JPH04234987A - Dna fragment - Google Patents
Dna fragmentInfo
- Publication number
- JPH04234987A JPH04234987A JP40881090A JP40881090A JPH04234987A JP H04234987 A JPH04234987 A JP H04234987A JP 40881090 A JP40881090 A JP 40881090A JP 40881090 A JP40881090 A JP 40881090A JP H04234987 A JPH04234987 A JP H04234987A
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- cancer
- cells
- mouse
- variable region
- 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.)
- Pending
Links
- 239000012634 fragment Substances 0.000 title claims abstract description 14
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 14
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 3
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 claims description 3
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 23
- 230000002494 anti-cea effect Effects 0.000 abstract description 16
- 201000011510 cancer Diseases 0.000 abstract description 10
- 210000004408 hybridoma Anatomy 0.000 abstract description 10
- 206010028980 Neoplasm Diseases 0.000 abstract description 9
- 150000001413 amino acids Chemical class 0.000 abstract description 8
- 238000007796 conventional method Methods 0.000 abstract description 8
- 239000000427 antigen Substances 0.000 abstract description 7
- 102000036639 antigens Human genes 0.000 abstract description 7
- 108091007433 antigens Proteins 0.000 abstract description 7
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 206010035226 Plasma cell myeloma Diseases 0.000 abstract description 6
- 201000000050 myeloid neoplasm Diseases 0.000 abstract description 6
- 108020004999 messenger RNA Proteins 0.000 abstract description 5
- 206010070834 Sensitisation Diseases 0.000 abstract description 3
- 230000008313 sensitization Effects 0.000 abstract description 3
- 239000000439 tumor marker Substances 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 2
- 230000001605 fetal effect Effects 0.000 abstract 3
- 239000003795 chemical substances by application Substances 0.000 abstract 2
- 239000002299 complementary DNA Substances 0.000 abstract 2
- 102100022019 Pregnancy-specific beta-1-glycoprotein 2 Human genes 0.000 abstract 1
- 230000003393 splenic effect Effects 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 13
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 10
- 239000004677 Nylon Substances 0.000 description 8
- 239000012528 membrane Substances 0.000 description 8
- 229920001778 nylon Polymers 0.000 description 8
- 239000011780 sodium chloride Substances 0.000 description 6
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 210000004988 splenocyte Anatomy 0.000 description 4
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000009396 hybridization Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 241000972773 Aulopiformes Species 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000701959 Escherichia virus Lambda Species 0.000 description 2
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 210000000683 abdominal cavity Anatomy 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000012790 confirmation Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000000032 diagnostic agent Substances 0.000 description 2
- 229940039227 diagnostic agent Drugs 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- WOSQESYFYGCGNS-UHFFFAOYSA-M lithium diaminomethylideneazanium chloride thiocyanate Chemical compound [Li+].[Cl-].[S-]C#N.NC(N)=[NH2+] WOSQESYFYGCGNS-UHFFFAOYSA-M 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 235000019515 salmon Nutrition 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- GEYOCULIXLDCMW-UHFFFAOYSA-N 1,2-phenylenediamine Chemical compound NC1=CC=CC=C1N GEYOCULIXLDCMW-UHFFFAOYSA-N 0.000 description 1
- TVZGACDUOSZQKY-LBPRGKRZSA-N 4-aminofolic acid Chemical compound C1=NC2=NC(N)=NC(N)=C2N=C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 TVZGACDUOSZQKY-LBPRGKRZSA-N 0.000 description 1
- LPXQRXLUHJKZIE-UHFFFAOYSA-N 8-azaguanine Chemical compound NC1=NC(O)=C2NN=NC2=N1 LPXQRXLUHJKZIE-UHFFFAOYSA-N 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 229960003896 aminopterin Drugs 0.000 description 1
- 238000000211 autoradiogram Methods 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 238000003163 cell fusion method Methods 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229960000633 dextran sulfate Drugs 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000013399 early diagnosis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000009169 immunotherapy Methods 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- QYSGYZVSCZSLHT-UHFFFAOYSA-N octafluoropropane Chemical compound FC(F)(F)C(F)(F)C(F)(F)F QYSGYZVSCZSLHT-UHFFFAOYSA-N 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 102000013415 peroxidase activity proteins Human genes 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 238000011470 radical surgery Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Abstract
Description
【０００１】0001
【産業上の利用分野】本発明は新規なＤＮＡ断片に関す
る。詳しくは癌細胞に特異的にみられるヒト癌胎児性抗
原（以下ＣＥＡと称することもある。）に対し特異的に
反応する抗体蛋白質をコードするＤＮＡ断片に関する。TECHNICAL FIELD The present invention relates to a novel DNA fragment. Specifically, the present invention relates to a DNA fragment encoding an antibody protein that specifically reacts with human carcinoembryonic antigen (hereinafter sometimes referred to as CEA), which is specifically found in cancer cells.
