JP2521496B2 - Anti-human cancer monoclonal antibody - Google Patents
Anti-human cancer monoclonal antibodyInfo
- Publication number
- JP2521496B2 JP2521496B2 JP62260498A JP26049887A JP2521496B2 JP 2521496 B2 JP2521496 B2 JP 2521496B2 JP 62260498 A JP62260498 A JP 62260498A JP 26049887 A JP26049887 A JP 26049887A JP 2521496 B2 JP2521496 B2 JP 2521496B2
- Authority
- JP
- Japan
- Prior art keywords
- human
- cancer
- lung adenocarcinoma
- monoclonal antibody
- human lung
- 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.)
- Expired - Fee Related
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は抗ヒト癌モノクローナル抗体、その製造法並
びにこれを利用したヒト癌診断薬に関する。TECHNICAL FIELD The present invention relates to an anti-human cancer monoclonal antibody, a method for producing the same, and a human cancer diagnostic agent using the same.
1975年、ケーラーとミルスタイン（Ｋhler,G.and M
ilstein,C.）は、マウスミエローマP3Kの誘導変異株で
あるP3X63−Ag8（HGPRT欠損株）と、抗ヒツジ赤血球マ
ウス抗体産生細胞よりハイブリドーマを作製し、このハ
イブリドーマが自立増殖性と抗ヒツジ赤血球マウス抗体
産生能を共に有していることを報告した。1979年Koprow
skiらは、悪性腫瘍に対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し、モノクローナル抗体の試
薬としての有用性を示すと共に治療薬としての可能性を
示唆して以来、特に腫瘍関連抗原に対するモノクローナ
ル抗体については、この技法が注目を浴びている。1975, Khler, G.and M
ilstein, C.) is a P 3 X 63 -Ag8 is induced mutant mouse myeloma P 3 K (HGPRT-deficient strain), to produce hybridomas from the anti-sheep erythrocyte mouse antibody-producing cells, the hybridoma autonomous proliferative It was also reported that they have the ability to produce anti-sheep erythrocyte mouse antibody. 1979 Koprow
Since ski et al. produced hybridomas that produce monoclonal antibodies against malignant tumors, showing the utility of monoclonal antibodies as reagents and suggesting their potential as therapeutic agents, especially regarding monoclonal antibodies against tumor-associated antigens, This technique is in the spotlight.
しかしながら、ヒト癌には極めて多くの種類があるこ
とから、その抗原性もまた多様であり、特定の癌抗原に
対して特異的に反応するモノクローナル抗体を創製する
ことは、特定の癌抗原を検出することすなわちヒト癌診
断において極めて重要である。However, since there are so many kinds of human cancers, their antigenicity is also diverse, and it is necessary to detect a specific cancer antigen by creating a monoclonal antibody that specifically reacts with the specific cancer antigen. That is, it is extremely important in human cancer diagnosis.
斯かる実状において本発明者らは、ヒト肺腺癌由来細
胞株を用い、細胞融合により該細胞株に特異的なモノク
ローナル抗体を産生するハイブリドーマを得、このハイ
ブリドーマを利用すればヒド肺腺癌由来細胞株に特異的
なモノクローナル抗体が得られること、さらに該モノク
ローナル抗体の標識体を利用すればヒト癌診断が極めて
的確かつ容易になることを見い出し、本発明を完成し
た。In such an actual situation, the present inventors have used a human lung adenocarcinoma-derived cell line to obtain a hybridoma that produces a monoclonal antibody specific to the cell line by cell fusion. It was found that a monoclonal antibody specific to a cell line can be obtained, and that the use of a labeled form of the monoclonal antibody makes diagnosis of human cancer extremely accurate and easy, and completed the present invention.
すなわち、本発明は下記の性質を有するヒト肺腺癌由
来細胞株PC−９に対するモノクローナル抗体；その製造
法；及び酵素若しくは放射性同位元素で標識された当該
モノクローナル抗体を含有するヒト癌診断薬を提供する
ものである。That is, the present invention provides a monoclonal antibody against human lung adenocarcinoma-derived cell line PC-9 having the following properties; a method for producing the same; and a human cancer diagnostic agent containing the monoclonal antibody labeled with an enzyme or a radioisotope. To do.
（１）ヒト肺腺癌由来細胞株PC−９と反応する。(1) Reacts with human lung adenocarcinoma-derived cell line PC-9.
（２）ヒト肺腺癌Ruweller、ヒト肺腺癌PC−３、ヒト肺
偏平上皮癌AOI、ヒト胃癌Kato−III、ヒト大腸癌Lovo、
ヒト前立腺癌PC−３（ATCC）、ヒト腎癌RCC−JA、ヒト
子宮頚癌Hela、ヒト悪性黒色腫MI及びヒト膀胱癌Ｔ−24
と反応しない。(2) Human lung adenocarcinoma Ruweller, human lung adenocarcinoma PC-3, human lung squamous cell carcinoma AOI, human gastric cancer Kato-III, human colon cancer Lovo,
Human Prostate Cancer PC-3 (ATCC), Human Renal Cancer RCC-JA, Human Cervical Cancer Hela, Human Malignant Melanoma MI and Human Bladder Cancer T-24
Does not react with.
