Applied Biosystems) , Beckman, etc.) and can be prepared by synthesizing a sequence complementary thereto. For siRNA, the sense strand and the antisense strand are respectively synthesized by a DNA / RNA automatic synthesizer, denatured at about 90 to about 95 ° C. for about 1 minute in an appropriate annealing buffer, and then about 30 to about 70 ° C. For about 1 to about 8 hours. In addition, longer double-stranded polynucleotides can be prepared by synthesizing complementary oligonucleotide strands so as to alternately overlap, annealing them, and then ligating with ligase. Alternatively, siRNA is synthesized as RNA (shRNA) in which a sense strand and an antisense strand are linked via a linker having an appropriate length (for example, about 3 to about 10 bases) and introduced into an enzyme dicer (dicer) in an animal cell to be introduced. ) And so on. Furthermore, an expression vector in which DNAs encoding the sense strand and the antisense strand are separately controlled by Pol III promoters such as U6 and H1, or the sense strand and the antisense strand are connected via a linker. The DNA encoding the linked RNA strand may be prepared as an expression vector placed under the control of a Pol III promoter, and expressed in animal cells to form siRNA.

  The inhibitory activity of the antisense nucleic acid of the present invention uses a transformant introduced with the LTBP-1L gene, an LTBP-1L gene expression system in vivo or in vitro, or an LTBP-1L protein translation system in vivo or in vitro. Can be examined.

The use of the above-described antibody of the present invention and the antisense nucleic acid of the present invention will be described below.
(1) Pharmaceutical containing the antibody of the present invention
The expression of LTBP-1L protein is increased in tissues of cancer patients with poor prognosis, and is considered to be a direct or indirect exacerbation factor particularly in advanced stage cancers. Since the antibody of the present invention can neutralize the activity of LTBP-1L protein, cancer (preferably ovarian cancer, cervical cancer, endometrial cancer, stomach cancer, colon cancer, lung cancer, other solid cancers (for example, , Liver cancer, pancreatic cancer, gallbladder cancer, kidney cancer, etc.), more preferably ovarian cancer, cervical cancer, endometrial cancer, stomach cancer, colon cancer and lung cancer, particularly preferably ovarian cancer, endometrial cancer and lung cancer) It can be used as an agent.
The therapeutic agent for cancer containing the antibody of the present invention has low toxicity and is orally or parenterally administered to humans (eg, intravascular administration, subcutaneous administration) as it is as a liquid or as a pharmaceutical composition of an appropriate dosage form. Etc.).
The antibody of the present invention may be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for administration may contain the antibody of the present invention and a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such pharmaceutical compositions are provided as dosage forms suitable for oral or parenteral administration.

  As a composition for parenteral administration, for example, injections, suppositories and the like are used. Injections are dosage forms such as intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, infusions, and the like. May be included. Such an injection can be prepared according to a known method. As a method for preparing an injection, it can be prepared, for example, by dissolving, suspending or emulsifying the antibody of the present invention or a salt thereof in a sterile aqueous liquid or oily liquid that is usually used for injection. As an aqueous solution for injection, for example, an isotonic solution containing physiological saline, glucose and other adjuvants, and the like are used, and suitable solubilizers such as alcohol (eg, ethanol), polyalcohol (eg, Propylene glycol, polyethylene glycol), nonionic surfactants [eg, polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] and the like may be used in combination. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. A suppository used for rectal administration may be prepared by mixing the above-described antibody or a salt thereof with an ordinary suppository base.

  Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including dragees and film-coated tablets), pills, granules, powders, capsules (including soft capsules), syrups Agents, emulsions, suspensions and the like. Such a composition is produced by a known method and may contain a carrier, a diluent or an excipient usually used in the pharmaceutical field. As the carrier and excipient for tablets, for example, lactose, starch, sucrose, and magnesium stearate are used.

  The above parenteral or oral pharmaceutical compositions are conveniently prepared in dosage unit form to suit the dosage of the active ingredient. Examples of the dosage form of such a dosage unit include tablets, pills, capsules, injections (ampoules), and suppositories. The antibody content is preferably 5 to 500 mg per dosage unit dosage form, in particular 5 to 100 mg for injections, and 10 to 250 mg for other dosage forms.