【０００２】0002
【従来の技術】１９６５年Ｇｏｌｄらにより見いだされ
たＣＥＡ（Ｊ．Ｅｘｐ．Ｍｅｄ．，１２２，４６７，（
１９６５））は、現在最も確立した腫瘍マーカーである
。その癌マーカーとしての有用性としては、（１）消化
器癌を中心としていろいろな悪性腫瘍に出現するマーカ
ーであること、（２）癌の早期診断に役立つ、もしくは
、悪性疾患か良性疾患かの診断には不可欠なものである
こと、（３）外科手術後の患者のモニターリング、こと
に根治手術の成否の判断や再発の早期発見にはなはだ有
用であること、（４）非観血的治療法の効果判定に有用
であること等が挙げられる（松岡，生化学，６２，３５
２，（１９９０））。[Prior Art] CEA (J. Exp. Med., 122, 467, (
(1965)) is currently the most established tumor marker. Its usefulness as a cancer marker is (1) it is a marker that appears in various malignant tumors, mainly gastrointestinal cancer, and (2) it is useful for early diagnosis of cancer or whether it is a malignant or benign disease. (3) It is extremely useful for monitoring patients after surgery, especially for determining the success or failure of radical surgery and for early detection of recurrence; (4) Non-invasive treatment. (Matsuoka, Biochemistry, 62, 35)
2, (1990)).
【０００３】このＣＥＡに対する抗体は上述の診断を可
能にするばかりでなく、治療薬としても利用することが
可能である。治療薬としての利用法としては、最初は受
動的免疫療法を期待したマウスモノクローナル抗体の単
独投与が行われたが、最近ではモノクローナル抗体の特
異的結合能を利用したいわゆるミサイル療法や、他薬剤
との併用が主流になるつつある（Ｄ．Ｓｃｈｅｉｂｅｒ
ｇ ＆ Ａ．Ｈｏｕｇｈｔｏｎ，Ｏｎｃｏｌｏｇｙ
，，１，３１（１９８７））。[0003] Antibodies against CEA not only enable the above-mentioned diagnosis, but can also be used as therapeutic agents. Initially, mouse monoclonal antibodies were administered alone with the hope of passive immunotherapy, but recently, monoclonal antibodies have been used in so-called missile therapy, which utilizes the specific binding ability of monoclonal antibodies, and in combination with other drugs. (D. Scheiber
g & a. Houghton, Oncology
, 1, 31 (1987)).
【０００４】0004
【発明の解決しようとする問題点】上述の目的に使用で
きるＣＥＡに特異的に結合するマウスモノクローナル抗
体は現在までに多数取得されているが、そのアミノ酸配
列、更には塩基配列を決定している例は、ジェネンテッ
ク社の特許（特開昭６０−１５５１３２号公報）がある
程度で、未だ非常に少ない。黒木らは約１５０種類の抗
ＣＥＡ抗体をＣＥＡのどの微小領域に結合するかをしら
べることにより、２５のサブグループに分類した（黒木
ら，Ｊｐｎ．Ｊ．Ｃａｎｃｅｒ ｒｅｓ．７８，３８
６，（１９８７））。このことは、実際に診断薬あるい
は治療薬としてモノクローナル抗体を使用する場合には
サブグループの違いによりその効果が著しく異なること
を示している。[Problems to be solved by the invention] A large number of mouse monoclonal antibodies that specifically bind to CEA that can be used for the above purpose have been obtained to date, but their amino acid and base sequences have not yet been determined. Examples include a patent by Genentech (Japanese Unexamined Patent Publication No. 60-155132), but there are still very few examples. Kuroki et al. classified approximately 150 types of anti-CEA antibodies into 25 subgroups by examining which microregion of CEA they bind to (Kuroki et al., Jpn. J. Cancer res. 78, 38
6, (1987)). This shows that when monoclonal antibodies are actually used as diagnostic or therapeutic agents, their effectiveness varies significantly depending on the subgroup.
【０００５】[0005]
【問題点を解決するための手段】本発明者らは、抗ＣＥ
Ａ特異抗体生産株について種々検討した。その結果、ジ
ェネンテック社が報告した抗ＣＥＡマウスモノクローナ
ル抗体とはＨ鎖で１２０アミノ酸中２４アミノ酸が、Ｌ
鎖で１０９アミノ酸中２０アミノ酸が異なっている新規
な抗ＣＥＡマウスモノクローナル抗体を産生する生産株
を得、更に、該抗体をコードするｃＤＮＡを取得するに
至り、本発明を完成するに至った。すなわち、本発明の
要旨は、ヒト癌胎児性抗原に特異的に反応する抗体の重
鎖可変領域及び軽鎖可変領域をコードするＤＮＡ断片で
あって、該抗体の重鎖可変領域及び軽鎖可変領域が、夫
々、配列番号：１の第１番〜第１２０番及び配列番号：
２の第１番〜第１０９番で表わされるアミノ酸配列をコ
ードすることを特徴とするＤＮＡ断片に存する。[Means for solving the problem] The present inventors have developed anti-CE
Various studies were conducted regarding A-specific antibody producing strains. As a result, the anti-CEA mouse monoclonal antibody reported by Genentech was found to have 24 amino acids out of 120 amino acids in the H chain.
We obtained a production strain that produces a novel anti-CEA mouse monoclonal antibody that differs in 20 out of 109 amino acids in the chain, and also obtained cDNA encoding the antibody, thus completing the present invention. That is, the gist of the present invention is a DNA fragment encoding the heavy chain variable region and light chain variable region of an antibody that specifically reacts with human carcinoembryonic antigen, The regions are Nos. 1 to 120 of SEQ ID NO: 1 and SEQ ID NO:
It consists of a DNA fragment characterized by encoding the amino acid sequence represented by No. 1 to No. 109 of No. 2.
