JP2005170815A - Monoclonal antibody to estrogen-binding protein - Google Patents
Monoclonal antibody to estrogen-binding protein Download PDFInfo
- Publication number
- JP2005170815A JP2005170815A JP2003410942A JP2003410942A JP2005170815A JP 2005170815 A JP2005170815 A JP 2005170815A JP 2003410942 A JP2003410942 A JP 2003410942A JP 2003410942 A JP2003410942 A JP 2003410942A JP 2005170815 A JP2005170815 A JP 2005170815A
- Authority
- JP
- Japan
- Prior art keywords
- estrogen
- binding protein
- monoclonal antibody
- immunoglobulin
- cells
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 101000841393 Candida albicans Probable NADPH dehydrogenase Proteins 0.000 title claims abstract description 83
- 210000004027 cell Anatomy 0.000 claims abstract description 53
- 210000004408 hybridoma Anatomy 0.000 claims abstract description 19
- 241001465754 Metazoa Species 0.000 claims abstract description 8
- 206010035226 Plasma cell myeloma Diseases 0.000 claims abstract description 7
- 201000000050 myeloid neoplasm Diseases 0.000 claims abstract description 7
- 210000000628 antibody-producing cell Anatomy 0.000 claims abstract description 4
- 102000018358 immunoglobulin Human genes 0.000 claims description 35
- 108060003951 Immunoglobulin Proteins 0.000 claims description 34
- 229940011871 estrogen Drugs 0.000 claims description 24
- 239000000262 estrogen Substances 0.000 claims description 24
- 241000124008 Mammalia Species 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 102000014914 Carrier Proteins Human genes 0.000 claims description 4
- 108091008324 binding proteins Proteins 0.000 claims description 4
- 229940039227 diagnostic agent Drugs 0.000 claims description 4
- 239000000032 diagnostic agent Substances 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 210000001124 body fluid Anatomy 0.000 claims description 2
- 239000010839 body fluid Substances 0.000 claims description 2
- 238000002331 protein detection Methods 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 238000001814 protein method Methods 0.000 claims 1
- 206010006187 Breast cancer Diseases 0.000 abstract description 32
- 208000026310 Breast neoplasm Diseases 0.000 abstract description 31
- 238000011161 development Methods 0.000 abstract description 7
- 108090000623 proteins and genes Proteins 0.000 description 22
- 102000004169 proteins and genes Human genes 0.000 description 19
- 239000000427 antigen Substances 0.000 description 17
- 102000036639 antigens Human genes 0.000 description 17
- 108091007433 antigens Proteins 0.000 description 17
- 210000004369 blood Anatomy 0.000 description 17
- 239000008280 blood Substances 0.000 description 17
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 14
- 230000010261 cell growth Effects 0.000 description 14
- 210000002966 serum Anatomy 0.000 description 11
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 10
- 239000000499 gel Substances 0.000 description 9
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000002835 absorbance Methods 0.000 description 8
- 239000000824 cytostatic agent Substances 0.000 description 8
- 229940072221 immunoglobulins Drugs 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 108700021154 Metallothionein 3 Proteins 0.000 description 7
- 102100028708 Metallothionein-3 Human genes 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 230000009471 action Effects 0.000 description 7
- 102000015694 estrogen receptors Human genes 0.000 description 7
- 108010038795 estrogen receptors Proteins 0.000 description 7
- 239000003966 growth inhibitor Substances 0.000 description 7
- 150000003431 steroids Chemical class 0.000 description 7
- 239000012228 culture supernatant Substances 0.000 description 6
- 238000011282 treatment Methods 0.000 description 6
- 108010010803 Gelatin Proteins 0.000 description 5
- 241000282412 Homo Species 0.000 description 5
- 108010044625 Proto-Oncogene Proteins c-mos Proteins 0.000 description 5
- 230000001093 anti-cancer Effects 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 5
- 230000004927 fusion Effects 0.000 description 5
- 239000008273 gelatin Substances 0.000 description 5
- 229920000159 gelatin Polymers 0.000 description 5
- 235000019322 gelatine Nutrition 0.000 description 5
- 235000011852 gelatine desserts Nutrition 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 102000009027 Albumins Human genes 0.000 description 4
- 108010088751 Albumins Proteins 0.000 description 4
- 238000002965 ELISA Methods 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 229960000890 hydrocortisone Drugs 0.000 description 4
- 230000003211 malignant effect Effects 0.000 description 4
- 230000010534 mechanism of action Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 230000003053 immunization Effects 0.000 description 3
- 239000003446 ligand Substances 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000012264 purified product Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 102000005963 steroid binding proteins Human genes 0.000 description 3
- 108020003178 steroid binding proteins Proteins 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 239000002202 Polyethylene glycol Substances 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 108091009879 estrogen binding proteins Proteins 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 206010003445 Ascites Diseases 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 208000002177 Cataract Diseases 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 241000699800 Cricetinae Species 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- -1 aminohexyl group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001718 carbodiimides Chemical class 0.000 description 1
- 210000003855 cell nucleus Anatomy 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000008260 defense mechanism Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 239000000598 endocrine disruptor Substances 0.000 description 1
- 231100000049 endocrine disruptor Toxicity 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 108091009323 immunoglobulin binding proteins Proteins 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000011419 induction treatment Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000003348 petrochemical agent Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 238000011176 pooling Methods 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 239000012151 protein quantification reagent Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000005932 reductive alkylation reaction Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Images
Abstract
Description
本発明は、ヒトなどの哺乳動物血液または組織中に存在する、エストロゲン結合性タンパク質に対するモノクローナル抗体、その製法、該抗体産生細胞、および該抗体を用いた検査薬、診断薬に関する。 The present invention relates to a monoclonal antibody against estrogen-binding protein present in blood or tissue of mammals such as humans, its production method, the antibody-producing cell, and a test or diagnostic agent using the antibody.
乳がんの発生や進行にはエストロゲンがカギとなっていることが知られている。研究者の間では、エストロゲンは標的細胞核内にある2種類のエストロゲンレセプターに結合することによって細胞に影響するとされている。
しかしながら、 いずれのエストロゲンレセプターも乳がん細胞の増殖に直接かかわっていないということが明らかにされており、他にエストロゲンの働きを仲介する因子が見つかっていないので、乳がんの発生と進行に関する研究は進んでいない。本発明者の1人はこれまでに、細胞外にもエストロゲンの働きを仲介する因子が存在している可能性があるという仮説を手掛かりに、ヒト血液からエストロゲンの働きを仲介する未知の乳ガン細胞増殖阻害因子を単離するとともに、この因子が、エストロゲンに結合性を有するタンパク質であることを明らかにし、さらに、エストロゲンの働きを仲介して細胞増殖を抑制する証拠として、血清もしくはこの因子を加えない培養液中ではエストロゲンが乳がん細胞の増殖に全く影響しないことを示した（非特許文献１）。
It is known that estrogen is the key to the development and progression of breast cancer. Among researchers, estrogen is thought to affect cells by binding to two types of estrogen receptors in the target cell nucleus.
However, it has been clarified that none of the estrogen receptors are directly involved in the growth of breast cancer cells, and no other factors that mediate the action of estrogen have been found, so research on the development and progression of breast cancer has advanced. Not in. One of the inventors of the present invention has hitherto known an unknown breast cancer cell that mediates the action of estrogen from human blood, based on the hypothesis that there may be a factor that mediates the action of estrogen outside the cell. In addition to isolating a growth inhibitory factor, it was clarified that this factor is a protein that binds to estrogen, and serum or this factor was added as evidence to inhibit cell growth by mediating the action of estrogen. It was shown that estrogen has no influence on the growth of breast cancer cells in the culture medium without any treatment (Non-patent Document 1).