  The dose of the above-mentioned preparation containing the antibody of the present invention varies depending on the administration subject, the target cancer type, symptoms, administration route, etc., for example, when used for the treatment of ovarian cancer in adults, The antibody of the present invention is usually about 0.01 to 20 mg / kg body weight, preferably about 0.1 to 10 mg / kg body weight, more preferably about 0.1 to 5 mg / kg body weight, about 1 to 5 times a day, preferably Is conveniently administered by intravenous injection about 1 to 3 times a day. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If symptoms are particularly severe, the dose may be increased according to the symptoms.

The antibody of the present invention can be administered per se or as an appropriate pharmaceutical composition. The pharmaceutical composition used for the administration comprises the antibody or a salt thereof and a pharmacologically acceptable carrier, diluent or excipient. Such compositions are provided as dosage forms suitable for oral or parenteral administration (eg, intravascular injection, subcutaneous injection, etc.).
Each of the above-described compositions may contain other active ingredients as long as they do not cause an unfavorable interaction by blending with the antibody.
Furthermore, the antibody of the present invention may be used for other drugs such as alkylating agents (eg, cyclophosphamide, ifosfamide, etc.), antimetabolites (eg, methotrexate, 5-fluorouracil, etc.), anticancer antibiotics (eg, Mitomycin, adriamycin, etc.), plant-derived anticancer agents (eg, vincristine, vindesine, taxol, etc.), cisplatin, carboplatin, etopoxide, irinotecan and the like. The antibody of the present invention and the drug may be administered to a patient at the same time or at different times.

(2) Medicine containing antisense nucleic acid
The antisense nucleic acid of the present invention, which can complementarily bind to the transcription product of the LTBP-1L gene and suppress the expression of the gene, has low toxicity, and is an LTBP-1L protein or LTBP-1L gene in vivo. Functions and actions can be suppressed, so cancer (ovarian cancer, cervical cancer, endometrial cancer, stomach cancer, colon cancer, lung cancer, other solid cancers (eg liver cancer, pancreatic cancer, gallbladder cancer, kidney cancer, etc.) ), More preferably ovarian cancer, cervical cancer, endometrial cancer, gastric cancer, colon cancer and lung cancer, particularly preferably ovarian cancer, endometrial cancer and lung cancer).

When the antisense nucleic acid of the present invention is used as a cancer therapeutic agent, it can be formulated and administered according to a method known per se.
In addition, for example, the above-described antisense nucleic acid is inserted alone or into an appropriate vector such as a retrovirus vector, adenovirus vector, adenovirus associated virus vector, etc., and then human or mammal (eg, rat, rabbit) according to conventional means. , Sheep, pigs, cattle, cats, dogs, monkeys, etc.) orally or parenterally. The antisense nucleic acid can be formulated as it is or with a physiologically recognized carrier such as an adjuvant for promoting ingestion and administered by a gene gun or a catheter such as a hydrogel catheter. Alternatively, it can be aerosolized and locally administered into the trachea as an inhalant.
Furthermore, for the purpose of improving pharmacokinetics, prolonging the half-life, and improving cellular uptake efficiency, the above-mentioned antisense nucleic acid is formulated alone (injection) with a carrier such as liposome and administered intravenously, subcutaneously, etc. May be.

  The dosage of the antisense nucleic acid varies depending on the target cancer type, administration subject, administration route, etc. For example, when the antisense nucleic acid of the present invention is administered for the purpose of treating ovarian cancer, In an adult (body weight 60 kg), about 0.1 to about 100 mg of the antisense nucleic acid is administered per day.

A short fragment (oligo DNA) of the human LTBP-1L gene containing the above-mentioned -202G and / or + 20A polymorphisms allows Sp1 binding to the LTBP-1L promoter in cancer patients with the -202G and / or + 20A alleles. Since it can be competitively inhibited, it can be used as a decoy nucleic acid that suppresses overexpression of the LTBP-1L gene. Therefore, the present invention also includes a partial base sequence of the human LTBP-1L gene, comprising about 15 to about 500 comprising the base represented by base numbers 2014 and / or 2235 in the base sequence represented by SEQ ID NO: 1. Cancer (ovarian cancer, cervical cancer, endometrial cancer, stomach cancer, colon cancer, lung cancer, other solid cancers (eg, liver cancer, pancreatic cancer, gallbladder) containing a nucleic acid comprising a continuous base sequence Cancer, kidney cancer, etc.), more preferably ovarian cancer, cervical cancer, endometrial cancer, stomach cancer, colon cancer and lung cancer, particularly preferably ovarian cancer, endometrial cancer and lung cancer).
The nucleic acid can be formulated in the same manner as the antisense nucleic acid of the present invention and can be administered to cancer patients.

