JPH04228089A - Agent for treatment and prevention of hypercalcemia - Google Patents
Agent for treatment and prevention of hypercalcemiaInfo
- Publication number
- JPH04228089A JPH04228089A JP3110565A JP11056591A JPH04228089A JP H04228089 A JPH04228089 A JP H04228089A JP 3110565 A JP3110565 A JP 3110565A JP 11056591 A JP11056591 A JP 11056591A JP H04228089 A JPH04228089 A JP H04228089A
- Authority
- JP
- Japan
- Prior art keywords
- monoclonal antibody
- gene
- pthrp
- fragment
- sequence
- 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.)
- Withdrawn
Links
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Abstract
Description
【０００１】0001
【産業上の利用分野】本発明は抗ヒト副甲状腺ホルモン
関連タンパクモノクローナル抗体（以下、抗ＰＴＨｒＰ
モノクローナル抗体とする）を有効成分とする抗高カル
シウム血症治療・予防剤に関する。さらに詳細には、本
発明は医療分野において副甲状腺ホルモン関連タンパク
（以下、ＰＴＨｒＰとする）の産生により発症する高カ
ルシウム血症の治療および予防、さらには再発の防止に
用いられる抗ＰＴＨｒＰモノクローナル抗体を有効成分
とする治療・予防剤に関する。本発明はさらに、上記モ
ノクローナル抗体の可変部領域のＨ鎖をコードする遺伝
子断片およびＬ鎖をコードする遺伝子断片；げっ歯類由
来の上記可変部領域とヒト由来の定常部領域とを有する
キメラモノクローナル抗体、該キメラモノクローナル抗
体を含有する高カルシウム血症治療・予防剤；および該
キメラモノクローナル抗体をコードする融合遺伝子に関
する。本発明は、さらに上記モノクローナル抗体を産生
するセルラインに関する。[Industrial Application Field] The present invention is an anti-human parathyroid hormone-related protein monoclonal antibody (hereinafter referred to as anti-PTHrP
The present invention relates to an antihypercalcemia treatment/prevention agent containing a monoclonal antibody) as an active ingredient. More specifically, the present invention provides an anti-PTHrP monoclonal antibody that is used in the medical field to treat and prevent hypercalcemia caused by the production of parathyroid hormone-related protein (hereinafter referred to as PTHrP), and to prevent its recurrence. It relates to therapeutic and preventive agents as active ingredients. The present invention further provides a gene fragment encoding the H chain and a gene fragment encoding the L chain of the variable region of the monoclonal antibody; a chimeric monoclonal antibody having the variable region derived from a rodent and the constant region derived from a human. The present invention relates to an antibody, a hypercalcemia treatment/prevention agent containing the chimeric monoclonal antibody; and a fusion gene encoding the chimeric monoclonal antibody. The present invention further relates to a cell line that produces the above monoclonal antibody.
【０００２】0002
【従来の技術】高カルシウム血症は、医療分野において
しばしば見い出される電解質異常症であり、患者に種々
の苦痛を与えるのみならず、重篤な場合は生命を脅かす
おそれがある。高カルシウム血症を惹起する原因疾患は
多様であるが、高頻度でかつ重篤な高カルシウム血症を
引き起こす原因疾患としては、悪性腫瘍および副甲状腺
ホルモン（以下、ＰＴＨとする）の過剰産生による副甲
状腺機能亢進症が挙げられる。BACKGROUND OF THE INVENTION Hypercalcemia is an electrolyte abnormality often found in the medical field, and it not only causes various types of pain to patients, but also threatens life in severe cases. There are various diseases that cause hypercalcemia, but the diseases that cause frequent and severe hypercalcemia include malignant tumors and overproduction of parathyroid hormone (hereinafter referred to as PTH). Examples include hyperparathyroidism.
【０００３】このうち、悪性腫瘍に伴う高カルシウム血
症は、その発生機序から、腫瘍細胞の骨への浸潤により
骨溶解が亢進し高カルシウム血症がもたらされる症例（
Ｌｏｃａｌ Ｏｓｔｅｏｌｙｔｉｃ Ｈｙｐｅｒｃａｌ
ｃｅｍｉａ）と、腫瘍から産生される体液性因子が全身
性に作用し高カルシウム血症が惹起される症例（Ｈｕｍ
ｏｒａｌ Ｈｙｐｅｒｃａｌｃｅｍｉａ ｏｆ Ｍａｌ
ｉｇｎａｎｃｙ，以下、ＨＨＭとする）との２つに大別
される。その代表的な前者の例としては、多発性骨髄腫
、乳癌などの骨転移などによる高カルシウム血症が挙げ
られ、後者（ＨＨＭ）の例としては、偏平上皮癌、腎尿
路系の癌などに伴う高カルシウム血症などが挙げられる
。[0003] Among these, hypercalcemia associated with malignant tumors is caused by cases in which bone infiltration of tumor cells accelerates osteolysis, resulting in hypercalcemia (
Local Osteolytic Hypercal
cemia) and cases in which humoral factors produced by tumors act systemically to induce hypercalcemia (Hum.
oral Hypercalcemia of Mal
ignency (hereinafter referred to as HHM). Typical examples of the former include hypercalcemia due to bone metastases such as multiple myeloma and breast cancer, while examples of the latter (HHM) include squamous cell carcinoma and cancer of the renal and urinary tract systems. Examples include hypercalcemia associated with this.
【０００４】このうち、ＨＨＭは、悪性腫瘍に伴う高カ
ルシウム血症の８０％以上を占める最も頻度の高い腫瘍
随伴性症候群である。このＨＨＭは、悪性腫瘍患者の末
期に急速に発症し進展することが多く、さらに特異的治
療法が存在しないことから多くの患者の死亡原因となっ
ている。[0004] Among these, HHM is the most frequent paraneoplastic syndrome, accounting for more than 80% of hypercalcemia associated with malignant tumors. This HHM often develops and progresses rapidly in the terminal stages of patients with malignant tumors, and furthermore, because no specific treatment exists, it is the cause of death for many patients.
【０００５】ＨＨＭの発症原因となる体液性因子につい
ては、多くの研究者により種々の研究がなされてきた。
Ｓｔｅｗａｒｔ，Ａ．Ｆ．らは、ＨＨＭ患者の血漿中に
ＰＴＨとは異なるがＰＴＨ類似の作用を示す物質の存在
を証明し、かつ、ＨＨＭを惹起する悪性腫瘍細胞がこの
物質を産生していることを明らかにした（Ｓｔｅｗａｒ
ｔ，Ａ．Ｆ．ら，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃ
ｉ．ＵＳＡ，８０巻，１４５４頁（１９８３）；および
Ｇｏｌｔｚｍａｎ，Ｄ．ら，Ｊ．Ｃｌｉｎ．Ｅｎｄｏｃ
ｒｉｎｏｌ．Ｍｅｔａｂ．，５３巻，８９９頁（１９８
１））。[0005] Many researchers have conducted various studies on humoral factors that cause the onset of HHM. Stewart, A. F. demonstrated the presence of a substance in the plasma of HHM patients that is different from PTH but exhibits an action similar to PTH, and also revealed that malignant tumor cells that induce HHM produce this substance ( Stewar
t, A. F. et al., Proc. Natl. Acad. Sc
i. USA, vol. 80, p. 1454 (1983); and Goltzman, D. et al., J. Clin. Endoc
rinol. Metab. , vol. 53, p. 899 (198
1)).
【０００６】Ｍａｒｔｉｎ，Ｔ．Ｊ．のグループは、高
カルシウム血症を呈した肺癌患者の癌細胞（ＢＥＮ細胞
）の培養上清からＰＴＨ様活性を有する物質を単離し、
該物質のＮ末端の一次構造を明らかにした。さらに、該
細胞のｍＲＮＡを用いてＰＴＨ様物質のｃＤＮＡを単離
し、ＰＴＨ様物質の前駆体の全一次構造を決定した（Ｍ
ｏｓｅｌｅｙ，Ｊ．Ｍ．ら，Ｐｒｏｃ．Ｎａｔｌ．Ａｃ
ａｄ．Ｓｃｉ．ＵＳＡ，８４巻，５０４８頁（１９８７
）；およびＳｕｖａ，Ｌ．Ｊ．ら，Ｓｃｉｅｎｃｅ，２
３７巻，８９３頁（１９８７））。そこではＰＴＨ様物
質はＰＴＨｒＰと称され、３６個のシグナルペプチドに
続く１４１個のアミノ酸残基よりなること、そして、Ｃ
末端の一次構造はＰＴＨと殆ど類似性がないが、Ｎ末端
の１−１３位のアミノ酸のうち８個は同一のアミノ酸か
らなると報告されている。ＰＴＨがその受容体と結合す
る機能；ＰＴＨが腎原性サイクリックＡＭＰの産生を亢
進する機能など、ＰＴＨがその生理活性を発現するには
Ｎ末端部分が不可欠であるとされている。従って、ＰＴ
ＨｒＰがＰＴＨと類似のＮ末端アミノ酸構造を有してい
るためにＰＴＨ様活性を有すると考えられる。[0006] Martin, T. J. The group isolated a substance with PTH-like activity from the culture supernatant of cancer cells (BEN cells) from a lung cancer patient who exhibited hypercalcemia.
The primary structure of the N-terminus of the substance was revealed. Furthermore, the cDNA of the PTH-like substance was isolated using the mRNA of the cells, and the entire primary structure of the precursor of the PTH-like substance was determined (M
oseley, J. M. et al., Proc. Natl. Ac
ad. Sci. USA, vol. 84, p. 5048 (1987
); and Suva, L. J. et al., Science, 2
Volume 37, page 893 (1987)). There, the PTH-like substance is called PTHrP and consists of 141 amino acid residues following 36 signal peptides, and C
Although the terminal primary structure has almost no similarity to PTH, it has been reported that 8 of the amino acids at positions 1-13 at the N-terminus are composed of the same amino acids. It is believed that the N-terminal portion is essential for PTH to exhibit its physiological activities, such as the function of PTH to bind to its receptor; the function of PTH to enhance the production of nephrogenic cyclic AMP. Therefore, P.T.
HrP is thought to have PTH-like activity because it has an N-terminal amino acid structure similar to that of PTH.
【０００７】Ｈｏｒｉｕｃｈｉ Ｎ．らは、ＰＴＨｒＰ
の１−３４位のペプチドを化学合成しラットに投与する
と、高カルシウム血症状態を示すことを報告している（
Ｓｃｉｅｎｃｅ，２３８巻，５６６頁（１９８７））。
さらに、Ｋｕｋｒｅｊａ，Ｓ．Ｃ．らは、高カルシウム
血症を呈した肺癌患者および喉頭癌患者のそれぞれの癌
細胞を無胸腺マウスに移植し、高カルシウム血症を示し
た時点でＰＴＨｒＰ（１−３４）およびＰＴＨｒＰ（１
−１６）ペプチドに対するウサギ抗血清を投与すると、
一部のマウスで血中カルシウムが正常レベルに減少した
ことを報告している（Ｊ．Ｃｌｉｎ．Ｉｎｖｅｓｔ，８
２巻，１７９８頁（１９８８））。このように、悪性腫
瘍に伴う高カルシウム血症の原因物質としてＰＴＨｒＰ
が重要な役割を果していることが知られている。Horiuchi N. et al., PTHrP
It has been reported that when a peptide at positions 1-34 of the peptide was chemically synthesized and administered to rats, a hypercalcemic state was observed (
Science, vol. 238, p. 566 (1987)). Furthermore, Kukreja, S. C. et al. transplanted cancer cells from a lung cancer patient and a laryngeal cancer patient with hypercalcemia into athymic mice, and at the time of hypercalcemia, PTHrP(1-34) and PTHrP(1) were transplanted into athymic mice.
-16) When rabbit antiserum against the peptide is administered,
reported that blood calcium decreased to normal levels in some mice (J. Clin. Invest, 8
2, 1798 (1988)). Thus, PTHrP is a causative agent of hypercalcemia associated with malignant tumors.
is known to play an important role.
【０００８】ところで、従来、悪性腫瘍に伴う高カルシ
ウム血症の治療には補液、およびフロセミドもしくはエ
タクリン酸を併用するが、一般にはこれだけでは不充分
であり、さらにカルシトニン、ミスラマイシンなどの追
加投与を必要とする。しかし、カルシトニンは連続投与
により効果が低下すること、そして、ミスラマイシンは
重篤な副作用を惹起するなどの欠点があり、充分な効果
を発揮するには至っていない。このような悪性腫瘍に伴
う高カルシウム血症の治療には、上記のようにＰＴＨｒ
Ｐ活性を特異的に阻害する薬剤が好ましいと考えられる
。ＰＴＨｒＰ活性だけを特異的に抑制する方法としては
、上記のＫｕｋｒｅｊａ，Ｓ．Ｃ．らの研究例があるが
、報告されている方法はウサギ抗血清あるいはそれを精
製して得られるポリクローナル抗体を用いているため、
治療成績が悪く、かつ、有効症例においてもその有効期
間は非常に短いという欠点がある。[0008] Conventionally, fluid replacement and furosemide or ethacrylic acid are used in combination to treat hypercalcemia associated with malignant tumors, but this alone is generally insufficient, and additional administration of calcitonin, mithramycin, etc. I need. However, calcitonin has disadvantages such as its effectiveness decreases with continuous administration, and mithramycin causes serious side effects, so it has not yet achieved sufficient efficacy. For the treatment of hypercalcemia associated with such malignant tumors, PTHr is used as described above.
Agents that specifically inhibit P activity are considered preferred. As a method for specifically suppressing only PTHrP activity, the method described by Kukreja, S. et al. C. There are examples of research by et al., but the reported method uses rabbit antiserum or polyclonal antibodies obtained by purifying it, so
The drawback is that the therapeutic results are poor, and even in effective cases, the effective period is very short.
【０００９】[0009]
【発明が解決しようとする課題】本発明は、上記従来の
問題点を解決するものであり、その目的とするところは
、高カルシウム血症に有効な治療・予防剤を提供するこ
とにある。本発明の他の目的は、悪性腫瘍に伴う高カル
シウム血症に有効な治療・予防剤を提供することにある
。SUMMARY OF THE INVENTION The present invention is intended to solve the above-mentioned conventional problems, and its object is to provide an effective therapeutic and preventive agent for hypercalcemia. Another object of the present invention is to provide an effective therapeutic and preventive agent for hypercalcemia associated with malignant tumors.
【００１０】0010
【課題を解決するための手段】本発明者らは、上記の問
題点を克服するため鋭意研究を重ねた結果、ヒトＰＴＨ
ｒＰに対して特異性を有するモノクローナル抗体を取得
することに成功し、該モノクローナル抗体を用いること
により、高カルシウム血症を高成績で、かつ比較的長期
間にわたり治療し得ることを見い出し、本発明を完成す
るに至った。[Means for Solving the Problems] As a result of extensive research to overcome the above problems, the present inventors have discovered that human PTH
We have succeeded in obtaining a monoclonal antibody having specificity for rP, and have discovered that by using the monoclonal antibody, hypercalcemia can be treated with high results and over a relatively long period of time, and the present invention I was able to complete it.
【００１１】本発明の高カルシウム血症治療・予防剤は
、抗ヒト副甲状腺ホルモン関連タンパクモノクローナル
抗体およびその関連物質でなる群から選択される少なく
とも１種を有効成分として含有し、そのことにより上記
目的が達成される。The hypercalcemia treatment/prevention agent of the present invention contains as an active ingredient at least one member selected from the group consisting of anti-human parathyroid hormone-related protein monoclonal antibodies and related substances, thereby achieving the above-mentioned effects. The purpose is achieved.
【００１２】本発明は、抗ヒト副甲状腺ホルモン関連タ
ンパクモノクローナル抗体のＨ鎖可変部領域をコードす
る遺伝子断片であって、配列番号１に記載のアミノ酸配
列をコードする遺伝子配列を少なくともその一部に有す
る遺伝子断片を包含する。[0012] The present invention provides a gene fragment encoding the H chain variable region of an anti-human parathyroid hormone-related protein monoclonal antibody, which comprises at least a portion of the gene sequence encoding the amino acid sequence set forth in SEQ ID NO: 1. This includes gene fragments with
【００１３】本発明は、抗ヒト副甲状腺ホルモン関連タ
ンパクモノクローナル抗体のＬ鎖可変部領域をコードす
る遺伝子断片であって、配列番号２に記載のアミノ酸配
列をコードする遺伝子配列を少なくともその一部に有す
る遺伝子断片を包含する。[0013] The present invention provides a gene fragment encoding the L chain variable region of an anti-human parathyroid hormone-related protein monoclonal antibody, which comprises at least a portion of the gene sequence encoding the amino acid sequence set forth in SEQ ID NO: 2. This includes gene fragments with
【００１４】本発明は、抗ヒト副甲状腺ホルモン関連タ
ンパクモノクローナル抗体であって、その可変部領域の
少なくとも一部に配列番号１に記載のアミノ酸配列を有
するモノクローナル抗体を包含する。The present invention includes an anti-human parathyroid hormone-related protein monoclonal antibody, which has the amino acid sequence set forth in SEQ ID NO: 1 in at least a portion of its variable region.
【００１５】本発明は、抗ヒト副甲状腺ホルモン関連タ
ンパクモノクローナル抗体であって、その可変部領域の
少なくとも一部に配列番号２に記載のアミノ酸配列を有
するモノクローナル抗体を包含する。The present invention includes an anti-human parathyroid hormone-related protein monoclonal antibody, which has the amino acid sequence set forth in SEQ ID NO: 2 in at least a portion of its variable region.
【００１６】本発明は、げっ歯類由来の可変部領域およ
びヒト由来の定常部領域からなり、ヒトの副甲状腺ホル
モン関連タンパクを認識するげっ歯類／ヒトキメラモノ
クローナル抗体、およびその誘導体を包含する。The present invention encompasses rodent/human chimeric monoclonal antibodies comprising a rodent-derived variable region and a human-derived constant region and recognizing human parathyroid hormone-related protein, and derivatives thereof. .
【００１７】本発明は、前記キメラモノクローナル抗体
をコードする融合遺伝子を包含する。[0017] The present invention includes a fusion gene encoding the chimeric monoclonal antibody.
【００１８】本発明は、上記キメラモノクローナル抗体
およびその誘導体の少なくとも１種を有効成分として含
有する高カルシウム血症治療・予防剤を包含する。The present invention includes a hypercalcemia treatment/prevention agent containing at least one of the chimeric monoclonal antibodies and derivatives thereof as an active ingredient.
【００１９】本発明は、上記モノクローナル抗体を産生
するセルラインを包含する。The present invention includes cell lines that produce the above monoclonal antibodies.
【００２０】本発明のモノクローナル抗体を調製するた
めには、従来からのモノクローナル抗体調製法を用いる
ことができる。つまり、抗原で免疫した動物から得られ
る抗体産生細胞と、ミエローマ細胞との細胞融合により
ハイブリドーマを調製し、得られたハイブリドーマの中
からＰＴＨｒＰ活性を特異的に阻害する抗体を産生して
いるクローンを選択することによって調製される。[0020] Conventional monoclonal antibody preparation methods can be used to prepare the monoclonal antibodies of the present invention. In other words, hybridomas are prepared by cell fusion of antibody-producing cells obtained from an animal immunized with an antigen and myeloma cells, and clones producing antibodies that specifically inhibit PTHrP activity are selected from among the hybridomas obtained. Prepared by selection.
【００２１】上記免疫に用いられる抗原としては、組換
えＤＮＡ法または化学合成法により調製したＰＴＨｒＰ
のアミノ酸配列の全部または一部のペプチドや、高カル
シウム血症を惹起した癌細胞の培養上清液由来のＰＴＨ
ｒＰなどが利用可能である。ＰＴＨｒＰのアミノ酸配列
の一部を含有するペプチドとしては、例えばＰＴＨｒＰ
のＮ末端部分に位置する、次の配列（配列番号７に示す
）を有するペプチドが挙げられる：Ａｌａ−Ｖａｌ−Ｓ
ｅｒ−Ｇｌｕ−Ｈｉｓ−Ｇｌｎ−Ｌｅｕ−Ｌｅｕ−Ｈｉ
ｓ−Ａｓｐ−Ｌｙｓ−Ｇｌｙ−Ｌｙｓ−Ｓｅｒ−Ｉｌｅ
−Ｇｌｎ−Ａｓｐ−Ｌｅｕ−Ａｒｇ−Ａｒｇ−Ａｒｇ−
Ｐｈｅ−Ｐｈｅ−Ｌｅｕ−Ｈｉｓ−Ｈｉｓ−Ｌｅｕ−Ｉ
ｌｅ−Ａｌａ−Ｇｌｕ−Ｉｌｅ−Ｈｉｓ−Ｔｈｒ−Ａｌ
ａ。
抗体産生細胞は、例えばＰＴＨｒＰまたはその一部に相
当するペプチド（同等の作用を有する物質）によってイ
ンビボあるいはインビトロで免疫された動物あるいはヒ
トの脾細胞由来のあるいはリンパ節細胞由来のＢリンパ
球や、患者末梢血由来のＢリンパ球が使用できる。[0021] The antigen used for the above immunization is PTHrP prepared by recombinant DNA method or chemical synthesis method.