その後、この研究をさらに進め、乳がんは病気の進行とともにエストロゲン感受性を失ってゆくことに着目し、これが細胞の形質維持の良いモデルであるため、引き続き乳がん細胞を用いた研究を行った。上記乳がん細胞のエストロゲン感受性の喪失に対し、上記細胞増殖阻害因子に対する感受性が関与していると考えられるが、そのメカニズムを明らかにするためには、上記細胞増殖阻害因子の機能を阻害する手段が必要になる。このため、上記細胞増殖阻害因子に対し特異的に結合する抗体が求められる。しかし、上記細胞増殖阻害因子については、その分子構造等についての知見が未だ充分に得られていないため、該特異抗体を得るのは難しい。したがって、本発明の課題は、このような困難性を克服し、上記細胞増殖阻害因子に対し特異的に結合する抗体を新たに提供することにある。 After that, we continued this research, focusing on the fact that breast cancer loses estrogen sensitivity as the disease progresses. Since this is a good model for maintaining the character of cells, we continued research using breast cancer cells. The loss of estrogen sensitivity of the breast cancer cells is thought to be related to the sensitivity to the cell growth inhibitory factor, but in order to clarify the mechanism, means for inhibiting the function of the cell growth inhibitory factor I need it. Therefore, an antibody that specifically binds to the cell growth inhibitory factor is required. However, it is difficult to obtain the specific antibody for the cell growth inhibitory factor, because sufficient knowledge about its molecular structure has not been obtained yet. Accordingly, an object of the present invention is to overcome such difficulties and provide a new antibody that specifically binds to the cell growth inhibitor.
本発明者等は、その後の研究の結果、上記細胞増殖阻害因子が、免疫グロブリンと非常に似た構造を有するとの知見を得た。このため、上記細胞増殖因子と生体内に多種多様に存在する免疫グロブリンとを識別しうる抗体を作製することは困難であると予想されたが、このような状況の下、上記細胞増殖阻害因子にのみ特異的に結合するモノクローナル抗体の作成を試み、種々の検討、実験を繰り返した後、上記増殖阻害因子に対し特異的に結合するモノクローナル抗体を得ることに初めて成功するとともに、該抗体が、乳ガンの発症、防御メカニズム解明あるい乳がんの発症の可能性を探るための薬剤等として、極めて有用であると確信し、本発明を完成するに至ったものである。 すなわち、本発明は以下（１）〜（１０）の構成を伴うものである。なお、以下においては、上記細胞増殖阻害因子を免疫グロブリン様エストロゲン結合性タンパク質という。 As a result of subsequent studies, the present inventors have found that the cell growth inhibitor has a structure very similar to that of immunoglobulin. For this reason, it was predicted that it would be difficult to produce an antibody capable of discriminating between the above-mentioned cell growth factor and a wide variety of immunoglobulins in the living body. Attempts to create a monoclonal antibody that specifically binds to only, and after various studies and experiments, succeeded for the first time in obtaining a monoclonal antibody that specifically binds to the growth inhibitory factor. The present invention has been completed with the belief that it is extremely useful as a drug for elucidating the onset and defense mechanisms of breast cancer or for exploring the possibility of onset of breast cancer. That is, the present invention involves the following configurations (1) to (10). Hereinafter, the cell growth inhibitor is referred to as an immunoglobulin-like estrogen binding protein.
（１） 免疫グロブリン様エストロゲン結合性タンパク質に対するモノクローナル抗体。
（２） 上記免疫グロブリン様エストロゲン結合性タンパク質が、哺乳動物由来のものである上記（１）に記載のモノクローナル抗体。
（３） 上記免疫グロブリン様エストロゲン結合性タンパク質が、ヒト由来のものである請求項１記載のモノクローナル抗体。
（４） 免疫グロブリン様エストロゲン結合タンパク質を動物に免役し、得られた抗体産生細胞と骨髄腫細胞とを融合してハイブリドーマを調製した後、免疫グロブリンと上記エストロゲン結合タンパク質とに対する結合性を指標に、上記エストロゲン結合性タンパク質に対するモノクローナル抗体産生ハイブリドーマを選別し、該選別されたハイブリドーマを培養し、免疫グロブリン様エストロゲン結合タンパク質に対するモノクローナル抗体を採取することを特徴とする、免疫グロブリン様結合性タンパク質に対するモノクローナル抗体の製造方法。
（５） 樹立ハイブリドーマ細胞株「1G6-3E-10D」を培養し、エストロゲン結合タンパク質に対するモノクローナル抗体を採取することを特徴とする、エストロゲン結合タンパク質に対するモノクローナル抗体の製造方法。
（６） 樹立ハイブリドーマ細胞株「1G6-3E-10D」。
（７） 上記（１）〜（３）のいずれかに記載のモノクローナル抗体を含有することを特徴とする、エストロゲン結合タンパク質の検出試薬
（８） 上記（１）〜（３）のいずれかに記載のモノクローナル抗体を含有することを特徴とする、エストロゲン結合タンパク質の定量試薬
（９） 上記（１）〜（３）のいずれかに記載のモノクローナル抗体を含有することを特徴とする、ヒトの体液、組織中に存在するエストロゲン結合タンパク質の濃度を定量するために使用する検査薬。
（１０） 上記（１）〜（３）のいずれかに記載のモノクローナル抗体を含有することを特徴とする診断薬。
(1) A monoclonal antibody against an immunoglobulin-like estrogen binding protein.
(2) The monoclonal antibody according to (1) above, wherein the immunoglobulin-like estrogen binding protein is derived from a mammal.
(3) The monoclonal antibody according to
(4) Immunoglobulin-like estrogen binding protein is immunized to animals, and the resulting antibody-producing cells and myeloma cells are fused to prepare a hybridoma, and then the binding property to the immunoglobulin and the estrogen binding protein is used as an index. A monoclonal antibody against an immunoglobulin-like binding protein, characterized by selecting a monoclonal antibody-producing hybridoma against the estrogen-binding protein, culturing the selected hybridoma, and collecting a monoclonal antibody against the immunoglobulin-like estrogen binding protein Antibody production method.
(5) A method for producing a monoclonal antibody against an estrogen binding protein, comprising culturing an established hybridoma cell line “1G6-3E-10D” and collecting the monoclonal antibody against the estrogen binding protein.
(6) The established hybridoma cell line “1G6-3E-10D”.
(7) The estrogen-binding protein detection reagent (8), which contains the monoclonal antibody according to any one of (1) to (3) above, or any one of (1) to (3) above. An estrogen-binding protein quantification reagent (9) characterized by containing the monoclonal antibody of (1) A human body fluid characterized by containing the monoclonal antibody according to any one of (1) to (3) above, A test drug used to quantify the concentration of estrogen binding protein present in tissues.
(10) A diagnostic agent comprising the monoclonal antibody according to any one of (1) to (3) above.
本発明のモノクローナル抗体の抗原は、エストロゲン依存性乳がん細胞増殖阻害因子として単離された免疫グロブリン様エストロゲン結合性タンパク質であり、本発明のモノクローナル抗体は、該タンパク質の検出および定量に極めて有用なものである。また、上記モノクローナル抗体を用いて、その生体内の該エストロゲン結合性タンパク質を定量することにより、健全な女性が乳がんになる可能性を検査することも可能となる。
さらに、乳がんは進行が進むと次第にエストロゲンに反応しなくなり、それと同時により悪性化することが知られている。悪性化した乳がん細胞にも細胞内に機能的に問題ないエストロゲン受容体が存在することが多く、細胞の形質変化にエストロゲン受容体が関係していないことが指摘されている。このような乳がん細胞の形質変化に対しては上記エストロゲン結合性タンパク質が関与している可能性が考えられ、本発明のモノクローナル抗体は、乳がんの進行を診断するために使用する診断薬としての使用が考えられる。また、基礎研究としては、エストロゲンの作用機序やホルモンがん発症の防御のメカニズムを調べるうえでも極めて重要な抗体である。
The antigen of the monoclonal antibody of the present invention is an immunoglobulin-like estrogen-binding protein isolated as an estrogen-dependent breast cancer cell growth inhibitor, and the monoclonal antibody of the present invention is extremely useful for the detection and quantification of the protein. It is. It is also possible to examine the possibility of a healthy woman to develop breast cancer by quantifying the estrogen binding protein in the living body using the monoclonal antibody.