（３）ヒト末梢白血球と反応しない。(3) Does not react with human peripheral leukocytes.
本発明のモノクローナル抗体は、ヒト肺腺癌由来細胞
株PC−９で免疫した動物から採取した抗体産生細胞と骨
髄腫細胞の細胞融合によって作製したハイブリドーマを
培地上で培養するか、又は動物腹腔内に投与して腹水中
に蓄積せしめ、該培養物又は腹水から採取することによ
り製造される。The monoclonal antibody of the present invention is prepared by culturing a hybridoma prepared by cell fusion of antibody-producing cells and myeloma cells collected from an animal immunized with human lung adenocarcinoma-derived cell line PC-9 on a medium, or intraperitoneally in the animal. It is produced by collecting the culture or ascites from the culture or the ascites.
本発明のモノクローナル抗体を産生するハイブリドー
マは、いわゆる細胞融合によって製造される。すなわ
ち、抗原としてヒト肺腺癌由来細胞株PC−９を用いて免
疫した動物から抗体産生細胞を調製し、これを骨髄腫細
胞と融合させ、得られたハイブリドーマを選択的に増殖
させ、該ハイブリドーマから抗体産生ハイブリドーマを
検索し、クローニングにより目的とするモノクローナル
抗体産生ハイブリドーマを得る。The hybridoma producing the monoclonal antibody of the present invention is produced by so-called cell fusion. That is, antibody-producing cells were prepared from an animal immunized with the human lung adenocarcinoma-derived cell line PC-9 as an antigen, fused with myeloma cells, and the obtained hybridoma was selectively proliferated. The desired antibody-producing hybridoma is obtained by cloning the antibody-producing hybridoma.
抗体産生細胞としては、例えばヒト肺腺癌由来細胞株
PC−９又はこれから得られる抗原を免疫させた動物から
得られる脾臓細胞、リンパ節細胞、Ｂ−リンパ球が挙げ
られる。免疫させる動物としてはマウス、ラット、ウサ
ギ、ヤギ、馬などが挙げられる。免疫は、例えばヒト肺
腺癌由来細胞株PC−９をそのまま動物の皮下、筋肉内あ
るいは腹腔内に約107個相当分／回を月１〜２回、２〜
４ケ月間投与することにより行われる。抗体産生細胞の
分離は、最終免疫から２〜４日後免疫動物から分取する
ことにより行われる。Examples of antibody-producing cells include human lung adenocarcinoma-derived cell lines.
Examples thereof include spleen cells, lymph node cells and B-lymphocytes obtained from animals immunized with PC-9 or an antigen obtained therefrom. Examples of animals to be immunized include mice, rats, rabbits, goats, horses and the like. For immunization, for example, the human lung adenocarcinoma-derived cell line PC-9 is subcutaneously, intramuscularly, or intraperitoneally administered to an animal as much as about 10 7 cells / dose once or twice a month, or 2 times a month.
It is performed by administering for 4 months. Separation of antibody-producing cells is performed by separating the immunized animals 2 to 4 days after the final immunization.
骨髄腫細胞としては、マウス、ラット、ヒト等由来の
ものが使用できる。抗体産生細胞と骨髄腫細胞とは同種
動物由来であることが好ましい。As the myeloma cells, those derived from mouse, rat, human and the like can be used. The antibody-producing cells and myeloma cells are preferably derived from the same species of animal.
細胞融合は、例えばケーターとミルスタインの方法
〔Ｋhler,G.and Milstein,C.,Nature,256,495（197
5）〕又はこれに準ずる方法によって行われる。すなわ
ち、RPMI1640培地などの培地中で抗体産生細胞と骨髄腫
細胞とを混合することにより行われる。このときポリエ
チレングリコール等の融合促進剤を添加することが好ま
しい。Cell fusion is performed by, for example, the method of Kater and Milstein [Khler, G. and Milstein, C., Nature, 256 , 495 (197
5)] or a method equivalent thereto. That is, it is performed by mixing antibody-producing cells and myeloma cells in a medium such as RPMI1640 medium. At this time, it is preferable to add a fusion promoter such as polyethylene glycol.
細胞融合終了後、RPMI1640培地等で適当に希釈し、遠
心分離し、沈渣をHAT培地等の選択培地に懸濁し、培養
することによりハイブリドーマを選択する。次いで培養
上清を用いて酵素抗体法により抗体産生ハイブリドーマ
を検索し、限界希釈法等によりクローニングを行い、本
発明モノクローナル抗体を産生するハイブリドーマを得
る。After completion of cell fusion, a hybridoma is selected by appropriately diluting with RPMI1640 medium or the like, centrifuging, suspending the precipitate in a selective medium such as HAT medium, and culturing. Next, the culture supernatant is used to search for antibody-producing hybridomas by the enzyme antibody method, and cloning is performed by the limiting dilution method or the like to obtain hybridomas that produce the monoclonal antibody of the present invention.