In this specification, when a base, an amino acid, or the like is indicated by an abbreviation, it is based on an abbreviation by the IUPAC-IUB Commission on Biochemical Nomenclature or a common abbreviation in the field. In addition, when there is an optical isomer with respect to an amino acid, the L form is shown unless otherwise specified.
DNA: Deoxyribonucleic acid
cDNA: complementary deoxyribonucleic acid
A: Adenine
T: Thymine
G: Guanine
C: cytosine
RNA: Ribonucleic acid
mRNA: Messenger ribonucleic acid
dATP: Deoxyadenosine triphosphate
dTTP: Deoxythymidine triphosphate
dGTP: Deoxyguanosine triphosphate
dCTP: Deoxycytidine triphosphate
ATP: Adenosine triphosphate
EDTA: Ethylenediaminetetraacetic acid
SDS: Sodium dodecyl sulfate
Gly: Glycine
Ala: Alanine
Val: Valine
Leu: Leucine
Ile: Isoleucine
Ser: Serine
Thr: Threonine
Cys: Cysteine
Met: methionine
Glu: Glutamic acid
Asp: Aspartic acid
Lys: Lysine
Arg: Arginine
His: histidine
Phe: Phenylalanine
Tyr: Tyrosine
Trp: Tryptophan
Pro: Proline
Asn: Asparagine
Gln: Glutamine
pGlu: pyroglutamic acid
Sec: selenocysteine

In addition, substituents, protecting groups and reagents frequently used in the present specification are represented by the following symbols.
Me: methyl group
Et: ethyl group
Bu: Butyl group
Ph: phenyl group
TC: thiazolidine-4 (R) -carboxamide group
Tos: p-toluenesulfonyl
CHO: Formyl
Bzl: benzyl
Cl ₂ -Bzl: 2,6-dichlorobenzyl
Bom: benzyloxymethyl
Z: benzyloxycarbonyl
Cl-Z: 2-chlorobenzyloxycarbonyl
Br-Z: 2-bromobenzyloxycarbonyl
Boc: t-Butoxycarbonyl
DNP: Dinitrophenyl
Trt: Trityl
Bum: t-Butoxymethyl
Fmoc: N-9-fluorenylmethoxycarbonyl
HOBt: 1-hydroxybenztriazole
HOOBt: 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine
HONB: 1-hydroxy-5-norbornene-2,3-dicarboximide
DCC: N, N'-dicyclohexylcarbodiimide

The sequence numbers in the sequence listing in the present specification indicate the following sequences.
[SEQ ID NO: 1]
The base sequence of the 5′-regulatory region of the human LTBP-1L gene is shown.
[SEQ ID NO: 2]
The nucleotide sequence of human LTBP-1L cDNA is shown.
[SEQ ID NO: 3]
1 shows the amino acid sequence of human LTBP-1L protein.

  The present invention will be described more specifically with reference to the following examples. However, these are merely examples and do not limit the present invention.

Example 1 Identification of novel SNPs in the 5'-regulatory region of LTBP-1L gene (1) Sample and DNA preparation Tissue specimens were 66 ovarian cancer patients, 66 endometrial cancer patients, gastric cancer patients at Kanazawa University Hospital. Surgically obtained from a series of 55 people, 81 colorectal cancer patients and 90 lung cancer patients, either stored at -80 ° C for DNA analysis or fixed with formaldehyde for immunohistochemical analysis. Written informed consent was obtained from patients for the use of patient tissue in this study. All patients were newly diagnosed and had no previous treatment (chemotherapy or radiation therapy) and were confirmed histologically. Healthy control DNA samples were obtained from oral epithelial cells of 156 Japanese individuals, including Kanazawa University staff and students, with written informed consent. Oral epithelial cells were collected by modifying the previously reported oral cleansing method (Lancet 1988, 1: 1356-6; Cancer Epidemiol. Biomarkers Prev. 1998, 7: 719-24). Briefly, at least 3 hours after brushing, participants rub the mouth vigorously for 10 seconds with 10 mL of mouthwash Mondamine (registered trademark; Earth Pharmaceutical), then spit it into a conical tube, 3,000 g Oral cells were collected by centrifugation for 10 minutes. Total genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega).