Furthermore, it is known that breast cancer gradually stops responding to estrogen as it progresses, and at the same time becomes more malignant. Malignant breast cancer cells often have estrogen receptors that are not functionally problematic in the cells, and it has been pointed out that estrogen receptors are not involved in cell phenotypic changes. The estrogen-binding protein may be involved in such a breast cancer cell trait change, and the monoclonal antibody of the present invention is used as a diagnostic agent for diagnosing the progression of breast cancer. Can be considered. In addition, as a basic research, it is an extremely important antibody for examining the mechanism of action of estrogen and the mechanism of defense against the onset of hormonal cancer.
本発明のモノクローナル抗体が認識する免疫グロブリン様エストロゲン結合性タンパク質の構造を70％決定したところ、リューマチ因子やＲｈ血液型を決定する抗原を認識する抗D免疫グロブリンと共通構造をもっていた。そこで本発明のモノクローナル抗体はエストロゲンが関係する乳がんなどのホルモンがん細胞の形質のモニター、内分泌かく乱物質の働き、骨粗鬆症、白内障の治療法の研究のほか、Rｈ血液型の研究、リューマチなどの自己免疫疾患のメカニズムを探る際にも用いられる可能性がある。 The structure of the immunoglobulin-like estrogen binding protein recognized by the monoclonal antibody of the present invention was determined to be 70%, and it had a common structure with the anti-D immunoglobulin that recognizes the antigen that determines rheumatoid factor and Rh blood group. Therefore, the monoclonal antibody of the present invention can be used to monitor the characteristics of hormone cancer cells such as breast cancer related to estrogen, to work endocrine disruptors, to treat osteoporosis and cataracts, to study Rh blood groups, to self such as rheumatism. It may also be used to explore the mechanisms of immune diseases.
本発明は、ヒトその他哺乳動物の血液中に存在する免疫グロブリンに類似する構造を有するエストロゲン結合性タンパク質に対するモノクローナル抗体に関するものであり、このエストロゲン結合性タンパク質は、エストロゲンの存在下、乳がん細胞の増殖を抑制する。本発明のモノクローナル抗体は、このような免疫グロブリン様エストロゲン結合性タンパク質の検出、分布、定量、単離、挙動のモニター、並びに該タンパク質のスクリーニングを可能とするものである。 The present invention relates to a monoclonal antibody against an estrogen binding protein having a structure similar to an immunoglobulin present in the blood of humans and other mammals, and the estrogen binding protein is capable of proliferating breast cancer cells in the presence of estrogen. Suppress. The monoclonal antibody of the present invention enables detection, distribution, quantification, isolation, behavior monitoring of such an immunoglobulin-like estrogen binding protein, and screening of the protein.
本発明の上記モノクローナル抗体を得るため、抗原として使用するエストロゲン結合性タンパク質は、例えば、ヒト等の哺乳動物の血液から、以前報告した方法（Anticancer Res. 20, 2785-90(2000)）もしくはその改良法である下記の実施例１の方法にしたがって単離することができる。また、得られるエストロゲン結合性タンパク質は、Ｈ鎖とＬ鎖からなるが、免疫グロブリンと非常に似た構造を有する。そして、血液中には多種多様の免疫グロブリンが存在するが、本発明のモノクローナル抗体はこれら免疫グロブリンと反応してはならず、かつそれと構造上80％以上の類似性をもつエストロゲン結合性タンパク質を確実に検出しなければならない。 In order to obtain the monoclonal antibody of the present invention, the estrogen-binding protein used as an antigen is, for example, a method previously reported (Anticancer Res. 20, 2785-90 (2000)) from the blood of mammals such as humans or the like. It can be isolated according to the method of Example 1 below, which is an improved method. The obtained estrogen-binding protein is composed of an H chain and an L chain, but has a structure very similar to that of an immunoglobulin. A wide variety of immunoglobulins exist in the blood, but the monoclonal antibody of the present invention must not react with these immunoglobulins, and an estrogen-binding protein having a structural similarity of 80% or more with it. It must be detected reliably.
以下に、ヒトの細胞増殖阻害因子であるエストロゲン結合性タンパク質を抗原とする場合を例にとり、本発明のモノクローナル抗体を得る方法についてさらに具体的に説明する。
まず、還元条件下のSDSポリアクリルアミドゲル電気泳動にて免疫グロブリン様エストロゲン結合性タンパク質をＨ鎖とＬ鎖に分離し、Ｈ鎖を含むゲルをかみそりで切り出してから電気的に溶出回収して生理食塩水に、一定時間、例えば一昼夜透析し、得られたエストロゲン結合性タンパク質のＨ鎖を抗原としてマウス、ラットその他の動物に注射する。免疫した動物から脾臓を取り出し、得られる脾細胞と骨髄腫細胞（ミエローマ細胞）とをポリエチレングリコール等の周知の手段により融合させる。
次に、融合細胞にＨＡＴ培地による選択を行ってモノクローナル抗体を産生する融合細胞を得る。この段階では多種多様な融合細胞が存在し、免疫グロブリンとエストロゲン結合性タンパク質で共通の部分構造を認識するモノクローナル抗体を産生する融合細胞も含まれるため、得られる抗体では免疫グロブリンと抗原である上記エストロゲン結合性タンパク質とを識別できない。
Hereinafter, the method for obtaining the monoclonal antibody of the present invention will be described more specifically by taking as an example the case where an estrogen-binding protein that is a human cell growth inhibitor is used as an antigen.
First, an immunoglobulin-like estrogen-binding protein is separated into H and L chains by SDS polyacrylamide gel electrophoresis under reducing conditions, and the gel containing the H chains is cut out with a razor and then electrically eluted and recovered for physiology. The solution is dialyzed against saline for a certain time, for example, all day and night, and the resulting estrogen-binding protein heavy chain is injected as an antigen into mice, rats and other animals. The spleen is removed from the immunized animal, and the resulting spleen cells and myeloma cells (myeloma cells) are fused by a known means such as polyethylene glycol.
Next, the fused cells are selected with HAT medium to obtain fused cells producing monoclonal antibodies. At this stage, there are a wide variety of fusion cells, including fusion cells that produce monoclonal antibodies that recognize a common partial structure between immunoglobulin and estrogen-binding protein. Indistinguishable from estrogen binding protein.
そこで、これらの融合細胞を希釈して多数の培養器に分散させ、各々の培養器中に生じるモノクローナル抗体と、抗原となる上記エストロゲン結合性タンパク質のみを除いたヒト免疫グロブリンの画分、及び上記エストロゲン結合性タンパク質とをそれぞれ反応させ、上記エストロゲン結合性タンパク質のみを除いたヒト免疫グロブリンの画分とは反応せず、エストロゲン結合性タンパク質とのみ反応する融合細胞を選択し、該融合細胞からモノクローナル抗体を得る。得られるモノクローナル抗体は、ヒト免疫グロブリンとは反応せず、免疫グロブリン様エストロゲン結合性タンパク質にのみ特異的に結合する抗体である。 Therefore, these fused cells are diluted and dispersed in a number of incubators, and the monoclonal antibody produced in each incubator, the fraction of human immunoglobulin excluding only the estrogen-binding protein as an antigen, and the above Select a fused cell that reacts with estrogen-binding protein, reacts only with estrogen-binding protein, and does not react with the human immunoglobulin fraction excluding only the estrogen-binding protein. Obtain an antibody. The resulting monoclonal antibody is an antibody that does not react with human immunoglobulin and specifically binds only to an immunoglobulin-like estrogen binding protein.