斯くして得られた抗体産生ハイブリドーマを利用して
本発明モノクローナル抗体を製造するには、該ハイブリ
ドーマが本発明モノクローナル抗体産生能を有している
ので、これを適当な培地上又は生体内で培養し、該培養
物から採取することによって行われる。就中、大量に製
造するには、該ハイブリドーマを骨髄腫細胞の由来細胞
と同種の動物の腹腔内に投与し、その腹水中に本発明モ
ノクローナル抗体を蓄積せしめ、該腹水から採取する方
法が好ましい。抗体産生ハイブリドーマの投与に先だっ
てプリステン等の鉱物油を投与するのが好ましい。In order to produce the monoclonal antibody of the present invention using the antibody-producing hybridoma thus obtained, since the hybridoma has the ability to produce the monoclonal antibody of the present invention, it is cultured in an appropriate medium or in vivo. And then harvested from the culture. In particular, for large-scale production, it is preferable to administer the hybridoma intraperitoneally to an animal of the same species as the myeloma cell-derived cells, allow the monoclonal antibody of the present invention to accumulate in the ascites, and collect from the ascites. . Prior to administration of the antibody-producing hybridoma, it is preferable to administer mineral oil such as pristine.
培養物又は腹水からの本発明モノクローナル抗体の採
取は、IgG精製に通常使用される硫安分画法、陰イオン
交換体もしくはプロテインＡカラムによるクロマトグラ
フィーによって実施される。Collection of the monoclonal antibody of the present invention from the culture or ascites is carried out by the ammonium sulfate fractionation method commonly used for IgG purification, or by chromatography on an anion exchanger or a protein A column.
斯くして得られる本発明モノクローナル抗体は、これ
を産生するハイブリドーマの種類により130−22および1
45−９の二種類存在する。これらのモノクローナル抗体
は、いずれもグロブリンクラスがIgG1であり、抗原であ
るPC−９細胞に特異的に反応し、他の癌細胞および白血
球とは反応しない。またPC−９細胞に対する結合親和定
数は130−22が145−９より高い。さらに本発明モノクロ
ーナル抗体は、正常ヒト組織とは反応せず、肺腺癌組織
および卵巣癌組織とのみ反応し、他の癌組織とも反応し
ない。The thus obtained monoclonal antibody of the present invention is 130-22 and 1 depending on the type of hybridoma producing it.
There are two types, 45-9. These monoclonal antibodies are all immunoglobulin classes is IgG 1, and specifically reacts with PC-9 cells an antigen and does not react with other cancer cells and leukocytes. The binding affinity constant for PC-9 cells of 130-22 is higher than that of 145-9. Furthermore, the monoclonal antibody of the present invention does not react with normal human tissues, only with lung adenocarcinoma tissues and ovarian cancer tissues, and does not react with other cancer tissues.
本発明モノクローナル抗体は、肺腺癌由来細胞と特異
的に反応するので、これを酵素、放射性同位元素、蛍光
物質で標識すれば癌診断薬として利用できる。Since the monoclonal antibody of the present invention specifically reacts with cells derived from lung adenocarcinoma, it can be used as a cancer diagnostic agent if it is labeled with an enzyme, a radioisotope or a fluorescent substance.
標識に用いられる酵素としては、パーオキシダーゼ、
アルカリフォスファターゼ、β−ガラクトシダーゼ等が
挙げられ、放射性同位元素としては、125I、131I、111I
n、99mTc、123I等が挙げられる。これらの標識物質によ
る本発明モノクローナル抗体の標識は常法に従って行わ
れる。The enzyme used for labeling is peroxidase,
Alkaline phosphatase, β-galactosidase and the like can be mentioned, and as the radioisotope, 125 I, 131 I, 111 I
n, 99m Tc, 123 I and the like. Labeling of the monoclonal antibody of the present invention with these labeling substances is carried out according to a conventional method.
