JP2761543B2 - Monoclonal antibody against human proto-oncogene product and hybridoma producing the same - Google Patents
Monoclonal antibody against human proto-oncogene product and hybridoma producing the sameInfo
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- JP2761543B2 JP2761543B2 JP1177392A JP17739289A JP2761543B2 JP 2761543 B2 JP2761543 B2 JP 2761543B2 JP 1177392 A JP1177392 A JP 1177392A JP 17739289 A JP17739289 A JP 17739289A JP 2761543 B2 JP2761543 B2 JP 2761543B2
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- monoclonal antibody
- cells
- erbb
- hybridoma
- human
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Description
【発明の詳細な説明】 ［産業上の利用分野］ この発明は、新規なモノクローナル抗体及びそれを産
生するハイブリドーマに関する。この発明のモノクロー
ナル抗体はヒト腺癌、特に乳癌、胃癌等の診断、治療薬
として用いることができる。Description: TECHNICAL FIELD The present invention relates to a novel monoclonal antibody and a hybridoma producing the same. The monoclonal antibody of the present invention can be used as a diagnostic or therapeutic agent for human adenocarcinoma, particularly breast cancer, gastric cancer and the like.
［従来の技術］ 癌の診断、治療法の開発のためにこれまで多方面から
研究が進められてきた。細胞の癌化は染色体DNAに傷が
つく等の異常が出発点となっていることが明らかになり
つつある。DNAの異常は大きく２つに分けることができ
る。１つはその異常のために特定の遺伝子の機能が欠損
し、その結果として細胞が癌化する異常であり、他方は
特定の遺伝子の機能を細胞の癌化に都合の良いように変
えてしまう異常である。後者の異常の原因になっている
のが癌遺伝子と呼ばれる。これまで約40種類知られてい
る特定の遺伝子がある。[Prior Art] Research has been carried out in various fields for the purpose of developing cancer diagnostics and therapeutic methods. It is becoming clear that canceration of cells starts from abnormalities such as damage to chromosomal DNA. DNA abnormalities can be broadly divided into two types. One is an abnormality in which the function of a specific gene is deficient due to the abnormality, and as a result, the cell becomes cancerous, and the other changes the function of the specific gene in a manner favorable to the canceration of the cell. It is abnormal. The cause of the latter abnormality is called an oncogene. There are about 40 specific genes known so far.
ニワトリに感染して癌をつくる代表的なウイルスにラ
ウス肉腫ウイルスがあり、1911年に発見されている。こ
のウイルスの発癌能がウイルスのゲノム上にあるsrc遺
伝子によることが分かったのは比較的最近である。Rous sarcoma virus is a typical virus that infects chickens and produces cancer, and was discovered in 1911. It is relatively recent that the oncogenic potential of this virus was found to be due to the src gene on the virus genome.
その後、今日まで癌研究の分野で飛躍的な進展が見ら
れるが、その１つにsrcと相同な遺伝子がニワトリ正常
細胞の染色体上に存在することが19767年にディー・ス
テーリンらによって見出された。その他数多くのRNA腫
瘍ウイルスについての分子生物学的解析からyes、erb
B、fps、abl、ras等の癌遺伝子が同定されたが、これら
のウイルスの癌遺伝子はsrc同様全て細胞由来癌原遺伝
子（以下ｃ−oncとする）に由来することが示された。To date, breakthroughs have been made in the field of cancer research, one of which was the discovery of a gene homologous to src on the chromosome of normal chicken cells by Dee Stalin et al. In 19767. Was. Yes, erb from molecular biological analysis of many other RNA tumor viruses
Oncogenes such as B, fps, abl, and ras were identified, but it was shown that the oncogenes of these viruses were all derived from cell-derived proto-oncogenes (hereinafter referred to as c-onc) like src.
普段正常細胞内に存在している限りでは、発癌活性を
示さない遺伝子ｃ−oncがRNA腫瘍ウイルスのゲノムに取
り込まれて発現することにより癌遺伝子としての性質を
示す。As long as the gene is normally present in normal cells, c-onc, which does not exhibit oncogenic activity, is incorporated into the genome of RNA tumor virus and expressed, thereby exhibiting properties as an oncogene.
正常な細胞は分化と増殖によって生命体を維持する機
能を担っているが、これらの癌原遺伝子は細胞の分化、
繁殖に溶接につながっている遺伝子であることもわかっ
ており、癌化のメカニズムに直接関連した癌の診断及び
治療法の開発につながることが期待される。Normal cells are responsible for maintaining life by differentiation and proliferation, but these proto-oncogenes are responsible for cell differentiation,
It is also known that this gene is linked to welding for reproduction, and is expected to lead to the development of cancer diagnosis and treatment methods directly related to the mechanism of canceration.