(2) Sequencing of LTBP-1L promoter
The LTBP-1L promoter region spanning from −2164 to +130 was analyzed by direct sequencing. The PCR primer sequences used are as follows.
F (Forward) -1: 5'-CGTCGACTCGATCTCAAAGTGTTGC-3 '(SEQ ID NO: 4) and
R (reverse) -1: 5'-GAGGATTGAGGTGAGTCACAAGG-3 '(SEQ ID NO: 5);
F-2: 5'-GTAGAACAAGGAATTGGATCCGT-3 '(SEQ ID NO: 6) and
R-2: 5'-TTGATTTGGCAGGCAGGGCCTC-3 '(SEQ ID NO: 7);
F-3: 5'-GTTCTCACAAGCAGCTAGTGCT-3 '(SEQ ID NO: 8) and
R-3: 5'-GAAAGTCCACAGTCATAGCAGTC-3 '(SEQ ID NO: 9);
F-4: 5'-CAAAGCCTTGGAAACACACCATC-3 '(SEQ ID NO: 10) and
R-4: 5'-TTAGGGTAGGACTAGAGTTCA-3 '(SEQ ID NO: 11);
F-5: 5'-GTCGGATTACGGTCCCGTGA-3 '(SEQ ID NO: 12) and
R-5: 5'-TTACTGAACGATCCTGTCCTTTC-3 '; (SEQ ID NO: 13) and
F-6: 5'-AGTATCACAGCAAACACGGAT-3 '(SEQ ID NO: 14) and
R-6: 5'-GGTGCACCACGTAGGTGATCCTCC-3 '(SEQ ID NO: 15)
All PCR products were purified and then sequenced directly from both ends. All sequencing reactions were performed using dye terminator chemistry in an ABI 310 DNA analyzer. As a result, -202G / C and + 20A / C novel SNPs were identified (FIG. 1A).

(3) Haplotype analysis
The -202G / C and + 20A / C haplotypes were analyzed by PCR-based restriction fragment length polymorphism (RFLP) method. The LTBP-1L promoter region spanning −392 to +154 was amplified using F-7: 5′-TTGGCTGCTCAGGTCTGACA-3 ′ (SEQ ID NO: 16) and R-6 primer. PCR was performed at 94 ° C for 2 minutes followed by 94 ° C for 1 minute; 56 ° C, 1 minute and 72 ° C for 1 minute under 31 cycles of conditions, with 72 ° C for 10 minutes at the final step. All PCR fragments were fully extended. This cycle number was within the range in which the PCR reaction was exponential. The 546 bp PCR product was double digested with EcoRII and CspI (both manufactured by Toyobo), and the resulting DNA fragments were separated on a 3% agarose gel. The haplotype was determined by detecting a band with the length shown in FIG. 1B.
As a result, only two haplotypes (GA and CC) of the four possible haplotypes were identified from the various cancer patient groups and the control group. Thus, three genotypes GA / GA, GA / CC, and CC / CC were detected. The CC haplotype is registered in GenBank (AF171934), but in the Japanese population, the allele frequency of CC is minor in the whole subject, ovarian cancer patient group, colon cancer patient group, and control group. The newly identified GA allele was a major allele in the Japanese population (Table 1). There was no significant difference in allelic frequency between the ovarian cancer patient group, the colon cancer patient group and the control group, thus indicating that the SNPs are not a susceptibility polymorphism for these cancers.
On the other hand, the frequency of CC haplotypes in the endometrial cancer patient group, gastric cancer patient group, and lung cancer patient group was significantly higher than that in the control group (Table 1).