PTH derived from the culture supernatant of cancer cells that caused hypercalcemia, or all or part of the amino acid sequence of
rP etc. are available. Examples of peptides containing part of the amino acid sequence of PTHrP include, for example, PTHrP
Mention may be made of a peptide having the following sequence (as shown in SEQ ID NO: 7), located in the N-terminal part of: Ala-Val-S
er-Glu-His-Gln-Leu-Leu-Hi
s-Asp-Lys-Gly-Lys-Ser-Ile
-Gln-Asp-Leu-Arg-Arg-Arg-
Phe-Phe-Leu-His-His-Leu-I
le-Ala-Glu-Ile-His-Thr-Al
a. Antibody-producing cells include, for example, B lymphocytes derived from splenocytes or lymph node cells of animals or humans immunized in vivo or in vitro with PTHrP or a peptide corresponding to a part thereof (a substance having an equivalent effect); B lymphocytes derived from patient peripheral blood can be used.
【００２２】細胞融合に用いるミエローマ細胞としては
、マウス、ラット、ヒト、およびその他の動物由来のミ
エローマ細胞を用いることができる。細胞融合は、例え
ばポリエチレングリコールを用いる方法（岩崎辰夫ら、
「単クローン抗体、ハイブリドーマとＥＬＩＳＡ」講談
社発行（１９８３）参照）により行うことができる。細
胞融合によって得られたハイブリドーマはスクリーニン
グ（例えば、酵素抗体法）に付され、抗体を産生するハ
イブリドーマが選択される。得られた抗体産生ハイブリ
ドーマはクローニング（例えば、限界希釈法）に付され
る。得られたクローンは、次いでスクリーニングに付さ
れ、目的とするモノクローナル抗体を産生するクローン
が選択される。例えば、ラット骨芽腫細胞ＲＯＳ１７／
２．８細胞またはＵＭＲ１０６細胞にＰＴＨｒＰを添加
することにより、アデニレートサイクレース活性が向上
する（アデニレートサイクレース促進活性が得られる）
が、これに対して、抗ＰＴＨｒＰモノクローナル抗体が
存在すると、その活性が阻害されることによってスクリ
ーニングが行われる。[0022] As myeloma cells used for cell fusion, myeloma cells derived from mice, rats, humans, and other animals can be used. Cell fusion can be achieved, for example, by using polyethylene glycol (Tatsuo Iwasaki et al.
(See "Monoclonal Antibodies, Hybridomas and ELISA" published by Kodansha (1983)). Hybridomas obtained by cell fusion are subjected to screening (eg, enzyme antibody method) to select hybridomas that produce antibodies. The obtained antibody-producing hybridoma is subjected to cloning (eg, limiting dilution method). The obtained clones are then subjected to screening to select clones that produce the desired monoclonal antibody. For example, rat osteoblastoma cell ROS17/
Adding PTHrP to 2.8 cells or UMR106 cells improves adenylate cyclase activity (obtains adenylate cyclase promoting activity)
However, if an anti-PTHrP monoclonal antibody is present, its activity is inhibited, and screening is therefore performed.
【００２３】得られたハイブリドーマのうちＥＶＯ３４
Ｂ１ＧおよびＥＶＯ１４１１Ｇの２種は、微生物工業技
術研究所特許微生物寄託センターにそれぞれ微工研菌寄
第１１４２９（ＦＥＲＭ Ｐ−１１４２９）および第１
１４３０号（ＦＥＲＭ Ｐ−１１４３０）として寄託さ
れている。ＥＶＯ３４Ｂ１Ｇは、ＰＴＨｒＰの１−３４
アミノ酸残基のペプチドを抗原として調製され、目的と
する抗体を産生するハイブリドーマクローンである。Ｅ
ＶＯ１４１１Ｇは、組換えＤＮＡ法により得られたＰＴ
ＨｒＰ（以下、ｒＰＴＨｒＰとする）を抗原として調製
され、目的抗体を産生するハイブリドーマクローンであ
る。[0023] Among the hybridomas obtained, EVO34
The two types, B1G and EVO1411G, were deposited with the Microbial Technology Research Institute Patent Microorganism Depositary No. 11429 (FERM P-11429) and No. 1 (FERM P-11429), respectively.
No. 1430 (FERM P-11430). EVO34B1G is PTHrP 1-34
This is a hybridoma clone that is prepared using a peptide of amino acid residues as an antigen and produces the desired antibody. E
VO1411G is a PT obtained by recombinant DNA method.
This is a hybridoma clone that is prepared using HrP (hereinafter referred to as rPTHrP) as an antigen and produces a target antibody.
【００２４】得られたハイブリドーマによる抗ＰＴＨｒ
Ｐモノクローナル抗体の産生量は、該ハイブリドーマを
培養し、その培養上清中の抗体価を、例えば酵素抗体法
で測定することによって検定することができる。得られ
たハイブリドーマから抗ＰＴＨｒＰモノクローナル抗体
を得るには、例えば、該ハイブリドーマを培養し、その
培養上清から適切な方法により抗体を分取するか、また
は、ハイブリドーマをあらかじめプリスタン処理したマ
ウスの腹腔内に移植し、増殖させた後、そのマウスの腹
水から抗体を分取する。抗ＰＴＨｒＰモノクローナル抗
体の調製には、組換えＤＮＡ法を用いた動物細胞または
微生物を用いることも可能である。上記抗体の精製は、
通常の生化学的手法を組み合わせることによって行われ
得る。例えば、硫安沈澱分画法、イオン交換クロマトグ
ラフィー、ゲル濾過法、ＰＥＧ分画法、アフィニティー
クロマトグラフィー、高速液体クロマトグラフィー、電
気泳動などを適宜組み合わせることによって行われ得る
。精製されたモノクローナル抗体は、生物学的製剤の製
剤化に通常用いられる方法によって製剤化される。例え
ば、メンブランフィルターなどによる濾過除菌操作を行
った後に、安定化剤と共に滅菌バイアルに凍結乾燥され
る。Anti-PTHr produced by the obtained hybridoma
The amount of P monoclonal antibody produced can be assayed by culturing the hybridoma and measuring the antibody titer in the culture supernatant, for example, by enzyme antibody method. To obtain an anti-PTHrP monoclonal antibody from the obtained hybridoma, for example, the hybridoma is cultured and the antibody is isolated from the culture supernatant by an appropriate method, or the hybridoma is intraperitoneally administered to a mouse that has been previously treated with pristane. After transplantation and proliferation, antibodies are collected from the ascites fluid of the mice. For the preparation of anti-PTHrP monoclonal antibodies, it is also possible to use animal cells or microorganisms using recombinant DNA methods. Purification of the above antibody
It can be carried out by a combination of conventional biochemical techniques. For example, it can be carried out by appropriately combining ammonium sulfate precipitation fractionation, ion exchange chromatography, gel filtration, PEG fractionation, affinity chromatography, high performance liquid chromatography, electrophoresis, and the like. Purified monoclonal antibodies are formulated by methods commonly used in the formulation of biological products. For example, after sterilization by filtration using a membrane filter or the like, it is freeze-dried into a sterilized vial together with a stabilizer.
【００２５】このようにして得られた抗ＰＴＨｒＰ抗体
の、高カルシウム血症の治療・予防効果は、高カルシウ
ム血症モデル動物に、抗ＰＴＨｒＰモノクローナル抗体
を投与し、血中カルシウムレベルの低下の有無によって
調べる実験系を用いることにより確認される。上記高カ
ルシウム血症モデル動物としては、例えば、発病の原因
等であるＰＴＨｒＰあるいはＰＴＨｒＰ（１−３４）ペ
プチドを１日当り約６ナノモル連続投与することによっ
て作製した高カルシウム血症モデル動物、あるいはＰＴ
ＨｒＰを産生するヒト癌細胞（例えばＰＣ−３細胞）を
ヌードマウスに移植することによって得られる担癌高カ
ルシウム血症モデル動物がある。本発明の抗ＰＴＨｒＰ
モノクローナル抗体は、このいずれの方法においても血
中カルシウムレベルを正常レベルまで下げることが確認
された。従って、本発明の抗ＰＴＨｒＰモノクローナル
抗体は、悪性腫瘍に伴う高カルシウム血症の治療・予防
剤として非常に好適であり、産業上の利用価値がきわめ
て高い。[0025] The therapeutic and preventive effects of the anti-PTHrP antibody thus obtained on hypercalcemia can be determined by administering the anti-PTHrP monoclonal antibody to hypercalcemia model animals and determining whether or not there is a decrease in blood calcium levels. This is confirmed by using an experimental system that examines Examples of the above-mentioned hypercalcemia model animals include hypercalcemia model animals prepared by continuously administering about 6 nanomoles per day of PTHrP or PTHrP (1-34) peptide, which is the cause of disease onset, or PT.
There is a tumor-bearing hypercalcemia model animal obtained by transplanting HrP-producing human cancer cells (eg, PC-3 cells) into nude mice. Anti-PTHrP of the present invention
It was confirmed that monoclonal antibodies lower blood calcium levels to normal levels using both methods. Therefore, the anti-PTHrP monoclonal antibody of the present invention is very suitable as a therapeutic/preventive agent for hypercalcemia associated with malignant tumors, and has extremely high industrial utility value.
【００２６】さらに、モノクローナル抗体そのものに限
定されず、その関連物質、つまり該モノクローナル抗体
の免疫学的特性を有するフラグメント、例えば、Ｆ（ａ
ｂ’）フラグメント、さらに、該フラグメントを含むペ
プチドを含有する製剤も高カルシウム血症治療・予防剤
として使用され得る。Furthermore, it is not limited to the monoclonal antibody itself, but also its related substances, that is, fragments having immunological properties of the monoclonal antibody, such as F(a
b') Fragments, as well as preparations containing peptides containing the fragments, can also be used as hypercalcemia therapeutic and preventive agents.
【００２７】発明者らは、さらに、上記抗ＰＴＨｒＰモ
ノクローナル抗体の抗原認識部位に関する検討を行った
。発明者らは、上記モノクローナル抗体のＨ鎖およびＬ
鎖の可変部領域の遺伝子配列を明かにすることを試みた
。上記Ｈ鎖の可変部領域をコードする遺伝子（ＶＤＪＨ
）およびＬ鎖の可変部領域をコードする遺伝子（ＶＪＬ
）は、該モノクローナル抗体を産生するハイブリドーマ
から、下記のようにｍＲＮＡを単離し、ｃＤＮＡを合成
して、これを用いたＰＣＲ法を行うことにより決定され
得る。The inventors further investigated the antigen recognition site of the anti-PTHrP monoclonal antibody. The inventors discovered that the H chain and L chain of the above monoclonal antibody
We attempted to clarify the gene sequence of the variable region of the chain. The gene encoding the variable region of the H chain (VDJH
) and the gene encoding the variable region of the light chain (VJL
) can be determined by isolating mRNA from a hybridoma producing the monoclonal antibody, synthesizing cDNA, and performing PCR using this.
【００２８】まず、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細胞、例えば、前記のＥＶＯ３４Ｂ
ＩＧまたはＥＶＯ３４ＢＩＧから、一般的手法により全
ＲＮＡを抽出する。さらに抽出された全ＲＮＡから、常
法により、例えば、市販のキットであるオリゴテックス
−ｄｔ３０（宝酒造社製）を用いて、ｍＲＮＡの単離を
行う。次いで、得られたｍＲＮＡからｃＤＮＡを合成し
、これをＰＣＲの鋳型とする。First, hybridoma cells producing the monoclonal antibody of the present invention, such as the above EVO34B
Total RNA is extracted from IG or EVO34BIG using standard techniques. Furthermore, mRNA is isolated from the extracted total RNA by a conventional method, for example, using a commercially available kit, Oligotex-dt30 (manufactured by Takara Shuzo Co., Ltd.). Next, cDNA is synthesized from the obtained mRNA and used as a template for PCR.
【００２９】次に，ＩｇＧの可変部領域中に存在する保
存的な配列をＫａｂａｔらのデータファイル（Ｓｅｑｕ
ｅｎｃｅｓ ｏｆ Ｐｒｏｔｅｉｎｓ Ｉｍｍｕｎｏｌ
ｏｇｉｃａｌ Ｉｎｔｅｒｅｓｔ， ＵＳ Ｄｅｐｔ．
ｏｆ Ｈｅａｌｔｈ ａｎｄ Ｈｕｍｍａｎ Ｓｅｒ
ｖｉｃｅｓ， ＵＳ Ｇｏｖｅｒｎｍｅｎｔ Ｐｒｉｎ
ｔｉｙ Ｏｆｆｉｃｅ （１９８７）から採用し、この
配列からプライマーを設計する。上記配列は成熟タンパ
ク質のアミノ末端付近に対応するＤＮＡ配列であり、シ
グナル配列が含まれていない。この配列の一部をＰＣＲ
に用いるプライマーとして選択する（プライマーの配列
を配列番号８〜１１に示す）。ＰＣＲにより得られる可
変部領域遺伝子の塩基配列を、例えば、Ｓａｎｇｅｒ，
Ｆら（Ｐｒｏｃ．Ｎａｔｌ． Ａｃａｄ． Ｓｃｉ．
ＵＳＡ ７４巻，５４６３頁 （１９７７））のジデ
オキシ法あるいはそれを改変した方法により決定する。Next, the conserved sequences present in the variable region of IgG were extracted from the data file of Kabat et al.
Ences of Proteins Immunol
logical Interest, US Dept.
of Health and Human Ser
vices, US Government Print
(1987), and primers are designed from this sequence. The above sequence is a DNA sequence corresponding to the vicinity of the amino terminus of the mature protein and does not contain a signal sequence. PCR part of this sequence
(The sequences of the primers are shown in SEQ ID NOS: 8 to 11). The nucleotide sequence of the variable region gene obtained by PCR is determined by, for example, Sanger,
F et al. (Proc. Natl. Acad. Sci.
USA Vol. 74, p. 5463 (1977)) or a modified method thereof.
【００３０】このようにして得られたＤＮＡ配列を配列
番号５（Ｈ鎖）および配列番号６（Ｌ鎖）に示す。プラ
イマー部分の配列を考慮すると、Ｈ鎖の可変部領域をコ
ードする遺伝子は、配列番号５の３４番目のＣから３５
４番目のＴまでのＤＮＡ配列を有することがわかる。プ
ライマー部分を除いた目的のＤＮＡ配列に対応するアミ
ノ酸配列を配列番号１に示す。Ｌ鎖の可変部領域をコー
ドする遺伝子は、配列番号６の３２番目のＣから３４０
番目のＡまでのＤＮＡ配列を有する。このＤＮＡ配列に
対応するプライマー部分を除いた目的のアミノ酸配列を
配列番号２に示す。The DNA sequences thus obtained are shown in SEQ ID NO: 5 (H chain) and SEQ ID NO: 6 (L chain). Considering the sequence of the primer part, the gene encoding the variable region of the H chain is from C to 35 of SEQ ID NO: 5.
It can be seen that it has a DNA sequence up to the fourth T. The amino acid sequence corresponding to the target DNA sequence excluding the primer portion is shown in SEQ ID NO: 1. The gene encoding the variable region of the L chain is from C to 340 of SEQ ID NO: 6.
It has a DNA sequence up to number A. The target amino acid sequence excluding the primer portion corresponding to this DNA sequence is shown in SEQ ID NO: 2.
【００３１】ところで、上記ハイブリドーマＥＶＯ３４
ＢＩＧ，ＥＶＯ１４１１Ｇなどにより産生される抗ＰＴ
ＨｒＰモノクローナル抗体は、高カルシウム血症の治療
・予防剤として有用であるが、これらのハイブリドーマ
はげっ歯類であるマウス由来であるため、これをヒトに
長期間投与するとアレルギー反応が起こる場合がある。
上記のように、このモノクローナル抗体の可変部領域の
配列が明かになったため、次に発明者らは、これをヒト
抗体の定常部領域に結合させたげっ歯類／ヒトキメラモ
ノクローナル抗体の調製を試みた。By the way, the above hybridoma EVO34
Anti-PT produced by BIG, EVO1411G, etc.
HrP monoclonal antibodies are useful as therapeutic and preventive agents for hypercalcemia, but since these hybridomas are derived from mice, which are rodents, allergic reactions may occur when administered to humans over a long period of time. . As mentioned above, since the sequence of the variable region of this monoclonal antibody was clarified, the inventors next attempted to prepare a rodent/human chimeric monoclonal antibody by binding this to the constant region of a human antibody. I tried.
【００３２】本発明のげっ歯類／ヒトキメラモノクロー
ナル抗体を作製するために必要なヒト免疫グロブリン定
常部領域遺伝子（Ｈ鎖の定常部領域に対応する遺伝子：
Ｃγｌ、およびＬ鎖の定常部領域に対応する遺伝子：Ｃ
к）は、例えば、ヒト免疫グロブリン産生ガン細胞ＡＲ
Ｈ−７７［ＡＴＣＣ（アメリカン タイプカルチャー
コレクション）より入手可能である］から得られう
る。
まず、ＡＲＨ−７７（ＡＴＣＣ ＣＲＬ−１６２
１）を培養し、得られた細胞からゲノムＤＮＡを得る。
このゲノムＤＮＡから目的とするＤＮＡ断片を得るため
に必要とされる制限酵素を用いて完全に分解した後、ア
ガロースゲル電気泳動で分子量分画し、該目的のＤＮＡ
断片（Ｃγｌを含むＤＮＡ断片およびＣкを含むＤＮＡ
断片）を得る。Human immunoglobulin constant region genes necessary for producing the rodent/human chimeric monoclonal antibody of the present invention (genes corresponding to the constant region of the H chain:
Gene corresponding to Cγl and L chain constant region: C
K), for example, human immunoglobulin-producing cancer cells AR
H-77 [available from ATCC (American Type Culture Collection)]. First, ARH-77 (ATCC CRL-162
1) and obtain genomic DNA from the resulting cells. After complete digestion using the restriction enzymes required to obtain the desired DNA fragment from this genomic DNA, molecular weight fractionation is performed by agarose gel electrophoresis to obtain the desired DNA fragment.
Fragments (DNA fragments containing Cγl and DNA fragments containing Cγl)
fragment).
【００３３】このようにして得られるＤＮＡ断片は、適
当なクローニングベクターに組み込まれて、ＤＮＡライ
ブラリーが構築される。例えば、ヒトＩｇＧのＣγｌを
含むＤＮＡ断片は、ＥｃｏＲＩで切断したラムダクロー
ニングベクターＥＭＢＬ４（ストラタジーン社製）にク
ローニングし、Ｈ鎖ファージライブラリーが構築される
。一方、ヒトＩｇＧのＣкを含むＤＮＡ断片は、Ｅｃｏ
ＲＩで切断したラムダクローニングベクターλｇｔ１０
（ストラタジーン社製）にクローニングし、同様にして
、Ｌ鎖ファージライブラリーが構築される。[0033] The DNA fragment thus obtained is inserted into an appropriate cloning vector to construct a DNA library. For example, a DNA fragment containing Cγl of human IgG is cloned into a lambda cloning vector EMBL4 (manufactured by Stratagene) cut with EcoRI to construct a heavy chain phage library. On the other hand, the DNA fragment containing C of human IgG is Eco
Lambda cloning vector λgt10 cut with RI
(manufactured by Stratagene), and a light chain phage library is constructed in the same manner.