【０００６】以下、本発明を説明するに、本発明のＤＮ
Ａ断片は、まず、抗ＣＥＡ特異抗体生産株（ミエローマ
細胞及び脾細胞より得られるハイブリドーマ）を得、該
生産株からチオシアン酸グアニジン−塩化リチウム法（
ｃａｓａｒａら，ＤＮＡ，２，３２９（１９８３））に
記載の方法で、ｍＲＮＡを調製して、アマーシャム社製
のｃＤＮＡ合成システムキット等によりそのｃＤＮＡラ
イブラリーを作製する。次いで、該ライブラリーからマ
ウス抗体重（Ｈ）鎖定常領域及び軽（Ｌ）鎖定常領域の
塩基配列部分をプローブとして常法に従いハイブリダイ
ゼーションを行い該プローブと反応するｃＤＮＡをクロ
ーニングすることにより得ることができる。該ｃＤＮＡ
が抗ＣＥＡ特異抗体のｃＤＮＡであることの確認は、別
に、上記ハイブリドーマが生産する抗体を常法に従い分
離精製し、常法に従いＮ末端アミノ酸配列を決定し、そ
の結果との一致により行った。[0006] Hereinafter, in order to explain the present invention, the DN of the present invention will be explained.
Fragment A was first obtained by obtaining an anti-CEA specific antibody producing strain (hybridoma obtained from myeloma cells and splenocytes), and then using the guanidine thiocyanate-lithium chloride method (
mRNA is prepared by the method described in Casara et al., DNA, 2, 329 (1983)), and a cDNA library thereof is prepared using a cDNA synthesis system kit manufactured by Amersham. Next, hybridization is performed in accordance with a conventional method using the base sequence portions of the mouse antibody heavy (H) chain constant region and light (L) chain constant region as probes from the library, and cDNA that reacts with the probes is cloned. I can do it. The cDNA
Confirmation that the cDNA was a cDNA of an anti-CEA-specific antibody was performed by separately separating and purifying the antibody produced by the above hybridoma according to a conventional method, determining the N-terminal amino acid sequence according to a conventional method, and checking the results based on the results.
【０００７】[0007]
１）培地
ＲＰＭＩ１６４０培地に、リラシリン１００μｇ／ｍｌ
、ストレプトマイシン１００μｇ／ｍｌ、グルタミン２
ｍＭ、炭酸水素ナトリウム１．６ｇ／ｍｌを加えた後、
二酸化炭素を吹き込みｐＨ７．２前後とし牛胎児血清（
ＦＣＳ）を１０％となるように加えて使用した。1) Liracillin 100μg/ml in RPMI1640 medium
, streptomycin 100 μg/ml, glutamine 2
After adding 1.6 g/ml of sodium bicarbonate,
Blow in carbon dioxide and adjust the pH to around 7.2 with fetal bovine serum (
FCS) was added to 10%.
【０００８】２）ミエローマ細胞株
Ｂａｌｂ／ｃマウス由来の骨髄細胞ＭＯ−ＰＣ−２１の
株化細胞δアザグアニン耐性のＰ３−Ｘ６３−Ａｇ−δ
Ｕ／（Ｐ３ＵＩ）を用いた。
３）抗原感作
ヒトＣＥＡ（三菱化成社製）をフロイント完全アジュバ
ンドと混合した抗原をＢａｌｂ／ｃマウスに１匹当り０
．２５ｍｇ／０．５ｍｌずつ腹腔に接種し、一次感作し
た。５週間後、更に抗原を０．５ｍｇ／０．５ｍｌずつ
腹腔または、０．３ｍｇ／０．３ｍｌずつ尾静脈に接種
二次感作した。その４日後の脾臓細胞を上記ミエローマ
細胞株と下記４）の様にして細胞融合させた。2) Myeloma cell line Balb/c mouse-derived bone marrow cell line MO-PC-21 cell line δ azaguanine-resistant P3-X63-Ag-δ
U/(P3UI) was used. 3) Antigen-sensitized human CEA (manufactured by Mitsubishi Kasei) mixed with complete Freund's adjuvant was administered to Balb/c mice at 0% per mouse.
．． 25 mg/0.5 ml was inoculated into the abdominal cavity for primary sensitization. Five weeks later, the antigen was further injected into the abdominal cavity at 0.5 mg/0.5 ml or into the tail vein at 0.3 mg/0.3 ml for secondary sensitization. Four days later, the spleen cells were fused with the myeloma cell line as described in 4) below.