本発明者らは癌原遺伝子（ｃ−onc）の機能を探るた
めに分子生物学的手法を用いて研究を行なってきた。既
に報告したようにヒト細胞由来癌原遺伝子（ヒトｃ−er
bBの遺伝子クローニングに続き、新たなヒト癌原遺伝子
erbB−２（以下、ヒトｃ−erbB−２とする）（Semba
ら、PNAS 82,6497（1985）、Yamamotoら、Nature 319、
230（1986）を発見した。The present inventors have conducted research using molecular biology techniques to probe the function of proto-oncogene (c-onc). As already reported, a human cell-derived proto-oncogene (human c-er
Following bB gene cloning, a new human proto-oncogene
erbB-2 (hereinafter referred to as human c-erbB-2) (Semba
Et al., PNAS 82 , 6497 (1985); Yamamoto et al., Nature 319 ,
230 (1986).
ヒトｃ−erbB−２は後に上皮成長因子（EGF）の受容
体の遺伝子に由来するこがわかったヒトｃ−erbBと極め
て相同性が高い遺伝子であり、その遺伝子産物はタンパ
ク質のチロシン残基を特異的にリン酸化するチロシンキ
ナーゼ活性を有している。ヒトｃ−erbB−２もヒトｃ−
erbBと同様、細胞表面に存在する受容体の一種と考えら
れているが、今日までそのリガンドは同定されていな
い。しかし、チロシンキナーゼ活性を有する増殖因子受
容体の過剰発現が癌の発症に何らかの役割を果たしてい
る可能性は十分に考えられる。この見地から本発明者ら
は手術時の摘出癌組織からDNAを調製し、ｃ−erbB−２
遺伝子に特異的なDNAとのハイブリッド形成法により予
測の確認実験を行なった。その結果、ヒトｃ−erbB−２
遺伝子は胃癌、乳癌等の腺上皮癌の２割程に増幅が見ら
れた。このことはヒトｃ−erbB−２が腺癌の発症、進展
に寄与しており、腺癌の診断に需要な情報を提供するこ
とを意味している。Human c-erbB-2 is a gene highly homologous to human c-erbB, which was later found to be derived from the epidermal growth factor (EGF) receptor gene. It has a tyrosine kinase activity that specifically phosphorylates. Human c-erbB-2 is also human c-
Like erbB, it is thought to be a type of cell surface receptor, but its ligand has not been identified to date. However, it is quite possible that overexpression of a growth factor receptor having tyrosine kinase activity may play a role in the development of cancer. From this point of view, the present inventors prepared DNA from excised cancer tissue at the time of surgery, and prepared c-erbB-2
An experiment to confirm the prediction was carried out by a hybridization method with DNA specific to the gene. As a result, human c-erbB-2
The gene was amplified in about 20% of glandular epithelial cancers such as gastric cancer and breast cancer. This means that human c-erbB-2 contributes to the development and progression of adenocarcinoma and provides information necessary for the diagnosis of adenocarcinoma.
erbB−２の発現機能に関してはWeinbergらのラットｃ
−erbB−２遺伝子（以下neuとする）（Science 235,17
7,（1984）、Nature 319、226（1986）、Nature 319,23
0（1986）,Cell 45,649（1986））が点変異を獲得した
ものであることがわかったが、neuは新生ラットに化学
発癌剤であるエチルニトロソウレアを投与して誘発した
腫瘍から培養細胞癌化能を持つ遺伝子として見出され
た。For the expression function of erbB-2, rat c from Weinberg et al.
-ErbB-2 gene (hereinafter referred to as neu) (Science 235 , 17
7, (1984), Nature 319 , 226 (1986), Nature 319 , 23
0 (1986), Cell 45 , 649 (1986)) was found to have acquired a point mutation, but neu was cultured from a tumor induced by the administration of the chemical carcinogen ethylnitrosourea to newborn rats. It was found as a gene with cell carcinogenic potential.
erbB−２の腫瘍組織での異常発現はDNA検出法でもコ
ピー数増大の診断情報を得ることができるが、この方法
は癌診断法としては一般的でなく、得られている情報も
DNAレベルのものに限定されている。Abnormal expression of erbB-2 in tumor tissue can provide diagnostic information of copy number increase by DNA detection, but this method is not general as a cancer diagnostic method, and the information obtained is
Limited to those at the DNA level.
もし、ヒトｃ−erbB−２の遺伝子産物を対応抗原とす
るモノクローナル抗体を得るこことができれば、乳癌や
胃癌等の腺癌の診断、治療に有利に用いることができ
る。If a monoclonal antibody using the gene product of human c-erbB-2 as a corresponding antigen can be obtained, it can be advantageously used for diagnosis and treatment of adenocarcinomas such as breast cancer and stomach cancer.
［発明が解決しようとする課題］ 従って、この発明の目的は、ヒトｃ−erbB−２の遺伝
子産物を対応抗原とするモノクローナル抗体及びそれを
産生するハイブリドーマを提供することである。[Problem to be Solved by the Invention] Accordingly, an object of the present invention is to provide a monoclonal antibody having a gene product of human c-erbB-2 as a corresponding antigen and a hybridoma producing the same.