本発明癌診断薬を用いて癌を診断するには、インビト
ロの方法として、本発明モノクローナル抗体標識体を利
用した種々の免疫測定法、例えば競合法やサンドイッチ
法による凝集反応や凝集阻止反応により抗原を定量また
は定性することができる。ここで、免疫測定法の実施に
あたっては、ポリスチレンビーズ等の担体に抗体を固定
化した固相化抗体を利用し、免疫反応を該固相化抗体上
で行わせることもできる。次にインビボの方法として、
本発明モノクローナル抗体に123I、131I、111In、99mTc
等の放射性同位元素を標識して、肺癌や卵巣癌等の患者
に投与すると、本発明モノクローナル抗体標識体は患者
の癌組織に集積する。そこで、患者の外部よりシンチカ
メラ等で、放射線を計測することにより癌の位置や大き
さを撮像することができる。To diagnose cancer using the cancer diagnostic agent of the present invention, as an in vitro method, various immunoassays using the labeled monoclonal antibody of the present invention, for example, an antigen by an agglutination reaction or an aggregation inhibition reaction by a competitive method or a sandwich method. Can be quantitative or qualitative. Here, in carrying out the immunoassay, it is also possible to use a solid-phased antibody in which the antibody is immobilized on a carrier such as polystyrene beads, and to carry out an immune reaction on the solid-phased antibody. Then as an in vivo method,
123 I, 131 I, 111 In, 99m Tc
When labeled with a radioactive isotope such as, and administered to a patient such as lung cancer or ovarian cancer, the monoclonal antibody-labeled product of the present invention accumulates in the cancer tissue of the patient. Therefore, the position and size of cancer can be imaged by measuring radiation from outside the patient with a scintillation camera or the like.
このように、本発明モノクローナル抗体標識体は、イ
ンビトロおよびインビボの方法により癌の診断が可能と
なる。As described above, the labeled monoclonal antibody of the present invention enables cancer diagnosis by in vitro and in vivo methods.
本発明モノクローナル抗体は肺前癌細胞膜に、存在す
る糖蛋白に特異的と考えられ、肺腺癌を認識し、かつ正
常組織由来培養細胞、白血球、赤血球及び正常組織とは
反応せず、癌特異な表面抗原を認識するものと推定され
る。INDUSTRIAL APPLICABILITY The monoclonal antibody of the present invention is considered to be specific to the glycoprotein present in the lung precancerous cell membrane, recognizes lung adenocarcinoma, and does not react with cultured cells derived from normal tissues, leukocytes, erythrocytes and normal tissues, and is cancer specific. It is presumed that it recognizes various surface antigens.
従って本発明癌診断薬を使用することにより、癌の早
期発見が可能になるとともに、癌の進行、治癒経過を的
確に把握することができる。Therefore, by using the cancer diagnostic agent of the present invention, it is possible to detect cancer at an early stage, and it is possible to accurately grasp the progress and healing process of cancer.
次に、実施例を挙げて本発明を詳細に説明する。 Next, the present invention will be described in detail with reference to examples.
実施例1.ハイブリドーマの作製 ヒト肺腺癌由来細胞株PC−９をそのままBalb/cマウス
の腹腔内に投与した。免疫はヒト肺腺癌由来細胞株PC−
９約107個相当分／回を月１回、２ケ月間投与、最終免
疫の３日後に脾臓を摘出し、脾臓細胞浮遊液を調製し
た。融合の親細胞として、マウス骨髄腫細胞（NS−１）
を培養して準備した。Example 1. Preparation of hybridoma Human lung adenocarcinoma-derived cell line PC-9 was intraperitoneally administered to Balb / c mice as it was. Immunity is derived from human lung adenocarcinoma cell line PC-
9 about 10 7 equivalent / dose once a month, two months administration, the spleen was removed 3 days after the final immunization, was prepared spleen cell suspension. Mouse myeloma cells (NS-1) as fusion parent cells
Were cultured and prepared.
上記で調製したマウス脾臓細胞を10:1の割合で混合し
1,300rpmで５分間遠心分離した。得られた沈渣を50％ポ
リエチレングリコール（4,000）を含むRPMI1640培地1ml
に浮遊させ、１分間放置した。次いで、RPMI1640倍地10
mlを徐々に加えて希釈後、900rpmで３分間遠心した。次
いで、得られた細胞をHAT培地50mlに懸濁し、0.1mlずつ
を96ウェルの組織培養プレートに分注した。この状態で
１週間培養すると、90〜95％程度の穴にコロニーが認め
られた。ある程度コロニーが生育したのちに（10〜13日
後）培養上清を用いて、抗体価を酵素抗体法により測定
し、適切なウェルから限界希釈法により、求めるハイブ
リドーマのクローニングを行った。The mouse spleen cells prepared above were mixed at a ratio of 10: 1.
Centrifuge at 1,300 rpm for 5 minutes. 1 ml of RPMI1640 medium containing 50% polyethylene glycol (4,000)
And left for 1 minute. Next, RPMI 1640
After gradually adding ml to dilute, the mixture was centrifuged at 900 rpm for 3 minutes. Then, the obtained cells were suspended in 50 ml of HAT medium, and 0.1 ml each was dispensed into a 96-well tissue culture plate. When cultured in this state for 1 week, colonies were observed in about 90 to 95% of the wells. After the colonies grew to some extent (after 10 to 13 days), the antibody titer was measured by the enzyme antibody method using the culture supernatant, and the desired hybridoma was cloned from the appropriate well by the limiting dilution method.