上記エストロゲン結合性タンパク質のみを除いたヒト免疫グロブリンの画分は、以下のように調製することができる。
例えば、新鮮な血液を一晩４℃で放置した後に遠心分離により血球を除いて上清を得、活性炭処理によりステロイド成分を除去してから、ステロイド、例えばヒドロコルチゾン（コルチゾール）をリガンドとしたアフィニティカラムを通過させる。
免疫グロブリン様エストロゲン結合性タンパク質は、ステロイドと結合するので、この操作により、アフィニティカラムに補足され、上記血液中の抗原となるエストロゲン結合性タンパク質を除去することができる。得られた素通り画分をプールし、免疫グロブリン画分をプロティンＡ−アフィニティカラムを用いて精製する。こうして抗原となるエストロゲン結合性タンパク質のみを除いたヒト免疫グロブリンの画分を得る。
A fraction of human immunoglobulin excluding only the estrogen-binding protein can be prepared as follows.
For example, after leaving fresh blood overnight at 4 ° C., blood cells are removed by centrifugation, a supernatant is obtained, steroid components are removed by activated carbon treatment, and then an affinity column using a steroid, for example, hydrocortisone (cortisol) as a ligand. Pass through.
Since the immunoglobulin-like estrogen-binding protein binds to the steroid, this operation can remove the estrogen-binding protein that is captured by the affinity column and becomes the antigen in the blood. The flow-through fractions obtained are pooled and the immunoglobulin fraction is purified using a protein A-affinity column. In this way, a fraction of human immunoglobulin excluding only the estrogen binding protein that becomes the antigen is obtained.
次いで、このヒト免疫グロブリン画分と、免疫グロブリン様エストロゲン結合性タンパク質との反応性（結合性）を指標として、上記の融合細胞を分散させた多数の各培養器のなかから、抗原となる免疫グロブリン様エストロゲン結合性タンパク質にのみ強く反応する抗体が産生されている培養器を選択し、さらにそれに含まれる融合細胞を希釈した多数の培養器で同じように選択を繰り返すというスクリーニング作業を行い、抗原となるエストロゲン結合性タンパク質とのみ特異的に反応する抗体を産生する融合細胞を単離する。この融合細胞が培養液中に放出する抗体分子が、本発明のモノクローナル抗体である。融合細胞は保存が可能であり、培養により、またはマウスの腹水中に産生させることにより、いつでも本発明のモノクローナル抗体を得ることができる。本発明においては、上記操作により、免疫グロブリン様エストロゲン結合性タンパク質に対するモノクローナル抗体を産生する融合細胞として、樹立ハイブリドーマ細胞株「1G6-3E-10D」を得ている。該融合細胞は、独立行政法人産業技術総合研究所特許生物寄託センターに２００３年１２月８日付けで受託番号ＦＥＲＭ Ｐ−１９６１０号として寄託されている。 Next, using the reactivity (binding property) of this human immunoglobulin fraction and immunoglobulin-like estrogen-binding protein as an index, the immunity that becomes the antigen from among each of the many incubators in which the above fused cells are dispersed. Select a culture vessel that produces antibodies that react only with globulin-like estrogen-binding protein, and then repeat the selection in a number of culture vessels in which the fused cells contained therein are diluted. A fusion cell producing an antibody that specifically reacts only with the estrogen-binding protein is isolated. The antibody molecule released from the fused cells into the culture solution is the monoclonal antibody of the present invention. The fused cells can be stored, and the monoclonal antibodies of the present invention can be obtained at any time by culturing or by producing them in mouse ascites. In the present invention, the established hybridoma cell line “1G6-3E-10D” has been obtained as a fused cell producing a monoclonal antibody against an immunoglobulin-like estrogen binding protein by the above-described operation. The fused cells have been deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology as deposit number FERM P-19610 dated December 8, 2003.
本発明において、動物に免疫する際、抗原として用いる免疫グロブリン様エストロゲン結合性タンパク質は、哺乳類由来のものであれば特に限定されず、例えば、ヒト、サル、ウシ、ウマ、ヤギ、ブタ、イヌ、ネコ、ウサギ、ハムスター、ラット、マウス由来の免疫グロブリン様エストロゲン結合性タンパク質を用いることができる。これら、哺乳動物から免疫グロブリン様エストロゲン結合性タンパク質は、以前発表した方法（（Anticancer Res. 20, 2785-90(2000)）、もしくはその変法、もしくはモノクローナル抗体を用いた免疫沈降法により調整しうる。
この本発明の免疫グロブリン様エストロゲン結合性タンパク質は、免疫グロブリンと類似した構造を有し、Ｈ鎖とL鎖からなる。Ｈ鎖はヒトの場合52ｋＤａ、L鎖は30ｋＤａである。動物種によって大きさが若干異なる。この免疫グロブリン様エストロゲン結合性タンパク質は、上記のように、以前、細胞増殖抑制因子として、ステロイド結合能を持ち、エストロゲン存在下で乳がん細胞の増殖を阻害することを報告している（Anticancer Res. 20, 2785-90(2000)）。
In the present invention, the immunoglobulin-like estrogen-binding protein used as an antigen when immunizing an animal is not particularly limited as long as it is derived from a mammal. For example, humans, monkeys, cows, horses, goats, pigs, dogs, Immunoglobulin-like estrogen-binding proteins derived from cats, rabbits, hamsters, rats, and mice can be used. These immunoglobulin-like estrogen binding proteins from mammals were prepared by the previously published method ((Anticancer Res. 20, 2785-90 (2000)), or a modification thereof, or immunoprecipitation using a monoclonal antibody. sell.
This immunoglobulin-like estrogen binding protein of the present invention has a structure similar to that of an immunoglobulin and consists of an H chain and an L chain. The H chain is 52 kDa for human and the L chain is 30 kDa. The size varies slightly depending on the animal species. As described above, this immunoglobulin-like estrogen-binding protein has previously been reported to have a steroid-binding ability as a cytostatic factor and inhibit the growth of breast cancer cells in the presence of estrogen (Anticancer Res. 20, 2785-90 (2000)).
抗原として用いるのは、分子を形成するＨ鎖とL鎖のうちＨ鎖である。さらに、遺伝子組み換え体によって作成した細胞増殖抑制因子のＨ鎖を免疫用として用いることもできる。該Ｈ鎖を遺伝子組み換え体によって作成するには、Ｈ鎖の遺伝子を含むDNAの全部もしくは一部を適当な制限酵素によって切り出し、リンカー配列と発現タンパク質判別用タグ配列を接続して適当なプロモーターの下流につないだ後、これを形質転換可能な宿主に導入する。ここで使用されるベクターDNAとしては、プラスミドベクター、シャトルベクター、ファージベクター、ウィルスベクターなどが挙げられ、宿主としては大腸菌、枯草菌、酵母、昆虫細胞、動物細胞が挙げられる。形質転換体は培養増幅後、プロモーターの性質に応じた発現誘導処理をして菌体もしくは培養上清より、発現したタンパク質をタグ配列を利用してアフィニティカラムクロマトグラフィーで回収、精製する。
以下に、本発明の実施例を示すが、本発明はこの実施例に限定されるものではない。
What is used as an antigen is the H chain of the H chain and L chain forming the molecule. Furthermore, the H chain of a cell growth inhibitor produced by a gene recombinant can also be used for immunization. In order to prepare the H chain using a gene recombinant, all or part of the DNA containing the H chain gene is excised with an appropriate restriction enzyme, and the linker sequence and the tag sequence for distinguishing the expressed protein are connected to form an appropriate promoter After being connected downstream, it is introduced into a transformable host. Examples of the vector DNA used here include plasmid vectors, shuttle vectors, phage vectors, and viral vectors. Examples of hosts include Escherichia coli, Bacillus subtilis, yeast, insect cells, and animal cells. The transformant is amplified after culture, and is subjected to expression induction treatment according to the nature of the promoter, and the expressed protein is recovered and purified from the cells or culture supernatant by affinity column chromatography using a tag sequence.