［課題を解決するための手段］ 抗原が単離されている場合には、それを抗原として用
いてケーラーとミルシュタインの常法に従い、その抗原
を対応抗原とするモノクローナル抗体を作製することは
比較的容易であるが、ヒトｃ−erbB−２の遺伝子産物は
未だ単離されていないので、これを対応抗原とするモノ
クローナル抗体の作製は容易ではない。そこで、本願発
明者らは、鋭意研究の結果、ヒトｃ−erbB−２遺伝子を
含むベクターを開発し、このベクターでマウス胎児由来
の腺維芽細胞を形質転換して、ヒトｃ−erbB−２遺伝子
を高率に発現する細胞を作製することに成功し（ヒトｃ
−erbB−２遺伝子産物は細胞膜上に存在する）、これを
免疫原として免疫した動物の抗体産生細胞とミエローマ
細胞とを融合してヒトｃ−erbB−２遺伝子産物を対応抗
原とするモノクローナル抗体を産生するハイブリドーマ
を作製することに成功し、この発明を完成した。[Means for solving the problem] When an antigen is isolated, it is comparatively possible to use the antigen as an antigen in accordance with Koehler and Milstein's standard method to produce a monoclonal antibody using the antigen as a corresponding antigen. Although the target is easy, since the gene product of human c-erbB-2 has not been isolated yet, it is not easy to prepare a monoclonal antibody using this as a corresponding antigen. Thus, the present inventors have developed a vector containing the human c-erbB-2 gene as a result of earnest studies, and transformed a mouse embryo-derived glandular fibroblast with this vector to obtain a human c-erbB-2 gene. We succeeded in producing cells expressing the gene at a high rate (human c
-ErbB-2 gene product is present on the cell membrane), and an antibody-producing cell of an animal immunized with the immunogenic product is fused with myeloma cells to produce a monoclonal antibody having the human c-erbB-2 gene product as a corresponding antigen. We succeeded in producing hybridomas and completed the present invention.
すなわち、この発明は、ヒトｃ−erbB−２産物を細胞
表面上に発現する細胞を免疫原とした動物の抗体産生細
胞とミエローマ細胞と融合して得られたハイブリドーマ
によって産生され、ヒトｃ−erbB−２遺伝子産物を対応
抗原とするモノクローナル抗体及びこれを産生するハイ
ブリドーマを提供する。That is, the present invention provides a human c-erbB produced by fusing an antibody-producing cell of an animal with a cell expressing the human c-erbB-2 product on the cell surface as an immunogen and a myeloma cell, and producing human c-erbB-2. Provided are a monoclonal antibody having a -2 gene product as a corresponding antigen and a hybridoma producing the same.
［発明の効果］ この発明により、ヒトｃ−erbB−２産物を細胞表面上
に発現する細胞を免疫原とした動物の抗体産生細胞とミ
エローマ細胞と融合して得られたハイブリドーマによっ
て産生され、ヒトｃ−erbB−２遺伝子産物を対応抗原と
する新規なモノクローナル抗体及びこれを産生する新規
なハイブリドーマが提供された。この発明のモノクロー
ナル抗体は、癌原遺伝子であるヒトｃ−erbB−２遺伝子
の産物を対応抗原とするものであり、また、上述のよう
に、ヒトｃ−erbB−２遺伝子は腺癌患者において増幅さ
れる場合が有意に存在するので、この発明のモノクロー
ナル抗体を用いてヒトｃ−erbB−２遺伝子の発現をチェ
ックすることにより、腺癌の診断を行なうことができ
る。また、この発明のモノクローナル抗体をヒトｃ−er
bB−２遺伝子産物と抗原抗体反応により結合させてヒト
ｃ−erbB−２遺伝子産物を不活性化することにより、腺
癌の治療を行なうことも可能応である。[Effects of the Invention] According to the present invention, human c-erbB-2 is produced by a hybridoma obtained by fusing an antibody-producing cell of an animal with a cell expressing the cell surface on the cell surface as an immunogen and a myeloma cell, and A novel monoclonal antibody using the c-erbB-2 gene product as a corresponding antigen and a novel hybridoma producing the same have been provided. The monoclonal antibody of the present invention uses the product of human c-erbB-2 gene which is a proto-oncogene as a corresponding antigen, and as described above, the human c-erbB-2 gene is amplified in adenocarcinoma patients. Since the expression of the human c-erbB-2 gene is checked using the monoclonal antibody of the present invention, adenocarcinoma can be diagnosed. Further, the monoclonal antibody of the present invention is
It is also possible to treat adenocarcinoma by inactivating the human c-erbB-2 gene product by binding to the bB-2 gene product by an antigen-antibody reaction.