実施例2.モノクローナル抗体の精製および特異性の検討 Balb/cマウスの腹腔内にプリステン0.5mlを投与し、
７日後にハイブリドーマ107個を腹腔内に摂取、10日か
ら14日後に腹水を得た。腹水はさらに既知のプロテイン
Ａセファロースカラムおよび硫安分画法を利用してモノ
クローナル抗体を精製した。Example 2. Purification of monoclonal antibody and examination of specificity Balb / c mice were intraperitoneally administered with 0.5 ml of pristine,
After 7 days, 10 7 hybridomas were intraperitoneally ingested, and ascites was obtained 10 to 14 days later. The ascites was further purified of a monoclonal antibody using a known protein A sepharose column and ammonium sulfate fractionation method.
得られた精製モノクローナル抗体のクラス、サブクラ
スをオクタロニー法により検討した結果、130−22、145
−９ともマウスIgG1であることが判明した。As a result of examining the class and subclass of the obtained purified monoclonal antibody by the Ouchterlony method, 130-22, 145
-9 both a mouse IgG 1 it has been found.
モノクローナル抗体の特異性の確認は癌細胞を固定化
した系および手術で患者より得られた癌組織標本を用
い、市販のキット（モノクローナルスクリーニングシス
テム、ニューイングランドニュークリア社製、米国）を
利用した酵素抗体法により行った。すなわち、表１に示
す11種類の癌細胞株およびヒト末梢白血球を96ウェルの
プレートに固定化して反応性を検討した。To confirm the specificity of the monoclonal antibody, an enzyme using a commercially available kit (monoclonal screening system, New England Nuclear Co., Ltd.) using a system in which cancer cells are immobilized and a cancer tissue specimen obtained from a patient by surgery The antibody method was used. That is, the 11 types of cancer cell lines and human peripheral leukocytes shown in Table 1 were immobilized on a 96-well plate and the reactivity was examined.
結果を表１に示す。 Table 1 shows the results.
その結果、抗体130−22、145−９はともに抗原である
PC−９細胞とのみ反応し、他のヒト癌細胞、白血球とは
反応しなかった。 As a result, the antibodies 130-22 and 145-9 are both antigens.
It reacted only with PC-9 cells and did not react with other human cancer cells and white blood cells.
本発明のモノクローナル抗体と正常組織、癌組織標本
との反応性は、既知のABC法（ベクタステイン ABCキッ
ト）により免疫組織染色により検討した。癌組織との反
応性についての結果を表２に示す。 Monoclonal antibody of the present invention and normal tissue, cancer tissue specimen
Reactivity with known ABC method (Vector stain ABC kit
It was examined by immunohistochemical staining. Anti-cancer tissue
The results of responsiveness are shown in Table 2.
その結果、患者より得られた正常の胃、腸、肺、腎
臓、肝臓、脾臓、膵臓、卵巣組織や胃癌、大腸癌、乳癌
組織とは反応しなかった。しかし、肺腺癌、卵巣癌組織
は強く染色された。 As a result, it did not react with normal stomach, intestine, lung, kidney, liver, spleen, pancreas, ovary tissue and gastric cancer, colon cancer, breast cancer tissue obtained from the patient. However, lung adenocarcinoma and ovarian cancer tissues were strongly stained.
実施例3.放射性同位元素で標識した抗体を用いる血清中
抗原濃度の測定 実施例２で示した酵素抗体法による検討と同じく、放
射性ヨード（Ｉ−125）で標識したモノクローナル抗体
も抗原であるPC−９細胞に結合する。その結合は非標識
抗体の添加で阻害され、スキャチャード解析ではＩ−12
5標識130−22抗体の方が145−９のそれよりも結合親和
定数が高い。130−22とPC−９細胞の結合親和定数は1.2
×109M-1だった。Example 3 Measurement of Serum Antigen Concentration Using Antibody Labeled with Radioisotope Similar to the examination by the enzyme antibody method shown in Example 2, a monoclonal antibody labeled with radioactive iodine (I-125) is also an antigen PC -9 binds to cells. The binding was inhibited by the addition of unlabeled antibody, and by Scatchard analysis I-12
The 5-labeled 130-22 antibody has a higher binding affinity constant than that of 145-9. The binding affinity constant between 130-22 and PC-9 cells is 1.2.
It was × 10 9 M -1 .
モノクローナル抗体と反応する抗原濃度の定量的な測
定は以下のサンドイッチ法により行った。The following sandwich method was used to quantitatively measure the concentration of the antigen that reacts with the monoclonal antibody.
まず検体100μと抗体を固相化したポリスチレンビ
ーズおよびＩ−125標識した抗体200μを室温にて20〜
24時間反応せしめた、洗浄を繰り返した後、ポリスチレ
ンビーズに結合した放射活性をガンマカウンターで計測
した。First, 100 μm of a sample, polystyrene beads having an antibody immobilized thereon, and 200 μm of an antibody labeled with I-125 are allowed to stand at room temperature for 20
After repeating the reaction for 24 hours and washing, the radioactivity bound to polystyrene beads was measured with a gamma counter.