Examples of the present invention are shown below, but the present invention is not limited to these examples.
１．抗原（免疫グロブリン様エストロゲン結合性タンパク質）の調製
1.5リットルの冷凍ヒト血清を４℃で解凍したら、３ｇ T-70デキストラン（ファルマシア社製）で処理した洗浄済み活性炭（シグマ社製）２０ｇを加えて、３４℃、２時間攪拌する。これを遠心分離機にかけて沈殿した活性炭を取り除き、新たにデキストラン処理済活性炭１０ｇを加えて、３４℃、１時間攪拌する。これを遠心分離機にかけて沈殿した活性炭を取り除き上精を得た。
上精に重量パーセントで２５％になるように硫酸アンモニウム結晶（ICN社製、タンパク精製グレード）を加えて、氷上で３０分攪拌する。これを遠心分離機にかけて沈殿を取り除き、上精にさらに硫酸アンモニウム結晶を加えて重量パーセントで４０％になるようにした。これを遠心分離機にかけて沈殿を回収し、２００ｍｌの０.２M食塩を含む０.０５Ｍピペラジン緩衝溶液（ｐＨ５）を加えて溶かした。
1. Preparation of antigen (immunoglobulin-like estrogen binding protein)
After thawing 1.5 liters of frozen human serum at 4 ° C., 20 g of washed activated carbon (manufactured by Sigma) treated with 3 g T-70 dextran (Pharmacia) is added and stirred at 34 ° C. for 2 hours. This is centrifuged to remove the precipitated activated carbon, 10 g of dextran-treated activated carbon is newly added, and the mixture is stirred at 34 ° C. for 1 hour. This was centrifuged to remove the precipitated activated carbon to obtain a fine powder.
Ammonium sulfate crystals (ICN, protein purification grade) are added to the finest so that the weight percentage is 25%, and the mixture is stirred on ice for 30 minutes. This was centrifuged to remove the precipitate, and ammonium sulfate crystals were further added to the upper fine so that the weight percentage became 40%. This was centrifuged to collect the precipitate, and 200 ml of 0.05 M piperazine buffer solution (pH 5) containing 0.2 M sodium chloride was added to dissolve the precipitate.
溶液のｐＨを６Ｎ塩酸で５.０に合わせたら、１００ｍｌのコルチゾールゲル（コルチゾールをカルボジイミドカップリング法で付加したファルマシア社製のアミノへキシル基付きアガロースゲルで、あらかじめ０.２M食塩を含む０.０５Ｍピペラジン緩衝溶液（ｐＨ５）で洗っておく）を加えて、一晩4℃で攪拌する。次の日にゲルをガラス管に詰め（ベッドサイズは直径２.５ｃｍ、高さ１８ｃｍになる）、５００ｍｌの４Ｍ尿素と０.２M食塩を含む０.０５Ｍピペラジン緩衝溶液（ｐＨ５）、続いて１０リットルの０.２M食塩を含む０.０５Ｍピペラジン緩衝溶液（ｐＨ５）で洗った。ゲルについたタンパク質は１リットルの０.５ｍｇ／ｍｌのコルチゾールを加えた０.２M食塩を含む０.０５Ｍピペラジン緩衝溶液（ｐＨ５）で溶出した。プールした溶出液（約９００ｍｌ）は、アミコン社製のＰＭ−１０膜を使って限外ろ過により約２ｍｌ（タンパク量約５ｍｇを含む）に濃縮した。 When the pH of the solution was adjusted to 5.0 with 6N hydrochloric acid, 100 ml of cortisol gel (an agarose gel with an aminohexyl group manufactured by Pharmacia, to which cortisol was added by a carbodiimide coupling method, and 0.2 M sodium chloride in advance was added. Wash with 05M piperazine buffer solution (pH 5)) and stir overnight at 4 ° C. The next day the gel is packed into a glass tube (bed size is 2.5 cm in diameter and 18 cm in height), 500 ml of 0.05 M piperazine buffer solution (pH 5) containing 4 M urea and 0.2 M salt, followed by 10 Washed with 0.05 M piperazine buffer solution (pH 5) containing 0.2 liter of 0.2 M salt. The protein attached to the gel was eluted with 0.05 M piperazine buffer solution (pH 5) containing 0.2 M sodium chloride to which 1 liter of 0.5 mg / ml cortisol was added. The pooled eluate (about 900 ml) was concentrated to about 2 ml (including about 5 mg of protein) by ultrafiltration using a PM-10 membrane manufactured by Amicon.
得られたサンプルは生理食塩水に対して一昼夜透析してベッド体積０.３ｍｌのプロテインＡ・アガロースゲルカラム（米国ＫＰＬ社製）にアプライし、製品の取り扱い説明書に従って免疫グロブリン画分を精製する。溶出用緩衝液を用いてカラムから回収されたサンプルは生理食塩水に対して一昼夜透析し、タンパク質濃度と純度を検定した上で、1ｍｇ／ｍｌになるように調整した。
この精製法で、１.５リットルの血清から約２.５ｍｇの免疫グロブリン様エストロゲン結合性タンパク質の完全精製標品が得られた。
精製表品をＳＤＳポリアクリルアミド電気泳動後、還元アルキル化後、トリプシンでゲル内消化を行い、得られたペプチド断片をＥＳＩ−ＱＵＡＤ−ＴＯＦでＭＳ／ＭＳ解析し、決定したシークエンスタグをもとにＭＡＳＣＯＴ（www.matrixscience.com）で検索した。その結果免疫グロブリンによく似たタンパク質であることが分かった。
The obtained sample was dialyzed against physiological saline for 24 hours, applied to a protein A / agarose gel column (manufactured by KPL, USA) having a bed volume of 0.3 ml, and the immunoglobulin fraction was purified according to the instruction manual of the product. . The sample collected from the column using the elution buffer was dialyzed overnight against physiological saline, tested for protein concentration and purity, and adjusted to 1 mg / ml.
This purification method yielded a fully purified preparation of approximately 2.5 mg of an immunoglobulin-like estrogen binding protein from 1.5 liters of serum.
The purified product is subjected to SDS polyacrylamide electrophoresis, reductive alkylation, in-gel digestion with trypsin, MS / MS analysis of the obtained peptide fragment with ESI-QUAD-TOF, and based on the determined sequence tag Searched by MASCOT (www.matrixscience.com). As a result, the protein was found to be similar to immunoglobulin.
２．免疫グロブリン様エストロゲン結合性タンパク質に対するモノクローナル抗体の作製
ステロイド結合性タンパク質のみを除いたヒト免疫グロブリンの画分を、以下のように調製した。新鮮な血液を一晩４℃で放置した後に遠心分離により血球を除いて上清を得、活性炭処理によりステロイド成分を除去してから、ヒドロコルチゾンをリガンドとしたアフィニティカラムを通過させた。得られた素通り画分をプールし、免疫グロブリン画分をプロティンＡ−アフィニティカラムを用いて精製した。
2. Preparation of monoclonal antibody against immunoglobulin-like estrogen binding protein A fraction of human immunoglobulin excluding only steroid binding protein was prepared as follows. Fresh blood was allowed to stand overnight at 4 ° C., blood cells were removed by centrifugation, a supernatant was obtained, steroid components were removed by activated carbon treatment, and then passed through an affinity column with hydrocortisone as a ligand. The flow-through fractions obtained were pooled, and the immunoglobulin fraction was purified using a protein A-affinity column.