［発明の具体的説明］ 上述したように、この発明のモノクローナル抗体はヒ
トｃ−erbB−２遺伝子産物を対応抗原とするものであ
り、これと特異的に抗原抗体反応を行なう。この発明の
モノクローナル抗体は、従来単離されていない物質を対
応抗原とするものである。すなわち、本発明者らによっ
て新たに作製された細胞を免疫原として用いることによ
って初めて作製されたものであるのが、後述するよう
に、本発明のモノクローナル抗体はヒトｃ−erbB−２遺
伝子産物と特異的に抗原抗体反応を行なう。[Specific Description of the Invention] As described above, the monoclonal antibody of the present invention uses the human c-erbB-2 gene product as a corresponding antigen, and specifically performs an antigen-antibody reaction with the same. The monoclonal antibody of the present invention uses a substance which has not been isolated so far as a corresponding antigen. That is, the monoclonal antibody of the present invention, which was first produced by using the cells newly produced by the present inventors as an immunogen, as described below, is a human c-erbB-2 gene product. Perform antigen-antibody reaction specifically.
この発明のモノクローナル抗体は以下のようにして得
ることができる。The monoclonal antibody of the present invention can be obtained as follows.
先ず、ヒトｃ−erbB−２遺伝子をベクターに組込んだ
組換えDNAであって、動物細胞内で増殖することがで
き、かつ動物細胞を形質転換することができるものを調
製する。本発明者らは、ベクターとしてpBR322を選び、
このベクターに4409塩基対のヒトｃ−erbB−２遺伝子の
DNA（第38番目の塩基〜第4466番目の塩基）にHind III
リンカーを接続し、この前後にSV−40転写プロモーター
（SV ori領域270〜5171の塩基配列）とポリＡシグナル
部分を含むSV−40 DNAの1782〜2771と4100〜4710の塩基
配列を組込んだpSVerB2と呼ぶ組変換えDNAを構築するこ
とにより行なった。なお、pSVerB2遺伝子地図を第１図
に示す。First, a recombinant DNA prepared by incorporating a human c-erbB-2 gene into a vector and capable of growing in animal cells and transforming animal cells is prepared. We chose pBR322 as the vector,
This vector contains the 4409 base pair human c-erbB-2 gene.
Hind III in DNA (bases 38 to 4466)
A linker was connected, and before and after this, the SV-40 transcription promoter (the nucleotide sequence of the SV ori region 270 to 5171) and the nucleotide sequence of the SV-40 DNA containing the poly A signal portion, 1782 to 2771 and 4100 to 4710, were incorporated. This was done by constructing a pair-converted DNA called pSVerB2. The pSVerB2 gene map is shown in FIG.
次にこのようにして得られた組換えDNAを適当な動物
細胞、例えばマウス胎児由来の線維芽細胞に導入してそ
の動物細胞を形質転換し、ヒトｃ−erbB−２遺伝子を発
現している細胞を選択することにより、この発明のモノ
クローナル抗体を作製するための免疫原として用いるこ
とができ、ヒトｃ−erbB−２遺伝子をその細胞表面に発
現している細胞を得ることができる。本発明者らは、上
記pSVerB2を常法に従ってカルシウム沈殿法により、マ
ウス胎児由来の線維芽細胞NIH/3T3（ATCC株番号CRL−16
58）に導入し、スクリーニングによりSV−11株を樹立し
た。SV−11株は工業技術院微生物工業技術研究所（微工
研）に寄託され、その受託番号は微工研菌寄第10197号
である。Next, the recombinant DNA thus obtained is introduced into a suitable animal cell, for example, a fibroblast derived from a mouse embryo, and the animal cell is transformed to express the human c-erbB-2 gene. By selecting the cells, the cells can be used as an immunogen for preparing the monoclonal antibody of the present invention, and cells expressing the human c-erbB-2 gene on the cell surface can be obtained. The present inventors carried out a calcium precipitation method on the above pSVerB2 by a calcium precipitation method according to a conventional method to obtain mouse embryonic fibroblast NIH / 3T3 (ATCC strain number CRL-16).
58), and the SV-11 strain was established by screening. The SV-11 strain has been deposited with the Institute of Microbial Industry and Technology (MIC) of the National Institute of Advanced Industrial Science and Technology, and its accession number is No. 10197 of MIC.
次に、このようにして作製した細胞を免疫原として用
いて免疫した動物の抗体産生細胞とミエローマ細胞とを
常法により融合してハイブリドーマを作製し、ヒトｃ−
erbB−２遺伝子産物を対応抗原とするモノクローナル抗
体を産生しているハイブリドーマを選択する。この選択
は、例えば、常法に従って蛍光抗体法により細胞株SV−
11に陽性であり、かつSV−11の野生株であるNIH3T3に陰
性の抗体を産生しているハイブリドーマを選択すること
により行なうことができる。Next, antibody-producing cells of an animal immunized using the cells thus prepared as an immunogen and myeloma cells were fused by a conventional method to prepare a hybridoma, and human c-
A hybridoma producing a monoclonal antibody having the erbB-2 gene product as a corresponding antigen is selected. This selection can be performed, for example, by a fluorescent antibody method using a cell line SV-
This can be performed by selecting hybridomas that are positive for No. 11 and that produce antibodies negative for NIH3T3, a wild strain of SV-11.
この発明のモノクローナル抗体はこのようにして得ら
れたハイブリドーマを培養し、その培養上清から回収す
ることができる。The monoclonal antibody of the present invention can be recovered from the culture supernatant by culturing the hybridoma thus obtained.