Example 2 Effect of SNPs on LTBP-1L Promoter Activity The effect of the polymorphism identified in Example 1 on the activity of LTBP-1L promoter was examined by luciferase reporter assay.
The LTBP-1L promoter-luciferase reporter plasmid was provided by Dr. Jorma Keski-Oja, University of Helsinki (J. Biol. Chem. 1999, 274: 32619-30). This plasmid is obtained by inserting the LTBP-1L gene 5′-regulatory region (-2211 to +54) into a luciferase reporter vector pGL3-Basic (Promega). Mutant plasmids were constructed using the GeneTailor Site-Directed Mutagenesis System (Invitrogen Life Technologies). 5'-TGCGCGGCCCGCTCCCCTGGCCCCTCCCCGCTCCC-3 '(SEQ ID NO: 17) and 5'-AGGGGAGCGGGCCGCGCAAGGTGAGGGTCC-3' (SEQ ID NO: 18) are used as primers for introducing -202G, and primers for introducing + 20A are used. 5′-GGCCGGGGGAGGGGGCCGGACAGCGCGCGACC-3 ′ (SEQ ID NO: 19) and 5′-GTCCGGCCCCCTCCCCCGGCCGTGCGGCTCGCCT-3 ′ (SEQ ID NO: 20) were used (see FIG. 2A). Plasmids pM, pM-Sp1 (Sp1 expression vector), and pCMV-DNsp3 (Sp3 dominant negative expression vector) were provided by Dr. Yoshihiro Tsuwa from Kyoto Prefectural University of Medicine.
The RMUG-S cell line derived from ovarian cancer (obtained from JCRB) was maintained in Ham F12 medium (Sigma-Aldrich) containing 10% FBS (containing 100 U / ml penicillin and 100 μg / ml streptomycin), and 1 day before transfection Were seeded in 24-well plates at a density of 5 × 10 ⁴ cells. Cells were transfected with 0.4 μg of each reporter plasmid alone or with the same amount of effector plasmid using FuGENE6 transfection reagent (Roche Molecular Biochemicals). To calibrate transfection efficiency, the Renilla luciferase plasmid phRL-tk was cotransfected. After 48 hours, cells were lysed using the Dual-lusiferase Reporter Assay System (Promega), and firefly and Renilla luciferase activities were measured using Lumat LB9507 (Berthold Technologies). All experiments were performed at least 3 times for each reporter plasmid and relative luciferase activity was calculated. Results are shown as mean ± SD (n = 3) (FIG. 2B). Transcriptional activity was highest for the GA haplotype, 3.6 times that of the CC haplotype. GC and CA haplotypes showed intermediate activity.

  The LTBP-1L promoter contains a GC box that is a Sp1 binding motif, one of which overlaps position -202 and the other adjacent to position +20 (FIG. 2A). Therefore, it was examined whether Sp1 controls the LTBP-1L promoter. When co-transfected with a reporter plasmid having a G-A haplotype and an Sp1 expression vector, transcriptional activity was significantly increased (FIG. 2C). In contrast, transcriptional activity was markedly inhibited when co-transfected with Sp3 dominant negative expression vectors known to inhibit Sp1 function. Similar effects with Sp1 were also observed in the C-C haplotype, but the efficiency was low. These results suggest that Sp1 activates the transcription of the LTBP-1L gene, especially in the G-A haplotype.

Example 3 Electrophoretic Mobility Shift Assay (EMSA)
EMSA was performed as previously described (Cancer Res. 1999, 59: 551-7). Using the MEGALABEL Kit (TaKaRa), double-stranded oligonucleotides 5'-GCGGCCCGCTCCCCTCGCCCCTCCCCGCT-3 '(SEQ ID NO: 21) and 5'-GCGGCCCGCTCCCCTGGCCCCTCCCCGCT-3' (SEQ ID NO: 22) respectively corresponding to -202C and -202G Labeled with [γ- ³² P] ATP. 1 μg of human Sp1 protein (Promega) containing 10% glycerol, 25 mM HEPES (pH 7.9), 50 mM KCl, 0.5 mM PMSF and 1 mM DTT in the presence and absence of a 100-fold molar excess of unlabeled competitor DNA In 25 μl of the reaction mixture, poly (dI-dC) was incubated on ice for 20 minutes. For the supershift assay, anti-Sp1 antibody (Santa Cruz Biotechnologies) was preincubated with recombinant Sp1 protein for 60 minutes on ice. After incubation, labeled oligonucleotide (> 30,000 cpm) was added and the reaction mixture was incubated for an additional 20 minutes at room temperature. The DNA-protein complex was separated from free DNA by 5% polyacrylamide gel electrophoresis, the gel was dried and subjected to autoradiography using a Fuji BAS-III bioimaging analyzer (Fuji Photo Film). To confirm the specific binding of Sp1, use a consensus oligonucleotide for Sp1 (5'-ATTCGATCGGGGCGGGGCGAGC-3 '(SEQ ID NO: 23)) or AP2 (5'-GATCGAACTGACCGCCCGCGGCCCGT-3' (SEQ ID NO: 24)) It was.