実施例1に従って免疫グロブリン様エストロゲン結合性タンパク質完全精製品10ｍｇを調整した。調整したタンパクの一部（4ｍｇ）は還元条件下のSDSポリアクリルアミドゲル電気泳動にてＨ鎖とＬ鎖に分離し、Ｈ鎖を含むゲルをかみそりで切り出してから電気的に溶出回収してプールした後、生理食塩水に一昼夜透析した。こうして免疫用Ｈ鎖1.5ｍｇを得た。
上で調整した免疫用Ｈ鎖を抗原としてマウスに3回に分けて注射した。免疫された動物から脾臓を取り出し、得られる脾細胞と骨髄腫細胞（ミエローマ細胞）とをポリエチレングリコールを用いて融合させた。
According to Example 1, 10 mg of an immunoglobulin-like estrogen binding protein complete purified product was prepared. A portion of the prepared protein (4 mg) is separated into H and L chains by SDS polyacrylamide gel electrophoresis under reducing conditions, and the gel containing the H chains is cut out with a razor and then electrically eluted and recovered for pooling. Then, it was dialyzed against physiological saline all day and night. In this way, 1.5 mg of H chain for immunization was obtained.
Mice were injected in three portions with the immunized heavy chain prepared above as an antigen. The spleen was removed from the immunized animal, and the resulting spleen cells and myeloma cells (myeloma cells) were fused using polyethylene glycol.
次に、得られたハイブリドーマ（融合細胞）にＨＡＴ培地による選択を行って、モノクローナル抗体を産生するハイブリドーマを得る。得られたハイブリドーマを希釈して多数の培養器に分散させ、各々の培養器中に生じる抗体と、上で調整したステロイド結合性タンパク質のみを除いたヒト免疫グロブリンの画分、及び免疫グロブリン様エストロゲン結合性タンパク質とをそれぞれ反応させ、ELISA（免疫蛍光測定法）試験の結果、前者と後者の吸光度の差が有意に高いハイブリドーマグループを選択し、多種類の融合細胞から目的のモノクローナル抗体を産生するクローンを得た。詳述すると、1次スクリーニングの結果、拡大培養した336グループのうち吸光度の差が有意に高い5グループを選び、それぞれを88グループに拡大培養した。 Next, the obtained hybridoma (fusion cell) is selected with a HAT medium to obtain a hybridoma producing a monoclonal antibody. The obtained hybridoma is diluted and dispersed in a number of incubators, the antibody produced in each incubator, the fraction of human immunoglobulin excluding only the steroid-binding protein prepared above, and the immunoglobulin-like estrogen React with each binding protein, and as a result of ELISA (immunofluorescence assay) test, select a hybridoma group with a significantly high difference in absorbance between the former and the latter, and produce the desired monoclonal antibody from many types of fused cells A clone was obtained. Specifically, as a result of the primary screening, 5 groups having significantly higher difference in absorbance were selected from the 336 groups expanded, and each was expanded to 88 groups.
２次スクリーニングの結果、それぞれからひとグループずつ吸光度の差が有意に高いクローンが得られた。それぞれを88グループに拡大培養した。３次スクリーニングの結果、３クローンのうち１クローン由来の拡大培養には細胞が見られなかったため、バックアップ用の予備培養細胞を再び拡大培養した（1G6-3E）。もうひとつのクローン由来の拡大培養には抗体産生能がなかった。最後のクローン由来の拡大培養からは吸光度の差が有意に高く、かつ細胞観察から１クローン／ウエルになっているクローンが２つ得られた（2H6-4E-4Aと2H6-4E-9C）。これら３クローンを拡大培養した。４次スクリーニングの結果、1G6-3Eクローンより吸光度の差が有意に高いクローン1G6-3E-10Dが得られた。細胞観察から1G6-3E-10D はこの時点で単一クローンとなっていた。2H6-4E-4A と2H6-4E-9Cについては２回撒きなおしを行ったが、吸光度の差が有意に高いクローンは得られなかった。以上より、1G6-3E-10Dを樹立クローンとした。 As a result of the secondary screening, clones having a significantly high difference in absorbance from each group were obtained. Each was expanded to 88 groups. As a result of the third screening, since no cells were found in the expanded culture derived from one of the three clones, the backup preculture cells were expanded again (1G6-3E). The expanded culture from the other clone lacked antibody-producing ability. From the expanded culture derived from the last clone, two clones having a significantly high difference in absorbance and 1 clone / well from cell observation were obtained (2H6-4E-4A and 2H6-4E-9C). These three clones were expanded and cultured. As a result of the fourth screening, clone 1G6-3E-10D having a significantly higher difference in absorbance than that of 1G6-3E clone was obtained. From the cell observation, 1G6-3E-10D became a single clone at this point. Regarding 2H6-4E-4A and 2H6-4E-9C, re-twisting was performed twice, but a clone having a significantly high difference in absorbance was not obtained. Based on the above, 1G6-3E-10D was used as an established clone.
ELISA法により、1G6-3E-10Dハイブリドーマの培養上清の反応特異性を確認した。ステロイド結合性タンパク質のみを除いたヒト免疫グロブリンの画分と免疫グロブリン様エストロゲン結合性タンパク質完全精製品を４μg/mlに希釈してELISA用96ウェルプレートに50μlずつ入れ、室温で1時間静置してから、液を捨て、0.5％ゼラチンを加えたPBST（Tween20を添加した生理食塩水）を２００μl加えて室温で1時間静置した。ゼラチン溶液を捨て、1G6-3E-10Dクローンの培養上清の希釈系列（ゼラチン溶液で1〜32768倍希釈）を50μlづつ加えて室温で1時間静置した。ウェルをゼラチン溶液で３回洗い、同溶液で2500倍希釈したHRP結合抗マウスIgG（γ）を50μlずつ入れ、室温で1時間静置した。ウェルをゼラチン溶液で３回洗い、26ｍｌの基質溶液100μlを加えて室温で発色反応し（約１０分）、100μlの停止液を加えてから、プレートリーダーで490nmの吸光度を測定した。結果を図２に示す。1G6-3E-10Dクローンの培養上清に含まれるモノクローナル抗体が免疫グロブリン様エストロゲン結合性タンパク質完に有意に高い吸光度の差を示すことが確認された。 The reaction specificity of the culture supernatant of 1G6-3E-10D hybridoma was confirmed by ELISA. Dilute human immunoglobulin fraction excluding steroid-binding protein alone and immunoglobulin-like estrogen-binding protein complete purified product to 4μg / ml, put 50μl each in a 96 well plate for ELISA, and let stand at room temperature for 1 hour. Then, the liquid was discarded, and 200 μl of PBST (physiological saline supplemented with Tween 20) supplemented with 0.5% gelatin was added and allowed to stand at room temperature for 1 hour. The gelatin solution was discarded, and a dilution series of the culture supernatant of 1G6-3E-10D clone (diluted 1 to 32768 times with gelatin solution) was added in 50 μl increments and allowed to stand at room temperature for 1 hour. Wells were washed 3 times with gelatin solution, 50 μl each of HRP-conjugated anti-mouse IgG (γ) diluted 2500 times with the same solution was added, and allowed to stand at room temperature for 1 hour. The wells were washed 3 times with gelatin solution, and 100 μl of 26 ml of substrate solution was added to cause color reaction at room temperature (about 10 minutes). After adding 100 μl of stop solution, the absorbance at 490 nm was measured with a plate reader. The results are shown in FIG. It was confirmed that the monoclonal antibody contained in the culture supernatant of the 1G6-3E-10D clone showed a significantly higher difference in absorbance than the immunoglobulin-like estrogen binding protein.