［実施例］ 以下、この発明を実施例に基きより具体的に説明す
る。なお、この発明は下記実施例に限定されるものでは
ない。EXAMPLES Hereinafter, the present invention will be described more specifically based on examples. The present invention is not limited to the following embodiments.
実施例１ モノクローナル抗体産生ハイブリドーマの調製 （ｉ）SV−11株の作製 ヒトｃ−erbB−２遺伝子を高発現する細胞株を得るた
めには、本発明者らは、動物細胞発現ベクターpSV2を選
び、このベクターにｃ−erbB−２遺伝子を組み込んだDN
Aを調製した。このベクターはSV40ウイルスの転写プロ
モーター（SV40 ori領域270〜5171塩基配列）とポリＡ
シグナル部位（SV40 DNAの1782〜2771と4100〜4710の塩
基配列）を有し、これにヒトｃ−erbB−２遺伝子のcDNA
からSma I及びAha3で切り出してきたｃ−erbB−２遺伝
子断片にHind IIIリンカーを接続したものを転写プロモ
ーターの下流のHind III部位に組み込むことにより構築
した。このベクターをpSVerbB−２と呼び、その遺伝子
地図を第１図に示した。Example 1 Preparation of Monoclonal Antibody-Producing Hybridoma (i) Preparation of SV-11 Strain In order to obtain a cell line that highly expresses the human c-erbB-2 gene, the present inventors selected the animal cell expression vector pSV2. , A DN having the c-erbB-2 gene incorporated into this vector.
A was prepared. This vector contains the SV40 virus transcription promoter (SV40 ori region 270-5171 nucleotide sequence) and polyA
It has a signal site (base sequence of 1782 to 2771 and 4100 to 4710 of SV40 DNA), and contains a cDNA of human c-erbB-2 gene.
Was constructed by incorporating a HindIII linker connected to a c-erbB-2 gene fragment cut out from SmaI and Aha3 into a HindIII site downstream of a transcription promoter. This vector is called pSVerbB-2, and its genetic map is shown in FIG.
次に、得られた組換えDNA pSVerbB−２をマウス胎児
由来の線維芽細胞NIH3T3（ATCC株番号CRL−1658）細胞
にリン酸カルシウム法を用いて導入して形質転換細胞を
得た。形質転換NIH3T3細胞からDNAハイブリダイゼーシ
ョン法でヒトｃ−erbB−２遺伝子発現株をスクリーニン
グすることによりSV−11株（微工研菌寄10197号）を樹
立した。Next, the obtained recombinant DNA pSVerbB-2 was introduced into NIH3T3 mouse embryo-derived fibroblasts (ATCC strain number CRL-1658) using the calcium phosphate method to obtain transformed cells. By screening a human c-erbB-2 gene-expressing strain from the transformed NIH3T3 cells by a DNA hybridization method, an SV-11 strain (Microtechnical Laboratories No. 10197) was established.
（ii）マウスの感作 上記細胞株SV−11を10％ウシ胎児血清添加DME培地で
培養し、0.05％EDTAで細胞をはがし取り、リン酸緩衝食
塩水で５回洗浄し、遠心分離（1000rpm x ５分）で細
胞を集め、細胞数を調製したものを用いた。(Ii) Mouse sensitization The above cell line SV-11 was cultured in a DME medium supplemented with 10% fetal bovine serum, the cells were detached with 0.05% EDTA, washed five times with phosphate buffered saline, and centrifuged (1000 rpm). x 5 minutes), and the cells were used for preparing the number of cells.
免疫操作は４週令のBALB/c雄マウスの尾静脈に1 x 10
7個の細胞株SV−11を静脈注射し、その後２週間の間隔
で、２〜４回尾静脈又は腹腔内に1 x 107個の細胞株SV
−11を追加免疫した。免疫の過程で、免疫原に対する抗
体価の上昇を追跡した。Immunization was performed at 1 x 10 in the tail vein of 4-week-old BALB / c male mice.
Seven cell lines SV-11 were injected intravenously and then 2 to 4 times at 2 week intervals in the tail vein or intraperitoneally 1 × 10 7 cell lines SV-11.
-11 was boosted. During the course of the immunization, the increase in the antibody titer against the immunogen was followed.
（iii）細胞融合 最終免疫より３日後に抗体価の高いマウスから脾細胞
を無菌的に取り出し、ステンレスメッシュで単細胞にほ
ぐし、脾細胞の1/10量の８−アザグアニン耐性骨髄腫細
胞Ｘ−63（Kohler G.とMilstein C,Nature 256,pp.495
−497（1975）、ATCCより入手）を混合し、洗浄塩沈
後、細胞のペレットに1mlの50％ポリエチレングリコー
ル（平均分子量1500）を加えて注意深く細胞融合操作を
行なった。(Iii) Cell fusion Three days after the final immunization, spleen cells were aseptically removed from the mouse with a high antibody titer, disintegrated into single cells with a stainless mesh, and 1/10 the amount of 8-azaguanine-resistant myeloma cells X-63 of the spleen cells. (Kohler G. and Milstein C, Nature 256 , pp.495
After washing and salt precipitation, 1 ml of 50% polyethylene glycol (average molecular weight 1500) was added to the cell pellet, and cell fusion was carefully performed.