【０００９】４）細胞融合法
ミエローマ細胞、脾細胞共に食塩燐酸緩衝液（１０ｍＭ
燐酸緩衝液ｐＨ７．５、０．９％食塩、ＰＢＳ）で３回
洗浄後、ＲＰＭＩ１６４０，１０％ＦＣＳに浮遊し、細
胞数を算定した。１×１０６ 個のＰ３Ｕ１に対して７
．５〜１０×１０８ 個の脾細胞を２〜３週間培養した
。次にＲＰＭＩ１６４０で遠心洗浄し、ＦＣＳを除きガ
ラススピッツ遠心管に細胞を集める。上清を完全に取り
去った後、ペレット上の細胞をほぐし、予め３７℃に温
めておいた４２．５％ポリエチレングリコール液（ＰＥ
Ｇ）を０．５ｍｌ加え、室温で１分間反応させた後、３
７℃のＲＰＭＩ１６４０ １ｍｌを３０秒毎に１０回
加えた。
その間、試験管をゆっくり回転し続ける。この様に、細
胞融合させた細胞を遠心洗浄し、Ｐ３Ｕ１細胞数が、５
〜１０×１０５ 個／ｍｌになるようにＲＰＭＩ１６４
０，１０％ＦＣＳを加える。その０．２ｍｌをマイクロ
タイタープレートに分注した。２４時間培養して上清を
半量捨てＨＡＴ（ヒポキサンチン、アミノプテリン及び
チミヂンを含有）培地を加える。以後この操作を４８時
間毎に２週間繰り返す。ミエローマ細胞及び脾細胞共に
ＨＡＴ培地中では増殖できないので増殖してくる細胞は
、ハイブリドーマと考えられる。従って、１０〜１４日
後増殖してきたハイブリドーマの認められる培養液につ
いて抗体活性を調べた。4) Cell fusion method Both myeloma cells and splenocytes were prepared using saline phosphate buffer (10mM
After washing three times with phosphate buffer (pH 7.5, 0.9% saline, PBS), the cells were suspended in RPMI 1640, 10% FCS, and the number of cells was counted. 7 for 1x106 P3U1
．． 5-10 x 10 splenocytes were cultured for 2-3 weeks. Next, the cells are washed by centrifugation with RPMI 1640, the FCS is removed, and the cells are collected in a glass Spitz centrifuge tube. After completely removing the supernatant, loosen the cells on the pellet and add 42.5% polyethylene glycol solution (PE) pre-warmed to 37°C.
After adding 0.5 ml of G) and reacting at room temperature for 1 minute,
1 ml of 7°C RPMI 1640 was added every 30 seconds 10 times. Meanwhile, continue to rotate the test tube slowly. The fused cells were centrifugally washed in this way, and the number of P3U1 cells was reduced to 5.
RPMI164 to ~10×105 cells/ml
Add 0.10% FCS. 0.2 ml of the solution was dispensed into a microtiter plate. After culturing for 24 hours, half of the supernatant is discarded and HAT (containing hypoxanthine, aminopterin, and thymidine) medium is added. Thereafter, this operation was repeated every 48 hours for 2 weeks. Since both myeloma cells and splenocytes cannot proliferate in HAT medium, the proliferating cells are considered to be hybridomas. Therefore, the antibody activity was examined in the culture solution in which hybridomas that had grown after 10 to 14 days were observed.
【００１０】５） 抗体活性のスクリーニング抗体活
性のスクリーニングは次に示すようなエンザイムイムノ
アッセイにより行った。
１ ＰＢＳに溶解した抗原（１ｍｇ／ｍｌ）を５０μ
ｌマイクロタイタープレート（９６穴、Ｆａｌｃｏｎ３
１２９）に吸着させた。（４℃、一晩）
２ 抗原溶液を除き、０．０５％Ｔｗｅｅｎ２０を含
んだＰＢＳ（ＰＢＳＴ）により４回洗浄した後、５％牛
アルブミン（ＢＳＡ）を含んだＰＢＳを１００μｌ加え
３７℃１時間放置した。5) Screening for antibody activity Screening for antibody activity was carried out by enzyme immunoassay as shown below. 1 50 μl of antigen (1 mg/ml) dissolved in PBS
l Microtiter plate (96 wells, Falcon3
129). (4℃, overnight) 2 After removing the antigen solution and washing 4 times with PBS containing 0.05% Tween 20 (PBST), add 100μl of PBS containing 5% bovine albumin (BSA) for 1 hour at 37℃. I left it alone.
【００１１】３ ＢＳＡ溶液を除いた後、ＰＢＳＴに
より４回洗浄する。次に、ハイブリドーマの培養上清を
５０μｌ加え、二時間反応させた。3 After removing the BSA solution, wash four times with PBST. Next, 50 μl of hybridoma culture supernatant was added and reacted for 2 hours.
【００１２】４ ＰＢＳＴで４回洗浄した後、１％Ｂ
ＳＡを含むＰＢＳで１０００倍に希釈したベルオキシダ
ーゼ結合抗マウスＩｇＧ抗体を５０μｌ加え、３７℃、
２時間反応させた。５ ＰＢＳＴにより４回洗浄した
後、０．５Ｍクエン酸１．２２ｍｌ，０．５Ｍ燐酸二ナ
トリウム ２．５６ｍｌ，オルトフェニレンジアミン
１０ｍｇ，３０％過酸化水素水 １０μｌ／２５
ｍｌを５０μｌ加え発色させた。十分発色した後、２Ｍ
硫酸を５０μｌ加え発色を停止させる。4 After washing four times with PBST, 1% B
Add 50 μl of peroxidase-conjugated anti-mouse IgG antibody diluted 1000 times with PBS containing SA, and heat at 37°C.
The reaction was allowed to proceed for 2 hours. 5 After washing 4 times with PBST, 0.5M citric acid 1.22ml, 0.5M disodium phosphate 2.56ml, orthophenylenediamine 10mg, 30% hydrogen peroxide solution 10μl/25
ml was added to develop color. After sufficient color development, 2M
Add 50 μl of sulfuric acid to stop color development.