３．上記モノクローナル抗体の抗原特異性試験
免疫グロブリン様エストロゲン結合性タンパク質とアルブミンを含まないヒトの処理血清を次のようにようにして調整した。新鮮な血液6ｍｌを一晩４℃で放置した後に18,000ｒｐｍ、1時間遠心分離により上清を血清として得た。得られた血清から３４℃、2時間と1時間の２回にわたる活性炭処理によりステロイドを除去してから、ステロイドをリガンドとしたアフィニティカラム（ベッドボリューム10ｍｌ）を通過させ、免疫グロブリン様エストロゲン結合性タンパク質を除去した。得られた素通り画分約10ｍｌをプールし、17ｍｌのバイオラッド社製ブルーゲルを加えてアルブミンを除去した。調整を終えた血清は生理食塩水で希釈してタンパク濃度を5mg/mlに調整した。
3. Antigen specificity test of the monoclonal antibody The human-treated serum containing no immunoglobulin-like estrogen-binding protein and albumin was prepared as follows. After 6 ml of fresh blood was left overnight at 4 ° C., the supernatant was obtained as serum by centrifugation at 18,000 rpm for 1 hour. Steroids are removed from the obtained serum by activated charcoal treatment at 34 ° C. for 2 hours and 2 hours, and then passed through an affinity column (bed volume 10 ml) containing steroid as a ligand to give an immunoglobulin-like estrogen-binding protein. Was removed. About 10 ml of the obtained flow-through fractions were pooled, and 17 ml of Bio-Rad Blue Gel was added to remove albumin. The serum after adjustment was diluted with physiological saline to adjust the protein concentration to 5 mg / ml.
こうして得られた処理済血清(タンパク量10μg)をネガティブサンプル（抗原を含まない）とした。
ポジティブサンプル（抗原を含む）としては部分精製したエストロゲン結合性タンパク質(純度10％以下、夾雑物は主としてアルブミン）7μgを用いた。ネガティブサンプル、及びポジティブサンプルの両方を還元条件下で変性させ、ＳＤＳポリアクリルアミドゲル電気泳動を行った。電気泳動後のゲルにｐＨ11のトランスファー緩衝液に浸したブロッティング用メンブレンを密着させ、さらにこれを同じｐＨ11のトランスファー緩衝液に浸したろ紙で挟んで、ゲル側にマイナス、メンブレン側にプラスの電荷をかけてタンパク質をメンブレン上に移動、吸着させた。
The treated serum thus obtained (protein amount 10 μg) was used as a negative sample (without antigen).
As a positive sample (including the antigen), 7 μg of partially purified estrogen-binding protein (purity of 10% or less, contaminants mainly albumin) was used. Both negative and positive samples were denatured under reducing conditions and subjected to SDS polyacrylamide gel electrophoresis. A blotting membrane soaked in pH 11 transfer buffer is in close contact with the gel after electrophoresis, and is further sandwiched between filter papers soaked in the same pH 11 transfer buffer so that a negative charge is charged on the gel side and a positive charge on the membrane side. The protein was transferred onto the membrane and adsorbed.
メンブレンを２枚作成し、一方は本発明のモノクローナル抗体（融合細胞株1G6-3E-10Dの培養上清を200倍希釈で用いた）と、もう一方は別途ウサギを用いて同じ抗原で免役することにより作成したポリクローナル抗体（2500倍希釈）と反応させた。次に前者に対しては、アルカリフォスファターゼ結合抗マウス免疫グロブリン（1000倍希釈）、後者に対しては、アルカリフォスファターゼ結合抗ウサギ免疫グロブリン（5000倍希釈）を反応させ、定法に従って発色反応を行った。 Two membranes were prepared, one was immunized with the same antigen using the monoclonal antibody of the present invention (the culture supernatant of the fusion cell line 1G6-3E-10D was used at 200-fold dilution) and the other using a separate rabbit. Was reacted with a polyclonal antibody (diluted 2500 times). Next, alkaline phosphatase-conjugated anti-mouse immunoglobulin (diluted 1000 times) was reacted with the former, and alkaline phosphatase-conjugated anti-rabbit immunoglobulin (diluted 5000 times) was reacted with the latter, and a color reaction was performed according to a conventional method. .
結果を図２に示す。これによれば、ポリクローナル抗体を用いた実験では精製した調整済み血清、上記エストロゲン結合性タンパク質ともに濃いバンドが出ている。調製済み血清に比べて精製したエストロゲン結合性タンパク質のバンドの位置が低いのは、多量に含まれるアルブミンがバンドを下に押し下げているためである。これは細胞増殖抑制因子が免疫グロブリンと構造が良く似ており、ポリクローナル抗体は調整済み血清に含まれる様々な免疫グロブリンとも反応するからである。このようにポリクローナル抗体は精製した細胞増殖抑制因子に含まれる免疫グロブリンと処理済血清に含まれる免疫グロブリンを区別できない。これに対してモノクローナル抗体は細胞増殖抑制因子のみにクロスしている。結論として本発明のモノクローナル抗体は、細胞増殖抑制因子のみを検出するという本発明の要求を満たしているとわかる。 The results are shown in FIG. According to this, in the experiment using a polyclonal antibody, both the purified sera purified and the estrogen-binding protein have dark bands. The reason why the band of the purified estrogen-binding protein is lower than that of the prepared serum is that albumin contained in a large amount pushes the band downward. This is because cytostatic factors are similar in structure to immunoglobulins, and polyclonal antibodies also react with various immunoglobulins contained in prepared serum. Thus, the polyclonal antibody cannot distinguish between the immunoglobulin contained in the purified cytostatic factor and the immunoglobulin contained in the treated serum. In contrast, the monoclonal antibody crosses only the cytostatic factor. In conclusion, it can be seen that the monoclonal antibody of the present invention satisfies the requirement of the present invention to detect only cytostatic factors.
上記のように、本発明のモノクローナル抗体は、ヒトその他哺乳動物の血液中にあるエストロゲン依存性の細胞増殖抑制因子、すなわち免疫グロブリン様エストロゲン結合性タンパク質を特異的に認識する。血液中では、このエストロゲン結合性タンパク質は混在する多種多様な免疫グロブリンと構造的に高い類似性を持つため、抗免疫グロブリンポリクローナル抗体で認識される。しかし、抗免疫グロブリンポリクローナル抗体では細胞増殖抑制因子と混在する免疫グロブリンを区別できない。これに対して本発明のモノクローナル抗体は両者を正確に区別する。このため、本発明のモノクローナル抗体を用いると、血液もしくは組織中の免疫グロブリン様エストロゲン結合性タンパク質を正確に検出、及び／又は定量できる。 As described above, the monoclonal antibody of the present invention specifically recognizes an estrogen-dependent cell growth inhibitory factor, ie, an immunoglobulin-like estrogen-binding protein, in the blood of humans and other mammals. In blood, this estrogen-binding protein is recognized by anti-immunoglobulin polyclonal antibodies because it has a high structural similarity with a wide variety of mixed immunoglobulins. However, anti-immunoglobulin polyclonal antibodies cannot distinguish immunoglobulins mixed with cytostatic factors. In contrast, the monoclonal antibody of the present invention accurately distinguishes between the two. Therefore, when the monoclonal antibody of the present invention is used, immunoglobulin-like estrogen binding protein in blood or tissue can be accurately detected and / or quantified.
したがって本発明のモノクローナル抗体は疫学的調査に用いると、例えば血中の上記エストロゲン結合性タンパク質のレベルと乳がんなどのホルモンがんの発生率の相関を調べるのに用いることが出来る。さらに、細胞の微細構造に注目すれば、多種多様な免疫グロブリン存在下でもこのエストロゲン結合性タンパク質の作用サイトや作用に伴う細胞の表面もしくは内部の局在位置を決定することが可能である。細胞の微視的局在位置を決定すると、細胞増殖抑制因子の作用メカニズムに対する知見が得られる。さらに本発明のモノクローナル抗体は免疫沈降法と組み合わせて用いることにより、エストロゲン結合性タンパク質それ自身、該タンパク質が結合する分子、もしくは該タンパク質にに結合する分子の単離にも利用できる。相互作用する分子を同定すれば、該タンパク質の作用メカニズムに対する知見が得られる。 Therefore, when the monoclonal antibody of the present invention is used in an epidemiological study, it can be used, for example, to examine the correlation between the level of the estrogen binding protein in the blood and the incidence of hormone cancer such as breast cancer. Furthermore, if attention is paid to the fine structure of the cell, it is possible to determine the site of action of this estrogen-binding protein and the localized position on the surface or inside of the cell accompanying the action even in the presence of various immunoglobulins. Determination of the microscopic location of the cell provides insight into the mechanism of action of the cytostatic factor. Furthermore, by using the monoclonal antibody of the present invention in combination with an immunoprecipitation method, the estrogen-binding protein itself, a molecule to which the protein binds, or a molecule that binds to the protein can be used. Identification of interacting molecules can provide insight into the mechanism of action of the protein.