その後融合細胞96穴マイクロプレートに１×106個の
割合で0.1mlづつ分注した。各マイクロプレートは５％C
O2、37℃（100％ RH）のインキュベーターで無血清培地
選択法で注意深く培養を続け、ハイブリドーマを得た。Thereafter, 0.1 ml was dispensed into a 96-well microplate of fused cells at a ratio of 1 × 10 6 cells. Each microplate is 5% C
The culture was carefully continued in a serum-free medium selection method in an incubator of O 2 and 37 ° C. (100% RH) to obtain a hybridoma.
（iv）このようにして選ばれたハイブリドーマは直ちに
限界希釈法によりクローニングを繰り返した。５回のク
ローニングにより安定したハイブリドーマの培養上清を
ヒト正常末梢血中の細胞と反応させ、リンパ球、単球と
それぞれ陰性のクローンを選びSV2−61及びSV2−61γと
命名した。モノクローナル抗体SV2−61及びSV2−61γを
産正するハイブリドーマ（ハイブリドーマSV2−61及び
ハイブリドーマSV2−61γ）はBALB/cマウスを用いてプ
レステンによる常法による腹水を作製した。ハイブリド
ーマSV2−61及びハイブリドーマSV2−61γは微工研に寄
託し、その受託番号はそれぞれ微工研菌寄第10162号及
び微工研菌寄第10777号である。(Iv) Cloning of the hybridomas selected in this manner was immediately repeated by the limiting dilution method. The culture supernatant of the hybridoma stabilized by cloning five times was reacted with cells in human normal peripheral blood, and clones negative for lymphocytes and monocytes were selected and named SV2-61 and SV2-61γ. Hybridomas producing monoclonal antibodies SV2-61 and SV2-61γ (hybridoma SV2-61 and hybridoma SV2-61γ) produced ascites by the usual method using Presten using BALB / c mice. The hybridoma SV2-61 and the hybridoma SV2-61γ are deposited with the Japan Institute of Micro-Technology, and their accession numbers are Micro-Technical Laboratories No. 10162 and No. 10777, respectively.
実施例２ モノクローナル抗体SV2−61及びSV2−61γの特徴づけ （ｉ）免疫グロブリンのサブクラス モノクローナル抗体SV2−61及びSV2−61γの免疫グロ
ブリンのサブクラスはオクタロニー法によりそれぞれマ
ウスIgM、マウスIgGと決定された。Example 2 Characterization of Monoclonal Antibodies SV2-61 and SV2-61γ (i) Immunoglobulin Subclass The immunoglobulin subclasses of monoclonal antibodies SV2-61 and SV2-61γ were determined to be mouse IgM and mouse IgG by the Ouchterlony method, respectively. .
（ii）特異性の決定 上記のようにして得られたモノクローナル抗体SV2−6
1及びSV2−61γは免疫原SV−11に陽性であり、野生株NI
H3T3に陰性であった。これは、具体的には以下のように
して試験した。すなわち、マイクロプレートで予め培養
した2 x 103個のSV−11細胞をマイクロプレートに付着
させ、フォルマリンPBSで固定した。次いで細胞に培養
上清を20μｌ加え、マイクロプレート上で常法に従い蛍
光抗体法を行ない、陽性ハイブリドーマをスクリーニン
グした。陽性細胞はさらに106個の細胞に対して常法に
より蛍光抗体法でフローサイトメトリー測定を行なっ
た。結果は第２図及び第３図のフローサイトグラムに示
されている。第２図は、モノクローナル抗体SV2−61に
ついての結果を示し、第３図はモノクローナル抗体SV2
−61γについての結果を示す。なお、第２図及び第３図
中、Ｎは陰性ピークを、Ｐは陽性ピークを示す。(Ii) Determination of specificity Monoclonal antibody SV2-6 obtained as described above
1 and SV2-61γ are positive for the immunogen SV-11 and the wild strain NI
H3T3 was negative. This was specifically tested as follows. That is, 2 × 10 3 SV-11 cells cultured in advance on a microplate were attached to the microplate and fixed with formalin PBS. Next, 20 μl of the culture supernatant was added to the cells, and a fluorescent antibody method was performed on a microplate according to a conventional method to screen for positive hybridomas. Positive cells were subjected to flow cytometry measurement on 10 6 cells by a fluorescent antibody method according to a conventional method. The results are shown in the flow cytograms of FIGS. 2 and 3. FIG. 2 shows the results for monoclonal antibody SV2-61, and FIG. 3 shows the results for monoclonal antibody SV2-61.
The results for -61γ are shown. 2 and 3, N indicates a negative peak and P indicates a positive peak.