【００３４】このようにして増幅されたＤＮＡ断片から
、目的とするＣγｌおよびＣк断片を、プローブを用い
たハイブリダイゼーション法によりスクリーニングする
。上記プローブは、Ｅｌｌｉｓｏｎ Ｊ．Ｗら（Ｎｕｃ
ｌｅｉｃ Ａｃｉｄｓ Ｒｅｓｅａｒｃｈ， １０巻、
４０７１頁（１９８２）が明かにしたＣγｌおよびＣк
の配列に基づいて作製される。例えばＣγｌの遺伝子か
ら２個のプローブ（プローブＨ１およびＨ２；それぞれ
配列番号１２および１３に示される）が設計される。プ
ローブＨ１およびＨ２は、ＡＲＨ−７７のＪＨ領域（Ｋ
ｕｄｏｕ Ａ． Ｊ． Ｉｍｎ． １３５巻、６４２頁
（１９８５））およびフレームワーク領域に対応する遺
伝子配列である。他方、Ｃк遺伝子からプローブＬ１（
配列番号１４に示す）が設計される。[0034] The DNA fragments thus amplified are screened for the desired Cγl and Cκ fragments by a hybridization method using a probe. The above probe was described by Ellison J. W et al.
leic acids research, vol. 10,
Cγl and Cк revealed by p. 4071 (1982)
is created based on the sequence of For example, two probes (probes H1 and H2; shown in SEQ ID NO: 12 and 13, respectively) are designed from the Cγl gene. Probes H1 and H2 probed the JH region (K
udou A. J. Immn. 135, p. 642 (1985)) and the gene sequence corresponding to the framework region. On the other hand, probe L1 (
(shown in SEQ ID NO: 14) is designed.
【００３５】上記ヒトＩｇＧ定常部領域遺伝子のスクリ
ーニングは、常法に従って、プラークハイブリダイゼー
ション法により行われる。まず上記各々のファージライ
ブラリーを適切な株に感染させ、上記プローブとハイブ
リダイズするプラークをスクリーニングする。次にジデ
オキシ法で塩基配列の決定を行い、タンパク質をコード
する部分の遺伝子が、すでに明かになっているＣγｌま
たはＣкの配列と一致することを確認する。それぞれの
挿入断片を適当な制限酵素を用いて切り出し、サブクロ
ーニングする。例えば、Ｃγｌ断片をＨｉｎｄＩＩＩと
ＸｂａＩとで切り出し、ｐＵＣ１９にサブクローニング
し、Ｃγｌ断片を有するベクターであるｐＵＣ−Ｃγｌ
を得る。同様にＣк断片をＥｃｏＲＩで切り出し，ｐＵ
Ｃ１９にサブクローニングし、Ｃкを有するベクターで
あるｐＬ１４−２を得る。[0035] Screening for the human IgG constant region genes is carried out by plaque hybridization according to a conventional method. First, appropriate strains are infected with each of the above phage libraries, and plaques that hybridize with the above probes are screened. Next, the base sequence is determined using the dideoxy method, and it is confirmed that the gene encoding the protein matches the already known sequence of Cγl or Cк. Each inserted fragment is excised using an appropriate restriction enzyme and subcloned. For example, the Cγl fragment is excised with HindIII and XbaI, subcloned into pUC19, and pUC-Cγl, which is a vector containing the Cγl fragment, is
get. Similarly, the Cк fragment was excised with EcoRI, and pU
By subcloning into C19, a vector containing CK, pL14-2, is obtained.
【００３６】上記抗ＰＴＨｒＰ抗体のＨ鎖の可変部領域
をコードするマウス由来のＤＮＡ配列、抗ＰＴＨｒＰ抗
体のＬ鎖の可変部領域をコードするマウス由来のＤＮＡ
配列、ヒトＩｇＧのＨ鎖の定常部領域をコードするＤＮ
Ａ発現ベクターを構築し、これを適当な宿主に導入して
得られる形質転換体により、上記マウス由来の抗ＰＴＨ
ｒＰ抗体の可変部領域とヒトＩｇＧの定常部領域とを有
するキメラモノクローナル抗体が生産され得る。Mouse-derived DNA sequence encoding the variable region of the H chain of the anti-PTHrP antibody, mouse-derived DNA encoding the variable region of the L chain of the anti-PTHrP antibody
Sequence, DN encoding the constant region of human IgG H chain
A transformant obtained by constructing an expression vector and introducing it into an appropriate host produces the anti-PTH
Chimeric monoclonal antibodies having rP antibody variable regions and human IgG constant regions can be produced.
【００３７】上記発現ベクターの構築を行うために、ま
ず、上記マウスＶＤＪＨ遺伝子の修飾を行い、タンパク
質の分泌に不可欠なシグナル配列を付加する。例えば、
Ｃａｂｉｌｌｙ，Ｓらの報告（Ｐｒｏｃ． Ｎａｔｌ．
Ｓｃｉ． ＵＳＡ．， ８１巻、３２７２頁（１９８
４））にある抗ＰＴＨｒＰ抗体（Ａｎｔｉ−ＣＥＡ
ＭｏＡｂ）のＨ鎖のシグナル配列の一部に対応する部分
を含む遺伝子を合成する。これをプライマー（配列番号
１５に示す）とし、上記ＶＤＪＨ遺伝子を鋳型としたＰ
ＣＲを行い、シグナル配列が付加されたＬＶＤＪＨ遺伝
子が得られる。同様に、マウスＶＪＬ遺伝子についても
上記Ａｎｔｉ−ＣＥＡ ＭｏＡｂのＬ鎖のシグナル配
列の一部に対応する部分を含む遺伝子を合成し、これを
プライマー（配列番号１６に示す）としてＰＣＲを行い
、シグナル配列が付加されたＬＶＪＬ遺伝子が得られる
。[0037] In order to construct the above expression vector, first, the above mouse VDJH gene is modified to add a signal sequence essential for protein secretion. for example,
Report by Cabilly, S. et al. (Proc. Natl.
Sci. USA. , vol. 81, p. 3272 (198
4)) Anti-PTHrP antibody (Anti-CEA
A gene containing a portion corresponding to a portion of the H chain signal sequence of MoAb) is synthesized. Using this as a primer (shown in SEQ ID NO: 15), P
CR is performed to obtain the LVDJH gene to which a signal sequence has been added. Similarly, for the mouse VJL gene, a gene containing a part corresponding to a part of the signal sequence of the L chain of the above Anti-CEA MoAb was synthesized, and PCR was performed using this as a primer (shown in SEQ ID NO: 16) to obtain the signal sequence. The LVJL gene to which is added is obtained.
【００３８】本発明のキメラ抗体発現ベクターを調製方
法の一例を次に説明する。An example of a method for preparing the chimeric antibody expression vector of the present invention will be described below.
【００３９】まず、ｐＳＶｅＳａｌＩ−ＨｉｎｄＩＩＩ
（ヨーロッパ特許出願公開Ｎｏ．０４２０５０２をＨｉ
ｎｄＩＩＩで切断して、平滑末端化し、ｐＸｂａＩリン
カーを付加し、Ｔ４ＤＮＡリガーゼで環状化するとｐＳ
ＶｅＸｂａＩが得られる（図２）。このベクターにはＸ
ｂａＩ認識部位の上流にＳＶ４０の複製起点を含む初期
プロモーター領域が存在している。次に、上記ヒトＨ鎖
Ｃγｌ遺伝子を含むプラスミドｐＵＣ−Ｃγ１から切り
出した、Ｃγｌ遺伝子を含むＤＮＡ断片と；上記マウス
Ｈ鎖のＬＶＤＪＨ遺伝子断片を含む断片と；ｐＳＶｅＸ
ｂａＩをＸｂａＩで切断したベクターとを連結すること
によりｐＳＶｅＨ２６が構築される（図２参照）。First, pSVeSalI-HindIII
(Hi European Patent Application Publication No. 0420502
Cutting with ndIII to make blunt ends, adding a pXbaI linker, and circularizing with T4 DNA ligase yields pS.
VeXbaI is obtained (Figure 2). This vector has an
There is an early promoter region containing the SV40 replication origin upstream of the baI recognition site. Next, a DNA fragment containing the Cγl gene cut out from the plasmid pUC-Cγ1 containing the human H chain Cγl gene; a fragment containing the mouse H chain LVDJH gene fragment; pSVeX
pSVeH26 is constructed by ligating baI with a vector cut with XbaI (see Figure 2).
【００４０】これとは別に、Ｃк遺伝子をｐＵＣ１９に
サブクローニングして得られるベクターｐＬ１４−２（
前出）由来のＣк遺伝子を含む断片；およびｐＳＶｅＸ
ｂａＩの切断断片をＴ４ＤＮＡリガーゼで連結すること
により、ｐＳＶｅＬ２６が得られる（図３）。Separately, vector pL14-2 (
A fragment containing the Cк gene derived from the above); and pSVeX
By ligating the baI cut fragment with T4 DNA ligase, pSVeL26 is obtained (Figure 3).
【００４１】上記ｐＳＶｅＬ２６由来のｍＬＶＪＬ遺伝
子およびＣк遺伝子を有するＤＮＡ断片；およびｐＳＶ
ｅＨ２６由来のｍＬＶＤＪＨ遺伝子およびＣγｌ遺伝子
とを有するＤＮＡ断片とを連結することにより、これら
４種の遺伝子を有する発現ベクターであるｐＳＶｅＨＬ
２６が構築される（図４）。さらに、この発現ベクター
を動物細胞に導入することを考慮し、該ベクターに適当
な薬剤耐性遺伝子、例えば、ＤＨＦＲおよびＥｃｏ−ｇ
ｐｔが導入され、ｐＳＶｅＨＬ２６−ＤＭが構築される
（図５）。[0041] A DNA fragment having the mLVJL gene and Cκ gene derived from the above pSVeL26; and pSV
By ligating a DNA fragment containing the eH26-derived mLVDJH gene and the Cγl gene, pSVeHL, an expression vector containing these four genes, was created.
26 is constructed (Figure 4). Furthermore, considering the introduction of this expression vector into animal cells, appropriate drug resistance genes, such as DHFR and Eco-g, are added to the vector.
pt is introduced and pSVeHL26-DM is constructed (Figure 5).
【００４２】このようにして得られた発現ベクターは、
適当な宿主、例えば、マウス骨髄腫細胞あるいは、ハム
スター細胞株に導入される。例えば、マウス骨髄腫細胞
であるＦＯ細胞にＤＥＡＥ−デキストラン法で発現ベク
ターが導入され得る（高井俊行、細胞工学、９巻、６５
２頁（１９９０））。あるいは、ハムスター細胞である
チャイニーズハムスター卵巣（ＣＨＯ）細胞（ＡＴＣＣ
ＣＣＬ−６１）にリン酸カルシウム法で、Ｃｈｅｎ
ら、（Ｍｏｌ． Ｃｅｌｌ． Ｂｉｏｌ．， ７巻、２
７４５頁 （１９８７）の方法により、発現ベクター
が導入され得る。[0042] The expression vector thus obtained is
The cells are introduced into a suitable host, such as mouse myeloma cells or hamster cell lines. For example, an expression vector can be introduced into FO cells, which are mouse myeloma cells, by the DEAE-dextran method (Toshiyuki Takai, Cell Engineering, Vol. 9, 65
2 (1990)). Alternatively, hamster cells, Chinese hamster ovary (CHO) cells (ATCC
CCL-61) using the calcium phosphate method, Chen
et al., (Mol. Cell. Biol., vol. 7, 2
Expression vectors can be introduced by the method of P. 745 (1987).
【００４３】宿主に発現ベクターが導入されたか否かは
、該ベクターが有する薬剤耐性マーカーを利用して調べ
ることが可能である、このようにして得られた形質転換
体が産生する抗体は、ＰＴＨｒＰに対する結合能を調べ
ることにより抗ＰＴＨｒＰモノクローナル抗体であるこ
とが確認される。産生された抗体量は、例えば、ＥＬＩ
ＳＡ法により測定され得る。本法により得られた代表的
な形質転換体の名称を後述の実施例１０の表２に示す。[0043] Whether or not the expression vector has been introduced into the host can be determined using the drug resistance marker possessed by the vector. It is confirmed that the antibody is an anti-PTHrP monoclonal antibody by examining its binding ability. The amount of antibody produced can be measured, for example, by ELI
It can be measured by the SA method. The names of typical transformants obtained by this method are shown in Table 2 of Example 10 below.
【００４４】このような形質転換体を適当な培地で培養
することにより抗ヒト副甲状腺ホルモン関連タンパクを
認識するマウス／ヒトキメラモノクローナル抗体が生産
される。このモノクローナル抗体は、上記マウス由来の
モノクローナル抗体と同等の性能を有し、高カルシウム
血症の治療・予防剤として好適に利用され得る。さらに
、このモノクローナル抗体をヒトに長期間にわたり投与
した場合もマウス由来のモノクローナル抗体の場合とは
異なり、アレルギー反応を引き起こすことがない。[0044] By culturing such a transformant in an appropriate medium, a mouse/human chimeric monoclonal antibody that recognizes anti-human parathyroid hormone-related protein is produced. This monoclonal antibody has performance equivalent to the mouse-derived monoclonal antibody described above, and can be suitably used as a therapeutic/preventive agent for hypercalcemia. Furthermore, even when this monoclonal antibody is administered to humans over a long period of time, it does not cause allergic reactions, unlike the case of mouse-derived monoclonal antibodies.
【００４５】上記組み換えによるキメラ抗体発現ベクタ
ーの構築において、例えばマウスモノクローナル抗体遺
伝子の可変部領域遺伝子の特定部位を変異することによ
り、生成する抗体の１個または数個のアミノ酸の置換、
欠失、付加あるいは転位を起こさせることが可能であり
、よりＰＴＨｒＰに対して特異性や親和性の高い抗体に
改良することができる。In the construction of the chimeric antibody expression vector by recombination, for example, by mutating a specific site of the variable region gene of the mouse monoclonal antibody gene, substitution of one or several amino acids in the resulting antibody;
It is possible to cause deletion, addition, or rearrangement, and it is possible to improve antibodies with higher specificity and affinity for PTHrP.
【００４６】[0046]
【実施例】以下に実施例を挙げて本発明を更に詳しく説
明するが、本発明はこれらに限定されるものではない。EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited thereto.
【００４７】［実施例１］（ｒＰＴＨｒＰの調製）Ｍａ
ｒｔｉｎ，Ｔ．Ｊ．ら（Ｊ．Ｂｉｏｌ．Ｃｈｅｍ．，２
６４巻，１４８０６頁（１９８９））の方法に準じ、次
のようにＰＴＨｒＰ遺伝子を合成し、大腸菌に組み込ん
で発現させてｒＰＴＨｒＰを得、これを後述の実施例４
で得られた抗ＰＴＨｒＰモノクローナル抗体で精製した
。[Example 1] (Preparation of rPTHrP) Ma
rtin, T. J. (J. Biol. Chem., 2
64, p. 14806 (1989)), the PTHrP gene was synthesized as follows, incorporated into E. coli and expressed to obtain rPTHrP, which was used in Example 4 described below.
It was purified using the anti-PTHrP monoclonal antibody obtained in .
【００４８】まず、ＤＮＡ合成機（アプライドバイオシ
ステムス社製）を用いて、約１１０ｍｅｒの大きさのＤ
ＮＡ（いずれもＰＴＨｒＰの１〜１４１位のＤＮＡのう
ちの一部）を８本合成し、各ＤＮＡをＯＤＳカラム（山
村化学研究所製）を用いた高速液体クロマトグラフィー
（島津製作所社製）で精製し、次いでアニールを行った
。その後、各ＤＮＡフラグメントをＴ４ＤＮＡリガーゼ
（宝酒造社製）により連結させ、目的とするＰＴＨｒＰ
（１−１４１）遺伝子を得た。このＰＴＨｒＰ遺伝子を
ベクターｐＫＫ２３３−２（ファルマシア社製）に組み
込んで発現ベクターを得た。これを大腸菌ＪＭ１０５に
導入して形質転換体を得た。このｒＰＴＨｒＰ発現大腸
菌を大量調製した後、超音波処理により大腸菌を溶菌さ
せた後、ｒＰＴＨｒＰを抽出した。ｒＰＴＨｒＰ抽出物
をセファクリルＳ−２００ＨＲ（ファルマシア社製）を
用いてゲル濾過し、分子量１万５千〜３万付近を分画し
た。この画分を、ＣＮＢｒ活性化セファロース４Ｂ（フ
ァルマシア社製）と、後述の実施例４で精製して得た抗
ＰＴＨｒＰ（１−３４）モノクローナル抗体とをカップ
リングさせた（５ｍｇ／担体）抗体カラムにチャージし
て精製した。一回の精製で取得されたｒＰＴＨｒＰは約
１０ｍｇであった。First, using a DNA synthesizer (manufactured by Applied Biosystems), D of approximately 110mer size was synthesized.
Eight NAs (all part of the DNA at positions 1 to 141 of PTHrP) were synthesized, and each DNA was subjected to high performance liquid chromatography (manufactured by Shimadzu Corporation) using an ODS column (manufactured by Yamamura Kagaku Kenkyusho). It was purified and then annealed. Thereafter, each DNA fragment was ligated using T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) to obtain the desired PTHrP.
(1-141) gene was obtained. This PTHrP gene was inserted into vector pKK233-2 (manufactured by Pharmacia) to obtain an expression vector. This was introduced into E. coli JM105 to obtain a transformant. After preparing a large amount of this rPTHrP-expressing E. coli, the E. coli was lysed by ultrasonication, and then rPTHrP was extracted. The rPTHrP extract was subjected to gel filtration using Sephacryl S-200HR (manufactured by Pharmacia), and fractions having a molecular weight of around 15,000 to 30,000 were fractionated. This fraction was coupled to an antibody column (5 mg/carrier) coupled with CNBr-activated Sepharose 4B (manufactured by Pharmacia) and the anti-PTHrP (1-34) monoclonal antibody purified in Example 4 described below. It was charged and refined. About 10 mg of rPTHrP was obtained in one purification.
【００４９】［実施例２］（抗ＰＴＨｒＰモノクローナ
ル抗体産生ハイブリドーマの調製）ＰＴＨｒＰのＮ末端
部分のペプチドであるＰＴＨｒＰ（１−３４）（ペプチ
ド研究所製）、または実施例１で得られたｒＰＴＨｒＰ
ペプチド５０μｇをフロイント完全アジュバントと混合
した後、８〜１２週令のＢａｌｂ／ｃマウスの皮下へ免
疫した。初回免疫後、３週間間隔で計３回の追加免疫を
行った。細胞融合に供する７日、５日、３日、２日、お
よび１日前に、マウス尾静脈より一匹当り５μｇの上記
ペプチドを投与した。その後、マウス脾を摘出し、免疫
脾細胞を得た。[Example 2] (Preparation of anti-PTHrP monoclonal antibody-producing hybridoma) PTHrP(1-34) (manufactured by Peptide Institute), which is a peptide of the N-terminal portion of PTHrP, or rPTHrP obtained in Example 1
After mixing 50 μg of the peptide with Freund's complete adjuvant, Balb/c mice aged 8 to 12 weeks were subcutaneously immunized. After the initial immunization, a total of three booster immunizations were performed at 3-week intervals. 7 days, 5 days, 3 days, 2 days, and 1 day before cell fusion, 5 μg of the above peptide was administered per mouse via the tail vein. Thereafter, the mouse spleen was removed to obtain immune splenocytes.
【００５０】次に、免疫脾細胞とマウスミエローマ細胞
（ＦＯ）とを２：１の割合で混合し、ポリエチレングリ
コール１５００の存在下で融合させ、１０％ＦＢＳを含
むイスコフ改変ダルベッコ培地に、脾細胞が２×１０６
個／ｍｌになるように調整し、９６穴マイクロプレート
に０．１ｍｌ／ウェルの割合で分注した。翌日、ＨＡＴ
培地を０．１ｍｌ／ウェルの割合で加え、以後１日おき
に培地の半量を新しいＨＡＴ培地と交換した。約２週間
後、ハイブリドーマのコロニーの形成が見られた。この
コロニーをさらに２４穴プレートで培養し、ＰＴＨｒＰ
（１−３４）由来のハイブリドーマクローン４２０株お
よびｒＰＴＨｒＰ由来のハイブリドーマクローン６１７
株を得た。Next, immunized splenocytes and mouse myeloma cells (FO) were mixed at a ratio of 2:1, fused in the presence of polyethylene glycol 1500, and the splenocytes were added to Iscove's modified Dulbecco's medium containing 10% FBS. is 2×106
The solution was adjusted to 0.1 ml/well and dispensed into a 96-well microplate at a rate of 0.1 ml/well. The next day, H.A.T.
Medium was added at a rate of 0.1 ml/well, and half of the medium was replaced with fresh HAT medium every other day thereafter. After about two weeks, formation of hybridoma colonies was observed. This colony was further cultured in a 24-well plate, and PTHrP
(1-34)-derived hybridoma clone 420 strain and rPTHrP-derived hybridoma clone 617 strain
I got the stock.