さらに、注目すべきは、上記エストロゲン結合性タンパク質は、ヒト血中に存在する唯一のエストロゲン依存性乳がん細胞増殖阻害因子として単離されたことである。乳がん細胞は血液が存在しないとエストロゲンに反応しないことが知られており、上記エストロゲン結合性タンパク質は、乳がんなどのホルモンがんの発生や増殖に関係していると考えられる。石油化学の発展とともに我が国あるいは欧米で乳がん患者は急速に増大しており、欧米では7人に1人の女性が乳がんに悩んでいる。しかしながら既知の細胞内エストロゲン受容体には乳がん発症や細胞の増殖促進作用がないとされており、乳がん発症と進展に対するエストロゲンの作用メカニズムは明らかにされていない。これに対して、本発明のモノクローナル抗体が認識する細胞増殖抑制因子にはエストロゲンの働きを仲介する能力があり（Anticancer Res. 20, 2779-84(2000)）、それを認識するモノクローナル抗体は乳がんのリスク管理と治療に応用することができる。 Furthermore, it should be noted that the estrogen binding protein has been isolated as the only estrogen-dependent breast cancer cell growth inhibitor present in human blood. Breast cancer cells are known not to respond to estrogen in the absence of blood, and the estrogen binding protein is thought to be involved in the development and growth of hormone cancers such as breast cancer. With the development of petrochemicals, breast cancer patients are rapidly increasing in Japan, Europe and the United States, and 1 in 7 women in Europe and the United States suffer from breast cancer. However, it is said that known intracellular estrogen receptors do not have the effect of promoting breast cancer development or cell proliferation, and the mechanism of action of estrogen on breast cancer development and progression has not been clarified. In contrast, the cytostatic factor recognized by the monoclonal antibody of the present invention has the ability to mediate the action of estrogen (Anticancer Res. 20, 2779-84 (2000)). It can be applied to risk management and treatment.
さらに乳がんは進行が進むと次第にエストロゲンに反応しなくなり、それと同時により悪性化することが知られている。悪性化した乳がん細胞にも細胞内に機能的に問題ないエストロゲン受容体が存在することが多く、細胞の形質変化にエストロゲン受容体が関係していないことが指摘されている。このような乳がん細胞の形質変化に対して上記エストロゲン結合性タンパク質が関与している可能性がある。この意味で、本発明の抗体は細胞の形質維持の研究に用いることができる。さらに、進行した乳がん患者の治療法開発にも本発明の抗体を用いることができる。 In addition, breast cancer is known to gradually stop responding to estrogen as it progresses, and at the same time become more malignant. Malignant breast cancer cells often have estrogen receptors that are not functionally problematic in the cells, and it has been pointed out that estrogen receptors are not involved in cell phenotypic changes. There is a possibility that the estrogen-binding protein is involved in such a phenotypic change of breast cancer cells. In this sense, the antibody of the present invention can be used for the study of cell trait maintenance. Furthermore, the antibody of the present invention can also be used for developing treatments for advanced breast cancer patients.
Claims (10)
A diagnostic agent comprising the monoclonal antibody according to any one of claims 1 to 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2003410942A JP4231923B2 (en) | 2003-12-09 | 2003-12-09 | Monoclonal antibody against estrogen binding protein. |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2003410942A JP4231923B2 (en) | 2003-12-09 | 2003-12-09 | Monoclonal antibody against estrogen binding protein. |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2008147641A Division JP4378520B2 (en) | 2008-06-05 | 2008-06-05 | Monoclonal antibody against estrogen binding protein. |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2005170815A true JP2005170815A (en) | 2005-06-30 |
| JP4231923B2 JP4231923B2 (en) | 2009-03-04 |
Family
ID=34731877
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2003410942A Expired - Lifetime JP4231923B2 (en) | 2003-12-09 | 2003-12-09 | Monoclonal antibody against estrogen binding protein. |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4231923B2 (en) |
-
2003
- 2003-12-09 JP JP2003410942A patent/JP4231923B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JP4231923B2 (en) | 2009-03-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2004081047A1 (en) | Monoclonal antibody and hybridoma producing the same | |
| EP3152231B1 (en) | Peptides, and methods and apparatus utilising same | |
| CN108473552B (en) | Antigenic composition for detecting specific autoantibody of exosome protein EIF3A and method for diagnosing liver cancer using same | |
| JPH10500408A (en) | Methods for detecting cancer with fragments of cytokeratin 18 and corresponding antibodies | |
| JP2017504812A (en) | Cell surface prostate cancer antigen for diagnosis | |
| JPH06502916A (en) | How to detect diabetic autoantibodies | |
| WO2011108628A1 (en) | Gastric cancer marker, and method for detecting gastric cancer | |
| KR101777259B1 (en) | Specific monoclonal antibody to EN2 protein or composition comprising the same for diagnosis of prostate cancer | |
| KR101777254B1 (en) | Specific monoclonal antibody from a specific antigen EN2 protein or composition comprising the same for diagnosis of prostate cancer | |
| US5534431A (en) | Hybridomas and monoclonal antibodies specific for unique determinants of nephropathy-related immunoglobulin G and complexes thereof | |
| JP6276992B2 (en) | Molecular markers for early detection of pleural mesothelioma patients and their expression analysis methods | |
| WO2018119626A1 (en) | Assay kit for neutrophil gelatinase-associated lipocalin | |
| JP4231923B2 (en) | Monoclonal antibody against estrogen binding protein. | |
| JP4378520B2 (en) | Monoclonal antibody against estrogen binding protein. | |
| JP2001122900A (en) | ANTI-DNASE gamma ANTIBODY AND ITS PREPARATION AND USE | |
| AU2005290997B2 (en) | Novel cancer associated antibodies and antigens and their use in cancer diagnosis | |
| CN114539401A (en) | Magnetic particle marking method and detection kit | |
| JP3920556B2 (en) | Abbreviated midkine (tMK) protein-specific monoclonal antibody and use thereof | |
| CN107163131B (en) | Antigenic polypeptide of tumor suppressor factor p16 and application thereof | |
| CN111434686A (en) | Anti-human PBX1 monoclonal antibody, preparation method thereof and application thereof in clinical diagnosis of recurrent abortion | |
| JPH07503141A (en) | Characteristics of the new anti-▲RB▼ monoclonal antibody | |
| CN110054688B (en) | anti-TTC 36 monoclonal antibody and application thereof | |
| Asadi Karam et al. | Extraction and purification of anti-proteinase 3 (PR3) antibodies from egg yolk | |
| WO2006121047A1 (en) | Method for determination of megsin in biological sample | |
| KR100996486B1 (en) | Monoclonal antibody against human MUDENG protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20050616 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20071016 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20071217 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20080122 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20071219 |
|
| A02 | Decision of refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A02 Effective date: 20080408 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20080605 |
|
| A911 | Transfer to examiner for re-examination before appeal (zenchi) |
Free format text: JAPANESE INTERMEDIATE CODE: A911 Effective date: 20080805 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20080826 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20081007 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20081111 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 4231923 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| S533 | Written request for registration of change of name |
Free format text: JAPANESE INTERMEDIATE CODE: R313533 |
|
| R350 | Written notification of registration of transfer |
Free format text: JAPANESE INTERMEDIATE CODE: R350 |
|
| EXPY | Cancellation because of completion of term |