【００１３】６ 発色は、イムノリーダーにより光学
的に測定した。
７ 抗原に特異的な抗体を生産している細胞のうち、
特に結合能力の強い抗体を生産している株一株を分離し
クローニング操作を重ねた上で抗ＣＥＡ特異抗体ｃＤＮ
Ａを取るハイブリドーマ細胞として保存した。6 Color development was optically measured using an immunoreader. 7 Among cells that produce antigen-specific antibodies,
After isolating one strain that produces an antibody with particularly strong binding ability and carrying out repeated cloning operations, an anti-CEA-specific antibody cDNA was created.
The cells were preserved as hybridoma cells designated as A.
【００１４】〔抗ＣＥＡ抗体遺伝子の取得〕１）ｍＲＮ
Ａの調製
該ハイブリドーマ細胞約１０９ 個の細胞よりチオシア
ン酸グアニジン−塩化リチウム法（Ｃａｓａｒａら、Ｄ
ＮＡ，２，３２９（１９８３））に従いポリＡを有する
ＲＮＡを下記のごとく調製した。該ハイブリドーマ細胞
約１０９ 個の細胞を直ちに液体窒素により凍結した。
これを液体窒素と共に、ワーリングブレンダーに入れ３
，０００ｒｐｍ ２分間にて粉砕した。これに５Ｍチオ
シアン酸グアニジン、１０ｍＭ ＥＤＴＡ，５０ｍＭ
トリス塩酸（ｐＨ７）及び８％β−メルカプトエタノー
ルからなる溶液１００ｍｌ中でホモゲナイザー（５ｒｐ
ｍ）にて更に破砕し、可溶化した。この可溶化物２０ｍ
ｌを遠心管中の５．７ＭＣｓＣｌ溶液１０ｍｌ上に静か
に乗せ、日立ＲＰＳ２８−２ローターにて２７，０００
ｒｐｍ ，２０時間遠心後ＲＮＡを沈澱として回収した
。このＲＮＡの沈澱を０．１％ラウリル硫酸ナトリウム
、１ｍＭＥＤＴＡ、１０ｍＭトリス塩酸（ｐＨ７．５）
からなる溶液１０ｍｌに溶解しフェノールクロロホルム
で注出後、エタノール沈澱により回収した。得られたＲ
ＮＡ約３．９５ｍｇを１０ｍＭトリス塩酸（ｐｈ８．０
）及び１ｍＭ ＥＤＴＡからなる溶液１ｍｌに溶かし
た。６５℃、５分間保温し０．１ｍｌの５Ｍ食塩を加え
た。混合物をオリゴｄＴセルロースカラム（ＰＬバイオ
ケミカル社）クロマトグラフィー（カラム体積０．５ｍ
ｌ）にかけた。吸着したポリＡ ＲＮＡを１ｍＭ
ＥＤＴＡ、１０ｍＭトリス塩酸（ｐＨ７．５）からなる
溶液で溶出し、ポリＡを有するｍＲＮＡ約１００μｇを
得た。[Obtaining anti-CEA antibody gene] 1) mRNA
A was prepared using the guanidine thiocyanate-lithium chloride method (Casara et al., D.
RNA having polyA was prepared as follows according to NA, 2, 329 (1983)). Approximately 10 9 of the hybridoma cells were immediately frozen in liquid nitrogen. Put this in a Waring blender with liquid nitrogen 3
,000 rpm for 2 minutes. To this, 5M guanidine thiocyanate, 10mM EDTA, 50mM
A homogenizer (5 rp.
The mixture was further crushed and solubilized using m). 20m of this solubilized material
1 was carefully placed on 10 ml of 5.7 MCsCl solution in a centrifuge tube, and the mixture was heated at 27,000 mL in a Hitachi RPS28-2 rotor.
After centrifugation at rpm for 20 hours, RNA was collected as a precipitate. This RNA precipitate was mixed with 0.1% sodium lauryl sulfate, 1mM EDTA, 10mM Tris-HCl (pH 7.5).
The sample was dissolved in 10 ml of a solution consisting of phenol, extracted with phenol chloroform, and recovered by ethanol precipitation. The obtained R
Approximately 3.95 mg of NA was added to 10 mM Tris-HCl (ph 8.0
) and 1 ml of a solution consisting of 1 mM EDTA. The mixture was kept warm at 65° C. for 5 minutes, and 0.1 ml of 5M sodium chloride was added. The mixture was chromatographed on an oligo dT cellulose column (PL Biochemical Co., Ltd.) (column volume 0.5 m).
l). Adsorbed polyA RNA at 1mM
About 100 μg of polyA-containing mRNA was obtained by elution with a solution consisting of EDTA and 10 mM Tris-HCl (pH 7.5).
【００１５】２）相補鎖ＤＮＡ（ｃＤＮＡ）の合成上記
のごとく分離精製したポリＡ ｍＲＮＡを用いアマー
シャム社製のｃＤＮＡ合成システムキットを用い、その
説明書に従いｃＤＮＡを合成した。３）ｃＤＮＡライブ
ラリーの作製
上記のごとく合成したｃＤＮＡを用い、アマーシャム社
製のｃＤＮＡクローニングシステムλｇｔ１０キットを
使用し、その説明書に従いｃＤＮＡライブラリーを作製
した。2) Synthesis of complementary strand DNA (cDNA) Using the polyA mRNA isolated and purified as described above, cDNA was synthesized using a cDNA synthesis system kit manufactured by Amersham in accordance with the instructions. 3) Preparation of cDNA library Using the cDNA synthesized as described above, a cDNA library was prepared using Amersham's cDNA cloning system λgt10 kit according to its instructions.