【００５１】これらのハイブリドーマクローンの培養上
清中に抗ＰＴＨｒＰモノクローナル抗体が産生されてい
るか否かを、次のように酵素抗体法により検討した。ま
ず、ｒＰＴＨｒＰを用い、１００ｎｇ／ウェルの割合で
コーティングした９６穴プレートにハイブリドーマの培
養上清を加え、３７℃で１時間反応させた。洗浄した後
、ペルオキシダーゼ標識抗マウスＩｇＧ＋ＩｇＭを加え
た。さらに洗浄した後、ｏ−フェニレンジアミン液を加
え、室温にて２０分間発色させた。４Ｎ硫酸を加えて反
応を停止させた後、４９２ｎｍで吸光度を測定した。[0051] Whether or not anti-PTHrP monoclonal antibodies were produced in the culture supernatants of these hybridoma clones was examined by the enzyme antibody method as follows. First, hybridoma culture supernatant was added to a 96-well plate coated with rPTHrP at a rate of 100 ng/well, and reacted at 37° C. for 1 hour. After washing, peroxidase-labeled anti-mouse IgG+IgM was added. After further washing, o-phenylenediamine solution was added and color was developed at room temperature for 20 minutes. After stopping the reaction by adding 4N sulfuric acid, absorbance was measured at 492 nm.
【００５２】以上の操作により、抗原としてＰＴＨｒＰ
（１−３４）を用いた場合には１９１株の、またｒＰＴ
ＨｒＰを用いた場合には３５８株の抗ＰＴＨｒＰモノク
ローナル抗体を産生しているハイブリドーマクローンが
得られたことがわかった。[0052] Through the above operations, PTHrP was used as an antigen.
(1-34), 191 strains and rPT
It was found that when HrP was used, 358 hybridoma clones producing anti-PTHrP monoclonal antibodies were obtained.
【００５３】次に、これらのハイブリドーマクローンの
培養上清を用いて、ｒＰＴＨｒＰに対する抗ＰＴＨｒＰ
モノクローナル抗体の中和活性能について検討した。Ｇ
ｕｔｉｅｒｒｅｚ，Ｇ．Ｅ．ら（Ｊ．Ｂｏｉｌ．Ｃｈｅ
ｍ．，２６２巻，１５８４５頁（１９８７））の方法に
準じて、次の方法により、〔Ｈ〕アデニンを取り込ませ
た、ＲＯＳ１７／２．８細胞またはＵＭＲ１０６細胞に
おける〔Ｈ〕ＡＴＰから〔Ｈ〕ｃＡＭＰへの変換を指標
として測定した。まず、４８穴マルチウェルプレートに
２．５×１０４個／ウェルのＲＯＳ１７／２．８細胞ま
たはＵＭＲ１０６細胞を接種し、ＦＢＳを１０％の割合
で添加したＤＭＥ培地中で３日間培養した。
培養液を除去した後、Ｈａｎｋｓの平衡塩類液で２回洗
浄し、新しい培地および１μＣｉ／ｍｌの〔Ｈ〕アデニ
ンを加え、３７℃で２時間インキュベートした。細胞を
さらに２回洗浄した後、１ｍＭ３−イソブチル−１−メ
チルキサンチン（ＩＢＭＸ；シグマ社製）を含み、ＦＢ
Ｓを２％の割合で添加したＤＭＥ培地５０μｌを加え３
７℃で５分間インキュベートした。次いでハイブリドー
マ培養上清２５μｌおよびｒＰＴＨｒＰ溶液（最終濃度
１０−７〜１０−１Ｍ）２５μｌを加え、さらに１０分
間インキュベートした。次いで、トリクロル酢酸（最終
濃度０．１２Ｍ）１ｍｌを加えて反応を終了させ、４Ｎ
ＮａＯＨ ２０μｌで中和した。合成されたｃＡＭＰ
は、Ｓａｌｏｍｏｎ，Ｙ．ら（Ａｎａｌ．Ｂｉｏｃｈｅ
ｍ．，５８巻，５４１頁（１９７４））の方法にしたが
って分離した後、その放射活性を測定し、ｒＰＴＨｒＰ
に対する抗ＰＴＨｒＰモノクローナル抗体の中和活性能
を評価した。[0053] Next, using the culture supernatants of these hybridoma clones, anti-PTHrP
The neutralizing activity of monoclonal antibodies was investigated. G
utierrez, G. E. (J. Boil. Che
m. [H]ATP to [H]cAMP in ROS17/2.8 cells or UMR106 cells, in which [H]adenine was incorporated, according to the method of [H]ATP to [H]cAMP. was measured using the conversion as an index. First, 2.5 x 104 cells/well of ROS17/2.8 cells or UMR106 cells were inoculated into a 48-well multiwell plate and cultured for 3 days in a DME medium supplemented with FBS at a rate of 10%. After removing the culture medium, the cells were washed twice with Hanks' balanced salt solution, fresh medium and 1 μCi/ml [H]adenine were added, and incubated at 37° C. for 2 hours. After washing the cells two more times, FB containing 1mM 3-isobutyl-1-methylxanthine (IBMX; manufactured by Sigma)
Add 50 μl of DME medium supplemented with S at a ratio of 2%.
Incubate for 5 minutes at 7°C. Next, 25 μl of hybridoma culture supernatant and 25 μl of rPTHrP solution (final concentration 10 −7 to 10 −1 M) were added, and the mixture was further incubated for 10 minutes. Next, 1 ml of trichloroacetic acid (final concentration 0.12M) was added to terminate the reaction, and 4N
Neutralized with 20 μl of NaOH. Synthesized cAMP
Salomon, Y. (Anal.Bioche
m. , vol. 58, p. 541 (1974)), its radioactivity was measured, and rPTHrP
The neutralizing activity of the anti-PTHrP monoclonal antibody against PTHrP was evaluated.
【００５４】以上の操作により、抗原としてＰＴＨｒＰ
（１−３４）を用いた場合には１０８株が、そしてｒＰ
ＴＨｒＰを用いた場合には３５株が、ｒＰＴＨｒＰを中
和する活性を有する抗ＰＴＨｒＰモノクローナル抗体を
産生しているハイブリドーマクローンであることがわか
った。[0054] Through the above operations, PTHrP was used as an antigen.
(1-34), 108 strains were used, and rP
When THrP was used, 35 strains were found to be hybridoma clones producing anti-PTHrP monoclonal antibodies having activity to neutralize rPTHrP.
【００５５】［実施例３］（ハイブリドーマにより産生
された抗ＰＴＨｒＰモノクローナル抗体のイムノグロブ
リンクラスおよびサブクラス）実施例２で得られた中和
活性能のある抗ＰＴＨｒＰモノクローナル抗体産生ハイ
ブリドーマ株が産生する抗ＰＴＨｒＰモノクローナル抗
体のイムノグロブリンクラスおよびサブクラスについて
検討した。イムノグロブリンクラスおよびサブクラスの
検討は、免疫グロブリンサブクラスポリクローナル抗体
を用い、実施例２で用いた酵素抗体法に従って行った。
代表例としてＰＴＨｒＰ（１−３４）由来およびｒＰＴ
ＨｒＰ由来の各々５株、計１０株の結果を表１に示す。[Example 3] (Immunoglobulin class and subclass of anti-PTHrP monoclonal antibody produced by hybridoma) Anti-PTHrP produced by the hybridoma strain producing the anti-PTHrP monoclonal antibody capable of neutralizing activity obtained in Example 2 We investigated the immunoglobulin classes and subclasses of monoclonal antibodies. Immunoglobulin classes and subclasses were investigated using immunoglobulin subclass polyclonal antibodies according to the enzyme antibody method used in Example 2. Typical examples include PTHrP(1-34) derived and rPT
Table 1 shows the results for a total of 10 strains, 5 strains each derived from HrP.
【００５６】[0056]
【表１】[Table 1]
【００５７】［実施例４］（抗ＰＴＨｒＰモノクローナ
ル抗体の調製）実施例３のハイブリドーマクローン１０
株の細胞を各々、２週間前にプリスタン０．５ｍｌを腹
腔内投与した１０〜１２週令Ｂａｌｂ／ｃマウスの腹腔
内に、５×１０６個／匹の割合で接種した。腹水が充分
に生産された後、この腹水を回収した。腹水中のモノク
ローナル抗体は、硫安塩析し、さらに１０ｍＭリン酸二
水素カリウム（ｐＨ６．０）で透析した後、ベーカーボ
ンドＡＢＸカラム（Ｊ．Ｔ．ベーカー社製）を用いて２
５０ｍＭリン酸二水素カリウム（ｐＨ６．８）のグラジ
エントにより精製した。得られた抗ＰＴＨｒＰモノクロ
ーナル抗体は２０．５〜５８．７ｍｇであった。[Example 4] (Preparation of anti-PTHrP monoclonal antibody) Hybridoma clone 10 of Example 3
Cells of each strain were intraperitoneally inoculated at a rate of 5 x 106 cells/mouse into 10-12 week old Balb/c mice that had been intraperitoneally administered with 0.5 ml of pristane two weeks earlier. After sufficient ascites was produced, the ascites was collected. Monoclonal antibodies in ascites were precipitated with ammonium sulfate, further dialyzed against 10 mM potassium dihydrogen phosphate (pH 6.0), and then purified using a Baker Bond ABX column (manufactured by J.T. Baker).
Purification was achieved by a gradient of 50 mM potassium dihydrogen phosphate (pH 6.8). The amount of the obtained anti-PTHrP monoclonal antibody was 20.5 to 58.7 mg.
【００５８】［実施例５］（抗ＰＴＨｒＰモノクローナ
ル抗体の高カルシウム血症動物モデルでの特性）実施例
４で得られた抗ＰＴＨｒＰモノクローナル抗体の高カル
シウム血症動物モデルでの治療・予防効果を、浸透圧ポ
ンプ（アルザ社製；モデル２００２）を用いた系で検討
した。まず、浸透圧ポンプに１％牛血清アルブミンを含
む生理食塩水に溶解させたｒＰＴＨｒＰ（１．７５ｍｇ
／ｍｌ）２００μｌを充填し、その浸透圧ポンプを１０
週令のＢａｌｂ／ｃマウスの腹腔内に埋め込み、２日毎
に尾静脈より血液を５０μｌずつ採取し、血清中のＣａ
レベルを原子吸光度計（日立製作所：モデルＺ６１００
）を用いて測定した。[Example 5] (Characteristics of anti-PTHrP monoclonal antibody in a hypercalcemic animal model) The therapeutic and preventive effects of the anti-PTHrP monoclonal antibody obtained in Example 4 in a hypercalcemic animal model were The study was conducted using a system using an osmotic pump (manufactured by ALZA, model 2002). First, rPTHrP (1.75 mg) dissolved in physiological saline containing 1% bovine serum albumin was placed in an osmotic pump.
/ml) and pump the osmotic pump to 10
It was implanted intraperitoneally into a week-old Balb/c mouse, and 50 μl of blood was collected from the tail vein every 2 days to determine the Ca content in the serum.
The level was measured using an atomic absorption spectrometer (Hitachi: Model Z6100)
).
【００５９】予備実験で浸透圧ポンプをマウス生体内に
埋め込んだ後、約５〜６日目で血清中のＣａレベルが１
９〜２０ｍｇ／ｄｌになることが判明したので、浸透圧
ポンプを埋め込んでから６日目に、実施例４で精製した
抗ＰＴＨｒＰモノクローナル抗体３００μｇをマウス尾
静脈内へ投与してその効果を検討した。その結果を図１
に示す。図１に示すように、実施例４で得られた精製抗
ＰＴＨｒＰモノクローナル抗体は高カルシウム血症治療
・予防剤として有効であることが判明した。[0059] In a preliminary experiment, after implanting an osmotic pump into a mouse, the serum Ca level reached 1 about 5 to 6 days later.
Since it was found that the concentration was 9 to 20 mg/dl, on the 6th day after implantation of the osmotic pump, 300 μg of the anti-PTHrP monoclonal antibody purified in Example 4 was administered into the tail vein of the mouse to examine its effect. . The results are shown in Figure 1.
Shown below. As shown in FIG. 1, the purified anti-PTHrP monoclonal antibody obtained in Example 4 was found to be effective as an agent for treating and preventing hypercalcemia.
【００６０】［実施例６］（抗ＰＴＨｒＰモノクローナ
ル抗体のＦ（ａｂ’）２断片の調製）０．０５Ｍクエン
酸ナトリウム／０．１５Ｍ ＮａＣｌ（ｐＨ４．０）の
緩衝液に実施例４で得られた精製抗ＰＴＨｒＰモノクロ
ナール抗体を溶解させ、これをペプシン（シグマ社製）
を用いて３７℃で５分間消化した。上記ペプシンは該抗
ＰＴＨｒＰモノクローナル抗体の３５分の１重量の量を
使用した。次いで、３Ｍ トリス（ｐＨ８．６）を１／
３容量添加して反応を停止させた。[Example 6] (Preparation of F(ab')2 fragment of anti-PTHrP monoclonal antibody) The protein obtained in Example 4 was added to a buffer of 0.05 M sodium citrate/0.15 M NaCl (pH 4.0). Dissolve the purified anti-PTHrP monoclonal antibody, and add it to pepsin (manufactured by Sigma).
was used for 5 minutes at 37°C. The amount of pepsin used was 1/35th the weight of the anti-PTHrP monoclonal antibody. Then, 3M Tris (pH 8.6) was added to
The reaction was stopped by adding 3 volumes.
【００６１】消化物を１０ｍＭ リン酸二水素カリウ
ム（ｐＨ６．０）で透析した後、ベーカーボンドＡＢＸ
カラム（Ｊ．Ｔ．ベーカー社製）を用いて、２５０ｍＭ
リン酸二水素カリウム（ｐＨ６．８）のグラジエントに
よるイオン交換クロマトグラフィーを行い、精製Ｆ（ａ
ｂ’）２断片を回収した。非還元条件下でのＳＤＳ−Ｐ
ＡＧＥゲル電気泳動により検討したところ、分子量１２
２，０００ダルトンの単一バンドが得られた。[0061] After dialyzing the digested product against 10mM potassium dihydrogen phosphate (pH 6.0),
Using a column (manufactured by J.T. Baker), 250mM
Purified F (a
b') Two fragments were recovered. SDS-P under non-reducing conditions
When examined by AGE gel electrophoresis, the molecular weight was 12.
A single band of 2,000 daltons was obtained.
【００６２】このＦ（ａｂ’）２断片精製物は、酵素抗
体法、アデニレートサイクレース促進活性の阻害活性お
よび高カルシウム血症動物モデルにおいて、消化前のＩ
ｇＧと同一の特異性および反応性を有していることが確
認された。[0062] This F(ab')2 fragment purified product was tested by enzyme antibody method, inhibitory activity of adenylate cyclase promoting activity, and hypercalcemic animal model.
It was confirmed that it has the same specificity and reactivity as gG.
【００６３】［実施例７］（抗ＰＴＨｒＰモノクローナ
ル抗体産生ハイブリドーマからの該モノクローナル抗体
可変部領域遺伝子［ＶＤＪＨ（Ｈ鎖）およびＶＪＬ（Ｌ
鎖）］の単離）以下のように、抗ＰＴＨｒＰモノクロー
ナル抗体産生ハイブリドーマからｍＲＮＡを単離しｃＤ
ＮＡを合成後、ＰＣＲ（ポリメラーゼチェインリアクシ
ョン）法を用いて、ＶＤＪＨ遺伝子およびＶＪＬ遺伝子
のクローニングを行った。[Example 7] (The monoclonal antibody variable region genes [VDJH (H chain) and VJL (L
Isolation of cD chain) mRNA was isolated from a hybridoma producing an anti-PTHrP monoclonal antibody as follows.
After synthesizing NA, the VDJH gene and VJL gene were cloned using PCR (polymerase chain reaction) method.
【００６４】７−１．全ＲＮＡの抽出
ハイブリドーマ細胞ＥＶＯ３４ＢＩＧの培養液を遠心分
離（１２００ｒｐｍ）にかけ、該ハイブリドーマ細胞を
約３Ｘ１０８個得た。これを、リン酸緩衝化生理食塩水
（以下ＰＢＳと略す）で洗浄し、この細胞からトータル
ＲＮＡセパレーター（クローンテック社製）を用いて全
ＲＮＡを抽出した。その方法は添付されたプロトコール
に従った。得られたＲＮＡをトリスーＥＤＴＡ緩衝液（
以下ＴＥと略す）（ｐＨ７．５）に溶解させて全ＲＮＡ
サンプルとした。7-1. Extraction of total RNA The culture solution of hybridoma cells EVO34BIG was centrifuged (1200 rpm) to obtain about 3×10 8 hybridoma cells. The cells were washed with phosphate buffered saline (hereinafter abbreviated as PBS), and total RNA was extracted from the cells using a total RNA separator (manufactured by Clontech). The method followed the attached protocol. The obtained RNA was added to Tris-EDTA buffer (
Total RNA was dissolved in TE (hereinafter abbreviated as TE) (pH 7.5).
It was used as a sample.
【００６５】７−２．ｍＲＮＡの精製
上記７−１項で抽出した全ＲＮＡからのｍＲＮＡの精製
は、オリゴテックスーｄｔ３０（宝酒造社製）を用いて
行った。方法は添付されたプロトコールに従い、フェノ
ールで抽出して、エタノール沈澱後、ＴＥに溶解させて
、ｍＲＮＡサンプルを得た。7-2. Purification of mRNA Purification of mRNA from the total RNA extracted in Section 7-1 above was performed using Oligotex dt30 (manufactured by Takara Shuzo Co., Ltd.). According to the attached protocol, the mRNA sample was extracted with phenol, precipitated with ethanol, and dissolved in TE.
【００６６】７−３．ｃＤＮＡの合成
可変部領域の遺伝子（ＶＤＪＨおよびＶＪＬ）をＰＣＲ
法によってクローニングするため、７ー２項で得られた
ｍＲＮＡからｃＤＮＡを合成した。合成はｃＤＮＡ合成
試薬（ファルマシア社製）を使用し、付属のプロトコー
ルに従った。このプロトコールはＧｕｂｌｅｒ，Ｕ．ら
（Ｇｅｎｅ、２５巻、２６３頁（１９８３）），Ｈａｎ
ａｈａｎ．Ｄ．（ＤＮＡＣｌｏｎｉｎｇ（Ｇｌｏｖｅｒ
，Ｄ．Ｍ．，編），ＩＲＬ Ｐｒｅｓｓ，Ｏｘｆｏｒｄ
，１巻，１０９頁（１９８５）），Ｓｃａｌｅｎｇｈｅ
，Ｆ．ら（Ｃｈｒｏｍｏｓｏｍａ ８２巻，２０５頁
（１９８５））、Ｏｋａｙａｍａ，Ｈ．ら（Ｍｅｔｈｏ
ｄｓ ｉｎ Ｅｎｚｙｍｏｌｏｇｙ １５４巻，３頁
（１９８７））などの文献を参考にしている。得られた
ｃＤＮＡをフェノール／クロロホルムで抽出し、スパン
カラムで精製することによりｃＤＮＡサンプルを得た。7-3. PCR of cDNA synthetic variable region genes (VDJH and VJL)
For cloning by the method, cDNA was synthesized from the mRNA obtained in Section 7-2. For synthesis, a cDNA synthesis reagent (manufactured by Pharmacia) was used and the attached protocol was followed. This protocol was described by Gubler, U. (Gene, vol. 25, p. 263 (1983)), Han
ahan. D. (DNA Cloning (Glover)
,D. M. , ed.), IRL Press, Oxford.
, vol. 1, p. 109 (1985)), Scalenghe
, F. (Chromosoma vol. 82, p. 205 (1985)), Okayama, H. et al. etho
ds in Enzymology, Vol. 154, p. 3 (1987)). The obtained cDNA was extracted with phenol/chloroform and purified using a span column to obtain a cDNA sample.