  As a result, it was found that the band showing the binding between the probe having -202C and the Sp1 protein was not detected or only faintly detected, whereas the probe having -202G strongly bound to the Sp1 protein. (Figure 3). As a result of the supershift assay, the complex of the probe having -202G and the Sp1 protein was supershifted by the anti-Sp1 antibody, but was not shifted by the antibody against the estrogen receptor or c-myc. It was confirmed that it was bound to protein. These bands disappeared with the addition of homologous unlabeled competitor DNA and Sp1 consensus oligonucleotides, but not with AP2 consensus oligonucleotides. Similar results were observed between the probe with + 20A and the probe with + 20C, but the binding affinity of + 20A to Sp1 was weaker than that of -202G.

Example 4 Immunohistochemical analysis of LTBP-1L expression in ovarian cancer
The effect of SNPs on the expression level of LTBP-1L protein was analyzed immunohistochemically using surgical specimens collected from 36 ovarian cancer patients with various genotypes.
The assay was performed on formalin-fixed and paraffin-embedded ovarian tissue using the VECTASTAIN ABC Elite kit (Vector Laboratories) according to the method described in Am. J. Pathol. 2003, 163: 859-67. Antigen retrieval was performed in 1 × antigen retrieval solution (Biogenex) for 10 minutes, and endogenous peroxidase was quenched in methanol containing 3% H ₂ O ₂ . Tissue sections were incubated with mouse anti-LTBP-1L monoclonal antibody (R & D Systems) (dilution 1: 1000) or whole serum of non-immunized mice for 16 hours at 4 ° C. After washing, the sections were reacted with a biotinylated secondary antibody, and sequentially with streptavidin-biotin-horseradish peroxidase complex, biotinylated tyramide, streptavidin-labeled peroxidase and 3,3′-diaminobenzidine (Dako Cytomation). Reaction was detected. Sections were counterstained with hematoxylin. Staining degree is negative 0 (no staining), low 1+ (5-25% of cells are positive or very weak in staining intensity), medium 2+ (25-75% of cells are positive, strong positive), high 3+ (75 % Or more cells were positive and the staining intensity was extremely strong). Two pathologists judged the results and excluded from the analysis if the results did not match.

As a result, LTBP-1L immunostaining was detected in both cancer and stromal cells in all examined tissue samples (Table 2). Of 13 ovarian cancers with GA homozygous genotype, 4 samples showed 3+ and 6 samples showed 2+ expression. Of the 14 cancer samples with heterozygous genotypes, 4 samples showed 3+ and 2 samples showed 2+ expression, but 8 samples had low 1+ expression. Out of 9 specimens with CC homozygous genotype, 1 specimen showed 3+ and 2 specimens had 2+ expression, but 5 specimens had low 1+ expression and 1 specimen showed expression. There wasn't. To summarize patients with 3+ and 2+ expression as an increased expression group, GA homozygous significantly correlated with increased expression of LTBP-1L compared to heterozygous and CC homozygous (P <0.05).

Example 5 Effect of LTBP-1L SNPs on clinical characteristics and results of various cancer patients
We investigated the relationship between LTBP-1L genotype and clinicopathological features (age, histological type, tumor grade and FIGO clinical stage) of ovarian, endometrial and lung cancers. There was no significant correlation between LTBP-1L genotype and these parameters. Next, the cumulative survival rate was compared for each genotype. Survival curves were plotted using the Kaplan-Meier method and compared using the log-rank test. The results are shown in FIGS. Compared to patients with heterozygous and CC homozygous genotypes, patients with GA homozygous genotypes had lower cumulative survival rates.