【００１６】４）プラークハイブリダイゼーション上記
のごとく作製したλファージよりなるｃＤＮＡライブラ
リーを１５ｃｍのシャーレにＬＢ軟寒天培地〔１％バク
トトリプトン、５％イーストイクストラクト、０．５％
塩化ナトリウム、及び０．７％寒天沫〕と共に、１６枚
のシャーレにおよそ２４万のクローンをまき、一晩培養
した後、培地中のλファージクローンをナイロン膜であ
るジーンスクリーニングプラス（デュポン社）上に移し
取った。一枚のシャーレあたりにナイロン膜２枚の割合
で移し取り、その様にしてできたナイロン膜を０．５Ｍ
水酸化ナトリウムが浸み込んだろ紙上に２分間静置し、
別に用意した乾いたろ紙上で水分を除いた後、同じ操作
を再び繰り返した。次に、同様に１Ｍトリス−塩酸（ｐ
Ｈ７．５）を浸み込ませたろ紙上でこのナイロン膜を静
置し、乾いたろ紙上で風乾した後、同じ操作を再び繰り
返した。こうして処理したナイロン膜は、次にナイロン
膜１枚当り５ｍｌのプレハイブリダイゼーション液〔１
Ｍ塩化ナトリウム、１％ＳＤＳ、２５０μｇ／ｍｌ鮭精
子ＤＮＡ〕に６５℃１時間浸した。次に既にその遺伝子
が取得されているマウス抗体Ｈ鎖遺伝子定常領域塩基配
列（５’ＡＧＡＴＧＧＧＧＧＴＧＴＣＧＴＴ３’、アプ
ライドバイオシステム３８０Ａ ＤＮＡ合成機により
常法に従い合成）、Ｌ鎖遺伝子定常領域塩基配列（５’
ＴＧＧＡＴＧＧＴＧＧＧＡＡＧＡＴＧ３’、アマーシャ
ム社より購入）から常法に従い作製した３２Ｐ標識プロ
ーブを含むハイブリダイゼーション液〔１Ｍ塩化ナトリ
ウム、１％ＳＤＳ、２５０μｇ／ｍｌ鮭***ＤＮＡ、１
０％硫酸デキストラン、１０ｎｇ／ｍｌ３２Ｐ標識プロ
ーブＤＮＡ〕中で５５℃で１８時間保温した。その後、
ナイロン膜を取り出し、０．１％のＳＤＳを含んだ２倍
の濃度のＳＳＣ溶液〔２０倍の濃度のＳＳＣ溶液；３Ｍ
塩化ナトリウム、０．３Ｍクエン酸ナトリウム〕中で室
温で３０分間２回洗い、０．１％のＳＤＳを含んだ０．
１倍の濃度のＳＳＣ溶液で４２℃、３０分間２回洗った
後、オートラジオグラフィーを行った。２枚１組のナイ
ロン膜のオートラジオグラム上のシグナルが一致したも
のはＨ鎖の場合１８個ありＬ鎖の場合１０個であった。
そのうちＨ鎖４個、Ｌ鎖６個は同じ制限酵素による切断
のパターンが同一のｃＤＮＡを含んでいた。4) Plaque Hybridization The cDNA library consisting of λ phage prepared as above was placed in a 15 cm Petri dish on LB soft agar medium [1% Bactotryptone, 5% Yeast Extract, 0.5%
Approximately 240,000 clones were sown in 16 Petri dishes with sodium chloride and 0.7% agar, and after culturing overnight, the λ phage clones in the medium were separated using a nylon membrane Gene Screening Plus (DuPont). I moved it above. Transfer two nylon membranes per petri dish, and transfer the resulting nylon membrane to 0.5M
Leave it for 2 minutes on filter paper soaked with sodium hydroxide.
After removing moisture on a separately prepared dry filter paper, the same operation was repeated again. Next, similarly, 1M Tris-HCl (p
The nylon membrane was left standing on a filter paper impregnated with H7.5), air-dried on the dry filter paper, and the same operation was repeated again. The nylon membrane treated in this way was then mixed with 5 ml of prehybridization solution [1 ml] per nylon membrane.
M sodium chloride, 1% SDS, 250 μg/ml salmon sperm DNA] at 65° C. for 1 hour. Next, the mouse antibody H chain gene constant region base sequence (5'AGATGGGGTGTCGTT3', synthesized using an Applied Biosystems 380A DNA synthesizer according to a conventional method), from which the gene has already been obtained, and the L chain gene constant region base sequence (5'
Hybridization solution containing a 32P-labeled probe prepared according to a conventional method from TGGATGGTGGGAAGATG3' (purchased from Amersham) [1M sodium chloride, 1% SDS, 250 μg/ml salmon sperm DNA, 1
The cells were incubated at 55° C. for 18 hours in 0% dextran sulfate, 10 ng/ml P-labeled probe DNA. after that,
Take out the nylon membrane and add 2 times the concentration of SSC solution containing 0.1% SDS [20 times the concentration of SSC solution; 3M
Sodium chloride, 0.3M sodium citrate] was washed twice for 30 minutes at room temperature, and washed twice in 0.3M sodium citrate containing 0.1% SDS.