【００６７】７−４．可変部領域遺伝子（ＶＤＪＨおよ
びＶＪＬ）のクローニング
ＩｇＧの可変部領域中の保存的なＤＮＡ配列を、Ｋａｂ
ａｔ らのデータファイル（Ｓｅｑｕｅｎｃｅｓ ｏｆ
Ｐｒｏｔｅｉｎｓ Ｉｍｍｕｎｏｌｏｇｉｃａｌ Ｉ
ｎｔｅｒｅｓｔ， ＵＳ Ｄｅｐｔ． ｏｆ Ｈｅａｌ
ｔｈ ａｎｄ Ｈｕｍａｎ Ｓｅｒｖｉｃｅｓ， ＵＳ
Ｇｏｖｅｒｎｍｅｎｔ Ｐｒｉｎｔｉｎｇ Ｏｆｆｉ
ｃｅ，（１９８７））から採用した。このヌクレオチド
配列は、成熟タンパク質のアミノ末端から始まるアミノ
酸配列をコードしており、このタンパクのシグナル配列
は含まない。このＤＮＡ配列をもとにし、ＰＣＲに用い
る次の４種のプライマー（配列番号８〜１１に塩基配列
を示す）を作成した。これらのプライマーは、ＤＮＡ合
成機（アプライドバイオシステムズ社製 Ｍｏｄｅｌ
３８１Ａ）にて合成した：ＶＤＪＨ５’５’ＴＣＴＡＧ
ＡＡＴＧＣ ＡＧＧＴ（ＣＴ）ＣＡＧＣＴ Ｇ（ＡＣ）
ＡＧＣＡＧＴＣ（ＡＴ） ＧＧ ３’ＶＤＪＨ３’５’
ＧＧＴＧＴＴＣＣＴＣ ＧＡＧＧＴＣＴＡＧＴ ＧＡＣ
ＣＧＴＧＧＴＣ ＣＣＴ（ＧＴ）（ＣＧ）（ＧＡ）ＣＣ
ＣＣ ＡＧ ３’ＶＪＬ５’５’ＣＧＴＧＧＣＴＣＴＡ
ＧＡＡＧＡＡＡＴＴＧ ＴＧ（ＡＣ）ＴＧＡＣＣＣＡ
ＧＴＣＴＣＣＡ ３’ＶＪＬ３’５’ＡＡＣＴＣＧＡ
ＧＣＣ ＡＧＣＴＴＧＧＴＣＣ Ｃ（ＣＡ）（ＣＧ）Ｃ
（ＡＴ）ＣＣＧＡＡ ＣＧＴＧ ３’。7-4. Cloning of variable region genes (VDJH and VJL) The conserved DNA sequences in the variable region of IgG were cloned into Kab
data file (Sequences of
Proteins Immunological I
Interested, US Dept. of Heal
th and Human Services, US
Government Printing Office
CE, (1987)). This nucleotide sequence encodes the amino acid sequence starting from the amino terminus of the mature protein and does not include the protein's signal sequence. Based on this DNA sequence, the following four types of primers (base sequences shown in SEQ ID NOS: 8 to 11) used for PCR were created. These primers were prepared using a DNA synthesizer (Applied Biosystems Model
381A): VDJH5'5'TCTAG
AATGC AGGT (CT) CAGCT G (AC)
AGCAGTC(AT) GG 3'VDJH3'5'
GGTGTTCCTC GAGGTCTAGT GAC
CGTGGTC CCT (GT) (CG) (GA) CC
CC AG 3'VJL5'5'CGTGGCTCTA
GAAGAAATTG TG(AC)TGACCCA
GTCTCCA 3'VJL3'5'AACTCGA
GCC AGCTTGGTCC C(CA)(CG)C
(AT) CCGAA CGTG 3'.
【００６８】上記ＶＤＪＨ５’およびＶＤＪＨ３’また
はＶＪＬ５’およびＶＪＬ３’をプライマーとし、上記
ｃＤＮＡを鋳型として、ＰＣＲを行うことにより抗体可
変部領域の特異的な配列（Ｈ鎖またはＬ鎖の）が増幅さ
れる。ＰＣＲ法によるＤＮＡ増幅についてはＥｒｌｉｃ
ｈ，Ｈ．Ａ．編（ ＰＣＲ Ｔｅｃｎｏｌｏｇｙ， Ｓ
ｔｏｃｋｔｏｎ Ｐｒｅｓｓ（１９８９））に記載され
ている。本実施例においては遺伝子増幅試薬（宝酒造社
製）を用いて上記ＰＣＲを行った。ｃＤＮＡクローニン
グに用いた上記プライマーは制限酵素切断部位を有して
いるので、ｃＤＮＡクローニングおよび増幅の結果、Ｖ
ＤＪＨおよびＶＪＬのそれぞれ５’側にＸｂａＩ或いは
３’側にＸｈｏＩが導入される。[0068] By performing PCR using the above VDJH5' and VDJH3' or VJL5' and VJL3' as primers and the above cDNA as a template, the specific sequence (H chain or L chain) of the antibody variable region is amplified. Ru. For information on DNA amplification using the PCR method, see Erlic.
h, H. A. Edited by PCR Technology, S.
Tockton Press (1989)). In this example, the above PCR was performed using a gene amplification reagent (manufactured by Takara Shuzo Co., Ltd.). Since the above primers used for cDNA cloning have restriction enzyme cleavage sites, as a result of cDNA cloning and amplification, V
XbaI or XhoI is introduced into the 5' side or the 3' side of DJH and VJL, respectively.
【００６９】７−５．可変部領域遺伝子の塩基配列７−
４でクローニングした可変部領域遺伝子の塩基配列をＳ
ａｎｇｅｒ，Ｆ．ら（Ｐｒｏｃ．Ｎａｔｌ． Ａｃａｄ
． Ｓｃｉ．ＵＳＡ．７４巻，５４６３頁（１９７７）
）のジデオキシ法を改変した試薬（東洋紡績社製）を用
いて塩基配列を決定した。その結果を配列番号５および
配列番号６に示す。さらに、それらの塩基配列から推定
されるアミノ酸配列を配列番号３および配列番号４に示
す。7-5. Base sequence of variable region gene 7-
The base sequence of the variable region gene cloned in step 4 is S
Anger, F. (Proc. Natl. Acad
．． Sci. USA. Volume 74, page 5463 (1977)
The base sequence was determined using a reagent (manufactured by Toyobo Co., Ltd.) that is a modified version of the dideoxy method (manufactured by Toyobo Co., Ltd.). The results are shown in SEQ ID NO: 5 and SEQ ID NO: 6. Furthermore, the amino acid sequences deduced from these base sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4.
【００７０】［実施例８］（ヒト免疫グロブリン定常部
領域遺伝子（Ｃγ１（Ｈ鎖）および、Ｃκ（Ｌ鎖））の
クローニング）マウス／ヒトキメラモノクローナル抗体
を作成するために必要なヒトのＣγ１およびＣκをヒト
免疫グロブリン産生癌細胞ＡＲＨー７７からクローニン
グした。[Example 8] (Cloning of human immunoglobulin constant region genes (Cγ1 (H chain) and Cκ (L chain))) Human Cγ1 and Cκ was cloned from the human immunoglobulin-producing cancer cell ARH-77.
【００７１】８−１． 染色体ＤＮＡの分離ヒト免疫
グロブリン産生癌細胞ＡＲＨ−７７（ＡＴＣＣ ＣＲ
Ｌ−１６２１）を、１０％牛胎児血清（以下ＦＣＳと略
する、大日本製薬社製）を含むＲＰＭＩ１６４０培地（
大日本製薬社製）で培養した。得られた細胞をＰＢＳで
洗浄し、ＴＮＥ溶液［１０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐ
Ｈ７．５），０．１Ｍ ＮａＣｌ，１ｍＭ ＥＤＴＡ］
に均一に懸濁した。そこに、プロテイナーゼＫ（ベーリ
ンガー社製）を、最終濃度が０．１ｍｇ／ｍｌにになる
ように、そしてＳＤＳを、その最終濃度が０．３％にな
るようにそれぞれを加えて攪拌後、６０℃に４時間保っ
た。等量のフェノールを加えて２時間攪拌した後、３０
００ｒｐｍで，１０分間遠心分離して水層を回収し、も
う１度フェノール抽出を繰り返した。水層に等量のクロ
ロホルムを加えて緩やかに浸盪後、３０００ｒｐｍで，
５分間遠心分離した。これに０．１容量の３Ｍ酢酸ナト
リウムおよび等容量のイソプロパノールを加えて、繊維
状になった染色体ＤＮＡをガラス棒に巻き取り、エタノ
ールで洗浄した後、風乾し、適当量のＴＥに溶解した。
１０分間煮沸した１ｍｇ／ｍｌのＲＮａｓｅを１／５０
量加えて１時間３７℃でインキュベートした後、再びフ
ェノール抽出、エタノール沈澱を行って精製した。最終
的にＴＥに溶解することにより染色体ＤＮＡを約３ｍｇ
得た。8-1. Isolation of chromosomal DNA Human immunoglobulin-producing cancer cells ARH-77 (ATCC CR
L-1621) in RPMI1640 medium (hereinafter abbreviated as FCS, manufactured by Dainippon Pharmaceutical Co., Ltd.) containing 10% fetal calf serum (FCS).
(manufactured by Dainippon Pharmaceutical Co., Ltd.). The obtained cells were washed with PBS and added to TNE solution [10mM Tris-HCl (p
H7.5), 0.1M NaCl, 1mM EDTA]
was homogeneously suspended. Proteinase K (manufactured by Boehringer) was added to the final concentration of 0.1 mg/ml, and SDS was added to the final concentration of 0.3%. After stirring, the mixture was stirred for 60 minutes. It was kept at ℃ for 4 hours. After adding an equal amount of phenol and stirring for 2 hours,
The aqueous layer was collected by centrifugation at 00 rpm for 10 minutes, and the phenol extraction was repeated once more. After adding an equal amount of chloroform to the aqueous layer and gently stirring, at 3000 rpm,
Centrifuged for 5 minutes. 0.1 volume of 3M sodium acetate and an equal volume of isopropanol were added thereto, and the fibrous chromosomal DNA was wound up onto a glass rod, washed with ethanol, air-dried, and dissolved in an appropriate amount of TE. 1/50 of 1 mg/ml RNase boiled for 10 minutes
After adding a certain amount of the product and incubating at 37°C for 1 hour, phenol extraction and ethanol precipitation were performed again for purification. Finally, approximately 3 mg of chromosomal DNA is obtained by dissolving it in TE.
Obtained.
【００７２】８−２．Ｃγ１を含むＤＮＡ断片およびＣ
кを含むＤＮＡ断片の調製
８−１項で得られた染色体ＤＮＡを、次のように制限酵
素で完全分解した後、アガロースゲル電気泳動で分子量
分画した。まず、染色体ＤＮＡ３００μｇを１０ｍＭ
Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５），１００ｍＭ 塩
化ナトリウム、１０ｍＭ塩化マグネシウム中で３００ｕ
ｎｉｔのＥｃｏＲＩとともに１晩３７℃に保持し、完全
切断した。これをフェノール抽出し、エタノール沈澱後
、ＴＥに溶解させた。このサンプルを０．７５％アガロ
ース電気泳動で、１００Ｖで，３時間泳動後、同時に泳
動した分子量マーカーをもとに約２ｋｂおよび約１５ｋ
ｂと思われる位置のゲルに切れ込みを入れた。そこにＤ
ＥＡＥペーパー（東洋濾紙社製ＤＥＡＥイオン交換フィ
ルター）を差し込み泳動を続けることで２ー４ｋｂおよ
び１５ー３０ｋｂのＤＮＡをペーパーに吸着させた。目
的のＤＮＡを吸着させた後、ゲルからペーパーを取り出
し１．５Ｍ 塩化ナトリウム溶液中でペーパーからＤ
ＮＡを溶出し、フェノール抽出およびエタノール沈澱を
行い、ＴＥに溶解させた。このようにしてＣγｌを含む
ＤＮＡ断片またはＣкを含むＤＮＡ断片が溶解したＴＥ
溶液が得られた。8-2. DNA fragment containing Cγ1 and C
Preparation of DNA fragment containing A The chromosomal DNA obtained in Section 8-1 was completely digested with restriction enzymes as follows, and then subjected to molecular weight fractionation by agarose gel electrophoresis. First, 300 μg of chromosomal DNA was added to 10 mM
300u in Tris-HCl (pH 7.5), 100mM Sodium Chloride, 10mM Magnesium Chloride
It was kept at 37°C overnight with EcoRI of nit to completely cleave it. This was extracted with phenol, precipitated with ethanol, and then dissolved in TE. This sample was electrophoresed on 0.75% agarose gel at 100V for 3 hours, and based on the molecular weight markers that were electrophoresed at the same time, it was determined that the sample was about 2 kb and about 15 kb.
An incision was made in the gel at the position believed to be b. There D
By inserting EAE paper (DEAE ion exchange filter manufactured by Toyo Roshi Co., Ltd.) and continuing electrophoresis, DNA of 2 to 4 kb and 15 to 30 kb was adsorbed to the paper. After adsorbing the target DNA, remove the paper from the gel and remove the DNA from the paper in a 1.5M sodium chloride solution.
NA was eluted, subjected to phenol extraction and ethanol precipitation, and dissolved in TE. In this way, the DNA fragment containing Cγl or the DNA fragment containing Cк was dissolved in TE.
A solution was obtained.
【００７３】８−３．染色体ＤＮＡファージライブラリ
ーの調製
８−２項で得られたＣγｌを含むＤＮＡ断片（１５−３
０ｋｂのＤＮＡ）１μｇと、ＥｃｏＲＩで切断したラム
ダクローニングベクターＥＭＢＬ４（ストラタジーン社
製）２．５μｇとをライゲーション試薬（宝酒造社製）
を用い、２６℃で１０分間反応させて連結した。このよ
うにして、Ｃγｌを含むＤＮＡ断片のクローニングを行
った。その後、ｉｎ ｖｉｔｒｏ パッケージング試薬
（ストラタジーン社製）のプロトコールに従ってパッケ
ージングしてＨ鎖ファージライブラリーとした。8-3. Preparation of chromosomal DNA phage library DNA fragment containing Cγl obtained in Section 8-2 (15-3
0 kb DNA) and 2.5 μg of lambda cloning vector EMBL4 (Stratagene) cut with EcoRI were combined with ligation reagent (Takara Shuzo).
were used to perform a reaction at 26° C. for 10 minutes to perform ligation. In this way, a DNA fragment containing Cγl was cloned. Thereafter, it was packaged according to the protocol of an in vitro packaging reagent (manufactured by Stratagene) to prepare a heavy chain phage library.
【００７４】これとは別に、８−２項で得られたＣкを
含むＤＮＡ断片（２−４ｋｂのＤＮＡ）１μｇとを、Ｅ
ｃｏＲＩで切断したラムダクローニングベクターλｇｔ
１０（ストラタジーン社製）２．５μｇとをライゲーシ
ョン試薬（宝酒造社製）を用いて、２６℃で１０分間反
応して連結した。このようにＣкを含むＤＮＡ断片のク
ローニングを行った。Separately, 1 μg of the DNA fragment (2-4 kb DNA) containing CK obtained in Section 8-2 was added to E.
Lambda cloning vector λgt cut with coRI
10 (manufactured by Stratagene) using a ligation reagent (manufactured by Takara Shuzo) at 26° C. for 10 minutes. In this manner, a DNA fragment containing C was cloned.
【００７５】８−４．ヒトＩｇＧ定常部遺伝子のプロー
ブの作製
Ｅｌｌｉｓｏｎ，Ｊ．Ｗ．ら（Ｎｕｃｌｅｉｃ Ａｃｉ
ｄｓ Ｒｅｓｅａｒｃｈ，１０巻、４０７１頁（１９８
２））が明らかにしたＡＲＨ−７７由来のＣγ１の配列
から次に示すプローブＨ１およびプローブＨ２を設計し
た。
プローブＨ１（配列番号１２に示す）
５’ ＡＧＧＴＧＣＡＧＣＴＡ
ＣＡＧＣＡＧＴＧＧＧＧＣＧＣＡＧＧＡＣＴＧＧＴＧＡ
ＡＧＣＣＴＴＣＧＧＡＧＡ ３’ プロー
ブＨ２（配列番号１３に示す）
５ ’ＡＣＴＡＣＴＡＣＴＡＴＧＧＴＡＴＧＧＡＣ
ＧＴＣＴＧＧＧＧＣＣＡＡＧＧＧＡＣＣＡＣＧＧＴＣＡ
ＣＣＧ ＴＣＴＣＣ
ＴＣＡ ３’これらのプローブは、ＤＮＡ合成機で合
成された。これらのプローブはそれぞれＡＲＨー７７の
ＪＨ領域およびフレームワーク領域の遺伝子配列である
。8-4. Preparation of probe for human IgG constant region gene Ellison, J.; W. (Nucleic Aci
ds Research, Volume 10, Page 4071 (198
The following probe H1 and probe H2 were designed from the sequence of ARH-77-derived Cγ1 revealed by 2)). Probe H1 (shown in SEQ ID NO: 12)
5'AGGTGCAGCTA
CAGCAGTGGGGGCGCAGGACTGGTGA
AGCCTTCGGAGA 3' probe H2 (shown in SEQ ID NO: 13)
5 'ACTACTACTATGGTATGGAC
GTCTGGGGCCAAGGGACCACCGGTCA
CCG TCTCC
TCA 3' These probes were synthesized on a DNA synthesizer. These probes are the gene sequences of ARH-77 JH region and framework region, respectively.
【００７６】これとは別に、Ｅｌｌｉｓｏｎ， Ｊ．Ｗ
． （前出）のＣκ遺伝子から次に示すプローブＬ１を
設計した。
プローブＬ１（配列番号１４に示す）
５ ’ＡＣＴＧＴＧＧＣＴＧＣＡＣ
ＣＡＴＣＴＧＴＣＴＴＣＡＴＣＴＴＣＣＣＧＣＣＡＴＣ
ＴＧＡＴＧＡＧＣＡＧＴＴ ３’このプローブもＤＮ
Ａ合成機で合成された。この配列はＣκ遺伝子の５’末
端側５０ｂｐの配列である。プローブの標識は、ＤＮＡ
５’末端標識用試薬（宝酒造社製）を用いて行った。６
５℃で１０分加熱して酵素を失活させた後、セファデッ
クスＧ−５０を詰めたカラム（ファルマシア社製）で精
製して使用した。Separately, Ellison, J. W
．． The following probe L1 was designed from the Cκ gene (described above). Probe L1 (shown in SEQ ID NO: 14)
5 'ACTGTGGCTGCAC
CATCTGTCTTCATCTTCCCGCCATC
TGATGAGCAGTT 3'This probe is also DN
Synthesized by A synthesizer. This sequence is a 50 bp sequence at the 5' end of the Cκ gene. The probe label is DNA
This was performed using a 5' end labeling reagent (manufactured by Takara Shuzo Co., Ltd.). 6
After inactivating the enzyme by heating at 5° C. for 10 minutes, it was purified using a column packed with Sephadex G-50 (manufactured by Pharmacia) and used.
【００７７】８−５．ヒトＩｇＧ定常部領域遺伝子のス
クリーニング
ＣγｌおよびＣκ遺伝子のスクリーニングは常法に従っ
てプラークハイブリダイゼーション法で行った。８ー２
項で作製したファージライブラリーのうちＣγｌを含む
ＤＮＡを有するλＥＭＢＬ４は大腸菌Ｐ２−３９２株に
、Ｃкを含むＤＮＡを有するλｇｔ１０はＮＭ５１４株
に感染させ、８ー４項で作製したプローブとハイブリダ
イズするプラークをスクリーニングした。その結果、プ
ローブＨ１およびＨ２とハイブリダイズする株Ｈ１−８
、そして、プローブＬ１とハイブリダイズする株Ｌ１４
−２を得た。それらから得られた挿入断片の塩基配列を
ジデオキシ法で決定した。タンパク質をコードする部分
の遺伝子は、どちらにおいても既知の配列と一致するこ
とを確認した。それぞれの挿入断片を制限酵素Ｈｉｎｄ
ＩＩＩおよびＸｂａＩ、あるいはＥｃｏＲＩを用いて切
り出し、ｐＵＣ１９にサブクローニングし、ｐＵＣーＣ
γ１あるいはｐＬ１４−２を得た。8-5. Screening for human IgG constant region genes Screening for Cγl and Cκ genes was performed by plaque hybridization according to a conventional method. 8-2
Of the phage libraries prepared in Section 8, λEMBL4, which has DNA containing Cγl, is used to infect Escherichia coli P2-392 strain, and λgt10, which contains DNA containing Cγl, is infected to NM514 strain, and hybridized with the probe prepared in Section 8-4. Screened for plaque. As a result, strain H1-8 hybridized with probes H1 and H2.
, and strain L14 that hybridizes with probe L1.