同様にして、胃癌組織から樹立され、erb−B2が約30
コピー発現していることが知られている樹立細胞株MKN
−７（文献:S.Fukushigeら、Molecular and Cellular B
iology,Mar.955（1986）、東京大学医化学研究所制癌部
より入手）でも陽性であった。Similarly, erb-B2 was established from gastric cancer tissue,
Established cell line MKN known to express copy
-7 (Literature: S. Fukushige et al., Molecular and Cellular B
iology, Mar. 955 (1986), obtained from the University of Tokyo Institute of Medical Science, Department of Cancer Research).
（iii）抗原の分子量 抗原の分子量は、免疫沈降と電気泳動を組み合わせた
方法により決定した。用いた細胞は、NIH3T3、SV−11、
ｃ−erbB−２遺伝子発現株であるSV227及び上記MKN−７
細胞であった。6cmシャーレ中でコンフルーエント状態
にある細胞を、メチオニンフリーのDMEM及び100μCiの
35S−メチニオンで４時間培養することによって細胞を
35S−メチニオンで標識し、これをRIPAバッファーで可
溶可した。これにモノクローナル抗体SV2−61又はSV2−
61γμｇを加え、氷冷下で反応させた後、プロテインＡ
−セファロース（登録商標）で沈降させた。電気泳動は
８％ポリアクリルアミドゲルで、30mAで約２時間泳動
し、ゲルを乾燥した後、オートラジオグラフィーを行な
った。(Iii) Molecular weight of antigen The molecular weight of the antigen was determined by a method combining immunoprecipitation and electrophoresis. The cells used were NIH3T3, SV-11,
SV227 which is a c-erbB-2 gene expression strain and the above MKN-7
Cells. Cells in a confluent state in a 6 cm Petri dish were washed with methionine-free DMEM and 100 μCi.
Cells are cultured for 4 hours in 35 S-methinion to
Labeled with 35 S-methinion, which was soluble in RIPA buffer. To this, the monoclonal antibody SV2-61 or SV2-
After adding 61γμg and reacting under ice cooling, protein A was added.
-Sedimentation on Sepharose®. Electrophoresis was performed on an 8% polyacrylamide gel at 30 mA for about 2 hours, and the gel was dried, followed by autoradiography.
その結果、モノクローナル抗体SV2−61又はSV2−61γ
と抗原抗体反応するタンパク質は分子量185kDの位置に
一本のバンドとして確認された。すなわち、MKN−７、S
V227及びSV−11細胞では185kDの位置にメインバンドが
見られた。これはヒトｃ−erbB−２遺伝子の塩基配列か
ら予測される抗原の分子量と一致していた。一方、陰性
コントロールとして、NRS正常ウサギ血清で同様の操作
を行なったが、185kDにバンドが見られなかった。As a result, the monoclonal antibody SV2-61 or SV2-61γ
A protein that reacted with the antigen and antibody was confirmed as a single band at a position of a molecular weight of 185 kD. That is, MKN-7, S
In V227 and SV-11 cells, a main band was observed at a position of 185 kD. This was consistent with the molecular weight of the antigen predicted from the nucleotide sequence of the human c-erbB-2 gene. On the other hand, the same operation was performed using NRS normal rabbit serum as a negative control, but no band was observed at 185 kD.
（iv）チロシンキナーゼ活性 SV−11細胞をモノクローナル抗体SV2−61で免疫沈降
したところ分子量185kDの位置にチロシンキナーゼ活性
が確認された。このことよりモノクローナル抗体SV2−6
1がｃ−erbB−２遺伝子産物を抗原として反応している
ことが示された。(Iv) Tyrosine kinase activity Immunoprecipitation of SV-11 cells with the monoclonal antibody SV2-61 revealed tyrosine kinase activity at a position of 185 kD molecular weight. This indicates that the monoclonal antibody SV2-6
It was shown that 1 reacted with the c-erbB-2 gene product as an antigen.
実施例３ 腺癌の診断 各種癌患者の外科手術時に摘出された腫瘍組織を本発
明のモノクローナル抗体SV2−61を用いる組織染色法
（ホルマリン固定パラフィン切片利用）で染色した結果
を表に示す。表より、モノクローナル抗体SV2−61は腺
癌と特異的に反応しており、腺癌の診断に優れた性能を
有するモノクローナル抗体であることがわかる。Example 3 Diagnosis of Adenocarcinoma The results of staining of tumor tissue excised during surgery on various cancer patients by the tissue staining method (using formalin-fixed paraffin sections) using the monoclonal antibody SV2-61 of the present invention are shown in the table. The table shows that the monoclonal antibody SV2-61 specifically reacts with adenocarcinoma and is a monoclonal antibody having excellent performance in diagnosing adenocarcinoma.