After washing twice for 30 minutes at 42°C with a 1x SSC solution, autoradiography was performed. There were 18 coincident signals on the autoradiogram of a pair of nylon membranes for the H chain and 10 for the L chain. Among them, 4 H chains and 6 L chains contained cDNAs with the same cleavage pattern with the same restriction enzyme.
【００１７】これらの中からＨ鎖、Ｌ鎖とも一つずつク
ローンを選びｃＤＮＡの塩基配列の決定をＳａｎｇｅｒ
らのジデオキシ法によって行った。求める抗体のｃＤＮ
Ａであることの確認は、別にこのハイブリドーマの生産
する抗体を常法に従い分離精製し、常法に従いＮ末端ア
ミノ酸配列を決定し、その結果との一致により行った。
決定した塩基配列から予想される重鎖及び軽鎖の可変領
域のアミノ酸配列を夫々、配列番号：１の第１番〜第１
２０番及び配列番号：２の第１番〜第１０９番で示す。
尚、配列番号：１の第−１番〜−第１９番及び配列番号
：２の第−１番〜第−２０番はシグナル配列を示す。
また特開昭６０−１５５１３２号公報記載の抗ＣＥＡマ
ウスモノクローナル抗体のアミノ酸配列との違いを図１
及び図２に示す。図１では、配列番号：１で表わされる
重鎖可変領域のアミノ酸の全配列を上段に示し、下段に
はジェネンテック社から報告されている抗ＣＥＡマウス
モノクローナル抗体のアミノ酸配列のうち配列番号：：
１で表わされるアミノ酸配列とは一致しないアミノ酸残
基のみを示した。図２では、軽鎖可変領域のアミノ酸配
列について同様の形式で示した。From these, one clone for each of the H chain and L chain was selected, and the cDNA base sequence was determined by Sanger
This was done by the dideoxy method of et al. cDNA of the desired antibody
Confirmation that it was A was performed by separately separating and purifying the antibody produced by this hybridoma according to a conventional method, determining the N-terminal amino acid sequence according to a conventional method, and checking the agreement with the results. The amino acid sequences of the variable regions of the heavy chain and light chain predicted from the determined nucleotide sequences were determined from No. 1 to No. 1 of SEQ ID NO: 1, respectively.
No. 20 and No. 1 to No. 109 of SEQ ID NO: 2. In addition, No. -1 to No. -19 of SEQ ID NO: 1 and No. -1 to No. -20 of SEQ ID NO: 2 represent a signal sequence. In addition, the difference from the amino acid sequence of the anti-CEA mouse monoclonal antibody described in JP-A-60-155132 is shown in Figure 1.
and shown in FIG. In FIG. 1, the entire amino acid sequence of the heavy chain variable region represented by SEQ ID NO: 1 is shown in the upper row, and the lower row shows the amino acid sequence of the anti-CEA mouse monoclonal antibody reported by Genentech, SEQ ID NO::
Only amino acid residues that do not match the amino acid sequence represented by 1 are shown. In FIG. 2, the amino acid sequence of the light chain variable region is shown in a similar format.
【００１８】本発明に於て我々が取得した抗ＣＥＡマウ
スモノクローナル抗体の塩基配列は、特開昭６０−１５
５１３２号公報記載の抗体ＣＥＡマウスモノクローナル
抗体とはＨ鎖で１２０アミノ酸中２４アミノ酸が、Ｌ鎖
で１０９アミノ酸中２０アミノ酸が異なっており、ＣＥ
Ａに特異的に結合する新規のマウスモノクローナル抗体
をコードするｃＤＮＡを取得したことが分る。かくして
、アミノ酸配列及び塩基配列が明らかにされた本発明の
ＤＮＡ断片は、上述の様な方法を繰返すことなく、ＤＮ
Ａ合成機等により再現性良く取得することができる。[0018] The base sequence of the anti-CEA mouse monoclonal antibody that we obtained in the present invention is disclosed in Japanese Patent Application Laid-Open No. 60-15
The antibody CEA mouse monoclonal antibody described in Publication No. 5132 differs from the CEA mouse monoclonal antibody in 24 out of 120 amino acids in the H chain and in 20 out of 109 amino acids in the L chain.
It can be seen that cDNA encoding a novel mouse monoclonal antibody that specifically binds to A was obtained. Thus, the DNA fragment of the present invention, whose amino acid sequence and base sequence have been revealed, can be converted into a DNA fragment without repeating the above-mentioned method.
It can be obtained with good reproducibility using an A-synthesizer or the like.
【００１９】[0019]
【発明の効果】本発明の新規なＤＮＡ断片は、ＣＥＡに
対し特異的に反応する抗体をコードしているので、その
診断薬あるいは治療薬として使用し得る。Effects of the Invention The novel DNA fragment of the present invention encodes an antibody that specifically reacts with CEA, and therefore can be used as a diagnostic or therapeutic agent for CEA.
【００２０】[0020]
【表１】[Table 1]
【００２１】[0021]
【表２】[Table 2]
【００２２】[0022]
【表３】[Table 3]
【００２３】[0023]
【表４】[Table 4]
【図１】本発明の抗ＣＥＡ抗体の重鎖可変領域をコード
するアミノ酸配列（上段）と公知の抗ＣＥＡ抗体の重鎖
可変領域をコードするアミノ酸配列（下段。相違する部
分のアミノ酸残基のみを記載。）を示す。FIG. 1: Amino acid sequence encoding the heavy chain variable region of the anti-CEA antibody of the present invention (upper row) and amino acid sequence encoding the heavy chain variable region of a known anti-CEA antibody (lower row; only the amino acid residues that differ) ).