-2 was obtained. The nucleotide sequences of the inserted fragments obtained from them were determined by the dideoxy method. It was confirmed that the gene encoding the protein in both cases matched the known sequence. Each insert fragment was digested with restriction enzyme Hind.
It was excised using III and XbaI or EcoRI, subcloned into pUC19, and pUC-C
γ1 or pL14-2 was obtained.
【００７８】［実施例９］（キメラ抗体発現ベクターの
作製）キメラ抗体発現ベクターは以下のようにして作製
した。[Example 9] (Preparation of chimeric antibody expression vector) A chimeric antibody expression vector was prepared as follows.
【００７９】９−１ マウス遺伝子の修飾７−４項で
クローニングしたマウスＶＤＪＨ遺伝子にはタンパク質
の分泌に不可欠なシグナル配列が含まれていない。従っ
て、Ｃａｂｉｌｌｙ，Ｓ．らの報告（Ｐｒｏｃ．Ｎａｔ
ｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ．，８１巻，３２７２頁
（１９８４））にある抗体（Ａｎｔｉ−ＣＥＡ ＭｏＡ
ｂ）のシグナル配列に対応する部分を含む遺伝子を合成
し、これをプライマー（プライマーＬＨ）としてＰＣＲ
を行った。その結果、該抗体のシグナル配列がＶＤＪＨ
遺伝子に付加されたＬＶＤＪＨ遺伝子を得た。そのプラ
イマーＬＨの配列を以下に示す。さらに、配列番号１５
に示す。
プライマーＬＨ ５’ＣＣＴＣＴＡＧＡＴＧＡＡＣ
ＴＴＣＧＧＧＣＴＣＡＧＣＴＴＧＡＴＴＴＡＣＣＴＴＧ
ＴＣＣＴＧＧＴＴＴＴＡＡ
ＡＡＧＴＴＧＴＣＣＡＧＴＧＴＣＡＧＧＴ
ＴＣＡＧＣＴＧＣＡＧＣＡＧＴＣＴＧＧＡＣ ３
’そして得られたＤＮＡ断片をｐＵＣ１９のＸｂａＩー
ＳａｌＩ部位にサブクローニングした。9-1 Modification of Mouse Gene The mouse VDJH gene cloned in Section 7-4 does not contain a signal sequence essential for protein secretion. Therefore, Cabilly, S. (Proc. Nat
l. Acad. Sci. USA. , Vol. 81, p. 3272 (1984)).
A gene containing a portion corresponding to the signal sequence of b) is synthesized, and this is used as a primer (primer LH) for PCR.
I did it. As a result, the signal sequence of the antibody was found to be VDJH
The LVDJH gene added to the gene was obtained. The sequence of primer LH is shown below. Furthermore, SEQ ID NO: 15
Shown below. Primer LH 5'CCTCTAGATGAAC
TTCGGGCTCAGCTTGATTTACCTTG
TCCTGGTTTTAA
AAGTTGTCCAGTGTCAGGT
TCAGCTGCAGCAGTCTGGAC 3
'The obtained DNA fragment was then subcloned into the XbaI-SalI site of pUC19.
【００８０】同様にマウスＶＪＬ遺伝子にも次のように
してシグナル配列を付加した。まず、Ａｎｔｉ−ＣＥＡ
ＭｏＡｂのＬ鎖シグナル配列に対応する部分を含む遺
伝子を合成し、これをプライマーとしてＰＣＲを行い、
シグナル配列がＶＪＬ遺伝子に付加されたＬＶＪＬ遺伝
子を得た。これをｐＵＣ１９のＸｂａＩ−ＳａｌＩ部位
にサブクローニングした。プライマーＬＬの配列を以下
に示す。さらに配列番号１６に示す：
プライマーＬＬ ５’ ＣＣＴＣＴＡＧＡＴＧＧ
ＧＣＡＴＣＡＡＧＡＴＧＧＡＧＡＣＡＣＡＴＴＣＴＣＡ
ＧＧＴＣＴＴＴＧＴＡＴＡＣＡ
ＴＧＴＴＧＣＴＧＴＧＧＴＴＧＴＣ
ＴＧＧＴＧＴＴＧＡＡＧＧＡＧＡＡＡＴＴＧＴＧＡＴＧ
ＡＣＣＣＡＧＴＣＴ
ＣＣＡＣＴＣＴＣＣ ３’。Similarly, a signal sequence was added to the mouse VJL gene as follows. First, Anti-CEA
A gene containing a portion corresponding to the L chain signal sequence of MoAb is synthesized, and PCR is performed using this as a primer.
An LVJL gene was obtained in which a signal sequence was added to the VJL gene. This was subcloned into the XbaI-SalI site of pUC19. The sequence of primer LL is shown below. Further shown in SEQ ID NO: 16: Primer LL 5' CCTCTAGATGG
GCATCAAGATGGAGACACATTCTCA
GGTCTTTGTATACA
TGTTGCTGTGGTTGTC
TGGTGTTGAAGGAGAAATTGTGATG
ACCCAGTCT
CCACTCTCC 3'.
【００８１】９ー２．キメラ抗体発現ベクターの調製ま
ず、ｐＳＶｅＳａｌＩ（特開昭６２ー１４７８３）を制
限酵素ＮｃｏＩで切断し、大腸菌内での複製起点（ｏｒ
ｉ）及びアンピシリン耐性を付与する約４．７ｋｂの断
片を単離した。Ｔ４ＤＮＡリガーゼを用いてこれを環状
化し、ｐＳＶｅＳａｌＩ−ＨｉｎｄＩＩＩ（ヨーロッパ
特許出願公開Ｎｏ．０４２０５０２）を作成した。これ
を大腸菌（Ｅ．ｃｏｌｉ）ＤＨ５αに導入して増幅させ
た。このベクターを図２に示すように、ＨｉｎｄＩＩＩ
で切断、平滑末端化し、ｐＸｂａＩリンカーを付加し、
Ｔ４ＤＮＡリガーゼで環状化しｐＳＶｅＸｂａＩを得た
。これを大腸菌ＤＨ５αに導入して増幅させた。図２に
示すように、このベクターｐＳＶｅＸｂａＩにはＸｂａ
Ｉ認識部位の上流にＳＶ４０の複製起点を含む初期プロ
モーター領域が存在している。次に、実施例８で得られ
たプラスミドｐＵＣーＣγ１（ヒトＨ鎖Ｃγ１遺伝子を
含む制限酵素ＨｉｎｄＩＩＩ＋ＸｂａＩ断片約６ｋｂが
サブクローニングされている）からＨｉｎｄＩＩＩとＸ
ｂａＩとでＣγｌ遺伝子を切り出した。このＣγ１遺伝
子と、上記９ー１項で得られたマウスＨ鎖のＬＶＤＪＨ
遺伝子断片と、ｐＳＶｅＸｂａＩをＸｂａＩで切断した
ベクターとをＴ４ＤＮＡリガーゼで連結してｐＳＶｅＨ
２６を構築した。その工程を図２に示す。9-2. Preparation of chimeric antibody expression vector First, pSVeSalI (Japanese Unexamined Patent Publication No. 14783-1983) was cut with the restriction enzyme NcoI, and the origin of replication (or
i) and an approximately 4.7 kb fragment conferring resistance to ampicillin. This was circularized using T4 DNA ligase to create pSVeSalI-HindIII (European Patent Application Publication No. 0420502). This was introduced into E. coli DH5α and amplified. This vector was transformed into HindIII as shown in FIG.
Cut it with
It was circularized with T4 DNA ligase to obtain pSVeXbaI. This was introduced into E. coli DH5α and amplified. As shown in Figure 2, this vector pSVeXbaI contains Xba
There is an early promoter region containing the SV40 replication origin upstream of the I recognition site. Next, HindIII and
The Cγl gene was excised with baI. This Cγ1 gene and the mouse H chain LVDJH obtained in Section 9-1 above
The gene fragment and the vector obtained by cutting pSVeXbaI with XbaI were ligated with T4 DNA ligase to create pSVeH.
26 was constructed. The process is shown in FIG.
【００８２】次に、実施例８で得られたｐＬ１４−２（
ヒトＣк遺伝子をｐＵＣ１９にサブクローニングして得
られる）の５’末端に近いＥｃｏＲＩ部位のみを部分分
解し、さらにｐＨｉｎｄＩＩＩリンカーを用いてＨｉｎ
ｄＩＩＩ部位に変換した（図３参照）。これをＥｃｏＲ
ＩおよびＨｉｎｄＩＩＩで切断してＣκ遺伝子を含む領
域を切り出した。次に、ｐＵＣ１９にサブクローニング
したマウスＬＶＪＬ遺伝子を、ＸｂａＩ並びにＨｉｎｄ
ＩＩＩで切り出した。上記ＥｃｏＲＩ−ＨｉｎｄＩＩＩ
断片と、ＸｂａＩ−ＨｉｎｄＩＩＩ断片と、ｐＳＶｅＸ
ｂａＩをＥｃｏＲＩおよびＸｂａＩで切断して得られる
断片とを、Ｔ４ＤＮＡリガーゼで連結して、ｐＳＶｅＬ
２６を作製した（第３図）。ｐＳＶｅＬ２６は、図３に
示すようにｍＬＶＪＬ遺伝子とヒトＣк遺伝子とを有す
る。Next, pL14-2 (obtained in Example 8)
(obtained by subcloning the human Cк gene into pUC19), only the EcoRI site near the 5' end was partially degraded, and then HindIII was added using a pHindIII linker.
dIII site (see Figure 3). EcoR this
The region containing the Cκ gene was excised by cutting with I and HindIII. Next, the mouse LVJL gene subcloned into pUC19 was
It was cut out in III. EcoRI-HindIII above
fragment, XbaI-HindIII fragment, pSVeX
The fragment obtained by cutting baI with EcoRI and XbaI was ligated with T4 DNA ligase to create pSVeL.
26 was prepared (Fig. 3). pSVeL26 has the mLVJL gene and the human C gene, as shown in FIG. 3.
【００８３】次に、このｐＳＶｅＬ２６のＴｔｈ１１１
Ｉ部位を平滑末端にし、ｐＢａｍＨＩリンカーを利用し
てＢａｍＨＩ認識部位を導入した（図４）。そしてＥｃ
ｏＲＩおよびＢａｍＨＩで切断することによってｍＬＶ
ＪＬ遺伝子およびヒトＣк遺伝子を含む断片を切り出し
た。得られた断片とｐＳＶｅＨ２６をＢａｍＨＩおよび
ＥｃｏＲＩで切断して得られる断片とをＴ４ＤＮＡリガ
ーゼを用いて連結することにより、キメラ抗体発現ベク
ターであるｐＳＶｅＨＬ２６を得た。上記工程を図４に
示す。Next, Tth111 of this pSVeL26
The I site was made blunt-ended, and a BamHI recognition site was introduced using a pBamHI linker (Figure 4). and Ec
mLV by cutting with oRI and BamHI
A fragment containing the JL gene and the human C gene was excised. The obtained fragment and the fragment obtained by cleaving pSVeH26 with BamHI and EcoRI were ligated using T4 DNA ligase to obtain pSVeHL26, which is a chimeric antibody expression vector. The above steps are shown in FIG.
【００８４】このキメラ抗体発現ベクターを動物細胞へ
導入するこも考慮し、次のようにして、このベクターに
薬剤耐性遺伝子を導入した。まず、Ｙａｍａｓｈｉｔａ
，Ｋ．らのｐＳＶｅＬＴｄｈ（Ａｇｒｉｃ．Ｂｉｏｌ．
Ｃｈｅｍ．，５４巻，２８０１頁（１９９０））のＴｔ
ｈ１１１Ｉ部位をＢａｍＨＩ認識部位に変換し、Ｂａｍ
ＨＩで切断することによって大腸菌キサンチンーグアニ
ンホスホリボシルトランスフェラーゼ（Ｅｃｏ−ｇｐｔ
）およびジヒドロ葉酸還元酵素（ＤＨＦＲ）の、それぞ
れ発現ユニットを含む遺伝子をＢａｍＨＩ断片として単
離した（図５）。この遺伝子断片をｐＳＶｅＨＬ２６の
ＢａｍＨＩ部位へ挿入、連結してｐＳＶｅＨ２６−ＤＭ
を得た。この工程を図５に示す。Considering the possibility of introducing this chimeric antibody expression vector into animal cells, a drug resistance gene was introduced into this vector as follows. First, Yamashita
, K. pSVeLTdh (Agric. Biol.
Chem. , vol. 54, p. 2801 (1990))
Convert h111I site to BamHI recognition site,
Escherichia coli xanthine-guanine phosphoribosyltransferase (Eco-gpt
) and dihydrofolate reductase (DHFR), respectively, were isolated as BamHI fragments (FIG. 5). This gene fragment was inserted into the BamHI site of pSVeHL26 and ligated to create pSVeH26-DM.
I got it. This process is shown in FIG.
【００８５】［実施例１０］（キメラ抗体発現ベクター
の動物細胞への導入および選択）実施例９ー２で作製し
たｐＳＶｅＨＬ２６−ＤＭをマウス骨髄腫細胞株、ある
いはハムスター細胞株に導入した。[Example 10] (Introduction of chimeric antibody expression vector into animal cells and selection) pSVeHL26-DM prepared in Example 9-2 was introduced into a mouse myeloma cell line or a hamster cell line.
【００８６】１０ー１．マウス骨髄腫細胞への遺伝子導
入
マウス骨髄腫細胞である、ＦＯ細胞に、ＤＥＡＥーデキ
ストラン法で発現ベクターを導入した。上記方法は、高
井ら（高井俊行、細胞工学、９巻，６５２頁（１９９０
））に従った。形質転換株の選択は、１０％ＦＣＳを含
むダルベッコ改変イーグルＭＥＭ培地（ＤＭＥＭ）にセ
レクション試薬（最終濃度：２００μｇ／ｍｌキサンチ
ン、５μｇ／ｍｌアデニン、５μｇ／ｍｌチミジン、２
．５μｇ／ｍｌミコフェノール酸、０．１μｇ／ｍｌア
ミノプテリン）を加えた培地で行った。10-1. Gene introduction into mouse myeloma cells An expression vector was introduced into FO cells, which are mouse myeloma cells, by the DEAE-dextran method. The above method is based on Takai et al. (Toshiyuki Takai, Cell Engineering, Vol. 9, p. 652 (1990).
)) followed. Selection of transformed strains was performed using selection reagents (final concentration: 200 μg/ml xanthine, 5 μg/ml adenine, 5 μg/ml thymidine, 2
．． The test was carried out using a medium supplemented with 5 μg/ml mycophenolic acid and 0.1 μg/ml aminopterin.
【００８７】得られた形質転換株の抗体産生量を、次の
ようにＥＬＩＳＡ法で測定した。まず、９６穴プレート
に抗ヒトＩｇＧを入れて４℃で１晩コートした後、０．
０５％Ｔｗｅｅｎ２０を含むＰＢＳで洗浄した。次に１
％ゼラチンを含むＰＢＳで非特異的反応をブロックした
。上記形質転換株の培養上清を各ウエルに入れて３７℃
に１時間保ち、０．０５％Ｔｗｅｅｎ２０を含むＰＢＳ
で洗浄した後、ペルオキシダーゼ標識モノクローナル抗
体（抗ヒトＩｇＧＦｃ断片、あるいは抗ヒトＩｇＧκ鎖
）を加えて３７℃で１時間インキュベートした。その後
、発色液（０．０４％ο−フェニレンジアミン、０．０
３３％過酸化水素、２５ｍＭクエン酸、５０ｍＭリン酸
水素２ナトリウム（ｐＨ５．０））を加えた。２０分後
に、４Ｎ硫酸を添加することで反応を停止し、４９２ｎ
ｍの吸光度を測定して、精製ヒト抗体を標準として生産
量を決定した。その結果、表２にその代表例を示すよう
に、抗体を発現している細胞が得られた。これらの株の
産生するキメラ抗体を用い、実施例２に従ってＰＴＨｒ
Ｐに対する結合能および中和能を調べたところ、マウス
モノクローナル抗体の場合といずれも同等の結果が得ら
れた。[0087] The amount of antibody produced by the obtained transformed strain was measured by ELISA method as follows. First, anti-human IgG was added to a 96-well plate and coated overnight at 4°C, followed by a 0.
The cells were washed with PBS containing 05% Tween20. Next 1
Non-specific reactions were blocked with PBS containing % gelatin. Add the culture supernatant of the above transformed strain to each well at 37°C.
for 1 hour in PBS containing 0.05% Tween20.
After washing with water, a peroxidase-labeled monoclonal antibody (anti-human IgG Fc fragment or anti-human IgG κ chain) was added and incubated at 37° C. for 1 hour. After that, coloring solution (0.04%ο-phenylenediamine, 0.0
33% hydrogen peroxide, 25mM citric acid, 50mM disodium hydrogen phosphate (pH 5.0)) were added. After 20 minutes, the reaction was stopped by adding 4N sulfuric acid and 492n
The production amount was determined by measuring the absorbance of m and using the purified human antibody as a standard. As a result, cells expressing antibodies were obtained, as shown in Table 2 as representative examples. Using chimeric antibodies produced by these strains, PTHr was detected according to Example 2.
When the P-binding ability and neutralizing ability were examined, results comparable to those of the mouse monoclonal antibody were obtained.
【００８８】１０−２．ハムスター細胞への遺伝子導入
ハムスター細胞であるチャイニーズハムスター卵巣（Ｃ
ＨＯ）細胞（ＡＴＣＣＣＣＬ−６１）（市販されている
）にＣｈｅｎ，Ｃ．ら（Ｍｏｌ．Ｃｅｌｌ．Ｂｉｏｌ．
，７巻．２７４５頁（１９８７））に従って、リン酸カ
ルシウム法で発現ベクターを導入した。形質転換株の選
択は、１０％ＦＣＳを含むＤＭＥＭにセレクション試薬
（最終濃度：２００μｇ／ｍｌキサンチン、５μｇ／ｍ
ｌアデニン、５μｇ／ｍｌチミジン、２５μｇ／ｍｌミ
コフェノール酸、０．１μｇ／ｍｌアミノプテリン）を
加えた培地で培養して行った。得られた形質転換株の抗
体産生量を、１０−１項と同様にＥＬＩＳＡ法で測定し
た。抗体を産生する株の代表例を表２に示す。これらの
株の産生するキメラ抗体を用い、実施例２に従ってＰＴ
ＨｒＰに対する結合能および中和能を調べたところ、マ
ウスモノクローナル抗体の場合といずれも同等の結果が
得られた。10-2. Gene introduction into hamster cellsChinese hamster ovary (C) which is a hamster cell
HO) cells (ATCCCCCL-61) (commercially available) by Chen, C. (Mol. Cell. Biol.
, 7 volumes. 2745 (1987)), the expression vector was introduced by the calcium phosphate method. For selection of transformed strains, add selection reagents (final concentration: 200 μg/ml xanthine, 5 μg/ml
1 adenine, 5 μg/ml thymidine, 25 μg/ml mycophenolic acid, and 0.1 μg/ml aminopterin). The amount of antibody produced by the obtained transformed strain was measured by the ELISA method in the same manner as in Section 10-1. Representative examples of strains producing antibodies are shown in Table 2. Using chimeric antibodies produced by these strains, PT
When the binding ability and neutralizing ability to HrP were examined, results comparable to those of the mouse monoclonal antibody were obtained.
【００８９】１０−３．キメラ抗体産生細胞の育種上記
１０−１項および１０−２項で得られたキメラ抗体発現
ベクターを含む形質転換株を、５０ｎＭから２００ｎＭ
のメソトレキセート（ＭＴＸ 武田薬品工業社製）を
含む培地で約１月間培養を続け、ＭＴＸ耐性を示す株（
ＤＨＦＲ遺伝子を有する株）を分離した。その結果得ら
れた株の代表例を表２に示す。10-3. Breeding of chimeric antibody-producing cells The transformed strain containing the chimeric antibody expression vector obtained in Sections 10-1 and 10-2 above was incubated at 50 nM to 200 nM.
The culture was continued for about one month in a medium containing methotrexate (MTX, manufactured by Takeda Pharmaceutical Co., Ltd.), and strains showing MTX resistance (
A strain containing the DHFR gene was isolated. Representative examples of the resulting strains are shown in Table 2.