第１図は、この発明のモノクローナル抗体を作製するた
めに用いた組換え体DNAであるpSVerbB2の遺伝子地図、 第２図は、この発明のモノクローナル抗体SV2−61の反
応性を示すフローサイトグラム、 第３図は、この発明のモノクローナル抗体SV2−61γの
反応性を示すフローサイトグラムである。FIG. 1 is a genetic map of pSVerbB2, which is a recombinant DNA used to prepare the monoclonal antibody of the present invention. FIG. 2 is a flow cytogram showing the reactivity of the monoclonal antibody SV2-61 of the present invention. FIG. 3 is a flow cytogram showing the reactivity of the monoclonal antibody SV2-61γ of the present invention.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.6 識別記号 ＦＩ Ｇ０１Ｎ 33/574 Ｃ１２Ｎ 5/00 Ｂ 33/577 Ｃ１２Ｎ 15/00 Ｃ (Ｃ１２Ｐ 21/08 Ｃ１２Ｒ 1:91） (72)発明者 益子 高 宮城県仙台市太白区太白２丁目９―３― 202 (72)発明者 白石 真人 東京都板橋区小茂根４―24―９ (72)発明者 平川 忠 神奈川県川崎市川崎区鈴木町１番１号 味の素株式会社中央研究所内 (72)発明者 房木 ノエミ 東京都杉並区善福寺２―14―24 (56)参考文献 Ｓｃｉｅｎｃｅ，232 ［4756］ （1986） Ｐ．1644−1646──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. 6 Identification code FI G01N 33/574 C12N 5/00 B 33/577 C12N 15/00 C (C12P 21/08 C12R 1:91) (72) Inventor Mashiko Taka 2-3-9-202, Taishiro-ku, Taishiro-ku, Sendai City, Miyagi Prefecture (72) Inventor Masato Shiraishi 4-24-9, Omone, Itabashi-ku, Tokyo (72) Inventor Tadashi Hirakawa, Suzukicho, Kawasaki-ku, Kawasaki-shi, Kanagawa No. 1 Inside the Central Research Laboratory of Ajinomoto Co., Inc. (72) Inventor Noemi Fugi 2-14-24 Zenfukuji, Suginami-ku, Tokyo (56) References Science, 232 [4756] (1986) 1644-1646
Claims (8)
に発現する細胞を免疫原とした動物の抗体産生細胞とミ
エローマ細胞と融合して得られたハイブリドーマによっ
て産生され、ヒト癌原遺伝子erbB−２産物を対応抗原と
するモノクローナル抗体。1. A human proto-oncogene which is produced by a hybridoma obtained by fusing an antibody-producing cell of an animal using cells expressing the human proto-oncogene erbB-2 product on the cell surface as an immunogen and myeloma cells, A monoclonal antibody having an erbB-2 product as a corresponding antigen.
がNIH3T3細胞とは反応しない請求項１記載のモノクロー
ナル抗体。2. The monoclonal antibody according to claim 1, which belongs to the IgM subgroup and reacts with SV-11 cells but does not react with NIH3T3 cells.
がNIH3T3細胞とは反応しない請求項１記載のモノクロー
ナル抗体。3. The monoclonal antibody according to claim 1, which belongs to the IgG subgroup and reacts with SV-11 cells but does not react with NIH3T3 cells.
れたハイブリドーマにより産生される請求項２記載のモ
ノクローナル抗体。4. The monoclonal antibody according to claim 2, wherein the monoclonal antibody is produced by a hybridoma deposited under Accession No.
れたハイブリドーマにより産生される請求項３記載のモ
ノクローナル抗体。5. The monoclonal antibody according to claim 3, wherein the monoclonal antibody is produced by a hybridoma deposited under Accession No. No. 10777 of Microbiken Bacteria.
れたモノクローナル抗体を産生するハイブリドーマ。6. A hybridoma which produces the monoclonal antibody according to any one of claims 1 to 5.
れた請求項６記載のハイブリドーマ。7. The hybridoma according to claim 6, which has been deposited under the accession number of No. 10162 of the microbe laboratory.
れた請求項６記載のハイブリドーマ。8. The hybridoma according to claim 6, wherein the hybridoma has been deposited under the accession number No. 10777 of Microbiological Research Laboratories.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63-204207 | 1988-08-17 | ||
| JP20420788 | 1988-08-17 |
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| Publication Number | Publication Date |
|---|---|
| JPH02150293A JPH02150293A (en) | 1990-06-08 |
| JP2761543B2 true JP2761543B2 (en) | 1998-06-04 |
Family
ID=16486606
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1177392A Expired - Lifetime JP2761543B2 (en) | 1988-08-17 | 1989-07-10 | Monoclonal antibody against human proto-oncogene product and hybridoma producing the same |
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| US6821515B1 (en) | 1995-07-27 | 2004-11-23 | Genentech, Inc. | Protein formulation |
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|---|---|---|---|---|
| AU662311B2 (en) | 1991-02-05 | 1995-08-31 | Novartis Ag | Recombinant antibodies specific for a growth factor receptor |
-
1989
- 1989-07-10 JP JP1177392A patent/JP2761543B2/en not_active Expired - Lifetime
Non-Patent Citations (1)
| Title |
|---|
| Ｓｃｉｅｎｃｅ，２３２ ［４７５６］ （１９８６） Ｐ．１６４４−１６４６ |
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|---|---|
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