Ｉ−125標識130−22抗体をトレーサーとし、130−22
をビーズに固相化したアッセイ、及びＩ−125標識した
抗体と145−９個相化抗体を用いるアッセイの２つの測
定方法により、PC−９細胞培養上清中の抗原濃度、患者
血清中の抗原濃度を測定することができる。Using I-125 labeled 130-22 antibody as a tracer, 130-22
The concentration of the antigen in the PC-9 cell culture supernatant and the patient serum were measured by two measurement methods: an assay in which beads were immobilized on a bead and an assay using an I-125-labeled antibody and a 145.9-phased antibody. The antigen concentration can be measured.
PC−９細胞培養上清中の抗原濃度を測定した結果を第
１図に示した。PC−９細胞を過増殖させた培養液1ml中
の抗原濃度を3,000単位として希釈し、上記のサンドイ
ッチ法により測定したところPC−９細胞培養液には大量
の抗原が分泌されていることが認められた。さらに各種
悪性腫瘍患者、正常人より得られた血清を用いて血清中
の抗原濃度を測定したところ、正常人の血液中には抗原
はほとんど検出されないが、卵巣癌、膵臓癌患者血清中
にも高い濃度の抗原が検出された。結果を第２図に示し
た。The results of measuring the antigen concentration in the PC-9 cell culture supernatant are shown in FIG. It was confirmed that a large amount of the antigen was secreted in the PC-9 cell culture medium when the antigen concentration was diluted to 3,000 units in 1 ml of the culture medium in which the PC-9 cells were overgrown and measured by the above sandwich method. Was given. Furthermore, when the antigen concentration in serum was measured using serum obtained from various malignant tumor patients and normal subjects, almost no antigen was detected in the blood of normal subjects, but it was also detected in the serum of patients with ovarian cancer and pancreatic cancer. High concentrations of antigen were detected. The results are shown in Fig. 2.
実施例4.肺癌細胞PC−９移植ヌードマウスにおける放射
性同位元素で標識した抗体の体内分布 肺癌細胞PC−９をヌードマウスに移植し、腫瘍が適当
な大きさになるまで飼った後、実施例３で使用したＩ−
125標識130−22抗体１μCi（Spec5mCi/mg）に非標識抗
体を加えて、抗体量がマウス一匹あたり20μｇになるよ
うにした液0.2mlを肺癌細胞（PC−９）移植ヌードマウ
スに尾静脈より投与した。Example 4. Biodistribution of antibody labeled with radioactive isotope in lung cancer cell PC-9 transplanted nude mouse Lung cancer cell PC-9 was transplanted to nude mouse and kept until the tumor had an appropriate size. I- used in 3
The unlabeled antibody was added to 1 μCi of 125-labeled 130-22 antibody (Spec5mCi / mg), and the amount of antibody was adjusted to 20 μg per mouse, and 0.2 ml of the solution was tail vein-administered to lung cancer cell (PC-9) nude mice. Administered more.
48時間後、肺癌細胞（PC−９）移植ヌードマウスを屠
殺し、解剖して各臓器の放射活性をガンマーカウンター
で計測した。After 48 hours, lung cancer cell (PC-9) -implanted nude mice were sacrificed, dissected and the radioactivity of each organ was measured with a gamma counter.
その結果を表３に示した。125I標識抗体は他の臓器よ
りも腫瘍に最も集積しており、肺癌や卵巣癌の診断に応
用出来る。Table 3 shows the results. 125 I-labeled antibodies are most concentrated in tumors than in other organs and can be applied to diagnose lung cancer and ovarian cancer.
【図面の簡単な説明】 第１図は本発明のモノクローナル抗体を用いたサンドイ
ッチ法により測定したPC−９細胞過増殖培養液中の抗原
濃度を示すグラフである。 第２図は本発明のモノクローナル抗体を用いたサンドイ
ッチ法により測定した健常人、悪性腫瘍患者血清中の抗
原濃度を示すグラフである。BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the antigen concentration in PC-9 cell overgrowth culture broth measured by the sandwich method using the monoclonal antibody of the present invention. FIG. 2 is a graph showing the antigen concentration in sera of healthy persons and patients with malignant tumors, which was measured by the sandwich method using the monoclonal antibody of the present invention.