【００９０】[0090]
【表２】[Table 2]
【００９１】１０−４ 形質転換細胞の培養および抗
体の精製
実施例１０ー３で得られた細胞のうち抗体生産量の高い
Ｆ２６−１０２株およびＦ２６−１０３株について、そ
れぞれ培養を行った。培養は、イスコフ改変ダルベッコ
培地（ＩＭＤＭ）に５μｇ／ｍｌトランスフェリンと１
ｍｇ／ｍｌＢＳＡを添加した無血清培地で行った。５％
ＣＯ２下で３７℃にて５日間培養して、約４リットルの
培養上清を得た。この培養上清からの抗体の精製は実施
例４と同様に行い、キメラ抗体をそれぞれ約１２ｍｇ得
た。10-4 Culture of transformed cells and purification of antibodies Among the cells obtained in Example 10-3, strains F26-102 and F26-103, which produced high amounts of antibodies, were cultured, respectively. Culture was carried out in Iscove's modified Dulbecco's medium (IMDM) with 5 μg/ml transferrin and 1
It was performed in serum-free medium supplemented with mg/ml BSA. 5%
The cells were cultured at 37° C. under CO2 for 5 days to obtain about 4 liters of culture supernatant. Antibodies from this culture supernatant were purified in the same manner as in Example 4, and about 12 mg of each chimeric antibody was obtained.
【００９２】［実施例１１］（キメラ抗体の高カルシウ
ム血症動物モデルでの特性）実施例１０−４で精製した
キメラ抗体を用いて実施例５と同様に高カルシウム血症
動物モデルでの治療および予防効果を測定した。その結
果、キメラ抗体はマウスモノクローナル抗体と同程度の
効果があり、高カルシウム血症の治療・予防剤として非
常に有効であることが明らかになった。[Example 11] (Characteristics of chimeric antibody in a hypercalcemic animal model) Treatment in a hypercalcemic animal model in the same manner as in Example 5 using the chimeric antibody purified in Example 10-4. and the preventive effect was measured. The results revealed that the chimeric antibody was as effective as a mouse monoclonal antibody and was highly effective as a therapeutic and preventive agent for hypercalcemia.
【００９３】さらに、ＰＴＨｒＰを産生する前立腺癌細
胞ＰＣ−３（大日本製薬より購入）をヌードマウスに移
植し、血中カルシウムレベルが１８〜２０ｍｇ／ｄｌに
なった時点で、実施例４または１０−４で調製した抗体
をそれぞれ３００μｇ尾静脈より投与（各ｎ＝５）し、
高カルシウム血症治療・予防効果を検討した。その結果
、各抗体投与区のマウスはすべて生存しており、１週間
以上にわたり正常カルシウムレベルが維持された。これ
に対して、対照区のマウスのカルシウムレベルは１８〜
２０ｍｇ／ｄｌと不変であり、平均３〜４日後にはほと
んどのマウスが死亡した。Furthermore, prostate cancer cells PC-3 (purchased from Dainippon Pharmaceutical Co., Ltd.) producing PTHrP were transplanted into nude mice, and when the blood calcium level reached 18 to 20 mg/dl, the cells of Example 4 or 10 were transplanted. -300 μg of each antibody prepared in step 4 was administered from the tail vein (n = 5 each),
The therapeutic and preventive effects of hypercalcemia were investigated. As a result, all mice in each antibody administration group survived and maintained normal calcium levels for over a week. In contrast, calcium levels in control mice ranged from 18 to
The concentration remained unchanged at 20 mg/dl, and most of the mice died after an average of 3 to 4 days.
【００９４】[0094]
【発明の効果】本発明によれば、このように抗ヒト副甲
状腺ホルモン関連タンパクモノクローナル抗体またはそ
れらのフラグメントを含む高カルシウム血症治療・予防
剤が得られる。これを用いると、従来の抗血清あるいは
ポリクローナル抗体を用いた方法に比べ、高成績でかつ
比較的長期間にわたり高カルシウム血症を治療すること
が可能となる。According to the present invention, a hypercalcemia treatment/prevention agent containing an anti-human parathyroid hormone-related protein monoclonal antibody or a fragment thereof can be obtained. Using this method, it becomes possible to treat hypercalcemia with high results and for a relatively long period of time compared to conventional methods using antiserum or polyclonal antibodies.
【００９５】さらに、本発明においては、上記抗体の可
変部領域の配列を解明させた。これを利用して、遺伝子
操作の技術により、ヒト副甲状腺ホルモン関連タンパク
を認識するげっ歯類／ヒトキメラモノクローナル抗体が
調製される。これを主成分とする高カルシウム血症予防
・治療剤を投与するとアレルギー反応がより低くおさえ
られるため、充分な治療効果が得られる。Furthermore, in the present invention, the sequence of the variable region of the above antibody was elucidated. Utilizing this, a rodent/human chimeric monoclonal antibody that recognizes human parathyroid hormone-related protein is prepared by genetic engineering techniques. When a hypercalcemia preventive/therapeutic agent containing this as a main ingredient is administered, allergic reactions are suppressed to a lower level, and a sufficient therapeutic effect can be obtained.
【００９６】[0096]
【００９７】[0097]
【配列番号：１】配列の長さ：９８
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：タンパク質
フラグメント型：中間部フラグメント
配列の特徴
特徴を決定した方法：Ｅ
配列
Ｐｒｏ Ｇｌｕ Ｌｅｕ Ｖａｌ Ｌｙｓ Ｐｒｏ Ｇ
ｌｙ Ａｌａ Ｓｅｒ Ｖａｌ Ｌｙｓ Ｉｌｅ Ｓｅ
ｒ Ｃｙｓ Ｌｙｓ Ａｌａ １
５
１０ １５Ｓｅｒ
Ｇｌｙ Ｔｙｒ Ｓｅｒ Ｐｈｅ Ｔｈｒ Ｇｌｙ
Ｔｙｒ Ｐｈｅ Ｍｅｔ Ａｓｎ Ｔｒｐ Ｖａｌ Ｍ
ｅｔ Ｇｌｎ Ｓｅｒ ２０
２５
３０Ｈｉｓ Ｇｌｙ Ｌｙ
ｓ Ｓｅｒ Ｌｅｕ Ｇｌｕ Ｔｒｐ Ｉｌｅ Ｇｌｙ
Ａｒｇ Ｉｌｅ Ａｓｎ Ｐｒｏ Ｔｙｒ Ａｓｎ
Ｇｌｙ ３５
４０
４５Ａｓｐ Ｔｈｒ Ｐｈｅ Ｔｙｒ Ａｓｎ Ｇ
ｌｎ Ｌｙｓ Ｐｈｅ Ｌｙｓ Ｇｌｙ Ｌｙｓ Ａｌ
ａ Ｔｈｒ Ｌｅｕ Ｔｈｒ Ｖａｌ ５０
５５
６０Ａｓｐ Ｌｙｓ Ｓｅｒ
Ｓｅｒ Ｓｅｒ Ｔｈｒ Ａｌａ Ｈｉｓ Ｍｅｔ
Ｇｌｕ Ｌｅｕ Ａｒｇ Ｓｅｒ Ｌｅｕ Ａｌａ Ｓ
ｅｒ ６５ ７０
７５
８０Ｇｌｕ Ａｓｐ Ｓｅ
ｒ Ａｌａ Ｖａｌ Ｔｙｒ Ｔｙｒ Ｃｙｓ Ａｌａ
Ａｒｇ Ｇｌｙ Ｇｌｙ Ｖａｌ Ｔｈｒ Ｔｈｒ
Ｇｌｕ ８５
９０
９５Ｐｈｅ Ｇｌｙ
９８[SEQ ID NO: 1] Sequence length: 98 Sequence type: Amino acid Topology: Linear Sequence type: Protein fragment type: Intermediate fragment Characteristics of the sequence Method for determining the characteristics: E Sequence Pro Glu Leu Val Lys Pro G
ly Ala Ser Val Lys Ile Se
r Cys Lys Ala 1
5
10 15Ser
Gly Tyr Ser Phe Thr Gly
Tyr Phe Met Asn Trp Val M
et Gln Ser 20
25
30His Gly Ly
s Ser Leu Glu Trp Ile Gly
Arg Ile Asn Pro Tyr Asn
Gly 35
40
45Asp Thr Phe Tyr Asn G
ln Lys Phe Lys Gly Lys Al
a Thr Leu Thr Val 50
55
60Asp Lys Ser
Ser Ser Thr Ala His Met
Glu Leu Arg Ser Leu Ala S
er 65 70
75
80Glu Asp Se
r Ala Val Tyr Tyr Cys Ala
Arg Gly Gly Val Thr Thr
Glu85
90
95Phe Gly 98
【００９８】[0098]
【配列番号：２】配列の長さ：９２
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：タンパク質
フラグメント型：中間部フラグメント
配列の特徴
特徴を決定した方法：Ｅ
配列
Ｌｅｕ Ｓｅｒ Ｌｅｕ Ｐｒｏ Ｖａｌ Ｓｅｒ Ｌ
ｅｕ Ｇｌｙ Ａｓｐ Ｇｌｎ Ａｌａ Ｓｅｒ Ｉｌ
ｅ Ｓｅｒ Ｃｙｓ Ａｒｇ １
５
１０ １５Ｓｅｒ
Ｓｅｒ Ｇｌｎ Ｓｅｒ Ｉｌｅ Ｖａｌ Ｈｉｓ
Ｓｅｒ Ａｓｎ Ｇｌｙ Ａｓｎ Ｔｈｒ Ｔｙｒ Ｌ
ｅｕ Ｇｌｕ Ｔｒｐ ２０
２５
３０Ｔｙｒ Ｌｅｕ Ｇｌ
ｎ Ｌｙｓ Ｐｒｏ Ｇｌｙ Ｇｌｎ Ｓｅｒ Ｐｒｏ
Ｌｙｓ Ｌｅｕ Ｌｅｕ Ｉｌｅ Ｔｙｒ Ｌｙｓ
Ｖａｌ ３５
４０
４５Ｓｅｒ Ａｓｎ Ａｒｇ Ｐｈｅ Ｓｅｒ Ｇ
ｌｙ Ｖａｌ Ｐｒｏ Ａｓｐ Ａｒｇ Ｐｈｅ Ｓｅ
ｒ Ｇｌｙ Ｓｅｒ Ｇｌｙ Ｓｅｒ ５０
５５
６０Ｇｌｙ Ｔｈｒ Ａｓｐ
Ｐｈｅ Ｔｈｒ Ｌｅｕ Ｌｙｓ Ｉｌｅ Ｓｅｒ
Ａｒｇ Ｖａｌ Ｇｌｕ Ａｌａ Ｇｌｕ Ａｓｐ Ｌ
ｅｕ ６５ ７０
７５
８０Ｇｌｙ Ｖａｌ Ｔｙ
ｒ Ｔｙｒ Ｃｙｓ Ｐｈｅ Ｇｌｎ Ｇｌｙ Ｓｅｒ
Ｈｉｓ Ｖａｌ Ｐｒｏ
８５ ９
０ ９２[SEQ ID NO: 2] Sequence length: 92 Sequence type: Amino acids Topology: Linear Sequence type: Protein fragment type: Intermediate fragment Characteristics of the sequence Method for determining the characteristics: E Sequence Leu Ser Leu Pro Val Ser L
eu Gly Asp Gln Ala Ser Il
e Ser Cys Arg 1
5
10 15Ser
Ser Gln Ser Ile Val His
Ser Asn Gly Asn Thr Tyr L
eu Glu Trp 20
25
30Tyr Leu Gl
n Lys Pro Gly Gln Ser Pro
Lys Leu Leu Ile Tyr Lys
Val 35
40
45Ser Asn Arg Phe Ser G
ly Val Pro Asp Arg Phe Se
r Gly Ser Gly Ser 50
55
60Gly Thr Asp
Phe Thr Leu Lys Ile Ser
Arg Val Glu Ala Glu Asp L
eu 65 70
75
80Gly Val Ty
r Tyr Cys Phe Gln Gly Ser
His Val Pro
85 9
0 92
【００９９】0099
【配列番号：３】配列の長さ：１１６
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：タンパク質
フラグメント型：中間部フラグメント
配列の特徴
特徴を決定した方法：Ｅ
配列
Ｍｅｔ Ｇｌｎ Ｖａｌ Ｇｌｎ Ｌｅｕ Ｇｌｎ Ｇ
ｌｎ Ｓｅｒ Ｇｌｙ Ｐｒｏ Ｇｌｕ Ｌｅｕ Ｖａ
ｌ Ｌｙｓ Ｐｒｏ Ｇｌｙ １
５
１０ １５Ａｌａ
Ｓｅｒ Ｖａｌ Ｌｙｓ Ｉｌｅ Ｓｅｒ Ｃｙｓ
Ｌｙｓ Ａｌａ Ｓｅｒ Ｇｌｙ Ｔｙｒ Ｓｅｒ Ｐ
ｈｅ Ｔｈｒ Ｇｌｙ ２０
２５
３０Ｔｙｒ Ｐｈｅ Ｍｅ
ｔ Ａｓｎ Ｔｒｐ Ｖａｌ Ｍｅｔ Ｇｌｎ Ｓｅｒ
Ｈｉｓ Ｇｌｙ Ｌｙｓ Ｓｅｒ Ｌｅｕ Ｇｌｕ
Ｔｒｐ ３５
４０
４５Ｉｌｅ Ｇｌｙ Ａｒｇ Ｉｌｅ Ａｓｎ Ｐ
ｒｏ Ｔｙｒ Ａｓｎ Ｇｌｙ Ａｓｐ Ｔｈｒ Ｐｈ
ｅ Ｔｙｒ Ａｓｎ Ｇｌｎ Ｌｙｓ ５０
５５
６０Ｐｈｅ Ｌｙｓ Ｇｌｙ
Ｌｙｓ Ａｌａ Ｔｈｒ Ｌｅｕ Ｔｈｒ Ｖａｌ
Ａｓｐ Ｌｙｓ Ｓｅｒ Ｓｅｒ Ｓｅｒ Ｔｈｒ Ａ
ｌａ ６５ ７０
７５
８０Ｈｉｓ Ｍｅｔ Ｇｌ
ｕ Ｌｅｕ Ａｒｇ Ｓｅｒ Ｌｅｕ Ａｌａ Ｓｅｒ
Ｇｌｕ Ａｓｐ Ｓｅｒ Ａｌａ Ｖａｌ Ｔｙｒ
Ｔｙｒ ８５
９０
９５Ｃｙｓ Ａｌａ Ａｒｇ Ｇ
ｌｙ Ｇｌｙ Ｖａｌ Ｔｈｒ Ｔｈｒ Ｇｌｕ Ｐｈ
ｅ Ｇｌｙ Ｔｙｒ Ｔｒｐ Ｇｌｙ Ｐｒｏ Ｇｌｙ
１００
１０５
１１０Ｔｈｒ Ｔｈｒ Ｖａｌ ＴＨｒ
１１５[SEQ ID NO: 3] Sequence length: 116 Sequence type: Amino acids Topology: Linear Sequence type: Protein fragment type: Intermediate fragment Characteristics of the sequence Method for determining the characteristics: E Sequence Met Gln Val Gln Leu Gln G
ln Ser Gly Pro Glu Leu Va
l Lys Pro Gly 1
5
10 15Ala
Ser Val Lys Ile Ser Cys
Lys Ala Ser Gly Tyr Ser P
he Thr Gly 20
25
30Tyr Phe Me
t Asn Trp Val Met Gln Ser
His Gly Lys Ser Leu Glu
Trp35
40
45Ile Gly Arg Ile Asn P
ro Tyr Asn Gly Asp Thr Ph
e Tyr Asn Gln Lys 50
55
60Phe Lys Gly
Lys Ala Thr Leu Thr Val
Asp Lys Ser Ser Ser Thr A
la 65 70
75
80His Met Gl
u Leu Arg Ser Leu Ala Ser
Glu Asp Ser Ala Val Tyr
Tyr 85
90
95Cys Ala Arg G
ly Gly Val Thr Thr Glu Ph
e Gly Tyr Trp Gly Pro Gly
100
105
110Thr Thr Val THr 115
【０１００】[0100]
【配列番号：４】配列の長さ：１１３
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：タンパク質
フラグメント型：中間部フラグメント
配列の特徴
特徴を決定した方法：Ｅ
配列
Ｌｅｕ Ｇｌｕ Ｇｌｕ Ｉｌｅ Ｖａｌ Ｍｅｔ Ｔ
ｈｒ Ｇｌｎ Ｓｅｒ Ｐｒｏ Ｌｅｕ Ｓｅｒ Ｌｅ
ｕ Ｐｒｏ Ｖａｌ Ｓｅｒ １
５
１０ １５Ｌｅｕ
Ｇｌｙ Ａｓｐ Ｇｌｎ Ａｌａ Ｓｅｒ Ｉｌｅ
Ｓｅｒ Ｃｙｓ Ａｒｇ Ｓｅｒ Ｓｅｒ Ｇｌｎ Ｓ
ｅｒ Ｉｌｅ Ｖａｌ ２０
２５
３０Ｈｉｓ Ｓｅｒ Ａｓ
ｎ Ｇｌｙ Ａｓｎ Ｔｈｒ Ｔｙｒ Ｌｅｕ Ｇｌｕ
Ｔｒｐ Ｔｙｒ Ｌｅｕ Ｇｌｎ Ｌｙｓ Ｐｒｏ
Ｇｌｙ ３５
４０
４５Ｇｌｎ Ｓｅｒ Ｐｒｏ Ｌｙｓ Ｌｅｕ Ｌ
ｅｕ Ｉｌｅ Ｔｙｒ Ｌｙｓ Ｖａｌ Ｓｅｒ Ａｓ
ｎ Ａｒｇ Ｐｈｅ Ｓｅｒ Ｇｌｙ ５０
５５
６０Ｖａｌ Ｐｒｏ Ａｓｐ
Ａｒｇ Ｐｈｅ Ｓｅｒ Ｇｌｙ Ｓｅｒ Ｇｌｙ
Ｓｅｒ Ｇｌｙ Ｔｈｒ Ａｓｐ Ｐｈｅ Ｔｈｒ Ｌ
ｅｕ ６５ ７０
７５
８０Ｌｙｓ Ｉｌｅ Ｓｅ
ｒ Ａｒｇ Ｖａｌ Ｇｌｕ Ａｌａ Ｇｌｕ Ａｓｐ
Ｌｅｕ Ｇｌｙ Ｖａｌ Ｔｙｒ Ｔｙｒ Ｃｙｓ
Ｐｈｅ ８５
９０
９５Ｇｌｎ Ｇｌｙ Ｓｅｒ Ｈ
ｉｓ Ｖａｌ Ｐｒｏ Ｔｙｒ Ｔｈｒ Ｐｈｅ Ｇｌ
ｙ Ａｌａ Ｇｌｙ Ｔｈｒ Ｌｙｓ Ｌｅｕ Ａｌａ
１００
１０５
１１０Ａｒｇ
１１３[SEQ ID NO: 4] Sequence length: 113 Sequence type: Amino acid Topology: Linear Sequence type: Protein fragment type: Intermediate fragment Characteristics of the sequence Characteristics were determined: E Sequence Leu Glu Glu Ile Val Met T
hr Gln Ser Pro Leu Ser Le
u Pro Val Ser 1
5
10 15 Leu
Gly Asp Gln Ala Ser Ile
Ser Cys Arg Ser Ser Gln S
er Ile Val 20
25
30His Ser As
n Gly Asn Thr Tyr Leu Glu
Trp Tyr Leu Gln Lys Pro
Gly 35
40
45Gln Ser Pro Lys Leu L
eu Ile Tyr Lys Val Ser As
n Arg Phe Ser Gly 50
55
60Val Pro Asp
Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr L
eu 65 70
75
80Lys Ile Se
r Arg Val Glu Ala Glu Asp
Leu Gly Val Tyr Tyr Cys
Phe 85
90
95Gln Gly Ser H
is Val Pro Tyr Thr Phe Gl
y Ala Gly Thr Lys Leu Ala
100
105
110Arg 113
【０１０１】[0101]
【配列番号：５】配列の長さ：３５６
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：ｃＤＮＡ ｔｏ ｍＲＮＡフラグメン
ト型：中間部フラグメント
配列
ＴＣＴＡＧＡ ＡＴＧ ＣＡＧ ＧＴＴ ＣＡＧ ＣＴ
Ｇ ＣＡＧ ＣＡＧ ＴＣＴ ＧＧＡ ＣＣＴ ＧＡＧ
ＣＴＧ ＧＴＧ ＡＡＧ ＣＣＴ ５１ＧＧＧ
ＧＣＴ ＴＣＡ ＧＴＧ ＡＡＧ ＡＴＡ ＴＣＣ
ＴＧＣ ＡＡＧ ＧＣＴ ＴＣＴ ＧＧＴ ＴＡＣ Ｔ
ＣＡ ＴＴＴ ＡＣＴ ９９ＧＧＣ ＴＡ
Ｃ ＴＴＴ ＡＴＧ ＡＡＣ ＴＧＧ ＧＴＧ ＡＴＧ
ＣＡＧ ＡＧＣ ＣＡＴ ＧＧＡ ＡＡＧ ＡＧＣ
ＣＴＴ ＧＡＧ １４７ＴＧＧ ＡＴＴ Ｇ
ＧＡ ＣＧＴ ＡＴＴ ＡＡＴ ＣＣＴ ＴＡＣ ＡＡ
Ｔ ＧＧＴ ＧＡＴ ＡＣＴ ＴＴＣ ＴＡＣ ＡＡＣ
ＣＡＧ １９５ＡＡＧ ＴＴＣ ＡＡＧ
ＧＧＣ ＡＡＧ ＧＣＣ ＡＣＡ ＴＴＧ ＡＣＴ Ｇ
ＴＡ ＧＡＣ ＡＡＡ ＴＣＣ ＴＣＴ ＡＧＣ ＡＣ
Ａ ２４３ＧＣＣ ＣＡＣ ＡＴＧ ＧＡＧ
ＣＴＣ ＣＧＧ ＡＧＣ ＣＴＧ ＧＣＡ ＴＣＴ
ＧＡＧ ＧＡＣ ＴＣＴ ＧＣＡ ＧＴＣ ＴＡＴ
２９１ＴＡＴ ＴＧＴ ＧＣＡ ＡＧＡ ＧＧ
Ｇ ＧＧＧ ＧＴＴ ＡＣＧ ＡＣＧ ＧＡＧ ＴＴＴ
ＧＧＴ ＴＡＣ ＴＧＧ ＧＧＴ ＣＣＡ
３３９ＧＧＧ ＡＣＣ ＡＣＧ ＧＴＣ ＡＣＴ Ａ
Ｇ
３５
６[SEQ ID NO: 5] Sequence length: 356 Sequence type: Number of nucleic acid strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Fragment type: Intermediate fragment sequence TCTAGA ATG CAG GTT CAG CT
G CAG CAG TCT GGA CCT GAG
CTG GTG AAG CCT 51GGG
GCT TCA GTG AAG ATA TCC
TGC AAG GCT TCT GGT TAC T
CA TTT ACT 99GGC TA
C TTT ATG AAC TGG GTG ATG
CAG AGC CAT GGA AAG AGC
CTT GAG 147TGG ATT G
GA CGT ATT AAT CCT TAC AA
T GGT GAT ACT TTC TAC AAC
CAG 195AAG TTC AAG
GGC AAG GCC ACA TTG ACT G
TA GAC AAA TCC TCT AGC AC
A 243GCC CAC ATG GAG
CTC CGG AGC CTG GCA TCT
GAG GAC TCT GCA GTC TAT