ADVANTAGE OF THE INVENTION According to this invention, the quality of prognosis in cancer, especially ovarian cancer, endometrial cancer, and cancers such as lung cancer can be determined. In addition, antibodies against LTBP-1L, antisense nucleic acids of LTBP-1L, and the like are useful as therapeutic agents for cancer, particularly cancer such as ovarian cancer.
Furthermore, according to the present invention, susceptibility to cancers such as endometrial cancer, gastric cancer and lung cancer can be predicted, which is useful for examining the risk of developing these cancers.

While the invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that the preferred embodiments can be modified. The present invention contemplates that the present invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications encompassed within the spirit and scope of the appended claims.
This application is based on Japanese Patent Application No. 2006-019859 filed in Japan, the contents disclosed therein being incorporated in full herein. In addition, the contents described in all publications including the patents and patent application specifications mentioned here are incorporated herein by reference to the same extent as if all of them were explicitly stated. It is.

Claims (9)

In the genomic DNA-containing sample taken from a cancer patient Japanese, SEQ ID NO: represented by human LTBP-1L gene 5'-regulatory in the nucleotide sequence of a region nucleotide numbers 201 4 or 1 is in the nucleotide represented by 2235 A test method for prognosis of cancer, characterized by testing a polymorphism. Cancer, ovarian cancer, Ru is selected from the group consisting of endometrial cancer and lung cancer, the method of claim 1. A partial nucleotide sequence of human LTBP-1L gene, SEQ ID NO: was the base sequence in the nucleotide number 201 4 or represented by 1 includes a base represented by 2235, 1 5-5 00 bases of contiguous nucleotide sequence A prognosis agent for cancer in Japanese comprising a nucleic acid having One or more sets of nucleic acid probes and / or primers capable of detecting each of the polymorphisms at the bases indicated by base numbers 2014 and 2235 in the base sequence of the human LTBP-1L gene 5′-regulatory region represented by SEQ ID NO: 1 A test kit for prognosis of Japanese cancer comprising Nucleic acid probe, SEQ ID NO: human LTBP-1L gene 5'-regulatory region was the nucleotide sequence in the nucleotide number 201 4 or the represented by 1 includes a polymorphic site bases represented by 2235, 1 5-5 00 a nucleic acid comprising a contiguous nucleotide sequence of nucleotide, nucleic acid primers, a partial nucleotide sequence of human LTBP-1L gene, SEQ ID NO: was the nucleotide sequence in the nucleotide number 201 4 or represented by 1 including 5 0-1 a polymorphic site bases represented by 2235, according to claim 4, kit of wherein the pair of nucleic acid capable of amplifying a contiguous nucleotide sequence of 000 bases. The kit according to claim 4 or 5 , wherein the cancer is ovarian cancer, endometrial cancer or lung cancer. In genomic DNA containing samples collected from subjects Japanese, SEQ ID NO: human LTBP-1L gene 5'-regulatory region was the nucleotide sequence in the nucleotide number 201 4 or that represented by 1 multi the base represented by 2235 A test method for susceptibility to cancer selected from the group consisting of endometrial cancer, gastric cancer and lung cancer, which comprises assaying a type. One or more sets of nucleic acid probes and / or primers capable of detecting each of the polymorphisms at the bases indicated by base numbers 2014 and 2235 in the base sequence of the human LTBP-1L gene 5′-regulatory region represented by SEQ ID NO: 1 A kit for testing susceptibility to endometrial cancer, gastric cancer or lung cancer in Japanese . Nucleic acid probe, SEQ ID NO: human LTBP-1L gene 5'-regulatory region was the nucleotide sequence in the nucleotide number 201 4 or the represented by 1 includes a polymorphic site bases represented by 2235, 1 5-5 00 a nucleic acid comprising a contiguous nucleotide sequence of nucleotide, nucleic acid primers, a partial nucleotide sequence of human LTBP-1L gene, SEQ ID NO: was the nucleotide sequence in the nucleotide number 201 4 or represented by 1 including 5 0-1 a polymorphic site bases represented by 2235, according to claim 8, wherein the kit of a contiguous nucleotide sequence pair of nucleic acid capable of amplifying a 000 base.