フロントページの続き (56)参考文献 特開 昭60−199830（ＪＰ，Ａ) Ｉｎｔ．Ｊ．Ｃａｎｃｅｒ，35［４ ］（1985）ｐ．449−455 Ｈｙｂｒｉｄｏｍａ，４［１ ］（1985）ｐ．76 Ｃａｎｃｅｒ Ｒｅｓ．，45［11 Ｐ ＡＲＴ２］（1985）ｐ．5808−5812 Ｃａｎｃｅｒ Ｒｅｓ．，45［11 Ｐ ＡＲＴ２］（1985）ｐ．5813−5817 Ｃａｎｃｅｒ Ｒｅｓ．，46［９ ］（1986）ｐ．4438−4443 ＪＰＮ Ｊ．Ｍｅｄ．，25［２ ］（1986）ｐ．127−134Continuation of front page (56) References JP-A-60-199830 (JP, A) Int. J. Cancer, 35 [4] (1985) p. 449-455 Hybridoma, 4 [1] (1985) p. 76 Cancer Res. , 45 [11 P ART2] (1985) p. 5808-5812 Cancer Res. , 45 [11 P ART2] (1985) p. 5813-5817 Cancer Res. , 46 [9] (1986) p. 4438-4443 JPN J. Med. 25 [2] (1986) p. 127-134
Claims (5)
−９に対するモノクローナル抗体。 （１）ヒト肺腺癌由来細胞株PC−９と反応する。 （２）ヒト肺腺癌Ruweller、ヒト肺腺癌PC−３、ヒト肺
偏平上皮癌AOI、ヒト胃癌Kato−III、ヒト大腸癌Lovo、
ヒト前立腺癌PC−３（ATCC）、ヒト腎癌RCC−JA、ヒト
子宮癌頚Hela、ヒト悪性黒色腫MI及びヒト膀胱癌Ｔ−24
と反応しない。 （３）ヒト末梢白血球と反応しない。1. A human lung adenocarcinoma-derived cell line PC having the following properties:
A monoclonal antibody against -9. (1) Reacts with human lung adenocarcinoma-derived cell line PC-9. (2) Human lung adenocarcinoma Ruweller, human lung adenocarcinoma PC-3, human lung squamous cell carcinoma AOI, human gastric cancer Kato-III, human colon cancer Lovo,
Human Prostate Cancer PC-3 (ATCC), Human Renal Cancer RCC-JA, Human Uterine Cancer Cervical Hela, Human Malignant Melanoma MI and Human Bladder Cancer T-24
Does not react with. (3) Does not react with human peripheral leukocytes.
求の範囲第１項記載のモノクローナル抗体。2. The monoclonal antibody according to claim 1, wherein the immunoglobulin class is IgG.
物から採取した抗体産生細胞と骨髄腫細胞の細胞融合に
より作製したハイブルドーマが産生するものである特許
請求の範囲第１項記載のモノクローナル抗体。3. A hybridoma produced by cell fusion of antibody-producing cells and myeloma cells collected from an animal immunized with human lung adenocarcinoma-derived cell line PC-9. Monoclonal antibody.
物から採取した抗体産生細胞と骨髄腫細胞の細胞融合に
より作製したハイブリドーマを培養し、培養物よりヒト
肺腺癌由来細胞株PC−９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次の性質を有するヒト肺腺癌
由来細胞株PC−９に対するモノクローナル抗体の製造
法。 （１）ヒト肺腺癌由来細胞株PC−９と反応する。 （２）ヒト肺腺癌Ruweller、ヒト肺腺癌PC−３、ヒト肺
偏平上皮癌AOI、ヒト胃癌Kato−III、ヒト大腸癌Lovo、
ヒト前立腺癌PC−３（ATCC）、ヒト腎癌RCC−JA、ヒト
子宮頚癌Hela、ヒト悪性黒色腫MI及びヒト膀胱癌Ｔ−24
と反応しない。 （３）ヒト末梢白血球と反応しない。4. A human lung adenocarcinoma-derived cell line is cultured from a hybridoma prepared by cell fusion of antibody-producing cells and myeloma cells collected from an animal immunized with the human lung adenocarcinoma-derived cell line PC-9. A method for producing a monoclonal antibody against a human lung adenocarcinoma-derived cell line PC-9 having the following properties, which comprises collecting a monoclonal antibody against PC-9. (1) Reacts with human lung adenocarcinoma-derived cell line PC-9. (2) Human lung adenocarcinoma Ruweller, human lung adenocarcinoma PC-3, human lung squamous cell carcinoma AOI, human gastric cancer Kato-III, human colon cancer Lovo,
Human Prostate Cancer PC-3 (ATCC), Human Renal Cancer RCC-JA, Human Cervical Cancer Hela, Human Malignant Melanoma MI and Human Bladder Cancer T-24
Does not react with. (3) Does not react with human peripheral leukocytes.