291TAT TGT GCA AGA GG
G GGG GTT ACG ACG GAG TTT
GGT TAC TGG GGT CCA
339GGG ACC ACG GTC ACT A
G
35
6
【０１０２】[0102]
【配列番号：６】配列の長さ：３４１
配列の型：核酸
鎖の数：二本鎖
トポロジー：直鎖状
配列の種類：ｃＤＮＡ ｔｏ ｍＲＮＡフラグメン
ト型：中間部フラグメント
配列
Ｔ ＣＴＡ ＧＡＡ ＧＡＡ ＡＴＴ ＧＴＧ ＡＴＧ
ＡＣＣ ＣＡＧ ＴＣＴ ＣＣＡ ＣＴＣ ＴＣＣ
ＣＴＧ ＣＣＴ ＧＴＣ ＡＧＴ ４９ＣＴＴ
ＧＧＡ ＧＡＴ ＣＡＡ ＧＣＣ ＴＣＣ ＡＴＣ
ＴＣＴ ＴＧＣ ＡＧＡ ＴＣＴ ＡＧＴ ＣＡＧ Ａ
ＧＣ ＡＴＴ ＧＴＡ ９７ＣＡＴ ＡＧ
Ｔ ＡＡＴ ＧＧＡ ＡＡＣ ＡＣＣ ＴＡＴ ＴＴＡ
ＧＡＡ ＴＧＧ ＴＡＣ ＣＴＧ ＣＡＧ ＡＡＡ
ＣＣＡ ＧＧＣ １４５ＣＡＧ ＴＣＴ Ｃ
ＣＡ ＡＡＧ ＣＴＣ ＣＴＧ ＡＴＣ ＴＡＣ ＡＡ
Ａ ＧＴＴ ＴＣＣ ＡＡＣ ＣＧＡ ＴＴＴ ＴＣＴ
ＧＧＧ １９９ＧＴＣ ＣＣＡ ＧＡＣ
ＡＧＧ ＴＴＣ ＡＧＴ ＧＧＣ ＡＧＴ ＧＧＡ Ｔ
ＣＡ ＧＧＧ ＡＣＡ ＧＡＴ ＴＴＣ ＡＣＡ ＣＴ
Ｃ ２４１ＡＡＧ ＡＴＣ ＡＧＣ ＡＧＡ
ＧＴＧ ＧＡＧ ＧＣＴ ＧＡＧ ＧＡＴ ＣＴＧ
ＧＧＡ ＧＴＴ ＴＡＴ ＴＡＣ ＴＧＣ ＴＴＴ
２８９ＣＡＡ ＧＧＴ ＴＣＡ ＣＡＴ ＧＴ
Ｔ ＣＣＧ ＴＡＣ ＡＣＧ ＴＴＣ ＧＧＴ ＧＣＴ
ＧＧＧ ＡＣＣ ＡＡＧ ＣＴＧ ＧＣＴ
３３７ＣＧＡ Ｇ
３４
１[SEQ ID NO: 6] Sequence length: 341 Sequence type: Number of nucleic acid strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Fragment type: Middle fragment sequence T CTA GAA GAA ATT GTG ATG
ACC CAG TCT CCA CTC TCC
CTG CCT GTC AGT 49CTT
GGA GAT CAA GCC TCC ATC
TCT TGC AGA TCT AGT CAG A
GC ATT GTA 97CAT AG
T AAT GGA AAC ACC TAT TTA
GAA TGG TAC CTG CAG AAA
CCA GGC 145CAG TCT C
CA AAG CTC CTG ATC TAC AA
A GTT TCC AAC CGA TTT TCT
GGG 199GTC CCA GAC
AGG TTC AGT GGC AGT GGA T
CA GGG ACA GAT TTC ACA CT
C 241AAG ATC AGC AGA
GTG GAG GCT GAG GAT CTG
GGA GTT TAT TAC TGC TTT
289CAA GGT TCA CAT GT
T CCG TAC ACG TTC GGT GCT
GGG ACC AAG CTG GCT
337CGA G
34
1
【０１０３】[0103]
【配列番号：７】配列の長さ：３４
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：ペプチド
フラブメント型：中間部フラグメント
配列の特徴
特徴を決定した方法：Ｓ
配列
Ａｌａ Ｖａｌ Ｓｅｒ Ｇｌｕ Ｈｉｓ
Ｇｌｎ Ｌｅｕ Ｌｅｕ Ｈｉｓ Ａｓｐ
Ｌｙｓ Ｇｌｙ Ｌｙｓ Ｓｅｒ Ｉｌｅ
Ｇｌｎ １
５
１０
１５Ａｓｐ Ｌｅｕ
Ａｒｇ Ａｒｇ Ａｒｇ Ｐｈｅ Ｐｈｅ
Ｌｅｕ Ｈｉｓ Ｈｉｓ Ｌｅｕ Ｉｌｅ
Ａｌａ Ｇｌｕ Ｉｌｅ Ｈｉｓ
２０
２５
３０Ｔｈｒ
Ａｌａ
３４[SEQ ID NO: 7] Sequence length: 34 Sequence type: Amino acid Topology: Linear Sequence type: Peptide fragment type: Intermediate fragment Features of the sequence Characteristics were determined: S Sequence Ala Val Ser Glu His
Gln Leu Leu His Asp
Lys Gly Lys Ser Ile
Gln 1
5
10
15Asp Leu
Arg Arg Arg Phe Phe
Leu His His Leu Ile
Ala Glu Ile His
20
25
30Thr
Ala 34
【０１０４】[0104]
【配列番号：８】配列の長さ：３２
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列
ＴＣＴＡＧＡＡＴＧＣ ＡＧＧＴＹＣＡＧＣＴ Ｇ
ＭＡＧＣＡＧＴＣＷ ＧＧ
３２[SEQ ID NO: 8] Sequence length: 32 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence TCTAGAATGC AGGTYCAGCT G
MAGCAGTCW GG
32
【０１０５】[0105]
【配列番号：９】配列の長さ：４２
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列
ＧＧＴＧＴＴＣＣＴＣ ＧＡＧＧＴＣＴＡＧＴ ＧＡＣ
ＣＧＴＧＧＴＣ ＣＣＴＫＳＲＣＣＣＣ ＡＧ
４２[SEQ ID NO: 9] Sequence length: 42 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence GGTGTTCCTC GAGGTCTAGT GAC
CGTGGTC CCTKSRCCC
42
【０１０６】[0106]
【配列番号：１０】配列の長さ：３７
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列
ＣＧＴＧＧＣＴＣＴＡ ＧＡＡＧＡＡＡＴＴＧ Ｔ
ＧＭＴＧＡＣＣＣＡ ＧＴＣＴＣＣＡ
３７[SEQ ID NO: 10] Sequence length: 37 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence CGTGGCTCTA GAAGAAATTG T
GMTGACCCA GTCTCCA
37
【０１０７】[0107]
【配列番号：１１】配列の長さ：３４
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：合成ＤＮＡ
配列
ＡＡＣＴＣＧＡＧＣＣ ＡＧＣＴＴＧＧＴＣＣ Ｃ
ＭＳＣＷＣＣＧＡＡ ＣＧＴＧ
３４[SEQ ID NO: 11] Sequence length: 34 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence AACTCGAGCC AGCTTGGTCC C
MSCWCCGAACGTG
34
【０１０８】[0108]
【配列番号：１２】配列の長さ：４８
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列
ＡＧＧＴＧＣＡＧＣＴ ＡＣＡＧＣＡＧＴＧＧ ＧＧＣ
ＧＣＡＧＧＡＣ ＴＧＧＴＧＡＡＧＣＣ ＴＴＣＧＧＡ
ＧＡ ４８[SEQ ID NO: 12] Sequence length: 48 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence AGGTGCAGCT ACAGCAGTGG GGC
GCAGGAC TGGTGAAGCC TTCGGA
GA 48
【０１０９】[0109]
【配列番号：１３】配列の長さ：６６
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列
ＡＣＴＡＣＴＡＣＴＡ ＴＧＧＴＡＴＧＧＡＣ Ｇ
ＴＣＴＧＧＧＧＣＣ ＡＡＧＧＧＡＣＣＡＣ ＧＧ
ＴＣＡＣＣＧＴＣ ＴＣＣＴＣＡ
６６[SEQ ID NO: 13] Sequence length: 66 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence ACTACTACTA TGGTATGGAC G
TCTGGGGCC AAGGGACCAC GG
TCACCGTC TCCTCA
66
【０１１０】[0110]
【配列番号：１４】配列の長さ：５０
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列
ＡＣＴＧＴＧＧＣＴＧ ＣＡＣＣＡＴＣＴＧＴ Ｃ
ＴＴＣＡＴＣＴＴＣ ＣＣＧＣＣＡＴＣＴＧ ＡＴ
ＧＡＧＣＡＧＴＴ
５０[SEQ ID NO: 14] Sequence length: 50 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence ACTGTGGCTG CACCATCTGT C
TTCATCTTC CCGCCATCTG AT
GAGCAGTT
50
【０１１１】[0111]
【配列番号：１５】配列の長さ：８９
配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列
ＣＣＴＣＴＡＧＡＴＧ ＡＡＣＴＴＣＧＧＧＣ ＴＣＡ
ＧＣＴＴＧＡＴ ＴＴＡＣＣＴＴＧＴＣ ＣＴＧＧＴＴ
ＴＴＡＡ ＡＡＧＴＴＧＴＣＣＡ ６０ＧＴＧ
ＴＣＡＧＧＴＴ ＣＡＧＣＴＧＣＡＧＣ ＡＧＴＣＴＧ
ＧＡＣ
８９[SEQ ID NO: 15] Sequence length: 89 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence CCTCTAGATG AACTTCGGGC TCA
GCTTGAT TTACCTTGTC CTGGTT
TTAA AAGTTGTCCA 60GTG
TCAGGTT CAGCTGCAGC AGTCTG
G.A.C.
89
【０１１２】[0112]
【配列番号：１６】配列の長さ：１０９配列の型：核酸
鎖の数：一本鎖
トポロジー：直鎖状
配列の種類：他の核酸 合成ＤＮＡ
配列
ＣＣＴＣＴＡＧＡＴＧ ＧＧＣＡＴＣＡＡＧＡ ＴＧＧ
ＡＧＡＣＡＣＡ ＴＴＣＴＣＡＧＧＴＣ ＴＴＴＧＴＡ
ＴＡＣＡ ＴＧＴＴＧＣＴＧＴＧ ６０ＧＴＴ
ＧＴＣＴＧＧＴ ＧＴＴＧＡＡＧＧＡＧ ＡＡＡＴＴＧ
ＴＧＡＴ ＧＡＣＣＣＡＧＴＣＴ ＣＣＡＣＴＣＴＣＣ
１０９[SEQ ID NO: 16] Sequence length: 109 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids Synthetic DNA Sequence CCTCTAGATG GGCATCAAGA TGG
AGACACA TTCTCAGGTC TTTGTA
TACA TGTTGCTGTG 60GTT
GTCTGGT GTTGAAGGAG AAATTG
TGAT GACCCAGTCT CCACTCTCC
109
【図１】高カルシウム血症動物モデルを用いた、抗ＰＴ
ＨｒＰ モノクローナル抗体の高カルシウム血症に対す
る治療・予防効果を示すグラフである。[Figure 1] Anti-PT using a hypercalcemic animal model
1 is a graph showing the therapeutic and preventive effects of HrP monoclonal antibodies on hypercalcemia.
【図２】本発明のキメラモノクローナル抗体をコードす
る遺伝子を有する発現ベクターを調製するために用いら
れるｐＳＶｅＨ２６の構築を示す説明図である。FIG. 2 is an explanatory diagram showing the construction of pSVeH26 used to prepare an expression vector containing a gene encoding the chimeric monoclonal antibody of the present invention.
【図３】本発明のキメラモノクローナル抗体をコードす
る遺伝子を有する発現ベクターを調製するために用いら
れるｐＳＶｅＬ２６の構築を示す説明図である。FIG. 3 is an explanatory diagram showing the construction of pSVeL26 used to prepare an expression vector containing a gene encoding the chimeric monoclonal antibody of the present invention.
【図４】本発明のキメラモノクローナル抗体をコードす
る遺伝子を有する発現ベクターであるｐＳＶｅＨＬ２６
の構築を示す説明図である。FIG. 4: pSVeHL26, an expression vector carrying a gene encoding the chimeric monoclonal antibody of the present invention.
FIG.
【図５】本発明のキメラモノクローナル抗体をコードす
る遺伝子を有する発現ベクターであるｐＳＶｅＨＬ２６
−ＤＭの構築を示す説明図である。FIG. 5: pSVeHL26, an expression vector carrying a gene encoding the chimeric monoclonal antibody of the present invention.
- It is an explanatory diagram showing construction of DM.
Claims (13)
クローナル抗体およびその関連物質でなる群から選択さ
れる少なくとも１種を有効成分として含有する高カルシ
ウム血症治療・予防剤。1. A hypercalcemia treatment/prevention agent containing as an active ingredient at least one member selected from the group consisting of anti-human parathyroid hormone-related protein monoclonal antibodies and substances related thereto.
ラグメントおよび／または該フラグメントを含むペプチ
ドを含む請求項１に記載の治療・予防剤。2. The therapeutic/prophylactic agent according to claim 1, which comprises an F(ab') fragment of the monoclonal antibody and/or a peptide containing the fragment.
ＶＯ１４１１Ｇの産生するモノクローナル抗体の群から
選択されるモノクローナル抗体を含有する請求項１に記
載の治療・予防剤。Claim 3: Hybridoma EVO34B1G or E
The therapeutic/prophylactic agent according to claim 1, which contains a monoclonal antibody selected from the group of monoclonal antibodies produced by VO1411G.
ラグメントおよび／または該フラグメントを含むペプチ
ドを含む請求項３に記載の治療・予防剤。4. The therapeutic/prophylactic agent according to claim 3, which comprises an F(ab') fragment of the monoclonal antibody and/or a peptide containing the fragment.
クローナル抗体のＨ鎖可変部領域をコードする遺伝子断
片であって、配列番号１に記載のアミノ酸配列をコード
する遺伝子配列を少なくともその一部に有する遺伝子断
片。5. A gene fragment encoding the H chain variable region of an anti-human parathyroid hormone-related protein monoclonal antibody, which has at least a portion of the gene sequence encoding the amino acid sequence set forth in SEQ ID NO: 1. piece.
クローナル抗体のＬ鎖可変部領域をコードする遺伝子断
片であって、配列番号２に記載のアミノ酸配列をコード
する遺伝子配列を少なくともその一部に有する遺伝子断
片。6. A gene fragment encoding the L chain variable region of an anti-human parathyroid hormone-related protein monoclonal antibody, which has at least a portion of the gene sequence encoding the amino acid sequence set forth in SEQ ID NO: 2. piece.
クローナル抗体であって、その可変部領域の少なくとも
一部に配列番号１に記載のアミノ酸配列を有する、モノ
クローナル抗体。7. An anti-human parathyroid hormone-related protein monoclonal antibody, which has the amino acid sequence set forth in SEQ ID NO: 1 in at least a portion of its variable region.
クローナル抗体であって、その可変部領域の少なくとも
一部に配列番号２に記載のアミノ酸配列を有する、モノ
クローナル抗体。8. An anti-human parathyroid hormone-related protein monoclonal antibody, which has the amino acid sequence set forth in SEQ ID NO: 2 in at least a portion of its variable region.
の定常部領域を有し、ヒトの副甲状腺ホルモン関連タン
パクを認識するげっ歯類／ヒトキメラモノクローナル抗
体、またはその誘導体。9. A rodent/human chimeric monoclonal antibody, or a derivative thereof, which has a variable region derived from a rodent and a constant region derived from a human, and which recognizes human parathyroid hormone-related protein.
シウム血症の治療効果を有する、請求項９に記載のモノ
クローナル抗体またはその誘導体。10. The monoclonal antibody or its derivative according to claim 9, wherein the chimeric monoclonal antibody has a therapeutic effect on hypercalcemia.
抗体をコードする融合遺伝子。11. A fusion gene encoding the chimeric monoclonal antibody according to claim 9.
ル抗体およびその誘導体の少なくとも１種を有効成分と
して含有する高カルシウム血症治療・予防剤。12. A hypercalcemia treatment/prevention agent containing at least one of the chimeric monoclonal antibody and its derivative according to claim 10 as an active ingredient.
れかに記載のモノクローナル抗体を産生するセルライン
。13. A cell line that produces the monoclonal antibody according to any one of claims 1, 3, 7, 9, or 11.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3110565A JPH04228089A (en) | 1990-05-15 | 1991-05-15 | Agent for treatment and prevention of hypercalcemia |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2-124581 | 1990-05-15 | ||
| JP12458190 | 1990-05-15 | ||
| JP3110565A JPH04228089A (en) | 1990-05-15 | 1991-05-15 | Agent for treatment and prevention of hypercalcemia |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04228089A true JPH04228089A (en) | 1992-08-18 |
Family
ID=26450171
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3110565A Withdrawn JPH04228089A (en) | 1990-05-15 | 1991-05-15 | Agent for treatment and prevention of hypercalcemia |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04228089A (en) |
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| WO1996039184A1 (en) * | 1995-06-06 | 1996-12-12 | The Regents Of The University Of California | THERAPEUTIC SEPSIS TREATMENT USING ANTAGONISTS TO PTHrP |
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| WO1998013388A1 (en) | 1996-09-26 | 1998-04-02 | Chugai Seiyaku Kabushiki Kaisha | Antibody against human parathormone related peptides |
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