【図２】本発明の抗ＣＥＡ抗体の軽鎖可変領域をコード
するアミノ酸配列（上段）と公知の抗ＣＥＡ抗体の軽鎖
可変領域をコードするアミノ酸配列（下段。相違する部
分のアミノ酸残基のみを記載。）を示す。FIG. 2: Amino acid sequence encoding the light chain variable region of the anti-CEA antibody of the present invention (upper row) and amino acid sequence encoding the light chain variable region of a known anti-CEA antibody (lower row; only the amino acid residues that differ) ).
Claims (2)
抗体の重鎖可変領域及び軽鎖可変領域をコードするＤＮ
Ａ断片であって、該抗体の重鎖可変領域及び軽鎖可変領
域が、夫々、配列番号：１の第１番〜第１２０番及び配
列番号：２の第１番〜第１０９番で表わされるアミノ酸
配列をコードすることを特徴とするＤＮＡ断片。Claim 1: A DNA encoding a heavy chain variable region and a light chain variable region of an antibody that specifically reacts with human carcinoembryonic antigen.
A fragment in which the heavy chain variable region and light chain variable region of the antibody are represented by No. 1 to No. 120 of SEQ ID NO: 1 and No. 1 to No. 109 of SEQ ID NO: 2, respectively. A DNA fragment characterized by encoding an amino acid sequence.
が、夫々、配列番号：３の及び配列番号：４で示される
ことを特徴とする請求項１記載のＤＮＡ断片。2. The DNA fragment according to claim 1, wherein the heavy chain variable region and light chain variable region of the antibody are shown by SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP40881090A JPH04234987A (en) | 1990-12-28 | 1990-12-28 | Dna fragment |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP40881090A JPH04234987A (en) | 1990-12-28 | 1990-12-28 | Dna fragment |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04234987A true JPH04234987A (en) | 1992-08-24 |
Family
ID=18518216
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP40881090A Pending JPH04234987A (en) | 1990-12-28 | 1990-12-28 | Dna fragment |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04234987A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2020510438A (en) * | 2017-03-15 | 2020-04-09 | オックスフォード ジェネティクス リミテッドＯｘｆｏｒｄ Ｇｅｎｅｔｉｃｓ Ｌｉｍｉｔｅｄ | How to select antibodies |
-
1990
- 1990-12-28 JP JP40881090A patent/JPH04234987A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2020510438A (en) * | 2017-03-15 | 2020-04-09 | オックスフォード ジェネティクス リミテッドＯｘｆｏｒｄ Ｇｅｎｅｔｉｃｓ Ｌｉｍｉｔｅｄ | How to select antibodies |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6291235B1 (en) | Isolated nucleic acid which encodes protein which binds to A33 antibody | |
| JP3194020B2 (en) | Tumor antigen-specific antibodies, pharmaceutical compositions containing the same and cell lines producing the antibodies | |
| JPS5845407B2 (en) | Method for producing malignant tumor antibodies | |
| JPH1070991A (en) | Chimeric immunogloblin gene for tumor-relating antigen | |
| JP2008118996A (en) | Cloning and characterization of napsin, one kind of aspartic protease | |
| JP2002543830A (en) | Novel Fab fragment libraries and methods of using them | |
| JP2935520B2 (en) | New high-affinity modified antibody families used for cancer treatment | |
| JPS60260597A (en) | Tumor specific monoclonal antibody | |
| WO1997008189A9 (en) | Colon cell and colon cancer cell associated nucleic acid molecules, protein and peptides | |
| WO1997004080A1 (en) | Novel protein and monoclonal antibody specific thereto | |
| EP0146050B1 (en) | Site selective plasminogen activator and method of making and using same | |
| EP0750037A2 (en) | TNF-convertase and method measuring inhibition of TNF convertase | |
| CA1341357C (en) | Platelet-specific chimeric immunoglobulin | |
| JP3504963B2 (en) | DNA fragment encoding amino acid sequence of anti-human high affinity IgE receptor monoclonal antibody | |
| US5677182A (en) | Cleavage site blocking antibody to prohormone proteins and uses thereof | |
| JPH07101999A (en) | Amino acid sequence of anti-idiotype antibody to anti-cancer human monoclonal antibody and dna base sequence coding for the same | |
| WO1998012227A1 (en) | Recombinant single chain antibodies directed against the gp54 cancer marker, composition comprising same and use thereof | |
| JPH04234987A (en) | Dna fragment | |
| JP3023469B2 (en) | Glycoprotein 39 gene | |
| JPH04500603A (en) | Cloned nephritis antigen | |
| US6491926B1 (en) | 35 kD tumor associated protein antigen: uses and methods of detection | |
| JP2888587B2 (en) | Monoclonal antibody specific for cancer-related human galactosyltransferase, hybridoma producing the same, and method for measuring cancer-related human galactosyltransferase in a specimen using the same | |
| JPH04346792A (en) | Amino acid sequence of anti-cancer human monoclonal antibody and dna base sequence coding the same antibody | |
| JPH04330295A (en) | Mouse-human chimera antibody | |
| CN109593132B (en) | Monoclonal antibodies for treating cancer and uses thereof |