次の性質を有するヒト肺腺癌由来細胞株PC−９に対する
モノクローナル抗体を含有するヒト癌診断薬。 （１）ヒト肺腺癌由来細胞株PC−９と反応する。 （２）ヒト肺腺癌Ruweller、ヒト肺腺癌PC−３、ヒト肺
偏平上皮癌AOI、ヒト胃癌Kato−III、ヒト大腸癌Lovo、
ヒト前立腺癌PC−３（ATCC）、ヒト腎癌RCC−JA、ヒト
子宮頚癌Hela、ヒト悪性黒色腫MI及びヒト膀胱癌Ｔ−24
と反応しない。 （３）ヒト末梢白血球と反応しない。5. A human cancer diagnostic agent containing a monoclonal antibody against a human lung adenocarcinoma-derived cell line PC-9 having the following properties, which is labeled with an enzyme or a radioisotope. (1) Reacts with human lung adenocarcinoma-derived cell line PC-9. (2) Human lung adenocarcinoma Ruweller, human lung adenocarcinoma PC-3, human lung squamous cell carcinoma AOI, human gastric cancer Kato-III, human colon cancer Lovo,
Human Prostate Cancer PC-3 (ATCC), Human Renal Cancer RCC-JA, Human Cervical Cancer Hela, Human Malignant Melanoma MI and Human Bladder Cancer T-24
Does not react with. (3) Does not react with human peripheral leukocytes.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP24620986 | 1986-10-16 | ||
| JP61-246209 | 1986-10-16 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS63240798A JPS63240798A (en) | 1988-10-06 |
| JP2521496B2 true JP2521496B2 (en) | 1996-08-07 |
Family
ID=17145140
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP62260498A Expired - Fee Related JP2521496B2 (en) | 1986-10-16 | 1987-10-15 | Anti-human cancer monoclonal antibody |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2521496B2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3623381A1 (en) * | 2011-05-19 | 2020-03-18 | The Regents Of The University Of Michigan | Integrin alpha-2 binding agents and use thereof to inhibit cancer cell proliferation |
-
1987
- 1987-10-15 JP JP62260498A patent/JP2521496B2/en not_active Expired - Fee Related
Non-Patent Citations (6)
| Title |
|---|
| ＣａｎｃｅｒＲｅｓ．，４５［１１ＰＡＲＴ２］（１９８５）ｐ．５８０８−５８１２ |
| ＣａｎｃｅｒＲｅｓ．，４５［１１ＰＡＲＴ２］（１９８５）ｐ．５８１３−５８１７ |
| ＣａｎｃｅｒＲｅｓ．，４６［９］（１９８６）ｐ．４４３８−４４４３ |
| Ｈｙｂｒｉｄｏｍａ，４［１］（１９８５）ｐ．７６ |
| Ｉｎｔ．Ｊ．Ｃａｎｃｅｒ，３５［４］（１９８５）ｐ．４４９−４５５ |
| ＪＰＮＪ．Ｍｅｄ．，２５［２］（１９８６）ｐ．１２７−１３４ |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS63240798A (en) | 1988-10-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0394277B1 (en) | Second generation monoclonal antibodies having binding specificity to tag-72 and human carcinomas and methods for employing the same | |
| JPH066067B2 (en) | Monoclonal antibody and method for producing the same | |
| Stein et al. | Murine monoclonal antibodies raised against human non-small cell carcinoma of the lung: specificity and tumor targeting | |
| US5486456A (en) | Method of diagnosing ovarian, endometrial, and colon cancers with monoclonal antibodies OXA and OXB | |
| EP0210970A2 (en) | Detection, imaging and therapy of renal cell carcinoma with monoclonal antibodies | |
| JPH08266273A (en) | Hybridoma for producing anti-human breast carcinoma monoclonal antibody | |
| JP2574269B2 (en) | Monoclonal antibodies having binding specificity for human prostate tumor associated antigen and methods of using them | |
| JPH0751062B2 (en) | Monoclonal antibody | |
| US5639622A (en) | Monoclonal antibodies against tumor-associated antigens, a process for the preparation thereof and the use thereof | |
| CA1308676C (en) | Anti-human igg (immunoglobulin g) monoclonal antibody and process for preparing the same | |
| Börner et al. | Selection of monoclonal antibodies for use in an immunometric assay for carcinoembryonic antigen | |
| JP2655830B2 (en) | Monoclonal antibody producing cell line for human non-small cell lung cancer | |
| JP2521496B2 (en) | Anti-human cancer monoclonal antibody | |
| JPH04507285A (en) | Anti-idiotope immunoassay | |
| US5206007A (en) | Anti-human myocardial c protein monoclonal antibody and hybridoma producing the antibody | |
| AU8071187A (en) | Monoclonal paratopic molecule directed to human ganglioside gd2 | |
| AU618209B2 (en) | Antigen recognized by mca 16-88 | |
| JP2792651B2 (en) | Monoclonal antibodies against human pancreatic cancer | |
| JP2021511068A (en) | Antibodies to Centrin-1, preparation methods, and their use | |
| US4962048A (en) | Monoclonal antibodies to human pancreatic cancer | |
| EP0827513B1 (en) | Reagents for treatment and diagnostic visualisation, particularly in cancer | |
| JPH03188099A (en) | Novel tumor-related antigen | |
| Kunimatsu et al. | Development of new immunoradiometric assay for CA 125 antigen using two monoclonal antibodies produced by immunizing lung cancer cells | |
| JPH02219594A (en) | Monochlonal antibody to cancer to lung and cell surface antigen | |
| CA1330545C (en) | Characteristics and use of the anti-cea antibody produced by the hybridoma having the atcc number hbl0029 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |